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11859096 | DETAILED DESCRIPTION Various embodiments will now be described more fully with reference to the accompanying drawings. The disclosure may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. FIG.1is a flow diagram of a process100for applying a gas impermeable coating.FIG.2(a)is a diagrammatic illustration of a polyelectrolyte complex.FIG.2(b)is a diagrammatic illustration of the process100. Referring toFIGS.1-2(b) collectively, the process100begins at block102. At block104, a polyelectrolyte complex (PEC) suspension is formed. PECs are formed by the entropy-driven association of oppositely-charged polyelectrolytes in water and can exist as stable colloids, flocculants, or metastable coacervates. Governed by conditions such as, for example, pH and ionic strength, PEC coacervation is marked by a liquid-liquid phase separation, where a polymer-rich coacervate phase is in equilibrium with a polymer-poor solution phase. PEC coacervates are comprised of weakly bound polyelectrolytes, and have viscous liquid-like behavior that can be exploited to quickly apply them as thin films. As illustrated inFIG.2(a), the PEC coacervate complex is comprised of at least two oppositely charged polyelectrolytes (202aand202b) in water204. In various embodiments, cationic polydiallyldimethylammonium chloride (PDDA) and anionic polyacrylic acid (PAA), for example, may be used to form the coacervate. Other embodiments may make use of strong polyelectrolytes. Other further embodiments may make use of, for example, Polyethylenimine, Poly(allyl amine), polyethylenimine (PEI), Chitosan, Functionalized starch, Poly(vinyl amine), Poly(vinylbenzyltrimethyl-ammonium) bromide, and Poly(4-vinyl-N-butyl-pyridinium) chloride as a polycation, and Poly(acrylic acid) and sodium polyacrylate, Poly(styrene sulfonic acid) and sodium poly(styrene sulfonate), Carboxymethylcellulose, Alginic acid, Hyaluronic acid, Heparin, and Polyphosphoric acid and Polyphosphate salts as a polyanion. The ionic strengths of the polycation and polyanion are controlled in order to make the polyanion and the polycation suitable for a coating process. The ionic strength is normally manipulated by the addition of salt such as, for example, NaCl. The pH of the PEC is controlled such that the polyelectrolytes dissolve and there is no hydrogen bonding or electrostatic interaction between the polyelectrolytes. In embodiments utilizing a strong polyanion and a weak polycation, the PEC is formed at a pH of, for example, 2. In embodiments utilizing a strong polycation and a weak polyanion, the pH of the PEC is controlled to have a basic (i.e. greater than 7) pH to dissolve the polyelectrolytes. As used herein, the term “strong” (e.g. strong polyelectrolyte, strong polycation, or strong polyanion) refers to a polyelectrolyte that dissolves completely in solution for most pH values. The term “weak” (e.g. weak polyelectrolyte, weak polycation, or weak polyanion) refers to a polyelectrolyte that are not fully charged in solution and are partially dissociated at intermediate pH. The fractional charge of a weak polyelectrolyte can be modified by changing the solution pH, counter-ion concentration, or ionic strength. As used herein, the term “ionic strength” refers to a measure of the concentration of ions in a solution. In various embodiments, regulation of ionic-strength via, for example, addition of salt, could be utilized in lieu of, or in addition to, pH regulation in order to control interaction of the polyelectrolytes. Other embodiments may utilize two weak polyelectrolytes. In embodiments utilizing two weak polyelectrolytes, the polyelectrolytes are controlled utilizing at least one of a low-pH solution, a high-pH solution, and ionic-strength control. At block106, the PEC suspension is applied to a substrate such as, for example, polyethylene terephthalate (PET), poly(lactic acid) (PLA), polyurethane (PU), polyethylene (PE), polystyrene (PS). The substrate208may be of any thickness as dictated by the particular application. As illustrated inFIG.2(b), in a typical embodiment, the PEC suspension206is applied to the substrate208using, for example, a dip process. In other embodiments, however, the PEC suspension could be applied to the substrate using, for example, gravure application, slot-die application, roll-to-roll application, or by use of an instrument such as, for example, a Mayer rod. During application of the PEC suspension206to the substrate208, the thickness of the applied PEC suspension206is controlled by viscosity and the presence of dissolved solids. At block108, the coated substrate209is dried. In a typical embodiment, the coated substrate208is dried, for example, for approximately 20 minutes at, for example, approximately 150° C.; however, in other embodiments, other temperatures and drying times could be utilized. Such drying evaporates excess water from the PEC206and has been demonstrated to improve optical clarity of the gas-impermeable coating when compared to processes where the drying block108is omitted. At block110, the coated substrate209is immersed in a buffering solution210. In a typical embodiment, the buffering solution210has a pH of a weak acid such as, for example, in the range of 3-5. In various embodiments, the buffering solution210may be, for example, acetic acid, citric acid, a phosphate buffer, or a trizma buffer as dictated by the polyelectrolytes used in the PEC206. In embodiments utilizing a strong polyanion and a weak polycation, a buffering solution210having a basic pH is desirable. During block110, the pH of the buffering solution210causes pH-induced ionic bonding in the PEC206, which results in curing of the PEC206. In a typical embodiment, utilization of the buffering solution210to result in a cured PEC coating211results in approximately a 10-fold improvement in gas-impermeability than if the buffering solution210were not utilized. In various embodiments, one or both of blocks108and110may be omitted from the process100depending on the needs of the particular application. The process100ends at block112. FIG.3is a table illustrating thickness of the gas-impermeable coating at various cure conditions. By way of example, the data shown inFIG.3utilizes a PEC having a 1:3 molar ratio of cation to anion; however, in various embodiments, other molar ratios could be utilized as dictated by design requirements. As shown inFIG.3, the presence of a higher weight percentage of solid content in the PEC206gives rise to thicker gas impermeable coatings. Group302illustrates 6 wt % solid PEC with no cure. Group304illustrates 4.5 wt % solid PEC with no cure. Group306illustrates 3 wt % solid PEC with no cure. Group308illustrates 1.5 wt % solid PEC with no cure. Group312illustrates 6 wt % solid PEC utilizing a buffering solution with a pH of 3 for curing. Group314illustrates 4.5 wt % solid PEC utilizing a buffering solution with a pH of 3 for curing. Group316illustrates 3 wt % solid PEC utilizing a buffering solution with a pH of 3 for curing. Group318illustrates 1.5 wt % solid PEC utilizing a buffering solution with a pH of 3 for curing. Group322illustrates 6 wt % solid PEC utilizing a buffering solution with a pH of 4 for curing. Group324illustrates 4.5 wt % solid PEC utilizing a buffering solution with a pH of 4 for curing. Group326illustrates 3 wt % solid PEC utilizing a buffering solution with a pH of 4 for curing. Group328illustrates 1.5 wt % solid PEC utilizing a buffering solution with a pH of 4 for curing. Group332illustrates 6 wt % solid PEC utilizing a buffering solution with a pH of 5 for curing. Group334illustrates 4.5 wt % solid PEC utilizing a buffering solution with a pH of 5 for curing. Group336illustrates 3 wt % solid PEC utilizing a buffering solution with a pH of 5 for curing. Group338illustrates 1.5 wt % solid PEC utilizing a buffering solution with a pH of 5 for curing. FIG.4is a flow diagram illustrating a process400for applying a gas impermeable coating.FIG.5is a diagrammatic illustration of the process400. Referring toFIGS.4-5in combination, the process400begins at block402. At block404, a polyelectrolyte complex (PEC) suspension is formed. As illustrated inFIG.5, the PEC coacervate complex is comprised of at least two oppositely charged polyelectrolytes (502aand502b) in water504. In various embodiments, cationic polyethylenimine (PEI) and anionic polyacrylic acid (PAA), for example, may be used to form the coacervate. Other embodiments may make use of strong polyelectrolytes. Other further embodiments may make use of Polyethylenimine, Poly(allyl amine), Poly(diallyldimethyl ammonium chloride), Chitosan, Functionalized starch, Poly(vinyl amine), Poly(vinylbenzyltrimethyl-ammonium) bromide, and Poly(4-vinyl-N-butyl-pyridinium) chloride as a polycation, and Poly(acrylic acid) and sodium polyacrylate, Poly(styrene sulfonic acid) and sodium poly(styrene sulfonate), Carboxymethylcellulose, Alginic acid, Hyaluronic acid, Heparin, and Polyphosphoric acid and Polyphosphate salts as a polyanion. The ionic strengths of the polycation and polyanion are controlled in order to make it suitable for a Meyer rod coating process. The ionic strength is normally manipulated by the addition of salt such as, for example, NaCl. At block406, the PEC suspension506is applied to a substrate. As illustrated inFIG.5, in a typical embodiment, the PEC suspension506is applied to the substrate508using, for example, a Meyer rod510. The Meyer rod510is drawn across the substrate, doctoring off coating fluid, using formed or wired grooves to deposit a specific wet-film thickness onto the substrate508. The PEC suspension506used for this process must have sufficient viscosity to resist dewetting and contain enough weight polymer to deposit a uniform layer. In a typical embodiment, NaCl concentration heavily influences the viscosity and maintaining a proper concentration is vital to the coating process. In certain embodiments, the substrate508may be corona-treated or plasma treated to increase surface energy. In certain embodiments, a wetting agent, such as, for example, a surfactant, may be added to the PEC suspension506to decrease surface tension and achieve more complete wetting of the substrate508by the PEC suspension506. At block408, the coating is treated. As illustrated inFIG.5, once the substrate508is coated the film512must be treated to allow the applied film to become solid. In a typical embodiment, salt must be driven out from the film512to allow the film512to solidify. Thus, any solution containing a lower salt concentration should be able to facilitate this process. In certain embodiments, a buffer514may also be used such as, for example, a citric acid/citrate buffer aqueous solution, where the buffer improves the film's512durability to rinsing and produces higher cohesive energy density, thereby preventing gas molecules from moving aside polymer chains. In some embodiments, and as illustrated inFIG.5, the treatment may be performed by immersion of the film512. Various other embodiments may make use of spraying the film or a combination of the immersion and spraying. Additionally, various embodiments may utilize various forms of drying such as air drying, oven drying, or suspension. Various other embodiments, however, may not dry the solid film512. In a typical embodiment, the resulting film thickness is less than 10 microns. In a typical embodiment, the oxygen transmission rate through the film is less than approximately 10 cm3/(m2day atm).FIG.6provides a graphical illustration of oxygen transmission rate versus pH. After treatment in block408, the film512is subjected to a high humidity treatment in block410in order to remove porosity and uncover fissures. Pores likely arise from fast evaporation of water from the film during air drying and are not present in layer-by-layer assembled films. Water acts as a plasticizer, allowing polymer to fill the coating's pores. In various embodiments, exposure to high humidity such as, for example, greater than approximately 95% relative humidity for approximately 12 hours helps to close pores present in the film512. In other embodiments, thermal crosslinking chemically bonds the PEI to the PAA to create amide bonds. Thermal crosslinking may be performed by high heat at, for example, approximately 150° C. for approximately 2 hours. Various other embodiments may make use of other types of crosslinking. Some crosslinking may occur, for example, via chemical reaction. In certain embodiments, a crosslinking agent is added to the original PEC suspension and later activated. The final film512would be exposed to a crosslinking agent to finish the process. The process400ends at block412. EXAMPLES Cationic branched polyethylenimine (Mw=25,000 g mol−1), anionic polyacrylic acid solution (Mw=100,000 g mol−1, 35 wt % in water), anhydrous sodium hydroxide pellets (reagent grade, ≥98%), sodium chloride, citric acid monohydrate, and sodium citrate dihydrate were purchased from Sigma-Aldrich (Milwaukee, Wisconsin). P-doped, single side polished (1 0 0) silicon wafers (University Wafer, South Boston, Massachusetts), with a thickness of 500 μm, were used as substrates408for profilometer thickness measurements and microscopy. Films for oxygen transmission testing were deposited on a 127 μm thick poly(ethylene terephthalate) (PET) film (ST505, Dupont-Teijin) purchased from Tekra (New Berlin, Wisconsin). All aqueous solutions were prepared with 18.2 MΩ·cm deionized water. The pH of individual 20 wt % solutions of PEI and PAA were adjusted to 8.0 using 5 M HCl and NaOH, respectively. After achieving pH 8.0, the solutions were diluted to 10 wt % polymer and the pH was adjusted to 8.0 again. The PEC suspensions506were prepared by taking equal volumes of the two solutions and adding sodium chloride to achieve the desired concentration NaCl. PEI was added dropwise to the PAA while stirring vigorously. A solution pH of 8 was used because it reduced localized flocculation during mixing relative to lower pH processing. The suspensions were stirred for 10 min and then allowed to sit for approximately 1 hour. Coacervates and solutions were “annealed” in an oven for approximately 2 hours at approximately 70° C. prior to characterization. A PEI/PAA coacervate, prepared using 1.0 M NaCl, was separated from the dilute phase by pipet. PET substrates508were corona treated immediately before coating. Silicon and PET substrates508were mounted on glass and coacervate fluid506was deposited using the Meyer rod510. The Meyer rod510was approximately 0.5 inches in diameter, approximately 16 inches long and had an “equivalent wire diameter” of approximately 0.05 mm. The substrate508was then dipped in 100 mM citric acid/citrate buffer for 1 minute, followed by spraying with water to rinse and drying with a stream of filtered air. Humidity post-treatment involved placing the coated substrate508in a chamber with humidity varying from 93-97% relative humidity for approximately 12 hour. Thermal crosslinking was accomplished by placing the coated substrate508in an oven at approximately 150° C. for approximately 2 hour. All films were stored in a drybox for approximately 24 hours or more prior to characterization. Film512thickness was measured on silicon wafers with, for example, a profilometer. Surface morphology was imaged using, for example, a JSM-7500F FESEM (JEOL, Tokyo, Japan). Prior to imaging, each film was sputter coated with approximately 5 nm of, for example, platinum/palladium to reduce surface charging of the film. Atomic force microscopy was done using a Bruker Dimension Icon atomic force microscope (“AFM”). All mapping measurements were conducted using the tapping mode imaging of the AFM, under ambient conditions such as, for example, approximately 24° C. and approximately 45% relative humidity. Oxygen transmission rate measurements were performed by using, for example, an Oxtran 2/21 ML oxygen permeability instrument, in accordance with ASTM Standard D-3985, at approximately 23° C. and at approximately 50% relative humidity. Viscosity (TO) was measured using, for example, an AR G2 Rheometer (TA Instruments, New Castle, Delaware) using a 40 mm, 2° steel cone. Shear-stress experiments were performed at approximately 25° C. over a frequency range of approximately 1 1-100 Hz. Transmittance of PEC films512was measured using a USB2000 UV-Vis spectrometer (Ocean Optics, Dunedin, Florida). As illustrated inFIG.7, viscosity decreases with higher salt concentration.FIG.10is a graphical illustration of transmittance versus wavelength for various pH films. The effect of salt concentration on the complexation of the polyelectrolytes was studied by varying the concentration of sodium chloride in PEI and PAA solutions at, for example, 10 wt % polymer at pH 8.0, before mixing. In a range of 0 to approximately 0.25 M NaCl, PEI and PAA strongly associate into larger networks and form a macroscopic precipitate that is unsuitable for the Meyer rod510coating process. At concentrations of approximately 1.50 M and above, the ionic strength is high enough for PEI and PAA to be dissolved as individual chains, creating a true solution of polyelectrolytes. Coacervation is achieved at intermediate NaCl concentrations of, for example, approximately, 0.50-1.00 M, where phase separation of the polymer-rich coacervate layer and a polymer-poor dilute layer is observed. Samples of varying salt concentration 1 hour after mixing are shown inFIG.6b. To further coalesce microphase droplets and better identify which suspensions phase separate, both the solutions and coacervates were “annealed” for approximately 2 hours at approximately 70° C., resulting in optically transparent phases. All coacervate phases were decanted for further characterization. All solutions have pH of approximately 8.0 and contain approximately 10% weight polymer with a 1:1 weight ratio of PEI to PAA. Rheology was performed on the PEI/PAA solutions as well as the lower concentration salt coacervate phases to determine which PEC sample was suitable as the coating fluid for coating with the Meyer rod510.FIG.7is a graphical illustration of the viscosity of coacervate phases at varying NaCl concentration. The free chain polyelectrolyte solutions (1.25-1.50 M) have viscosities much lower than those of the coacervates (0.50 M-1.0 M), where there is stronger interaction between polymer chains. The viscosities of the free chain polyelectrolyte solutions were considerably higher than water such as, for example, 1 mPa·s, indicating critical overlap concentration was achieved. To utilize the Meyer rod510for coating with minimal defects, the viscosity of the fluid should be high enough to resist secondary flows induced by dewetting and surface tension. The coacervate suspensions are suitable candidates for these coatings as they remain within the range of viscosity suitable for the Meyer rod510technique such as, for example, in the range of approximately 300-800 mPa·s. The complex coacervate with 1.0 M NaCl was used for the gas barrier film because its viscosity remained suitable over a wide range of shear rates such as, for example, 0.2-100.0 s−1. After separating from the top dilute phase of the polyelectrolyte complex, the coacervate prepared with 1.0 M NaCl was applied to substrates using the Meyer rod510illustrated inFIG.5. The PEC film512was then dipped in water to extract sodium chloride, allowing the polyelectrolyte chains to more strongly associate and solidify the complex. Because the polyelectrolyte coacervate is above critical overlap concentration, the complex solidifies into a coherent film rather than individual colloidal particles. Subsequent spray rinsing appeared to erode the film512and there was noticeable undesirable stickiness, likely due to the incomplete ionization of PEI at pH 8 in the film. When PEI is not completely protonated, there are less ammonium groups to ionically bond with the carboxylate ions of PAA. After rod coating, the films were immersed into citric acid/citrate buffer solutions at pH 6, 4, and 2 to fully protonate PEI, which improved its association with PAA. In addition to improving the film's durability to rinsing, acid buffer treatment produces higher cohesive energy density, which prevents gas molecules from moving aside polymer chains. Spray rinsing removed buffer, persistent salt and excess polymer and the films were finally dried by a stream of dry air. PEC films on silicon treated by pH 6 buffer were depleted in some areas and coatings treated at pH 2 could not remain adhered after spray rinsing. Using pH 4 buffer proved to be the most effective treatment and produced highly conformal films, with average thickness of approximately 1.63±0.09 μm. As illustrated inFIG.8a, scanning electron micrographs of pH 4 treated PEC films revealed a considerable amount of porosity (FIG.8a). Atomic force microscopy (AFM) revealed pores spanning the thickness of the film, inhibiting the film's ability to reduce gas permeability. Pores likely arise from the fast evaporation of water from the film during air drying and are not present in films assembled layer-by-layer. To remove these pores, films were post-treated for approximately 12 hours in a 95% relative humidity chamber. Water acts as a plasticizer, allowing polymer to fill the coating's pores. As shown inFIGS.8d-8f, both SEM and AFM of humidity-treated films512reveal very smooth coatings. Roughness of treated films512was reduced two orders of magnitude (from approximately 395 nm to approximately 2.60 nm and thickness increased to 1.91±0.08 μm. Although unsuccessful on silicon, PEC films treated by a pH 2 and 6 buffer were successfully deposited on PET substrates508along with pH 4 treated films. pH 4 and pH 6 treatment resulted in hazy films, with visible light transmittance of approximately 14 and approximately 11%, respectively. Films512treated with pH 2 buffer were completely opaque having approximately 3% visible light transmittance.FIG.10shows that pH 4 treatment results in the best oxygen barrier on PET, reducing the oxygen transmission rate (OTR) of 0.127 mm thick PET from 9.51 to 1.46 cm3/(m2·day·atm). Humidity treatment of the PEC coating reduced the porosity of the film512, marked by the substantial reduction of coating haziness (98% transparent), resulting in further reduction of OTR to 0.384 cm3/(m2·day·atm). An even further reduction in OTR resulted with thermal cross-linking of the humidified film512, achieving an OTR of 0.08 cm3/(m2·day·atm). Thermal cross-linking chemically bonds the PEI to PAA to create amide bonds. Unlike an earlier study incorporating thermal crosslinking, no contraction was observed here, with the film maintaining a thickness of 1.99±0.06 μm. The rod coating of polyelectrolyte coacervate suspensions provides a framework in which multi-polyelectrolyte films can be deposited in a single step. With this technique, it is likely that many multilayer gas barrier coatings can be more quickly and simply deposited. By affecting salt concentration, coacervates of sufficient viscosity can be formed to resist fluid flow and the film can be cured by using an appropriate pH buffer. Post-treatments of the film improved the oxygen barrier enormously and by combining humidity and cross-linking treatments, these PEC-based films provided PET with a two order of magnitude reduction in oxygen transmission rate, while also achieving optical transparency. This environmentally benign process offers the opportunity for scalable, less-costly barrier films. Although various embodiments of the method and system of the present disclosure have been illustrated in the accompanying Drawings and described in the foregoing Specification, it will be understood that the disclosure is not limited to the embodiments disclosed, but is capable of numerous rearrangements, modifications, and substitutions without departing from the spirit and scope of the disclosure as set forth herein. For example, although the area102has been described herein as being an agricultural field, one skilled in the art will recognized that the area102could be any geographic area on which remote sensing could be performed. It is intended that the Specification and examples be considered as illustrative only. | 24,402 |
11859097 | MODES OF THE INVENTION Hereinafter, specific exemplary embodiments of the present invention will be described in more detail. Prior to describing the present invention, it should be noted that the following description of specific structures or functions is merely illustrative and provided only for the purpose of describing embodiments according to the concept of the present invention, and that the embodiments according to the concept of the present invention may be implemented in various forms and should not be construed as being limited to the exemplary embodiments described in the present specification. In addition, the embodiments according to the concept of the present invention may be variously modified and may be implemented in various forms, and specific exemplary embodiments will be described in detail in the present specification. However, there is no intention to limit the embodiments according to the concept of the present invention to the specific disclosure forms, and it should be understood that all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention are encompassed. In the present invention, there is provided a functional water-based paint composition, which has been researched and developed to have enhanced fire-retardant properties and have a waterproofing effect and prevent condensation by having excellent thermal insulation and heat-shielding effects. For this, one aspect of the present invention provides a paint composition including a main raw material and a liquid raw material in the ratio of 1:1, wherein the main raw material includes calcium sulfoaluminate (3Ca3Al2O3CaSO4) having a fineness of 6,000 to 12,000 cm2/g at 10 to 30 wt %, expandable graphite at 20 to 40 wt %, calcium carbonate (CaCO3) passed through a 325 mesh at 10 to 40 wt %, a foaming agent at 1 to 2 wt %, a fluidizing agent at 0.1 to 1 wt %, a preservative at 0.1 to 1 wt %, an antifoaming agent at 0.1 to 1 wt %, hydroxymethyl cellulose or methyl cellulose having a viscosity of 5,000 to 30,000 cP at 0.01 to 0.5 wt %, anhydrous gypsum at 10 wt % or less, a microporous ceramic having a particle size of 325 mesh or less at 30 wt % or less, a phosphorus-based flame retardant at 20 wt % or less, and TiO2at 5 wt % or less, and the liquid raw material includes one or a mixture of two or more of an epoxy resin, an EVA resin, and an acrylic resin at 20 to 50 wt % and water at 50 to 80 wt %. The paint composition of the present invention includes, among the above-described inorganic additives, an inorganic additive exhibiting a hydraulic reaction in order to compensate for the disadvantage that the existing paints cannot form enough thickness with one-time application. The paint composition of the present invention includes calcium sulfoaluminate or alumina cement as an inorganic binder and may further include anhydrous gypsum in order to ensure the hardness and waterproofing properties of a paint and the adhesion to an object to be painted and maintain an application thickness. In this case, the inorganic binder may increase paint hardness by being hydrated by water, realize excellent waterproofing properties by reacting with water and a resin, and maintain a thickness by forming a hydrate. The paint composition of the present invention includes expandable graphite in order to have a thermal insulation effect and particularly to prevent the generation of smoke from a fire. When exposed to heat of 200° C. or more, the expandable graphite may expand and thus prevent flames from being transmitted to a building or structure, thereby providing protection for the building or structure. The paint composition of the present invention includes a foaming agent in order to improve foaming performance. The paint composition of the present invention includes a microporous ceramic fired and foamed at high temperature in order to achieve thermal superinsulation properties. In this case, the microporous ceramic may be fired and foamed at a temperature of 1,200 to 1,400° C. When firing is performed at a temperature below the above-described temperature range, the ceramic may be insufficiently fired or very slowly fired. On the other hand, when firing is performed at a temperature above the above-described temperature range, the unnecessarily high temperature may lead to high energy loss and adversely affect the firing device. The paint composition of the present invention includes a resin for the effect of enhancing the adhesion to an object to be painted and improving waterproofing properties by reacting with the inorganic binder included in the composition of the present invention. In particular, when used for a steel structure or the like, that is, when sufficient adhesion is required, the paint composition may include a two-component epoxy resin, and when used for a concrete building or when moderate adhesion is required, the paint composition may include a one-component EVA resin or acrylic resin. According to one embodiment of the present invention, the paint composition of the present invention includes one or a mixture of two or more of the above-described resins. The paint composition of the present invention includes a flame retardant in order to prevent the resin included in the composition of the present invention from being affected by flames and improve fire-retardant properties. The paint composition of the present invention includes an antifoaming agent, a fluidizing agent, a preservative, and the like as additives. The antifoaming agent is added to suppress the generation of foam which causes surface defects, such as cratering or the weakening of coating, in a dry coating, and serves to control the amount of gas and reduce air bubbles. Accordingly, the effects of ensuring quality stability and improving strength are provided. In the present invention, for example, AGITAN 295, AGITAN P803, AGITAN P833, or the like which is generally known may be used as the antifoaming agent, but the present invention is not limited thereto, and any of various other types of antifoaming agents may be used. The fluidizing agent, which is a blending agent used for softening a paint by improving the flow characteristics of the paint, is added to disperse particles in the paint composition and serves to improve fluidity while maintaining the quality of the prepared paint composition by forming a lubricating film between particles and thereby reducing the adhesion between components. In addition, the fluidizing agent increases the levelness of a resulting lubricating film and improves workability in forming the lubricating film. In the present invention, for example, CMC/PC, Peramin SMF 30, Peramin CONPAC 149S, or the like which is generally known may be used as the fluidizing agent, but the present invention is not limited thereto, and any of other types of fluidizing agents may be used. In addition, the preservative is added to increase the preservability of the product. The paint composition of the present invention may further include a general pigment in order to impart color to the paint, and the pigment may be an organic or inorganic pigment and may be a black or colored pigment. Hereinafter, the present invention will be described in more detail by way of exemplary embodiments. However, the exemplary embodiments are merely illustrative of the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by the exemplary embodiments. Example 1 Preparation of Functional Water-Based Paint Composition For the preparation of a functional water-based paint composition of the present invention, a main raw material was prepared by mixing high-fineness calcium sulfoaluminate (3Ca3Al2O3CaSO4) satisfying a fineness of 6,000 to 12,000 cm2/g at 25 wt %, anhydrous gypsum at 7 wt %, a microporous ceramic fired and foamed at 1,300° C. and passed through a 325 mesh at 20 wt %, expandable graphite at 30 wt %, calcium carbonate (CaCO3) passed through a 325 mesh at 9 wt %, a foaming agent at 1.5 wt %, a phosphorus-based flame retardant at 5 wt %, a fluidizing agent at 0.2 wt %, a preservative at 0.1 wt %, TiO2at 2 wt %, an antifoaming agent at 0.18 wt %, and methyl cellulose satisfying a viscosity of 5,000 to 30,000 cP at 0.02 wt %. Subsequently, a liquid raw material for the functional water-based paint composition of the present invention was prepared by mixing an acrylic resin at 40 wt % and water at 60 wt %. The prepared main raw material and liquid raw material were mixed at the ratio of 1:1, and thereby the functional water-based paint composition of the present invention was finally obtained. Experimental Example 1 Evaluation of Thermal Insulation Effect In order to confirm that the functional water-based paint composition prepared according to one exemplary embodiment of the present invention has a thermal insulation effect, the thermal insulation effect was evaluated by the Korea Testing & Research Institute in accordance with KS L 9016:2010 (flat-plate heat flux sensor method). The results are shown inFIG.1. Referring toFIG.1, it can be confirmed that the functional water-based paint composition of the present invention has an excellent thermal insulation effect, by having a thermal conductivity of 0.078 W/(m·K). Experimental Example 2 Evaluation of Heat-Shielding Effect In order to confirm that the functional water-based paint composition prepared according to one exemplary embodiment of the present invention has a heat-shielding effect, solar radiation reflectance was evaluated by the Korea Testing & Research Institute in accordance with KS L 2514:2011. The results are shown inFIG.2. Referring toFIG.2, it can be confirmed that the functional water-based paint composition of the present invention has an excellent heat-shielding effect, by having a solar radiation reflectance of 91.5%. Experimental Example 3 Evaluation of Fire-Retardant Properties In order to confirm that the functional water-based paint composition prepared according to one exemplary embodiment of the present invention has fire-retardant properties, the fire-retardant properties were evaluated by the KS F ISO 1182 test, which is a method for testing the fire-retardant properties of building materials. As a result, it was determined that the functional water-based paint composition of the present invention is a first-grade fire retardant. Experimental Example 4 Comparison of Coating Thickness According to Number of Coatings and Thermal Insulation Effect The coating thicknesses formed by the one-time application of a coating and thermal insulation effects of the functional water-based paint composition prepared according to one exemplary embodiment of the present invention and commercially available thermal insulation waterproofing paint compositions were evaluated and compared. In the case of the paint composition of the present invention, a thickness of about 10 mm was ensured even with the one-time application of a coating. On the other hand, in the case of the paint compositions commercially available from Company A and Company B, it was only possible to ensure a thickness of 10 mm when the paint compositions were applied 17 times and 18 times, respectively. In addition, in order to compare the thermal insulation effects of the paint compositions upon obtaining a thickness of 10 mm, thermal conductivity was evaluated by the Korea Testing & Research Institute in accordance with KS L 9016:2010 (flat-plate heat flux sensor method). The results of evaluating the number of coatings and thermal conductivity of the paint composition of the present invention and the paint compositions commercially available from Company A and Company B applied to a thickness of 10 mm are shown in the following Table 1. TABLE 1ClassificationExample 1Company ACompany BNumber of coatings to reach 1011718mm thicknessThermal conductivity (units:0.650.740.68N/(m · K)) Referring to Table 1, it can be confirmed that in the case of the functional water-based paint composition of the present invention, it is possible to achieve, even with the one-time application a coating, the coating thickness only obtainable by applying the existing paint compositions several times and have excellent thermal insulation and heat-shielding effects. | 12,455 |
11859098 | DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION The compositions (synonymously, formulas), kits, systems, and methods of the present invention will be described in detail by reference to various non-limiting embodiments. This description will enable one skilled in the art to make and use the invention, and it describes several embodiments, adaptations, variations, alternatives, and uses of the invention. These and other embodiments, features, and advantages of the present invention will become more apparent to those skilled in the art when taken with reference to the following detailed description of the invention in conjunction with any accompanying figures. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated, all numbers expressing conditions, concentrations, dimensions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending at least upon a specific analytical technique. All references to “room temperature” should be understood as about 25° C., which for purposes of this patent application means 25° C.±5° C. Unless otherwise indicated, all references to Mnin this disclosure mean number-average molecular weight. In this specification, “antimicrobial agent” is synonymous with “antimicrobial active” and such terms may be used interchangeably. The term “comprising,” which is synonymous with “including,” “containing,” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named claim elements are essential, but other claim elements may be added and still form a construct within the scope of the claim. As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of” (or variations thereof) appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified elements or method steps, plus those that do not materially affect the basis and novel characteristic(s) of the claimed subject matter. With respect to the terms “comprising,” “consisting of,” and “consisting essentially of,” where one of these three terms is used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms, except when used in a Markush group. Thus in some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of” or, alternatively, by “consisting essentially of.” The present invention provides a two-part formula with two precursor parts that may be in a kit, in which the two-part formula is designed to form a biphasic polymer, preferably an antimicrobial biphasic polymer. In the case of antimicrobial polymers, it is preferred that a formula can be distributed to end users and is stable for many months before use. The present disclosure describes various two-part formulas that are shelf-stable while being designed to produce the desired polymers after application and curing on a substrate. The polymers are preferably solventborne polyurethanes or polyureas and are pre-packaged in two-part formula kits, in which an isocyanate is physically and spatially separated from a polyol or polyamine, so that coating does not cure prior to application on a surface. In preferred embodiments, the two-part formula is designed to produce a biphasic polymer containing a discrete structural phase and a continuous transport phase bound together via crosslinking agents. Commercial applications of the disclosed two-part formulas include, but are not limited to, antimicrobial surfaces in cars, especially shared-ride vehicles, to inhibit the transfer of microbes from one person to another; antimicrobial surfaces in airplanes where UV light cannot reach to sanitize contaminated surfaces; antimicrobial surfaces inside and outside vehicles that may be used to rescue or move people who have been exposed to diseases and pandemics; for antimicrobial surfaces in homes (e.g., kitchens and bathrooms); in restaurants; and on clothing and personal protective equipment. All of these use cases require a convenient formulation to be available for application on surfaces. Some variations of the invention provide a two-part formula for fabricating a biphasic polymer, wherein the two-part formula consists essentially of:(A) a first liquid volume, wherein the first liquid volume comprises:(A)(i) a structural phase containing a solid structural polymer;(A)(ii) a transport phase containing a solid transport polymer;(A)(iii) a chain extender;(A)(iv) a curing catalyst;(A)(v) a first solvent; and(A)(vi) optionally, first additives; and(B) a second liquid volume that is volumetrically isolated from the first liquid volume, wherein the second liquid volume comprises:(B)(i) a crosslinker that is capable of crosslinking the solid structural polymer with the solid transport polymer;(B)(ii) a second solvent; and(B)(iii) optionally, second additives. In some embodiments, the solid structural polymer is selected from non-fluorinated carbon-based polymers. The non-fluorinated carbon-based polymers may be selected from the group consisting of polycarbonates, polyacrylates, polyalkanes, polyurethanes, polyethers, polyureas, polyesters, polyepoxides, and combinations thereof. In some embodiments, the non-fluorinated carbon-based polymers contain hydroxyl reactive groups, amine reactive groups, or both hydroxyl reactive groups and amine reactive groups. In some embodiments, the solid structural polymer is selected from fluorinated polymers. The fluorinated polymers may be selected from the group consisting of fluorinated polyols, perfluorocarbons, perfluoropolyethers, polyfluoroacrylates, polyfluorosiloxanes, polyvinylidene fluoride, polytrifluoroethylene, and combinations thereof. In some embodiments, the fluorinated polymers contain hydroxyl reactive groups, amine reactive groups, or both hydroxyl reactive groups and amine reactive groups. The fluorinated polymers may be branched with a functionality greater than 2. In some embodiments, the structural phase has a glass-transition temperature Tgof greater than 20° C., such as from about 25° C. to about 300° C. The glass-transition temperature Tgof a material characterizes temperatures at which a glass transition is observed. A glass transition is the gradual and reversible transition in amorphous materials (or in amorphous regions within semicrystalline materials) from a hard and relatively brittle “glassy” state into a viscous or rubbery state as the temperature is increased. A glass transition generally occurs over a temperature range and depends on the thermal history; therefore, a test method needs to be defined in order to ascertain a value of Tgfor a given material. In this patent application, the glass-transition temperature Tgis measured according to the equal-areas method described in International Standard ISO 11357-2, “Plastics—Differential scanning calorimetry (DSC)—Part 2: Determination of glass transition temperature and step height”, Third Edition, March 2020, which is hereby incorporated by reference. The measurement of Tguses the energy release on heating in differential scanning calorimetry (DSC). The change in heat flow rate as a function of temperature is recorded and the glass-transition temperature and step height are determined from the curve thus obtained. The glass transition is assigned to the temperature obtained by drawing a vertical line such that the areas between DSC trace and baselines below and above the curve are equal. As the glass transition is a kinetic phenomenon, the glass-transition temperature depends on the actual cooling rate and annealing conditions below Tg. Unperturbed glass transitions are obtained only if cooling and subsequent heating rate are the same and no significant physical aging occurs due to annealing below Tg. If a sample is cooled significantly slower or annealed below Tg, enthalpy relaxations can occur, resulting in endotherm peaks just above Tg. Peaks due to enthalpy relaxation will disappear by extrapolating to zero heating rates. The equal-areas method provides the best procedure to obtain an accurate Tgin the case of occurrence of enthalpy relaxations. The equal-areas method is described in section 10.1.2 of ISO 11357-2, which is hereby incorporated by reference. Examples of polymers with Tg<20° C. include silicones, polyvinylidene fluoride, polyvinyl fluoride, polychloroprene, polyethylene, polypropylene, and poly(butyl acrylate). Many examples of polymers with Tg≥20° C. are provided below. The preference for Tg≥20° C. is based on the use temperature of the antimicrobial structure being about 20° C. If the use temperature is higher, such as 40° C., then the Tgof the solid structural polymer may be about 40° C. or higher. Likewise, in certain situations where the antimicrobial-structure use temperature is lower, such as 0° C., then the Tgof the solid structural polymer may be about 0° C. or higher. In various embodiments, the glass-transition temperature Tgof the solid structural polymer is about, at least about, or at most about 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 60° C., 70° C., 80° C., 90° C., 100° C., 110° C., 120° C., 130° C., 140° C., 150° C., 160° C., 170° C., 180° C., 190° C., 200° C., 210° C., 220° C., 230° C., 240° C., 250° C., 260° C., 270° C., 280° C., 290° C., or 300° C., including any intervening ranges (e.g., Tg=30-150° C., Tg=50-200° C., etc.). Reference to a range of Tgmeans that a solid structural polymer may be selected such that its single value of Tg, measured pursuant to ISO 11357-2, falls within the specified range. In some embodiments, a high glass-transition temperature (i.e., Tg≥20° C.) of the solid structural polymer improves the anti-fouling performance of the antimicrobial structure (e.g., a coating). Structural phases such as poly(butadiene) or poly(tetrahydrofuran) have Tg<20° C. and typically include at least 10 vol % of a fluorinated polyol added to the structural phase to reject stains. Non-fluorinated solid structural polymers are preferred for resisting penetration of external soils into the coating, such as a fluorine-free anti-fouling coating. In some embodiments, the solid transport polymer is a hygroscopic solid transport polymer selected from the group consisting of poly(acrylic acid), poly(ethylene glycol), poly(2-hydroxyethyl methacrylate), poly(vinyl imidazole), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(vinylpyrolidone), modified cellulosic polymers, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, and combinations thereof. In some embodiments, the solid transport polymer is a hydrophobic, non-lipophobic solid transport polymer selected from the group consisting of poly(propylene glycol), poly(tetramethylene glycol), polybutadiene, polycarbonate, polycaprolactone, acrylic polyols, and combinations thereof. In some embodiments, the solid transport polymer is a hydrophilic solid transport polymer with ionic charge, and wherein the ionic charge is optionally present within the hydrophilic solid transport polymer as carboxylate groups, amine groups, sulfate groups, or phosphate groups. In some embodiments, the solid transport polymer is an electrolyte solid transport polymer selected from the group consisting of polyethylene oxide, polypropylene oxide, polycarbonates, polysiloxanes, polyvinylidene difluoride, and combinations thereof. In some embodiments, the transport phase consists essentially of the solid transport polymer. In other embodiments, the transport phase consists essentially of a liquid mixed with the solid transport polymer. In some embodiments, the crosslinker includes at least one moiety selected from the group consisting of amine, hydroxyl, isocyanate, a blocked isocyanate, epoxide, carbodiimide, and combinations thereof. In some embodiments, each of the first solvent and the second solvent is independently selected from the group consisting of water, alcohols, polyols, ethers, esters, ketones, aldehydes, carbonates, lactones, sulfoxides, ionic liquids, and combinations thereof. Exemplary first and second solvents include methyl ethyl ketone, methyl isobutyl ketone, methyl propyl ketone, toluene, and xylenes. In some embodiments, each of the first additives and the second additives is independently selected from the group consisting of buffers, UV stabilizers, fillers, pigments, flattening agents, flame retardants, salts, surfactants, defoamers, dispersants, wetting agents, antioxidants, adhesion promoters, leveling agents, and combinations thereof. In some embodiments, an antimicrobial agent is present within the first liquid volume, within the second liquid volume, or within both the first liquid volume and the second liquid volume. In these embodiments, the biphasic polymer to be fabricated from the two-part formula is an antimicrobial biphasic polymer. When the antimicrobial agent is present, it may be dissolved or suspended in the first solvent. Alternatively, or additionally, the antimicrobial agent may be dissolved or suspended in second solvent. In certain embodiments, the antimicrobial agent may be contained within the first liquid volume but not dissolved in the first solvent. In certain embodiments, the antimicrobial agent may be contained within the second liquid volume but not dissolved in the second solvent. In specific embodiments, the antimicrobial agent may be contained within the first and second liquid volumes but not necessarily dissolved or suspended in either the first solvent or the second solvent. In some embodiments, there initially is no antimicrobial agent. The antimicrobial agent may be added at a later time, such as right before use of the biphasic polymer as an antimicrobial biphasic polymer. Also, the antimicrobial agent may be replenished at various times during or after use of the antimicrobial biphasic polymer. In some embodiments, the antimicrobial agent is selected from quaternary ammonium molecules. The quaternary ammonium molecules may be selected from benzalkonium chloride, benzethonium chloride, methylbenzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, cetyl trimethylammonium chloride, alkyltrimethylammonium chloride, tetraethylammonium chloride, didecyldimethylammonium chloride, dodecyl-dimethyl-(2-phenoxyethyl)azanium chloride, bromide versions thereof, or a combination of the foregoing. Other salts of quaternary ammonium may be employed as quaternary ammonium molecules. In some embodiments, the antimicrobial agent is selected from metal ions. The metal ions are optionally selected from the group consisting of silver, copper, zinc, and combinations thereof. In some embodiments, the antimicrobial agent is selected from metal oxides. The metal oxides are optionally selected from copper (I) oxide, copper (II) oxide, zinc oxide, silver oxide, and combinations thereof. The metal oxides may be in the form of metal oxide nanoparticles, microparticles, or a combination thereof. In some embodiments, the antimicrobial agent is selected from acids. The acids are optionally selected from the group consisting of citric acid, acetic acid, peracetic acid, glycolic acid, lactic acid, succinic acid, pyruvic acid, oxalic acid, hydrochloric acid, and combinations thereof. In some embodiments, the antimicrobial agent is selected from bases. The bases are optionally selected from the group consisting of ammonia, ammonium hydroxide, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, and combinations thereof. In some embodiments, the antimicrobial agent is selected from salts. The salts are optionally selected from the group consisting of copper chloride, copper nitrate, copper citrate, copper acetate, zinc chloride, zinc nitrate, zinc citrate, zinc acetate, silver chloride, silver nitrate, silver citrate, silver acetate, and combinations thereof. In some embodiments, the antimicrobial agent is selected from peroxides. The peroxides are optionally selected from the group consisting of hydrogen peroxide, organic peroxides, and combinations thereof. In some embodiments, the antimicrobial agent is selected from oxidizing molecules. The oxidizing molecules are optionally selected from the group consisting of hypochlorous acid, hydrogen peroxide, sodium hypochlorite, sodium chlorite, sodium chlorate, calcium hypochlorite, calcium chlorite, calcium chlorate, calcium perchlorate, and combinations thereof. In some embodiments, the antimicrobial agent is selected from N-halamines that are halogenated with chlorine, bromine, iodine, or a combination thereof. The two-part formula is preferably shelf-stable. By “shelf-stable” it is meant that at room temperature (about 25° C.) and atmospheric pressure (about 1 bar), the two-part formula is capable of being stored for 30 days without substantial chemical reactions spontaneously occurring. Preferably, the two-part formula is capable of being stored at 25° C. and 1 bar for 60 days, and even more preferably at least 90 days, such as at least 120, 150, or 180 days, without substantial chemical reactions spontaneously occurring. Some variations of the invention provide a multi-part formula for fabricating a biphasic polymer, wherein the multi-part formula comprises:(A) a first liquid volume, wherein the first liquid volume comprises:(A)(i) a structural phase containing a solid structural polymer;(A)(ii) a transport phase containing a solid transport polymer;(A)(iii) a chain extender;(A)(iv) a curing catalyst;(A)(v) a first solvent; and(A)(vi) optionally, first additives; and(B) a second liquid volume that is volumetrically isolated from the first liquid volume, wherein the second liquid volume comprises:(B)(i) a crosslinker that is capable of crosslinking the solid structural polymer with the solid transport polymer;(B)(ii) a second solvent; and(B)(iii) optionally, second additives. The multi-part formula may be a two-part formula, a three-part formula, or a four-part formula, for example. In addition to part (A) and part (B), there may be another part, such as a part for additives. In other variations, part (A) is split into sub-parts, which are designed to remain separated from each other as well as from part (B). For example, the structural phase, the transport phase, the chain extender, the curing catalyst, or additives may be included in one or more distinct sub-parts, if desired, within a multi-part formula. The two-part formula may be contained with a kit. The kit may contain instructions for converting the two-part formula into the biphasic polymer. For example, the instructions may direct a user to make the biphasic polymer by carrying out the following steps:(a) start with the two-part formula;(b) combine the first liquid volume with the second liquid volume, thereby forming a combined liquid volume; and(c) cure the combined liquid volume to react the crosslinker with the solid structural polymer and the solid transport polymer, thereby generating a biphasic polymer. The instructions in the kit may be printed physical instructions, such as a paper insert in the kit. The instructions may be provided on the Internet, with a link to a web site provided in the kit. The instructions may be provided from a mobile-device app, with download directions provided in the kit. The instructions may be recorded to an audio file that is on a disk or sound card provided in the kit. Some variations of the invention provide a kit for fabricating a biphasic polymer, wherein the kit comprises:(A) a first liquid volume, wherein the first liquid volume comprises:(A)(i) a structural phase containing a solid structural polymer;(A)(ii) a transport phase containing a solid transport polymer;(A)(iii) a chain extender;(A)(iv) a curing catalyst;(A)(v) a first solvent; and(A)(vi) optionally, first additives; and(B) a second liquid volume that is volumetrically isolated from the first liquid volume, wherein the second liquid volume comprises:(B)(i) a crosslinker that is capable of crosslinking the solid structural polymer with the solid transport polymer;(B)(ii) a second solvent; and(B)(iii) optionally, second additives; and(C) user instructions for converting parts (A) and (B) into a biphasic polymer. Other variations of the invention provide a method of making a biphasic polymer, the method comprising:(a) providing a two-part formula for fabricating a biphasic polymer, wherein the two-part formula consists essentially of:(A) a first liquid volume, wherein the first liquid volume comprises:(A)(i) a structural phase containing a solid structural polymer;(A)(ii) a transport phase containing a solid transport polymer;(A)(iii) a chain extender;(A)(iv) a curing catalyst;(A)(v) a first solvent; and(A)(vi) optionally, first additives; and(B) a second liquid volume that is volumetrically isolated from the first liquid volume, wherein the second liquid volume comprises:(B)(i) a crosslinker that is capable of crosslinking the solid structural polymer with the solid transport polymer;(B)(ii) a second solvent; and(B)(iii) optionally, second additives; and(b) combining the first liquid volume with the second liquid volume, thereby forming a combined liquid volume; and(c) curing the combined liquid volume to react the crosslinker with the solid structural polymer and the solid transport polymer, thereby generating a biphasic polymer,wherein the biphasic polymer comprises a discrete solid structural phase comprising the solid structural polymer, wherein the biphasic polymer further comprises a continuous transport phase comprising the solid transport polymer, wherein the solid structural polymer is crosslinked, via the crosslinker, with the solid transport polymer, wherein the continuous transport phase is interspersed within the discrete solid structural phase, and wherein the discrete solid structural phase and the continuous transport phase are separated by an average phase-separation length selected from about 100 nanometers to about 500 microns. In some methods, the biphasic polymer is formed as a coating on a bulk object made from a different material. In some methods, the biphasic polymer is formed at a surface of a bulk object. The bulk object itself may be fabricated from the biphasic polymer itself. In some embodiments, the structural phase (which may also be an anti-fouling phase) is made from solid structural polymers terminated with alcohol and/or amine groups. The solid structural polymers may be selected from non-fluorinated carbon-based polymers including alkanes, polyurethanes, polyureas, or polyesters. The solid structural polymers may be selected from silicones such as polydimethyl siloxane, polytrifluoropropylmethyl siloxane, aminopropylmethyl siloxane, aminoethylaminopropylmethyl siloxane, or aminoethylaminoisobutylmethyl siloxane. The solid structural polymers may be selected from fluorinated polymers including fluorinated polyols, perfluorocarbons, perfluoropolyethers, polyfluoroacrylates, polyfluorosiloxanes, polyvinylidene fluoride, or polytrifluoroethylene. The solid structural polymers may be selected from polycarbonate diols. The solid structural polymers may be selected from epoxide functional polymers. In some embodiments, the transport phase is made from solid transport polymers terminated with alcohol and/or amine groups. The solid transport polymers may be selected from hygroscopic polymers, such as poly(acrylic acid), a poly(ethylene glycol), poly(2-hydroxyethyl methacrylate), poly(vinyl imidazole), poly(2-methyl-2-oxazoline), and poly(2-ethyl-2-oxazoline), poly(vinylpyrolidone), or modified cellulosic polymers such as carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, or methyl cellulose. In certain embodiments, the solid transport polymers include Carbopol® poly(acrylic acid) that can bind antimicrobial actives. The solid transport polymers may be selected from hydrophobic but not lipophobic polymers, such as poly(propylene glycol), poly(tetramethylene glycol), polybutadiene, polycarbonate, polycaprolactone, or polyacrylic polyol. The solid transport polymers may be selected from hydrophilic polymers created with ionic charge. Monomers containing ionic charge, such as pendent carboxylate, amine, sulfate, and phosphate may be inserted along the hydrophilic polymer backbone. The hydrophilic polymers may bind antimicrobial actives. The solid transport polymers may be selected from electrolyte polymers, such as polyethylene oxide, polypropylene oxide, polycarbonates, or polysiloxanes. The biphasic polymer may contain chain extenders (difunctional) or crosslinkers (trifunctional or greater functionality) that are alcohol- or amine-terminated. Preferred chain extenders include aromatic diamine species such as Ethacure® 100, Ethacure® 300, and Ethacure® 410. Preferred crosslinkers include multifunctional alcohol species such as trimethylolpropane ethoxylate, trimethyloyl propane, pentaerythritol ethoxylate, or glycerol ethoxylate. The biphasic polymer also typically contains an isocyanate crosslinker, or a reacted form thereof. Typically, in the two-part formula, the isocyanate crosslinker is contained in Part B. When Parts A and B are combined, the isocyanate crosslinker crosslinks the polymer network to create the biphasic polymer. Various additives may be introduced to the two-part formula. Additives include, but are not limited to, wetting agents, buffers, UV stabilizers, particulate fillers, flammability suppressants, and antimicrobial actives. Any of these additives may also be added to the biphasic polymer after it is fabricated. Wetting agents are surfactants that produce a smooth coating surface and aid substrate adhesion. Examples of surfactants include nonionic surfactants, cationic surfactants, anionic surfactants, silicone surfactants, and fluorosurfactants. Buffers are inorganic or organic molecules that maintain a pH value through acid/base reactions. Buffers can be discrete or bonded to one of the phases of the biphasic polymer. UV stabilizers may be antioxidants (e.g., thiols), hindered amines (e.g., tetramethylpiperidine derivatives), or UV-absorbing nanoparticles, for example. Examples of UV-absorbing nanoparticles include TiO2, ZnO, CdS, CdTe, ZnS, Ag, or a combination thereof. Particulate fillers may be selected from silica, alumina, silicates, talc, aluminosilicates, barium sulfate, mica, diatomite, calcium carbonate, calcium sulfate, carbon, wollastonite, or a combination thereof. The particulate filler is optionally surface-modified with a compound selected from the group consisting of fatty acids, silanes, alkylsilanes, fluoroalkylsilanes, silicones, alkyl phosphonates, alkyl phosphonic acids, alkyl carboxylates, alkyldisilazanes, and combinations thereof. Flammability suppressants are additives that suppress flammability of the biphasic polymer. Exemplary flammability suppressants include ammonium salts, phosphate salts, phosphines, halogenated compounds, carbonate salts, hydroxide salts, borate salts, and high-surface-area silica that inhibits flame propagation. Specific examples of flammability suppressants are ammonium polyphosphate, magnesium hydroxide, zinc hydroxystannate, antimony trioxide, magnesium hydroxycarbonate, zinc borate, magnesium aluminum hydroxycarbonate, aluminum trihydroxide, tetrabromobisphenol A, tetrabromobisphenol A bis (2,3-dibromopropyl ether), bisphenol-A bis(diphenyl phosphate), brominated polyols, melamine resins, and chlorinated paraffin. Antimicrobial actives may be selected from quaternary ammonium molecules, metal ions (e.g., silver, copper, and/or zinc), metal oxide nanoparticles, acids (e.g., citric acid, acetic acid, peracetic acid, glycolic acid, or hydrochloric acid), bases (e.g., ammonia, potassium hydroxide, sodium hydroxide, or sodium bicarbonate), peroxides (e.g., hydrogen peroxide or organic peroxides such as benzoic peroxide), oxidizing molecules (e.g., such as sodium hypochlorite, hydrogen peroxide, or hypochlorous acid), N-halamines, or a combination thereof. The selected of antimicrobial actives is further discussed later in this specification. The method may further comprise introducing an antimicrobial agent to the continuous transport phase of the biphasic polymer. The antimicrobial agent may be introduced for the first time, or the antimicrobial agent may be replenished after a period of time. Some embodiments utilize a method of filling or replenishing an antimicrobial agent in the biphasic polymer, the method comprising:(a) selecting an antimicrobial agent;(b) providing a biphasic polymer that is designed to contain the antimicrobial agent;(c) optionally measuring a concentration of the antimicrobial agent within the biphasic polymer;(d) providing a replenishment solution comprising a quantity of the antimicrobial agent and an antimicrobial-agent solvent;(e) applying the replenishment solution to the biphasic polymer; and(f) removing excess replenishment solution, if any, from the biphasic polymer, thereby generating an antimicrobial-agent-replenished biphasic polymer. In some embodiments, the antimicrobial agent is contained in the first liquid volume (Part A) of the two-part formula. In other embodiments, the antimicrobial agent is contained in the second liquid volume (Part B) of the two-part formula. In still other embodiments, the same antimicrobial agent is contained in the first and second liquid volumes of the two-part formula. In yet other embodiments, a first antimicrobial agent is contained in the first liquid volume, and a second antimicrobial agent is contained in the second liquid volume. In certain embodiments, a first antimicrobial agent precursor is contained in the first liquid volume, and a second antimicrobial agent precursor is contained in the second liquid volume; when the liquid volumes are combined, the first and second precursors react to form a selected antimicrobial agent. In other embodiments, no antimicrobial agent is contained in two-part formula. An antimicrobial agent may be introduced, such as via wiping or spraying, into the biphasic polymer after it is formed from the two-part formula. Some variations provide an antimicrobial structure comprising:(a) a discrete solid structural phase comprising a solid structural polymer, wherein the solid structural polymer is characterized by a glass-transition temperature from about 20° C. to about 300° C.;(b) a continuous transport phase that is interspersed within the discrete solid structural phase, wherein the continuous transport phase comprises a solid transport material; and(c) an antimicrobial agent contained within the continuous transport phase, wherein the antimicrobial agent is at least partially dissolved in a fluid and/or wherein the antimicrobial agent is in a solid solution with the continuous transport phase,wherein the discrete solid structural phase and the continuous transport phase are separated by an average phase-separation length selected from about 100 nanometers to about 500 microns. The concentration of antimicrobial agent in the biphasic polymer may be from about 1 ppm to about 10 wt % (based on all components present), depending on the specific antimicrobial agent and/or other factors. In various embodiments, the concentration of antimicrobial agent in the biphasic polymer is about, at least about, or at most about 1 ppm, 10 ppm, 25 ppm, 50 ppm, 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 0.1 wt %, 0.2 wt %, 0.5 wt %, 1 wt %, 2 wt %, 5 wt %, or 10 wt %, including any intervening ranges. The biphasic polymer may be a polymer described in U.S. Pat. No. 10,689,542, issued on Jun. 23, 2020; U.S. Pat. No. 11,225,589, issued on Jan. 18, 2022; and/or U.S. Pat. No. 11,369,109, issued on Jun. 28, 2022, each of which is hereby incorporated by reference herein for all purposes. In some embodiments, the discrete solid structural phase is covalently bonded to the continuous transport phase. In certain embodiments, the discrete solid structural phase is crosslinked, via a crosslinking molecule, with the continuous transport phase. In some embodiments, the solid structural polymer is a non-fluorinated carbon-based polymer. The non-fluorinated carbon-based polymer may be selected from the group consisting of polycarbonates, polyacrylates, polyalkanes, polyurethanes, polyethers, polyureas, polyesters, and combinations thereof. In some preferred embodiments, the solid structural polymer is a polycarbonate, a polyacrylate, or a combination thereof. In certain embodiments, the solid structural polymer is a polycarbonate, such as a polycarbonate that is end-terminated with hydroxyl groups (—OH), amino groups (—NH2), and/or epoxide groups (—O—). In certain embodiments, the solid structural polymer is a polyacrylate, such as a polyacrylate functionalized with alkanes, alkenes, and/or aromatic groups. The continuous transport phase may include a hygroscopic solid transport polymer as a solid transport material. The solid transport material may be a hygroscopic solid transport polymer selected from the group consisting of poly(acrylic acid), poly(ethylene glycol), poly(2-hydroxyethyl methacrylate), poly(vinyl imidazole), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(vinylpyrolidone), modified cellulosic polymers, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, and combinations thereof, for example. The continuous transport phase may include a hydrophobic, non-lipophobic solid transport polymer. The solid transport material may be a hydrophobic, non-lipophobic solid transport polymer selected from the group consisting of poly(propylene glycol), poly(tetramethylene glycol), polybutadiene (or other unsaturated polyolefins), polycarbonate, polycaprolactone, acrylic polyols, and combinations thereof, for example. The continuous transport phase may include a hydrophilic solid transport polymer with ionic charge. The solid transport material may be a hydrophilic solid transport polymer with ionic charge, wherein the ionic charge is optionally present within the hydrophilic solid transport polymer as carboxylate groups, amine groups, ammonium groups, sulfate groups, or phosphate groups, for example. The continuous transport phase may include an electrolyte solid transport polymer. The solid transport material may be an electrolyte solid transport polymer selected from the group consisting of polyethylene oxide, polypropylene oxide, polycarbonates, polysiloxanes, polyvinylidene difluoride, and combinations thereof, for example. In some embodiments, the continuous transport phase contains a fluid, wherein the antimicrobial agent is at least partially dissolved in the fluid. The fluid may be selected from the group consisting of water, alcohols, polyols, ketones, ethers, esters, carbonates, sulfoxides, ionic liquids, and combinations thereof, for example. Exemplary fluids are water, dialkyl carbonate, propylene carbonate, γ-butyrolactone, 2-phenoxyethanol, dimethyl sulfoxide, t-butanol, glycerol, propylene glycol, and ionic liquids. In some embodiments, a solid transport material is a solid transport polymer. The solid structural polymer may be crosslinked, via a crosslinking molecule, with the solid transport polymer. The crosslinking molecule may include at least one moiety selected from the group consisting of amine, hydroxyl, isocyanate, epoxide, carbodiimide, and combinations thereof, for example. Exemplary isocyanates include Vestanat® 1890 and Desmodur® 3300. The crosslinking molecule may also function as a chain extender. Alternatively, or additionally, a separate chain extender may be used. In some embodiments, a crosslinker or chain extender is selected from polyol or polyamine crosslinkers or chain extenders that possess a functionality of 2, 3, or greater. In various embodiments, polyol or polyamine crosslinkers or chain extenders are selected from the group consisting of 1,3-butanediol, 1,4-butanediol, 1,3-propanediol, 1,2-ethanediol, diethylene glycol, triethylene glycol, tetraethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, neopentyl glycol, 1,6-hexanediol, 1,4-cyclohexanedimethanol, ethanol amine, diethanol amine, methyldiethanolamine, phenyldiethanolamine, glycerol, trimethylolpropane, 1,2,6-hexanetriol, triethanolamine, pentaerythritol propoxylate, ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, diethyltoluenediamine, dimethylthiotoluenediamine, isophoronediamine, diaminocyclohexane, N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine, and homologues, derivatives, or combinations thereof. The average phase-separation length, between the discrete solid structural phase and the continuous transport phase, may vary widely. In some embodiments, the average phase-separation length is selected from about 100 nanometers to about 100 microns. In some embodiments, the average phase-separation length is selected from about 200 nanometers to about 50 microns. In some embodiments, the average phase-separation length is selected from about 1 micron to about 100 microns. In some embodiments, the average phase-separation length is selected from about 1 micron to about 50 microns. In various embodiments, the average phase-separation length is selected from about, at least about, or at most about 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 250 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, or 500 μm, including any intervening ranges (e.g., 150 nm-5 μm, 500 nm-45 μm, etc.). There may be narrow or broad distribution of phase-separation lengths. Exemplary imaging techniques to measure phase separation include, but are not limited to, confocal laser scanning microscopy, scanning electron microscopy, scanning tunneling microscopy, and atomic force microscopy. In some methods, the biphasic polymer is transparent or partially transparent for optical frequencies of ordinary light. Transparent, antimicrobial biphasic polymers are useful because they do not change the appearance of underlying substrates being coated (e.g., a door handle). The biphasic polymer may be characterized by an optical transparency of about 80% or greater. In various embodiments, the optical transparency of the biphasic polymer is about, or at least about, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, for example. In this disclosure, the optical transparency of a biphasic polymer is the light transmittance, averaged across light wavelengths from 400 nm to 800 nm, through a 100-micron film of the antimicrobial structure at 25° C. and 1 bar. When the biphasic polymer has a transparency less than 50%, the biphasic polymer may be characterized as translucent. The optical transparency of the biphasic polymer is a function of the optical transparency, the nature, and extent of individual components—the structural phase, the transport phase, the antimicrobial agent, and any other additives. In some embodiments, each component is at least partially transparent. Antimicrobial agent liquids or solutions are typically clear. When one component is relatively opaque, the overall antimicrobial structure may still have an acceptable transparency, depending on the amount of the relatively opaque component, for example. In certain embodiments, different phases of the biphasic polymer are selected such that the respective refraction indices are matched or substantially similar. One example is polytetrahydrofuran with poly(ethylene glycol), which are index-matched to within about 1%. Another example is polytetrahydrofuran with poly(propylene glycol), which are index-matched to within 2%. Another example is polycarbonate with poly(ethylene glycol), which are index-matched to within 10%. In some embodiments, a continuous transport phase and a discrete solid structural phase are selected such that the index of refraction matches to within ±10%, preferably within ±5%, more preferably with ±2%, and most preferably with ±1%. Note, however, that refractive-index matching is not a requirement of the present invention. The optical transparency of the biphasic polymer may temporarily deviate from its initial value when dirt or debris contaminate the surface, before the surface is wiped or cleaned. Antifouling biphasic polymers are beneficial to avoid permanent decrease in optical transparency in the case of non-cleanable fouling, for example. In some embodiments, a biphasic-polymer coating is stain-resistant due to the presence of fluorinated polymers or additives. Fluorinated materials have low surface energies which reduce the penetration of liquid contaminants, thereby enhancing stain resistance. In some embodiments, a biphasic-polymer coating is stain-resistant without the use of fluorinated polymers or additives. The stain resistance arises from the incorporation of materials with a glass-transition temperature above room temperature, instead of requiring fluorinated materials to avoid soil infiltration. In some embodiments, a discrete solid structural phase provides mechanical integrity and anti-fouling characteristics (stain resistance). The continuous transport phase acts as a medium for the fast diffusion of antimicrobial agents. Preferred variations utilize polymeric coatings that are solid but have fast transport rates of antimicrobial agents, enabled by a two-phase architecture with a discrete solid structural phase combined with an antimicrobial-containing continuous transport phase that is phase-separated with the discrete solid structural phase. In this patent application, “fast transport” means a specific conductivity of at least 10−5mS/cm. “Antimicrobial agents” or synonymously “antimicrobial actives” include germicides, bactericides, virucides (antivirals), antifungals, antiprotozoals, antiparasites, and biocides. In some embodiments, antimicrobial agents are specifically bactericides, such as disinfectants, antiseptics, and/or antibiotics. In some embodiments, antimicrobial agents are specifically virucides, or include virucides. Some embodiments overcome the conventional trade-off between antifouling and fluorinated material content. Fluorinated materials are usually employed in order to reject stains and fluids. By contrast, in some embodiments, a biphasic polymer incorporates a solid structural polymer having a glass-transition temperature above the use temperature. The crystallized nature of the solid structural polymer being below its Tgresults in the material not being penetrated by stains. A second phase, which is a continuous transport phase, enables removal of stains on the surface, in these embodiments. In certain embodiments, the entire biphasic polymer is non-fluorinated, i.e., contains essentially no fluorine. In these embodiments, there is no fluorine content in the two-part formula. Some embodiments overcome the conventional trade-off between transport of absorbed molecules and transparency. Phase separation of 0.1-500 μm results in up to 1000× faster diffusion compared to nanoscale (<100 nm) phase separation. Fast transport of antimicrobial agents is retained without creating an optically opaque antimicrobial structure. In some embodiments, the antimicrobial agent is selected from quaternary ammonium molecules, such as (but not limited to) benzalkonium chloride, benzethonium chloride, methylbenzethonium chloride, cetalkonium chloride, or combinations thereof. In some embodiments, the antimicrobial agent is selected from metal ions, such as (but not limited to) silver, copper, zinc, or combinations thereof. In some embodiments, the antimicrobial agent is selected from metal oxide nanoparticles, containing a metal oxide such as (but not limited to) ZnO, CuO, Cu2O, Ag2O, or a combination thereof. In some embodiments, the antimicrobial agent is selected from acids, such as (but not limited to) citric acid, acetic acid, peracetic acid, glycolic acid, lactic acid, succinic acid, pyruvic acid, oxalic acid, hydrochloric acid, or combinations thereof. In some embodiments, the antimicrobial agent is selected from bases, such as (but not limited to) ammonia, ammonium hydroxide, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, or combinations thereof. In some embodiments, the antimicrobial agent is selected from salts, such as (but not limited to) copper chloride, copper nitrate, copper citrate, copper acetate, zinc chloride, zinc nitrate, zinc citrate, zinc acetate, silver chloride, silver nitrate, silver citrate, silver acetate, or combinations thereof. In some embodiments, the antimicrobial agent is selected from peroxides, such as (but not limited to) hydrogen peroxide, organic peroxides (e.g., benzoic peroxide), or combinations thereof. In some embodiments, the antimicrobial agent is selected from oxidizing molecules, such as (but not limited to) sodium hypochlorite, calcium hypochlorite, hypochlorous acid, hydrogen peroxide, or combinations thereof. The antimicrobial agent preferably is not in the form of purely solid particles. In preferred embodiments, the antimicrobial agent is not in the form of solid particles at temperatures of use (e.g., 20-40° C.). Quaternary ammonium salts are deliquescent and will advantageously form a concentrated solution that does not dry out. Quaternary ammonium salts may absorb moisture from the air or may be dissolved in a solvent, such as ethylene glycol or oligomers thereof. Hypochlorous acid, sodium hypochlorite, calcium hypochlorite, and hydrogen peroxide only exist as solutions or liquids, practically speaking. Hypochlorous acid and sodium hypochlorite are never found dry because they decompose with increasing concentration before they dry out. Hydrogen peroxide is a liquid above −0.4° C. at 1 bar pressure. The antimicrobial agent may be at least partially dissolved in a fluid that is contained within the continuous transport phase. The fluid may be selected from the group consisting of water, dialkyl carbonate, propylene carbonate, γ-butyrolactone, 2-phenoxyethanol, dimethyl sulfoxide, t-butanol, glycerol, propylene glycol, ionic liquids, and combinations thereof, for example. In some embodiments, the biphasic polymer is characterized in that the antimicrobial agent has a diffusion coefficient from about 10−18m2/s to about 10−9m2/s, measured at 25° C. and 1 bar, within the continuous transport phase. In certain embodiments, the antimicrobial agent has a diffusion coefficient from about 10−16m2/s to about 10−11m2/s measured at 25° C. and 1 bar, within the continuous transport phase. In various embodiments, the antimicrobial agent has a diffusion coefficient, measured at 25° C. and 1 bar, within the continuous transport phase, of about, or at least about 10−17m2/s, 10−16m2/s, 10−15m2/s, 10−14m2/s, 10−13m2/s, 10−12m2/s, 10−11m2/s, 10−10m2/s, or 10−9m2/s, including any intervening ranges. The biphasic polymer may further contain one or more additives selected from the group consisting of buffers, UV stabilizers, fillers, pigments, flattening agents, flame retardants, salts, surfactants, dispersants, defoamers, wetting agents, antioxidants, and combinations thereof, for example. Other additives are possible as well. Additional variations of the present disclosure will now be described, without limiting the scope of the invention defined by the claims. The disclosed biphasic polymer is capable of resolving the technical tradeoffs between antimicrobial solutions and solid surfaces. Conventional liquid solutions are fast but not persistent. Liquid solutions can reduce the population of bacteria and viruses on a timescale of minutes, but the liquid solutions do not stay on surfaces and have a one-time effect. Conventional solid antimicrobial surfaces reduce bacteria and virus populations quite slowly, causing bacteria and virus to remain on surfaces for extended times. See Behzadinasab et al., “A Surface Coating that Rapidly Inactivates SARS-CoV-2”, ACS Appl. Mater. Interfaces2020, 12, 31, as an example of an antimicrobial coating that requires at least 1 hour for effectiveness. The slow activity of conventional solid antimicrobial materials is due to the time needed for antimicrobial agents to diffuse to the surface. These surfaces also fail to work if they are dirty, because soil blocks the transport of antimicrobial agents to the surface. By contrast, a biphasic polymer can break the tradeoff between activity and persistence. The discrete solid structural phase provides persistence on a surface while the continuous transport phase allows antimicrobial agents to move to microbes (e.g., viruses or bacteria) on the surface at order-of-magnitude faster rates than is possible with diffusion through a single solid material. A biphasic structure simultaneously provides durability and fast transport to the surface where antimicrobial agents can kill or deactivate microbes at the surface. The continuous transport phase may contain an aqueous or non-aqueous solvent or electrolyte to further enhance transport rates of antimicrobial agents. In some embodiments, the continuous transport phase passively absorbs water from the environment, which water may enhance transport rates of antimicrobial agents and/or improve the effectiveness of the antimicrobial agents. In preferred biphasic polymers, the solid structural polymer is covalently bonded to the solid transport polymer. In preferred embodiments, a solid structural polymer is crosslinked, via a crosslinking molecule, with a solid transport polymer. The crosslinking is preferably covalent crosslinking, but can alternatively be ionic crosslinking. When the discrete and continuous phases are covalently crosslinked, an abrasion-resistant structure is established within the continuous transport phase. Additionally, when the structural polymer and the transport polymer are crosslinked, the length scales of the different phases can be controlled, such as to enhance transport rates of the antimicrobial agent. A crosslinking molecule (when present) may include at least one moiety selected from the group consisting of an amine moiety, a hydroxyl moiety, an isocyanate moiety, and a combination thereof, for example. Other crosslinking molecules may be employed. In certain embodiments, at least one moiety is an isocyanate moiety, which may be a blocked isocyanate. In some embodiments, the continuous transport phase is a solid solution or solid suspension of the solid transport material and the antimicrobial agent. For example, when the antimicrobial agent is a liquid, the continuous transport phase may be a solution of the solid transport material and the antimicrobial agent. When the antimicrobial agent is a solid, the continuous transport phase may be a suspension of the solid transport material and the antimicrobial agent. In certain embodiments, the solid transport material and the antimicrobial agent form a true solid solution, which means that each material is dissolved in the other material such that a single solid phase results. In other embodiments, the continuous transport phase contains a transport-phase liquid that at least partially dissolves the antimicrobial agent. The transport-phase liquid may be selected from the group consisting of water, dialkyl carbonate, propylene carbonate, γ-butyrolactone, 2-phenoxyethanol, and combinations thereof. Alternatively, or additionally, the transport-phase liquid is selected from polar solvents. Polar solvents may be protic polar solvents or aprotic polar solvents. Exemplary polar solvents include, but are not limited to, water, alcohols, ethers, esters, ketones, aldehydes, carbonates, and combinations thereof. In some embodiments, the transport-phase liquid is water that is passively incorporated from atmospheric humidity. Alternatively, or additionally, the transport-phase liquid is selected from ionic liquids. Exemplary ionic liquids include, but are not limited to, ammonium-based ionic liquids synthesized from substituted quaternary ammonium salts. In some embodiments, the antimicrobial agent is selected from quaternary ammonium molecules (whether or not classified as an ionic liquid). Exemplary quaternary ammonium molecules include, but are not limited to, benzalkonium chloride, benzethonium chloride, methylbenzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, cetrimonium, cetrimide, tetraethylammonium bromide, didecyldimethylammonium chloride, dioctyldimethylammonium chloride, and domiphen bromide. Quaternary ammonium molecules or eutectic mixtures of quaternary ammonium molecules that are liquids at room temperature—ionic liquids or ionic liquid eutectics, respectively—enable liquid-state rates of transport with negligible vapor pressure. A specific example is tetrabutylammonium heptadecafluorooctanesulfonate (C24H36F17NO3S), which has a melting point <5° C. Another specific example is tetraoctylammonium chloride (C32H68ClN) with a melting point of 50-54° C. mixed with tetraheptylammonium chloride (C28H60ClN) with a melting point of 38-41° C. in a eutectic composition ratio that forms a liquid at room temperature (25° C.). Quaternary ammonium molecules may be mixed with imidazolium-based ionic liquids, pyridinium-based ionic liquids, pyrrolidinium-based ionic liquids, and/or phosphonium-based ionic liquids. In certain embodiments, the transport-phase liquid contains one or more water-soluble salts, one or more of which may function as an antimicrobial agent. Exemplary water-soluble salts include, but are not limited to, copper chloride, copper nitrate, zinc chloride, zinc nitrate, silver chloride, silver nitrate, or combinations thereof. Other exemplary water-soluble salts include quaternary ammonium salts, such as (but not limited to) the quaternary ammonium molecules recited above. In certain embodiments, the transport-phase liquid is a eutectic liquid salt, which is optionally derived from ammonium salts. The eutectic liquid salt may contain an antimicrobial agent or otherwise be antimicrobially active. In some embodiments, the antimicrobial agent is selected from oxidizing molecules, such as (but not limited to) those selected from the group consisting of sodium hypochlorite, calcium hypochlorite, hypochlorous acid, hydrogen peroxide, and combinations thereof. In some embodiments, the antimicrobial agent is selected from metal ions, such as (but not limited to) silver, copper, zinc, cobalt, nickel, or combinations thereof. Any metal ion with at least some antimicrobial activity itself, or which confers antimicrobial activity to a compound which the metal ion binds to, may be employed. The metal ion may be present in a metal complex or a metal salt, for example. Metal ions may be present in oxides. In certain embodiments, the antimicrobial agent contains a neutral metal (e.g., zero-valent silver, copper, or zinc) which may be dissolved in a liquid and/or may be present as nanoparticles, for example. The antimicrobial biphasic polymer disclosed herein is not limited to transport of antimicrobial agent exclusively by pure diffusion. Depending on the specific choice of materials, antimicrobial agent, and method of using the structure, the actual transport may occur by various mass-transfer mechanisms including, but not limited to, Fickian diffusion, non-Fickian diffusion permeation, sorption transport, solubility-diffusion, charge-driven flow, convection, capillary-driven flow, and so on. As just one example, when the antimicrobial biphasic polymer is employed in an automobile, the polymer can move around quickly in space such that the antimicrobial agent undergoes some amount of centrifugal convection. Even when the mass transport is dominated by diffusion, the actual transport rate (flux) of antimicrobial agent through the structure depends not only on the diffusion coefficient, but also on the three-dimensional concentration gradient, temperature, and possibly other factors such as pH. In various embodiments, the actual flux of antimicrobial agent through the structure is about, or at least about, 2×, 3×, 4×, 5×, 10×, 20×, 30×, 40×, 50×, 100×, 200×, 300×, 400×, 500×, or 1000× higher than the flux through a solid-state material. A person of ordinary skill in the art can calculate or estimate transport fluxes for a given structure geometry and materials, or carry out experiments to determine such fluxes. The antimicrobial biphasic polymer may be characterized by an original concentration of antimicrobial agent (prior to exposure to microbes). The original concentration of antimicrobial agent may be selected based on the type of antimicrobial agent, and intended use of the antimicrobial biphasic polymer, and/or other factors. In various embodiments, the original concentration of antimicrobial agent is about, at least about, or at most about 0.00001 wt %, 0.0001 wt %, 0.001 wt %, 0.01 wt %, 0.1 wt %, 1 wt %, 5 wt %, 10 wt %, 20 wt %, 30 wt %, 40 wt %, or 50 wt %, on the basis of mass of antimicrobial agent divided by total mass of all components within 0.1%, 1%, 5%, or 10% depth from the surface into the bulk structure. An electrolyte may be included in the continuous transport phase, to increase transport rates of the antimicrobial agent. An exemplary electrolyte is a complex formed between poly(ethylene oxide) and metal salts, such as poly(ethylene oxide)-Cu(CF3SO3)2which is a known copper conductor. Cu(CF3SO3)2is the copper(II) salt of trifluoromethanesulfonic acid. See Bonino et al., “Electrochemical properties of copper-based polymer electrolytes”,Electrochimica Acta, Vol. 37, No. 9, Pages 1711-1713 (1992), which is incorporated by reference. When an electrolyte is included in the continuous transport phase, one or more solvents for the electrolyte may be present. Solvents for the electrolyte may be selected from the group consisting of sulfoxide, sulfolane, ethylene carbonate, propylene carbonate, dimethyl carbonate, diethyl carbonate, methyl ethyl carbonate, 1,2-dimethoxyethane, 1,2-diethoxyethane, γ-buterolactone, γ-valerolactone, 1,3-dioxolane, tetrahydrofuran, 2-methyltetrahydrofuran, acetonitrile, proprionitrile, diglyme, triglyme, methyl formate, trimethyl phosphate, triethyl phosphate, and mixtures thereof, for example. When an electrolyte is included in the continuous transport phase, there may be a salt within an aqueous or non-aqueous solvent. Exemplary salts are salts of transition metals (e.g., V, Ti, Cr, Co, Ni, Cu, Zn, Tb, W, Ag, Cd, or Au), salts of metalloids (e.g., Al, Ga, Ge, As, Se, Sn, Sb, Te, or Bi), salts of alkali metals (e.g., Li, Na, or K), salts of alkaline earth metals (e.g., Mg or Ca), or a combination thereof. In some embodiments, a gel electrolyte is included in the continuous transport phase. A gel electrolyte contains a liquid electrolyte including an aqueous or non-aqueous solvent as well as a salt, in a polymer host. The solvent and salt may be selected from the lists above. The polymer host may be selected from the group consisting of poly(ethylene oxide), poly(vinylidene fluoride), poly(acrylonitrile), poly(methyl methacrylate), poly(vinylidene fluoride—hexafluoropropylene) (PVdF-co-HFP), polycarbonate, polysiloxane, and combinations thereof. N-halamines may be incorporated into the backbone of the biphasic polymer, in the structural phase, the transport phase, or both phases. N-halamines are compounds that stabilize an oxidizing agent (such as chlorine contained within the N-halamine molecule) and may be used to kill or deactivate microbes. N-halamines remain stable over long time periods and may be recharged by exposure to an oxidizer such as dilute bleach or ozone. Exemplary N-halamines include, but are not limited to, hydantoin (imidazolidine-2,4-dione); 1,3-dichloro-5,5-dimethylhydantoin; 3-bromo-1-chloro-5,5-dimethylhydantoin; 5,5-dimethylhydantoin; 4,4-dimethyl-2-oxazalidinone; tetramethyl-2-imidazolidinone; and 2,2,5,5-tetramethylimidazo-lidin-4-one. Examples of antimicrobial N-halamines are also disclosed in Lauten et al.,Applied and Environmental MicrobiologyVol. 58, No. 4, Pages 1240-1243 (1992), which is incorporated by reference. In certain embodiments, the antimicrobial structure further contains one or more layers of an antimicrobial-agent storage phase that is distinct from the continuous transport phase and the discrete solid structural phase. In these or other certain embodiments, the antimicrobial structure further contains inclusions of an antimicrobial-agent storage phase that is distinct from the continuous transport phase and the discrete solid structural phase. An antimicrobial-agent storage phase may be fabricated from the same material as the solid transport material, or from a different material. For example, both the solid transport material and the antimicrobial-agent storage phase (when present) may be made from a hydrophobic, non-lipophobic polymer. The antimicrobial-agent storage phase may contain an antimicrobial agent that is released initially, continuously, or periodically into the continuous transport phase. The antimicrobial structure may further contain one or more additives, such as (but not limited to) salts, buffers, UV stabilizers, particulate fillers, pigments, flattening agents, surfactants, dispersants, flame retardants, or combinations thereof. Additives, when present, may be incorporated into the discrete solid structural phase, the continuous transport phase, both of these phases, or neither of these phases but within a separate phase. When an additive is a salt, there will be a cation and anion forming the salt. The cation element may be Li, Na, K, Mg, and/or Ca, for example. The anion element or group may be F, Cl, Br, I, SO3, SO4, NO2, NO3, CH3COO, and/or CO3, for example. When an additive is a buffer, it may be an inorganic or organic molecule that maintains a pH value or pH range via acid-base reactions. A buffer may be discrete or may be bonded to the solid transport material, for example. When an additive is a UV stabilizer, it may be an antioxidant (e.g., a thiol), a hindered amine (e.g., a derivative of tetramethylpiperidine), UV-absorbing nanoparticles (e.g., TiO2, ZnO, CdS, CdTe, or ZnS—Ag nanoparticles), or a combination thereof, for example. When an additive is a particulate filler, it may be selected from the group consisting of silica, alumina, silicates, talc, aluminosilicates, barium sulfate, mica, diatomite, calcium carbonate, calcium sulfate, carbon, wollastonite, and a combination thereof, for example. A particulate filler is optionally surface-modified with a compound selected from the group consisting of fatty acids, silanes, alkylsilanes, fluoroalkylsilanes, silicones, alkyl phosphonates, alkyl phosphonic acids, alkyl carboxylates, alkyldisilazanes, and combinations thereof, for example. When an additive is a pigment, it may be selected from the group consisting of metal-complex pigments, azo pigments, polycyclic pigments, and anthraquinone pigments. Metal-oxide pigments include titanium dioxide, cobalt oxide, and iron oxide, for example. When an additive is a flame retardant for the suppression of flammability (e.g., to inhibit flame propagation), the flame retardant may be selected from the group consisting of ammonium salts, phosphate salts, phosphines, halogenated compounds, carbonate salts, hydroxide salts, borate salts, high-surface-area silicas, expandable graphite, and combinations thereof. Specific examples of flame retardants are ammonium polyphosphate, magnesium hydroxide, zinc hydroxystannate, antimony trioxide, magnesium hydroxycarbonate, zinc borate, magnesium aluminum hydroxycarbonate, aluminum trihydroxide, tetrabromobisphenol A, tetrabromobisphenol A bis(2,3-dibromopropyl ether), bisphenol-A bis(diphenyl phosphate), brominated polyols, melamine resins, chlorinated paraffins, and combinations thereof. Some embodiments will now be further described in reference to exemplary synthesis of a discrete solid structural phase and a continuous transport phase, a preferred biphasic architecture, and selective incorporation of an antimicrobial agent within the continuous transport phase. Some embodiments are premised on the preferential incorporation of an antimicrobial agent within one phase of a multiphase polymer coating. The structure of a microphase-separated polymer network provides a reservoir for antimicrobial agents within the continuous phase. As intended herein, “microphase-separated” means that the first and second solid materials (e.g., soft segments) are physically separated on a microphase-separation length scale from about 0.1 microns to about 500 microns. Unless otherwise indicated, all references to “phases” in this patent application are in reference to solid phases or fluid phases. A “phase” is a region of space (forming a thermodynamic system), throughout which all physical properties of a material are essentially uniform. Examples of physical properties include density and chemical composition. A solid phase is a region of solid material that is chemically uniform and physically distinct from other regions of solid material (or any liquid or vapor materials that may be present). Solid phases are typically polymeric and may melt or at least undergo a glass transition at elevated temperatures. Reference to multiple solid phases in a composition or microstructure means that there are at least two distinct material phases that are solid, without forming a solid solution or homogeneous mixture. In some embodiments, the antimicrobial agent is in a fluid. Preferably, the fluid is not solely in a vapor phase at 25° C., since vapor is susceptible to leaking from the structure. However, the fluid may contain vapor in equilibrium with liquid, at 25° C. Also, in certain embodiments, a fluid is in liquid form at 25° C. but at least partially in vapor form at a higher use temperature, such as 30° C., 40° C., 50° C., or higher. By a liquid being “disposed in” a solid material, it is meant that the liquid is incorporated into the bulk phase of the solid material, and/or onto surfaces of particles of the solid material. The liquid will be in close physical proximity with the solid material, intimately and/or adjacently. The disposition is meant to include various mechanisms of chemical or physical incorporation, including but not limited to, chemical or physical absorption, chemical or physical adsorption, chemical bonding, ion exchange, or reactive inclusion (which may convert at least some of the liquid into another component or a different phase, including potentially a solid). Also, a liquid disposed in a solid material may or may not be in thermodynamic equilibrium with the local composition or the environment. Liquids may or may not be permanently contained in the structure; for example, depending on volatility or other factors, some liquid may be lost to the environment over time. By “selectively” disposed in the continuous transport phase, or the “selectivity” into the continuous transport phase, it is meant that of the antimicrobial agent that is disposed within the structure overall, at least 51%, preferably at least 75%, and more preferably at least 90% of the antimicrobial agent is disposed in only the continuous transport phase. In various embodiments, the selectivity into the continuous transport phase is about, or at least about, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In some embodiments, a liquid is added to a polymer such as by submerging and soaking into the polymer. In these embodiments, the liquid may be absorbed into a solid polymer. In certain embodiments, the liquid absorption swells a polymer, which means that there is an increase of volume of polymer due to absorption of the liquid. The liquid may be, but does not need to be, classified as a solvent for the solid polymer which it swells. The phase-separated microstructure preferably includes discrete islands of one material (the discrete solid structural phase) within a continuous sea of the other material (the continuous transport phase). The continuous phase provides unbroken channels within the material for transport of mass and/or electrical charge. In some embodiments, there are both phase-separated inclusions of the same chemical material, as well as physically and chemically distinct materials as additional inclusions. The discrete solid structural phase and the continuous transport phase may be present as phase-separated regions of a copolymer, such as a block copolymer. As intended herein, a “block copolymer” means a copolymer containing a linear arrangement of blocks, where each block is defined as a portion of a polymer molecule in which the monomeric units have at least one constitutional or configurational feature absent from the adjacent portions. Segmented block copolymers are preferred, providing two (or more) phases. An exemplary segmented copolymer is a urethane-urea copolymer. In some embodiments, a segmented polyurethane includes a microphase-separated structure of fluorinated and non-fluorinated species. In some embodiments, a segmented copolymer is employed in which first soft segments form a continuous matrix and second soft segments are a plurality of discrete inclusions. In other embodiments, the first soft segments are a plurality of discrete inclusions and the second soft segments form a continuous matrix. Segmented copolymers are typically created by combining a flexible oligomeric soft segment terminated with an alcohol or amine reactive groups and a multifunctional isocyanate. When the isocyanate is provided in excess relative to the alcohol/amine reactive groups, a viscous prepolymer mixture with a known chain length distribution is formed. This can then be cured to a high-molecular-weight network through the addition of amine or alcohol reactive groups to bring the ratio of isocyanate to amine/alcohol groups to unity. The product of this reaction is a chain backbone with alternating segments: soft segments of flexible oligomers and hard segments of the reaction product of low-molecular-weight isocyanates and alcohol/amines. Due to the chemical immiscibility of these two phases, the material typically phase-separates on the length scale of these individual molecular blocks, thereby creating a microstructure of flexible regions adjacent to rigid segments strongly associated through hydrogen bonding of the urethane/urea moieties. This combination of flexible and associated elements typically produces a physically crosslinked elastomeric material. Some variations of the invention utilize a segmented copolymer composition comprising:(a) one or more first soft segments selected from fluoropolymers having an average molecular weight from about 500 g/mol to about 20,000 g/mol, wherein the fluoropolymers are (α,ω)-hydroxyl-terminated, (α,ω)-amine-terminated, and/or (α,ω)-thiol-terminated;(b) one or more second soft segments selected from polyesters or polyethers, wherein the polyesters or polyethers are (α,ω)-hydroxyl-terminated, (α,ω)-amine-terminated, and/or (α,ω)-thiol-terminated;(c) one or more isocyanate species possessing an isocyanate functionality of 2 or greater, or a reacted form thereof; and(d) one or more polyol or polyamine chain extenders or crosslinkers, or a reacted form thereof,wherein the first soft segments and the second soft segments may (in some embodiments) be microphase-separated on a microphase-separation length scale from about 0.1 microns to about 500 microns, andoptionally wherein the molar ratio of the second soft segments to the first soft segments is less than 2.0. In some embodiments, fluoropolymers are present in the triblock structure: wherein:X, Y═CH2—(O—CH2—CH2)p-T, and X and Y are independently selected;p=1 to 50;T is a hydroxyl, amine, or thiol terminal group;m=0 to 100 (in some embodiments, m=1 to 100); andn=0 to 100 (in some embodiments, n=1 to 100). Some variations of the invention utilize a segmented copolymer composition comprising:(a) one or more first soft segments selected from polycarbonates having an average molecular weight from about 500 g/mol to about 20,000 g/mol, wherein the polycarbonates are (α,ω)-hydroxyl-terminated, (α,ω)-amine-terminated, and/or (α,ω)-thiol-terminated;(b) one or more second soft segments selected from polyesters or polyethers, wherein the polyesters or polyethers are (α,ω)-hydroxyl-terminated, (α,ω)-amine-terminated, and/or (α,ω)-thiol-terminated;(c) one or more isocyanate species possessing an isocyanate functionality of 2 or greater, or a reacted form thereof; and(d) one or more polyol or polyamine chain extenders or crosslinkers, or a reacted form thereof,wherein the first soft segments and the second soft segments may (in some embodiments) be microphase-separated on a microphase-separation length scale from about 0.1 microns to about 500 microns. In some embodiments, the continuous transport phase includes a polyelectrolyte and a counterion to the polyelectrolyte. The polyelectrolyte may be selected from the group consisting of poly(acrylic acid) or copolymers thereof, cellulose-based polymers, carboxymethyl cellulose, chitosan, poly(styrene sulfonate) or copolymers thereof, poly(acrylic acid) or copolymers thereof, poly(methacrylic acid) or copolymers thereof, poly(allylamine), and combinations thereof, for example. The counterion may be selected from the group consisting of H+, Li+, Na+, K+, Ag+, Ca2+, Mg2+, La3+, C16N+, F−, Cl−, Br−, I−, BF4−, So42−, PO42−, C12SO3−, and combinations thereof, for example. Other ionic species, combined with counterions, may be employed as well in the continuous transport phase. Generally, in some embodiments, ionic species may be selected from the group consisting of an ionizable salt, an ionizable molecule, a zwitterionic component, a polyelectrolyte, an ionomer, and combinations thereof. An “ionomer” is a polymer composed of ionomer molecules. An “ionomer molecule” is a macromolecule in which a significant (e.g., greater than 1, 2, 5, 10, 15, 20, or 25 mol %) proportion of the constitutional units have ionizable or ionic groups, or both. The classification of a polymer as an ionomer versus polyelectrolyte depends on the level of substitution of ionic groups as well as how the ionic groups are incorporated into the polymer structure. For example, polyelectrolytes also have ionic groups covalently bonded to the polymer backbone, but have a higher ionic group molar substitution level (such as greater than 50 mol %, usually greater than 80 mol %). Polyelectrolytes are polymers whose repeating units bear an electrolyte group. Polyelectrolyte properties are thus similar to both electrolytes (salts) and polymers. Like salts, their solutions are electrically conductive. Like polymers, their solutions are often viscous. In some embodiments, the continuous transport phase includes a polymer such as a polyurethane, a polyurea, a polysiloxane, or a combination thereof, with at least some charge along the polymer backbone. Polymer charge may be achieved through the incorporation of ionic monomers such as dimethylolpropionic acid, or another ionic species. The degree of polymer charge may vary, such as about, or at least about, 1, 2, 5, 10, 15, 20, or 25 mol % of the polymer repeat units being ionic repeat units. In some embodiments, the continuous transport phase includes an ionic species selected from the group consisting of (2,2-bis-(1-(1-methyl imidazolium)-methylpropane-1,3-diol bromide), 1,2-bis(2′-hydroxyethyl)imidazolium bromide, (3-hydroxy-2-(hydroxymethyl)-2-methylpropyl)-3-methyl-1H-3λ4-imidazol-1-ium bromide, 2,2-bis(hydroxymethyl)butyric acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, N-methyl-2,2′-iminodiethanol, 3-dimethylamino-1,2-propanediol, 2,2-bis(hydroxymethyl)propionic acid, 1,4-bis(2-hydroxyethyl)piperazine, 2,6-diaminocaproic acid, N,N-bis(2-hydroxyethyl)glycine, 2-hydroxypropanoic acid hemicalcium salt, dimethylolpropionic acid, N-methyldiethanolamine, N-ethyldiethanolamine, N-propyldiethanolamine, N-benzyldiethanolamine, N-t-butyldiethanolamine, bis(2-hydroxyethyl) benzylamine, bis(2-hydroxypropyl) aniline, and homologues, combinations, derivatives, or reaction products thereof. A liquid may be introduced into the continuous transport phase actively, passively, or a combination thereof. In some embodiments, a liquid is actively introduced to the continuous transport phase by spraying of the liquid, deposition from a vapor phase derived from the liquid, liquid injection, bath immersion, or other techniques. In some embodiments, a liquid is passively introduced to the continuous transport phase by letting the liquid naturally be extracted from the normal atmosphere, or from a local atmosphere adjusted to contain one or more desired liquids in vapor or droplet (e.g., mist) form. In certain embodiments, a desired additive is normally a solid at room temperature and is first dissolved or suspended in a liquid that is then disposed in the continuous transport phase. In other certain embodiments, a desired additive is normally a solid at room temperature and is first melted to produce a liquid that is then disposed in the continuous transport phase. Within the continuous transport phase, the desired additive may partially or completely solidify back to a solid, or may form a multiphase material, for example. Some potential additives contain reactive groups that unintentionally react with chemical groups contained in the polymer precursors. Therefore, in some cases, there exists an incompatibility of liquid species in the resin during chemical synthesis and polymerization. Addition of reactive fluid additives into the reaction mixture during synthesis can dramatically alter stoichiometry and backbone structure, while modifying physical and mechanical properties. One strategy to circumvent this problem is to block the reactive groups (e.g., alcohols, amines, and/or thiols) in the fluid additive with chemical protecting groups to render them inert to reaction with other reactive chemical groups (e.g., isocyanates) in the coating precursors. In particular, it is possible to temporarily block a reactive position by transforming it into a new functional group that will not interfere with the desired transformation. That blocking group is conventionally called a “protecting group.” Incorporating a protecting group into a synthesis requires at least two chemical reactions. The first reaction transforms the interfering functional group into a different one that will not compete with (or compete at a lower reaction rate with) the desired reaction. This step is called protection. The second chemical step transforms the protecting group back into the original group at a later stage of synthesis. This latter step is called deprotection. In some embodiments in which an additive contains alcohol, amine, and/or thiol groups, the additive thus contains chemical protecting groups to prevent or inhibit reaction of the alcohol, amine, and/or thiol groups with isocyanates. The protecting groups may be designed to undergo deprotection upon reaction with atmospheric moisture, for example. In the case of an additive containing alcohol groups, the protecting groups may be selected from the silyl ether class of alcohol protecting groups. For example, the protecting groups may be selected from the group consisting of trimethylsilyl ether, isopropyldimethylsilyl ether, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether, tribenzylsilyl ether, triisopropylsilyl ether, and combinations thereof. In these or other embodiments, the protecting groups to protect alcohol may be selected from the group consisting of 2,2,2-trichloroethyl carbonate, 2-methoxyethoxymethyl ether, 2-naphthylmethyl ether, 4-methoxybenzyl ether, acetate, benzoate, benzyl ether, benzyloxymethyl acetal, ethoxyethyl acetal, methoxymethyl acetal, methoxypropyl acetal, methyl ether, tetrahydropyranyl acetal, triethylsilyl ether, and combinations thereof. In the case of an additive containing amine groups, the protecting groups may be selected from the carbamate class of amine protecting groups, such as (but not limited to) vinyl carbamate. Alternatively, or additionally, the protecting groups may be selected from the ketamine class of amine protecting groups. In these or other embodiments, the protecting groups to protect amine may be selected from the group consisting of 1-chloroethyl carbamate, 4-methoxybenzenesulfonamide, acetamide, benzylamine, benzyloxy carbamate, formamide, methyl carbamate, trifluoroacetamide, tert-butoxy carbamate, and combinations thereof. In the case of an additive containing thiol groups, the protecting groups may be selected from S-2,4-dinitrophenyl thioether and/or S-2-nitro-1-phenylethyl thioether, for example. The typical reaction mechanism when water is the deprotecting reagent is simple hydrolysis. Water is often nucleophilic enough to kick off a leaving group and deprotect a species. One example of this is the protection of an amine with a ketone to form a ketamine. These can be mixed with isocyanates when the amine alone would react so quickly as to not be able to be practically mixed. Instead the ketamine reagent is inert but after mixing and casting as a film, atmospheric moisture will diffuse into the coating, remove the ketone (which vaporizes itself) and leaves the amine to rapidly react with neighboring isocyanates in situ. Many deprotecting agents require high pH, low pH, or redox chemistry to work. However, some protecting groups are labile enough that water alone is sufficient to cause deprotection. When possible, a preferred strategy to spontaneously deprotect the molecules is through reaction with atmospheric moisture, such as an atmosphere containing from about 10% to about 90% relative humidity at ambient temperature and pressure. A well-known example is the room-temperature vulcanization of silicones. These systems have silyl ethers that are deprotected with moisture and in doing so the free Si—OH reacts with other silyl ethers to create Si—O—Si covalent bonds, forming a network. In other embodiments, a chemical deprotection step is actively conducted, such as by introducing a deprotection agent and/or adjusting mixture conditions such as temperature, pressure, pH, solvents, electromagnetic field, or other parameters. This specification hereby incorporates by reference herein Greene and Wuts,Protective Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons, New York, 2007, for its teachings of the role of protecting groups, synthesis of protecting groups, and deprotection schemes including for example adjustment of pH by addition of acids or bases, to cause deprotection. As intended in this patent application, “hygroscopic” means that a material is capable of attracting and holding water molecules from the surrounding environment. The water uptake of various polymers is described in Thijs et al., “Water uptake of hydrophilic polymers determined by a thermal gravimetric analyzer with a controlled humidity chamber”J. Mater. Chem., (17) 2007, 4864-4871, which is hereby incorporated by reference herein. In some embodiments, a hygroscopic material is characterized by a water absorption capacity, at 90% relative humidity and 30° C., of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt % uptake of H2O. In some embodiments employing segmented copolymers, one of the first soft segments and second soft segments is oleophobic. An oleophobic material has a poor affinity for oils. As intended herein, the term “oleophobic” means a material with a contact angle of hexadecane greater than 90°. An oleophobic material may also be classified as lipophobic. In some embodiments employing segmented copolymers, one of the first soft segments and the second soft segments may be a “low-surface-energy polymer” which means a polymer, or a polymer-containing material, with a surface energy of no greater than 50 mJ/m2. In some embodiments, one of the first soft segments and the second soft segments has a surface energy from about 5 mJ/m2to about 50 mJ/m2. In some embodiments employing segmented copolymers, the first soft segments or the second soft segments may be or include a fluoropolymer, such as (but not limited to) a fluoropolymer selected from the group consisting of polyfluoroethers, perfluoropolyethers, fluoroacrylates, fluorosilicones, polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polyvinylfluoride (PVF), polychlorotrifluoroethylene (PCTFE), copolymers of ethylene and trifluoroethylene, copolymers of ethylene and chlorotrifluoroethylene, and combinations thereof. In these or other embodiments, the first soft segments or the second soft segments may be or include a siloxane. A siloxane contains at least one Si—O—Si linkage. The siloxane may consist of polymerized siloxanes or polysiloxanes (also known as silicones). One example is polydimethylsiloxane. In some embodiments, the molar ratio of the second soft segments to the first soft segments is about 2.0 or less. In various embodiments, the molar ratio of the second soft segments to the first soft segments is about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 1.95. It is noted that (α,ω)-terminated polymers are terminated at each end of the polymer. The α-termination may be the same or different than the ω-termination on the opposite end. The fluoropolymers and/or the polyesters or polyethers may terminated with a combination of hydroxyl groups, amine groups, and thiol groups, among other possible termination groups. Note that thiols can react with an —NCO group (usually catalyzed by tertiary amines) to generate a thiourethane. Also it is noted that in this disclosure, “(α,ω)-termination” includes branching at the ends, so that the number of terminations may be greater than 2 per polymer molecule. The polymers herein may be linear or branched, and there may be various terminations and functional groups within the polymer chain, besides the end (α,ω) terminations. In this description, “polyurethane” is a polymer comprising a chain of organic units joined by carbamate (urethane) links, where “urethane” refers to N(H)—(C═O)—O—. Polyurethanes are generally produced by reacting an isocyanate containing two or more isocyanate groups per molecule with one or more polyols containing on average two or more hydroxyl groups per molecule, in the presence of a catalyst. Polyols are polymers with on average two or more hydroxyl groups per molecule. For example, a,w-hydroxyl-terminated perfluoropolyether is a type of polyol. “Isocyanate” is the functional group with the formula —N═C═O. For the purposes of this disclosure, O—C(═O)—N(H)—R is considered a derivative of isocyanate. “Isocyanate functionality” refers to the number of isocyanate reactive sites on a molecule. For example, diisocyanates have two isocyanate reactive sites and therefore an isocyanate functionality of 2. Triisocyanates have three isocyanate reactive sites and therefore an isocyanate functionality of 3. “Polyfluoroether” refers to a class of polymers that contain an ether group—an oxygen atom connected to two alkyl or aryl groups, where at least one hydrogen atom is replaced by a fluorine atom in an alkyl or aryl group. “Perfluoropolyether” (PFPE) is a highly fluorinated subset of polyfluoroethers, wherein all hydrogen atoms are replaced by fluorine atoms in the alkyl or aryl groups. “Polyurea” is a polymer comprising a chain of organic units joined by urea links, where “urea” refers to N(H)—(C═O)—N(H)—. Polyureas are generally produced by reacting an isocyanate containing two or more isocyanate groups per molecule with one or more multifunctional amines (e.g., diamines) containing on average two or more amine groups per molecule, optionally in the presence of a catalyst. A “chain extender or crosslinker” is a compound (or mixture of compounds) that link long molecules together and thereby complete a polymer reaction. Chain extenders or crosslinkers are also known as curing agents, curatives, or hardeners. In polyurethane/urea systems, a curative is typically comprised of hydroxyl-terminated or amine-terminated compounds which react with isocyanate groups present in the mixture. Diols as curatives form urethane linkages, while diamines as curatives form urea linkages. The choice of chain extender or crosslinker may be determined by end groups present on a given prepolymer. In the case of isocyanate end groups, curing can be accomplished through chain extension using multifunctional amines or alcohols, for example. Chain extenders or crosslinkers can have an average functionality greater than 2 (such as 2.5, 3.0, or greater), i.e. beyond diols or diamines. In some embodiments, polyesters or polyethers are selected from the group consisting of poly(oxymethylene), poly(ethylene glycol), poly(propylene glycol), poly(tetrahydrofuran), poly(glycolic acid), poly(caprolactone), poly(ethylene adipate), poly(hydroxybutyrate), poly(hydroxyalkanoate), and combinations thereof. In some embodiments, the isocyanate species is selected from the group consisting of 4,4′-methylenebis(cyclohexyl isocyanate), hexamethylene diisocyanate, cycloalkyl-based diisocyanates, tolylene-2,4-diisocyanate, 4,4′-methylenebis(phenyl isocyanate), isophorone diisocyanate, and combinations or derivatives thereof. The polyol or polyamine chain extender or crosslinker possesses a functionality of 2 or greater, in some embodiments. At least one polyol or polyamine chain extender or crosslinker may be selected from the group consisting of 1,4-butanediol, 1,3-propanediol, 1,2-ethanediol, glycerol, trimethylolpropane, ethylenediamine, isophoronediamine, diaminocyclohexane, and homologues, derivatives, or combinations thereof. In some embodiments, polymeric forms of polyol chain extenders or crosslinkers are utilized, typically hydrocarbon or acrylic backbones with hydroxyl groups distributed along the side groups. The one or more chain extenders or crosslinkers (or reaction products thereof) may be present in a concentration, in the segmented copolymer composition, from about 0.01 wt % to about 25 wt %, such as from about 0.05 wt % to about 10 wt %. First soft segments may be present in a concentration from about 5 wt % to about 95 wt % based on total weight of the composition. In various embodiments, the first soft segments may be present in a concentration of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95 wt % based on total weight of the composition. Second soft segments may be present in a concentration from about 5 wt % to about 95 wt % based on total weight of the composition. In various embodiments, the second soft segments may be present in a concentration of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95 wt % based on total weight of the composition. In some embodiments, fluorinated polyurethane oligomers are terminated with silane groups. The end groups on the oligomers (in the prepolymer) may be modified from isocyanate to silyl ethers. This can be accomplished through reaction of an isocyanate-reactive silane species (e.g., aminopropyltriethoxysilane) to provide hydrolysable groups well-known in silicon and siloxane chemistry. Such an approach eliminates the need for addition of a stoichiometric amount of curative to form strongly associative hard segments, while replacing the curative with species that possess the ability to form a covalently crosslinked network under the influence of moisture or heat. Such chemistry has been shown to preserve beneficial aspects of urethane coatings while boosting scratch resistance. In addition, the reactivity of the terminal silane groups allows for additional functionality in the form of complimentary silanes blended with the prepolymer mixture. The silanes are able to condense into the hydrolysable network upon curing. This strategy allows for discrete domains of distinct composition. A specific embodiment relevant to anti-fouling involves the combination of fluoro-containing urethane prepolymer that is endcapped by silane reactive groups with additional alkyl silanes. In some embodiments employing segmented copolymers, the microphase-separated microstructure containing the first and second soft segments may be characterized as an inhomogeneous microstructure. As intended in this patent application, “phase inhomogeneity,” “inhomogeneous microstructure,” and the like mean that a multiphase microstructure is present in which there are at least two discrete phases that are separated from each other. The two phases may be one discrete solid structural phase in a continuous solid phase, two co-continuous solid phases, or two discrete solid structural phases in a third continuous solid phase, for example. In some embodiments, the length scale of phase inhomogeneity refers to the average size (e.g., effective diameter) of discrete inclusions of one phase dispersed in a continuous phase. In some embodiments, the length scale of phase inhomogeneity refers to the average center-to-center distance between nearest-neighbor inclusions of the same phase. The average length scale of phase inhomogeneity (which may also be referred to as an average phase-separation length) may generally be from about 0.1 microns to about 500 microns. In some embodiments, the average length scale of phase inhomogeneity is from about 0.5 microns to about 100 microns, such as about 1 micron to about 50 microns. In various embodiments, the average length scale of phase inhomogeneity is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 microns, including any intermediate values not explicitly recited, and ranges starting, ending, or encompassing such intermediate values. These are average values, noting that a portion of phase inhomogeneity may be present on a length scale less than 0.1 micron or greater than 500 microns (e.g., about 1000 microns), with the overall average falling in the range of 0.1-500 microns. Note that in this disclosure, “about 0.1 microns” is intended to encompass 0.05-0.149 microns (50-149 nanometers), i.e. ordinary rounding. The antimicrobial structure may also be characterized by hierarchical phase separation. For example, when segmented copolymers are utilized, first soft segments and second soft segments—in addition to being microphase-separated—are typically nanophase-separated. As intended herein, two materials being “nanophase-separated” means that the two materials are separated from each other on a length scale from about 1 nanometer to about 100 nanometers. For example, the nanophase-separation length scale may be from about 10 nanometers to about 100 nanometers. The nanophase separation between first solid material (or phase) and second solid material (or phase) may be caused by the presence of a third solid material (or phase) disposed between regions of the first and second solid materials. For example, in the case of first and second solid materials being soft segments of a segmented copolymer also with hard segments, the nanophase separation may be driven by intermolecular association of hydrogen-bonded, dense hard segments. In these cases, in some embodiments, the first soft segments and the hard segments are nanophase-separated on an average nanophase-separation length scale from about 10 nanometers to less than 100 nanometers. Alternatively, or additionally, the second soft segments and the hard segments may be nanophase-separated on an average nanophase-separation length scale from about 10 nanometers to less than 100 nanometers. The first and second soft segments themselves may also be nanophase-separated on an average nanophase-separation length scale from about 10 nanometers to less than 100 nanometers, i.e., the length scale of the individual polymer molecules. The nanophase-separation length scale is hierarchically distinct from the microphase-separation length scale. With traditional phase separation in block copolymers, the blocks chemically segregate at the molecular level, resulting in regions of segregation on the length scale of the molecules, such as a nanophase-separation length scale from about 10 nanometers to about 100 nanometers. See Petrovic et al., “POLYURETHANE ELASTOMERS”Prog. Polym. Sci., Vol. 16, 695-836, 1991. The extreme difference of the two soft segments means that in the reaction pot the soft segments do not mix homogeneously and so create discrete region that are rich in fluoropolymer or rich in non-fluoropolymer (e.g., PEG) components, distinct from the molecular-level segregation. These emulsion droplets contain a large amount of polymer chains and are thus in the micron length-scale range. These length scales survive the curing process, so that the final material contains the microphase separation that was set-up from the emulsion, in addition to the molecular-level (nanoscale) segregation. In some embodiments, therefore, the larger length scale of separation (0.1-500 microns) is driven by an emulsion process, which provides microphase separation that is in addition to classic molecular-level phase separation. Chen et al., “Structure and morphology of segmented polyurethanes: 2. Influence of reactant incompatibility”POLYMER,1983, Vol. 24, pages 1333-1340, is hereby incorporated by reference herein for its teachings about microphase separation that can arise from an emulsion-based procedure. In some embodiments, discrete inclusions have an average size (e.g., effective diameter) from about 50 nm to about 150 μm, such as from about 100 nm to about 100 μm. In various embodiments, discrete inclusions have an average size (e.g., effective diameter) of about 50 nm, 100 nm, 200 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 50 μm, 100 μm, or 200 μm. In these or other embodiments, discrete inclusions (of discrete solid structural phase) have an average center-to-center spacing between adjacent inclusions, through a continuous matrix (of continuous transport phase), from about 50 nm to about 150 μm, such as from about 100 nm to about 100 μm. In various embodiments, discrete inclusions have an average center-to-center spacing between adjacent inclusions of about 50 nm, 100 nm, 200 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 50 μm, 100 μm, or 200 μm. In some variations of the invention, the antimicrobial structure forms a coating disposed on a substrate. The coating may have a thickness from about 1 μm to about 10 mm, for example. In various embodiments, the coating thickness is about, at least about, or at most about 100 nm, 1 μm, 10 μm, 100 μm, 1 mm, or 10 mm, including any intervening ranges. Thicker coatings provide the benefit that even after surface abrasion, the coating still functions because the entire depth of the coating (not just the outer surface) contains the functional materials. The coating thickness will generally depend on the specific application. Note that the definition of optical transparency in this disclosure, which averages the transparency across light wavelengths from 400 nm to 800 nm through a 100-micron film of the antimicrobial structure at 25° C. and 1 bar, does not mean that the coating thickness must be 100 μm. An optional substrate may be disposed on the back side of the antimicrobial structure. A substrate will be present when the material forms a coating or a portion of a coating (e.g., one layer of a multilayer coating). Many substrates are possible, such as a metal, polymer, wood, or glass substrate. Essentially, the substrate may be any material or object for which antimicrobial protection is desirable. In some embodiments, an adhesion layer is disposed on a substrate, wherein the adhesion layer is configured to promote adhesion of the antimicrobial structure to the selected substrate. An adhesion layer contains one or more adhesion-promoting materials, such as (but not limited to) primers (e.g., carboxylated styrene-butadiene polymers), alkoxysilanes, zirconates, and titanium alkoxides. Various strategies are possible to form the materials of the biphasic polymer, as will be appreciated by a skilled artisan. In some embodiments, the biphasic polymer is made in the form of an applique that may be adhered to a surface at the point of use. The two-part formula may be solventborne or waterborne. When the two-part formula is solventborne, the first and second solvents are both organic solvents or inorganic solvents other than water. When the two-part formula is waterborne, the first and second solvents are both aqueous solvents, such as water or a solution containing water. In certain embodiments, one of the first or second solvents contains water, while the other solvent does not contain water. In some waterborne embodiments, first or second soft segments may be derived from an aqueous dispersion of a linear crosslinkable polyurethane containing charged groups, and the other soft segments may be derived from a crosslinking agent containing charged groups, for example. In some embodiments, a precursor includes a silane, a silyl ether, a silanol, an alcohol, or a combination or reaction product thereof, and optionally further includes a protecting group that protects the precursor from reacting with other components. Some embodiments employ waterborne polyurethane dispersions. A successful waterborne polyurethane dispersion sometimes requires the specific components to contain ionic groups to aid in stabilizing the emulsion. Other factors contributing to the formulation of a stable dispersion include the concentration of ionic groups, concentration of water or solvent, and rate of water addition and mixing during the inversion process. An isocyanate prepolymer may be dispersed in water. Subsequently, a curative component may be dispersed in water. Water evaporation then promotes the formation of a microphase-separated polyurethane material. A composition or precursor composition may generally be formed from a precursor material (or combination of materials) that may be provided, obtained, or fabricated from starting components. The precursor material is capable of hardening or curing in some fashion, to form a precursor composition containing the first soft segments and second soft segments, microphase-separated on a microphase-separation length scale from about 0.1 microns to about 500 microns. The precursor material may be a liquid; a multiphase liquid; a multiphase slurry, emulsion, or suspension; a gel; or a dissolved solid (in solvent), for example. In some embodiments, an emulsion sets up in the reaction mixture based on incompatibility between the two blocks (e.g., PEG and PC). The emulsion provides microphase separation in the precursor material. The precursor material is then cured from casting or spraying. The microphase separation survives the curing process (even if the length scales change somewhat during curing), providing the benefits in the final materials (or precursor compositions) as described herein. The microphase separation in this invention is not associated with molecular length-scale separation (5-50 nm) that many classic block-copolymer systems exhibit. Rather, the larger length scales of microphase separation, i.e. 0.1-500 μm, arise from the emulsion that was set-up prior to curing. Xu et al., “Structure and morphology of segmented polyurethanes: 1. Influence of incompatibility on hard-segment sequence length”POLYMER,1983, Vol. 24, pages 1327-1332 and Chen et al., “Structure and morphology of segmented polyurethanes: 2. Influence of reactant incompatibility”POLYMER,1983, Vol. 24, pages 1333-1340, are each hereby incorporated by reference herein for their teachings about emulsion set-up in polyurethane systems prior to curing. In some variations of the invention, a precursor material is applied to a substrate and allowed to react, cure, or harden to form a final composition (e.g., coating). In some embodiments, a precursor material is prepared and then dispensed (deposited) over an area of interest. Any known methods to deposit precursor materials may be employed. A fluid precursor material allows for convenient dispensing using spray coating or casting techniques. The fluid precursor material may be applied to a surface using any coating technique, such as (but not limited to) spray coating, dip coating, doctor-blade coating, air knife coating, curtain coating, single and multilayer slide coating, gap coating, knife-over-roll coating, metering rod (Meyer bar) coating, reverse roll coating, rotary screen coating, extrusion coating, casting, or printing. Because relatively simple coating processes may be employed, rather than lithography or vacuum-based techniques, the fluid precursor material may be rapidly sprayed or cast in thin layers over large areas (such as multiple square meters). When a solvent or carrier fluid is present in a fluid precursor material, the solvent or carrier fluid may include one or more compounds selected from the group consisting of water, alcohols (such as methanol, ethanol, isopropanol, or tert-butanol), ketones (such as acetone, methyl ethyl ketone, or methyl isobutyl ketone), hydrocarbons (e.g., toluene), acetates (such as tert-butyl acetate), acids (such as organic acids), bases, and any mixtures thereof. When a solvent or carrier fluid is present, it may be in a concentration of from about 10 wt % to about 99 wt % or higher, for example. The precursor material may be converted to an intermediate material or the final composition using any one or more of curing or other chemical reactions, or separations such as removal of solvent or carrier fluid, monomer, water, or vapor. Curing refers to toughening or hardening of a polymeric material by physical crosslinking, covalent crosslinking, and/or covalent bonding of polymer chains, assisted by electromagnetic waves, electron beams, heat, and/or chemical additives. Chemical removal may be accomplished by heating/flashing, vacuum extraction, solvent extraction, centrifugation, etc. Physical transformations may also be involved to transfer precursor material into a mold, for example. Additives may be introduced during the hardening process, if desired, to adjust pH, stability, density, viscosity, color, or other properties, for functional, ornamental, safety, or other reasons. EXAMPLES Materials. Polyethylene glycol (Mn=600 g/mol, referred to as PEG 600), pentaerythritol propoxylate (5/4 PO/OH ratio), 2-butanone, and dibutyltin dilaurate (C32H64O4Sn, catalyst) are obtained from MilliporeSigma (Darmstadt, Germany). 2-Butanone is dried over sieves prior to use. CPX 2012 is obtained from TRiiSO LLC (Del Mar, California, USA). Ethacure 100-LC is obtained from Albemarle. Desmodur N3300A (aliphatic polyisocyanate, HDI trimer) is obtained from Covestro (Leverkusen, Germany). BYK-054 (defoamer) is obtained from BYK (Wesel, Germany). Zeffle GK-570 (butyl acetate solution of a copolymer of tetrafluoroethylene and hydrocarbon olefins with pendant OH groups) is obtained from Daikin (Changshu, Jiangsu, China). DisperByk 2008 (acrylic copolymer) is obtained from ALTANA (Wesel, Germany). Acematt 3300 (silica) is obtained from Evonik (Essen, Germany). Declam (The Boeing Company, Chicago, Illinois, USA) is a laminate with a poly(vinyl fluoride) top surface, used as a substrate for coatings. Example 1A: Preparation of Part A of Amine-Cured System PEG 600 (10.00 g) is added to a mixer cup. CPX 2012 (polycarbonate precursor, 20.54 g) is melted at 70° C. and added to the mixer cup. The solution is centrifugally mixed for one minute at 2000 revolutions per minute (RPM). The mixer cup is placed in an oven and heated to 70° C. The cup is removed from the oven followed by the addition of 2-butanone (45.16 g), Ethacure 100-LC (4.84 g), and dibutyltin dilaurate (0.24 g, 4000 ppm). The solution is mixed for two minutes at 2000 RPM. The resultant solution is homogenous and mostly clear with a light yellow tint. The solution is poured into a glass jar, an inert gas layer is added, and the container is sealed tightly. This forms Part A of the two-part formula for an amine-curable system. Example 1B: Preparation of Part B of Amine-Cured System 2-Butanone (45.16 g) and Desmodur N3300A (24.83 g) are added to a mixer cup and centrifugally mixed for two minutes at 2000 RPM. The resultant solution is homogenous and clear. The solution is poured into a glass jar, an inert gas layer is added, and the container is sealed tightly. This forms Part B of the two-part formula for an amine-curable system. Example 1C: Spray Coating of Amine-Cured System Part A (Example 1A, 8.00 g) and Part B (Example 1B, 6.96 g) are combined in a mixer cup and centrifugally mixed at 2000 RPM for one minute. The resultant solution is added to a spray gun (Anest Iwata LPH-80) and sprayed onto a substrate containing areas of both aluminum and Declam. Five coats are applied in total. The substrate is placed into an oven and allowed to cure for four hours at 60° C., forming an amine-cured biphasic polymer from the two-part formula. The biphasic polymer coating is approximately 2.5 mils thick (about 64 microns). Example 1D: Aging and Spray Coating of Amine-Cured System Part A (Example 1A, 8.00 g) and Part B (Example 1B, 6.96 g) are each aged for 23 days. Following this aging, Part A and Part B are then combined in a mixer cup and centrifugally mixed at 2000 RPM for one minute. The resultant solution is immediately added to a spray gun (Anest Iwata LPH-80) and sprayed onto a substrate containing Declam. Five coats are applied in total. The substrate is placed into an oven and allowed to cure for four hours at 60° C., forming an amine-cured biphasic polymer from the two-part formula. The biphasic polymer coating is approximately 2.5 mils thick (about 64 microns). Example 2A: Preparation of Part A of Alcohol-Cured System PEG 600 (10.00 g) is added to a mixer cup. CPX 2012 (polycarbonate precursor, 20.54 g) is melted at 70° C. and added to the mixer cup. The solution is centrifugally mixed for one minute at 2000 RPM. The cup is placed in an oven and heated to 70° C. The cup is removed from the oven followed by the addition of 2-butanone (45.19 g), pentaerythritol propoxylate (5.47 g), BYK-054 (0.12 g), and dibutyltin dilaurate (0.24 g, 4000 ppm). The solution is mixed for four minutes at 2000 RPM. The resultant solution is homogenous and clear. The solution is poured into a glass jar, an inert gas layer is added, and the container is sealed tightly. This forms Part A of the two-part formula for an alcohol-curable system. Example 2B: Preparation of Part B of Alcohol-Cured System 2-Butanone (45.19 g) and Desmodur N3300A (24.25 g) are added to a mixer cup and centrifugally mixed for two minutes at 2000 RPM. The resultant solution is homogenous and clear. The solution is poured into a glass jar, an inert gas layer is added, and the container is sealed tightly. This forms Part B of the two-part formula for an alcohol-curable system. Example 2C: Spray Coating of Alcohol-Cured System Part A (Example 2A, 8.00 g) and Part B (Example 2B, 6.84 g) are added to a mixer cup and centrifugally mixed at 2000 RPM for one minute. The resultant solution is added to a spray gun (Anest Iwata LPH-80) and sprayed onto a substrate containing areas of both aluminum and Declam. Five coats are applied in total. The substrate is placed into an oven and allowed to cure for four hours at 60° C., forming an alcohol-cured biphasic polymer from the two-part formula. The biphasic polymer coating is approximately 2.5 mils thick (about 64 microns). Example 2D: Aging and Spray Coating of Alcohol-Cured System Part A (Example 2A, 8.00 g) and Part B (Example 2B, 6.84 g) are each aged for 11 days. Following this aging, Part A and Part B are then combined in a mixer cup and centrifugally mixed at 2000 RPM for one minute. The resultant solution is added to a spray gun (Anest Iwata LPH-80) and sprayed onto a substrate containing Declam. Five coats are applied in total. The substrate is placed into an oven and allowed to cure for four hours at 60° C., forming an alcohol-cured biphasic polymer from the two-part formula. The biphasic polymer coating is approximately 2.5 mils thick (about 64 microns). Example 3A: Preparation of Part A of Alcohol-Cured Mixed Fluorinated and Non-Fluorinated System PEG 600 (4.50 g), Zeffle GK-570 (2.06 g), and pentaerythritol propoxylate (2.56 g) are added to a mixer cup. CPX 2012 (polycarbonate precursor, 7.89 g) is melted at 70° C. and added to the mixer cup. The solution is centrifugally mixed for one minute at 2000 RPM. The cup is placed in an oven and heated to 70° C. The cup is removed from the oven followed by the addition of 2-butanone (6.78 g). The solution is mixed for two minutes at 2000 RPM. Dibutyltin dilaurate (0.11 g), BYK-054 (0.05 g), DisperByk 2008 (0.27 g), and Bardac 208M (0.81 g) (quaternary ammonium compounds, as antimicrobial agent) are added to the mixer cup. This solution is mixed for 1 minute at 2000 RPM. Acematt 3300 (2.44 g) is added to the mixer cup and the solution is mixed for two minutes at 2000 RPM. Butyl acetate (13.47 g) is added to the mixer cup and the solution is mixed for one minute at 2000 RPM. The resultant solution is homogenous and opaque. The solution is poured into a glass jar, an inert gas layer is added, and the container is sealed tightly. This forms Part A of the two-part formula for an alcohol-curable mixed fluorinated and non-fluorinated system. Example 3B: Preparation of Part B of Alcohol-Cured Mixed Fluorinated and Non-Fluorinated System 2-Butanone (5.43 g) and Desmodur N3300A (10.86 g) are added to a mixer cup and centrifugally mixed for two minutes at 2000 RPM. The resultant solution is homogenous and clear. The solution is poured into a glass jar, an inert gas layer is added, and the container is sealed tightly. This forms Part B of the two-part formula for an alcohol-curable mixed fluorinated and non-fluorinated system. Example 3C: Spray Coating of Alcohol-Cured Mixed Fluorinated and Non-Fluorinated System Part A (Example 3A, 40.23 g) and Part B (Example 3B, 16.28 g) are added to a mixer cup and centrifugally mixed at 2000 RPM for one minute. The resultant solution is added to a spray gun (Anest Iwata LPH-80) and sprayed onto a substrate containing areas of both aluminum and Declam. Four coats are applied in total. The substrate is placed into an oven and allowed to cure for four hours at 65° C., forming an alcohol-cured biphasic polymer from the two-part formula. The biphasic polymer coating is approximately 2.5 mils thick (about 64 microns). Example 3D: Aging and Spray Coating of Alcohol-Cured Mixed Fluorinated and Non-Fluorinated System Part A (Example 3A, 8.00 g) and Part B (Example 3B, 3.24 g) are each aged for 91 days. Following this aging, Part A and Part B are then combined in a mixer cup and centrifugally mixed at 2000 RPM for one minute. The resultant solution is added to a spray gun (Anest Iwata LPH-80) and sprayed onto a substrate containing Declam. Four coats are applied in total. The substrate is placed into an oven and allowed to cure for four hours at 60° C., forming an alcohol-cured mixed fluorinated and non-fluorinated biphasic polymer from the two-part formula. The biphasic polymer coating is approximately 2.5 mils thick (about 64 microns). Example 4: Hardness Results of Example 1C, 1D, 2C, 2D, 3C, and 3D Spray Coatings Hardness is measured with a BYK Pencil Hardness Test Kit in accordance with ASTM D3363-22, “Standard Test Method for Film Hardness by Pencil Test”, which is incorporated by reference. When two materials of different hardness are forced against each other, one of the materials yields or crumbles, while the other is unaffected. Thus a scale of relative hardness can be established on the basis of the ability of one material to scratch or deform another. In ASTM D3363-22, the test method specifies that pencils with a known hardness grade are moved over the surface to be tested at a fixed angle and pressure. The result is a grade from 9B (softest) to 9H (hardest), on a spectrum. Measurements are taken on the coatings formed from spraying onto a Declam substrate directly after the solutions are prepared according to Examples 1C, 2C, and 3C, as well as following aging of the solutions, according to Examples 1D, 2D, and 3D. The hardness results are shown in Table 1. It is noted that the hardness of the alcohol-cured biphasic polymer is essentially unchanged after aging (Example 2D versus 2C), and the hardness of the alcohol-cured mixed fluorinated and non-fluorinated biphasic polymer is essentially unchanged after approximately 3 months of aging (Example 3D versus 3C). Example 5: Visual Inspection of Example 1C, 1D, 2C, 2D, 3C, and 3D Spray Coatings The spray coatings are visually inspected for cosmetic appearance. The visual inspections are done for the coatings formed from spraying onto a Declam substrate directly after the solutions are prepared according to Examples 1C, 2C, and 3C, as well as following aging of the solutions, according to Examples 1D, 2D, and 3D. As summarized in Table 2, all three sets of samples look good and appear almost identical with very glossy finishes and no large scale visual defects (uncured coating, hazing, cratering, bubbling, etc.). Example 6: Stability of Two-Part Formula Kit The stability of the formulations is assessed visually after initial preparation and out to 61 days for Part A (Example 1A) and Part B (Example 1B) of the amine-curable biphasic polymer. Likewise, the stability of the formulations is assessed visually after initial preparation and out to 49 days for Part A (Example 2A) and Part B (Example 2B) of the alcohol-curable biphasic polymer. After these time periods, no settling, separation, or curing is observed in either Part A or Part B for either of the amine-curable two-part formula or the alcohol-curable two-part formula. However, the initial slight yellow tint of the amine-curable Part A (Example 1A) has darkened considerably over time. This result is consistent with the storage characteristics of the Ethacure 100-LC component itself. TABLE 1Hardness Results for Example Coatings via ASTM D3363-22Solution Aging Prior toHardness ValueFormulationSpraying (Days)(ASTM D3363-22)Example 1C07HExample 1D23HExample 2C03HExample 2D112HExample 3C03HExample 3D914H TABLE 2Cosmetic Results for Example Coatings via ASTM D3363-22Solution Aging Prior toFormulationSpraying (Days)Visual AppearanceExample 1C0GoodExample 1D23GoodExample 2C0GoodExample 2D11GoodExample 3C0GoodExample 3D91Good There are many commercial applications of antimicrobial surfaces in homes, in restaurants, on clothing and personal protective equipment, in cars, and in airplanes, for example. In one commercial example, the antimicrobial structure is a coating disposed on an automotive dash board. In another commercial example, the antimicrobial structure is a coating disposed on an overhead stowage bin in an aerospace cabin. In this detailed description, reference has been made to multiple embodiments which show by way of illustration specific exemplary embodiments of the invention. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that modifications to the various disclosed embodiments may be made by a skilled artisan. Where methods and steps described above indicate certain events occurring in certain order, those of ordinary skill in the art will recognize that the ordering of certain steps may be modified and that such modifications are in accordance with the variations of the invention. Additionally, certain steps may be performed concurrently in a parallel process when possible, as well as performed sequentially. All publications, patents, and patent applications cited in this specification are herein incorporated by reference in their entirety as if each publication, patent, or patent application were specifically and individually put forth herein. This patent application hereby incorporates by reference the following patents: U.S. Pat. No. 10,689,542, issued on Jun. 23, 2020; U.S. Pat. No. 11,225,589, issued on Jan. 18, 2022; and U.S. Pat. No. 11,369,109, issued on Jun. 28, 2022. The embodiments and variations described above should provide an indication of the utility and versatility of the present invention. Other embodiments that do not provide all of the features and advantages set forth herein may also be utilized, without departing from the spirit and scope of the present invention. Such modifications and variations are considered to be within the scope of the invention defined by the claims. | 122,229 |
11859099 | DESCRIPTION OF THE INVENTION The invention is directed to curable polyurethane coating compositions, which may be used to form fouling release (FR) coatings, e.g., for use in protecting boat hulls. A curable coating composition of the invention comprises, consists essentially of, or consists of:a) at least one amphiphilic additive, comprising, consisting essentially of, or consisting of the reaction product of:a1) at least one polyisocyanate witha2) at least one monocarbinol-terminated poly(dimethylsiloxane) (PDMS) and/ora3) at least one poly(ethylene glycol) methyl ether (mPEG);b) at least one polyisocyanate;c) at least one polyol; andd) optionally, at least one amphiphilic PEG-PDMS isocyanate prepolymer, comprising, consisting essentially of, or consisting of the reaction product of:d1) at least one polyisocyanate,d2) at least one monocarbinol-terminated PDMS, andd3) at least one mPEG,wherein the at least one amphiphilic PEG-PDMS isocyanate prepolymer, d), if present, has an overall isocyanate to total hydroxyl equivalent ratio such that isocyanate is in excess. Another curable coating composition of the invention comprises, consists essentially of, or consists of the at least one polyisocyanate, b), the at least one polyol, c), and the at least one amphiphilic PEG-PDMS isocyanate prepolymer, d), wherein the at least one amphiphilic PEG-PDMS isocyanate prepolymer, d), has an overall isocyanate to total hydroxyl equivalent ratio such that isocyanate is in excess. Methods of inhibiting fouling on a surface exposed to aquatic conditions, such as a boat hull, comprising applying the curable coating compositions of the invention to at least a portion of said surface hull are also provided. The application also describes surfaces designed to be exposed to aqueous conditions (e.g., salt water conditions, such as seawater or brackish water, or fresh water conditions, such as found with materials in contact with lake or stream water), which are protected with a coating including the curable coating compositions of the invention. The amphiphilic additive, a), for use in the curable coating compositions is made by reacting at least one polyisocyanate, a1), with at least one monocarbinol-terminated PDMS, a2), and/or at least one mPEG a3). To prepare the amphiphilic additive, the polyisocyanate, a1), and the monocarbinol-terminated PDMS, a2), plus (+) the mPEG, a3), are reacted in a 1:1 equivalent ratio of isocyanate group (NCO): hydroxyl group (OH). Similarly, the amphiphilic PEG-PDMS isocyanate prepolymer, d), is made by reacting at least one polyisocyanate, d1), with at least one monocarbinol-terminated PDMS, d2), and/or at least one mPEG d3). However, in contrast to the amphiphilic additive, a), the amphiphilic PEG-PDMS isocyanate prepolymer, d), has an overall isocyanate (NCO) to total hydroxyl (OH) equivalent ratio such that isocyanate is in excess. For example, the isocyanate to total hydroxyl equivalent ratio in the at least one amphiphilic PEG-PDMS isocyanate prepolymer, d), may range from 1.5:1 to 1.1:1. By way of another example, in the at least one amphiphilic PEG-PDMS isocyanate prepolymer, d), the polyisocyanate, d1), and the monocarbinol-terminated PDMS, d2), plus (+) the mPEG, a3), are reacted in an isocyanate to hydroxyl ratio where the moles of hydroxyl reacted are less than the stoichiometric amount of the isocyanate present while maintaining 5-10 wt. % of PDMS and PEG content based on the overall coating formulation. For example, IPDI trimer may be reacted with monocarbinol-terminated PDMS and mPEG) in a 1.5:1 isocyanate to hydroxyl ratio while maintaining 5-10 wt. % of PDMS and PEG content based on the overall coating formulation. FIG.1depicts a general structure proposed for both the amphiphilic additive and amphiphilic PEG-PDMS isocyanate prepolymer synthesized (collectively, referred to as “pre-polymer” inFIG.1). In the structure depicted with an IPDI trimer as the exemplary polyisocyanate, X can be either an isocyanate group, PDMS chain linked through urethane or mPEG linked through urethane. The integer “m” connotes the repeating units in the PDMS and the integer “n” the repeating units in the mPEG. The polyisocyanates, a1), b), and d1), are, independent of one another, selected from the group consisting of an aliphatic polyisocyanate, a cycloaliphatic polyisocyanate, an araliphatic polyisocyanate, an aromatic polyisocyanate, and mixtures thereof. For example, the polyisocyanate, a1), b), and d1), may be, independent of one another, selected from the group consisting of: wherein R is independently an optionally substituted, divalent C1-C15alkyl, an optionally substituted C3-C15cycloalkyl, or a group selected from: Preferably, R is a C2-C10straight chain or branched alkyl. Polyisocyanates based on methylene diphenyl diisocyanate (“MDI”) and trimers thereof, hexamethylene diisocyanate (“HMDI”) and trimers thereof, isophorone diisocyanate (“IPDI”) and trimers thereof, and the like can be used. Preferably, the polyisocyanate is an isophorone-based polyisocyanate. The polyisocyanate may be a polyisocyanate having at least three isocyanate groups such as an MDI trimer, an IPDI trimer (Desmodur Z4470 BA), and an HDI trimer (Desmodur N3300 A). Other polyisocyanates known in the art may also be used. Examples include Desmodur HL, Desmodur IL, triisocyanatononane, Desmodur RE, Desmodur RFE. The monocarbinol-terminated PDMS, a2) and d2), may have, independent of one another, a molecular weight ranging from 400Mnto 50,000Mn(e.g., 5000Mnto 10,000Mn). For example, the monocarbinol-terminated PDMS may have molecular weight of 1000, of 5000, or of 10,000Mn. The monocarbinol-terminated PDMS, a2) and d2), may have, independent of one another, the following structure: wherein R, independent of one another, is a C3-C12straight chain alkyl or an alkylene ether; and wherein n ranges from 0 to about 270. The mPEG, a3) and d3), may have, independent of one another, a molecular weight ranging from 350Mnto 20,000Mn(e.g., 500Mnto 800Mn, 550Mnto 750Mn). Preferably, the mPEG has a molecular weight of 550 or of 750 g/mole. The polyisocyanate, a1), may be present in an amount ranging from about 0.01 to 50 wt. % (e.g., 0.1 to 40 wt. %, 1 to 35 wt. %, 5 to 30 wt. %, 15 to 25 wt. %), based on the solid content of the amphiphilic additive, a). The monocarbinol-terminated PDMS, a2), may be present in an amount ranging from about 0.01 to 50 wt. % (e.g., 5 to 45 wt. %, 10 to 30 wt. %, 15 to 25 wt. %), based on the solid content of the amphiphilic additive, a). The mPEG, a3), may be present in an amount ranging from about 0.01 to 75 wt. % (e.g., 0.1 to 70 wt. %, 1 to 68 wt. %, 20 to 65 wt. %, 30 to 60 wt. %, 40 to 50 wt. %), based on the solid content of the amphiphilic additive, a). The mole % ratio of monocarbinol-terminated PDMS, a2): mPEG, a3), in the amphiphilic additive, a), may range from 0:100 to 100:0 (e.g., 10:90, 20:80, 30:70, 33:66, 40:60, 50:50, 60:40, 66:33, 70:30, 80:20, 90:10). The amphiphilic additive, a), and the amphiphilic PEG-PDMS isocyanate prepolymer, d), may be, independent of one another, present in an amount ranging from about 0.1 to 40 wt. % (e.g., 1 to 35 wt. %, 5 to 30 wt. %, 10 to 25 wt. %, 15 to 20 wt. %), based on the solid content of the curable coating composition. The polyisocyanate, b), may be present in an amount ranging from about 10 to 40 wt. % (e.g., 15 to 35 wt. %, 20 to 30 wt. %), based on the solid content of the curable coating composition. The amount of polyisocyanate, b), added should maintain an overall isocyanate to total hydroxyl equivalents where the isocyanate is in a slight molar excess, for example an isocyanate to hydroxyl equivalent ratio of 1.2:1 or 1.1:1, for the final curable coating composition. The polyol, c), may be present in an amount ranging from about 20 to 60 wt. % (e.g., 25 to 55 wt. %, 30 to 50 wt. %, 35 to 45 wt. %). The curable coating composition may also contain a solvent which, if present, may be present in an amount from about 0.5 to 75 wt. % (e.g., 1 to 65 wt. %, 2 to 45 wt. %, 5 to 40 wt. %, 10 to 35 wt. %, 15 to 30 wt. %, 20 to 25 wt. %). The amphiphilic additive, a), and/or the one amphiphilic PEG-PDMS isocyanate prepolymer, d), may be prepared by first dissolving the polyisocyanate in a suitable organic solvent or mixture of organic solvents. Suitable organic solvents include, but are not limited to, ethyl-3-ethoxypropionate (EEP), butyl acetate, t-butyl acetate, amyl acetate, acetone, methylethyl ketone, methyl amyl ketone, N,N-dimethyl formamide, N-methyl pyrollidinone, dimethyl sulfoxide, and the like. The monocarbinol-terminated PDMS and/or mPEG may be added to the solution together with a suitable catalyst, for example organometallic compounds or organic bases, and other such catalysts known in the art. Examples of organometallic compounds are dibutyl tin dilaurate, dibutyl tin diacetate (DBTDAc), bismuth carboxylate, and compounds of zirconium and aluminum such as K-Kat 4205, K-Kat-5218, and K-Kat-XC-6212. Examples of organic base catalysts are sold under the DABCO trade name by Air Products. DABCO is 1,4-diazabicyclo[2.2.2]octane. The reaction typically takes place at room temperature with stirring for several hours, for example, 4-12 hours. The polyol, c), may be selected from the group consisting of polyester polyols, polyether polyols, polycarbonate polyols, acrylic polyols, and mixtures thereof. Preferably, the polyol, c), is an acrylic polyol. The polyol may include polyol having at least three hydroxyl groups. A mixture of polyols can also be used in formulating a polyurethane coating. Polyester polyols can include those made from the melt polycondensation of polyfunctional acids with polyfunctional alcohols or those made from the ring opening polymerization of cyclic monomers such as epsilon-caprolactone. Examples of suitable polyester polyols include, for example, poly(caprolactone) polyols, poly(hexamethylene adipate), and the like. Examples of suitable polyether polyols include, for example, poly(ethyleneglycol), poly(propylene glycol), poly(butylene glycol), poly(tetramethylene oxide), and the like. Acrylic polyols may be synthesized, typically by free radical polymerization, from a mixture of at least one hydroxy functional monomer plus one or more non-functional monomers. Suitable hydroxy-functional monomers include, for example, hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, hydroxypropyl methacrylate, and the like. Examples of non-functional monomers include, for example, styrene, methyl methacrylate, methyl acrylate, butyl methacrylate, butyl acrylate, lauryl methacrylate, lauryl acrylate, 2-ethylhexyl acrylate, 2-ethyl hexyl methacrylate, and the like. The acrylic polyol may be synthesized in solution using a thermally-activated free radical initiator. The polyol can be synthesized in either a batch, semi-batch or continuous process. Examples of free radical initiators are benzoyl peroxide, t-amyl peroxy-2-ethylhexanoate, t-butyl hydroperoxide, di-t-butyl peroxide, azobisisobutyronitrile, azobisisovaleronitrile, and the like. The acrylic polyol may be made by free radical polymerization and then diluted in a solvent, such as toluene, xylene, methylisobutyl ketone, and the like. In one embodiment, the polyol may include a polycaprolactone polyol such as a polycaprolactone triol. One example of an acrylic polyol for use in a coating composition of the invention is an acrylic polyol composed of 80% butyl acrylate and 20% 2-hydroxy ethyl acrylate by weight. Catalysts for the crosslinking of the curable coating compositions can be either organometallic complexes or organic bases, and other such catalysts known in the art. Examples of organometallic compounds are dibutyl tin dilaurate, dibutyl tin diacetate, bismuth carboxylate, and compounds of zirconium and aluminum such as K-Kat 4205, K-Kat-5218, and K-Kat-XC-6212. Examples of organic base catalysts are sold under the DABCO trade name by Air Products. DABCO is 1,4-diazabicyclo[2.2.2]octane. Suitable examples of isocyanate reaction catalysts include diethyl tin diacetate, dibutyltin diacetate, dibutyltin dilaurate, dibutyltin maleate, dilauryltin diacetate, dioctyltin diacetate, or a mixture thereof. In one embodiment the isocyanate reaction catalyst includes a tin catalyst. The curable coating compositions may also be formulated with or without solvents. The curable coating compositions of the invention, as mentioned above, may be a solvent-free coating composition or may optionally contain a solvent such as, for example, acetone, THF, methyl ethyl ketone (MEK), xylene, acetone, acetylacetone, benzene, toluene, methyl amyl ketone (MAK), methyl isobutyl ketone, butyl acetate, t-butyl acetate, ethyl 3-ethoxypropionate (EEP), isopropanol, aromatic 100, aromatic 150, tetrahydrofuran, diethyl ether, butanol, butoxyethanol, etc. The curable coating compositions may be a solution in such a solvent or mixture of solvents. The curable coating compositions may also include a pot life extender, such as, for example, alkane-2,4-dione (e.g., 2,4-pentadione), N,N-dialkyl acetoacetamide, alkyl acetoacetate, and the like. These, and the other common additives discussed below, may be included in amounts known in the art for their use. The invention also relates to the use of the curable coating compositions of the invention which may be coated onto a substrate and cured using techniques known in the art. The substrate can be any common substrate such as paper, polyester films such as polyethylene and polypropylene, metals such as aluminum and steel, glass, urethane elastomers, primed (painted) substrates, and the like. The invention further relates to an article of manufacture or object (e.g., a boat hull) comprising the curable coating compositions of the invention. The curable coating compositions of the invention may be cured at room temperature (ambient cure) or at elevated temperatures (thermal cure). The invention also relates to a method for reducing or preventing biofouling of a surface exposed to an aqueous environment comprising the steps of: coating the surface with the curable coating compositions to form a coated surface, and curing the coating composition on the coated surface. The invention also relates to a marine fouling-release coating comprising the curable coating compositions. The curable coating compositions of the invention may further contain a pigment (organic or inorganic), if a coating having a particular color is desired, and/or additives and fillers known in the art. For example, the curable coating compositions of the invention may further contain coating additives. Examples of such coating additives include, but are not limited to, one or more leveling, rheology, and flow control agents such as silicones, fluorocarbons, or cellulosics; extenders; reactive coalescing aids such as those described in U.S. Pat. No. 5,349,026, incorporated herein by reference; plasticizers; flatting agents; pigment wetting and dispersing agents and surfactants; ultraviolet (UV) absorbers; UV light stabilizers; tinting pigments; colorants; defoaming and antifoaming agents; anti-settling, anti-sag and bodying agents; anti-skinning agents; anti-flooding and anti-floating agents; biocides, fungicides, and mildewcides; corrosion inhibitors; thickening agents; or coalescing agents. Specific examples of such additives can be found in Raw Materials Index, published by the National Paint & Coatings Association, 1500 Rhode Island Avenue, N.W., Washington, D.C. 20005. Further examples of such additives may be found in U.S. Pat. No. 5,371,148, incorporated herein by reference. Examples of flatting agents include, but are not limited to, synthetic silica, available from the Davison Chemical Division of W. R. Grace & Company as SYLOID®; polypropylene, available from Hercules Inc., as HERCOFLAT®; synthetic silicate, available from J. M. Huber Corporation, as ZEOLEX®. Examples of viscosity, suspension, and flow control agents include, but are not limited to, polyaminoamide phosphate, high molecular weight carboxylic acid salts of polyamine amides, and alkylene amine salts of an unsaturated fatty acid, all available from BYK Chemie U.S.A. as ANTI TERRA®. Further examples include, but are not limited to, polysiloxane copolymers, polyacrylate solution, cellulose esters, hydroxyethyl cellulose, hydroxypropyl cellulose, polyamide wax, polyolefin wax, hydroxypropyl methyl cellulose, polyethylene oxide, and the like. EXAMPLES Example 1 As described below, polydimethyl siloxane and polyethylene glycol modified Isophorone diisocyanate (IPDI) pre-polymers were synthesized. Later, the pre-polymers were used to prepare siloxane polyurethane FR coatings according to the invention. IPDI trimer (Desmodur Z4470 BA) was diluted with EEP and reacted with polyethylene glycol methyl ether (m-PEG) and monocarbinol-terminated polydimethyl siloxane (PDMS). Three siloxane molecular weight were used (1000, 5000, and 10000 g/mole). Two molecular weight variations (550, 750 g/mole) were used for m-PEG. 1.1 Materials Monocarbinol-terminated polydimethyl siloxane (PDMS) with three molecular weights (MCR-C12:1000, MCR-C18:5000, MCR-C22:10000 g/mole) were purchased from Gelest Inc. Isophorone diisocyanate (IPDI) polyisocyanate Desmodur Z4470 BA was generously provided by Bayer MaterialScience. Acetylacetone, methyl amyl ketone (MAK), ethyl-3-ethoxypropionate (EEP), polyethylene glycol methyl ether (m-PEG 550 and 750 g/mole), and dibutyltin diacetate (DBTDAc) were purchased from Sigma Aldrich. An acrylic polyol composed of 80% butyl acrylate and 20% 2-hydroxylethyl acrylate was synthesized via conventional free radical polymerization and diluted in 50% toluene. Aminopropyl-terminated polydimethyl siloxane (APT-PDMS) with molecular weight 20000 g/mole that was synthesized through ring opening equilibration reaction was used for internal control. Intersleek 700 (IS 700), Intersleek 900 (IS 900), Intersleek 1100 SR (IS 1100SR) commercial FR coatings and Intergard 264 marine primer were provided by International Paint. Hempasil X3 commercial FR coating was provided by Hempel. Silicone elastomer, Silastic® T2 (T2) was provided by Dow Corning. Aluminum panels (4×8 in., 0.6 mm thick, type A, alloy 3003 H14) purchased from Q-lab were sand blasted and primed with Intergard 264 using air-assisted spray application. Microtiter plates were modified using circular disks (1 inch diameter) of primed aluminum. 1.2 Pre-Polymer Formulation Table 1 contains formulation for IPDI-10%-1kPDMS-550PEG pre-polymer modified with PDMS (MW=1000) and m-PEG (MW=750). IPDI trimer Desmodur Z4470 BA, which contains 70% solids, was used as the isocyanate. First isocyanate (3.4299 g) and EEP (1.6000 g) was weighed into a 40 mL glass vial with magnetic stir bar. The content was thoroughly mixed using the vortex for 5 mins. Next, PDMS (1.6000 g), PEG (1.6000 g), and catalyst solution (0.3200 g) were added. The content was thoroughly mixed using the vortex followed by overnight mixing using magnetic stirring. The isocyanate to total hydroxyl equivalence ratio was maintained at 3:2 for all pre-polymers.FIG.1depicts a general structure proposed for the pre-polymers synthesized. In the structure, X can be either an isocyanate group, PDMS chain linked through urethane or m-PEG linked through urethane. The integer “m” connotes the repeating units in the PDMS and the integer “n” the repeating units in the m-PEG. TABLE 1Composition of the IPDI-PDMS-PEG-prepolymer Part 1IPDI-5%-1KPDMS-550PEG-prepolymerMwAmountEq. WtF1AmountIngredients(g/mol)(g)Wt. %(g/eq)Eq% SolidsAdded (g)Isocyanate (Desmodur Z 4470 BA)2.401015.00603556.76E−03703.4299EEP——————1.6000Monocarbinol-terminated PDMS10001.600010.000010001.60E−031001.6000Hydroxyl-terminated PEG-7505501.600010.00005502.91E−031001.6000DBTDAc—0.00320.0200——10.3200 Table 2 describes exemplary pre-polymer compositions made according to the invention. X2/X1ranged from (0.0347-0.2857) and X3/X1ranged from (0.6318-0.3810), respectively. TABLE 2Pre-polymer CompositionsWt. ofOH Eq.OH Eq.IPDIWt. offromWt. offromtrimerNCO Eq.MW ofPDMSPDMSMW ofm-PEGm-PEGPre-polymer(g)(X1)PDMS(g)(X2)m-PEG(g)(X3)EEP (g)IPDI-5-1kPDMS-550PEG1.71523.38E−0310000.80008.00E−045500.80001.45E−031.6000IPDI-5-5kPDMS-550PEG1.22832.42E−0350000.80001.60E−045500.80001.45E−031.6000IPDI-5-10kPDMS-550PEG1.16752.30E−03100000.80008.00E−055500.80001.45E−031.6000IPDI-5-1kPDMS-750PEG1.41992.80E−0310000.80008.00E−047500.80001.07E−031.6000IPDI-5-5kPDMS-750PEG0.93301.84E−0350000.80001.60E−047500.80001.07E−031.6000IPDI-5-10kPDMS-750PEG0.87221.72E−03100000.80008.00E−057500.80001.07E−031.6000IPDI-10-1kPDMS-550PEG3.42996.76E−0310001.60001.60E−035501.60002.91E−031.6000IPDI-10-5kPDMS-550PEG2.45674.84E−0350001.60003.20E−045501.60002.91E−031.6000IPDI-10-10kPDMS-550PEG2.33514.60E−03100001.60001.60E−045501.60002.91E−031.6000IPDI-10-1kPDMS-750PEG2.84005.60E−0310001.60001.60E−037501.60002.13E−031.6000IPDI-10-5kPDMS-750PEG1.86633.68E−0350001.60003.20E−047501.60002.13E−031.6000IPDI-10-10kPDMS-750PEG1.74463.44E−03100001.60001.60E−047501.60002.13E−031.6000 1.3 Characterization Fourier Transformed Infrared (FTIR) spectroscopy was used to characterize the pre-polymers prepared. The liquid pre-polymer was spread on a potassium bromide (KBr) plate as a thin film prior to obtaining the spectrum. Attenuated Total Reflectance Fourier Transformed Infrared spectroscopy (ATR-FTIR) was utilized to characterize the coating surfaces after water aging. Bruker Vertex 70 with Harrick's ATR™ accessory using a hemispherical Ge crystal was used to obtain ATR-FTIR spectrum of coatings. FIG.2shows the FTIR of the liquid pre-polymer IPDI-10%-5kPDMS-550PEG. The peak at approximately 2200 cm−1indicates the presence of residual, unreacted isocyanate which is used for crosslinking with the acrylic polyol later. The peak at 3300-3400 cm−1due to N—H stretching shows successful reaction of isocyanate with OH-PDMS and m-PEG. This could also be supported by the presence of carbamate (C═O) peak at 1690 cm−1. The ether stretching (—C—O—C—) due to ethylene glycol is also present in the FTIR spectrum at 1210 cm−1. Presence of siloxane (—Si—O—Si—) stretching can be seen at 1000-1100 cm−1. ATR-FTIR provided information about chemical functional groups present on the top surface of solid materials. Typical penetration depth of ATR-FTIR varies from 0.5 to 2 μm depending on the angle of incidence, wavelength of light, and the refractive indices of ATR crystal and the material of interest.FIG.3shows the normalized ATR-FTIR for IPDI-10%-PDMS-550PEG pre-polymer containing coating formulations 7, 8, and 9 (Table 5) after water Immersion. These coatings have 10% PDMS and PEG content (based on the total solids) with m-PEG 550 being used in all three. However, the PDMS molecular weight was varied from 1000, 5000, to 10000. FTIR spectrum shows the presence of —C—O—C— (1180 cm−1) and —Si—O—Si— (1020-1100 cm−1) functionalities, suggesting the presence of both PEG and PDMS. However, the intensity of —Si—O—Si— (1020-1100 cm−1) and Si—CH3(790 cm−1) peaks are slightly lower in coatings 8 and 9 compared to coating 7. Simultaneously, the peaks corresponding to PEG are slightly lower in intensity for coating 7 compared to the other two coatings. Therefore, coating 7 may have higher concentration of siloxane closer to the surface compared to other two coatings. The spectrum shows presence of two types of carbonyl groups C═O (1750 cm−1) and C═O (1690 cm−1). The C═O″ corresponds to the carbonyl on acrylic polyol and the C═O* corresponds to the carbamate group. The peak for R—O—C(O)—NH—R′ is weak but visible at (3350-3450 cm−1), suggesting very lower concentration closer to the coating surface. 1.4 Coating Formulation After prepolymer formulation, additional isocyanate (5.0039 g) was added along with acrylic polyol (13.7926 g, BA: HEA 80:20 in 50% toluene) and pot life extender acetylacetone (0.3200 g). The overall isocyanate to total hydroxyl equivalence was maintained at 1.1:1 for the final formulation. The content was thoroughly mixed using the vortex followed by magnetic stirring for 1 hour. Table 3 describes a composition of the coating formulations of the invention. TABLE 3Composition of Coating Formulation (Part 2)IPDI-10-1KPDMS-550PEG-coating formulation part 2MwAmountEq. WtAmountIngredients(g/mol)(g)Wt. %(g/eq)Eq% SolidsAdded (g)Isocyanate (Desmodur Z 4470 BA)3.502721.89203559.87E−03705.0039Acrylic Polyol6.896343.10206501.06E−025013.7926Acetylacetone0.32002.0000——1000.3200 Formulation was deposited into microtiter plates and drawdowns were prepared on primed aluminum panels. For depositions, 250 μL of formulation were dispensed using an automatic pipette to each well. Drawdowns were made using a wire-wound drawdown bar with a wet film thickness of 80 μm on 8″×4″ primed aluminum panels. Both microtiter plates and coated panels were allowed to cure under ambient conditions for 24 hrs. The following day, all the coatings were cured at 80° C. for 45 min. Table 4 describes exemplary coating compositions made according to the invention. TABLE 4Coating CompositionsTypeOverallTypeOverallFormulationType of pre-polymerofWt. %ofWt. %#usedPDMSPDMSPEGPEG1IPDI-5-1kPDMS-550PEGPDMS-1k5m-PEG-55052IPDI-5-5kPDMS-550PEGPDMS-5k5m-PEG-55053IPDI-5-10kPDMS-550PEGPDMS-10k5m-PEG-55054IPDI-5-1kPDMS-750PEGPDMS-1k5m-PEG-75055IPDI-5-5kPDMS-750PEGPDMS-5k5m-PEG-75056IPDI-5-10kPDMS-750PEGPDMS-10k5m-PEG-75057IPDI-10-1kPDMS-550PEGPDMS-1k10m-PEG-550108IPDI-10-5kPDMS-550PEGPDMS-5k10m-PEG-550109IPDI-10-10kPDMS-550PEGPDMS-10k10m-PEG-5501010IPDI-10-1kPDMS-750PEGPDMS-1k10m-PEG-7501011IPDI-10-5kPDMS-750PEGPDMS-5k10m-PEG-7501012IPDI-10-10kPDMS-750PEGPDMS-10k10m-PEG-75010 All other consequent formulations were also prepared following a similar procedure. PDMS and PEG levels of 5% and 10% were used based on the overall coating formulation.FIG.4shows the composition of basic ingredients based on solids of the main ingredients in formulation 7 (described above) based on weight percent. Additional solvent, catalyst (solution), and pot life extender were also included. 1.5 Control Coatings All commercially available coatings were prepared following the technical data sheets provided by the suppliers. A brief description of the procedure followed to prepare siloxane polyurethane FR coatings is described here. The non-reactive components, such as, APT-PDMS (20% by wt.), acrylic polyol, and pot life extender, were combined in a glass container and allowed to mix overnight. On the next day, isocyanate (Desmodur 4470BA) and catalyst (0.05% by wt. from a 1% MAK solution) were added. The formulation was allowed to mix for about an hour. The isocyanate to other functional group (hydroxyl and amine) ratio was kept at 1.1:1. Drawdowns were made using a wire wound drawdown bar with 80 μm dry film thickness on 8″×4″ aluminum panels previously primed with Intergard 264 primer. Formulation was deposited into microtiter plates, 250 μL of formulation were dispensed using an automatic pipette to each well. The coatings were allowed to cure for 24 hours under ambient conditions inside a dust free cabinet, followed by force curing in the oven at 80° C. for 45 minutes. Table 5 describes the list of control coatings. TABLE 5List of Control CoatingsCoatingControl NameDescription13A4-20%Internal Siloxane-PU FR Control14Hempasil X3Silicone Hydrogel based CommercialFR Control15NDSU-PUPure Polyurethane16Dow T2Silicone Elastomer17IS 700Intersleek Commercial FR Control18IS 900Intersleek Commercial FR Control19IS 1100SRIntersleek Commercial FR (SlimeRelease) Control 1.6 Water Aging and Biological Assay Tests All the coatings were subjected to a pre-leaching process for 28 days. Coated plates and panels were placed in a water tank that was automatically emptied and refilled every hour. Following the pre-leaching process, a leachate toxicity study was conducted to ensure non-toxicity of the coatings. All the experimental coatings displayed non-toxicity. Next, bacteria (C. lytica), algae (N. incerta), barnacle (A. amphitrite), and mussel (G. demissa) assays were conducted. Detailed description of the assay tests use can be found in Casse et al.,Biofouling2007, 23 (¾), 267-276; Stafslien et al.,Biofouling2007, 23 (1), 37-44; and Webster et al.,Biofouling2007, 23 (¾), 179-192, which are incorporated herein by reference. FR performance towards marine bacteriumCellulophaga lyticafor experimental and control coatings were evaluated by a retention and retraction assay followed by water-jet treatment to evaluate adhesion (FIGS.5and6).FIG.5shows the bacterial biofilm (Cellulophaga lytica) growth and retention after water-jet treatment at 20 Psi pressure.FIG.6shows the percent removal of bacterial biofilm (Cellulophaga lytica) after water-jet treatment at 20 Psi pressure. Absorbance of crystal violet at 600 nm wavelength is directly proportional to the biomass present. Bacterial biofilm growth and retention for coatings with IPDI-10-1kPDMS-750PEG, IPDI-10-5kPDMS-750PEG, and IPDI-10-10kPDMS-750PEG pre-polymers showed comparable results to Hempasil X3 commercial FR coating. These compositions show almost complete removal of biofilms after water-jet. Also, the coatings 7 and 9 displayed >90% biofilm removal upon treatment of water-jet at 20 psi pressure. In general, coatings 7, 9, 10, 11, and 12 were on par with commercial FR coatings from International Paint in terms of bacterial biofilm removal. Overall, the several IPDI-PEG-PDMS pre-polymer modified coatings demonstrated significantly improved FR performance towardsCellulophaga lyticacompared to A4-20% 1stgeneration siloxane polyurethane formulation. Results from bacterial biofilm assay suggests thatCellulophaga lyticahave lower affinity towards IPDI-PEG-PDMS pre-polymer modified siloxane polyurethane coatings. FR performance towards slime forming diatomsNavicula incertawere evaluated using a similar assay (FIGS.7and8).FIG.7shows the microalgae (Navicula incerta) attachment and retention after water-jet treatment at 20 Psi pressure.FIG.8shows the percent removal of microalgae (Navicula incerta) after water-jet treatment at 20 Psi pressure. The amount of biomass was determined using the chlorophyll extraction. Coating compositions 7, 10, and 11 showed the lowest retention of diatoms after 20 psi water-jet treatment, which is similar to the performance of Intersleek 1100 SR, Intersleek 900, Polyurethane, and Hempasil X3. Coatings with IPDI-10-1kPDMS-750PEG and IPDI-10-5kPDMS-750PEG pre-polymers showed significant improvement in diatom removal compared to A4-20 siloxane polyurethane formulation. Thus suggesting that micro algaeNavicula incertahave lower adhesion towards pre-polymers with longer PEG chains. In general, it has been a challenge to find a coating composition that provides good FR performance towards bothCellulophaga lyticaandNavicula incerta, mainly due to their opposite preference for surface wettability.Cellulophaga lyticapreferentially adheres to hydrophilic surfaces whereasNavicula incertapreferentially adheres to hydrophobic surfaces. Bodkhe et al.,Prog. Org. Coat.2012, 75 (1-2), 38-48. Coating formulations 10 and 11 appeared to address that issue since both showed the best FR performance towards bacteria and diatoms. Several experimental coatings with IPDI-PEG-PDMS pre-polymer coatings showed no mussel attachment, suggesting that mussels did not prefer to settle on these coatings (FIGS.9and10).FIG.9shows the attachment efficiency of mussels (Geukensia demissa) based on 6 mussel attachment attempts.FIG.10shows the mussel adhesion (Geukensia demissa) based on attached mussels, 6 mussel attachment attempts. Similarly, Intersleek 900, Hempasil X3, and A4-20 control showed no mussel attachment. However, the coating compositions that demonstrated excellent FR performance towards bacteria and diatoms showed some mussel attachment, although they were easily removed with approximately 10 N force. Out of the coatings that displayed some mussel attachments, coatings 10 and 11 showed the lowest number of mussel attachment and lower force for removal, suggesting good overall FR performance towards all three organisms. Barnacle adhesion strength towards coatings was evaluated using a two week reattachment assay followed by a push off test. Adhesion strength (or critical removal stress) was quantified by shear force for removal divided by barnacle basal plate area. The effects of PDMS MW was clearly seen by the barnacle adhesion strength for AmSiPU coatings (FIG.11). Coatings containing pre-polymers modified with shorter PDMS chains showed high barnacle adhesion strength. The opposite behavior was observed for coatings modified with longer PDMS chains (10000). Also the coatings with higher PDMS MW had no broken barnacles, which is further evidence that PDMS MW had a significant effect on easy release of barnacles attached to surfaces. Several experimental coatings showed non-attached barnacles and lower adhesion strengths that were comparable to Intersleek® 900 performance. Coatings 3, 6, 9, 11, and 12 displayed the best performance allowing removal of all reattached barnacles with lower adhesion strengths. On these coatings, several barnacles were unable to re-attach; further indicator of good FR performance. Coatings consisting of pre-polymer with 10% concentration of PDMS and PEG provided the better FR performance towards barnacles compared to those with 5%. Surface wettability and surface charge play an important role in barnacle settlement. It is often observed that PDMS based materials show low critical removal stress of barnacles (A. amphitrite) which is attributed to their low surface energy. However AmSiPU coatings with both hydrophilic PEG and hydrophobic PDMS displayed lower barnacle adhesion strengths. This assay demonstrates the important role of PDMS being an essential component in amphiphilic FR system. Compared to previous attempts of amphiphilic siloxane-PU coatings, IPDI-PDMS-PEG pre-polymer modified coatings were able to maintain good FR towards barnacles while improving performance towards microfoulers. The control coating polyurethane (no PDMS), showed the worst performance towards barnacles on which all reattached barnacles broke. Hempasil® X3 and Intersleek 1100SR showed the best performance by not allowing any barnacle to reattach during the two weeks of immersion in artificial sea water.FIG.11shows reattached barnacle (Amphibalanus amphitrite) adhesion strength. Six barnacles were used for each reattachment study, out of which italicized numbers represent the non-attached barnacles. The ratio represents the number of released barnacles versus the number of broken/damaged barnacles during push off measurements. Each bar represents the average adhesion strength based on the number of successfully pushed barnacles. 1.7 Conclusion Isophorone diisocyanate-Polyethylene glycol-Polydimethyl siloxane (IPDI-PEG-PDMS) pre-polymers of the invention were synthesized by reacting IPDI trimer with monocarbinol-terminated polydimethyl siloxane and m-PEG. Later, these pre-polymers were incorporated in to siloxane polyurethane coatings. ATR-FTIR suggests that both siloxane and PEG are pre-dominant on the surface after water aging. Several coatings showed excellent FR performance towards bacteria (Cellulophaga lytica) having >90% removal. Coatings 10 and 11 with 10-1kPDMS-750PEG and IPDI-10-5kPDMS-750PEG pre-polymers displayed significantly better FR performance towards microalgae (Navicula incerta) surpassing 1stgeneration siloxane polyurethane formulation and on par with newest Intersleek FR coatings. Interestingly, coatings that had average FR performance towards bacteria showed no mussel attachments. Although coating 10 and 11 displayed few mussel (Geukensia demissa) attachments, they were easily removed with smaller force. Overall, the IPDI-PEG-PDMS pre-polymers of the invention improve FR performance of non-toxic siloxane polyurethane marine coatings. Example 2 2.1 Materials Polyisocyanates Desmodur Z4470 BA and Desmodur N3300 A were provided by Covestro LLC. An acrylic polyol composed of 80% butyl acrylate and 20% 2-hydroxyethyl acrylate was synthesized via conventional free radical polymerization and diluted up to 50% with toluene. Polyester diol CAPA™ 2054 was provided by Perstorp. Monocarbinol terminated polydimethyl siloxane (PDMS) in two molecular weights (MCR-C18:5000, MCR-C22:10000 g/mole) was purchased from Gelest Inc. Amino-propyl terminated polydimethyl siloxane (APT-PDMS) with a molecular weight of 20000 g/mole was synthesized at NDSU through a ring opening equilibration reaction. Ethyl-3-ethoxypropionate (EEP), methyl amyl ketone, acetylacetone, dibutyltin diacetate, polyethylene glycol methyl ether (PEG 750), and butyl acetate were purchased from Sigma Aldrich. 2.2 Synthesis Commercially available IPDI trimer (Desmodur Z4470 BA) and HDI trimer (Desmodur N3300 A) were modified using PDMS and PEG to design pre-polymers with many different compositions. A general procedure for pre-polymer IPDI-10PEG-10PDMS5k-5PDMS10k (Formulation 3) modified by PDMS (MW=5000 and 10000) and PEG (MW=750) will be described here (The amount of PDMS 5000 and PEG are each 10% by wt. and PDMS 10000 is 5% by weight based on the total weight of the polyurethane). First PEG (1.6000 g) was dissolved in EEP (1.6000 g) in a 20 mL vial using mixing by vortex for 5 minutes. Next, a magnetic stir bar and PDMS 5000 (1.6000 g) and PDMS 10000 (0.8000 g) were added and vortexed for an additional 2 minutes. Isocyanate (1.9271 g) and DBTDAc catalyst solution (1% by wt. in MAK) (0.3200 g) were then added to the vial. Contents of the vial were then mixed using a vortex mixer for 5 minutes followed by stirring for 24 hours using a magnetic stir plate. Throughout the pre-polymers, the total isocyanate to hydroxyl equivalents ratio was sustained at 3:2. Details of other formulations can be found in Table 6. 2.3 Coating Formulation and Curing Coating formulation for the pre-polymer described is included here. Acrylic polyol (13.1606 g) and acetyl acetone pot life extender (0.3200 g) were added into the vial containing the pre-polymer. The vial was then mixed via vortex for 2 minutes followed by additional isocyanate (5.8152 g) and DBTDAc (0.1600 g). The overall isocyanate to hydroxyl ratio was maintained at 1.1:1 for final formulation. The contents were then thoroughly mixed using a vortex mixer and followed by magnetic stirring for 1 hour. Formulations were then deposited into multi-well plates and drawdowns were done on primed aluminum panels. The coating formulations (250 μL) were deposited using an automatic repeat pipette for each well. Drawdowns were made using a wire drawdown bar leaving a wet film thickness at 80 μm on 8″×4″ primed aluminum panels. Coatings were cured at ambient conditions for 24 hours then oven cured for 45 minutes at 80° C. All other formulations were prepared following similar procedure as outlined above. For this study, PDMS levels of 5%, 10%, and 15% along with PEG levels of 10% and 15% based on coating formulation were considered. Table 6 shows the 11 experimental coatings evaluated in this study. TABLE 6Pre-polymer compositions considering formulation variables.PolyurethanePre-polymer compositionbulk compositionType ofType ofAmountType ofAmountType ofType ofNameLabelNCOPEGof PEGPDMSof PDMSPolyolNCO1IPDI-10PEG-10PDMS5kIPDIPEG 75010%PDMS 5k10%APIPDI2IPDI-15PEG-15PDMS5kIPDIPEG 75015%PDMS 5k15%APIPDI3IPDI-10PEG-10PDMS5k-IPDIPEG 75010%PDMS 5k10%APIPDI5PDMS10kPDMS 10k5%4IPDI-15PEG-10PDMS5k-IPDIPEG 75015%PDMS 5k10%APIPDI5PDMS10kPDMS 10k5%5IPDI-15PEG-15PDMS5kIPDIPEG 75015%PDMS 5k15%PCLPIPDI6HDI-15PEG-15PDMS5kHDIPEG 75015%PDMS 5k15%APIPDI7HDI-15PEG-10PDMS5k-HDIPEG 75015%PDMS 5k10%APIPDI5PDMS10kPDMS 10k5%8HDI-15PEG-15PDMS5kHDIPEG 75015%PDMS 5k15%APHDI9HDI-10PEG-10PDMS5k-HDIPEG 75010%PDMS 5k10%APHDI5PDMS10kPDMS 10k5%10HDI-15PEG-10PDMS5k-HDIPEG 75015%PDMS 5k10%APHDI5PDMS10kPDMS 10k5%11IPDI-15PEG-10PDMS5k-IPDIPEG 75015%PDMS 5k10%APHDI5PDMS10kPDMS 10k5%12A4-20Control13Hempasil X3Control14Dow T2Control15PUControl16IS900Control17IS1100SRControl Table 7 shows the isocyanate values for several pre-polymers. Isocyanate values for these prepolymers were similar to the theoretical values indicating the successful synthesis. TABLE 7Percent isocyanate for chosen pre-polymersPre-polymer FormulationTheoretical % NCOExperimental % NCOIPDI-10PEG750-2.202.10 ± 0.44710PDMS5kIPDI-15PEG750-2.182.21 ± 0.21515PDMS5kIPDI-15PEG750-2.121.81 ± 0.18010PDMS5k-5PDMS10kHDI-15PEG750-2.482.23 ± 0.18815PDMS5kHDI-15PEG750-2.672.03 ± 0.20410PDMS5k-5PDMS10k FIG.12shows theC. lyticacell attachment and biomass remaining after water jet treatment. AmSiPU coatings displayed low cell attachment compared to commercial standards. Also coatings 1-5 showed excellent FR properties towardsC. lyticabiofilm.FIG.13features the percent removal of bacterial biofilm using the amount of cell attachment and biomass remaining. Coatings 1-5 showed ≈100% bacterial biofilm removal on par with Hempasil® X3 and exceeded the Intersleek® standards. All the AmSiPU coatings showed better FR properties compared to A4-20 internal control. Overall compositional changes in polyisocyanate pre-polymers and in polyurethane bulk did not seems to have significant effect on FR properties of AmSiPU coatings towardsC. lytica. FIG.14shows the diatom (N. incerta) cell attachment and biomass remaining after water jet treatment at 20 psi. InitialN. incertacell attachment of AmSiPU coatings were comparable to T2 silicone elastomer and Intersleek® 1100SR standard. However Hempasil® X3 demonstrated the lowest amount ofN. incertacell attachment and biomass left after cleaning. Following water jet treatment, many AmSiPU coatings had low biomass remaining which was comparable to standards Intersleek® 700, 900, and 1100SR.FIG.15shows the percent removal of diatom. All AmSiPU coatings show significant improvement inN. incertaFR properties compared to the internal control A4-20. Coatings 1, 2, 3, 6 and 7 show ≈80% removal of diatom, comparable to commercial standards Hempasil® X3, and all Intersleek standards. Slight decrease in diatom removal for coatings 4 and 5 may be attributed to slight variation in composition. Examples 3 and 4 3.1 Materials Isophorone diisocyanate (IPDI) polyisocyanate Desmodur Z4470 BA was provided by Covestro LLC. Monocarbinol-terminated polydimethylsiloxane (PDMS) of molecular weight 5,000Mnand 10,000Mn(MCR-C22) was purchased from Gelest, Inc. Poly(ethylene glycol) methyl ether (550Mnand 750Mn) (mPEG), ethyl-3-ethoxy propionate, methyl ethyl ketone (MEK), acetylacetone, methyl amyl ketone (MAK), and dibutyltin diacetate (DBTDAc) were purchased from Sigma Aldrich. Toluene and isopropanol were purchased from VWR. Following a detailed description elsewhere, an acrylic polyol made of 80% butyl acrylate and 20% 2-hydroxyethyl acrylate was prepared via conventional free radical polymerization and diluted to 50% in toluene. Bodkhe et al.,J. Coating. Tech. Res.2012, 9 (3), 235-249. Aminopropyl terminated polydimethylsiloxane (APT-PDMS) with molecular weight (MW) of 20,000Mnwas also synthesized through a ring-opening equilibration reaction. Both synthesized polymers were prepared following guidelines from elsewhere. Bodkhe et al.,J. Coating. Tech. Res.2012, 9 (3), 235-249. Amphiphilic prepolymer based on PEG 750Mnand PDMS 10,000Mnfor the AmpSiPU coating system were also prepared following procedure elsewhere, called R0 in this study. Galhenage et al.,J. Coating. Tech. Res.2017, 14 (2), 307-322. AkzoNobel International Paint provided the commercial FR standards Intersleek® 700 (IS 700), Intersleek® 900 (IS 900), and Intersleek® 1100SR (IS 1100). Silicone elastomer, Silastic® T2 (T2), and control thermoplastic polystyrene (PS) was provided by Dow Corning. Hydrophobic A4-20 coating (A4-20), a siloxane-polyurethane system, was prepared as an internal control following the procedures described elsewhere. Bodkhe et al.,J. Coating. Tech. Res.2012, 9 (3), 235-249. Amphiphilic T-10 coating, internal coating control, was prepared following the procedure elsewhere for a formulation that contained 10 wt. % PEG 750Mnand PDMS 10,000Mn. Galhenage et al.,J. Coating. Tech. Res.2017, 14 (2), 307-322. Also, a pure polyurethane formulation without APT-PDMS was also prepared to be included as a control. Aluminum panels (4″×8″ in., 0.64 mm thick, type A, alloy 3003 H14) and steel panels (3″×6″ in., 0.51 mm thick, type QD) were purchased from Q-lab and were sandblasted and primed with Intergard 264 (International Paint) using air-assisted spray application. Multi-well microtiter plates were modified using circular disks (1-inch diameter) of primed aluminum. Stafslien et al.,Biofouling2007, 23 (1), 45-54. 3.2 Methods of Characterization 3.2.1 Isocyanate Titrations Isocyanate titration was used to monitor the reaction progress and confirm the complete conversion of the isocyanate groups after the synthesis of the additive. An additive sample (0.3-0.5 g) was weighed in an Erlenmeyer flask and diluted with isopropanol. Then, 25 mL of 0.1 N dibutyl amine solution and an additional 25 mL of isopropanol were added to the flask and the mixture was stirred for 15 minutes. Several drops (3-5 drops) of bromophenol blue indicator were added to the flask. The content of the flask was titrated using a standardized 0.1 N hydrochloric acid until the endpoint blue to yellow was observed. A blank prepared with only 25 mL of dibutyl amine solution was also titrated following the same procedure. The recorded amount of hydrochloric acid for both titrations was used to calculate the amount of isocyanate remaining. 3.2.2 Percent Solids Determination The non-volatile content of the additive was determined following ASTM D2369. Briefly, a weighed empty aluminum pan was filled with additive sample (1-2 g). Isopropyl alcohol was used to cover the sample. The pan was placed in an oven at 120° C. for 1 hour. After removal from the oven, the pan was weighed again to determine the percent solids. Three replicates were recorded. 3.2.3 Fourier Transform Infrared Spectroscopy Fourier transform infrared (FTIR) spectroscopy was used to characterize the additive, using a Thermo Scientific Nicolet 8700 FTIR. The additive was applied as a thin layer on a potassium bromide (KBr) plate to collect the spectrum. 3.2.4 Surface Characterization A Kruss® DSA 100 (Drop Shape Analyzer) was utilized to measure the surface wettability and surface energy of the coatings. Three replicate water and diiodomethane contact angles were measured for each sample. For each replicate, the static contact angle was measured over 9 minutes. Surface energy for each surface was calculated using the Owens-Wendt method. Owens et al.,J. Appl. Polym. Sci.1969, 13 (8), 1741-1747. Slip angle, advancing and receding water contact angles for surface were evaluated using a tilting stage where a 25-4 water droplet was viewed on a coating surface (tilted at 10°/min) and values were recorded at the degree that the droplet started to roll off. The measured angles and surface energies were calculated using Kruss® Advance software. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to characterize the surfaces of the coatings. A Bruker Vertex 70 with Harrick's ATR™ accessory using a hemispherical Ge crystal was utilized to collect ATR-FTIR spectra for a coating. X-Ray Photoelectron spectroscopy (XPS) was carried out using a Thermo Scientific™ K-Alpha™ system to determine the elemental composition of the coatings. The instrument is equipped with a monochromatic Al Kα(1486.68 eV) X-ray source and Ar+ion source (up to 4000 eV). Depth profiling of a coating was evaluated using argon ion with 30 etch cycles. For each etch cycle, the ion beam was set to 1,000 eV Monatomic Mode with low current and 30 s etch time. After each etching cycle, five replicate survey spectra were collected, at low resolution, with a constant analyzer pass energy of 200 eV, for a total of 20 ms. For each run, photoemission lines for C1s, N1s, 01s, and Si2p were observed. Spectra were collected at an angle normal to the surface (90° of a 400-μm area. The chamber pressure was maintained below 1.5×10−7torr and samples were analyzed at ambient temperature. Atomic concentrations were quantified by the instrument's software as a representation of the atomic intensities as a percentage of the total intensity of all elements. Two internal PU systems without (pure PU) and with AmpAdd-1 additive (modified PU) were examined to verify the self-stratification of AmpAdd additives into the surface of coatings. Atomic force microscopy (AFM) was utilized to study the surface topography of the coatings. A Dimension 3100 microscope with Nanoscope controller scanned the surface of experimental coatings, collecting images on a sample area of 100 μm×100 μm in the tapping mode. The experiment was run in air, under ambient conditions, using a silicon probe with a spring constant of 0.1-0.6 N/m and resonant frequency of 15-39 kHz. For each surface, three replicates at varying spots were collected to ensure consistency and accuracy of the data. 3.2.5 Water Aging All the prepared coatings were pre-leached for 28 days in running tap water. The water tanks were equipped to automatically fill and empty every 4 hours. Water aging of the coatings is carried out to meet two objectives: 1) to leach out any impurities that may interfere with FR assessments; and 2) to determine if there are any surface rearrangements of the coatings or whether the additives leach out to a significant degree. All biological laboratory assays were carried out after the pre-leaching water aging process was completed. 3.2.6 Biological Laboratory Assays Growth and Release of Macroalgae (Ulva linza) A set of multiwall plates was sent to Newcastle University, following water-immersion for 28 days, to evaluate FR performance of coatings againstU. linza. The detailed description about the assessment can be found elsewhere. Cassé et al.,Biofouling2007, 23 (2), 121-130. Briefly, after leachate collection, all multi-well plates were equilibrated in 0.22 μm filtered artificial seawater (Tropic Marin®) for 2 hours. To each well, a 1-mL suspension ofU. linzaspores was added, adjusted to 3.3×105spores/mL (0.05 OD at absorbance 660 nm) in enriched seawater medium. Starr et al.,Phycol.1987, 23 (suppl.), 1-47. Spores that settled on the discs were grown for 7 days inside an illuminated incubator at 18° C. with a 16:8 light: dark cycle (photon flux density 45 μmol·m−2·s−1). There was no washing to remove unsettled spores after settlement. After 7 days, the biomass generated prior to water jetting was assessed from a single row of wells (6) from each plate. Two other rows of wells were exposed to water jet pressures of 67 kPa (9.7 psi) and 110 kPa (16 psi) for 10 seconds per well.U. linzabiofilm biomass was determined before and after water jetting by extracting chlorophyll with 1 mL DMSO to each water-pressured well followed by measuring the fluorescence at 360 nm excitation and 670 nm emission. Fluorescence from the extracted chlorophyll is directly proportional to the biomass present on each coating surface. Mieszkin et al.,Biofouling2012, 28 (9), 953-968. The removal ofU. linzaat each pressure was compared with the unsprayed wells that were used to determine initial biomass. Bacterial (Cellulophaga lytica) Biofilm Adhesion FR properties towards bacteria were evaluated using retention and adhesion assays described previously. Stafslien et al.,Biofouling2007, 23 (1), 45-54; Stafslien et al.,Rev. Sci. Instrum.2007, 78 (7), 072204. Briefly, a solution of the marine bacteriumCellulophaga lyticaat 107cells/mL concentration in artificial seawater (ASW) containing 0.5 g/L peptone and 0.1 g/L yeast extract was deposited into 24-well plates (1 mL/well). The plates were then incubated statically at 28° C. for 24 hours. The ASW growth medium was then removed and the coatings were subjected to water-jet treatments. On each plate, the first column of coatings was not treated and showed the initial amount of bacterial biofilm growth. The second and third columns were subjected to water-jetting at 10 psi and 20 psi, respectively, for 5 seconds. Following water-jet treatments, the coating surfaces were stained with 0.5 mL of a crystal violet solution (0.3 wt. % in deionized water) for 15 minutes and then rinsed three times with deionized water. After 1 hour of drying at ambient laboratory conditions, the crystal violet dye was extracted from the coating surfaces by adding 0.5 mL of 33% acetic acid solution for 15 minutes. The resulting eluates were transferred to a 96-well plate (0.15 mL/coating replicate) and subjected to absorbance measurements at 600 nm wavelength using a multi-well plate spectrophotometer. The absorbance values were directly proportional to the amount of bacterial biofilm present on coating surfaces before and after water-jetting treatments. Percent removal of bacterial biofilm was quantified by comparing the mean absorbance values of the non-jetted and water-jetted coating surfaces. Selim et al.,Mater. Des.2016, 101, 218-225. Growth and Release of Microalgae (Navicula incerta) The laboratory biological assay with the diatom (Navicula incerta) was conducted following a similar procedure to that described previously. Callow et al.,Nat. Commun.2011, 2 (1), 244-244; Cassé et al.,Biofouling2007, 23 (2), 121-130; Casse et al.,Biofouling2007, 23 (4), 267-276. Briefly, a suspension with 4×105cells/mL ofN. incerta(adjusted to 0.03 OD at absorbance 660 nm) in Guillard's F/2 medium was deposited into each well (1 mL per well) and cell attachment was stimulated by static incubation for 2 hours under ambient conditions in the dark. Coating surfaces were then subjected to water-jet treatments. Stafslien et al.,Rev. Sci. Instrum.2007, 78 (7), 072204. First column of wells was not water-jetted so that initial cell attachment could be determined and the next two-columns of wells were water-jetted at 10 psi and 20 psi, respectively, for 10 seconds. Microalgae biomass was quantified by extracting chlorophyll using 0.5 mL of DMSO and measuring fluorescence of the transferred extracts at an excitation wavelength of 360 nm and emission wavelength at 670 nm. The relative fluorescence (RFU) measured from the extracts was considered to be directly proportional to the biomass remaining on the coating surfaces after water-jetting. Percent removal of attached microalgae was determined using relative fluorescence of non-jetted and water-jetted wells. Adult Barnacle (Amphibalanus amphitrite) Adhesion Adult barnacle reattachment test was carried out to assess FR of coatings against macrofoulants. Stafslien et al.,J. Coating. Tech. Res.2012, 9 (6), 651-665; Rittschof et al.,Biofouling2008, 24 (1), 1-9. Coatings prepared on 4″×8″ panels after water aging were utilized for this laboratory assay. Barnacles were dislodged from silicone substrates sent from Duke University and immobilized on experimental coatings (6 barnacles per coating) using a custom-designed immobilization template. The immobilized barnacles were allowed to reattach and grow for 2 weeks while immersed in an ASW aquarium tank system with daily feedings of brine shrimpArtemia nauplii(Florida Aqua Farms). After the 2-week attachment period, the number of non-attached barnacles was recorded, and the attached barnacles were pushed off (in shear) using a hand-held force gauge mounted onto a semi-automated stage. Once the barnacles were dislodged, their basal plate areas were determined from scanned images using Sigma Scan Pro 5.0 software program. Barnacle adhesion strength (MPa) was calculated by taking the ratio of peak force of removal to the basal plate area for each reattached barnacle. To ensure consistency, barnacles of similar sizes were tested. The average barnacle adhesion strength for each coating was reported as a function of the number of barnacles released with a measurable force and that exhibited no visible damage to the basis or shell plates. 3.2.7 Coating Property Evaluation Stability, adhesion, strength, and flexibility are desirable properties for organic coatings. A double-rub test, according to ASTM D 5402, evaluated the resistance of coatings against solvents. A hammer (0.75 kg) with three-fold cheesecloth wrapped around its head was soaked in MEK or 3.5 wt. % NaCl water solution and rubbed against the coating. The head of hammer was rewet after each 25 double rubs. The number of double rubs was noted when marks were observed on the surface of coatings. Impact test, according to ASTM D 2794, was used to assess strength of coatings using a Gardner impact tester. The maximum drop height was 43 inches with a weight of 4 pounds. Coated steel panels were placed in the testing location, and the load at varying heights was dropped on the coating. The results were recorded in inch-pounds (in-lb). Crazing and/or loss of adhesion from the substrate were observed as a failure point. Coatings that did not fail were reported as having an impact strength of >172 in-lb. The test was run in both forward (front) and reverse modes. The weight was dropped on top of the coating film in forward impact mode, while the weight was dropped on the back of the coated substrate in the reverse mode. A crosshatch adhesion test, according to ASTM D 3359, assessed the adhesion of the coatings to the substrate by applying and removing pressure-sensitive tape over cuts made in the film. The results were reported on a scale of 0 B to 5 B, where 0 B indicates complete removal of the coating and 5 B indicates no removal of the coatings from the substrate as a result of this test. The conical mandrel test, according to ASTM D 522, was used to determine the flexibility of the coatings on the substrate. In principle, ideal flexible coatings should not have any cracks when undergoing the bending test. The results of flexibility were reported as the length of a formed crack in cm on the coating after the bending test. 3.2.8 Statistical Analysis Statistical analysis were performed in SAS software, version 9.4. The GLM procedure with Tukey's method were utilized to determine the difference mean for each treatment group under a completely randomized experimental design. The assessed response for this analysis were the biomass remaining of marine organisms of interest. 3.3 Example 3 3.3.1 Design of Experiment An amphiphilic additive based on 10,000MnPDMS and 750MnPEG was synthesized and incorporated in a polyurethane coating system. These molecular weights offer desirable FR performance according to the literature. Galhenage et al.,J. Coating. Tech. Res.2017, 14 (2), 307-322; Bodkhe et al.,Prog. Org. Coat.2012, 75 (1-2), 38-48. Here, only one variable factor was examined: the amount of the amphiphilic additive in the PU coating system. The additive was added in varying amounts, ranging from 10 wt. % up to 40 wt. % (the highest amount that could be added before the PU film lost its integrity in response to mechanical coating tests). Thus, a total of six formulations were prepared as outlined in Table 8. The table outlines the amount of additive in each system and content of PEG and PDMS in the solids content of the final coating system. TABLE 8Coating CompositionsFormulationAdditive Amount (wt. %)PDMS Wt. %PEG Wt. %F00 (Pure PU00unmodifiedsystem)F101044F202088F25251010F30301212F40401717F50*502121*F50 was not included in surface and biological assay characterizations as it lacked the desired mechanical integrity. 3.3.2 Control and Standard Coatings Commercial standards were prepared following the respective manufacturers' guidelines. Internal control hydrophobic SiPU (A4) was prepared following the procedure outlined in the literature. Bodkhe et al.,J. Coating. Tech. Res.2012, 9 (3), 235-249. T-10 coating, internal amphiphilic control, containing a covalently incorporated 10,000MnPDMS and 750MnPEG prepolymer was also prepared following a previously reported method. Galhenage et al.,J. Coating. Tech. Res.2017, 14 (2), 307-322. Similar to the experimental coatings, the control and standards were also prepared on 4″×8″ primed aluminum panels and multi-well plates. Table 9 contains detailed descriptions of the control and standard coatings used for this study. TABLE 9List of Control and standard reference Coatings*Control NameControl IDDescriptionSiPU A4-20A4Internal SiPU FR ControlAmphiphilic SiPUT-10Internal AmphiphilicSiPU ControlCommercialPUPure Polyurethane StandardPolyurethaneDow ® T2T2Silicone Elastomer StandardIntersleek ® 700IS 700Intersleek ® CommercialFR StandardIntersleek ® 900IS 900Intersleek ® CommercialFR StandardIntersleek ® 1100SRIS 1100Intersleek ® CommercialFR Standard*Commercial PS was used as an interna standard to check thatU. linzawas behaving within expectations. 3.3.3 Synthesis of Amphiphilic Additive The amphiphilic additive was synthesized by reacting mono-hydroxy-terminated PEG and PDMS with the polyisocyanate IPDI trimer Desmodur Z4470 (Scheme 1). The molar ratio of NCO groups to the combined OH groups of PEG and PDMS was 1:1. The functional isocyanate groups were fully converted to urethane linkages by attachment of PEG and PDMS chains. PEG and PDMS were added in equal weight ratios to meet the required one molar ratio. Specifically, to synthesize the amphiphilic additive (AmpAdd), PEG 750Mn(1.00 g) was mixed with toluene (3.00 g) in a 25-mL flask. PDMS 10,000Mn(1.00 g) was added to the flask and mixed robustly with vortex for 2 minutes. IPDI trimer resin (0.56 g) and DBTDAc catalyst solution (1% by wt. in MAK) (0.128 g) were then added to the flask. The reaction was run at 80° C. for 2 hours. As another method, the reaction could also be completed at ambient conditions for 24 hours. A reflux condenser was used when heat was applied. The flask was equipped with a magnetic stirrer, nitrogen inlet, and temperature controller. In theory, the synthesized prepolymer contained 41.37 wt. % PEG and 41.37 wt. % PDMS. 3.3.4 Synthesis of the Curable Coating Compositions and Their Curing All coating formulations were prepared similarly, except the amount of added additive varied. To prepare the unmodified polyurethane F0 formulation, acrylic polyol (8.00 g; 50% solid) and acetylacetone (0.62 g) (potlife extender) were added in a vial and stirred under ambient conditions for 24 hours. IPDI isocyanate trimer Desmodur Z4470 BA resin (2.96 g) and DBTDAc catalyst solution (0.25 g) were added to the vial, and the mixture was stirred for another hour before application to the substrate. To prepare an additive-modified polyurethane formulation, for example F25, acrylic polyol (8.00 g; 50% solid), acetylacetone (0.62 g) (potlife extender), and the 10kPDMS-750PEG additive (4.18 g; 60% solid) were added to a vial and stirred under ambient conditions for 24 hours. IPDI isocyanate trimer Desmodur Z4470 BA resin (2.96 g) and DBTDAs catalyst solution (0.25 g) were added to the vial, and the mixture was stirred for another hour before application to the substrate. Coating formulations were applied on primed 8″×4″ aluminum and 6″×3″ steel panels using a wire-round drawdown bar with a film thickness of 80 μm. All coatings were allowed to cure under ambient conditions for 24 hours, followed by oven curing at 80° C. for 45 minutes. Coatings were cut out in circular shapes and glued to 24-well plates for biological assays test. 3.3.5 Results and Discussion Amphiphilic coatings have been recognized as a promising path to address biofouling issues. While several amphiphilic systems having been extensively investigated, there is a lack of knowledge about these systems that ranges from the mechanism of performance to design parameters. To this effect, it was determined whether there is a threshold concentration of amphiphilic moieties where a system attains desirable FR performance and then additional amounts of such moieties do not further improve the performance. The AmpAdd additive based on 10,000MnPDMS and 750Mnwas added at increasing amounts to a polyurethane coating system (developed internally with IPDI isocyanate trimer and acrylic polyol) and the relationship between the concentration of the AmpAdd and FR performance was established accordingly. The amphiphilic additive, AmpAdd, was prepared by reacting mono-hydroxyl-terminated PEG (750Mn) and PDMS (10,000Mn) with an IPDI isocyanate trimer resin. The complete conversion of the isocyanate groups to urethane linkage was confirmed with FTIR and isocyanate titrations. An FTIR spectrum of the AmpAdd (FIG.16) shows the absence of the isocyanate peak at 2250 cm−1and stretching for secondary amine of the formed urethane linkage at 3350 cm−1. Additionally, the appearance of overlapping peaks for PDMS (Si—O—Si) at 1030 cm−1and PEG (C—O—C) at 1105 cm−1confirmed the attachment of amphiphilic chains on the additive. Furthermore, isocyanate titrations validated the complete conversion of the isocyanate groups since the titrations indicated the absence of isocyanate. A series of coatings was then made where the AmpAdd was incorporated into a polyurethane coating at a range of concentrations as indicated in Table 8. Surface characterization of the coatings was completed with ATR-FTIR, contact angle measurements, XPS, and AFM. ATR-FTIR was used to assess the presence of the chemical moieties on the surfaces of the coatings. Although the spectra for all the modified PU coatings were generally similar, the only differences observed were changes to the intensities of peaks associated with PEG at 1030 cm−1and PDMS at 1105 cm−1(FIG.17—red and green highlights, respectively) with respect to the C—O—C peak of the urethane linkage (FIG.17—yellow highlight). Also, an overlapped broad stretching peak for hydroxyl group (due to urethane linkage from the AmpAdd and crosslinking reaction) is present at ca. 3350 cm−1. These data indicate that both PDMS and PEG are present at the PU surfaces containing the AmpAdd and are thus amphiphilic, while the unmodified PU lacks this property. Overall, as more amphiphilic additive was present in a system, the intensities for PEG and PDMS peaks increased accordingly, signaling a direct correlation between the availability of amphiphilic moieties on the surfaces with the amount of additive. Contact angle measurements were utilized as another method to characterize the surfaces. The contact angle data were collected as static measurements over time and dynamic measurements using a tilting stage. The additives resulted in a dynamic surface, meaning the contact angles for both water and diiodomethane decreased as a function of time (FIG.18A). This dynamic nature is attributed to the amphiphilicity of the surfaces where the hydrophilic domains cause the water droplet to spread as they swell. The observed dynamic behavior for the additively modified PU coatings was similar to the T-10 amphiphilic control coating, while the hydrophobic A4 system did not possess such a feature (due to lack of hydrophilic domains). The change in values was more prominent for water contact angles (WCA) than diiodomethane contact angles (MICA). However, the extent of changes in contact angle values was similar regardless of the amount of additive. Additionally, as the amount of AmpAdd was increased for the modified PU coatings, the initial water contact angle decreased until a plateau was observed for coatings with 25 wt. % or a higher amount of the additive (formulations F25, F30, F40). This trend is attributed to the increasing amount of hydrophilic moieties on the surface due to AmpAdd. When the concentration of these moieties on the surface became saturated, additional amounts of AmpAdd did not further impact the surface, displaying a leveling trend. Also, the contact angles of modified PU coatings were generally lower than that of the control coatings, which can be related to the addition of AmpAdd. Surface energy for the experimental and control coatings was calculated using WCA and MICA values (FIG.18B). The surface energy values for modified PU coatings were between 40-45 mN/m initially and increased as a function of time, showing a dynamic nature similar to contact angle values. Mostly, the greatest change was observed for coatings with higher amounts of AmpAdd (coatings F25 and F40). The surface energy values for the modified coatings were different from the control coatings at 25-30 mN/m. The slip angle (water droplet roll-off angle) for the studied coatings showed a declining trend as the amount of the AmpAdd increased for the systems (FIG.18C). Similar to the WCA values, the slip angle becomes relatively constant once it reaches a 25 wt. % concentration of AmpAdd. In comparison, the hydrophobic A4 showed a considerably higher slip angle while the amphiphilic T-10 displayed a value within the range of the assessed coatings. Furthermore, the tilting experiment provided advancing contact angle (Adv CA) and receding contact angle (Rec CA) values, and a trend similar to slip angle was observed (FIG.18D). The hysteresis (difference between Adv CA and Rec CA) was higher for coatings F10 and F20 than coatings with higher amounts of AmpAdd in their composition (coatings F25, F30, F40). The lower the hysteresis, the smoother a surface is, and typically this increases the ease of “roll off” from its surface. Relating these results to the control coatings, the A4 system showed a hysteresis similar to the systems with lower amounts of AmpAdd (i.e., F20) and the T-10 showed a similar value to the higher AmpAdd-containing systems (i.e., F30). Contact angle measurements for the coatings after 28 days of water aging increased, which was relatable to the T-10 control system (FIG.19). This change was attributed to rearrangement of the surface as hydrophilic and hydrophobic domains interacted with water and to the probability that some amount of AmpAdd may have leached out. XPS was utilized to quantify the elemental compositions of materials on the surface and as a function of depth of the coatings. As expected, the results showed that the AmpAdd additive self-stratified onto the surface, so that there was a higher concentration of Si than C on the surface, while this trend was reversed throughout the bulk of a coating (FIG.20). The XPS depth profiling analysis suggests that the concentration of the amphiphilic moieties on the surface was directly related to the amount of incorporated AmpAdd. The initial Si concentration was higher (C concentration was lower) as the amount of AmpAdd was increased (except for F10). The data indicated that the concentration of Si rapidly declined as a function of thickness for the F10 coating and plateaued at ˜2%. A less drastic decreasing trend was also noticed for coatings F20, F25, and F30, and all these systems eventually leveled at a Si concentration around 5-6%. However, the decreasing trend was not observed for coating F40, indicating the concentration of Si atoms was almost uniform until the assessed thickness of 36 nm (FIG.20B). The XPS data for the C atom showed an increasing trend for coatings F10, F20, F25, and F30 (FIG.20A), in accord with the decreasing Si atom trend for each system. The increasing C atom trend was not observed for coating F40, which correlated with the unchanging Si atom concentration of this formulation. As expected, the unmodified PU system showed a uniform concentration of the C atom throughout the coating while there was no Si in its composition. The XPS data confirms that the amount of AmpAdd has a direct effect on the composition of the system both at the surface and in the bulk of the coating. AFM was employed to study the morphology of the developed surfaces. The general notation is that soft materials like PDMS appear lighter (high phase angles) and harder materials like PEG appear darker (low phase angles). The AmpAdd-modified PU coatings displayed heterogeneous surfaces in both height and phase AFM images that were composed of light and dark patterns, implying the formation of a complex amphiphilic morphology (FIG.21). The unmodified PU system exhibited a uniform homogenous surface (free of patterns) that was relatively similar to the hydrophobic A4 system (since it has solely PDMS on the surface) (FIG.22). As the AmpAdd was incorporated into the PU system, the presence of spherical micro-domains on the surface was observed. The AmpAdd-modified coatings displayed a surface that was relatable to the morphology of control T-10 amphiphilic coating (FIG.22)—this system uses the same molecular weights of PEG (750Mn) and PDMS (10,000Mn) that are used for the synthesized AmpAdd additive, but instead the PEG and PDMS chains are covalently bound into the coating system. The area of these surface domains increased as the concentration of AmpAdd in a formulation increased from 10 wt. % (F10) to 20 wt. % (F20) and 25 wt. % (F25). The F30 formulation (containing 30 wt. % AmpAdd) exhibited a very similar morphology to F25, but many smaller domains were seen among the micro-domains. Coating F40 showed domains that were larger in comparison to F25 and F30, which may be due to the saturated surface by AmpAdd (note that capturing AFM images for the F40 coating was more challenging than other systems due to its increased slippery nature and limitations of the instrument). The AFM images support the evidence from ATR-FTIR and XPS that the AmpAdd self-stratified into the surface. Furthermore, the increasing trend of the amount of the observed heterogeneous domains is in direct correlation with the incorporated amount of AmpAdd; the higher the additive amount, the higher area coverage of domains on the surface. The AFM images for coatings were taken after water immersion. Overall, the coatings experienced a slight decrease in number of the domains on their surface. This change is noticeable inFIG.23, exhibiting the AFM images for F25 coating after and before water immersion. The AFM images indicate that the AmpAdd rearranges on the surface as it is not crosslinked into the system, and this observation corresponds with increased water contact angle values after the water immersion period. Biological assays were conducted to evaluate FR properties of the studied coatings using a range of marine fouling organisms. All the assessments were carried out after 28 days of water leaching to ensure that toxic impurities did not interfere with the results. The coatings were evaluated for leachate toxicity usingC. lytica, N. incerta, andU. linzaas described elsewhere (Cassé et al.,Biofouling2007, 23 (2), 121-130; Majumdar et al.,ACS Comb. Sci.2011, 13 (3), 298-309) prior to any FR experiments. All the coatings were non-toxic, opening the way for biological assessments. U. linzais a known biofouling macroalga species. The algal spores produced byU. linzaexplore surfaces searching for areas that are most favorable for attachment. They respond to a number of cues including surface chemistry, wettability and roughness. In general, the spores tend to settle at lower densities on hydrophilic than on hydrophobic surfaces, but when presented with amphiphilic coatings the ambiguous nature of the surfaces can delay settlement and therefore also result in lower settlement densities. Finlay et al.,Integr. Comp. Biol.2002, 42 (6), 1116-1122; Callow et al.,Appl. Environ. Microbiol.2000, 66 (8), 3249-3254; Callow et al.,J. R. Soc. Interface2005, 2 (4), 319-325. Settlement across the range of additive containing coatings was broadly similar which probably reflects the narrow range of surface energies and the fact that unsettled spores were not removed from the system. After settlement, the spores germinate and develop into sporelings (young plants) on the surfaces of the coatings. To assess FR potential the biofouled surfaces were water-jetted at two pressure levels—10 psi and 16 psi—and the biomass remaining was determined (FIG.24A—blue bars 10 psi; green bars 16 psi). Adhesion strength is generally stronger on moderately hydrophilic surfaces, such as the polyurethane standard coating (PU) and the polyurethane base coating (F0), as can be seen in the results (FIG.24A). For the additive containing coatings the release trend was similar at both water pressures. At 16 psi, the introduction of 10 wt. % AmpAdd (4 wt. % PEG and PDMS each) and 20 wt. % of AmpAdd (8 wt. % PDMS and PEG each) improved the release ofU. linzaalmost two times that of the unmodified PU system (F0 coating) (comparison P-values<0.05, Tukey's method). The release was improved further by adding 25 wt. % (10 wt. % PEG and PDMS each) of AmpAdd, however, the addition of further amounts of AmpAdd did not result in further Ulva removal (FIG.24B) and/or less biomass remaining (FIG.24A). At 10 psi, the release in relation to the amount of AmpAdd followed a similar trend; however, the critical concentration of AmpAdd needed to be at 30 wt. % (12 wt. % PDMS and PEG each) to offer the optimum performance. These findings are on par with other studies where an activity threshold was also identified for FR activity against sporelings ofU. linzain coatings containing amphiphilic diblock copolymers of PDMS and PEGylated-fluoroalkyl polystyrene blocks. Martinelli et al.,Biofouling2011, 27 (5), 529-541. The activity similarly increased with the weight content of the block copolymer and was related to surface segregation. Fluorine-free amphiphilic block copolymers containing PDMS and PEG also showed a marked threshold for FR activity against sporelings ofU. linza. Sundaram et al.,Biofouling2011, 27 (6), 589-602. These coatings reconstructed on immersion in water bringing the PEG chains to the surface which correlated with the performance against sporelings. The AmpAdd-modified coatings F25, F30, and F40 outperformed all the internal controls and commercial standards, and the release data forU. linzasuggests that a critical amphiphilic concentration (CAC) is required to optimize performance. This CAC for optimal release ofU. linzawas approximately 10-12 wt. % PEG and PDMS each (25-30 wt. % AmpAdd) as illustrated inFIG.24. C. lyticais another biofouling organism that is recognized for its affinity to settle on a variety of surfaces that range from hydrophilic to hydrophobic. Lejars et al.,Chem. Rev.2012, 112 (8), 4347-4390 The extent of biofouling among the studied and control systems varied greatly (FIG.25A—red bars). Overall, experimental coatings F0, F30, and F40 and control coatings A4 and T-10 showed the lowestC. lyticabiofouling, while commercial controls such as IS 700, IS 900, and IS 1100 showed the highest amount ofC. lyticabiofouling. The FR experiments were carried out at two pressure levels and the biomass ofC. lyticaremaining was reported at 10 psi (FIG.25A—blue bars) and 20 psi (FIG.25A—green bars). Generally, the release ofC. lyticafilm was higher at 20 psi than 10 psi resulting in a lower amount of biomass remaining on the surface, but the trends were alike between the two pressure levels (FIG.25A). At 20 psi, an amount of AmpAdd additive between 10 wt. % to 25 wt. % resulted in improved release ofC. lyticain contrast to the unmodified system (comparison P-values<0.05, Tukey's method), but the extent of biomass remaining was almost the same regardless of the amount of additive within this range (comparison P-values>0.05, Tukey's method). Once the amount of AmpAdd additive in a system reached 30 wt. % for F30 coating (12 wt. % PEG and PDMS each), the release ofC. lyticasignificantly improved over the observed performance for the F10, F20, and F25 systems (comparison P-values<0.05, Tukey's method). However, the addition of more AmpAdd in F40 system (40 wt. %; 17 wt. % PEG and PDMS each) did not enhance the release or percent removal and showed a similar performance to F30 (FIG.25) (comparison P-values>0.05, Tukey's method). At 10 psi, the addition of the AmpAdd additive did not result in better FR performance up to 25 wt. % of AmpAdd, thus coatings F10, F20, and F25 displayedC. lyticarelease that was comparable to the unmodified F0 system. However, once the amount of AmpAdd reached 30 wt. % and higher, it showed an improved release for theC. lyticafilm. Coatings F30 and F40 were compared with both internal controls and commercial coatings as these two demonstrated the best results among the AmpAdd-modified coatings. The data presented inFIG.25Ashows that F30 and F40 coatings outperformed both the internal and commercial systems either significantly or marginally (FIG.25A). As forU. linza, the FR data ofC. lyticasuggests that a critical amphiphilic concentration (CAC) was needed to deliver the minimum biomass remaining (FIG.25A) and/or maximum release/percent removal (FIG.25B) until a plateaued performance was observed. This CAC forC. lyticawas at 12 wt. % PEG and PDMS each (30 wt. % AmpAdd), being in the range of the CAC forU. linza. N. incertais well-known as another major biofouling organism that adheres more strongly to hydrophobic surfaces. Finlay et al.,Integrative and Comparative Biology2002, 42 (6), 1116-1122; Callow et al.,Applied Environmental Microbiology2000, 66 (8), 3249-3254. The extent ofN. incertabiofouling varied among the studied coatings, internal controls, and commercial controls (FIG.26A—red bars). Overall, coatings T2, PU and IS 700 showed the highest biofouling; commercial IS 900 and IS 1100 SR showed the lowest amount of biofouling; and the studied additively modified PU coatings showed intermediate biofouling forN. incerta. The release of formedN. incertabiofilm was evaluated at two pressure levels and the remaining biofilm ofN. incertawas assessed at 10 psi (FIG.26A—blue bars) and 20 psi (FIG.26A—green bars). The remaining biomass of theN. incertafilms was noticeably lower at 20 psi than 10 psi. Even though the extent of release/removal was different due to the water pressure levels used, the observed trends at both levels were similar among the additively modified PU coatings. Generally, the addition of AmpAdd clearly improved release of theN. incertabiofilm (FIG.26A). At 20 psi pressure level, the removal of the film improved until 25 wt. % (10 wt. % PEG and PDMS each) of the AmpAdd was added into the system, comparing F25, F20, and F10 systems with unmodified F0 formulation (FIG.26A) (comparison P-values<0.05, Tukey's method). The further addition of additive after this point resulted in a plateau and negligible further improvement per observed performance of F30 and F40 systems where 30 wt. % and 40 wt. % of the amphiphilic additive was incorporated, respectively (comparison P-values>0.05, Tukey's method). A similar trend was noticed at the 10 psi pressure level, except the plateau point was determined to be at 30 wt. % of AmpAdd (12 wt. % PEG and PDMS each). Coating systems F25, F30 and F40 that contained 25 wt. % or more additives released theN. incertafilms better than internal controls (A4 and T-10), indicating the effect of amphiphilic concentration at the surface on release performance (comparison P-values<0.05, Tukey's method). The only two coatings that outperformed the AmpAdd-modified PU systems were IS 900 and IS 1100. TheN. incertarelease data followed a similar trend to those obtained forU. linzaandC. lytica, in that a critical CAC needs to be met for attaining the maximum FR performance in a plateaued region (FIG.26B). This CAC forN. incertawas at 10-12 wt. % PEG and PDMS each (25-30 wt. % AmpAdd), on par with the CAC forU. linzaandC. lyticamarine organisms. Mechanical tests were performed on the PU coatings to assess their integrity as the amount of AmpAdd increased in the system. The properties started to drop at 50 wt. % of the AmpAdd; thus, 40 wt. % concentration of AmpAdd was marked as the highest limit before properties declined (Table 10). Coatings showed desirable stability against MEK and salt water double rubs until 40 wt. % of AmpAdd. Additionally, the additive did not impact the performance of the coating in response to the rapid deformation impact test. The conical mandrel bend test showed that the additive did not affect the flexibility of the coatings. Although it was expected that the long PEG and PDMS chains would contribute to better flexibility, it did not occur because these moieties were mostly present on the surface of a coating rather than throughout the bulk due to their self-stratification. The adhesion of the coatings to the substrate remained consistent and unchanged until 40 wt. % of AmpAdd. Generally, the introduction of the AmpAdd was not detrimental to the PU system until 40 wt. %. Thus, coatings with 40 wt. % or less of AmpAdd were selected to be investigated for this study. TABLE 10Results of mechanical tests on unmodified and modified PU CoatingsMEK DoubleWater DoubleForwardReverseConicalRub (Numberrubs (NumberImpactImpactMandrelCrosshatchFormulationof rubs)of rubs)(in-lb)(in-lb)(mm)AdhesionF0>400>4006412904BF10>400>40068161005BF20>400>4007212905BF25>400>40068121004BF30>400>40068161205BF40380>40076201305BF5029232076201303B 3.3.6 Conclusions The results showed that amphiphilic moieties are able migrate to the surface of a coating and modify it until a point of saturation, and then additional surface-active agents do not change the surface or impact the FR performance. This point of surface saturation is the critical amphiphilic concentration (CAC). This behavior shares similarities with what occurs when surfactants are added to a liquid. In this case, the surfactant reduces the surface tension until the interface is saturated and then additional surfactant does not change the surface tension, but forms micelles, with the concentration at which this occurs being known as the critical micelle concentration. Dominguez et al.,J. Chem. Educ.1997, 74 (10), 1227; Van Oss et al., Colloids Surf. A-Colloids and Surfaces A: Physicochem. Eng. Asp. 1993, 78, 1-49. This work explored the effect of incorporating an amphiphilic additive into a polyurethane coating system. The amphiphilic additive was made by attaching hydroxyl-terminated PEG and PDMS chains on a polyisocyanate. Amphiphilic coating systems are being widely investigated as marine coatings, but there continues to be a lack of knowledge about these recently developed systems. Thus, it was determined at what concentration of amphiphilicity a conventional PU system develops FR properties. Generally, as the amount of the amphiphilic additive in the PU coating increased, the surface of the coating system became more amphiphilic. The FR data of the coatings against all biological assays (C. lytica, U. linza, andN. incerta) demonstrated that the systems performed best when a specific amount of amphiphilicity was present in a composition (a performance comparable or better than both internal controls and commercial coatings). The amount of amphiphilicity that resulted in the desired performance towards all marine organisms was between 10-12 wt. % PEG and PDMS each (25-30 wt. % AmpAdd), and a further amount of AmpAdd above this concentration did not boost the FR performance. Surface characterization provided further insight into these surfaces as well. ATR-FTIR showed the presence of an amphiphilic surface. Contact angle measurements indicated that the amphiphilic concentration had a direct impact on the surface considering that coatings with 25 wt. % AmpAdd or higher were more dynamic, possessed lower slip-off angles, and displayed the lowest contact angle hysteresis (difference between advancing and receding contact angles). XPS showed that the AmpAdd self-stratified onto the surfaces, and the presence of the amphiphilic moieties on the surface was directly correlated to the amount of AmpAdd in a system. XPS data indicated that for coatings containing 25 wt. % or higher AmpAdd, the additives were well distributed on the surface and extended to a higher thickness within the bulk of the coating. AFM images clearly showed the presence of heterogenous micro-sized domains after AmpAdd was introduced to the PU system, and the sum of domains increased as the amount of AmpAdd increased in a formulation. Once AmpAdd was introduced at 25 wt. % or higher amounts in a formulation, the surfaces were saturated by these spherical micro-domains. Mechanical integrity of the coatings was assessed too, and it was determined that the coatings maintained their integrity until 40 wt. % of AmpAdd was added. Overall, the FR data and surface characterizations go hand in hand. Both suggest that at a critical amphiphilic concentration there are noticeable changes in contact angles, surface morphology, and extent of removal of the biological films. The critical amphiphilic concentration (CAC) that resulted in the maximum FR performance was between 10-12 wt. % PEG and PDMS each (25-30 wt. % AmpAdd). This invention opens the door for an unexplored area of marine coating technology; namely, to better understand amphiphilicity to improve the design of FR coatings. 3.4 Example 4 3.4.1 Design of Experiment A series of amphiphilic additives (AmpAdd) containing chains of PDMS and PEG were synthesized and added into an amphiphilic siloxane-polyurethane coating system, called AmpSiPU. The AmpSiPU is a system that is formulated with IPDI trimer polyisocyanate, amphiphilic PEG-PDMS-isocyanate prepolymers, and acrylic polyol. The selected AmpSiPU formulation is called R0 in this example, containing 10 wt. % of 10,000MnPDMS and 10 wt. % of 750MnPEG as self-stratifying crosslinked prepolymers; this formulation was selected as it performed the best in the results published elsewhere. Galhenage et al.,J. Coating. Tech. Res.2017, 14 (2), 307-322. AmpAdds were prepared by installing PEG and PDMS chains on IPDI trimer polyisocyanate resin. The ratio of isocyanate groups to the combined OH groups of PEG and PDMS was a 1:1 molar ratio. To attain types of AmpAdds (Table 11), PEG and PDMS were used in varying molecular weights and amounts to complete the required one molar hydroxyl ratio. The molecular weights of 5,000Mnand 10,000Mnfor PDMS, and 550Mnand 750Mnfor PEG were chosen to synthesize amphiphilic additives in accordance with optimal chain lengths for FR performance. Galhenage et al.,J. Coating. Tech. Res.2017, 14 (2), 307-322; Bodkhe et al.,Prog. Org. Coat.2012, 75 (1-2), 38-48. Also, the amount of attached PEG and PDMS on an additive was another variable to access new AmpAdds. For example, “50:50 PDMS: PEG” for Amp1 additive means that both PEG and PDMS were added in equal weight (i.e., 2 g PEG and 2 g PDMS) to synthesize the additive. It should be noted that there was a wt. % for IPDI polyisocyanate resin as part of the additive structure too since the amphiphilic chains were grafted on its backbone. Therefore, the wt. % values of PEG and PDMS on an additive could be calculated and were used to determine the final content of PEG and PDMS moieties in a formulation (Table 11). TABLE 11List of prepared additives and their compositional detailsIPDIPDMS TypePEG TypePDMS:PEGPDMSPEGisocyanateAdditive(Mn)(Mn)(% ratio)(wt. %)(wt. %)(wt. %)Amp-15,00075050:50424216Amp-25,00055050:50424216Amp-310,00075050:50424216Amp-410,00055050:50424216Amp-510,00075033:66156123Amp-610,00075010:9086725 Knowing that the R0 coating of AmpSiPU contains up to 10 wt. % each PDMS and PEG, these additives were added at 15 wt. %, 10 wt. % and 20 wt. %. A total of 15 formulations were investigated: 8 experimental (Table 12) and 7 controls (both internal and commercial) (Table 13). For the experimental systems, the first four formulations (R1-R4) were designed as a 22experimental design to evaluate two factors for the designed AmpAdds including the molecular weight of PDMS (5,000Mnand 10,000Mn) and molecular weight of PEG (550Mnand 750Mn), while keeping the amphiphilic balance unchanged (amount of hydrophilic and hydrophobic moieties in a system). Formulations R5 and R6 were considered to evaluate the effect of shifting the amphiphilic balance towards more hydrophilicity via AmpAdds, comparing these formulations with R3 and R0. Formulations R7 and R8 were considered to evaluate the effect of the amount of an AmpAdd, comparing these formulations with R3 and R0. The table outlines type of the added AmpAdd, amount of the added AmpAdd, wt. % of PEG and PDMS based on the added AmpAdd, and overall wt. % of PEG and PDMS in a formulation (including 10 wt. % of each PDMS and PEG in R0 base formulation). TABLE 12Coating CompositionsFormulationAdded AmpAdd DetailsDetailsAdditiveAdditive AmountPDMSPEGPDMSPEGFormulationType(wt. %)(wt. %)(wt. %)(wt. %)(wt. %)R0————10.010.0R1Amp-115.06.26.216.216.2R2Amp-215.06.26.216.216.2R3Amp-315.06.26.216.216.2R4Amp-415.06.26.216.216.2R5Amp-515.05.010.015.020.0R6Amp-615.01.513.511.523.5R7Amp-320.08.38.318.318.3R8Amp-310.04.14.114.114.1 3.4.2 Control and Standard Coatings Commercial standards were prepared following the manufacturer's instructions. A4 SiPU coating, internal hydrophobic control, containing 20,000MnPDMS as crosslinker was prepared following directions elsewhere; this formulation was selected since it showed the best performance among the studied systems. Bodkhe et al.,J. Coating. Tech. Res.2012, 9 (3), 235-249. Similar to experimental coatings all control and standards were also prepared on 4″×8″ primed aluminum panels and multi-well plates. Table 13 contains detailed descriptions of the control and standard coatings used for this study. TABLE 13List of Control CoatingsControl NameControl IDDescriptionA4 SiPUA4Internal Hydrophobic SiPU ControlPolyurethanePUPure Polyurethane StandardPolystyrenePSPure Polystyrene Standard(used forU. linzatest)Dow T2T2Silicone Elastomer StandardIntersleek ® 700IS 700Intersleek Commercial FR StandardIntersleek ® 900IS 900Intersleek Commercial FR StandardIntersleek ® 1100 SRIS 1100Intersleek Commercial FR Standard 3.4.3 Synthesis of Amphiphilic Additive The AmpAdd additives were produced via reacting hydroxyl-terminated PEG and PDMS chains with the IPDI trimer polyisocyanate (Scheme 1 above). The molar ratio of NCO groups to the combined OH groups of PEG and PDMS was 1:1. The functional isocyanate groups were fully converted to urethane linkages by reacting with PEG and PDMS chains. PEG and PDMS were added in weight ratios that met the required molar ratio. To synthesize Amp-2 (containing weight ratio of PDMS: PEG 50:50), PEG 550Mn(8.00 g) was diluted in toluene (8.00 g) in a 50-mL flask. PDMS 5,000Mn(8.00 g) was added to the flask and mixed robustly with vortex for 2 minutes. IPDI trimer resin (6.32 g) and DBTDAc catalyst solution (1% by wt. in MAK) (1.12 g) were then added to the flask. The reaction was carried out at 80° C. for 2 hours. The reaction could also be carried out at ambient conditions for 24 hours. The flask was equipped with a magnetic stirrer, nitrogen inlet, and temperature controller. The reflux condenser was used when the heat was applied. The synthesized Amp-2 additive contained 42.0 wt. % PEG and 42.0 wt. % PDMS (Table 11), calculated based on solid contents utilized to synthesize the additive and the final solid content. All other AmpAdds were synthesized following the same procedure. 3.4.4 Synthesis of the Curable Coating Compositions and Their Curing A synthesized AmpAdd was added to R0 (an AmpSiPU formulation). The R0 system is an amphiphilic marine FR coating that mainly composed of acrylic polyol, IPDI trimer isocyanate resin, and amphiphilic PEG-PDMS-isocyanate prepolymers. For example, to formulate coating R2, acrylic polyol (12.24 g; 50% solid), acetylacetone (1.74 g) (potlife extender), amphiphilic PEG-PDMS-isocyanate prepolymer (5.10 g; 70% solid), and Amp-2 additive (3.73 g; 50% solid) were added to a vial and stirred ambiently for 24 hours. IPDI polyisocyanate trimer Desmodur Z4470 BA resin (2.95 g) and DBTDAs catalyst solution (0.28 g) were added to the vial, and the mixture was stirred for another hour. Coating formulations were drawn down on primed 8′×4′ aluminum panels using a wire-round drawdown bar with a film thickness of 80 μm. All coatings were cured at ambient laboratory conditions for 24 hours, followed by oven curing at 80° C. for 45 minutes. All experimental coatings were prepared following the same procedure. Coatings were cut out in circular shapes and glued to 24-well plates for biological assays test. Furthermore, two internal model PU systems without amphiphilic prepolymers were prepared using the same materials and following the same procedure (except no PEG-PDMS-isocyanate prepolymers were added). One system was the unmodified model PU and the other one was modified model PU with Amp3 additive is its composition. These two model PU systems were used to substantiate the self-stratification of AmpAdd additives into the surface of coatings via X-ray photoelectron spectroscopy (XPS) experiments (these controls avoided interference of PDMS signals of the prepolymer in R0 and PDMS signals of an added AmpAdd). 3.4.5 Results and Discussion Amphiphilic FR coatings have shown promising results to mitigate marine biofouling. These systems offer better performance than traditional hydrophobic FR coatings. As discussed above, when a critical amphiphilic concentration (CAC) is achieved for a system, its marine performance improves considerably. Thus, this concept was utilized to further investigate how increasing the amphiphilicity for an established amphiphilic marine coating using additives affects its surface and FR properties. Amphiphilic siloxane-polyurethane (AmpSiPU) was selected to be modified by additives in this example, specifically R0 formulation containing 10 wt. % 10,000MnPDMS and 10 wt. % 750MnPEG (a top system in another study). Galhenage et al.,J. Coating. Tech. Res.2017, 14 (2), 307-322. A series of amphiphilic additives were introduced to R0 to evaluate their effect on this system. The amphiphilic additives (AmpAdds) were synthesized by attaching hydroxyl-terminated PEG and PDMS chains on IPDI isocyanate trimer resin through the facile reaction of isocyanate and alcohol. A dried toluene solvent was used to ensure water did not react with isocyanate during the reaction. The complete conversion of isocyanate groups due to the reaction was confirmed with FTIR and isocyanate titration of the additives. All FTIR spectrum displayed disappearance of isocyanate peak at 2250 cm−1and a broadened signal for secondary amine (from the formed urethane linkage) at 3350 cm−1(FIG.27), supporting the reaction of isocyanate groups. Also, the signature peaks for Si—O—Si at 1035 cm−1(FIG.27—Green highlights) and C—O—C of PEG at 1105 cm−1(FIG.27—Red highlights) demonstrate the reaction of PDMS and PEG chains onto the isocyanate-based backbone, respectively. The intensity of the C—O—C peak increased, and the intensity of the Si—O—Si signal decreased as a higher amount of PEG and a lower amount of PDMS was attached to an additive, respectively (FIG.27A; comparing from bottom to top). Also, 750MnPEGs appear to show stronger peaks (Amp-1 and Amp-3 additives) than 550MnPEGs (Amp-2 and Amp-4 additives) in comparison with their neighboring Si—O—Si signals (FIG.27B). Overall, the FTIR data qualitatively indicates the amount of PEG and PDMS and their incorporated molecular weights (MW) results in a different additive. Isocyanate titrations were carried out on additives, and the results implied the presence of no remaining isocyanate groups. The surfaces of unmodified and modified AmpSiPU R0 coatings were analyzed with a series of techniques including ATR-FTIR, contact angle measurements, and AFM. Also, XPS was used to validate the self-stratification of amphiphilic additives into a surface for model PU coatings. ATR-FTIR of modified coatings showed the signals for both C—O—C (FIG.28—Green highlights) and Si—O—Si (FIG.28—Red highlights) with higher intensity than the unmodified R0 coating. Like additives of different weight ratios of PEG and PDMS, the intensity of ether and siloxane peaks for the coatings followed a similar trend; the C—O—C signal increased as an additive with higher PEG content was used (FIG.28A; comparing from bottom to top). The intensity of PEG and PDMS peaks also gradually increased as a higher amount of amphiphilic additives was introduced (FIG.28B; comparing from bottom to top). The ATR-FTIR data suggests amphiphilic additives self-stratified into the surface, comparing PEG and PDMS signals of modified and unmodified R0 coatings. Contact angle measurements, both static and dynamic, were conducted to assess surface properties of coatings. The static contact angles monitored water and methylene iodide droplets on a surface as a function of time up to 9 minutes (values plateaued at this point). The unmodified R0 AmpSiPU coating shows a dynamic surface, meaning the contact angle values change over time (i.e., droplets spread). In comparison to R0, the initial water contact angle (WCA) and methylene iodide contact angle (MICA) values for all AmpAdd-modified coatings decreased, and the change over time for contact angles was more noticeable than R0 (FIG.29A). The dynamic change of contact angles (specially WCA) over time is attributed to the addition of AmpAdds, resulting in a higher density of PEG chains on the surface that facilitates spreading a water droplet. The non-dynamic behavior of hydrophobic A4 (WCA or MICA does not change) due to its PDMS rich surface further reconfirms the surfaces of R0 and its modified versions are amphiphilic. The static data indicates the degree of changes among AmpAdd-modified formulations vary slightly (FIG.29A), suggesting the MW of PEG and PDMS or amount of additive in a system does not remarkably impact the dynamic behavior for a system. Surface energies (SE) of coatings were calculated using WCAs and MICAS values (FIG.29B); SEs are typically higher when WCA and MICA values are lower than 90°. The data shows SE for R0 jumps significantly after the addition of amphiphilic additives, increasing from 27-30 mN/m to a 40-70 mN/m. The SE for hydrophobic A4 is similar to control R0, suggesting R0 is more hydrophobic on the surface than its modified versions. Dynamic contact angle experiments were carried out using a tilting stage to determine slip angle (water droplet roll-off angle) and advancing/receding contact angles. The AmpAdds decreased the slip angle of the R0 coating by almost 50%, from nearly 6° to 3°-3.5° (FIG.29C). The decrease of the roll-off angle implies that AmpAdds contribute to the easier removal of objects from the surface of R0. There was no significant difference between roll-off angles of modified R0 coatings. In comparison, the hydrophobic A4 system had a slip angle at 9°, implying amphiphilic surfaces improved roll-off performance. Advancing contact angles (Adv CA) and receding contact angles (Rec CA) were remarkably lower for modified R0 coatings (except R7 which held relatively higher values) than both the unmodified R0 and hydrophobic A4 systems (FIG.29D). There was no major difference between CA values due to MW of PEG and PDMS or amphiphilicity balance. The hysteresis (numerical difference between Adv CA and Rec CA) remained unchanged and less than 10° for unmodified and modified R0 coatings, demonstrating that the additives did not roughen the surface of R0 system. WCAs were recorded for coatings after water immersion, and the results indicated the modified R0 coatings were as stable as control systems including unmodified R0 and SiPU A4 (FIG.30). Overall, WCAs slightly increased for all systems to a relatively equal extent. The changes are associated with several indicators such as rearrangement of surface domains due to interaction with water. AFM was used to characterize the surface morphologies of the studied coatings. AFM phase images typically show lighter appearance (high phase angle) for soft materials like PDMS while dark appearance (low phase angle) for harder materials like PEG. AFM images for R0 AmpSiPU (FIG.31—R0;FIG.32—R0) and A4 SiPU (FIG.33) control systems display a microdomain-containing heterogeneous surface and a microdomain-free homogenous surface, respectively, indicating the amphiphilic R0 system possesses a patterned surface (due to its PEG-PDMS prepolymers). Galhenage et al.,J. Coating. Tech. Res.2017, 14 (2), 307-322. The addition of AmpAdds into R0 further modified its heterogenous surface. The data indicates that the MW of PDMS impacts the surface morphology of R0, while MW of PEG does not affect it necessarily—systems with 5kMnPDMS (R1 and R2) contain smaller domains than systems with 10 kMnPDMS (R3 and R4). The introduction of AmpAdds with higher contents of PEG (Amp-5 and Amp-6) retained a comparable morphology to R3 and R4 coatings, indicating no major changes (except the images were harder to capture). The amount of additive did alter the morphology of R0, comparing Amp-3 at 10 wt. % (R8), 15 wt. % (R3), and 20 wt. % (R7). The comparison implies surface of R7 was highly saturated where domains were merged and very near to each other, while R3 and R8 showed fewer domains on their surfaces in the order mentioned. As a sum, AmpAdds appeared to result in the surface domains being more organized and more narrowly dispersed in size. Additionally, AFM images were recorded for coatings after 28-days of water immersion (FIG.34). The comparison of images before and after water-immersion demonstrated the surface domains were stable, slight rearrangements occurred, and no major depletion/leaching of domains was observed. The post-immersion rearrangement of domains may be the reason that contact angle data slightly changed as well (FIG.32). Overall, the AFM images demonstrated that amphiphilic additives modified the surfaces and several factors influence the change such as the MW of moieties or the amount of additive. The AFM images correlate with ATR-FTIR and contact angle data that AmpAdds self-stratified into surfaces and caused a dynamic interaction with assessed droplets. XPS was utilized to confirm the self-stratification of the amphiphilic additives into the surface of R0. XPS depth analysis was initially conducted on modified and unmodified R0 coatings as a function of coating thickness up to 30 nm, but the data was not conclusive due to difficulties in differentiating between PEG and PDMS of R0 itself and incorporated additives. Therefore, an internal model PU system was used to validate the self-stratification of AmpAdds. The model PU system was similar to the R0 AmpSiPU system but without amphiphilic PEG-PDMS-based prepolymers, and for confirmation purposes, Amp-3 was added to the model PU system at 20 wt. %. XPS depth analysis of unmodified model PU and AmpAdd-modified model PU supported the theory that amphiphilic additives self-stratify into a surface (FIG.35). The unmodified model PU showed a constant concentration of carbon (FIG.35A) and nitrogen (FIG.35B) throughout the evaluated 30 nm depth, indicating a relatively homogenous network by composition. Comparatively, the modified model PU system showed the surface contained silicon which its concentration gradually decreased as a function of depth (FIG.35B) while the concentration of nitrogen and carbon atoms increased (FIG.35). The depth-dependent concentrations of Si, C, and N supported the self-stratification of amphiphilic additives to the surface, correlating with data from ATR-FTIR, contact angle analysis, and AFM. To assess the FR performance of the coatings, biological assays using four representative marine fouling organisms were carried out withU. linza, C. lytica, N. incerta, and barnacles. All assessments were carried out after 28 days of water aging. All coatings were non-toxic as they were tested through leachate toxicity ofC. lytica, N. incerta, andU. linza, following the procedure described elsewhere. Casse et al.,Biofouling2007, 23 (2), 121-130; Majumdar et al.,ACS Comb. Sci.2011, 13 (3), 298-309. U. linzais a macroalgae that prefers hydrophobic surfaces for settlement more than hydrophilic surfaces. Finlay et al.,Integr. Comp. Biol.2002, 42 (6), 1116-1122; Callow et al.,Appl. Environ. Microbiol.2000, 66 (8), 3249-3254; Callow et al.,J. R. Soc. Interface2005, 2 (4), 319-325. The extent of biofouling among all coatings (both exemplified and control) was almost equal, and no significant difference was observed. The biofouled surfaces were water-jetted at 10 psi and 16 psi to assess their FR performance under hydrodynamic pressure. The extent ofU. linzaremoval (percent removal) was higher at 16 psi (FIG.36—Green bars) than 10 psi (FIG.36—Blue bars), but the overall trend of removal was similar at both pressure levels. Among coatings R1-R4, the MW of PDMS and PEG caused negligible differences in FR performance—Amp-3 composed of 10 kMnPDMS and 750MnPEG was relatively the best-performing additive. Comparing R3, R7, and R8 systems, it is suggested that the amount of added AmpAdd did not have a major impact on FR properties (though Amp-3 at 15 wt. % showed the relative best performance). Alternatively, the data implied that the degree of additive hydrophilicity influenced the FR performance, where system R6 with the most hydrophilic additive (Amp-6) outperformed R5 and R3 with Amp-5 and Amp-3 additives, respectively. Not only did the AmpAdds improve performance of the R0 coating in respect to the A4 system, some of AmpAdds such as Amp-3, Amp-5, and Amp-6 also pushed FR of R0 system to be on par with well-performing materials such as T2 and IS 1100. Conclusively, the amphiphilic additives improved FR performance of R0 againstU. linzaas the comparison between unmodified and modified R0 systems demonstrated. The FR results ofU. linzaare in correlation with literature that the hydrophilic surfaces weaken the adhesion of this organism. C. lyticais a micro-biofouling bacterium that can settle on both hydrophobic and hydrophilic surfaces, challenging the traditional hydrophobic FR surfaces. Thus, amphiphilic surfaces are considered as a feasible solution to fight against biofouling ofC. lytica. Lejars et al.,Chem. Rev.2012, 112 (8), 4347-4390; Galhenage et al.,J. Coating. Tech. Res.2017, 14 (2), 307-322. The extent of biofouling among all coatings were relatively similar except for IS 700, IS 900, and IS 1100 that showed almost twiceC. lyticabiofouling, and it was observed AmpAdds did not result in an increased initial film formation. The FR performance of surfaces was evaluated at two water pressure levels, 10 psi and 20 psi. The biomass remaining ofC. lyticaafter 10 psi (FIG.37—Blue bars) and 20 psi (FIG.37—Green bars) water jetting showed that 10 psi water pressure level released lessC. lyticathan the 20 psi but the overall trend for both pressures was similar. Coatings R1 with Amp-1 and R7 and R8 with Amp-3 (at 20 wt. % and 10 wt. %, respectively) displayed better FR performance than unmodified R0 and control A4 systems, while the remaining modified counterparts were comparable to R0 and A4. Additionally, the three systems (R1, R7, and R8) pushed the performance of R0 to be on par with IS 900 and IS 1100, allowing for the least amount ofC. lyticato remain settled on their surfaces after water jetting. While the removal is greater for IS 900 and IS 1100 systems due to their very high initial biofouling, it should be noted the AmpAdd-modified R0 coatings experienced less initial biofouling. Coating R6 delaminated and could not be tested, which was attributed to its most hydrophilic matrix swollen under water. Overall, AmpAdds did not have a detrimental effect on FR performance of R0; the additives either improved the FR performance or did not alter it. N. incertais a micro-biofoulant that settles preferably on hydrophobic surfaces. Finlay et al.,Integr. Comp. Biol.2002, 42 (6), 1116-1122; Callow et al.,Appl. Environ. Microbiol.2000, 66 (8), 3249-3254. The extent of biofouling settlement was alike among all studied and control coatings, and no remarkable change was noticed due to the addition of AmpAdds. Biofouled surfaces withN. incertawere water-jetted at 10 psi and 20 psi pressured levels. The higher water pressure level released more organisms (FIG.38—Green bars) than the lower pressure level (FIG.38—Blue bars), while the overall trends remained alike. The FR data indicates that AmpAdds improved the performance of the R0 base system by almost 60% at both pressure levels. However, the release performance was very similar among the modified R0 coatings, resulting in no substantial conclusion regarding the effect of MW of PEG and PDMS, the extent of additive hydrophilicity, and the amount of additive in a system. All the AmpAdd-modified R0 systems remarkably outperformed several internal and commercial controls including A4, PU, and IS 700. Additionally, several systems including R2, R5, R7, and R8 showed matching performance in respect to the top-performing commercial systems such as IS 900 and 1100 SR. Coating R6 delaminated and could not be tested, which was attributed to its highly hydrophilic matrix swollen underwater (this was the most hydrophilic formulation). The FR data ofN. incertasuggests that the designed AmpAdds impart better performance to the base AmpSiPU R0 coating, indicating that a higher degree of amphiphilicity for this system is beneficial; however, it could not be concluded which particular AmpAdd was better over another. Barnacles, a macrofoulant, is another known marine organism that is infamous for its detrimental biofouling effects. Callow et al., Biologist 2002, 49 (1), 10-14; Aldred et al.,Biofouling2010, 26 (6), 673-683. Different species of barnacle settle on different surfaces, for example,Amphibalanus amphitritesettles both hydrophilic and hydrophobic surfaces. Stafslien et al.,J. Coating. Tech. Res.2012, 9 (6), 651-665; Rittschof et al.,Biofouling2008, 24 (1), 1-9; Huggett et al.,Biofouling2009, 25 (5), 387-399; Rittschof et al.,Sci. Mar.1989, 53 (2), 411-416. Consequently, it is challenging to propose a universal rule predicting their behavior. Lejars et al.,Chem. Rev.2012, 112 (8), 4347-4390; Petrone et al.,Biofouling2011, 27 (9), 1043-1055; Di Fino et al.,Biofouling2014, 30 (2), 143-152; Gatley-Montross et al.,Biointerphases2017, 12 (5), 051003; Aldred et al.,Biofouling2019, 35 (2), 159-172. AmpAdds resulted in both beneficial and detrimental effects in terms of the adhesion strength of the reattached barnacles (FIG.39). For example, in respect to the unmodified R0 system, Amp-2 at 15 wt. % (R2 formulation) and Amp-3 at 20 wt. % (R7 formulation) reduced the adhesion strength of barnacles, while Amp-3 at 15 wt. % (R3 formulation) increased the adhesion strength of the barnacles. These two additives (Amp-3 and Amp-3) increased the overall amphiphilicity balance of the R0 system equally, meaning the wt. % of PEG and PDMS in solid contents were same. However, when AmpAdds with varying hydrophilic-hydrophobic balance shifted the systems to be more hydrophilic (increasing from R0 to R5 to R6), the adhesion strength of barnacles slightly increased, which may be due to affinity ofA. Amphitriteto more hydrophilic surfaces in this case. Overall, this data suggested the amount of an additive or its design parameters influences the performance of a system, which can be either favorable or unfavorable. 3.4.6 Conclusions A novel series of amphiphilic additives by attaching PEG and PDMS chains on a polyisocyanate resin via the facile isocyanate and alcohol reaction, allowing for the easy synthesis of amphiphilic additives having varied molecular weights of amphiphilic chains and tunable hydrophilic-hydrophobic balance, are described herein. The introduction of amphiphilic additives to an amphiphilic marine coating system, known as the amphiphilic siloxane-polyurethane (AmpSiPU), was beneficial overall, boosting the performance of this system to tackle marine biofoulants better. The surface characterizations confirmed that the amphiphilic additives modified the surface of the control amphiphilic coatings. Contact angle measurements displayed modified AmpSiPU systems were more dynamic in their interaction with water and methylene iodide droplets in respect to the control AmpSiPU coating. ATR-FTIR confirmed the presence of PEG and PDMS moieties on the surfaces that were attributed to amphiphilic additives. In respect to the base AmpSiPU system, modified surfaces exhibited highly saturated surfaces containing heterogenous microdomains under AFM. Additionally, the XPS experiment on model PU systems confirmed that the additives self-stratified to the surfaces. Biological assays demonstrated that amphiphilic additives improved the FR performance of the base AmpSiPU system. Overall, the PEG-PDMS additives boosted the performance of AmpSiPU system againstU. linzaandN. incertaand advanced it slightly againstC. lytica, while they presented both advantageous and hampering effects against barnacles. The results indicated systems where the hydrophilic balance was slightly more than hydrophobic offered a more desirable performance against some organisms such asU. linzaandN. incerta. | 117,471 |
11859100 | DETAILED DESCRIPTION Definitions The term “polymer” includes, but is not limited to, homopolymers, copolymers, for example, block, random, and alternating copolymers, terpolymers, quaterpolymers, etc., and blends and modifications thereof. Furthermore, unless otherwise specifically limited, the term “polymer” shall include all possible configurational isomers of the material. These configurations include, but are not limited to isotactic, syndiotactic, and atactic symmetries. A polymer is a molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units (i.e. repeating units) derived, actually or conceptually, from molecules of low relative mass (i.e. monomers). Typically, the number of repeating units is higher than 10, preferably higher than 20, in polymers. If the number of repeating units is less than 10, the polymers may also be referred to as oligomers. The term “monomer” as used herein, refers to a molecule which can undergo polymerization thereby contributing constitutional units (repeating units) to the essential structure of a polymer. The term “homopolymer” as used herein, stands for a polymer derived from one species of (real, implicit or hypothetical) monomer. The term “copolymer” as used herein, generally means any polymer derived from more than one species of monomer, wherein the polymer contains more than one species of corresponding repeating unit. In one embodiment the copolymer is the reaction product of two or more species of monomer and thus comprises two or more species of corresponding repeating unit. It is preferred that the copolymer comprises two, three, four, five or six species of repeating unit. Copolymers that are obtained by copolymerization of three monomer species can also be referred to as terpolymers. Copolymers that are obtained by copolymerization of four monomer species can also be referred to as quaterpolymers. Copolymers may be present as block, random, and/or alternating copolymers. The term “block copolymer” as used herein, stands for a copolymer, wherein adjacent blocks are constitutionally different, i.e. adjacent blocks comprise repeating units derived from different species of monomer or from the same species of monomer but with a different composition or sequence distribution of repeating units. Further, the term “random copolymer” as used herein, refers to a polymer formed of macromolecules in which the probability of finding a given repeating unit at any given site in the chain is independent of the nature of the adjacent repeating units. Usually, in a random copolymer, the sequence distribution of repeating units follows Bernoullian statistics. The term “alternating copolymer” as used herein, stands for a copolymer consisting of macromolecules comprising two species of repeating units in alternating sequence. The term “polysilazane” as used herein, refers to a polymer in which silicon and nitrogen atoms alternate to form the basic backbone. Since each silicon atom is bound to at least one nitrogen atom and each nitrogen atom to at least one silicon atom, both chains and rings of the general formula —[SiR1R2—NR3-]m(silazane repeating unit) occur, wherein R1to R3may be hydrogen atoms, organic substituents or heteroorganic substituents; and m is an integer. If all substituents R1to R3are hydrogen atoms, the polymer is designated as perhydropolysilazane, polyperhydrosilazane or inorganic polysilazane (—[SiH2—NH—]m). If at least one substituent R1to R3is an organic or heteroorganic substituent, the polymer is designated as organopolysilazane. The term “polysiloxazane” as used herein, refers to a polysilazane which additionally contains sections in which silicon and oxygen atoms alternate. Such sections may be represented, for example, by —[O—SiR7R8—]n, wherein R7and R8may be hydrogen atoms, organic substituents, or heteroorganic substituents; and n is an integer. If all substituents of the polymer are hydrogen atoms, the polymer is designated as perhydropolysiloxazane. If at least one substituents of the polymer is an organic or heteroorganic substituent, the polymer is designated as organopolysiloxazane. The term “functional coating” as used herein refers to coatings which impart one or more specific properties to a surface. Generally, coatings are needed to protect surfaces or impart specific effects to surfaces. There are various effects which may be imparted by functional coatings. For example, mechanical resistance, surface hardness, scratch resistance, abrasion resistance, anti-microbial effect, anti-fouling effect, wetting effect (towards water), hydro- and oleophobicity, smoothening effect, durability effect, antistatic effect, anti-staining effect, anti-fingerprint effect, easy-to-clean effect, anti-graffiti effect, chemical resistance, corrosion resistance, anti-oxidation effect, physical barrier effect, sealing effect, heat resistance, fire resistance, low shrinkage, UV-barrier effect, light fastness, and/or optical effects. The term “cure” means conversion to a crosslinked polymer network (for example, through irradiation or catalysis). The term “aromatic unit” as used herein, relates to a monocyclic or polycyclic aromatic or heteroaromatic system which forms part of the molecular structure of a chemical compound. Polycyclic aromatic systems include two or more connected aromatic ring systems which are fixed in one plane. Heteroaromatic systems contain one or more heteroatoms selected from N, O, S and P. The aromatic unit may be unsubstituted or substituted, preferably with one or more substituents selected from hydroxyl, alkyl, phenyl and fluorine. The aromatic unit is typically linked via one or more chemical bonds to adjacent structural moieties of the chemical compound. The person skilled in the art is familiar with the terms and concepts “aromatic” and “non-aromatic”. The term “aryl” as used herein, means a mono-, bi- or tricyclic aromatic or heteroaromatic group which may be substituted. Heteroaromatic groups contain one or more heteroatoms (e.g. N, O, S and/or P) in the heteroaromatic system. Preferred Embodiments The present invention relates to a coating composition, comprising: (i) a silazane-containing polymer; and (ii) a non-polymeric phenolic compound; wherein the non-polymeric phenolic compound comprises at least two and not more than four aromatic units, preferably two, three or four aromatic units, in its molecular structure. Non-Polymeric Phenolic Compound The non-polymeric phenolic compound is a low molecular weight compound having phenolic hydroxyl groups (i.e. hydroxyl groups bound to an aromatic moiety). Some of the non-polymeric phenolic compounds may be regarded as a condensation product of a phenol component and formaldehyde. However, the compounds do not represent polymers, but low molecular weight compounds. The non-polymeric phenolic compound comprises at least two and not more than four aromatic units, preferably two, three or four aromatic units, in its molecular structure. It is preferred that at least two of the aromatic units in the non-polymeric phenolic compound have in each case at least one hydroxyl group directly attached thereto. It is further preferred that the aromatic units are six-membered aromatic ring systems, which may be substituted or unsubstituted. It is more preferred that the aromatic units are phenyl systems, which may be substituted or unsubstituted. Preferred substituents for said aromatic units are selected from hydroxyl, alkyl, phenyl and fluorine groups. If a phenyl group is present as a substituent on an aromatic unit, the phenyl group likewise represents a further independent aromatic unit. In any case, it needs to be ensured that the total number of aromatic units in the non-polymeric phenolic compound does not exceed four. It is preferred that the non-polymeric phenolic compound has a molecular weight in the range from 180 to 1,000 g/mol, preferably from 220 to 750 g/mol, more preferably from 240 to 600 g/mol, and most preferably from 250 to 480 g/mol. It is further preferred that the non-polymeric phenolic compound contains 12 to 56 (preferably 12 to 32) carbon atoms, 2 to 8 (preferably 2 to 6) oxygen atoms and 10 to 32 hydrogen atoms. In a preferred embodiment of the present invention, the non-polymeric phenolic compound is represented by one of the following Formulae (I), (II) and (III): wherein in Formula (I): X represents —CO—, —SO2—, —O— or a linear or branched alkylene group having 1 to 5 carbon atoms, which may be fluorinated, wherein the alkylene group is preferably selected from —CH2—, —CF2—, —CH(CH3)—, —CF(CF3)—, —C(CH3)2—, —C(CF3)2—, —C(CH3)(CH2CH3)—, —C(CF3)(CF2CF3)—, —C(CH2CH3)2— or —C(CF2CF3)2—, or X is absent; R1represents at each occurrence independently from each other hydroxyl, alkyl having 1 to 5 carbon atoms, preferably methyl, ethyl, propyl, butyl or pentyl, or phenyl; k is an integer from 0 to 4, preferably 0, 1 or 2; and p is an integer from 1 to 3, preferably 1, 2 or 3, more preferably 1 or 2; wherein in Formula (II): Y represents a saturated or unsaturated hydrocarbyl moiety, which may be fluorinated and/or may contain one or more hydroxyl groups, wherein Y may be linked to one or more of the adjacent aromatic units independently from each other via a second bond to form a bi- or multicyclic system; RIIrepresents at each occurrence independently from each other hydroxyl, alkyl having 1 to 5 carbon atoms, preferably methyl, ethyl, propyl, butyl or pentyl, or phenyl; m is at each occurrence independently from each other an integer from 1 to 5, preferably 1, 2 or 3; n is at each occurrence independently from each other an integer from 0 to 4, preferably 0, 1 or 2; and q is an integer from 2 to 4, preferably 2, 3 or 4; and wherein in Formula (III): RIIIrepresents at each occurrence independently from each other alkyl having 1 to 5 carbon atoms, preferably methyl, ethyl, propyl, butyl or pentyl; s is an integer from 0 to 3, preferably 0, 1, 2 or 3; and r is 3 or 4, preferably 4. The hydrocarbyl moiety in Formula (II) forms the central part (backbone) of the non-polymeric phenolic compound around which q of the individual substituted aromatic units, which are shown in parentheses ( ), are arranged. The hydrocarbyl moiety is composed of carbon and hydrogen atoms and may be substituted with one or more fluorine atoms and/or hydroxyl groups. In this case, there is then a fluorinated and/or hydroxylated hydrocarbyl moiety. The hydrocarbyl moiety may have a linear, branched, cyclic and/or polycyclic structure. The hydrocarbyl moiety may be saturated or unsaturated. In addition to C—C single bonds, unsaturated hydrocarbyl moieties may contain one or more C═C double bonds and/or one or more C≡C triple bonds. The C═C double bonds may be isolated, conjugated and/or cumulated. The C≡C triple bonds may be isolated and/or conjugated. If the hydrocarbyl moiety contains a plurality of C═C double bonds which together form one or more aromatic units within the hydrocarbyl moiety, these aromatic units are taken into account for the total number or aromatic units to be present in the non-polymeric phenolic compound and the upper limit of q is reduced, correspondingly, so that the total number of aromatic units in the molecular structure of the non-polymeric phenolic compound does not exceed the maximum number. The compounds according to Formula (III) are cyclic compounds containing 3 or 4 repeating units shown in parentheses. The cyclic structure of the compounds is illustrated by In a more preferred embodiment of the present invention, the non-polymeric phenolic compound of Formula (I) is represented by Formula (I-A) or (I-B): wherein X represents —CO—, —SO2—, —O—, or a linear or branched alkylene group having 1 to 5 carbon atoms, which may be fluorinated, wherein the alkylene group is preferably selected from —CH2—, —CF2—, —CH(CH3)—, —CF(CF3)—, —C(CH3)2—, —C(CF3)2—, —C(CH3)(CH2CH3)—, —C(CF3)(CF2CF3)—, —C(CH2CH3)2— and —C(CF2CF3)2—; RIrepresents at each occurrence independently from each other hydroxyl, alkyl having 1 to 5 carbon atoms, preferably methyl, ethyl, propyl, butyl or pentyl, or phenyl; and k is an integer from 0 to 4, preferably 0, 1 or 2. In a more preferred embodiment of the present invention, the non-polymeric phenolic compound of Formula (II) is represented by Formula (II-A), (II-B), (II-C), (II-D), (II-E), (II-F) or (II-G): wherein in Formula (II-A): RII=Me or Ph, preferably Ph; RIIA=Ph, —C6H4—OH, —CH2—C6H4—OH or —CH2CH2—C6H4—OH; or both RIIAtogether form a fluorenylene group or cyclohexylene group, which may be substituted with —OH or —C6H4—OH; and n=0 or 1; wherein in Formula (II-B): RIIB=H or Me; RIIB′=-Ph, —C6H4—OH or —C6H4—C(CH3)2—C6H4—OH; and n=0 or 1; wherein in Formula (II-C): RIICrepresents alkyl having 1 to 3 carbon atoms, preferably methyl, ethyl or propyl; n=0 or 1; r=0, 1 or 2; and s=0, 1, 2 or 3; wherein in Formula (II-D): RII=Me or Ph, preferably Me; RIIDrepresents alkyl having 1 to 3 carbon atoms, preferably methyl, ethyl or propyl; Sp represents a linear or branched alkylene group having 1 to 5 carbon atoms, preferably —CH2—, —C(CH3)2—, —CH2—CH2— or —CH2—CH2—CH2—; n=0 or 1; t=0 or 1; and u=0 or 1; wherein in Formula (II-E): RIIE=H or Me; and v=0, 1, 2 or 3; wherein in Formula (II-F): RIIF=H or Me; and m=1, 2 or 3; and wherein in Formula (II-G): RIIG=H or Me; and m=1, 2 or 3. In a more preferred embodiment of the present invention, the non-polymeric phenolic compound of Formula (III) is represented by Formula (III-A) or (III-B): wherein RIIIrepresents at each occurrence independently from each other alkyl having 1 to 5 carbon atoms, preferably methyl, ethyl, propyl, butyl or pentyl. In a most preferred embodiment of the present invention, the non-polymeric phenolic compound is selected from: Preferably, the total content of the non-polymeric phenolic compound in the coating composition is in the range from 10 to 90 weight-%, preferably from 15 to 75 weight-%, based on the total weight of silazane-containing polymer in the coating composition. Silazane-Containing Polymer In a preferred embodiment, the silazane-containing polymer comprises a repeating unit M1which is represented by the following Formula (1): —[SiR1R2—NR3—] (1) wherein R1, R2and R3are the same or different from each other and independently selected from hydrogen, an organic group, a heteroorganic group, or a combination thereof. Suitable organic and heteroorganic groups for R1, R2and R3include alkyl, alkylcarbonyl, alkenyl, cycloalkyl, aryl, arylalkyl, alkylsilyl, alkylsilyloxy, arylsilyl, arylsilyloxy, alkylamino, arylamino, alkoxy, alkoxycarbonyl, alkylcarbonyloxy, aryloxy, aryloxycarbonyl, arylcarbonyloxy, arylalkyloxy, and the like, and combinations thereof (preferably, alkyl, alkenyl, cycloalkyl, aryl, arylalkyl, alkoxy, aryloxy, arylalkyloxy, and combinations thereof); the groups preferably having from 1 to 30 carbon atoms (more preferably, 1 to 20 carbon atoms; even more preferably, 1 to 10 carbon atoms; most preferably, 1 to 6 carbon atoms (for example, methyl, ethyl or vinyl)). The groups can be further substituted with one or more substituent groups such as halogen (fluorine, chlorine, bromine, and iodine), alkoxy, alkoxycarbonyl, amino, carboxyl, hydroxyl, nitro, and the like, and combinations thereof. In a preferred embodiment, R1and R2are the same or different from each other and independently selected from hydrogen, alkyl having 1 to 30 (preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 6) carbon atoms, alkenyl having 2 to 30 (preferably 2 to 20, more preferably 2 to 10, most preferably 2 to 6) carbon atoms, or aryl having 2 to 30 (preferably 3 to 20, more preferably 4 to 10, most preferably 6) carbon atoms, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by fluorine; and R3is selected from hydrogen, alkyl having 1 to 30 (preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 6) carbon atoms, alkenyl having 2 to 30 (preferably 2 to 20, more preferably 2 to 10, most preferably 2 to 6) carbon atoms, or aryl having 2 to 30 (preferably 3 to 20, more preferably 4 to 10, most preferably 6) carbon atoms, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by fluorine or OR′, wherein R′ is selected from alkyl having 1 to 30 (preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 6) carbon atoms. In a more preferred embodiment, R1and R2are the same or different from each other and independently selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl or phenyl, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by fluorine; and R3is selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, vinyl or phenyl, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by —F, —OCH3, —OCH2CH3, —OCH2CH2CH3, or —OCH(CH3)2. Most preferably, R1, R2and R3are the same or different from each other and independently selected from the list consisting of —H, —CH3, —CH2CH3, —CH2CH2CH3, —CH(CH3)2, —CH═CH2, and —C6H5, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by fluorine. In a preferred embodiment, the silazane-containing polymer comprises a repeating unit M2which is represented by the following Formula (2): —[SiR4R5—NR6—] (2) wherein R4, R5and R6are the same or different from each other and independently selected from hydrogen, an organic group, a heteroorganic group, or a combination thereof. Suitable organic and heteroorganic groups for R4, R5and R6include alkyl, alkylcarbonyl, alkenyl, cycloalkyl, aryl, arylalkyl, alkylsilyl, alkylsilyloxy, arylsilyl, arylsilyloxy, alkylamino, arylamino, alkoxy, alkoxycarbonyl, alkylcarbonyloxy, aryloxy, aryloxycarbonyl, arylcarbonyloxy, arylalkyloxy, and the like, and combinations thereof (preferably, alkyl, alkenyl, cycloalkyl, aryl, arylalkyl, alkoxy, aryloxy, arylalkyloxy, and combinations thereof); the groups preferably having from 1 to 30 carbon atoms (more preferably, 1 to 20 carbon atoms; even more preferably, 1 to 10 carbon atoms; most preferably, 1 to 6 carbon atoms (for example, methyl, ethyl or vinyl)). The groups can be further substituted with one or more substituent groups such as halogen (fluorine, chlorine, bromine, and iodine), alkoxy, alkoxycarbonyl, amino, carboxyl, hydroxyl, nitro, and the like, and combinations thereof. In a preferred embodiment, R4and R5are the same or different from each other and independently selected from hydrogen, alkyl having 1 to 30 (preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 6) carbon atoms, alkenyl having 2 to 30 (preferably 2 to 20, more preferably 2 to 10, most preferably 2 to 6) carbon atoms, or aryl having 2 to 30 (preferably 3 to 20, more preferably 4 to 10, most preferably 6) carbon atoms, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by fluorine; and R6is selected from hydrogen, alkyl having 1 to 30 (preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 6) carbon atoms, alkenyl having 2 to 30 (preferably 2 to 20, more preferably 2 to 10, most preferably 2 to 6) carbon atoms, or aryl having 2 to 30 (preferably 3 to 20, more preferably 4 to 10, most preferably 6) carbon atoms, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by fluorine or OR″, wherein R″ is selected from alkyl having 1 to 30 (preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 6) carbon atoms. In a more preferred embodiment, R4and R5are the same or different from each other and independently selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl or phenyl, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by fluorine; and R6is selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, vinyl or phenyl, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by —F, —OCH3, —OCH2CH3, —OCH2CH2CH3, or —OCH(CH3)2. Most preferably, R4, R5and R6are the same or different from each other and independently selected from the list consisting of —H, —CH3, —CH2CH3, —CH2CH2CH3, —CH(CH3)2, —CH═CH2, and —C6H5, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by fluorine. In a preferred embodiment, the silazane-containing polymer comprises a repeating unit M3which is represented by the following Formula (3): —[SiR7R8—O—] (3) wherein R7and R8are the same or different from each other and independently selected from hydrogen, an organic group, a heteroorganic group, or a combination thereof. Suitable organic and heteroorganic groups for R7and R8include alkyl, alkylcarbonyl, alkenyl, cycloalkyl, aryl, arylalkyl, alkylsilyl, alkylsilyloxy, arylsilyl, arylsilyloxy, alkylamino, arylamino, alkoxy, alkoxycarbonyl, alkylcarbonyloxy, aryloxy, aryloxycarbonyl, arylcarbonyloxy, arylalkyloxy, and the like, and combinations thereof (preferably, alkyl, alkenyl, cycloalkyl, aryl, arylalkyl, alkoxy, aryloxy, arylalkyloxy, and combinations thereof); the groups preferably having from 1 to 30 carbon atoms (more preferably, 1 to 20 carbon atoms; even more preferably, 1 to 10 carbon atoms; most preferably, 1 to 6 carbon atoms (for example, methyl, ethyl or vinyl)). The groups can be further substituted with one or more substituent groups such as halogen (fluorine, chlorine, bromine, and iodine), alkoxy, alkoxycarbonyl, amino, carboxyl, hydroxyl, nitro, and the like, and combinations thereof. In a preferred embodiment, R7and R8are the same or different from each other and independently selected from hydrogen, alkyl having 1 to 30 (preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 6) carbon atoms, alkenyl having 2 to 30 (preferably 2 to 20, more preferably 2 to 10, most preferably 2 to 6) carbon atoms, or aryl having 2 to 30 (preferably 3 to 20, more preferably 4 to 10, most preferably 6) carbon atoms, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by fluorine. In a more preferred embodiment, R7and R8are the same or different from each other and independently selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl or phenyl, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by fluorine. Most preferably, R7and R8are the same or different from each other and independently selected from the list consisting of —H, —CH3, —CH2CH3, —CH2CH2CH3, —CH(CH3)2, —CH═CH2, and —C6H5, wherein one or more hydrogen atoms bonded to carbon atoms may be replaced by fluorine. It is preferred that the silazane-containing polymer comprises a repeating unit M1and a further repeating unit M2, wherein M1and M2are silazane repeating units which are different from each other. It is also preferred that the silazane-containing polymer comprises a repeating unit M1and a further repeating unit M3, wherein M1is a silazane repeating unit and M3is a siloxane repeating unit. It is also preferred that the silazane-containing polymer comprises a repeating unit M1, a further repeating unit M2and a further repeating unit M3, wherein M1and M2are silazane repeating units which are different from each other and M3is a siloxane repeating unit. In one embodiment, the silazane-containing polymer is a polysilazane which may be a perhydropolysilazane or an organopolysilazane. Preferably, the polysilazane contains a repeating unit M1and optionally a further repeating unit M2, wherein M1and M2are silazane repeating units which are different from each other. In an alternative embodiment, the silazane-containing polymer is a polysiloxazane which may be a perhydropolysiloxazane or an organopolysiloxazane. Preferably, the polysiloxazane contains a repeating unit M1and a further repeating unit M3, wherein M1is a silazane repeating unit and M3is a siloxane repeating unit. Preferably, the polysiloxazane contains a repeating unit M1, a further repeating unit M2and a further repeating unit M3, wherein M1and M2are silazane repeating units which are different from each other and M3is a siloxane repeating unit. Preferably, the silazane-containing polymer is a copolymer such as a random copolymer or a block copolymer or a copolymer containing at least one random sequence section and at least one block sequence section. More preferably, the silazane-containing polymer is a random copolymer or a block copolymer. It is preferred that the silazane-containing polymers used in the present invention do not have a monocyclic structure. More preferably, the silazane-containing polymers have a mixed polycyclic, linear and/or branched-chain structure. The silazane-containing polymers have a molecular weight distribution. Preferably, the silazane-containing polymers used in the present invention have a mass average molecular weight Mw, as determined by GPC, of at least 1,000 g/mol, more preferably of at least 1,200 g/mol, even more preferably of at least 1,500 g/mol. Preferably, the mass average molecular weight Mwof the silazane-containing polymers is less than 100,000 g/mol. More preferably, the molecular weight Mwof the silazane-containing polymers is in the range from 1,500 to 50,000 g/mol. Preferably, the total content of the silazane-containing polymer in the coating composition is in the range from 10 to 90 weight-%, preferably from 25 to 85 weight-%, based on the total weight of the coating composition. Further Components It is preferred that the coating composition of the present invention comprises one or more solvents. Suitable solvents are organic solvents such as, for example, aliphatic and/or aromatic hydrocarbons, which may be halogenated, such as 1-chloro-4-(trifluoromethyl)benzene, esters such as ethyl acetate, n-butyl acetate or tert-butyl acetate, ketones such as acetone or methyl ethyl ketone, ethers such as tetrahydrofuran or dibutyl ether, and also mono- or polyalkylene glycol dialkyl ethers (glymes), or mixtures thereof. Moreover, the coating composition according to the present invention may comprise one or more additives, preferably selected from the list consisting of additives influencing evaporation behavior, additives influencing film formation, adhesion promoters, anti-corrosion additives, cross-linking agents, dispersants, fillers, functional pigments (e.g. for providing functional effects such as electric or thermal conductivity, magnetic properties, etc.), nanoparticles, optical pigments (e.g. for providing optical effects such as color, refractive index, pearlescent effect, etc.), particles reducing thermal expansion, primers, rheological modifiers (e.g. thickeners), surfactants (e.g. wetting and leveling agents or additives for improving hydro- or oleophobicity and anti-graffiti effects), viscosity modifiers, and other kinds of resins or polymers. Nanoparticles may be selected from nitrides, titanates, diamond, oxides, sulfides, sulfites, sulfates, silicates and carbides which may be optionally surface-modified with a capping agent. Preferably, nanoparticles are materials having a particle diameter of <100 nm, more preferably <80 nm, even more preferably <60 nm, even more preferably <40 nm, and most preferably <20 nm. The particle diameter may be determined by any standard method known to the skilled person. It is possible to accelerate the curing of the coating composition by the addition of one or more catalysts. Examples of useful catalysts are Lewis acids such as boron-, aluminum-, tin- or zinc-alkyls, aryls or carboxylates, Brönsted acids such as carboxylic acids, bases such as primary, secondary or tertiary amines or phosphazenes, or metal salts such as Pd, Pt, Al, B, Sn or Zn salts of carboxylates, acetylacetonates or alkoxylates. If Silazanes having both Si—H and Si—CH═CH2groups are used, well known hydrosilylation catalysts such as Pt or Pd salts or complexes can be used. If Silazanes having only Si—CH═CH2or both Si—H and Si—CH═CH2groups are used, UV or thermal radical initiators like peroxides or azo compounds can be used. In a preferred embodiment, the coating composition according to the present invention comprises one or more of the above-mentioned catalysts. It is preferred that the mass ratio between the silazane-containing polymer and the non-polymeric phenolic compound in the coating composition of the present invention is in the range from 1:100 to 100:1, preferably from 1:50 to 50:1, more preferably from 1:10 to 10:1, even more preferably from 1:8 to 8:1, and most preferably from 1:3 to 6:1. It is to be understood that the skilled person can freely combine the above-mentioned preferred, more preferred, particularly preferred and most preferred embodiments relating to the coating composition and definitions of its components in any desired way. Method The present invention further relates to a method for preparing a coated article, wherein the method comprises the following steps:(a) applying a coating composition according to the present invention to a surface of an article; and(b) curing said coating composition to obtain a coated article. In a preferred embodiment, the coating composition, which is applied in step (a), is previously provided by mixing a first component comprising a silazane-containing polymer with a second component comprising a non-polymeric phenolic compound, wherein the silazane-containing polymer and the non-polymeric phenolic compound are defined as indicated above for the coating composition according to the present invention. It is preferred that the mixing of the first component with the second component takes place at an elevated temperature, preferably at a temperature between 60 and 150° C., more preferably at a temperature between 80 and 140° C. and most preferably at a temperature between 100 and 130° C. Preferably, the coating composition, which is applied in step (a), is a homogeneous liquid having a viscosity in the range from 2 to 1,000 mPas. The viscosity of the composition may be adjusted by the type and content of solvent as well as the type, ratio and molecular weight of the silazane-containing polymer and non-polymeric phenolic compound. It is preferred that the coating composition is applied in step (a) by an application method suitable for applying liquid compositions to a surface of an article. Such methods include, for example, wiping with a cloth, wiping with a sponge, dip coating, spray coating, flow coating, roller coating, slit coating, slot coating, spin coating, dispensing, screen printing, stencil printing or ink-jet printing. Dip coating and spray coating are particularly preferred. The coating composition of the invention may be applied to the surface of various articles such as, for example, buildings, dentures, furnishings, furniture, sanitary equipment (toilets, sinks, bathtubs, etc.), signs, signboard, plastic products, glass products, ceramics products, metal products, wood products and vehicles (road vehicles, rail vehicles, watercrafts and aircrafts). It is preferred that the surface of the article is made of any one of the base materials as described for the use below. Typically, the coating composition is applied in step (a) as a layer in a thickness of 1 μm to 1 cm, preferably 10 μm to 1 mm, to the surface of the article. In a preferred embodiment, the coating composition is applied as a thin layer having a thickness of 1 to 200 μm, more preferably 5 to 150 μm and most preferably 10 to 100 μm. In an alternative preferred embodiment, the coating composition is applied as a thick layer having a thickness of 200 μm to 1 cm, more preferably 200 μm to 5 mm and most preferably 200 μm to 1 mm. The curing of the coating in step (b) may be carried out under various conditions such as e.g. by ambient curing, thermal curing and/or irradiation curing. The curing is optionally carried out in the presence of moisture, preferably in the form of water vapor. For this purpose, a climate chamber may be used. Ambient curing preferably takes place at temperatures in the range from 10 to 40° C. Thermal curing preferably takes place at temperatures in the range from 100 to 200° C., preferably from 120 to 180° C. Irradiation curing preferably takes place with IR irradiation or UV irradiation. Preferred IR irradiation wavelengths are in the range from 7 to 15 μm or from 1 to 3 μm for substrate absorption. Preferred UV irradiation wavelengths are in the range from 200 to 300 nm (short wavelength range). Preferably, the curing in step (b) is carried out in a furnace or climate chamber. Alternatively, if articles of very large size are coated (e.g. buildings, vehicles, etc.), the curing is preferably carried out under ambient conditions. Preferably, the curing time for step (b) is from 0.01 to 24 h, more preferably from 0.10 to 16 h, still more preferably from 0.15 to 8 h, and most preferably from 0.20 to 5 h, depending on the coating composition and coating thickness. After curing in step (b), the silazane-containing polymer and the non-polymeric phenolic compound are chemically linked to form a coating on the surface of the article. The coating obtained by the above method forms a rigid and dense functional coating which is excellent in adhesion to the surface and imparts at least one of the following improved properties to the article: improved mechanical resistance and durability (including improved surface hardness, improved scratch resistance and/or improved abrasion resistance); improved wetting and adhesion properties (including hydro- and oleophobicity, easy-to-clean effect and/or anti-graffiti effect); improved chemical resistance (including improved corrosion resistance (e.g. against solvents, acidic and alkaline media and corrosive gases) and/or improved anti-oxidation effect); improved optical effects (improved light fastness); and improved physical barrier or sealing effects. Article Moreover, a coated article is provided, which is obtainable or obtained by the above-mentioned preparation method. Use The present invention further relates to the use of the coating composition according to the present invention for forming a functional coating on the surface of a base material. It is preferred that by the use according to the present invention one or more of the following surface properties is improved: mechanical resistance and durability (including surface hardness, scratch resistance and/or abrasion resistance); wetting and adhesion properties (including hydro- and oleophobicity, easy-to-clean effect and/or anti-graffiti effect); chemical resistance (including corrosion resistance (e.g. against solvents, acidic and alkaline media and corrosive gases) and/or anti-oxidation effect); optical effects (light fastness); and physical barrier or sealing effects. Preferred base materials, to which the coating composition according to the present invention is applied, include a wide variety of materials such as, for example, metals (such as iron, steel, silver, zinc, aluminum, nickel, titanium, vanadium, chromium, cobalt, copper, zirconium, niobium, molybdenum, ruthenium, rhodium, silicon, boron, tin, lead or manganese or alloys thereof provided, if necessary, with an oxide or plating film); plastics (such as polymethyl methacrylate (PMMA), polyurethane, polyesters (PET), polyallyldiglycol carbonate (PADC), polycarbonate, polyimide, polyamide, epoxy resin, ABS resin, polyvinyl chloride, polyethylene (PE), polypropylene (PP), polythiocyanate, or polytetrafluoroethylene (PTFE)); glass (such as fused quartz, soda-lime-silica glass (window glass), sodium borosilicate glass (Pyrex®), lead oxide glass (crystal glass), aluminosilicate glass, or germanium-oxide glass); and construction materials (such as brick, cement, ceramics, clay, concrete, gypsum, marble, mineral wool, mortar, stone, or wood and mixtures thereof). The base materials may be treated with a primer to enhance the adhesion of the functional coating. Such primers are, for instance, silanes, siloxanes, or silazanes. If plastic materials are used, it may be advantageous to perform a pretreatment by flaming, corona or plasma treatment which might improve the adhesion of the functional coating. If construction materials are used, it may be advantageous to perform a precoating with lacquers, varnishes or paints such as, for example, polyurethane lacquers, acrylic lacquers and/or dispersion paints. The present invention is further illustrated by the examples following hereinafter which shall in no way be construed as limiting. The skilled person will acknowledge that various modifications, additions and alternations may be made to the invention without departing from the spirit and scope of the present invention. Examples In the examples, the Bisphenols AF and S (available from Sigma-Aldrich Chemie GmbH, Germany) are reacted with Durazane 1033 and Durazane 1800 (available from MERCK KGaA, Germany). Preparation of Coating Compositions 100 g silazane are dissolved in 400 g mesitylene and heated under nitrogen atmosphere to 120° C. Then 25 g of the solid bisphenol are added in 5 portions of 5 g each in time intervals of 90 min (see Table 1). After each addition it takes about 60 min until the solid bisphenol is completely dissolved. After the last addition the reaction mixture is heated to 140° C. for 3 h. Then the solution is cooled down to room temperature, filtrated over a 1 μm PTFE filter and evaporated under reduced pressure at 60° C. to final mass of 250 g. The result is a slightly viscous, clear, colorless to slightly yellow solution. TABLE 1Preparation of coating compositionsCompositionSilazaneBisphenol1Durazane 1033Bisphenol AF2Durazane 1800Bisphenol S To demonstrate the performance of the prepared compositions as coatings, the compositions were applied on different substrates and subjected to different tests. Coating on silicon wafers was performed by spin coating with film thickness of 5-6 μm for Application Tests 1 and 2. Coating on silicon wafers was performed by spin coating with film thickness adjustment by controlled spinning speed (rpm) and solvent content (viscosity) of: 5-6 μm, 8-10 μm, 18-20 μm, 40-50 μm and 70-80 μm for Application Test 3. Coating on steel panels (available from Q-Panel) was performed by dip coating with a film thickness of 20-25 μm for Application Tests 4 and 6. Coating on aluminum panels (available from Q-Panel) was performed by dip coating with a film thickness of 20-25 μm for Application Test 5. Application Test 1 Curing Time at Ambient Conditions of 25° C. and 50% Relative Humidity. After coating, the wafers were stored in controlled atmosphere of 25° C. and a relative humidity of 50%. After 15 min and subsequently in time periods of 15 min the surface of the coating was checked for stickiness. The first time, when the surface was not sticky anymore, was defined as the minimum dry-to-touch time (see Table 2). TABLE 2Application Test 1Dry-to-touch time@ 25° C. and 50%Materialrelative humidityComposition 145minComposition 245minReference: Durazane 1033>24hReference: Durazane 1800>24hComposition 1 + 0.5% Al(AcAc)330minComposition 1 + 0.5% DBU*30minReference: Durazane 1033 + 0.5% Al(AcAc)3120minReference: Durazane 1033 + 0.5% DBU*120min*DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene Application Test 1 demonstrates a very fast dry-to-touch curing of the inventive coating compositions. Quick dry-to-touch curing is very important for some industrial processes, e.g. in coil coating and other processes where a roll-to-roll process is used. Another advantage of fast drying is reduced absorption of air-born dust on the wet coating which results in less surface defects. The addition of well-known curing catalysts may further reduce the dry-to-touch time. Application Test 2 Film Hardness after Curing at Elevated Temperature of 120° C. for 4 h. The scratch resistance was analyzed with an “Elcometer 3000 Manual Clement Unit” equipped with a “0.04 inch tungsten carbide ball” pin (available from Elcometer Instruments GmbH, Germany). The test was started with a weight of 200 g and the weight load was increase in steps of 50 g. After scratching the specimen was investigated if a scratch was visible with the naked eye. The highest weight without scratch was noted (see Table 3). TABLE 3Application Test 2Maximum weightwithout scratchMaterial[g]Composition 1950Composition 2950Reference: Durazane 1033500Reference: Durazane 1800550Composition 2 + 0.5% Luperox*1150Reference: Durazane 1800 + 0.5% Luperox*700*Luperox = Luperox 531M80 (1,1-bis(tert-amylperoxy)cyclohexane) Application Test 2 demonstrates an improved scratch resistance, hardness and mechanical stability of the coatings obtained from the inventive compositions compared to pure silazane resins. Especially, the combination of a vinyl group containing silazane and a bisphenol with thermal curing and the addition of a radical initiator showed excellent scratch resistance. Application Test 3 Maximum Film Thickness without Crack Formation. After spin coating of the materials on silicon wafers in different film thicknesses, the wafers were cured for 4 h@120° C. and then for 4 h@180° C. on a hot plate. After cooling to room temperature the coating was inspected on crack formation with the naked eye (see Table 4). TABLE 4Application Test 35-68-1018-2040-5070-80Materialμm*μm*μm*μm*μm*Composition 1nonononocrackcrackcrackcrackcrackComposition 2nononononocrackcrackcrackcrackcrackReference:nonocrack——Durazane 1033crackcrackReference:nonocrack——Durazane 1800crackcrackComposition 2 + 0.5% Luperox**nononononocrackcrackcrackcrackcrackReference:nononocrack—Durazane 1800 + 0.5% Luperox**crackcrackcrack*film thickness in micrometer*Luperox = Luperox 531M80 (1,1-bis(tert-amylperoxy)cyclohexane) Application Test 3 demonstrates a high possible film thicknesses achievable with the inventive coating compositions. Pure silazanes cannot form crack-free films with thicknesses of 50 μm or more. However, for some applications, as for example in heavy duty corrosion protection, film thicknesses of >50 μm are common. The new compositions can form crack-free films with film thicknesses of significantly more than 50 μm. Application Test 4 HCl Resistance of Steel Panels. After dip coating of the materials on steel substrates, the substrates were cured for 4 h@120° C. and then for 4 h@150° C. on a hot plate. After cooling to room temperature a drop of 10% aqueous HCl or a drop of 37% aqueous HCl was placed on the substrate for 16 h. Then the surface was cleaned with water, dried and the coating was inspected on corrosion or spots with the naked eye (see Table 5). TABLE 5Application Test 410%37%Materialaqueous HClaqueous HClComposition 1no spotno spotComposition 2no spotno spotReference:severe corrosionsevere corrosionDurazane 1800Composition 2 + 0.5% Luperox*no spotno spotReference:some corrosionsome corrosionDurazane 1800 + 0.5% Luperox**Luperox = Luperox 531M80 (1,1-bis(tert-amylperoxy)cyclohexane) Application Test 5 NaOH Resistance of Aluminum Panels. After dip coating of the materials on aluminum substrates, the substrates were cured for 4 h@120° C. and then for 4 h@150° C. on a hot plate. After cooling to room temperature a drop of 2.5% aqueous NaOH or a drop of 5% aqueous NaOH was placed on the substrate for 16 h. Then the surface was cleaned with water, dried and the coating was inspected on corrosion or spots with the naked eye (see Table 6). TABLE 6Application Test 52.5%5%Materialaqueous NaOHaqueous NaOHComposition 1no spotsome corrosionComposition 2no spotsome corrosionReference:some corrosionsevere corrosionDurazane 1800Composition 2 + 0.5% Luperox*no spotno spotReference:some corrosionsevere corrosionDurazane 1800 + 0.5% Luperox**Luperox = Luperox 531M80 (1,1-bis(tert-amylperoxy)cyclohexane) Application Tests 4 and 5 demonstrate the improved chemical resistance of the coatings obtained from the inventive compositions. Protection of steel, aluminum and other metals against alkaline, acidic or other aggressive conditions is for example important in the architectural and automotive area. Other areas, where protective coatings are needed, are on a variety of sensitive surfaces to prevent corrosion by aggressive environments. Examples are the silver mirror background in LED packages or copper circuits in IC, devices which have to operate under harsh conditions, as for example sensors in the automotive industry. Application Test 6 Flexibility by Bend Test. After dip coating of the materials on steel substrates, the substrates were cured for 4 h@120° C. and then for 4 h@150° C. on a hot plate. After cooling to room temperature, the panels were subjected to a bending test with an “Elcometer 1510 Conical Mandrel Bend Tester” (available from Elcometer Instruments GmbH, Germany) with a mandrel diameter from 3.2 to 38.1 mm and a mandrel length of 303 mm. The coatings on the bended substrates were inspected on cracks or delamination with the naked eye (see Table 7). TABLE 7Application Test 6Result ofMaterialbending testComposition 1no delaminationComposition 2no delaminationReference: Durazane 1033severe delaminationReference: Durazane 1800severe delaminationComposition 2 + 0.5% Luperox*no delaminationReference: Durazane 1800 + 0.5% Luperox*some delamination*Luperox = Luperox 531M80 (1,1-bis(tert-amylperoxy)cyclohexane) Application Test 6 demonstrates the good flexibility of the coatings obtained from the inventive compositions. Good flexibility in combination with high hardness is needed for example in coil-coating. | 45,670 |
11859101 | DETAILED DESCRIPTION OF THE INVENTION A “curable composition” as used in this specification refers to a composition that has one or more components that can participate in a curing transformation. The composition can undergo a change in its physical properties, over a period of time, as a result of chemical and/or physical processes. A curable composition may be curable at room temperature or a lower temperature, or may require exposure to elevated temperature such as a temperature above room temperature or other condition(s) to initiate and/or to accelerate the curing transformation. Once a curable composition is applied to a surface (and during application), the curing reaction can proceed to provide a cured composition. A cured composition develops a tack-free surface, cures, and then fully cures over a period of time. A composition is considered fully cured when the hardness no longer increases. Curable compositions provided by the present disclosure may be applied directly onto the surface of a substrate as a single layer (often referred to as monocoat) or a multi-layer and/or over an underlayer such as a primer by any suitable process. The curable compositions provided by the present disclosure may be used, for example, in sealants, coatings, adhesives, encapsulants, and potting compositions. A sealant refers to a composition capable of producing a film that has the ability to resist operational conditions, such as moisture and temperature, and at least partially block the transmission of materials, such as water, fuel, and other liquids and gases. A sealant can be used to seal surfaces, smooth surfaces, fill gaps, seal joints, seal apertures, and other features. A coating refers to a curable composition that is deposited on an article that serves to protect the article and/or improve the appearance of the coated article. Examples include pigmented coatings that provide color to automobiles, airplanes, ships or free-standing structures such as metallic cans, buildings or bridges. An encapsulant refers to a curable composition that is applied on at least a portion of a material to increase durability and/or modulate the workable life of the material. Examples of encapuslants include films of polymeric ethylvinyl actetate used to cover parts of photovoltaic modules to protect the modules from harsh environmental factors. A “putty” or “potting composition” refers to curable composition that is applied to a surface as a filler to smoothen surface irregularities and/or improve appearance. Examples of the application of putty include, but are not limited to, the repair of scratches, holes, deformities, and dents in automobile parts. Other examples of the application of putty include filling cracks in woodwork, securing glass and/or smoothening surfaces in buildings, particularly, walls and ceilings. Adhesives refer to curable compositions utilized to bond together two or more substrate materials. For example, structural adhesives may be used for binding together automotive or industrial components. As used in this disclosure the phrase “curable composition layer” is meant to include sealant, coating, adhesive, encapsulant, and potting composition layers that may be applied over a substrate or over other curable composition layers. For convenience and/or illustration purposes, this disclosure may refer to single or multiple “coating composition layers.” However, the use of the phrase “coating composition layer” is used herein for illustrative purposes only, and should be understood to include the various other sealant, adhesive, encapsulant, and potting composition layers that are contemplated as possible alternatives. Accordingly, except as described in the present examples, the phrase “coating composition layer” can be used interchangeably to mean any other curable composition layer contemplated herein, such as a sealant, coating, adhesive, encapsulant, and potting composition layer, as determined by one of ordinary skill in the art. In addition, when used in the specification, a “first coating layer” or “second coating layer” may include, separately, one or more coating applications to form either the first or second coating layer. Accordingly a “coating layer” as identified herein does not preclude the presence of one or more other coating applications of the same or different composition to form that layer. For example, where the first coating layer is a basecoat layer it is contemplated at one, two, or more basecoat applications may be used together to form the “first coating layer.” Similarly, a clearcoat can be contemplated to have one, two or more clearcoat applications to form the “second coating layer”. As used in this specification, particularly in connection with coating layers or films, the terms “on,” “onto,” “over,” and variants thereof (e.g., “applied over,” “formed over,” “deposited over,” “provided over,” “located over,” and the like), mean applied, formed, deposited, provided, or otherwise located over a surface of a substrate, but not necessarily in contact with the surface of the substrate. For example, a coating layer “applied over” a substrate does not preclude the presence of one or more other coating layers of the same or different composition located between the applied coating layer and the substrate. Likewise, a second coating layer “applied over” a first coating layer does not preclude the presence of one or more other coating layers of the same or different composition located between the applied second coating layer and the applied first coating layer. As used in this specification, the terms “polymer” and “polymeric” means prepolymers, oligomers, and both homopolymers and copolymers. As used in this specification, “prepolymer” means a polymer precursor capable of further reactions or polymerization by one or more reactive groups to form a higher molecular mass or cross-linked state. As used in this specification, the prefix “poly” refers to two or more. For example, a “polyfunctional” molecule (whether a polymer, monomer, or other compound) comprises two or more reactive functional groups such as hydroxyl groups, amine groups, mercapto groups, carbamate groups, and the like. More specifically, “polyol” means a compound comprising two or more hydroxyl groups, “polyamine” means a compound comprising two or more amine groups, “polythiol” means a compound comprising two or more mercapto groups, and “polycarbamate” means a compound comprising two or more carbamate groups. A polyfunctional compound such as a polyol, polyamine, polythiol, or polycarbamate may be a polymer, but does not have to be a polymer, and may comprise, for example, non-polymeric compounds. A polymeric polyol, polymeric polyamine, polymeric polythiol, or polymeric polycarbamate respectively comprises two or more pendant and/or terminal hydroxyl, amine, mercapto, or carbamate functional groups on the polymer molecules. A “pendant group” refers to a group that comprises an offshoot from the side of a polymer backbone and which does not comprise part of the polymer backbone, whereas “terminal group” refers to a group on an end of a polymer backbone and which comprises part of the polymer backbone. Additionally, the terms polyol, polyamine, polythiol, and polycarbamate may encompass compounds comprising combinations of different types of functional groups. For example, a compound comprising two or more hydroxyl groups and two or more carbamate groups may be referred to as a polyol, a polycarbamate, or a polyol/polycarbamate. Furthermore, polyol, polyamine, polythiol, and polycarbamate compounds may comprise either or both the neutral functional groups (hydroxyl, amine, mercapto, or carbamate) and/or a salt of an ionized form of the functional group (e.g., alkoxide salts, ammonium salts, and the like). As used in this specification, the term “1,1-di-activated vinyl compound” means a compound comprising a vinyl group having two electron withdrawing groups (EWG) covalently bonded to one of the π-bonded carbons and no substituents covalently bonded to the other π-bonded carbon (i.e., -EWG-C(═CH2)-EWG-), wherein the electron withdrawing groups independently comprise halogen groups, haloalkyl groups, carbonyl-containing groups (e.g., esters, amides, aldehydes, ketones, acyl halides, carboxylic/carboxylate groups), cyano groups, sulfonate groups, ammonium groups, quaternary amine groups, or nitro groups. The term “multifunctional form” means a compound comprising two or more 1,1-di-activated vinyl groups covalently bonded in one molecule. For instance, a dialkyl methylene malonate is an example of a 1,1-di-activated vinyl compound, and a transesterification adduct of a dialkyl methylene malonate and a polyol is an example of a multifunctional form of a dialkyl methylene malonate. The curable compositions described in this specification comprise a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof, and a 1,1-di-activated vinyl compound, or a multifunctional form thereof, or a combination thereof. The 1,1-di-activated vinyl compounds can comprise methylene dicarbonyl compounds, dihalo vinyl compounds, dihaloalkyl disubstituted vinyl compounds, or cyanoacrylate compounds, or multifunctional forms of any thereof, or combinations of any thereof. Examples of 1,1-di-activated vinyl compounds and multifunctional forms thereof that can be used in the coating compositions are described in U.S. Pat. Nos. 8,609,885; 8,884,051; 9,108,914; 9,181,365; and 9,221,739, which are incorporated by reference into this specification. Additional examples of 1,1-di-activated vinyl compounds and multifunctional forms thereof that can be used in the coating compositions are described in U.S. Publication Nos. 2014/0288230; 2014/0329980; and 2016/0068618, which are incorporated by reference into this specification. The curable compositions can comprise a 1,1-di-activated vinyl compound comprising a methylene malonate. Methylene malonates are compounds having the general formula (I): wherein R and R′ may be the same or different and may represent nearly any substituent or side-chain, such as substituted or unsubstituted alkyl or aryl groups. For example, the curable compositions can comprise a dialkyl methylene malonate, a diaryl methylene malonate, a multifunctional form of a dialkyl methylene malonate, or a multifunctional form of a diaryl methylene malonate, or a combination of any thereof. A multifunctional form of a methylene malonate can comprise a transesterification adduct of the methylene malonate and a polyol. A multifunctional form of a methylene malonate can thus have the general formula (II): wherein X is a polyol residue and each R may be the same or different, as described above. As used herein the term “residue” refers to a group derived from the respective compound. For instance, in the above formula, X is an n-valent group derived from a polyol by a transesterification reaction involving methylene malonate and n hydroxyl groups of said polyol. Likewise, a polymer comprising residues of a certain compound is obtained from polymerizing said compound. In some examples, a multifunctional form of a methylene malonate can comprise a transesterification adduct of the methylene malonate and a diol, and thus have the general formula (III): wherein X is a diol residue and R and R′ may be the same or different, as described above. Polyols that are suitable for the production of a transesterification adduct with a methylene malonate include, for example, polymeric polyols (such as polyether polyols, polyester polyols, acrylic polyols, and polycarbonate polyols) and monomeric polyols (such as alkane polyols, including alkane diols such as 1,5-pentanediol and 1,6-hexanediol). The transesterification adduct can be formed by the reaction of a methylene malonate and a polyol, in the presence of a catalyst, in a suitable reaction medium. Examples of transesterification adducts of methylene malonates and polyols that may be used in the coating compositions are described in U.S. Publication No. 2014/0329980 and U.S. Pat. No. 9,416,091, which are incorporated by reference herein. Further, the concentration of the transesterification adduct can be influenced by ratio of the reactants and/or distillation or evaporation of the reaction medium. In some examples, the curable compositions can comprise dimethyl methylene malonate (D3M), a multifunctional form of D3M, or both. In some examples, the curable compositions can comprise diethyl methylene malonate (DEMM), a multifunctional form of DEMM, or both. The multifunctional forms of D3M or DEMM can comprise transesterification adducts of D3M or DEMM and a polyol, such as, for example, 1,5-pentanediol or 1,6-hexanediol. In some examples, the curable compositions can comprise a combination of a dialkyl methylene malonate and a multifunctional form of a dialkyl methylene malonate. The curable compositions can comprise, for example, DEMM and a multifunctional form of DEMM comprising a transesterification adduct of DEMM and at least one polyol. The DEMM can be transesterified with polyol comprising, for example, an alkane diol such as 1,5-pentanediol or 1,6-hexanediol. As described above, the curable compositions comprise a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof, and a 1,1-di-activated vinyl compound, or a multifunctional form thereof, or a combination thereof. While not intending to be bound by any theory, it is believed that the vinyl group(s) in the 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof can react via a Michael addition mechanism with the hydroxyl, amine, mercapto, and/or carbamate groups in polyol, polyamine, polythiol, and/or polycarbamate resins or other compounds (i.e., polyfunctional polymeric resins or polyfunctional monomeric compounds), and thereby form stable covalent linkages. Additionally, the 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof can undergo self-polymerization reactions, thereby forming polymers, which may covalently bond to polyfunctional polymeric resins or polyfunctional monomeric compounds through the linkages formed by the Michael addition reactions with the hydroxyl, amine, mercapto, and/or carbamate groups. Therefore, the 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof can function as crosslinking/curing agents for polyfunctional polymeric resins or polyfunctional monomeric compounds. In some example, the 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof can provide a multiple-cure mechanism comprising both Michael addition reactions and polymerization reactions that crosslink and cure polyfunctional polymeric resins or polyfunctional monomeric compounds. Polyfunctional polymeric resins that can be formulated in the curable compositions and crosslinked and cured with 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof include polymeric resins comprising pendant and/or terminal hydroxyl, amine, mercapto, and/or carbamate groups, such as, for example, polyether polyols, polyester polyols, acrylic polyols, polycarbonate polyols, polyether polyamines, polyester polyamines, acrylic polyamines, polycarbonate polyamines, polyether polythiols, polyester polythiols, acrylic polythiols, polycarbonate polythiols, polyether polycarbamates, polyester polycarbamates, acrylic polycarbamates, polycarbonate polycarbamates, and combinations of any thereof. Additional polyfunctional polymeric resins that can be formulated in the curable compositions and crosslinked and cured with 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof include any polyfunctional polymeric resins that incorporate hydroxyl, amine, mercapto, or carbamate groups, or combinations of any thereof, including for example, polyester resins, polyurethane resins, polyurea resins, polyether resins, polythioether resins, polycarbonate resins, polycarbamate resins, epoxy resins, phenolic resins, and aminoplast resins (urea-formaldehyde and/or melamine-formaldehyde). In addition to, or in lieu of, polyfunctional polymeric resins, polyfunctional monomeric compounds can be formulated in the curable compositions and crosslinked and cured with 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof. If polyol compounds are used, they may be the same as or different from those used for forming the transesterification adducts described above. Examples of monomeric polyol compounds include, but are not necessarily limited to, glycols such as ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol, 1,3-butylene glycol, tetramethylene glycol, hexamethylene glycol, neopentyl glycol, pentaerythritol, and combinations of any thereof. Other suitable hydroxyl-containing polyfunctional monomeric compounds include, but are not limited to, 1,5-pentandiol, 1,6-hexanediol, cyclohexanedimethanol, 2-ethyl-1,6-hexanediol, 1,4-butanediol, 1,3-propanediol, trimethylol propane, 1,2,6-hexanetriol, glycerol, and combinations of any thereof. Additionally, monomeric amino alcohols that can be formulated in the curable compositions and crosslinked and cured with 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof include, but are not limited to, ethanolamine, propanolamine, butanolamine, and combinations of any thereof. Examples of monomeric polyamine compounds that can be formulated in the curable compositions and crosslinked and cured with 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof include, for example, diamines such as, for example, ethylenediamine, hexamethylenediamine, 1,2-propanediamine, 2-methyl-1,5-penta-methylenediamine, 2,2,4-trimethyl-1,6-hexanediamine, isophoronediamine, diaminocyclohexane, xylylenediamine, 1,12-diamino-4,9-dioxadodecane, and combinations of any thereof. Other suitable monomeric and polymeric polyamine compounds include polyetheramines such as the Jeffamine® products available from Huntsman Chemical Company. Examples of monomeric and polymeric polythiol compounds that can be formulated in the curable compositions and crosslinked and cured with 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof include, for example, resins and compounds produced by the esterification of a polyol with a mercapto organic acid. Examples of suitable polyols include the polyols described above, and examples of suitable mercapto organic acids include thioglycolic acid and mercaptopropionic acid. Examples of monomeric polythiol compounds include, but are not limited to, glyceryl dithioglycolate, glyceryl trithioglycolate, glycol dimercaptoacetate, pentaerythritol tetramercaptoacetate, glycol di-(3-mercaptopropionate), pentaerythritol tetra(3-mercaptoproprionate), dipentaerythritol hexa(3-mercaptopropionate), trimethylolpropane tris-(thioglycolate), pentaerythritol tetrakis-(thioglycolate), ethyleneglycol dithioglycolate, trimethylolpropane tris(β-thiopropionate), pentaerythritol tetrakis-(β-thiopropionate), dipentaerythritol poly(β-thiopropionate), and combinations of any thereof. Other suitable monomeric and polymeric polythiol compounds include the Thiocure® products available from Bruno Bock Chemische Fabrik GmbH & Co. KG. Examples of monomeric and polymeric polycarbamate compounds that can be formulated in the curable compositions and crosslinked and cured with 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof include, for example, resins and compounds produced by the transcarbamylation of a polyol with an alkyl carbamate (i.e., the transesterification of the alkyl carbamate with the polyol). In addition to (1) the polyol, polyamine, polythiol, or polycarbamate, or combinations of any thereof, and (2) the 1,1-di-activated vinyl compound, or multifunctional form thereof, or combination thereof, the curable compositions can further comprise an acid promoter. In some examples, the acid promoter can comprise a strong acid. As used in this specification, the term “strong acid” means an acid having a pKain water at 25° C. of less than −1.3 and, for protic acids, at least one proton (H+) that completely dissociates in aqueous solution. Strong acid promoters that can be formulated in the curable compositions include, for example, inorganic strong acids and organic strong acids. Suitable inorganic strong acids include, for example, mineral acids (e.g., hydrochloric acid, perchloric acid, sulfuric acid, and nitric acid) and heteropoly acids (e.g., phosphotungstic acid, phosphomolybdic acid, silicotungstic acid, and silicomolybdic acid). Suitable organic strong acids include, for example, sulfonic acids (e.g., p-toluenesulfonic acid, methanesulfonic acid, and dodecylbenzenesulfonic acid). Combinations of any strong acids (e.g., a mixture of a sulfonic acid and a heteropoly acid) may also be formulated in the curable compositions. Without intending to be bound by any theory, it is believed that acids may function as Lewis acids in the curable compositions and complex to the 1,3-dicarbonyl motif, thereby promoting a Michael addition reaction between the functional groups on the polyfunctional components and the vinyl groups on the 1,1-di-activated vinyl compound and/or multifunctional form thereof. Accordingly, a strong acid component in a curable composition may shift the crosslinking and curing reactions away from self-polymerization of the 1,1-di-activated vinyl compound and/or multifunctional form thereof and toward Michael addition reactions forming covalent linkages between the polyfunctional components and the 1,1-di-activated vinyl compound and/or multifunctional form thereof. In addition to (1) the polyol, polyamine, polythiol, or polycarbamate, or combinations of any thereof, and (2) the 1,1-di-activated vinyl compound, or multifunctional form thereof, or combination thereof, the curable compositions can further comprise an activator. As used in this specification, the term “activator” means a compound or other agent capable of initiating and/or catalyzing (i) polymerization of 1,1-di-activated vinyl compounds or multifunctional forms thereof and/or (ii) addition reactions between 1,1-di-activated vinyl compounds or multifunctional forms thereof and polyfunctional components (e.g., polyol, polyamine, polythiol, and/or polycarbamate resins or compounds). The term “activator” includes (1) active forms of activator compounds and (2) latent precursor forms of activator compounds that are capable of conversion from the latent precursor form into the active form (e.g., by exposure to an effective amount of heat, electromagnetic radiation, pressure, or a chemical co-activator). Additionally, latent precursor forms of activator compounds that are capable of conversion into the active form include activators associated with a volatile or otherwise removable neutralizing agent or inhibitor compound that can evaporate or otherwise be removed from the curable composition when applied as a coating layer, thereby activating the activator. The activator can comprise a base. As used in this specification, the term “base” means an electronegative compound or functional group capable of initiating the anionic polymerization of a 1,1-di-activated vinyl compound. Suitable activators include organic bases (e.g., amine-containing compounds and carboxylate salts), inorganic bases (e.g., hydroxide salts and carbonate salts), organometallic compounds, and combinations of any thereof. Suitable activators also include polymers comprising pendant and/or terminal amine, carboxylate salt, or other base functionality capable of initiating the anionic polymerization of a 1,1-di-activated vinyl compound. In some examples, the activator comprises a strong base (pH over 9), a moderate base (pH from 8-9), or a weak base (pH from over 7 to 8), or a combination of any thereof. The activator may comprise, for example, sodium acetate; potassium acetate; acid salts of sodium, potassium, lithium, copper, or cobalt; tetrabutyl ammonium fluoride, chloride, or hydroxide; an amine, including primary, secondary, and tertiary amines; an amide; salts of polymer bound acids; benzoate salts; 2,4-pentanedionate salts; sorbate salts; propionate salts; secondary aliphatic amines; piperidene, piperazine, N-methylpiperazine, dibutylamine, morpholine, diethylamine, pyridine, triethylamine, tripropylamine, triethylenediamine, N,N-dimethylpiperazine, butylamine, pentylamine, hexylamine, heptylamine, nonylamine, decylamine; 1,4-diazabicyclo[2.2.2]octane (DABCO); 1,1′-iminobis-2-propanol (DIPA); 1,2-cyclohexaneamine; 1,3-cyclohexandimethanamine; 2-methylpentamethylenediamine; 3,3-iminodipropylamine; triacetone diamine (TAD); salts of amines with organic monocarboxylic acids; piperidine acetate; metal salt of a lower monocarboxylic acid; copper(II) acetate, cupric acetate monohydrate, potassium acetate, zinc acetate, zinc chloracetate, magnesium chloracetate, magnesium acetate; salts of acid containing polymers; salts of polyacrylic acid co-polymers; and combinations of any thereof. In some examples, the curable compositions can comprise a tertiary amine activator such as, for example, DABCO; 2-(dimethylamino)ethanol (DMAE/DMEA); 2-piperazin-1-ylethylamine; N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine; 2-[2-(dimethylamino)ethoxy]ethanol; 1-[bis[3-(dimethylamino)propyl]amino]-2-propanol; N,N,N′,N″,N″-pentamethyldiethylenetriamine; N,N,N′,N′-tetraethyl-1,3-propanediamine; N,N,N′,N′-tetramethyl-1,4-butanediamine; N,N,N′,N′-tetramethyl-1,6-hexanediamine; 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane; 1,3,5-trimethylhexahydro-1,3,5-triazine; methyl dicocoamine; 1,8-diazabicycloundec-7-ene (DBU); 1,5-diazabicyclo-[4,3,0]-non-5-ene (DBN); 1,1,3,3-tetramethylguanidine; or combinations of any thereof. The activator can comprise an ionic liquid. As used herein, the term “ionic liquid” means a salt having a melting point temperature of less than 100° C. at 1 atmosphere of pressure. Ionic liquids can be in a liquid state at room temperature (approximately 23° C.) and atmospheric pressure. Ionic liquids comprise a cation ionically associated with an anion. The cations can comprise, for example, heterocyclic nitrogen-containing organic cations such as imidazolium cations, pyrazolium cations, pyrrolidinium cations, pyridinium cations, pyrazinium cations, or pyrimidinium cations, including derivatives thereof; or other cations such as, for example, C1-C32tetraalkylphosphonium cations, C1-C32tetraalkylammonium cations, or C1-C32trialkylsulfonium cations. The anions can comprise, for example, a halide (F−, Cl−, Br−, I−), formate, acetate, nitrate, phosphate, sulfonate, tetrafluoroborate, hexfluorophosphate, triflate (trifluoromethane sulfonate), bis(trifluoromethylsulfonyl)imide, tosylate, an alkyl sulfonate anion (e.g., methyl sulfonate), an alkylsulfate anion, a carboxylate anion, or a phthalate anion. The ionic liquid activators used in the compositions, coatings, and processes described in this specification can comprise any combination of the above-described cations and anions that initiate and/or catalyze (i) polymerization of 1,1-di-activated vinyl compounds or multifunctional forms thereof and/or (ii) addition reactions between 1,1-di-activated vinyl compounds or multifunctional forms thereof and polyfunctional components (e.g., polyol, polyamine, polythiol, and/or polycarbamate resins or compounds). The ionic liquid activator can comprise an imidazolium salt of the formula: wherein R1and R2are each independently a C1-C12alkyl group; R3, R4, and R5are each independently a hydrogen or a C1-C12alkyl group, and X−is an anion. In some examples, R1and R2are each a C1-C12alkyl group; and R3, R4, and R5are each a hydrogen atom. In some examples, R1, R2, and R3are each a C1-C12alkyl group; and R4and R5are each a hydrogen atom. The ionic liquid activator can comprise a pyrazolium salt of the formula: wherein R1and R2are each independently a C1-C12alkyl group; R3, R4, and R5are each independently a hydrogen or a C1-C12alkyl group, and X−is an anion. In some examples, R1and R2are each a C1-C12alkyl group; and R3, R4, and R5are each a hydrogen atom. In some examples, R1, R2, and R3are each a C1-C12alkyl group; and R4and R5are each a hydrogen atom. The ionic liquid activator can comprise a pyridinium salt of the formula: wherein R1is a C1-C12alkyl group; R2are each independently a C1-C12alkyl group, n is 0 to 5, and X−is an anion. The ionic liquid activator can comprise a pyrimidinium salt and/or a pyrazinium salt of the formulas: wherein R1is a C1-C12alkyl group; R2are each independently a C1-C12alkyl group, n is 0 to 4, and X−is an anion. The ionic liquid activator can comprise an ammonium salt and/or a phosphonium salt of the formulas: wherein R1, R2, R3, and R4are each independently a C1-C12alkyl group; and X−is an anion. The anion (X−) in the salts described above can comprise, for example, a halide (F−, Cl−, Br−, I−), formate, acetate, nitrate, phosphate, sulfonate, tetrafluoroborate, hexfluorophosphate, triflate (trifluoromethane sulfonate), bis(trifluoromethylsulfonyl)imide, tosylate, an alkyl sulfonate anion (e.g., methyl sulfonate), an alkylsulfate anion, a carboxylate anion, or a phthalate anion. The ionic liquid activators used in the compositions, coatings, and processes described in this specification can comprise any combination of the above-described cations and anions, and can also comprise combinations of any two or more ionic liquids each independently comprising the above-described cations and anions. The curable compositions can comprise an activator in amounts, based on total composition weight, ranging from a non-zero amount up to 10%, up to 5%, up to 2%, up to 1%, up to 0.5%, or up to 0.1%, or any sub-range subsumed within such ranges. The activators may be maintained separate from the 1,1-di-activated vinyl compounds or multifunctional forms thereof (e.g., in separate container) until a time sufficiently close to the application of the curable composition over a substrate in order to prevent premature curing of the curable composition. The activator may then be mixed with all of the other components of the curable composition and applied over a substrate using a suitable application technique (e.g., spraying, electrostatic spraying, dipping, rolling, brushing, troweling electrocoating, and the like). In other examples, described below, activators may be applied over and/or under layers of the curable compositions to (1) activate addition reactions between the polyfunctional components and the 1,1-di-activated vinyl compounds or multifunctional forms thereof, and/or (2) activate polymerization reactions among the 1,1-di-activated vinyl compounds or multifunctional forms thereof. Additional examples of activators and activation methods that can be used in the present curable compositions are described in U.S. Pat. No. 9,181,365, which is incorporated by reference into this specification. In addition to (1) the polyol, polyamine, polythiol, or polycarbamate, or combinations of any thereof, and (2) the 1,1-di-activated vinyl compound, or multifunctional form thereof, or combination thereof, the curable compositions can further comprise an extender. As used in this specification, the term “extender” means a compound or other agent capable of decreasing the reaction rate of (i) polymerization of 1,1-di-activated vinyl compounds or multifunctional forms thereof and/or (ii) addition reactions between 1,1-di-activated vinyl compounds or multifunctional forms thereof and polyfunctional components (e.g., polyol, polyamine, polythiol, and/or polycarbamate resins or compounds). Accordingly, extenders function to extend the pot life of the curable compositions and can be used in combination with activators and/or acid promoters, as described above, to control the pot life and cure response of the curable compositions comprising (1) the polyol, polyamine, polythiol, or polycarbamate, or combinations of any thereof, and (2) the 1,1-di-activated vinyl compound, or multifunctional form thereof, or combination thereof. The extenders used in the compositions, coatings, and processes described in this specification can comprise, for example, a carboxylic anhydride compound and/or a carboxylic acid compound. Suitable carboxylic anhydride compounds include, for example, unsaturated anhydrides such as maleic anhydride; citraconic anhydride; itaconic anhydride; aconitic anhydride; bicyclo[2.2.2]oct-7-ene-2,3,5,6-tetracarboxylic dianhydride; crotonic anhydride; 1-cyclopentene-1,2-dicarboxylic anhydride; methacrylic anhydride; or combinations of any thereof. Suitable carboxylic anhydride compounds also include, for example, saturated anhydrides such as the saturated homologues of any of the above-described unsaturated anhydrides (e.g., succinic anhydride). Suitable carboxylic acid compounds include, for example, short-chain (e.g., C2to C20) saturated and unsaturated carboxylic acids such as oxalic acid, acetic acid, propionic acid, octanoic, stearic acid, isostearic acid, benzoic acid, citric acid, (meth)acrylic acid, crotonic acid, isocrotonic acid, vinylacetic acid, 2-pentenoic acid, 3-pentenoic acid, allylacetic acid, maleic acid, fumaric acid, citraconic acid, itaconic acid, aconitic acid, saturated and unsaturated fatty acids (e.g., palmitoleic acid, vaccenic acid, and/or oleic acid), and combinations of any thereof. Alternatively, or in addition, the extenders used in the compositions, coatings, and processes described in this specification can comprise, for example, an anhydride-containing vinyl polymer and/or a carboxylic acid-containing vinyl polymer. As used in this specification, the term “vinyl polymer” means any polymer produced by addition reactions between carbon-carbon double bonds. Anhydride-containing vinyl polymers can be produced from monomer mixtures comprising an ethylenically unsaturated carboxylic acid anhydride such as, for example, maleic anhydride; citraconic anhydride; itaconic anhydride; aconitic anhydride; bicyclo[2.2.2]oct-7-ene-2,3,5,6-tetracarboxylic dianhydride; crotonic anhydride; 1-cyclopentene-1,2-dicarboxylic anhydride; methacrylic anhydride; or combinations of any thereof. Anhydride-containing vinyl polymers can be produced from monomer mixtures further comprising ethylenically unsaturated monomers such as, for example, styrene and derivatives thereof, vinyl acetate, vinyl chloride, (meth)acrylate esters, and the like. Anhydride-containing vinyl polymers suitable for use as extenders in the compositions, coatings, and processes described in this specification are described, for example, in U.S. Pat. No. 4,798,745 at column 7, line 27 to column 8, line 3, and at column 10, line 40 to column 12, line 59, which is incorporated by reference into this specification. Carboxylic acid-containing vinyl polymer suitable for use as extenders in the compositions, coatings, and processes described in this specification can be produced from monomer mixtures comprising an ethylenically unsaturated carboxylic acid such as, for example, (meth)acrylic acid, crotonic acid, isocrotonic acid, vinylacetic acid, 2-pentenoic acid, 3-pentenoic acid, allylacetic acid, maleic acid, fumaric acid, citraconic acid, itaconic acid, aconitic acid, saturated and unsaturated fatty acids (e.g., palmitoleic acid, vaccenic acid, and/or oleic acid), and combinations of any thereof. Carboxylic acid-containing vinyl polymers can be produced from monomer mixtures further comprising ethylenically unsaturated monomers such as, for example, styrene and derivatives thereof, vinyl acetate, vinyl chloride, (meth)acrylate esters, and the like. Additionally, vinyl polymers containing both carboxylic acid groups and carboxylic acid anhydride groups can be used as extenders in the compositions, coatings, and processes described in this specification. Such vinyl polymer can be produced from monomer mixtures comprising both an ethylenically unsaturated carboxylic acid and an ethylenically unsaturated carboxylic acid anhydrides, as described above. In some examples, the curable compositions can comprise (1) the polyol, polyamine, polythiol, or polycarbamate, or combinations of any thereof, (2) the 1,1-di-activated vinyl compound, or multifunctional form thereof, or combination thereof, and (3) any combination of an acid promoter, an activator, and/or an extender, as described above. In addition to (1) the polyol, polyamine, polythiol, or polycarbamate, or combinations of any thereof, (2) the 1,1-di-activated vinyl compound, or multifunctional form thereof, or combination thereof, and (3) any promoter, activator, and/or extender (e.g., an acid and/or base and/or anhydride-containing vinyl polymer), if present, the curable compositions can further comprise additional materials such as additional resins, solvents, reactive diluents, colorants, and the like. As used herein, “colorant” means any substance that imparts color and/or other opacity and/or other visual effect to the coating composition, particularly when applied over a substrate and cured. A colorant can be added to the coating composition in any suitable form, such as discrete particles, dispersions, solutions, and/or flakes. A single colorant or a mixture of two or more colorants can be used in the coatings compositions described in this specification. Example colorants include pigments (organic or inorganic), dyes, and tints, such as those used in the paint industry and/or listed by the Dry Color Manufacturers Association (DCMA), as well as special effect compositions. A colorant may include, for example, a finely divided solid powder that is insoluble, but wettable under the conditions of use. A colorant can be organic or inorganic and can be agglomerated or non-agglomerated. Colorants can be incorporated into the coating by use of a grind vehicle, such as an acrylic grind vehicle, the use of which will be familiar to persons skilled in the art. Example pigments and/or pigment compositions include, but are not limited to, carbazole dioxazine crude pigment, azo, monoazo, diazo, naphthol AS, salt type (flakes), benzimidazolone, isoindolinone, isoindoline and polycyclic phthalocyanine, quinacridone, perylene, perinone, diketopyrrolo pyrrole, thioindigo, anthraquinone, indanthrone, anthrapyrimidine, flavanthrone, pyranthrone, anthanthrone, dioxazine, triarylcarbonium, quinophthalone pigments, diketo pyrrolo pyrrole red (“DPPBO red”), titanium dioxide, carbon black, and mixtures of any thereof. The terms “pigment” and “colored filler” can be used interchangeably. Example dyes include, but are not limited to, soluble in organic solvents and/or water such as phthalo green or blue, iron oxide, bismuth vanadate, anthraquinone, perylene, and quinacridone. Example tints include, but are not limited to, pigments dispersed in water-based or water miscible carriers such as AQUA-CHEM 896 (available from Degussa, Inc.), and CHARISMA COLORANTS and MAXITONER INDUSTRIAL COLORANTS (available from the Accurate Dispersions Division of Eastman Chemical Company). A colorant optionally formulated in the coating compositions can also comprise a special effect composition or pigment. As used herein, a “special effect composition or pigment” means a composition or pigment that interacts with visible light to provide an appearance effect other than, or in addition to, a continuous unchanging color. Example special effect compositions and pigments include those that produce one or more appearance effects such as reflectance, pearlescence, metallic sheen, texture, phosphorescence, fluorescence, photochromism, photosensitivity, thermochromism, goniochromism, and/or color-change. Examples of special effect compositions can include transparent coated mica and/or synthetic mica, coated silica, coated alumina, aluminum flakes, a transparent liquid crystal pigment, a liquid crystal coating, and combinations of any thereof. Other examples of materials that can be formulated in the curable compositions include plasticizers, abrasion resistant particles, anti-oxidants, hindered amine light stabilizers, UV light absorbers and stabilizers, surfactants, flow and surface control agents, thixotropic agents, solvents and co-solvents, reactive diluents, catalysts, reaction inhibitors, and other customary auxiliaries in the paint and coating industry. The invention described in this specification includes the use of the curable compositions described above. For example, a process for coating a substrate can comprise applying a first coating layer over at least a portion of a substrate, applying a second coating layer over at least a portion of the first coating layer, and curing the first coating layer and/or the second coating layer. The curing of the first coating layer and the second coating layer can be performed sequentially or simultaneously with or without intermediate flashing, drying, or dehydrating steps. The first coating layer and/or the second coating layer is formed from a curable composition comprising (1) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof, and (2) a 1,1-di-activated vinyl compound, or a multifunctional form thereof, or a combination thereof. The curable composition, whether applied as the first coating layer and/or the second coating layer, can comprise, in any combination, any of the features or characteristics described above. For example, the 1,1-di-activated vinyl compound can comprise a methylene dicarbonyl compound, a dihalo vinyl compound, a dihaloalkyl disubstituted vinyl compound, or a cyanoacrylate compound, or a multifunctional form of any thereof, or a combination of any thereof. The polyol, polyamine, polythiol, or polycarbamate can comprise a polyfunctional polymeric resin or a polyfunctional monomeric compound or a combination thereof. The curable composition can include a promoter (e.g., a strong acid such as a sulfonic acid and/or a heteropoly acid) and/or an activator (e.g., an amine such as a tertiary amine like DABCO or DMAE/DMEA, or an ionic liquid) and/or an extender (e.g., an anhydride-containing vinyl polymer). As described above, the curable composition is not necessarily limited to the sole use of an acid promotor or an activator or an extender. In some examples, a combination of both an acid promoter and an activator compound can be used in a curable composition. Similarly, a combination of an acid promoter and an extender, or a combination of an activator compound and an extender, or a combination of all three, can be used in a curable composition. Without intending to be bound by any theory, the presence of an acid promoter, an activator compound, and/or an extender in a curable composition can influence the final coating properties by modulating the cure kinetics and/or the extent of Michael addition reactions versus self-polymerization of the 1,1-di-activated vinyl compound and/or multifunctional form thereof. In some examples, the acid catalyst can comprise a “strong acid” as described above and/or weaker acids. Suitable weaker acids that can be formulated in the curable compositions include, for example, inorganic weak acids and organic weak acids. In this context, weak acids are defined as having pKa in the range of −1.3 to 7 in water at 25° C. Suitable inorganic weak acids include, for example, sulfamic acid, phosphoric acid, hypochlorous acid, and boric acid. Suitable organic weak acids include, for example, carboxylic acids such as oxalic acid, acetic acid, propionic acid, octanoic, stearic acid, isostearic acid, benzoic acid, and citric acid. As used in this specification, the terms “cure” and “curing” refer to the progression of a liquid curable composition from the liquid state to a cured state and encompass physical drying of curing compositions through solvent or carrier evaporation (e.g., thermoplastic curing compositions) and/or chemical crosslinking of components in the curable compositions (e.g., thermosetting curing compositions). In this regard, the term “cured,” as used in this specification, refers to the condition of a liquid curable composition in which a film or layer formed from the liquid curable composition is at least set-to-touch. In some examples, the curing of the first coating layer and/or the second coating layer can comprise spraying an activator solution over and/or under at least a portion of the first coating layer and/or the second coating layer. The activator solution can comprise an activator (as described above) dissolved or otherwise dispersed in a liquid carrier. The activator solution can comprise an activator compound such as amine activator (e.g., a tertiary amine compound such as DABCO or DMAE/DMEA) dissolved in an aqueous or organic solvent (e.g., an ester solvent such as n-butyl acetate). The activator solution can be sprayed or otherwise applied over a substrate and the curable composition applied over the pre-applied activator solution. Alternatively, or in addition, the activator solution can be sprayed or otherwise applied over a pre-applied layer or film of the curable composition. The activator solution may initiate Michael addition reactions and/or polymerization reactions at the interface of the applied curable composition layer or film and may migrate into the layer or film to further initiate curing reactions. In some examples, the curing of the first coating layer may be initiated by activator compounds present in the second coating layer, or the curing of the second coating layer may be initiated by activator compounds present in the first coating layer. For instance, the first coating layer may comprise an activator compound, and the curing of the second coating layer comprises activating an addition reaction and/or a polymerization reaction in the second coating layer with the activator compound in the first coating layer. In this manner, the activator compound in the first coating layer may initiate Michael addition reactions and/or polymerization reactions in the second coating layer at the interface between the two layers. The activator compound in the first coating layer may also migrate through the interface and into the second coating layer to further initiate curing reactions. In this example, the chemical composition of the first coating layer may be such that the activator does not function to initiate crosslinking or other curing reactions in the first coating layer, but does so initiate curing reaction in the second coating layer upon application of the second coating layer over and in direct contact with the first coating layer. Alternatively, the second coating layer may comprise an activator compound, and the curing of the first coating layer comprises activating an addition reaction and/or a polymerization reaction in the first coating layer with the activator compound in the second coating layer. In this manner, the activator compound in the second coating layer may initiate Michael addition reactions and/or polymerization reactions in the first coating layer at the interface between the two layers. The activator compound in the second coating layer may also migrate through the interface and into the first coating layer to further initiate curing reactions. In this example, the chemical composition of the second coating layer may be such that the activator does not function to initiate crosslinking or other curing reactions in the second coating layer, but does so initiate curing reaction in the first coating layer upon application of the second coating layer over and in direct contact with the first coating layer. The activator present in either the first coating layer or the second coating layer which initiates crosslinking or other curing reactions in the other coating layer can comprise an activator compound such as amine activator (e.g., a tertiary amine compound such as DABCO or DMAE/DMEA). In some examples, the first coating layer and/or the second coating layer can be applied over at least a portion of a bare substrate or a pre-applied coating (e.g., a primer coating) using application techniques such as spraying, electrostatic spraying, dipping, rolling, brushing, electrocoating, and the like. Once applied, the first coating layer and the second coating layer can be dehydrated and/or cured. As described above, the curing of the first coating layer and the second coating layer can be performed sequentially (i.e., the first coating layer is cured before the application of the second coating layer) or simultaneously with or without intermediate flashing, drying, or dehydrating steps. For example, the first coating layer can be applied and dehydrated, the second coating layer can be applied over the dehydrated first coating layer, and both the first and second coating layers baked or otherwise treated to cure the multi-layer system. The specific curing conditions of the coating layers will be based, at least in part, on the chemical formulation of the curable composition forming the layers. In some examples, the first coating layer and/or the second coating layer can be dehydrated and/or cured, independently or together, at temperatures ranging from ambient temperature (about 20° C. to 25° C.) to 500° C., or any sub-range subsumed therein, for example, from ambient temperatures to 200° C., from ambient temperatures to 150° C., from ambient temperatures to 140° C., from ambient temperatures to 130° C., from ambient temperatures to 120° C., from ambient temperatures to 100° C. from ambient temperatures to 90° C., from ambient temperatures to 80° C., from ambient temperatures to 60° C., or from ambient temperatures to 50° C. As described above, 1,1-di-activated vinyl compounds and/or multifunctional forms thereof can function as crosslinking/curing agents for polyfunctional polymeric resins or polyfunctional monomeric compounds. Again not intending to be bound by any theory, it is believed that the vinyl group(s) in the 1,1-di-activated vinyl compounds and/or the multifunctional forms thereof can react via a Michael addition mechanism with the hydroxyl, amine, mercapto, and/or carbamate groups in polyfunctional polymeric resins or polyfunctional monomeric compounds) and thereby form stable covalent linkages. Accordingly, after curing, at least one of the cured first coating layer and/or the cured second coating layer may comprise an addition reaction product of (1) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof, and (2) a 1,1-di-activated vinyl compound and/or a multifunctional forms thereof. The 1,1-di-activated vinyl compound and/or a multifunctional forms thereof may comprise a di alkyl methylene malonate, a diaryl methylene malonate, a multifunctional form of a dialkyl methylene malonate, or a multifunctional form of a diaryl methylene malonate, or a combination of any thereof. The “Addition Reaction product” refers to the adduct formed by the reaction of 1,1′-di-activated vinyl compound and/or multifunctional form thereof with a nucleophile (such as an amine, thiol or alcohol and/or their polymeric form). Without being bound to any theory, this may be the result of an addition of the nucleophile to the conjugate double bond (‘the Michael Addition Reaction’), or displacing the alcohol of the ester of a 1,1-diactivated vinyl ester with a another alcohol (a trans-esterification reaction), an amine, a thiol and/or a polymeric form of them. For example, the reaction of an amine can result in an amide product and the reaction with a thiol can result in a thioester product. A “polymeric addition product” refers to the product of polymerization reaction, wherein a multitude of reactants react repetitively. Without being bound by any theory, this could be done through a variety of reaction mechanisms, such as anionic polymerization, condensation polymerization, chain growth or radical polymerization. For example, a primary amine can react 1,1′-diethyl methylene malonate (DEMM) via anionic polymerization to form polymeric-DEMM. In some examples, after curing, at least one of the cured first coating layer and/or the cured second coating layer may comprise an addition reaction product of (1) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof, and (2) diethyl methylene malonate and a multifunctional form of diethyl methylene malonate. The multifunctional form of the diethyl methylene malonate may comprise a transesterification adduct of diethyl methylene malonate and at least one polyol. The transesterification adduct of the diethyl methylene malonate and the at least one polyol may comprise a transesterification adduct of the diethyl methylene malonate and a diol (e.g., an alkane diol such as 1,5-pentanediol or 1,6-hexanediol). The invention described in this specification includes coatings formed from the curable compositions described above. For example, a multi-layer coating can comprise a first coating layer applied over at least a portion of a substrate, and a second coating layer applied over at least a portion of the first coating layer. The first coating layer and/or the second coating layer can comprise an addition reaction product of: (1a) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof; and (1b) a 1,1-di-activated vinyl compound, or a multifunctional form thereof, or a combination thereof. The first coating layer and/or the second coating layer can additionally or alternatively comprise a polymerization reaction product of the 1,1-di-activated vinyl compound, or a multifunctional form thereof, or a combination thereof. In examples where only the first coating layer or the second coating layer is formed from a curable composition comprising a polyfunctional resin or compound and a 1,1-di-activated vinyl compound and/or a multifunctional form thereof, the other coating layer can be formed from a different curable composition comprising any useful formulation. Other types of curable compositions than can be used with the coating compositions described in this specification to produce multi-layer coatings include, for example, polyurethane-based coating compositions, polyurea-based coating compositions, acrylic-based coating compositions, epoxy-based coating compositions, polyester-based coating compositions, polyether-based coating compositions, polythioether-based coating compositions, polyamide-based coating compositions, polycarbonate-based coating compositions, polycarbamate-based coating compositions, and aminoplast-based coating compositions (including coating compositions comprising urea-formaldehyde and/or melamine-formaldehyde resins). The coating compositions described in this specification can be used to form basecoats, topcoats, tiecoats, and the like, in combination with other coating chemistries that form other coating layers in a multi-layer coating system. As used in this specification, the term “basecoat” means a coating layer that is deposited onto a primer and/or directly onto a substrate, optionally including components (such as pigments) that impact the color and/or provide other visual impact. As used in this specification, the term “topcoat” means a coating layer that is deposited over another coating layer such as a basecoat. Topcoats are often, but not always, “clearcoats,” which as used in this specification means a coating layer that is at least substantially transparent or fully transparent to visible. As used in this specification, the term “substantially transparent” refers to a coating wherein a surface beyond the coating is at least partially visible to the naked eye when viewed through the coating. As used in this specification, the term “fully transparent” refers to a coating wherein a surface beyond the coating is completely visible to the naked eye when viewed through the coating. It is appreciated that a clearcoat can comprise colorants, such as pigments, provided that the colorants do not interfere with the desired transparency of the clearcoat layer. In some examples, a clearcoat layer is free of added colorants such as pigments. As used in this specification, the term “tiecoat” means a coating layer that is located between two other coating layers, such as, for example, a coating layer located between a basecoat layer and a topcoat layer. The multi-layer coatings described in this specification can comprise a primer coating layer, which can correspond to a first coating layer. As used in this specification, a “primer coating layer” means an undercoating that may be deposited onto a substrate in order to prepare the surface for application of a protective or decorative coating system. A primer coating layer can be formed over at least a portion of the substrate as a first coating layer and a second coating layer (e.g., a basecoat) can be formed over at least a portion of the primer coating layer. As such, the multi-layer coating of the present invention can comprise a primer coating layer and one or more of a basecoat layer and a topcoat layer. A first coating layer comprising a primer coating layer can be formed from a curable composition that comprises a film-forming resin such as a cationic based resin, an anionic based resin, and/or any of the additional film-forming resins previously described. The curable composition used to form the primer coating composition can include a corrosion inhibitor, particularly in coating formulations intended for use on metallic substrates. As used in this specification, a “corrosion inhibitor” means a component reduces the rate or severity of corrosion of a surface on a metal or metal alloy substrate. Also, the first coating layer can be a direct gloss coating. A direct gloss coating, in this context, refers to a pigmented top coat layer is either glossy or has a matte finish. A corrosion inhibitor can include, but is not limited to, an alkali metal component, an alkaline earth metal component, a transition metal component, or combinations of any thereof. The term “alkali metal” refers to an element in Group 1 (International Union of Pure and Applied Chemistry (IUPAC)) of the periodic table of the chemical elements, and includes, e.g., cesium (Cs), francium (Fr), lithium (Li), potassium (K), rubidium (Rb), and sodium (Na). The term “alkaline earth metal” refers to an element of Group 2 (IUPAC) of the periodic table of the chemical elements, and includes, e.g., barium (Ba), beryllium (Be), calcium (Ca), magnesium (Mg), and strontium (Sr). The term “transition metal” refers to an element of Groups 3 through 12 (IUPAC) of the periodic table of the chemical elements, and includes, e.g., titanium (Ti), zirconium (Zr), chromium (Cr), and zinc (Zn), among various others. Examples of inorganic components that can function as corrosion inhibitors in primer coating compositions include magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium phosphate, magnesium silicate, zinc oxide, zinc hydroxide, zinc carbonate, zinc phosphate, zinc silicate, zinc dust, and combinations thereof. The components of a primer coating composition can be selected to form an electrodepositable coating composition. As used in this specification, the term “electrodepositable coating composition” refers to a curable composition that is capable of being deposited onto an electrically conductive substrate under the influence of an applied electrical potential. Examples of electrodepositable coating compositions include anionic and cationic electrodepositable coating compositions, such as epoxy or polyurethane-based coatings, such as the electrodepositable coatings described in U.S. Pat. No. 4,933,056 at column 2, line 48 to column 5, line 53; U.S. Pat. No. 5,530,043 at column 1, line 54 to column 4, line 67; U.S. Pat. No. 5,760,107 at column 2, line 11 to column 9, line 60; and U.S. Pat. No. 5,820,987 at column 3, line 48 to column 10, line 63, each of which is incorporated by reference into this specification. Suitable electrodepositable coating compositions also include those commercially available from PPG Industries, Inc., such as the POWERCRON® series of anodic and cathodic epoxy and acrylic coatings, ED-6060C, ED-6280, ED-6465, and ED-7000, for example. As described above, a primer coating composition can be deposited as a first coating layer directly over at least a portion of a substrate before application of a second coating layer. Alternatively, a first coating layer can be deposited over a cured primer coating layer where the first coating layer functions as a basecoat layer, and a second coating layer deposited over the first coating layer where the second coating layer functions as a topcoat layer or a tiecoat layer (when a subsequent layer is applied over the second coating layer). Once a primer coating composition is applied to at least a portion of a substrate, the primer coating layer can be dehydrated and/or cured before applying an overcoating layer, whether a basecoat or a topcoat. A primer coating composition can be dehydrated and/or cured, for example, at a temperature of 175° C. to 205° C. to form a primer coating layer. When the curable composition described in this specification is used to form a basecoat layer or a tiecoat layer, the multi-layer coating can comprise a topcoat layer formed from a different coating composition such as, for example a coating composition formulated to produce an isocyanate-crosslinked polyurethane clearcoat. Additional examples of topcoat layers that can be used with the multi-layer coating of the present invention include those described in U.S. Pat. No. 4,650,718 at column 1, line 62 to column 10, line 16; U.S. Pat. No. 5,814,410 at column 2, line 23 to column 9 line 54; and U.S. Pat. No. 5,891,981 at column 2, line 22 to column 12, line 37, each of which is incorporated by reference into this specification. Suitable topcoat coating compositions that can be used to form a topcoat layer over the coating compositions described in this specification also include those commercially available from PPG Industries, Inc. under the trademarks NCT®, DIAMOND COAT®, and CERAMICLEAR®. As described above, in the multi-layer coatings of the present invention, at least one of the first coating layer and/or the second coating layer can comprise an addition reaction product of: (1a) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof; and (1b) a 1,1-di-activated vinyl compound, or a multifunctional form thereof, or a combination thereof. The first coating layer and/or the second coating layer can additionally or alternatively comprise a polymerization reaction product of the 1,1-di-activated vinyl compound, or a multifunctional form thereof, or a combination thereof. As noted, the 1,1-di-activated vinyl compound can comprise, for example, a methylene dicarbonyl compound, a dihalo vinyl compound, a dihaloalkyl disubstituted vinyl compound, or a cyanoacrylate compound, or a multifunctional form of any thereof, or a combination of any thereof. In some examples of the multi-layer coating, the first coating layer and/or the second coating layer can comprise an addition reaction product of (1) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof, and (2) a dialkyl methylene malonate, a diaryl methylene malonate, a multifunctional form of a dialkyl methylene malonate, or a multifunctional form of a diaryl methylene malonate, or a combination of any thereof. For instance, the first coating layer and/or the second coating layer can comprise an addition reaction product of (1) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof, and (2) diethyl methylene malonate and a multifunctional form of diethyl methylene malonate. The multifunctional form of diethyl methylene malonate can comprise a transesterification adduct of diethyl methylene malonate and at least one polyol. The transesterification adduct of the diethyl methylene malonate and the at least one polyol can comprise a transesterification adduct of diethyl methylene malonate and a diol (e.g., an alkane diol such as 1,5-pentanediol or 1,6-hexanediol). In some examples of the multi-layer coating, the first coating layer and/or the second coating layer can be formed from a curable composition including a promoter (e.g., a strong acid such as a sulfonic acid and/or a heteropoly acid) and/or an activator (e.g., an amine such as a tertiary amine like DABCO or DMAE/DMEA, or an ionic liquid) and/or an extender (e.g., an anhydride-containing vinyl polymer). In some examples, the first coating layer comprises an activator compound that activated addition reactions in the second coating layer when the second coating layer was applied over the first coating layer. In other examples, the second coating layer comprises an activator compound that activated addition reactions in the first coating layer when the second coating layer was applied over the first coating layer. In some examples, the second coating layer comprises (1) an addition reaction product of the polyfunctional resin or polyfunctional compound and the 1,1-di-activated vinyl compound and/or multifunctional form thereof, and/or (2) a polymerization reaction product of the 1,1-di-activated vinyl compound and/or multifunctional form thereof, and the first coating layer is formed from a curable composition that cures when heated at a temperature of less than 500° C., less than 200° C., less than 150° C., less than 140° C., less than 130° C., less than 120° C., or less than 100° C. The curable composition that forms the first coating layer can comprise, for example, polyurethane-based coating compositions, polyurea-based coating compositions, acrylic-based coating compositions, epoxy-based coating compositions, polyester-based coating compositions, polyether-based coating compositions, polythioether-based coating compositions, polyamide-based coating compositions, polycarbonate-based coating compositions, polycarbamate-based coating compositions, and aminoplast-based coating compositions (including coating compositions comprising urea-formaldehyde and/or melamine-formaldehyde resins). In some examples, the first coating layer is formed from a curable composition that does not comprise (i.e., is substantially free of) melamine resin and formaldehyde condensates. The term “substantially free,” as used in this specification, means that the described materials are present, if at all, at incidental impurity levels, generally less than 1000 parts per million (ppm) by weight based on total curable composition weight. In some examples, the first coating layer comprises (1) an addition reaction product of the polyfunctional resin or polyfunctional compound and the 1,1-di-activated vinyl compound and/or multifunctional form thereof, and/or (2) a polymerization reaction product of the 1,1-di-activated vinyl compound and/or multifunctional form thereof, and the second coating layer comprises a clearcoat layer (e.g., an isocyanate-crosslinked polyurethane clearcoat layer). The curable compositions can be applied to a wide range of substrates including vehicle components and components of free-standing structures such as buildings, bridges, or other civil infrastructures. More specific substrates include, but are not limited to, automotive substrates (e.g., body panels and other parts and components), industrial substrates, aircraft components, watercraft components, packaging substrates (e.g., food and beverage cans), wood flooring and furniture, apparel, electronics (e.g., housings and circuit boards), glass and transparencies, sports equipment (e.g., golf balls, and the like), appliances (e.g., dish washing machines, clothes washing machines, clothes drying machines). Substrates can be, for example, metallic or non-metallic. Metallic substrates include, but are not limited to, tin, steel (including electrogalvanized steel, cold rolled steel, hot-dipped galvanized steel, among others), aluminum, aluminum alloys, zinc-aluminum alloys, steel coated with a zinc-aluminum alloy, and aluminum plated steel. Non-metallic substrates include polymeric, plastic, polyester, polyolefin, polyamide, cellulosic, polystyrene, polyacrylic, poly(ethylene naphthalate), polypropylene, polyethylene, nylon, EVOH, polylactic acid, other “green” polymeric substrates, poly(ethyleneterephthalate) (PET), polycarbonate, polycarbonate acrylonitrile butadiene styrene (PC/ABS), polyamide, wood, veneer, wood composite, particle board, fiberboard, cement, concrete, brick, stone, paper, cardboard, textiles, leather (both synthetic and natural), glass or fiberglass composites, carbon fiber composites, mixed fiber (e.g., fiberglass and carbon fiber) composites, and the like. The substrate can be one that has been already treated in some manner, such as to impart visual and/or color effect, a protective pretreatment or primer coating layer, or other coating layer, and the like. The present invention further includes an article comprising the multi-layer coatings formed from the curable compositions described in this specification. For example, the curable compositions of the present invention are also suitable for use as packaging coatings. The application of various pretreatments and coatings to packaging is well established. Such treatments and/or coatings, for example, can be used in the case of metal cans, wherein the treatment and/or coating is used to retard or inhibit corrosion, provide a decorative coating, provide ease of handling during the manufacturing process, and the like. Coatings can be applied to the interior of such cans to prevent the contents from contacting the metal of the container. The coatings applied to the interior of metal cans also help prevent corrosion in the headspace of the cans, which is the area between the fill line of the product and the can lid; corrosion in the headspace is particularly problematic with food products having a high salt content. Coatings can also be applied to the exterior of metal cans. Certain coatings of the present invention are particularly applicable for use with coiled metal stock, such as the coiled metal stock from which the ends of cans are made (“can end stock”), and end caps and closures are made (“cap/closure stock”). Since coatings designed for use on can end stock and cap/closure stock are typically applied prior to the piece being cut and stamped out of the coiled metal stock, they are typically flexible and extensible. Coatings for cans subjected to relatively stringent temperature and/or pressure requirements should also be resistant to popping, corrosion, blushing and/or blistering. Accordingly, the present invention is further directed to a package coated at least in part with any of the curable compositions described above. A “package” is anything used to contain another item, particularly for shipping from a point of manufacture to a consumer, and for subsequent storage by a consumer. A package will be therefore understood as something that is sealed so as to keep its contents free from deterioration until opened by a consumer. Thus, the present “package” is distinguished from a storage container or bakeware in which a consumer might make and/or store food; such a container would only maintain the freshness or integrity of the food item for a relatively short period. A package according to the present invention can be made of metal or non-metal, for example, plastic or laminate, and be in any form. An example of a suitable package is a laminate tube. Another example of a suitable package is a metal can. The term “metal can” includes any type of metal can, container, or any type of receptacle or portion thereof that is sealed by the food/beverage manufacturer to minimize or eliminate spoilage of the contents until such package is opened by the consumer. One example of a metal can is a food can; the term “food can(s)” is used herein to refer to cans, containers or any type of receptacle or portion thereof used to hold any type of food and/or beverage. The term “metal can(s)” specifically includes food cans and also specifically includes “can ends” including “E-Z open ends,” which are typically stamped from can end stock and used in conjunction with the packaging of food and beverages. The term “metal cans” also specifically includes metal caps and/or closures such as bottle caps, screw top caps and lids of any size, lug caps, and the like. The metal cans can be used to hold other items as well, including, but not limited to, personal care products, bug spray, spray paint, and any other compound suitable for packaging in an aerosol can. The cans can include “two piece cans” and “three-piece cans” as well as drawn and ironed one-piece cans; such one piece cans often find application with aerosol products. Packages coated according to the present invention can also include plastic bottles, plastic tubes, laminates and flexible packaging, such as those made from PE, PP, PET and the like. Such packaging could hold, for example, food, toothpaste, personal care products and the like. The coating can be applied to the interior and/or the exterior of the package. In some examples, the curable compositions prepared and used according to the present invention may be substantially free, may be essentially free, and/or may be completely free of bisphenol A and epoxy compounds derived from bisphenol A (“BPA”), such as bisphenol A diglycidyl ether (“BADGE”). The term “substantially free” as used in this context means the coatings compositions contain less than 1000 parts per million (ppm), “essentially free” means less than 100 ppm, and “completely free” means less than 20 parts per billion (ppb) of any of the above mentioned compounds, derivatives, or residues thereof. WORKING EXAMPLES The following working examples are intended to further describe the invention. It is understood that the invention described in this specification is not necessarily limited to the examples described in this section. In particular, the curable compositions of the disclosure may take various forms, including sealants, coatings, adhesives, encapsulants, and potting compositions, as set forth is some of the following examples presented herein. Example 1 A Curable Composition Comprising a Polyol and a 1,1-Di-Activated Vinyl Compound An acrylic polyol was prepared by copolymerizing, in percent by weight, 22.4% isostearic acid, 23.3% hydroxypropyl acrylate, 10.7% methyl methacrylate, 32.4% styrene, and 11.2% glycidyl methacrylate. The acrylic polyol was dissolved in xylene at 58.8% solids by weight. A crosslinker composition was provided comprising a 1,1-di-activated vinyl compound and a multifunctional form thereof (a combination of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,6-hexanediol). The acrylic polyol, the crosslinker composition, and an activator solution (comprising 5% 1,4-diazabicyclo[2.2.2]octane in n-butyl acetate) were combined together to form test samples as provided in Table 1. TABLE 1AcrylicCrosslinkerActivatorPolyol1Composition2OH:eneSolution3Sample(g)(g)Ratio(g)Gel TimeA00.20.000.015ImmediateB1.87510.470.0157 min. 30 sec.C1.87510.470.055 min.D1.87510.470.11 min. 20 sec.E1.87510.470.210 sec.F1.8750.50.940.0276 mins.G1.8750.50.940.0322 mins.H1.8750.50.940.0417 mins.I1.8750.50.940.0510 mins.J1.8750.50.940.13 mins.K1.8750.50.940.51 min. 15 sec.L1.8750.251.890.05>24 hr.M1.8750.251.890.1>24 hr.N1.8750.251.890.5>24 hr.O1.8750.251.890.8>24 hr.P1.8750.251.891.5>24 hr.1Acrylic polyol as described in U.S. Publication No. 2004/0234698, Example 4 (Table 5, Footnote 5).2A mixture of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,6-hexanediol, as described in U.S. Publication No. 2014/0329980.35 wt % 1,4-diazabicyclo[2.2.2]octane in n-butyl acetate. The samples were each combined in 20 mL scintillation vials in the order of (1) acrylic polyol, (2) activator solution, and (3) crosslinker composition with mixing before and after the addition of the crosslinker composition. The gel time was recorded as the time elapsed after combining all components until the composition did not demonstrate a visually observable flow when the vial was inverted. In samples where a gel formed, the gel was visually clear and homogeneous, indicating addition reaction between the hydroxyl groups on the acrylic polyol when present and the vinyl groups on the diethyl methylene malonate and/or the transesterification adduct of diethyl methylene malonate and 1,6-hexanediol. It was observed that gel time decreased as the amount of activator solution increased. It was also observed that gel time increased as the hydroxyl-to-vinyl group ratio (OH:ene ratio) increased (i.e., as the amount of crosslinker composition decreased). The results of this Example 1 indicate that curable compositions can be formulated based on curable compositions comprising a polyol and a 1,1-di-activated vinyl compound. Example 2 A Curable Composition Comprising a Polyamine and a 1,1-Di-Activated Vinyl Compound A trifunctional polyether polyamine was provided comprising a primary amine-terminated polyoxypropylene. (Jeffamine® T-403, available from Huntsman Corporation). A crosslinker composition was provided comprising a 1,1-di-activated vinyl compound and a multifunctional form thereof (a combination of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,5-pentanediol). The polyether polyamine and the crosslinker composition were combined together to form test samples as provided in Table 2. The samples were each combined in 20 mL scintillation vials and mixed. The gel time was recorded as the time elapsed after combining all components until the composition did not demonstrate a visually observable flow when the vial was inverted. TABLE 2PolyetherCrosslinkerPolyamine1Composition2NH:eneSample(g)(g)ratioGel TimeA0.8220.5<1 min.3B1.6221ImmediateC2.4321.5ImmediateD2.4313<2 min.1Jeffamine ® T-403, available from Huntsman Corporation.2A mixture of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,5-pentanediol, as described in U.S. Publication No. 2014/0329980.3Gel as defined above was not obtained but a very viscous liquid was formed quickly, the viscosity was such that very slow flow could be observed upon inversion of the vial. The gels that formed in Samples B and C, with amine-to-vinyl group ratios (NH:ene ratio) of 1 or 1.5 (slightly amine rich), were visually clear and homogenous, indicating addition reaction between the amine groups on the polyether polyamine and the vinyl groups on the diethyl methylene malonate and/or the transesterification adduct of diethyl methylene malonate and 1,5-pentanediol. The gels that formed in Samples A and D were relatively amine poor and amine rich, respectively, and contained small localized gel domains within an overall softer gel. Without intending to be bound by any theory, the amine groups on the polyether polyamine likely can function both as a reactant in addition reactions with the vinyl groups and as a catalyst for the addition reactions and the homopolymerization of the crosslinker composition (i.e., base-catalyzed polymerization of the vinyl groups in the diethyl methylene malonate and the transesterification adduct). The multiple functions of the amine groups (i.e., polymerization catalyst and crosslinkable functional group) can lead to inhomogenous gels comprising both polymerization and addition reaction products when the stoichiometry is shifted away from unity. The results of this Example 2 indicate that curable compositions can be formulated based on curable compositions comprising a polyamine and a 1,1-di-activated vinyl compound Example 3 A Curable Composition Comprising a Polythiol and a 1,1-Di-Activated Vinyl Compound A tetrafunctional polythiol was provided comprising pentaerythritol tetra(3-mercaptoproprionate) (THIOCURE® PETMP, available from Bruno Bock Chemische Fabrik GmbH & Co KG). Two crosslinker compositions were provided, each comprising a 1,1-di-activated vinyl compound and a multifunctional form thereof: (1) a combination of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,6-hexanediol (referred to below as the “HD” crosslinker composition); and (2) a combination of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,5-pentanediol (referred to below as the “PD” crosslinker composition). The polythiol, an HD or PD crosslinker composition, and, in some samples, an activator solution (comprising 5% 1,4-diazabicyclo[2.2.2]octane in n-butyl acetate) were combined together to form test samples as provided in Table 3. The samples were each combined in 20 mL scintillation vials in the order of (1) polythiol, (2) activator solution (if used), and (3) crosslinker composition with mixing before (if activator solution was used) and after the addition of the crosslinker composition. The gel time was recorded as the time elapsed after combining all components until the composition did not demonstrate a visually observable flow when the vial was inverted. TABLE 3CrosslinkerComposition2ActivatorPolythiol1(type/massthiol:eneSolution3Sample(g)(g))ratio(g)Gel TimeA1.0PD/0.21.0045min.B2.0PD/1.00.5045min.C1.0HD/1.520.940>24hrs.D1.0HD/1.520.940.01526min.E1.0HD/1.520.940.0520sec.1Pentaerythritol tetra(3-mercaptoproprionate).2A mixture of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,6-hexanediol (HD), or a mixture of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,5-pentanediol (PD), as described in U.S. Publication No. 2014/0329980.35 wt % 1,4-diazabicyclo[2.2.2]octane in n-butyl acetate. In addition to the in-vial gel time measurements, samples A and B were used to form coatings applied over cold-rolled steel substrates pre-coated with ED-6060C electrocoat (electrocoat available from PPG Industries, Inc., and substrate panels available in pre-coated form from ACT Test Panels LLC). The coatings were applied by drawdown over the electrocoat on the substrate panels using a wooden applicator to obtain a dry film thickness of 2-4 mils (approximately 50-100 microns). The coatings formed from samples A and B were tested for film drying and curing properties. Tack-free time was measured as the amount of time required for an applied coating film to achieve a level of dryness, such that, upon the application and removal of a cotton ball, no cotton fibers were transferred to the coating surface. The cotton ball was applied in the following manner:1. With the substrate panel in a horizontal position, hold a cotton ball approximately 3 inches above and drop the cotton ball onto the applied coating film.2. Hold the substrate panel coating side up for 5±2 seconds with the cotton ball in contact with the coating film.3. After the 5±2 seconds, flip the substrate panel coating side down.a. If the cotton ball drops off leaving no fibers on the film, the coating is tack-free.b. If the cotton ball does not drop off or leaves fibers, repeat steps 1-3 at appropriate time intervals (e.g., every 15 minutes) until coating is tack-free.MEK double rub tests were also performed. The MEK double rub test reports the number of double (back-and-forth) rubs, performed by hand with a methyl ethyl ketone (MEK) soaked rag, required to dissolve the applied coating such that the substrate is visible. This MEK double rub test was performed 1 hour after achieving a tack-free coating. The double rubs were performed up to a maximum number of 100 and discontinued. A gel formed for all of samples A-E, and the gels were visually clear and homogeneous, indicating addition reaction between the mercapto groups on the polythiol and the vinyl groups on the diethyl methylene malonate and/or the transesterification adduct of the diethyl methylene malonate. Likewise, the coatings formed from samples A and B were also visually clear and homogeneous. Sample A took 65 minutes and Sample B took 75 minutes to become tack-free after application over the pre-electrocoated steel substrates, and both coatings reached the maximum of 100 MEK double rubs as tested one hour after achieving a tack-free state. Comparing samples A and C, it was observed that with similar mercapto-to-vinyl group ratios (thiol:ene ratio), the use of the crosslinker composition comprising greater transesterification adduct content (i.e., the PD crosslinker composition used in sample A) correlated with more rapid gel time. Comparing samples C, D, and E, it was also observed that gel time decreased as the amount of activator solution increased. The results of this Example 3 indicate that coating compositions can be formulated based on curable compositions comprising a polythiol and a 1,1-di-activated vinyl compound. Example 4 A Coating Composition Comprising a Polyol, a 1,1-Di-Activated Vinyl Compound, and a Strong Acid An acrylic polyol having a weight average molecular weight of 8,600 (Determined by gel permeation chromatography using a Waters 2695 separation module equipped with a Waters 2414 differential refractometer (RI detector). Tetrahydrofuran (THF) was used as the eluent at a flow rate of 1 ml min-1, and two PLgel Mixed-C (300×7.5 mm) columns were used for separation) and a hydroxyl equivalent weight of 438 grams/equivalent (determined by titration with excess acetic anhydride, followed by back-titration with a standard potassium hydroxide solution) was prepared by polymerizing the following monomers (pbw=parts by weight): 140 pbw hydroxypropyl acrylate, 70 pbw styrene, 66.5 pbw butyl acrylate, 64.7 pbw butyl methacrylate, 7 pbw glacial acrylic acid, and 1.75 pbw methyl methacryalate. The acrylic polyol was dissolved at 67% solids by weight in a mixture of ShellSol A100 solvent (a predominantly C9 aromatic hydrocarbon solvent available from Shell Chemicals) and propylene glycol monomethyl ether acetate (PM Acetate available from Eastman Chemical Company). A crosslinker composition was provided comprising a 1,1-di-activated vinyl compound and a multifunctional form thereof (a combination of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,5-pentanediol). The acrylic polyol, the crosslinker composition, and a strong acid promoter (phosphotungstic acid) were combined together in the amounts provided in Table 4 to form a coating composition. TABLE 4ComponentPart by weight (grams)Acrylic Polyol152.9Crosslinker Cornposition242.1Phosphotungstic Acid51Acrylic polyol with Mw of 8,600 hydroxyl equivalent weight of 438 g/eq., 67% solids in A100/PM acetate solvent.2A mixture of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,5-pentanediol, as described in U.S. Publication No. 2014/0329980. A multi-layer coating was prepared by applying the coating composition shown in Table 4 over a steel substrate pre-coated with ED-6465 electrocoat (electrocoat available from PPG Industries, Inc., and substrate panels available in pre-coated form from ACT Test Panels LLC). The components of the coating composition were mixed together in a vial at room temperature then applied by drawdown over the electrocoat on the substrate panels using a drawdown bar with a 3 mil gap. The coated panel was allowed to flash for 7 minutes at ambient conditions and baked for 30 mins at 140° C. The resulting coating was solvent resistant as indicated by surviving 100 MEK double rubs. The coating was visually homogeneous and glossy. Without intending to be bound by any theory, it is believed that the inorganic strong acid facilitated an addition reaction between the hydroxyl groups on the acrylic polyol and the vinyl groups on the diethyl methylene malonate and/or the transesterification adduct of diethyl methylene malonate and 1,5-pentanediol. Example 5 A Coating Composition Comprising a Polycarbamate, a 1,1-Di-Activated Vinyl Compound, and a Strong Acid A polyester polycarbamate having a weight average molecular weight of 2,200 (Determined by gel permeation chromatography using a Waters 2695 separation module equipped with a Waters 2414 differential refractometer (RI detector). Tetrahydrofuran (THF) was used as the eluent at a flow rate of 1 ml min-1, and two PLgel Mixed-C (300×7.5 mm) columns were used for separation) and a carbamate/hydroxyl combined equivalent weight of 413 grams/equivalent was prepared as described in Example 3 of U.S. Pat. No. 6,228,953 B1. The polyester polycarbamate was dissolved at 66% solids by weight in a mixture of Dowanol PM (glycol ether available from The Dow Chemical Company), propylene glycol monomethyl ether acetate (PM Acetate available from Eastman Chemical Company), ShellSol A100 solvent (a predominantly C9 aromatic hydrocarbon solvent available from Shell Chemicals), and xylene in the ratio 12.7:13.3:7.4:0.6, by weight. A crosslinker composition was provided comprising a 1,1-di-activated vinyl compound and a multifunctional form thereof (a combination of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,5-pentanediol). The polyester polycarbamate, the crosslinker composition, and a strong acid promoter (dodecylbenzenesulfonic acid) were combined together in the amounts provided in Table 5 to form a coating composition. TABLE 5ComponentPart by weight (grams)Polyester Polycarbamate151.5Crosslinker Composition243.5Dodecylbenzenesulfonic Acid51Polyester with Mw of 2,300, hydroxyl and carbamate functionality combined equivalent weight of 413 g/eq., 66% solids in Dowanol PM/PM acetate/A100/xylene solvent.2A mixture of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,5-pentanediol, as described in U.S. Publication No. 2014/0329980. A multi-layer coating was prepared by applying the coating composition shown in Table 5 over a steel substrate pre-coated with ED-6465 electrocoat (electrocoat available from PPG Industries, Inc., and substrate panels available in pre-coated form from ACT Test Panels LLC). The components of the coating composition were mixed together in a vial at room temperature then applied by drawdown over the electrocoat on the substrate panels using a drawdown bar with a 3 mil gap. The coated panel was allowed to flash for 7 minutes at ambient conditions and baked for 30 mins at 140° C. The resulting coating was solvent resistant as indicated by surviving 100 MEK double rubs. The coating was visually homogeneous and glossy. Without intending to be bound by any theory, it is believed that the organic strong acid facilitated an addition reaction between the carbamate groups and hydroxyl groups on the polyester polycarbamate and the vinyl groups on the diethyl methylene malonate and/or the transesterification adduct of diethyl methylene malonate and 1,5-pentanediol. Example 6 A Curable Composition Comprising a Polythiol, a 1,1-Di-Activated Vinyl Compound, and a Strong Acid A tetrafunctional polythiol was provided comprising pentaerythritol tetra(3-mercaptoproprionate) (THIOCURE® PETMP, available from Bruno Bock Chemische Fabrik GmbH & Co KG). A crosslinker composition was provided comprising a 1,1-di-activated vinyl compound and a multifunctional form thereof (a combination of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,5-pentanediol). The polythiol, the crosslinker composition, and a strong acid promoter (methanesulfonic acid) were combined together in the amounts provided in Table 6 to form a curable composition. TABLE 6ComponentPart by weight (grams)Polythiol157Crosslinker Composition241Methanesulfonic Acid21Pentaerythritol tetra(3-mercaptoproprionate).2A mixture of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,5-pentanediol, as described in U.S. Publication No. 2014/0329980. Under ambient conditions, the methanesulfonic acid was added to a 20 mL glass vial containing the crosslinker composition. The vial was shaken for several minutes. The polythiol was then added, immediately stirred with a wooden applicator stick, then shaken. Within seconds the vial was hot to the touch, the viscosity of the mixture rapidly increased, and a homogenous gel was formed within 5 minutes. The homogeneity of the resulting gel indicated the occurrence of addition reactions between the mercapto groups on the polythiol and the vinyl groups on the diethyl methylene malonate and/or the transesterification adduct of diethyl methylene malonate and 1,5-pentanediol Example 6 was repeated without the addition of the methanesulfonic acid for comparison. The polythiol and the crosslinker composition were combined together in the amounts provided in Table 7. TABLE 7ComponentPart by weight (grams)Thiocure PETMP158DEMM Pentanediol Crosslinker2421Pentaerythritol tetra(3-mercaptoproprionate).2A mixture of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,5-pentanediol, as described in U.S. Publication No. 2014/0329980. Under ambient conditions, the polythiol was added to a 20 mL glass vial containing the crosslinker composition. The vial was shaken for several minutes. There was no observed exotherm from the mixing of the two reactants. The viscosity of the mixture increased steadily over the course of 3 hours resulting in a homogenous gel. Without intending to be bound by any theory, it is believed that the organic strong acid facilitated an addition reaction between the mercapto groups on the polythiol and the vinyl groups on the diethyl methylene malonate and/or the transesterification adduct of diethyl methylene malonate and 1,5-pentanediol. This addition reaction is believed to occur without the strong acid promoter, but at a slower reaction rate. Example 7 A Coating Composition Comprising a Polyol and a 1,1-Di-Activated Vinyl Compound An acrylic polyol was prepared by copolymerizing, in percent by weight, 22.4% isostearic acid, 23.3% hydroxypropyl acrylate, 10.7% methyl methacrylate, 32.4% styrene, and 11.2% glycidyl methacrylate. The acrylic polyol was dissolved in xylene at 58.8% solids by weight. A crosslinker composition was provided comprising a 1,1-di-activated vinyl compound and a multifunctional form thereof (a combination of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,6-hexanediol). The acrylic polyol and the crosslinker composition were combined together under ambient conditions in the amounts provided in Table 8 to form a coating composition. TABLE 8ComponentPart by weight (grams)Acrylic Polyol179DEMM Hexanediol Crosslinker2211Acrylic polyol as described in U.S. Publication No. 2004/0234698, Example 4 (Table 5, Footnote 5).2A mixture of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,6-hexanediol, as described in U.S. Publication No. 2014/0329980. A multi-layer coating was prepared by applying the coating composition shown in Table 8 over a steel substrate pre-coated with ED-6060C electrocoat (electrocoat available from PPG Industries, Inc., and substrate panels available in pre-coated form from ACT Test Panels LLC). The components of the coating composition were mixed together in a vial at room temperature then applied by drawdown over the electrocoat on the substrate panels using a drawdown bar with a 3 mil gap. Then 8 g of an activator solution of 1,4-diazabicyclo[2.2.2] octane (DABCO) in n-butyl acetate (0.3% solution by weight) was spray applied over the coating film using a SATA Jet 4000 B HVLP with a 1.3 mm nozzle at 10 psi. The resultant cured coating film was tack-free in 25 minutes as measured by the time from the activator solution spray at which a cotton ball leaves no fibers behind when applied onto the surface of the applied coating film as described above in Example 3. Example 8 Curable Compositions Comprising a 1,1-Di-Activated Vinyl Compound and an Ionic Liquid Activator, Optionally a Polythiol, and Optionally an Extender A crosslinker composition was provided comprising a 1,1-di-activated vinyl compound and a multifunctional form thereof (a combination of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,6-hexanediol). An ionic liquid was provided comprising an alkylimidazolinium phthalate. A tetrafunctional polythiol was provided comprising pentaerythritol tetra(3-mercaptoproprionate). An extender was provided comprising a vinyl polymer prepared using an ethylenically unsaturated carboxylic acid anhydride. The crosslinker composition (or unreacted diethyl methylene malonate), the ionic liquid, the polythiol, and the extender were combined together in the amounts provided in Table 9 to form curable compositions. TABLE 9CrosslinkerIonicComposition1DEMM2Liquid3Polythiol4Extender5Sample(g)(g)(g)(g)(g)A—2.00.03—B2.0—0.04—C—2.00.07—1.00D2.0—0.09—0.50E2.0——1.39F2.0—0.021.39G2.0—0.071.391.001A mixture of diethyl methylene malonate and a transesterification adduct of diethyl methylene malonate and 1,6-hexanediol, as described in U.S. Publication No. 2014/0329980.2Unreacted diethyl methylene malonate (monomeric).3An alkylimidazolinium phthalate, available as IL-002 from Sanyo Chemical Industries, Ltd.4Pentaerythritol tetra(3-mercaptoproprionate), available as THIOCURE ® PETMP, available from Bruno Bock Chemische Fabrik GmbH & Co KG.5Example 1 in U.S. Pat. No. 4,798,745, column 10, line 40-column 11, line, 22, incorporated by reference into this specification. The components of the curable compositions listed in Table 9 were mixed together in vials at room temperature. The curable compositions were evaluated for gel time, and select formulations were evaluated for coating film drying and curing properties (tack-free time) and solvent resistance (MEK double rub test). Gel time was measured as the time elapsed after combining all ingredients until the composition did not demonstrate a visually observable flow when the vial containing the composition was inverted. Coating films were prepared by applying the curable compositions listed in Table 9 over 10.16 cm by 30.18 cm cold-rolled steel substrate panels pre-coated with ED-6060 electrocoat (electrocoat available from PPG Industries, Inc., and substrate panels available in pre-coated form from ACT Test Panels LLC). The coatings were applied immediately upon mixing by drawdown over the electrocoat on the substrate panels using a drawdown bar with a 2-4 mil gap (50-102 micrometers). Tack-free time was measured as the amount of time required for a coating to achieve a level of dryness such that upon the application and removal of a cotton ball no cotton fibers were transferred to the coating surface. MEK double rubs (MEK DR) are reported as the number of double rubs performed by hand with a methyl ethyl ketone soaked rag required to dissolve the coating such that the substrate is visible, up to a maximum number of 100 MEK DR. The gel time, tack-free time, and MEK DR results are reported in Table 10. TABLE 10Tack-Free TimeTack-Free Time(after 60° C. bakeSampleGel Time(ambient temperature)for 10 minutes)MEK DRA3min.3min.——B6min.7min.——C>1hour>1hour0 min.0D30min.—0 min.100E3hour>3hour——F<1min.———G10min.>1hour—— The gel time and tack-free time of Samples A and B show that the ionic liquid was effective at polymerizing the DEMM and the crosslinker composition both in the vial and as a coating film applied onto a panel. It is noted that the short gel time and tack-free time of Samples A and B indicate rapid polymerization of the DEMM or DEMM crosslinker. Sample C was similar to Sample A but it further comprised the extender, which provided for a longer gel time and tack-free time at ambient conditions. Upon a brief bake (60° C. for 10 minutes), Sample C formed a tack-free coating, but did not survive any MEK DR, which was unsurprising because the reaction product is believed to be an un-crosslinked linear polymer formed from anionic polymerization of the DEMM monomer. Sample D was similar to Sample B but it further comprised the extender, which provided for a longer gel time and tack-free time at ambient conditions. Upon a brief bake (60° C. for 10 minutes), Sample D formed a tack-free coating that survived 100 MEK DR, indicating it formed a solvent-resistant crosslinked coating. Sample E, which contained no ionic liquid, exhibited a relatively slow reaction of the crosslinker composition and the polythiol as indicated by the long gel time and tack-free time. Sample F, which was similar to Sample E but contained added ionic liquid, exhibited a substantially faster reaction as indicated by the gel time under one minute. In fact, the reaction of Sample F upon initial mixing was so fast that it was not possible to apply the composition as a coating film on a substrate panel before the composition was too viscous to apply. Sample G was similar to Sample F but further comprised added extender. The addition of the extender resulted in an intermediate gel time of 10 minutes, as compared to over 3 hours for Sample E and less than one minute for Sample F, which provides a more practical pot life for the composition. These examples demonstrate the utility of ionic liquid for activating the cure of 1,1-di-activated vinyl compounds and multifunctional forms thereof, alone or in combination with polyfunctional materials such as polyols, polyamines, polythiols, and/or polycarbamates. The use of an extender to control the reaction rate was also demonstrated, thereby providing control over pot life and cure response, and facilitating longer gel times that extend the usable application time of the compositions while still maintaining reasonably fast curing kinetics. ASPECTS OF THE INVENTION Aspects of the invention include, but are not limited to, the following numbered clauses. 1. A curable composition comprising: a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof; and a 1,1-di-activated vinyl compound, or a multifunctional form thereof, or a combination thereof. 2. The curable composition of clause 1, wherein the 1,1-di-activated vinyl compound comprises a methylene dicarbonyl compound, a dihalo vinyl compound, a dihaloalkyl disubstituted vinyl compound, or a cyanoacrylate compound, or a multifunctional form of any thereof, or a combination of any thereof. 3. The curable composition of clause 1 or clause 2, wherein the 1,1-di-activated vinyl compound comprises a dialkyl methylene malonate, a diaryl methylene malonate, a multifunctional form of a dialkyl methylene malonate, or a multifunctional form of a diaryl methylene malonate, or a combination of any thereof. 4. The curable composition of any one of clauses 1-3, wherein the 1,1-di-activated vinyl compound comprises diethyl methylene malonate and a multifunctional form of diethyl methylene malonate comprising a transesterification adduct of diethyl methylene malonate and at least one polyol. 5. The curable composition of clause 4, wherein the transesterification adduct of diethyl methylene malonate and at least one polyol comprises a transesterification adduct of diethyl methylene malonate and a diol. 6. The curable composition of clause 5, wherein the diol comprises an alkane diol. 7. The curable composition of clause 6, wherein the alkane diol comprises 1,5-pentane diol and/or 1,6-hexanediol. 8. The curable composition of any one of clauses 1-7, further comprising a strong acid. 9. The curable composition of clause 8, wherein the strong acid comprises a sulfonic acid and/or a heteropoly acid. 10. The curable composition of any one of clauses 1-9, further comprising an activator compound. 11. The curable composition of clause 10, wherein the activator compound comprises a tertiary amine. 12. The curable composition of clause 10 or 11, wherein the activator compound comprises an ionic liquid. 13. The curable composition of any one of clauses 1-12, further comprising an acid promoter and an activator compound. 14. The curable composition of clause 13, wherein the acid promoter comprises a strong acid and the activator compound comprises a tertiary amine and/or an ionic liquid. 15. The curable composition of clause 14, wherein the strong acid comprises a sulfonic acid and/or a heteropoly acid. 16. The curable composition of any one of clauses 1-15, further comprising an extender. 17. The curable composition of clause 16, wherein the extender comprises an anhydride-containing vinyl polymer. 18. The curable composition of clause 17, wherein the anhydride-containing vinyl polymer comprises maleic anhydride monomer residues. 19. A multi-layer curable composition comprising: a first curable composition layer applied over at least a portion of a substrate; and a second curable composition layer applied over at least a portion of the first coating layer; wherein the first curable composition layer and/or the second curable composition layer comprises one or more of: (1) an addition reaction product of: (1a) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof; and (1b) a 1,1-di-activated vinyl compound, or a multifunctional form thereof, or a combination thereof; and/or (2) a polymerization reaction product of the 1,1-di-activated vinyl compound, or a multifunctional form thereof, or a combination thereof. 20. The multi-layer curable composition of clause 19, wherein the 1,1-di-activated vinyl compound comprises a methylene dicarbonyl compound, a dihalo vinyl compound, a dihaloalkyl disubstituted vinyl compound, or a cyanoacrylate compound, or a multifunctional form of any thereof, or a combination of any thereof. 21. The multi-layer curable composition of clause 19 or clause 20, wherein the first curable composition layer and/or the second curable composition layer comprises an addition reaction product of: (1a) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof, and (1b) a dialkyl methylene malonate, a diaryl methylene malonate, a multifunctional form of a dialkyl methylene malonate, or a multifunctional form of a diaryl methylene malonate, or a combination of any thereof. 22. The multi-layer curable composition of any one of clauses 19-21, wherein the first curable composition layer and/or the second curable composition layer comprises an addition reaction product of: (1a) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof; and (1b) diethyl methylene malonate and a multifunctional form of diethyl methylene malonate, wherein the multifunctional form of diethyl methylene malonate comprises a transesterification adduct of diethyl methylene malonate and at least one polyol. 23. The multi-layer curable composition of clause 22, wherein the transesterification adduct of diethyl methylene malonate and at least one polyol comprises a transesterification adduct of diethyl methylene malonate and a diol. 24. The multi-layer curable composition of any one of clauses 19-23, wherein: (i) the first curable composition layer comprises an activator compound that activated addition reactions in the second curable composition layer when the second curable composition layer was applied over the first curable composition layer; or (ii) the second curable composition layer comprises an activator compound that activated addition reactions in the first curable composition layer when the second curable composition layer was applied over the first curable composition layer. 25. The multi-layer curable composition of clause 24, wherein the activator compound comprises a tertiary amine compound. 26. The multi-layer curable composition of clause 25, wherein the activator compound comprises 2-(dimethylamino)ethanol and/or 1,4-diazabicyclo[2.2.2]octane. 27. The multi-layer coating of clause 24-26, wherein the activator compound comprises an ionic liquid. 28. The multi-layer curable composition of any one of clauses 19-27, wherein the first coating layer and/or the second curable composition layer is formed from a curable composition comprising a strong acid. 29. The multi-layer curable composition of clause 28, wherein the strong acid comprises a sulfonic acid and/or a heteropoly acid. 30. The multi-layer curable composition of any one of clauses 19-29, further comprising an extender comprising an anhydride-containing vinyl polymer. 31. The multi-layer curable composition of any one of clauses 19-30, wherein the first curable composition layer is formed from a curable composition composition that cures when heated at a temperature of less than 500° C.; and wherein the second curable composition layer comprises (1) the addition reaction product and/or (2) the polymerization reaction product. 32. The multi-layer curable composition of any one of clauses 19-31, wherein the curable composition that forms the first coating layer is substantially free of melamine resin and formaldehyde condensates. 33. The multi-layer curable composition of any one of clauses 19-32, wherein the second curable composition layer comprises a clearcoat layer. 34. An article comprising the multi-layer curable composition of any one of clauses 19-33 deposited over a surface of the article. 35. The article of clause 34, wherein the article comprises a vehicle component or a component of a free-standing structure. 36. A process for coating a substrate comprising: applying a first curable composition layer over at least a portion of a substrate; applying a second curable composition layer over at least a portion of the first curable composition layer; and curing the first curable composition layer and/or the second curable composition layer; wherein the first c curable composition layer and/or the second curable composition layer is formed from a curable composition comprising: a polyol, a polyamine, polythiol, or a polycarbamate, or a combination of any thereof; and a 1,1-di-activated vinyl compound, or a multifunctional form thereof, or a combination thereof. 37. The process of clause 36, wherein the 1,1-di-activated vinyl compound comprises a methylene dicarbonyl compound, a dihalo vinyl compound, a dihaloalkyl disubstituted vinyl compound, or a cyanoacrylate compound, or a multifunctional form of any thereof, or a combination of any thereof. 38. The process of clause 36 or clause 37, wherein the curing of the first curable composition layer and/or the second curable composition layer comprises spraying an activator solution over and/or under at least a portion of the first curable composition layer and/or the second curable composition layer. 39. The process of clause 38, wherein the activator solution comprises an amine activator. 40. The process of clause 39, wherein the amine activator comprises 2-(dimethylamino)ethanol and/or 1,4-diazabicyclo[2.2.2]octane. 41. The process of any one of clauses 36-40, wherein: (i) the first curable composition layer comprises an activator compound, and wherein the curing of the second curable composition layer comprises activating an addition reaction in the second curable composition layer with the activator compound in the first curable composition layer; or (ii) the second curable composition layer comprises an activator compound, and wherein the curing of the first curable composition layer comprises activating an addition reaction in the first curable composition layer with the activator compound in the second curable composition layer. 42. The process of clause 41, wherein the activator compound comprises a tertiary amine compound. 43. The process of clause 42, wherein the activator compound comprises 2-(dimethylamino)ethanol and/or 1,4-diazabicyclo[2.2.2]octane. 44. The process of clause 41-43, wherein the activator compound comprises an ionic liquid. 45. The process of any one of clauses 36-44, wherein at least one of the first curable composition layer and/or the second curable composition layer, when cured, comprises an addition reaction product of: (1a) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof; and (1b) a dialkyl methylene malonate, a diaryl methylene malonate, a multifunctional form of a dialkyl methylene malonate, or a multifunctional form of a diaryl methylene malonate, or a combination of any thereof. 46. The process of any one of clauses 36-45, wherein at least one of the first curable composition layer and/or the second curable composition layer, when cured, comprises an addition reaction product of: (1a) a polyol, a polyamine, a polythiol, or a polycarbamate, or a combination of any thereof; and (1b) diethyl methylene malonate and a multifunctional form of diethyl methylene malonate, wherein the multifunctional form of diethyl methylene malonate comprises a transesterification adduct of diethyl methylene malonate and at least one polyol. 47. The process of clause 46, wherein the transesterification adduct of diethyl methylene malonate and at least one polyol comprises a transesterification adduct of diethyl methylene malonate and a diol. 48. The process of any one of clauses 36-47, wherein the curable composition further comprises a strong acid. 49. The process of clause 48, wherein the strong acid comprises a sulfonic acid and/or a heteropoly acid. 50. The process of any one of clauses 36-49, wherein the curable composition further comprises an extender comprising an anhydride-containing vinyl polymer. 51. An article comprising the curable composition of clause 1 deposited over a surface of the article. 52. The article of clause 51, wherein the article comprises a vehicle component or a component of a free-standing structure. Various features and characteristics are described in this specification to provide an understanding of the composition, structure, production, function, and/or operation of the invention, which includes the disclosed compositions, coatings, and processes. It is understood that the various features and characteristics of the invention described in this specification can be combined in any suitable manner, regardless of whether such features and characteristics are expressly described in combination in this specification. The Inventors and the Applicant expressly intend such combinations of features and characteristics to be included within the scope of the invention described in this specification. As such, the claims can be amended to recite, in any combination, any features and characteristics expressly or inherently described in, or otherwise expressly or inherently supported by, this specification. Furthermore, the Applicant reserves the right to amend the claims to affirmatively disclaim features and characteristics that may be present in the prior art, even if those features and characteristics are not expressly described in this specification. Therefore, any such amendments will not add new matter to the specification or claims, and will comply with written description, sufficiency of description, and added matter requirements, including the requirements under 35 U.S.C. § 112(a) and Article 123(2) EPC. Any numerical range recited in this specification describes all sub-ranges of the same numerical precision (i.e., having the same number of specified digits) subsumed within the recited range. For example, a recited range of “1.0 to 10.0” describes all sub-ranges between (and including) the recited minimum value of 1.0 and the recited maximum value of 10.0, such as, for example, “2.4 to 7.6,” even if the range of “2.4 to 7.6” is not expressly recited in the text of the specification. Accordingly, the Applicant reserves the right to amend this specification, including the claims, to expressly recite any sub-range of the same numerical precision subsumed within the ranges expressly recited in this specification. All such ranges are inherently described in this specification such that amending to expressly recite any such sub-ranges will comply with written description, sufficiency of description, and added matter requirements, including the requirements under 35 U.S.C. § 112(a) and Article 123(2) EPC. Also, unless expressly specified or otherwise required by context, all numerical parameters described in this specification (such as those expressing values, ranges, amounts, percentages, and the like) may be read as if prefaced by the word “about,” even if the word “about” does not expressly appear before a number. Additionally, numerical parameters described in this specification should be construed in light of the number of reported significant digits, numerical precision, and by applying ordinary rounding techniques. It is also understood that numerical parameters described in this specification will necessarily possess the inherent variability characteristic of the underlying measurement techniques used to determine the numerical value of the parameter. The invention(s) described in this specification can comprise, consist of, or consist essentially of the various features and characteristics described in this specification. The terms “comprise” (and any form of comprise, such as “comprises” and “comprising”), “have” (and any form of have, such as “has” and “having”), “include” (and any form of include, such as “includes” and “including”), and “contain” (and any form of contain, such as “contains” and “containing”) are open-ended linking verbs. Thus, a composition, coating, or process that “comprises,” “has,” “includes,” or “contains” one or more features and/or characteristics possesses those one or more features and/or characteristics, but is not limited to possessing only those one or more features and/or characteristics. Likewise, an element of a composition, coating, or process that “comprises,” “has,” “includes,” or “contains” one or more features and/or characteristics possesses those one or more features and/or characteristics, but is not limited to possessing only those one or more features and/or characteristics, and may possess additional features and/or characteristics. The grammatical articles “a,” “an,” and “the,” as used in this specification, including the claims, are intended to include “at least one” or “one or more”, unless otherwise indicated. Thus, the articles are used in this specification to refer to one or more than one (i.e., to “at least one”) of the grammatical objects of the article. By way of example, “a component” means one or more components, and thus, possibly, more than one component is contemplated and can be employed or used in an implementation of the described compositions, coatings, and processes. Nevertheless, it is understood that use of the terms “at least one” or “one or more” in some instances, but not others, will not result in any interpretation where failure to use the terms limits objects of the grammatical articles “a,” “an,” and “the” to just one. Further, the use of a singular noun includes the plural, and the use of a plural noun includes the singular, unless the context of the usage requires otherwise. Any patent, publication, or other document identified in this specification is incorporated by reference into this specification in its entirety unless otherwise indicated, but only to the extent that the incorporated material does not conflict with existing descriptions, definitions, statements, illustrations, or other disclosure material expressly set forth in this specification. As such, and to the extent necessary, the express disclosure as set forth in this specification supersedes any conflicting material incorporated by reference. Any material, or portion thereof, that is incorporated by reference into this specification, but which conflicts with existing definitions, statements, or other disclosure material set forth herein, is only incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material. Applicant reserves the right to amend this specification to expressly recite any subject matter, or portion thereof, incorporated by reference. The amendment of this specification to add such incorporated subject matter will comply with written description, sufficiency of description, and added matter requirements, including the requirements under 35 U.S.C. § 112(a) and Article 123(2) EPC. | 121,115 |
11859102 | DETAILED DESCRIPTION InFIG.1, two metallic steel pipes1are apparent, which are joined in the abutment region3with a welded connection2. In order to be able to apply the welded connection2, a plastic sheathing, or a so-called factory jacket, in the form of a polyethylene (PE) layer S, has initially been removed in the abutment region, allowing the exposed surface regions3at the end of the metallic inner pipes to be subsequently joined with the welded connection2. So that the pipes1do not subsequently undergo corrosion in the region of their abutment and of the surface3exposed there, an anticorrosion wrapping is applied to the surface3in accordance with the invention, as elucidated in detail hereinafter. For this purpose, first of all, in accordance with the representation inFIG.1, the surface3is cleaned and freed as and where necessary from residual moisture, as shown inFIG.2. Operation for this purpose may take place with a hot air fan or, as represented, with an open propane gas flame. In the subsequentFIG.3, as and where necessary, the transition of the surface3to the wrapping or to the PE layer S is then smoothed and cleaned, this being illustrated by a suggested abrasive paper. Subsequently, in the context ofFIG.4, an elastomer-based undercoat composition4is applied. The undercoat composition4used in accordance with the invention is an aqueous and also solvent-free dispersion, which is applied to the surface3to be protected. For details regarding the chemical constitution and the physical properties, especially the peel resistance, of the adhesion promoter4, reference may be made to the observations above. When the undercoat composition4has dried, which generally requires drying times of less than 10 min at room temperature, the actual wrapping can then be applied. The drying of the undercoat composition4also entails a change in color. In fact, while still wet, the adhesion promoter4predominantly possesses a white color and becomes transparent as drying progresses, meaning that the drying process can be verified visually and possibly by touch. After the drying of the adhesion promoter4, in accordance with the representation inFIG.5, an inner tape5is first applied to the surface3by winding with a slight tension. The inner tape5in this case is wound helically around the surface3of the steel pipe that is to be sheathed, and also around the adjacent regions of the PE layer S. An overlap Ü of at least 30% is observed, as shown in enlarged form inFIG.5. In other words, the overlap Ü of at least 30% means that a subsequent wrapping or turn covers a leading wrap with at least 30% of its width, as depicted in the enlarged representation inFIG.5. When the inner tape5has been applied to the surface3to be sheathed, the outer tape6is subsequently wound in a comparable way, with similar overlap, likewise helically around the surface3or around the inner tape5, as represented byFIG.6. The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are to be included within the scope of the following claims. | 3,263 |
11859103 | DETAILED DESCRIPTION The present disclosure relates to thermoplastic polymer particles suitable for use in additive manufacturing and related methods where warping in additive manufacturing methods like SLS may be mitigated. More specifically, the present disclosure includes thermoplastic polymer particles that are highly spherical and comprise a thermoplastic polymer and a nucleating agent. The nucleating agent may, advantageously, increase the crystallization temperature of the thermoplastic polymer, which may allow the object (or layer thereof) to sufficiently solidify before warping (or significant warping) occurs. Further, in some instances, the process of melt emulsification may reduce the crystallization temperature of the thermoplastic polymer. That is, the polymer particles produced by melt emulsification of a thermoplastic polymer may have a lower crystallization temperature than the thermoplastic polymer starting material. Advantageously, the inclusion of nucleating agents may cause the crystallization temperature to be substantially maintained when comparing (a) the thermoplastic polymer material without a nucleating agent and prior to melt emulsification and (b) the polymer particles produced by melt emulsification of said thermoplastic polymer with a nucleating agent present. For example, the polymer particles may have a crystallization temperature within about 10° C. (or about 7° C., or about 5° C., or about 3° C.) of the thermoplastic polymer starting material without a nucleating agent. Accordingly, by mitigating a reduction in crystallization temperature, the warping in subsequent additive manufacturing methods like SLS may be mitigated. Additionally, the highly spherical nature of the thermoplastic polymer particles described herein may provide better flow characteristics and, consequently, better consolidation in SLS methods, especially as compared to cryo-milled particles. Definitions and Test Methods Numerical ranges used herein include the numbers recited in the range. For example, the numerical range “from 1 wt % to 10 wt %” includes 1 wt % and 10 wt % within the recited range. As used herein, the term “immiscible” refers to a mixture of components that, when combined, form two or more phases that have less than 5 wt % solubility in each other at ambient pressure and at room temperature or the melting point of the component if it is solid at room temperature. For example, polyethylene oxide having 10,000 g/mol molecular weight is a solid room temperature and has a melting point of 65° C. Therefore, said polyethylene oxide is immiscible with a material that is liquid at room temperature if said material and said polyethylene oxide have less than 5 wt % solubility in each other at 65° C. A “polymer” has two or more of the same or different mer units. A “homopolymer” is a polymer having mer units that are the same. The term “polymer” as used herein includes, but is not limited to, homopolymers, copolymers, terpolymers, etc. The term “polymer” as used herein also includes impact, block, graft, random, and alternating copolymers. The term “polymer” shall further include all possible geometrical configurations unless otherwise specifically stated. Such configurations may include isotactic, syndiotactic, and random symmetries. As used herein, the term “thermoplastic polymer” refers to a plastic polymer material that softens and hardens reversibly on heating and cooling. Thermoplastic polymers encompass thermoplastic elastomers. As used herein, the term “elastomer” refers to a copolymer comprising a crystalline “hard” section and an amorphous “soft” section. In the case of a polyurethane, the crystalline section may include a portion of the polyurethane comprising the urethane functionality and optional chain extender group, and the soft section may include the polyol, for instance. As used herein, the term “oxide” refers to both metal oxides and non-metal oxides. For purposes of the present disclosure, silicon is considered to be a metal. An “olefin,” alternatively referred to as “alkene,” is a linear, branched, or cyclic compound of carbon and hydrogen having at least one double bond. Further, when a polymer is referred to as “comprising an olefin” or as a “polyolefin,” the olefin present in the polymer is the polymerized form of the olefin. As used herein, when a polymer is referred to as “comprising, consisting of, or consisting essentially of” a monomer, the monomer is present in the polymer in the polymerized form of the monomer or is the derivative form of the monomer. For example, when a copolymer is said to have an “ethylene” content of 35 wt % to 55 wt %, it is understood that the mer unit in the copolymer is derived from ethylene in the polymerization reaction and said derived units are present at 35 wt % to 55 wt %, based upon the weight of the copolymer. As used herein, the term “embed” relative to particles (e.g., nanoparticles) and a surface of a polymer particle refers to the particle being at least partially extending into the surface of the polymer particle such that polymer is in contact with the nanoparticle to a greater degree than would be if the nanoparticle were simply laid on the surface of the polymer particle. Herein, D10, D50, D90, and diameter span are primarily used herein to describe particle sizes. As used herein, the term “D10” refers to a diameter below which 10% (on a volume-based distribution, unless otherwise specified) of the particle population is found. As used herein, the terms “D50”, “average particle diameter,” and “average particle size” refers to a diameter below which 50% (on a volume-based median average, unless otherwise specified) of the particle population is found. As used herein, the term “D90” refers to a diameter below which 90% (on a volume-based distribution, unless otherwise specified) of the particle population is found. As used herein, the terms “diameter span” and “span” and “span size” when referring to diameter provides an indication of the breadth of the particle size distribution and is calculated as (D90-D10)/D50. Particle diameters and particle size distributions are determined by light scattering techniques using a Malvern MASTERSIZER™ 3000. For light scattering techniques, the control samples were glass beads with a diameter within the range of 15 μm to 150 μm under the tradename Quality Audit Standards QAS4002™ obtained from Malvern Analytical Ltd. Samples were analyzed as dry powders, unless otherwise indicated. The particles analyzed were dispersed in air and analyzed using the AERO S™ dry powder dispersion module with the MASTERSIZER™ 3000. The particle sizes were derived using instrument software from a plot of volume density as a function of size. As used herein, when referring to sieving, pore/screen sizes are described per U.S.A. Standard Sieve (ASTM E11-17). As used herein, the terms “circularity” relative to the particles refer to how close the particle is to a perfect sphere. To determine circularity, optical microscopy images using flow particle imaging are taken of the particles. The perimeter (P) and area (A) of the particle in the plane of the microscopy image is calculated (e.g., using a SYSMEX FPIA 3000 particle shape and particle size analyzer, available from Malvern Instruments). The circularity of the particle is CEA/P, where CEAis the circumference of a circle having the area equivalent to the area (A) of the actual particle. Herein, the circularity is based on three runs through a SYSMEX FPIA 3000 particle shape and particle size analyzer, where 6,000 to 10,000 particles are analyzed per run. The reported circularity is the median average circularity based on particle number. In the analysis, a threshold for distinguishing the greyscale levels between the background pixels and the particle pixels (e.g., to correct for non-uniform illumination conditions) was set at 90% of the background modal value. As used herein, the term “shear” refers to stirring or a similar process that induces mechanical agitation in a fluid. As used herein, the term “aspect ratio” refers to length divided by width, wherein the length is greater than the width. The melting point of a polymer, unless otherwise specified, is determined by ASTM E794-06(2018) with 10° C./min ramping and cooling rates. The softening temperature or softening point of a polymer, unless otherwise specified, is determined by ASTM D6090-17. The softening temperature can be measured by using a cup and ball apparatus available from Mettler-Toledo using a 0.50 gram sample with a heating rate of 1° C./min. The crystallization temperature is the temperature at which a polymer crystallizes (i.e., solidification) into a structured form, naturally or in an artificially initiated process, wherein atoms or molecules are highly organized into a crystal. The crystallization temperature may be measured by Differential Scanning Calorimetry (DSC). DSC provides a rapid method for determining polymer crystallinity based on the heat required to melt the polymer. The crystallization temperature (° C.) is measured according to ASTM E794-06(2018) with 10° C./min ramping and cooling rates where the crystallization temperature is determined based on the second heating and cooling cycle. The crystallinity (%) of a polymer, unless otherwise specified, is determined by ASTM D3418-15. For crystallinity calculations, a 100% crystalline TPU is considered to have an enthalpy of 196.8 J/g. Mw is the weight-average molecular weight. Unless otherwise noted, Mw has units of g/mol or kDa (1,000 g/mol=1 kDa) and is measured by gel permeation chromatography. The melt flow index (MFI) is the measure of resistance to flow of polymer melt under defined set of conditions (unit: g/10 min) and is measured by ASTM 1238-20 Standard Procedure A at 195° C. using a 2 mm orifice and a 2.16 kg load. Being a measure at low shear rate condition, MFI is inversely related to molecular weight of the polymer. As used herein, “tensile modulus” (MPa) of a solid material is a mechanical property that measures its stiffness. It is defined as the ratio of its tensile stress (force per unit area) to its strain (relative deformation) when undergoing elastic deformation. It can be expressed in Pascals or pounds per square inch (psi). ASTM D638-14 can be used to determine tensile modulus of a polymer. Angle of repose is a measure of the flowability of a powder. Angle of repose measurements were determined using a Hosokawa Micron Powder Characteristics Tester PT-R using ASTM D6393-14 “Standard Test Method for Bulk Solids” Characterized by Carr Indices.” Aerated density (ρaer) is measured per ASTM D6393-14. Bulk density (ρbulk) is measured per ASTM D6393-14. Tapped density (ρtap) is measured per ASTM D6393-14. Hausner ratio (Hr) is a measure of the flowability of a powder and is calculated by Hr=ρtap/ρbulk, where ρbulkis the bulk density per ASTM D6393-14 and ρtapis the tapped density per ASTM D6393-14. As used herein, viscosity of carrier fluids are the kinematic viscosity at 25° C., unless otherwise specified, measured per ASTM D445-19. For commercially procured carrier fluids (e.g., polydimethylsiloxane oil (PDMS)), the kinematic viscosity data cited herein was provided by the manufacturer, whether measured according to the foregoing ASTM or another standard measurement technique. The dimensional accuracy of SLS part (%) is a quantitative measure of the accuracy of a 3D printed sintered parts of SLS. Thermoplastic Polymer Particles and Methods of Making The methods and compositions described herein relate to highly spherical polymer particles that comprise one or more thermoplastic polymers and one or more nucleating agents. Without being limited by theory, it is believed that having the nucleating agent may increase the crystallization temperature of the thermoplastic polymer and mitigate warping of objects (or portions thereof) produced by additive manufacturing methods using said polymer particles. At least one of the one or more thermoplastic polymers may have a crystallization temperature (Tc) and a melting temperature (Tm) that satisfies Tm≤Tc+60° C. (or Tm≤Tc+50° C., or Tm≤Tc+40° C., or Tm≤Tc+30° C., or Tc+20° C.≤Tm≤Tc+60° C., or Tc+20° C.≤Tm≤Tc+50° C., or Tc+20° C.≤Tm≤Tc+40° C., or Tc+20° C.≤Tm≤Tc+30° C.). Where no crystallization temperature can be measured, the polymer is considered to satisfy any of the foregoing. For example, the present disclosure includes methods that comprise: mixing a mixture comprising: (a) a thermoplastic polymer(s), (b) a nucleating agent, (c) a carrier fluid that is immiscible with the thermoplastic polymer, and optionally (d) an emulsion stabilizer at a temperature greater than a melting point or softening temperature of each of the thermoplastic polymer(s) and at a shear rate sufficiently high to disperse the thermoplastic polymer in the carrier fluid; cooling the mixture to below the melting point or softening temperature of the thermoplastic polymer to form spherical polymer particles; and separating the spherical polymer particles from the carrier fluid. The polymer particles (comprising a thermoplastic polymer and a nucleating agent) produced by melt emulsification may have a crystallization temperature that is substantially the same as a crystallization temperature of the thermoplastic polymer prior to mixing. When comparing crystallization temperatures, crystallization temperature may be substantially the same when said crystallization temperatures are within for example about 10° C. (or about 7° C., or about 5° C., or about 3° C.). FIG.1is a flow chart of a nonlimiting example method100of the present disclosure. Thermoplastic polymer102, nucleating agent104, carrier fluid106, and optionally other additives108(e.g., an emulsion stabilizer, a compatibilizer, etc.) are combined110to produce a mixture112. The components102,104,106, and108can be added individually or in a blend of components in any order and include mixing and/or heating during the process of combining110the components102,104,106, and108. For example, the thermoplastic polymer102and the nucleating agent104may be premixed before combining110. In another example, the nucleating agent104may be added while combining110and after addition of the thermoplastic polymer102. In another example, the emulsion stabilizer may first be dispersed in the carrier fluid, optionally with heating said dispersion, before adding the thermoplastic polymer102and the nucleating agent104. In yet another example, the thermoplastic polymer102may be heated to produce a polymer melt to which the carrier fluid106and the nucleating agent104are added together or in either order. At least a portion of combining110occur in a mixing apparatus used for the processing and/or another suitable vessel. By way of nonlimiting example, the thermoplastic polymer102may be heated to a temperature greater than the necessary melting point or softening temperature described herein in the mixing apparatus used for the processing, and the emulsion stabilizer may be dispersed in the carrier fluid in another vessel. Then, said dispersion may be added to the melt in the mixing apparatus used for the processing. The mixture112is then processed114by applying sufficiently high shear to the mixture112at a temperature greater than the melting point or softening temperature of the polymer of the thermoplastic polymer102to form a melt emulsion116. The shear rate should be sufficient enough to disperse the polymer melt (e.g., comprising the thermoplastic polymer102and the nucleating agent104) in the carrier fluid106as droplets (i.e., the melt emulsion116). Without being limited by theory, it is believed that, all other factors being the same, increasing shear should decrease the size of the droplets of the polymer melt in the carrier fluid106. However, at some point there may be diminishing returns on increasing shear and decreasing droplet size or there may be disruptions to the droplet contents that decrease the quality of particles produced therefrom. The mixing apparatuses used for the processing114to produce the melt emulsion116should be capable of maintaining the melt emulsion116at a temperature greater than the necessary melting point or softening temperature of the polymer(s) in the mixture112(e.g., the one or more polymers of the thermoplastic polymer102) described herein and applying a shear rate sufficient to disperse the polymer melt in the carrier fluid as droplets. Examples of mixing apparatuses used for the processing to produce the melt emulsion may include, but are not limited to, extruders (e.g., continuous extruders, batch extruders, and the like), stirred reactors, blenders, reactors with inline homogenizer systems, and the like, and apparatuses derived therefrom. The processing and forming the melt emulsion at suitable process conditions (e.g., temperature, shear rate, and the like) for a set period of time. The temperature of the processing and forming the melt emulsion should be a temperature greater than the necessary melting point or softening temperature of the polymer(s) in the mixture112described herein and less than the decomposition temperature of any components102,104,106,108in the mixture. For example, the temperature of processing114and forming the melt emulsion116may be about 1° C. to about 50° C. (or about 1° C. to about 25° C., or about 5° C. to about 30° C., or about 20° C. to about 50° C.) greater than the melting point or softening temperature of the polymer(s) in the mixture described herein provided the temperature of processing and forming the melt emulsion is less than the decomposition temperature of any components102,104,106,108in the mixture. The shear rate of processing114and forming the melt emulsion116should be sufficiently high to disperse the polymer melt in the carrier fluid as droplets. Said droplets should comprise droplets having a diameter of about 1000 μm or less (or about 1 μm to about 1000 μm, or about 1 μm to about 50 μm, or about 10 μm to about 100 μm, or about 10 μm to about 250 μm, or about 50 μm to about 500 μm, or about 250 μm to about 750 μm, or about 500 μm to about 1000 μm). The time for maintaining said temperature and shear rate for processing114and forming the melt emulsion116may be 10 seconds to 18 hours or longer (or 10 seconds to 30 minutes, or 5 minutes to 1 hour, or 15 minutes to 2 hours, or 1 hour to 6 hours, or 3 hours to 18 hours). Without being limited by theory, it is believed that a steady state of droplet sizes will be reached at which point processing can be stopped. That time may depend on, among other things, the temperature, shear rate, and the components102,104,106,108in the mixture112. The melt emulsion116inside and/or outside the mixing vessel is then cooled118to solidify the droplets into polymer particles124. Cooling112can be slow (e.g., allowing the melt emulsion to cool under ambient conditions) to fast (e.g., quenching). For example, the rate of cooling may range from about 10° C./hour to about 100° C./second to almost instantaneous with quenching (for example in dry ice) (or about 10° C./hour to about 60° C./hour, or about 0.5° C./minute to about 20° C./minute, or about 1° C./minute to about 5° C./minute, or about 10° C./minute to about 60° C./minute, or about 0.5° C./second to about 10° C./second, or about 10° C./second to about 100° C./second). During cooling118, little to no shear may be applied to the melt emulsion. In some instances, the shear applied during heating may be applied during cooling118. The cooled mixture resulting from cooling118the melt emulsion116may comprise solidified polymer particles and other components (e.g., the carrier fluid, excess emulsion stabilizer, and the like). The cooled mixture120can then be treated122to isolate the polymer particles124from other components126(e.g., the carrier fluid106, excess emulsion stabilizer, and the like) and wash or otherwise purify the polymer particles124. The solidified polymer particles124may be dispersed in the carrier fluid and/or settled in the carrier fluid. The polymer particles124comprise the thermoplastic polymer102, the nucleating agent104, and the other additives108(e.g., an emulsion stabilizer, a compatibilizer, etc.), when included. The polymer particles124may optionally be further purified or otherwise treated128to yield purified polymer particles130. Suitable treatments include, but are not limited to, washing, filtering, centrifuging, decanting, and the like, and any combination thereof. Solvents used for washing the polymer particles should generally be (a) miscible with the carrier fluid and (b) nonreactive (e.g., non-swelling and non-dissolving) with the polymer(s) of the polymer particles. Examples of solvents include, but are not limited to, hydrocarbon solvents (e.g., pentane, hexane, heptane, octane, cyclohexane, cyclopentane, decane, dodecane, tridecane, and tetradecane), aromatic hydrocarbon solvents (e.g., benzene, toluene, xylene, 2-methyl naphthalene, and cresol), ether solvents (e.g., diethyl ether, tetrahydrofuran, diisopropyl ether, and dioxane), ketone solvents (e.g., acetone and methyl ethyl ketone), alcohol solvents (e.g., methanol, ethanol, isopropanol, and n-propanol), ester solvents (e.g., ethyl acetate, methyl acetate, butyl acetate, butyl propionate, and butyl butyrate), halogenated solvents (e.g., chloroform, bromoform, 1,2-dichloromethane, 1,2-dichloroethane, carbon tetrachloride, chlorobenzene, and hexafluoroisopropanol), water, and the like, and any combination thereof. Solvent may be removed from the polymer particles by drying using an appropriate method such as air-drying, heat-drying, reduced pressure drying, freeze drying, or a hybrid thereof. The heating may be performed preferably at a temperature lower than the glass transition point of the polymer (e.g., about 50° C. to about 150° C.). Advantageously, carrier fluids and washing solvents of the systems and methods described herein can be recycled and reused. One skilled in the art will recognize any necessary cleaning of used carrier fluid and solvent necessary in the recycling process. The polymer particles, after separation from the other components, may optionally be further purified or otherwise treated. For example, to narrow the particle size distribution (or reduce the diameter span), the polymer particles can be passed through a sieve having a pore size of about 10 μm to about 250 μm (or about 10 μm to about 100 μm, or about 50 μm to about 200 μm, or about 150 μm to about 250 μm). In another example, the polymer particles may be washed with water to remove surfactant while maintaining substantially all of the nanoparticles associated with the surface of the polymer particles. In yet another example purification technique, the polymer particles may be blended with additives to achieve a desired final product. For clarity, because such additives are blended with the polymer particles described herein after the particles are solidified, such additives are referred to herein as “external additives.” Examples of external additives include flow aids, other polymer particles, fillers, and the like, and any combination thereof. In some instances, a surfactant used in making the polymer particles may be unwanted in downstream applications. Accordingly, yet another example purification technique may include at least substantial removal of the surfactant from the polymer particles (e.g., by washing and/or pyrolysis). The polymer particles and/or purified polymer particles may be characterized by composition, physical structure, and the like. The polymer particles may have a BET surface area of about 10 m2/g to about 500 m2/g (or about 10 m2/g to about 150 m2/g, or about 25 m2/g to about 100 m2/g, or about 100 m2/g to about 250 m2/g, or about 250 m2/g to about 500 m2/g). The polymer particles may have a D10 of about 0.1 μm to about 125 μm (or about 0.1 μm to about 5 μm, about 1 μm to about 10 μm, about 5 μm to about 30 μm, or about 1 μm to about 25 μm, or about 25 μm to about 75 μm, or about 50 μm to about 85 μm, or about 75 μm to about 125 μm), a D50 of about 0.5 μm to about 200 μm (or about 0.5 μm to about 10 μm, or about 5 μm to about 50 μm, or about 30 μm to about 100 μm, or about 30 μm to about 70 μm, or about 25 μm to about 50 μm, or about 50 μm to about 100 μm, or about 75 μm to about 150 μm, or about 100 μm to about 200 μm), and a D90 of about 3 μm to about 300 μm (or about 3 μm to about 15 μm, or about 10 μm to about 50 μm, or about 25 μm to about 75 μm, or about 70 μm to about 200 μm, or about 60 μm to about 150 μm, or about 150 μm to about 300 μm), wherein D10<D50<D90. The polymer particles may also have a diameter span of about 0.2 to about 10 (or about 0.2 to about 0.5, or about 0.4 to about 0.8, or about 0.5 to about 1, or about 1 to about 3, or about 2 to about 5, or about 5 to about 10). Without limitation, diameter span values of 1.0 or greater are considered broad, and diameter spans values of 0.75 or less are considered narrow. Preferable, the polymer particles have a diameter span of about 0.2 to about 1. In a first nonlimiting example, the polymer particles may have a D10 of about 0.1 μm to about 10 μm, a D50 of about 0.5 μm to about 25 μm, and a D90 of about 3 μm to about 50 μm, wherein D10<D50<D90. Said polymer particles may have a diameter span of about 0.2 to about 2. In a second nonlimiting example, the polymer particles may have a D10 of about 5 μm to about 30 μm, a D50 of about 30 μm to about 70 μm, and a D90 of about 70 μm to about 120 μm, wherein D10<D50<D90. Said polymer particles may have a diameter span of about 1.0 to about 2.5. In a third nonlimiting example, the polymer particles may have a D10 of about 25 μm to about 60 μm, a D50 of about 60 μm to about 110 μm, and a D90 of about 110 μm to about 175 μm, wherein D10<D50<D90. Said polymer particles may have a diameter span of about 0.6 to about 1.5. In a fourth nonlimiting example, the polymer particles may have a D10 of about 75 μm to about 125 μm, a D50 of about 100 μm to about 200 μm, and a D90 of about 125 μm to about 300 μm, wherein D10<D50<D90. Said polymer particles may have a diameter span of about 0.2 to about 1.2. In a fifth nonlimiting example, the polymer particles may have a D10 of about 1 μm to about 50 μm (or about 5 μm to about 30 μm, or about 1 μm to about 25 μm, or about 25 μm to about 50 μm), a D50 of about 25 μm to about 100 μm (or about 30 μm to about 100 μm, or about 30 μm to about 70 μm, or about 25 μm to about 50 μm, or about 50 μm to about 100 μm), and a D90 of about 60 μm to about 300 μm (or about 70 μm to about 200 μm, or about 60 μm to about 150 μm, or about 150 μm to about 300 μm), wherein D10<D50<D90. The polymer particles may also have a diameter span of about 0.4 to about 3 (or about 0.6 to about 2, or about 0.4 to about 1.5, or about 1 to about 3). The polymer particles may have a circularity of about 0.9 or greater (or about 0.90 to about 1.0, or about 0.93 to about 0.99, or about 0.95 to about 0.99, or about 0.97 to about 0.99, or about 0.98 to 1.0). The polymer particles may have an angle of repose of about 25° to about 45° (or about 25° to about 35°, or about 300 to about 40°, or about 350 to about 45°). The polymer particles may have a Hausner ratio of about 1.0 to about 1.5 (or about 1.0 to about 1.2, or about 1.1 to about 1.3, or about 1.2 to about 1.35, or about 1.3 to about 1.5). The polymer particles may have a bulk density of about 0.3 g/cm3to about 0.8 g/cm3(or about 0.3 g/cm3to about 0.6 g/cm3, or about 0.4 g/cm3to about 0.7 g/cm3, or about 0.5 g/cm3to about 0.6 g/cm3, or about 0.5 g/cm3to about 0.8 g/cm3). The polymer particles may have an aerated density of about 0.5 g/cm3to about 0.8 g/cm3(or about 0.5 g/cm3to about 0.7 g/cm3, or about 0.55 g/cm3to about 0.80 g/cm3). The polymer particles may have a tapped density of about 0.6 g/cm3to about 0.9 g/cm3(or about 0.60 g/cm3to about 0.75 g/cm3, or about 0.65 g/cm3to about 0.80 g/cm3, or about 0.70 g/cm3to about 0.90 g/cm3). Depending on the temperature and shear rate of processing and the composition and relative concentrations of the components (e.g., thermoplastic polymer, the carrier fluid, excess emulsion stabilizer, and the like) different shapes of the structures that compose the polymer particles may be produced. Typically, the polymer particles comprise substantially spherical particles (having a circularity of about 0.97 or greater). However, other structures including disc and elongated structures may be observed in the polymer particles. Therefore, the polymer particles may comprise one or more of: (a) substantially spherical particles having a circularity of 0.97 or greater, (b) disc structures having an aspect ratio of about 2 to about 10, and (c) elongated structures having an aspect ratio of 10 or greater. Each of the (a), (b), and (c) structures have emulsion stabilizers dispersed on an outer surface of the (a), (b), and (c) structures and/or embedded in an outer portion of the (a), (b), and (c) structures. At least some of the (a), (b), and (c) structures may be agglomerated. For example, the (c) elongated structures may be laying on the surface of the (a) substantially spherical particles. The polymer particles may have a sintering window that is within 10° C., preferably within 5° C., of the sintering window of the thermoplastic polymer or blend thereof used in the mixture. At least one of the one or more thermoplastic polymers in the mixture (e.g., mixture112ofFIG.1) or the polymer particles (e.g., polymer particles124/130ofFIG.1) may have a crystallization temperature (Tc) and a melting temperature (Tm) that satisfies Tm≤Tc+60° C. (or Tm≤Tc+50° C., or Tm≤Tc+40° C., or Tm≤Tc+30° C., or Tc+20° C.≤Tm≤Tc+60° C., or Tc+20° C.≤Tm≤Tc+50° C., or Tc+20° C.≤Tm≤Tc+40° C., or Tc+20° C.≤Tm≤Tc+30° C.). The thermoplastic polymer may be present in the mixture (e.g., mixture112ofFIG.1) at about 5 wt % to about 60 wt % (or about 5 wt % to about 25 wt %, or about 10 wt % to about 30 wt %, or about 20 wt % to about 45 wt %, or about 25 wt % to about 50 wt %, or about 40 wt % to about 60 wt %) of the mixture. The thermoplastic polymer may be present in the polymer particles (e.g., polymer particles124/130ofFIG.1) at about 40 wt % to about 99.95 wt % (or about 40 wt % to about 80 wt %, or about 60 wt % to about 90 wt %, or about 80 wt % to about 95 wt %, or about 85 wt % to about 98 wt %, or about 90 wt % to about 99.95 wt %) of the polymer particles. Examples of said thermoplastic polymers may include, but are not limited to, thermoplastic polyolefins, polyamides, polyurethanes, polyacetals, polycarbonates, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), ethylene vinyl acetate copolymer (EVA), polyhexamethylene terephthalate, polystyrenes, polyvinyl chlorides, polytetrafluoroethenes, polyesters (e.g., polylactic acid), polyethers, polyether sulfones, polyetherether ketones, polyacrylates, polymethacrylates, polyimides, acrylonitrile butadiene styrene (ABS), polyphenylene sulfides, vinyl polymers, polyarylene ethers, polyarylene sulfides, polysulfones, polyether ketones, polyamide-imides, polyetherimides, polyetheresters, copolymers comprising a polyether block and a polyamide block (PEBA or polyether block amide), functionalized or nonfunctionalized ethylene/vinyl monomer polymer, functionalized or nonfunctionalized ethylene/alkyl (meth)acrylates, functionalized or nonfunctionalized (meth)acrylic acid polymers, functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymers, ethylene/vinyl monomer/carbonyl terpolymers, ethylene/alkyl (meth)acrylate/carbonyl terpolymers, methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymers, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymers, chlorinated or chlorosulphonated polyethylenes, polyvinylidene fluoride (PVDF), phenolic resins, poly(ethylene/vinyl acetate), polybutadienes, polyisoprenes, styrenic block copolymers, polyacrylonitriles, silicones, and the like, and any combination thereof. Copolymers comprising one or more of the foregoing may also be used in the methods and systems of the present disclosure. In some cases, copolymers of PE with polar monomers, such as poly(ethylene-co-vinyl acetate), poly(ethylene-co-methyl acrylate), poly(ethylene-co-glycidyl methacrylate), and poly(ethylene-co-vinyl alcohol) may improve compatibility in polyethylene-poly(methylmethacrylate) (PE/PMMA) blends. The thermoplastic polymers in the compositions and methods of the present disclosure may be elastomeric or non-elastomeric. Some of the foregoing examples of thermoplastic polymers may be elastomeric or non-elastomeric depending on the exact composition of the polymer. Thermoplastic elastomers generally fall within one of six classes: styrenic block copolymers, thermoplastic vulcanizates (also referred to as elastomeric alloys), thermoplastic polyurethanes, thermoplastic copolyesters, and thermoplastic polyamides (typically block copolymers comprising polyamide). Examples of thermoplastic elastomers can be found in Handbook of Thermoplastic Elastomers, 2nd ed., B. M. Walker and C. P. Rader, eds., Van Nostrand Reinhold, New York, 1988. Examples of thermoplastic elastomers include, but are not limited to, elastomeric polyamides, polyurethanes, copolymers comprising a polyether block and a polyamide block (PEBA or polyether block amide), methyl methacrylate-butadiene-styrene (MBS)-type core-shell polymers, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymers, polybutadienes, polyisoprenes, styrenic block copolymers, and polyacrylonitriles), silicones, and the like. Elastomeric styrenic block copolymers may include at least one block selected from the group of: isoprene, isobutylene, butylene, ethylene/butylene, ethylene-propylene, and ethylene-ethylene/propylene. More specific elastomeric styrenic block copolymer examples include, but are not limited to, poly(styrene-ethylene/butylene), poly(styrene-ethylene/butylene-styrene), poly(styrene-ethylene/propylene), styrene-ethylene/propylene-styrene), poly(styrene-ethylene/propylene-styrene-ethylene-propylene), poly(styrene-butadiene-styrene), poly(styrene-butylene-butadiene-styrene), and the like, and any combination thereof. Examples of polyamides include, but are not limited to, polycaproamide (nylon 6, polyamide 6, or PA6), poly(hexamethylene succinamide) (nylon 4,6, polyamide 4,6, or PA4,6), polyhexamethylene adipamide (nylon 6,6, polyamide 6,6, or PA6,6), polypentamethylene adipamide (nylon 5,6, polyamide 5,6, or PA5,6), polyhexamethylene sebacamide (nylon 6,10, polyamide 6,10, or PA6,10), polyundecaamide (nylon 11, polyamide 11, or PA11), polydodecaamide (nylon 12, polyamide 12, or PA12), and polyhexamethylene terephthalamide (nylon 6T, polyamide 6T, or PA6T), nylon 10,10 (polyamide 10,10 or PA10,10), nylon 10,12 (polyamide 10,12 or PA10,12), nylon 10,14 (polyamide 10,14 or PA10,14), nylon 10,18 (polyamide 10,18 or PA10,18), nylon 6,18 (polyamide 6,18 or PA6,18), nylon 6,12 (polyamide 6,12 or PA6,12), nylon 6,14 (polyamide 6,14 or PA6,14), nylon 12,12 (polyamide 12,12 or PA12,12), and the like, and any combination thereof. Copolyamides may also be used. Examples of copolyamides include, but are not limited to, PA 11/10,10, PA 6/11, PA 6,6/6, PA 11/12, PA 10,10/10,12, PA 10,10/10,14, PA 11/10,36, PA 11/6,36, PA 10,10/10,36, PA 6T/6,6, and the like, and any combination thereof. A polyamide followed by a first number comma second number is a polyamide having the first number of backbone carbons between the nitrogens for the section having no pendent ═O and the second number of backbone carbons being between the two nitrogens for the section having the pendent ═O. By way of nonlimiting example, nylon 6,10 is [NH—(CH2)6—NH—CO—(CH2)8—CO]n. A polyamide followed by number(s) backslash number(s) are a copolymer of the polyamides indicated by the numbers before and after the backslash. Examples of polyurethanes include, but are not limited to, polyether polyurethanes, polyester polyurethanes, mixed polyether and polyester polyurethanes, and the like, and any combination thereof. Examples of thermoplastic polyurethanes include, but are not limited to, poly[4,4′-methylenebis(phenylisocyanate)-alt-1,4-butanediol/di(propylene glycol)/polycaprolactone], ELASTOLLAN® 1190A (a polyether polyurethane elastomer, available from BASF), ELASTOLLAN® 1190A10 (a polyether polyurethane elastomer, available from BASF), and the like, and any combination thereof. Polyolefins may be polymers of one or more monomers that may include, but are not limited to, substituted or unsubstituted C2to C40alpha olefins, preferably C2to C20alpha olefins, preferably C2to C12alpha olefins, preferably ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, decene, undecene, dodecane, and isomers thereof. For example, the polyolefin may comprise propylene and an optional comonomers comprising one or more ethylene or C4to C40olefins, preferably C4to C20olefins, or preferably C6to C12olefins. The C4to C40olefin monomers may be linear, branched, or cyclic. The C4to C40cyclic olefins may be strained or unstrained, monocyclic or polycyclic, and may optionally include heteroatoms and/or one or more functional groups. In another example, the polyolefin may comprise ethylene and an optional comonomers comprising one or more C3to C40olefins, preferably C4to C20olefins, or preferably C6to C12olefins. The C3to C40olefin monomers may be linear, branched, or cyclic. The C3to C40cyclic olefins may be strained or unstrained, monocyclic or polycyclic, and may optionally include heteroatoms and/or one or more functional groups. Examples of C2to C40olefins may include, but are not limited to, ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, decene, undecene, dodecene, norbornene, norbornadiene, dicyclopentadiene, cyclopentene, cycloheptene, cyclooctene, cyclooctadiene, cyclododecene, 7-oxanorbornene, 7-oxanorbornadiene, substituted derivatives thereof, and isomers thereof, preferably hexene, heptene, octene, nonene, decene, dodecene, cyclooctene, 1,5-cyclooctadiene, 1-hydroxy-4-cyclooctene, 1-acetoxy-4-cyclooctene, 5-methylcyclopentene, cyclopentene, dicyclopentadiene, norbornene, norbornadiene, and their respective homologs and derivatives, preferably norbornene, norbornadiene, and dicyclopentadiene. Examples of specific polyolefins may include, but are not limited to, polyethylene (as a homopolymer or a copolymer having 35 wt % or less of a C2to C40alpha olefin comonomer), polypropylene (as a homopolymer or a copolymer having 35 wt % or less of a C4to C40alpha olefin comonomer), ethylene-propylene copolymers, ethylene-propylene-diene copolymers, polybutene, polyisobutylene, polymethylpentene, poly (4-methyl-1-pentene), and the like, and any combination thereof. The thermoplastic polymer may have a melting point or softening temperature of about 50° C. to about 450° C. (or about 50° C. to about 125° C., or about 100° C. to about 175° C., or about 150° C. to about 280° C., or about 200° C. to about 350° C., or about 300° C. to about 450° C.). The thermoplastic polymer may have a glass transition temperature (ASTM E1356-08 (2014) with 10° C./min ramping and cooling rates) of about −50° C. to about 400° C. (or about −50° C. to about 0° C., or about −25° C. to about 50° C., or about 0° C. to about 150° C., or about 100° C. to about 250° C., or about 150° C. to about 300° C., or about 200° C. to about 400° C.). The thermoplastic polymer may optionally comprise an additive. Typically, the additive would be present before addition of said polymers to the mixture. Therefore, in the polymer melt droplets and resultant polymer particles, the additive is dispersed throughout the thermoplastic polymer. Accordingly, for clarity, this additive is referred to herein as an “internal additive.” The internal additive may be blended with the said polymer just prior to making the mixture or well in advance. When describing component amounts in the compositions described herein (e.g., the mixture and the polymer particles), a weight percent based on the polymer not inclusive of the internal additive. For example, a composition comprising 1 wt % of emulsion stabilizer by weight of 100 g of a polymer comprising 10 wt % internal additive and 90 wt % polymer is a composition comprising 0.9 g of emulsion stabilizer, 90 g of polymer, and 10 g of internal additive. The internal additive may be present in the thermoplastic polymer at about 0.1 wt % to about 60 wt % (or about 0.1 wt % to about 5 wt %, or about 1 wt % to about 10 wt %, or about 5 wt % to about 20 wt %, or about 10 wt % to about 30 wt %, or about 25 wt % to about 50 wt %, or about 40 wt % to about 60 wt %) of the thermoplastic polymer. For example, the thermoplastic polymer may comprise about 70 wt % to about 85 wt % of a thermoplastic polymer and about 15 wt % to about 30 wt % of an internal additive like glass fiber or carbon fiber. Examples of internal additives include, but are not limited to, fillers, strengtheners, pigments, pH regulators, and the like, and combinations thereof. Examples of fillers include, but are not limited to, glass fibers, glass particles, mineral fibers, carbon fiber, oxide particles (e.g., titanium dioxide and zirconium dioxide), metal particles (e.g., aluminum powder), and the like, and any combination thereof. Examples of pigments include, but are not limited to, organic pigments, inorganic pigments, carbon black, and the like, and any combination thereof. For example, fillers used herein may include exfoliated graphite (EG), exfoliated graphite nanoplatelets (xGnP), carbon black, carbon nanofibers (CNF), carbon nanotubes (CNT), graphenes, graphene oxides, graphite oxides, graphene oxide nanosheets, fullerenes. Examples of nucleating agents may include, but are not limited to, salts of benzoic acid, lithium benzoate, sodium benzoate, aluminum benzoate, potassium benzoate, sodium salts of organophosphates, finely divided organoclays, carbon nanotubes, silica, calcium carbonate, talc, benzene trisamides, nonitol derivatives, 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-sorbitol, sodium 2,4,8,10-tetra-tert-butyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin-6-olate-6-oxide, bicyclo[2.2.1]heptane-2,3-dicarboxylic acid disodium salt, 2,2′-methylene-bis-(4,6-di-tert-butylphenyl) phosphate, and the like, and any combination thereof. The nucleating agents may be present in the mixture (e.g., mixture112ofFIG.1) or the polymer particles (e.g., polymer particles124/130ofFIG.1) at about 0.05 wt % to about 5 wt % (or about 0.05 wt % to about 2 wt %, or about 0.1 wt % to about 1 wt %, or about 0.1 wt % to about 2 wt %, or about 1 wt % to about 5 wt %) of the thermoplastic polymer. The carrier fluid should be chosen such that at the various processing temperatures (e.g., from room temperature to process temperature) the thermoplastic polymer and the carrier fluid are immiscible. An additional factor that may be considered is the differences in (e.g., a difference or a ratio of) viscosity at process temperature between the thermoplastic polymer and the carrier fluid. The differences in viscosity may affect droplet breakup and particle size distribution. Without being limited by theory, it is believed that when the viscosities of the thermoplastic polymer and the carrier fluid are too similar, the circularity of the product as a whole may be reduced where the particles are more ovular and more elongated structures are observed. Suitable carrier fluids may have a viscosity at 25° C. of about 1,000 cSt to about 150,000 cSt (or about 1,000 cSt to about 60,000 cSt, or about 40,000 cSt to about 100,000 cSt, or about 75,000 cSt to about 150,000 cSt). For example, suitable carrier fluids may have a viscosity at 25° C. of about 10,000 cSt to about 60,000 cSt. Examples of carrier fluids may include, but are not limited to, silicone oil, fluorinated silicone oils, perfluorinated silicone oils, polyethylene glycols, alkyl-terminal polyethylene glycols (e.g., C1-C4 terminal alkyl groups like tetraethylene glycol dimethyl ether (TDG)), paraffins, liquid petroleum jelly, vison oils, turtle oils, soya bean oils, perhydrosqualene, sweet almond oils, calophyllum oils, palm oils, parleam oils, grapeseed oils, sesame oils, maize oils, rapeseed oils, sunflower oils, cottonseed oils, apricot oils, castor oils, avocado oils, jojoba oils, olive oils, cereal germ oils, esters of lanolic acid, esters of oleic acid, esters of lauric acid, esters of stearic acid, fatty esters, higher fatty acids, fatty alcohols, polysiloxanes modified with fatty acids, polysiloxanes modified with fatty alcohols, polysiloxanes modified with polyoxy alkylenes, and the like, and any combination thereof. Examples of silicone oils include, but are not limited to, polydimethylsiloxane (PDMS), methylphenylpolysiloxane, an alkyl modified polydimethylsiloxane, an alkyl modified methylphenylpolysiloxane, an amino modified polydimethylsiloxane, an amino modified methylphenylpolysiloxane, a fluorine modified polydimethylsiloxane, a fluorine modified methylphenylpolysiloxane, a polyether modified polydimethylsiloxane, a polyether modified methylphenylpolysiloxane, and the like, and any combination thereof. When the carrier fluid comprises two or more of the foregoing, the carrier fluid may have one or more phases. For example, polysiloxanes modified with fatty acids and polysiloxanes modified with fatty alcohols (preferably with similar chain lengths for the fatty acids and fatty alcohols) may form a single-phase carrier fluid. In another example, a carrier fluid comprising a silicone oil and an alkyl-terminal polyethylene glycol may form a two-phase carrier fluid. In at least one embodiment, the carrier fluid is polydimethylsiloxane (PDMS). The carrier fluid may be present in the mixture at about 40 wt % to about 95 wt % (or about 75 wt % to about 95 wt %, or about 70 wt % to about 90 wt %, or about 55 wt % to about 80 wt %, or about 50 wt % to about 75 wt %, or about 40 wt % to about 60 wt %) of the mixture. In some instances, the carrier fluid may have a density of about 0.6 g/cm3to about 1.5 g/cm3, and the thermoplastic polymer may have a density of about 0.7 g/cm3to about 1.7 g/cm3, wherein the thermoplastic polymer may have a density similar, lower, or higher than the density of the carrier fluid. Other additives like emulsion stabilizers, thermoplastic polymers, compatibilizers, and the like, and any combination thereof may be included in the mixture and resultant polymer particles. The emulsion stabilizers used in the methods and compositions of the present disclosure may comprise nanoparticles (e.g. oxide nanoparticles, carbon black, polymer nanoparticles, and combinations thereof), surfactants, and the like, and any combination thereof. Oxide nanoparticles may be metal oxide nanoparticles, non-metal oxide nanoparticles, or mixtures thereof. Examples of oxide nanoparticles include, but are not limited to, silica, titania, zirconia, alumina, iron oxide, copper oxide, tin oxide, boron oxide, cerium oxide, thallium oxide, tungsten oxide, and the like, and any combination thereof. Mixed metal oxides and/or non-metal oxides, like aluminosilicates, borosilicates, and aluminoborosilicates, are also inclusive in the term metal oxide. The oxide nanoparticles may by hydrophilic or hydrophobic, which may be native to the particle or a result of surface treatment of the particle. For example, a silica nanoparticle having a hydrophobic surface treatment, like dimethyl silyl, trimethyl silyl, and the like, may be used in methods and compositions of the present disclosure. Additionally, silica with functional surface treatments like methacrylate functionalities may be used in methods and compositions of the present disclosure. Unfunctionalized oxide nanoparticles may also be suitable for use as well. Commercially available examples of silica nanoparticles include, but are not limited to, AEROSIL® particles available from Evonik (e.g., AEROSIL® R812S (about 7 nm average diameter silica nanoparticles having a hydrophobically modified surface and a BET surface area of 260±30 m2/g), AEROSIL® RX50 (about 40 nm average diameter silica nanoparticles having a hydrophobically modified surface and a BET surface area of 35±10 m2/g), AEROSIL® 380 (silica nanoparticles having a hydrophilically modified surface and a BET surface area of 380±30 m2/g), and the like, and any combination thereof. Carbon black is another type of nanoparticle that may be present as an emulsion stabilizer in the compositions and methods disclosed herein. Various grades of carbon black will be familiar to one having ordinary skill in the art, any of which may be used herein. Other nanoparticles capable of absorbing infrared radiation may be used similarly. Polymer nanoparticles are another type of nanoparticle that may be present as an emulsion stabilizer in the disclosure herein. Suitable polymer nanoparticles may include one or more polymers that are thermosetting and/or crosslinked, such that they do not melt when processed by melt emulsification according to the disclosure herein. High molecular weight thermoplastic polymers having high melting or decomposition points may similarly comprise suitable polymer nanoparticle emulsion stabilizers. Surfactants may be anionic, cationic, nonionic, or zwitterionic. Examples of surfactants include, but are not limited to, sodium dodecyl sulfate, sorbitan oleates, poly[dimethylsiloxane-co-[3-(2-(2-hydroxyethoxy)ethoxy)propylmethylsiloxane]], docusate sodium (sodium 1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate), and the like, and any combination thereof. Commercially available examples of surfactants include, but are not limited to, CALFAX® DB-45 (sodium dodecyl diphenyl oxide disulfonate, available from Pilot Chemicals), SPAN® 80 (sorbitan maleate non-ionic surfactant), MERPOL® surfactants (available from Stepan Company), TERGITOL™ TMN-6 (a water-soluble, nonionic surfactant, available from DOW), TRITON™ X-100 (octyl phenol ethoxylate, available from SigmaAldrich), IGEPAL® CA-520 (polyoxyethylene (5) isooctylphenyl ether, available from SigmaAldrich), BRIJ® S10 (polyethylene glycol octadecyl ether, available from SigmaAldrich), and the like, and any combination thereof. Surfactants may be included in the mixture (e.g., mixture112ofFIG.1) or the polymer particles (e.g., polymer particles124/130ofFIG.1) in an amount of about 0.01 wt % to about 10 wt % (or about 0.01 wt % to about 1 wt %, or about 0.5 wt % to about 2 wt %, or about 1 wt % to about 3 wt %, or about 2 wt % to about 5 wt %, or about 5 wt % to about 10 wt %) based on a total weight of thermoplastic polymer in the mixture or thermoplastic polymer in the polymer particles. Alternatively, the mixture may comprise no (or be absent of) surfactant. A weight ratio of nanoparticles to surfactant in the emulsion stabilizer in the mixture (e.g., mixture112ofFIG.1) or the polymer particles (e.g., polymer particles124/130ofFIG.1) may be about 1:10 to about 10:1 (or about 1:10 to about 1:1, or about 1:5 to about 5:1, or about 1:1 to about 10:1). The emulsion stabilizer may be included in the mixture (e.g., mixture112ofFIG.1) or the polymer particles (e.g., polymer particles124/130ofFIG.1) in an amount of about 0.01 wt % to about 10 wt % (or about 0.01 wt % to about 1 wt %, or about 0.1 wt % to about 3 wt %, or about 1 wt % to about 5 wt %, or about 5 wt % to about 10 wt %) based on a total weight of thermoplastic polymer in the mixture or thermoplastic polymer in the polymer particles. The emulsion stabilizers may be at the interface between the polymer melt and the carrier fluid in the melt emulsion. As a result, when the mixture is cooled, the emulsion stabilizers remain at, or in the vicinity of, said interface. Therefore, the structure of the polymer particles is, in general when emulsion stabilizers are used, includes emulsion stabilizers (a) dispersed on an outer surface of the polymer particles and/or (b) embedded in an outer portion (e.g., outer 1 vol %) of the polymer particles. That is, emulsion stabilizers, when included, may be deposited as coating, perhaps a uniform coating, on the polymer particles. In some instances, which may be dependent upon nonlimiting factors such as the temperature (including cooling rate), the type of thermoplastic polymer, and the types and sizes of emulsion stabilizers, the nanoparticles of emulsion stabilizers may become at least partially embedded within the outer surface of polymer particles. Even without embedment taking place, at least a portion of the nanoparticles within emulsion stabilizers may remain robustly associated with polymer particles to facilitate their further use. In contrast, dry blending already formed polymer particulates (e.g., formed by cryogenic grinding or precipitation processes) with a flow aid like silica nanoparticles does not result in a robust, uniform coating of the flow aid upon the polymer particulates. At least a portion of the surfactant, if used, may be associated with the outer surface as well. The coating of the emulsion stabilizer (e.g., comprising surfactants and/or nanoparticles) may be disposed substantially uniformly upon the outer surface. As used herein with respect to a coating, the term “substantially uniform” refers to even coating thickness in surface locations covered by the coating composition (e.g., nanoparticles and/or surfactant), particularly the entirety of the outer surface. The emulsion stabilizers may form a coating that covers at least 5% (or about 5% to about 100%, or about 5% to about 25%, or about 20% to about 50%, or about 40% to about 70%, or about 50% to about 80%, or about 60% to about 90%, or about 70% to about 100%) of the surface area of the polymer particles. When purified to at least substantially remove surfactant or another emulsion stabilizer, the emulsion stabilizers may be present on an outer surface of the polymer particles at less than 25% (or 0% to about 25%, or about 0.1% to about 5%, or about 0.1% to about 1%, or about 1% to about 5%, or about 1% to about 10%, or about 5% to about 15%, or about 10% to about 25%) of the surface area of the polymer particles. The coverage of the emulsion stabilizers on an outer surface of the polymer particles may be determined using image analysis of the scanning electron microscope images (SEM micrographs). The emulsion stabilizers may form a coating that covers at least 5% (or about 5% to about 100%, or about 5% to about 25%, or about 20% to about 50%, or about 40% to about 70%, or about 50% to about 80%, or about 60% to about 90%, or about 70% to about 100%) of the surface area of the polymer particles. When purified to at least substantially remove surfactant or another emulsion stabilizer, the emulsion stabilizers may be present on an outer surface of the polymer particles at less than 25% (or 0% to about 25%, or about 0.10% to about 5%, or about 0.10% to about 10%, or about 10% to about 5%, or about 10% to about 10%, or about 5% to about 15%, or about 10% to about 25%) of the surface area of the polymer particles. The coverage of the emulsion stabilizers on an outer surface of the polymer particles may be determined using image analysis of the SEM micrographs. Further, where voids form inside the polymer melt droplets, emulsion stabilizers should generally be at (and/or embedded in) the interface between the interior of the void and the polymer. The voids generally do not contain polymer. Rather, the voids may contain, for example, carrier fluid, air, or be void. The polymer particles described herein may comprise carrier fluid at about 5 wt % or less (or about 0.001 wt % to about 5 wt %, or about 0.001 wt % to about 0.1 wt %, or about 0.01 wt % to about 0.5 wt %, or about 0.1 wt % to about 2 wt %, or about 1 wt % to about 5 wt %) of the polymer particles. Compatibilizers may optionally be used to improve the blending efficiency and efficacy when two or more thermoplastic polymers are used. Examples of polymer compatibilizers include, but not limited to, PROPOLDER™ MPP2020 20 (polypropylene, available from Polygroup Inc.), PROPOLDER™ MPP2040 40 (polypropylene, available from Polygroup Inc.), NOVACOM™ HFS2100 (maleic anhydride functionalized high density polyethylene polymer, available from Polygroup Inc.), KEN-REACT™ CAPS™ L™ 12/L (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPOW™ L™ 12/H (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ LICA™ 12 (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPS™ KPR™ 12/LV (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPOW™ KPR™ 12/H (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ titanates & zirconates (organometallic coupling agent, available from Kenrich Petrochemicals), VISTAMAXX™ (ethylene-propylene copolymers, available from ExxonMobil), SANTOPRENE™ (thermoplastic vulcanizate of ethylene-propylene-diene rubber and polypropylene, available from ExxonMobil), VISTALON™ (ethylene-propylene-diene rubber, available from ExxonMobil), EXACT™ (plastomers, available from ExxonMobil) EXXELOR™ (polymer resin, available from ExxonMobil), FUSABOND™ M603 (random ethylene copolymer, available from Dow), FUSABOND™ E226 (anhydride modified polyethylene, available from Dow), BYNEL™ 41E710 (coextrudable adhesive resin, available from Dow), SURLYN™ 1650 (ionomer resin, available from Dow), FUSABOND™ P353 (a chemically modified polypropylene copolymer, available from Dow), ELVALOY™ PTW (ethylene terpolymer, available from Dow), ELVALOY™ 3427AC (a copolymer of ethylene and butyl acrylate, available from Dow), LOTADER™ AX8840 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3210 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3410 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3430 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 4700 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ AX8900 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 4720 (ethylene acrylate-based terpolymer, available from Arkema), BAXXODUR™ EC 301 (amine for epoxy, available from BASF), BAXXODUR™ EC 311 (amine for epoxy, available from BASF), BAXXODUR™ EC 303 (amine for epoxy, available from BASF), BAXXODUR™ EC 280 (amine for epoxy, available from BASF), BAXXODUR™ EC 201 (amine for epoxy, available from BASF), BAXXODUR™ EC 130 (amine for epoxy, available from BASF), BAXXODUR™ EC 110 (amine for epoxy, available from BASF), styrenics, polypropylene, polyamides, polycarbonate, EASTMAN™ G-3003 (a maleic anhydride grafted polypropylene, available from Eastman), RETAIN™ (polymer modifier available from Dow), AMPLIFY TY™ (maleic anhydride grafted polymer, available from Dow), INTUNE™ (olefin block copolymer, available from Dow), and the like and any combination thereof. Applications of Polymer Particles The present disclosure also relates to methods of selective laser sintering where the method may comprise: depositing (a) highly spherical polymer particles (e.g., melt emulsified polymer particles) comprising (a1) one or more thermoplastic polymers, (a2) one or more nucleating agents, and optionally (a3) additives described herein (e.g., emulsion stabilizer, compatibilizers, and the like) and optionally (b) thermoplastic polymer particles different than the particles (a) onto a surface; and once deposited, exposing at least a portion of the particles (a) and (b) (if included) to a laser to fuse the particles (a) and (b) (if included) and form a consolidated body. The polymer particles described herein (e.g., melt emulsified polymer particles) may be used to produce a variety of articles. By way of nonlimiting example, 3-D printing processes of the present disclosure may comprise: depositing particles (e.g., the foregoing particles (a) and (b) (if included)) described herein upon a surface (e.g., in layers and/or in a specified shape), and once deposited, heating at least a portion of the particles to promote consolidation thereof and form a consolidated body (or object). The consolidated body may have a void percentage of about 5% or less (e.g., 0% to about 5%, or about 0.5% to about 2%, or about 1% to about 3%, or about 2% to about 5%) after being consolidated. For example, heating and consolidation of the polymer particles (e.g., polymer particles124/130and other thermoplastic polymer particles) may take place in a 3-D printing apparatus employing a laser, such that heating and consolidation take place by selective laser sintering. Advantageously, the inclusion of the nucleating agent may mitigate warping of the object or layers thereof during SLS methods. Examples of articles that may be produced by such methods where the polymer particles may be used to form all or a portion of said articles include, but are not limited to, particles, films, packaging, toys, household goods, automotive parts, aerospace/aircraft-related parts, containers (e.g., for food, beverages, cosmetics, personal care compositions, medicine, and the like), shoe soles, furniture parts, decorative home goods, plastic gears, screws, nuts, bolts, cable ties, jewelry, art, sculpture, medical items, prosthetics, orthopedic implants, production of artifacts that aid learning in education, 3D anatomy models to aid in surgeries, robotics, biomedical devices (orthotics), home appliances, dentistry, electronics, sporting goods, and the like. Further, particles may be useful in applications that include, but are not limited to, paints, powder coatings, ink jet materials, electrophotographic toners, 3D printing, and the like. Example Embodiments A first nonlimiting example embodiment of the present disclosure is a method comprising: mixing a mixture comprising: a thermoplastic polymer, a nucleating agent, a carrier fluid, and optionally an emulsion stabilizer at a temperature at or greater than a melting point or softening temperature of the thermoplastic polymer to emulsify a thermoplastic polymer melt in the carrier fluid; cooling the mixture to below the melting point or softening temperature to form polymer particles; and separating the polymer particles from the carrier fluid, wherein the polymer particles have a crystallization temperature that is substantially the same as a crystallization temperature of the thermoplastic polymer prior to mixing. A second nonlimiting example embodiment of the present disclosure is a method comprising: mixing a mixture comprising: a thermoplastic polymer, a nucleating agent, a carrier fluid, and optionally an emulsion stabilizer at a temperature at or greater than a melting point or softening temperature of the thermoplastic polymer to emulsify a thermoplastic polymer melt in the carrier fluid, wherein the thermoplastic polymer prior to mixing has a crystallization temperature (Tc) and a melting temperature (Tm) that satisfies Tm≤Tc+60° C.; cooling the mixture to below the melting point or softening temperature to form polymer particles; and separating the polymer particles from the carrier fluid, wherein the polymer particles have a crystallization temperature within about 10° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. The first and second nonlimiting example embodiments may further include one or more of: Element 1: wherein the thermoplastic polymer prior to mixing has a crystallization temperature (Tc) and a melting temperature (Tm) that satisfies Tm≤Tc+60° C. (or Tc+20° C.≤Tm≤Tc+60° C., or Tc+20° C.≤Tm≤Tc+50° C., or Tc+20° C.≤Tm≤Tc+40° C., or Tc+20° C. Tm≤Tc+30° C.); Element 2: wherein polymer the particles have a crystallization temperature within about 10° C. of a crystallization temperature of the thermoplastic polymer prior to mixing; Element 3: wherein the polymer particles have a crystallization temperature within about 7° C. of a crystallization temperature of the thermoplastic polymer prior to mixing; Element 4: wherein the polymer particles have a crystallization temperature within about 5° C. of a crystallization temperature of the thermoplastic polymer prior to mixing; Element 5: wherein the polymer particles have a crystallization temperature within about 3° C. of a crystallization temperature of the thermoplastic polymer prior to mixing; Element 6: wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyurethane, a polyacetal, a polycarbonate, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), ethylene vinyl acetate copolymer (EVA), polyhexamethylene terephthalate, a polystyrene, a polyvinyl chloride, a polytetrafluoroethene, a polyester (e.g., polylactic acid), a polyether, a polyether sulfone, a polyetherether ketone, a polyacrylate, a polymethacrylate, a polyimide, acrylonitrile butadiene styrene (ABS), a polyphenylene sulfide, a vinyl polymer, a polyarylene ether, a polyarylene sulfide, a polysulfone, a polyether ketone, a polyamide-imide, a polyetherimide, a polyetherester, a copolymer comprising a polyether block and a polyamide block (PEBA or polyether block amide), a functionalized or nonfunctionalized ethylene/vinyl monomer polymer, a functionalized or nonfunctionalized ethylene/alkyl (meth)acrylate, a functionalized or nonfunctionalized (meth)acrylic acid polymer, a functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymer, an ethylene/vinyl monomer/carbonyl terpolymer, an ethylene/alkyl (meth)acrylate/carbonyl terpolymer, a methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymer, a polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymer, a chlorinated or chlorosulphonated polyethylene, polyvinylidene fluoride (PVDF), a phenolic resin, poly(ethylene/vinyl acetate), a polybutadiene, a polyisoprene, a styrenic block copolymer, a polyacrylonitrile, and a silicone; Element 7: wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyacetal, a polycarbonate, a polybutylene terephthalate (PBT), a polyethylene terephthalate (PET), a polyethylene naphthalate (PEN), a polytrimethylene terephthalate (PTT), a ethylene vinyl acetate copolymer (EVA), a polyhexamethylene terephthalate, and a polystyrene; Element 8: wherein the thermoplastic polymer comprises one or more substituted or unsubstituted C2to C40alpha olefins; Element 9: wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, decene, undecene, dodecene, norbornene, norbornadiene, dicyclopentadiene, cyclopentene, cycloheptene, cyclooctene, cyclooctadiene, cyclododecene, 7-oxanorbornene, 7-oxanorbornadiene, substituted derivatives thereof, and isomers thereof; Element 10: wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: polyethylene, polypropylene, ethylene-propylene copolymers, ethylene-propylene-diene copolymers, polybutene, polyisobutylene, polymethylpentene, poly (4-methyl-1-pentene), and any combination thereof; Element 11: wherein the nucleating agent comprises one or more selected from the group consisting of: a salt of benzoic acid, lithium benzoate, sodium benzoate, aluminum benzoate, potassium benzoate, sodium salts of organophosphates, finely divided organoclays, carbon nanotubes, silica, calcium carbonate, talc, benzene trisamides, nonitol derivatives, 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-sorbitol, sodium 2,4,8,10-tetra-tert-butyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin-6-olate-6-oxide, bicyclo[2.2.1]heptane-2,3-dicarboxylic acid disodium salt, 2,2′-methylene-bis-(4,6-di-tert-butylphenyl) phosphate, and any combination thereof; Element 12: wherein the nucleating agent is present in the polymer particle in an amount of at about 0.05 wt % to about 5 wt % of the thermoplastic polymer; Element 13: wherein the polymer particles have a circularity of about 0.90 to about 1.0; Element 14: wherein the polymer particles have an angle of repose of about 25° to about 45°; Element 15: wherein the polymer particles have a Hausner ratio of about 1.0 to about 1.5; Element 16: wherein the polymer particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90; Element 17: wherein the polymer particles have a diameter span of about 0.2 to about 10; Element 18: wherein the polymer particles have an aerated density of about 0.55 g/cm3to about 0.8 g/cm3; Element 19: wherein the polymer particles have a bulk density of about 0.3 g/cm3to about 0.8 g/cm3; Element 20: wherein the polymer particles have a tapped density of about 0.6 g/cm3to about 0.9 g/cm3; Element 21: wherein the polymer particles have a BET surface area of about 10 m2/g to about 500 m2/g; Element 22: wherein the polymer particles further comprise an emulsion stabilizer covering at least a portion of a surface of the polymer particles; and Element 23: wherein the polymer particles further comprise a nanoparticle emulsion stabilizer embedded in a surface of the polymer particles. Examples of combinations may include, but are not limited to, Element 1 in combination with one of Elements 2-5 and optionally in further combination with one or more of Elements 6-23; one of Elements 2-5 in combination with one or more of Elements 6-23; two or more of Elements 6-10 in combination; one or more of Elements 6-10 in combination with one or more of Elements 11-23; Elements 11 and 12 in combination; one or more of Elements 11-12 in combination with one or more of Elements 13-23; two or more of Elements 13-23 in combination; Elements 1, 5, and 8 in combination; Elements 1, 5, and 9 in combination; and Elements 1, 5, and 10 in combination. A third nonlimiting example embodiment of the present disclosure is a selective laser sintered article comprising before being laser sintering: polymer particles comprising: a thermoplastic polymer, and a nucleating agent, wherein the polymer particles have a crystallization temperature that is substantially the same as a crystallization temperature of the thermoplastic polymer prior to formation of the polymer particles. The third nonlimiting example embodiment may further include one or more of: Element 1; Element 2; Element 3; Element 4; Element 5; Element 6; Element 7; Element 8; Element 9; Element 10; Element 11; Element 12; Element 13; Element 14; Element 15; Element 16; Element 17; Element 18; Element 19; Element 20; Element 21; Element 22; and Element 23. Examples of combinations may include, but are not limited to, Element 1 in combination with one of Elements 2-5 and optionally in further combination with one or more of Elements 6-23; one of Elements 2-5 in combination with one or more of Elements 6-23; two or more of Elements 6-10 in combination; one or more of Elements 6-10 in combination with one or more of Elements 11-23; Elements 11 and 12 in combination; one or more of Elements 11-12 in combination with one or more of Elements 13-23; two or more of Elements 13-23 in combination; Elements 1, 5, and 8 in combination; Elements 1, 5, and 9 in combination; and Elements 1, 5, and 10 in combination. A fourth nonlimiting example embodiment of the present disclosure is an article comprising: a consolidated body produced by selective laser sintering of polymer particles comprising melt emulsified polymer particles that comprise: a thermoplastic polymer, and a nucleating agent, wherein the melt emulsified polymer particles have a crystallization temperature is substantially the same as a crystallization temperature of the thermoplastic polymer prior to melt emulsification. The fourth nonlimiting example embodiments may further include one or more of: Element 24: wherein the thermoplastic polymer prior to melt emulsification satisfies Tm≤Tc+60° C. (or Tc+20° C.≤Tm≤Tc+60° C., or Tc+20° C.≤Tm≤Tc+50° C., or Tc+20° C.≤Tm≤Tc+40° C., or Tc+20° C.≤Tm≤Tc+30° C.); Element 25: wherein melt emulsified polymer particles have a crystallization temperature within about 10° C. of a crystallization temperature of the thermoplastic polymer prior to mixing; Element 26: wherein the melt emulsified polymer particles have a crystallization temperature within about 7° C. of a crystallization temperature of the thermoplastic polymer prior to mixing; Element 27: wherein the melt emulsified polymer particles have a crystallization temperature within about 5° C. of a crystallization temperature of the thermoplastic polymer prior to mixing; Element 28: wherein the melt emulsified polymer particles have a crystallization temperature within about 3° C. of a crystallization temperature of the thermoplastic polymer prior to mixing; Element 29: wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyurethane, a polyacetal, a polycarbonate, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), ethylene vinyl acetate copolymer (EVA), polyhexamethylene terephthalate, a polystyrene, a polyvinyl chloride, a polytetrafluoroethene, a polyester (e.g., polylactic acid), a polyether, a polyether sulfone, a polyetherether ketone, a polyacrylate, a polymethacrylate, a polyimide, acrylonitrile butadiene styrene (ABS), a polyphenylene sulfide, a vinyl polymer, a polyarylene ether, a polyarylene sulfide, a polysulfone, a polyether ketone, a polyamide-imide, a polyetherimide, a polyetherester, a copolymer comprising a polyether block and a polyamide block (PEBA or polyether block amide), a functionalized or nonfunctionalized ethylene/vinyl monomer polymer, a functionalized or nonfunctionalized ethylene/alkyl (meth)acrylate, a functionalized or nonfunctionalized (meth)acrylic acid polymer, a functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymer, an ethylene/vinyl monomer/carbonyl terpolymer, an ethylene/alkyl (meth)acrylate/carbonyl terpolymer, a methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymer, a polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymer, a chlorinated or chlorosulphonated polyethylene, polyvinylidene fluoride (PVDF), a phenolic resin, poly(ethylene/vinyl acetate), a polybutadiene, a polyisoprene, a styrenic block copolymer, a polyacrylonitrile, and a silicone; Element 30: wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyacetal, a polycarbonate, a polybutylene terephthalate (PBT), a polyethylene terephthalate (PET), a polyethylene naphthalate (PEN), a polytrimethylene terephthalate (PTT), a ethylene vinyl acetate copolymer (EVA), a polyhexamethylene terephthalate, and a polystyrene; Element 31: wherein the thermoplastic polymer comprises one or more substituted or unsubstituted C2to C40alpha olefins; Element 32: wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, decene, undecene, dodecene, norbornene, norbornadiene, dicyclopentadiene, cyclopentene, cycloheptene, cyclooctene, cyclooctadiene, cyclododecene, 7-oxanorbornene, 7-oxanorbornadiene, substituted derivatives thereof, and isomers thereof, Element 33: wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: polyethylene, polypropylene, ethylene-propylene copolymers, ethylene-propylene-diene copolymers, polybutene, polyisobutylene, polymethylpentene, poly (4-methyl-1-pentene), and any combination thereof, Element 34: wherein the nucleating agent comprises one or more selected from the group consisting of: a salt of benzoic acid, lithium benzoate, sodium benzoate, aluminum benzoate, potassium benzoate, sodium salts of organophosphates, finely divided organoclays, carbon nanotubes, silica, calcium carbonate, talc, benzene trisamides, nonitol derivatives, 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-sorbitol, sodium 2,4,8,10-tetra-tert-butyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin-6-olate-6-oxide, bicyclo[2.2.1]heptane-2,3-dicarboxylic acid disodium salt, 2,2′-methylene-bis-(4,6-di-tert-butylphenyl) phosphate, and any combination thereof; Element 35: wherein the nucleating agent is present in the polymer particle in an amount of at about 0.05 wt % to about 5 wt % of the thermoplastic polymer; Element 36: wherein the melt emulsified polymer particles have a circularity of about 0.90 to about 1.0; Element 37: wherein the melt emulsified polymer particles have an angle of repose of about 25° to about 45°; Element 38: wherein the melt emulsified polymer particles have a Hausner ratio of about 1.0 to about 1.5; Element 39: wherein the melt emulsified polymer particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90; Element 40: wherein the melt emulsified polymer particles have a diameter span of about 0.2 to about 10; Element 41: wherein the melt emulsified polymer particles have an aerated density of about 0.55 g/cm3to about 0.8 g/cm3; Element 42: wherein the melt emulsified polymer particles have a bulk density of about 0.3 g/cm3to about 0.8 g/cm3; Element 43: wherein the melt emulsified polymer particles have a tapped density of about 0.6 g/cm3to about 0.9 g/cm3; Element 44: wherein the melt emulsified polymer particles have a BET surface area of about 10 m2/g to about 500 m2/g; Element 45: wherein the melt emulsified polymer particles further comprise an emulsion stabilizer covering at least a portion of a surface of the melt emulsified polymer particles; and Element 46: wherein the melt emulsified polymer particles further comprise a nanoparticle emulsion stabilizer embedded in a surface of the melt emulsified polymer particles. Examples of combinations may include, but are not limited to, Element 23 in combination with one of Elements 25-28 and optionally in further combination with one or more of Elements 29-46; one of Elements 25-28 in combination with one or more of Elements 29-46; two or more of Elements 29-33 in combination; one or more of Elements 29-33 in combination with one or more of Elements 34-46; Elements 34 and 35 in combination; one or more of Elements 34-35 in combination with one or more of Elements 36-46; two or more of Elements 36-46 in combination; Elements 24, 28, and 31 in combination; Elements 24, 28, and 32 in combination; and Elements 24, 28, and 33 in combination. A fifth nonlimiting example embodiment of the present disclosure is a composition comprising: polymer particles comprising a thermoplastic polymer, a nucleating agent, optionally an emulsion stabilizer, and optionally a compatibilizer. The fifth nonlimiting example embodiment may further include one or more of: Element 6; Element 7; Element 8; Element 9; Element 10; Element 11; Element 12; Element 13; Element 14; Element 15; Element 16; Element 17; Element 18; Element 19; Element 20; Element 21; Element 22; Element 23; Element 47: wherein the polymer particles are produced by a melt emulsification method wherein the thermoplastic polymer prior to mixing has a crystallization temperature (Tc) and a melting temperature (Tm) that satisfies Tm≤Tc+60° C. (or Tc+20° C.≤Tm≤Tc+60° C., or Tc+20° C.≤Tm≤Tc+50° C., or Tc+20° C.≤Tm≤Tc+40° C., or Tc+20° C.≤Tm≤Tc+30° C.); Element 48: Element 47 in combination with Element 2; Element 49: Element 47 in combination with Element 3; Element 50: Element 47 in combination with Element 4; and Element 51: Element 47 in combination with Element 5. Examples of combinations may include, but are not limited to, two or more of Elements 6-10 in combination; one or more of Elements 6-10 in combination with one or more of Elements 11-23; Elements 11 and 12 in combination; one or more of Elements 11-12 in combination with one or more of Elements 13-23; two or more of Elements 13-23 in combination; Element 47 (optionally in combination with one or more of Elements 48-51) in combination with one or more of Elements 6-23; and Element 47 in combination with one of Elements 48-51. A sixth nonlimiting example embodiment of the present disclosure is a method comprising: depositing first polymer particles of the fifth nonlimiting example embodiment (optionally with one or more Elements as described above) and optionally second polymer particles different than the first polymer particles onto a surface; and once deposited, exposing at least a portion of the first and second, when included, polymer particles to a laser to fuse the first and second, when included, polymer particles and form a consolidated body. CLAUSES Clause 1. A method comprising: mixing a mixture comprising: a thermoplastic polymer, a nucleating agent, a carrier fluid, and optionally an emulsion stabilizer at a temperature at or greater than a melting point or softening temperature of the thermoplastic polymer to emulsify a thermoplastic polymer melt in the carrier fluid; cooling the mixture to below the melting point or softening temperature to form polymer particles; and separating the polymer particles from the carrier fluid, wherein the polymer particles have a crystallization temperature that is substantially the same as a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 2. A method comprising: mixing a mixture comprising: a thermoplastic polymer, a nucleating agent, a carrier fluid, and optionally an emulsion stabilizer at a temperature at or greater than a melting point or softening temperature of the thermoplastic polymer to emulsify a thermoplastic polymer melt in the carrier fluid, wherein the thermoplastic polymer prior to mixing has a crystallization temperature (Tc) and a melting temperature (Tm) that satisfies Tm≤Tc+60° C.; cooling the mixture to below the melting point or softening temperature to form polymer particles; and separating the polymer particles from the carrier fluid, wherein the polymer particles have a crystallization temperature that is substantially the same as a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 3. The method of Clause 1 or 2, wherein the thermoplastic polymer prior to mixing has a crystallization temperature (Tc) and a melting temperature (Tm) that satisfies Tm≤Tc+60° C. (or Tc+20° C.≤Tm≤Tc+60° C., or Tc+20° C.≤Tm≤Tc+50° C., or Tc+20° C.≤Tm≤Tc+40° C., or Tc+20° C.≤Tm≤Tc+30° C.). Clause 4. The method of Clause 1 or 2, wherein the polymer particles have a crystallization temperature within about 10° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 5. The method of Clause 1 or 2, wherein the polymer particles have a crystallization temperature within about 7° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 6. The method of Clause 1 or 2, wherein the polymer particles have a crystallization temperature within about 5° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 7. The method of Clause 1 or 2, wherein the polymer particles have a crystallization temperature within about 3° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 8. The method of Clause 1 or 2, wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyurethane, a polyacetal, a polycarbonate, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), ethylene vinyl acetate copolymer (EVA), polyhexamethylene terephthalate, a polystyrene, a polyvinyl chloride, a polytetrafluoroethene, a polyester (e.g., polylactic acid), a polyether, a polyether sulfone, a polyetherether ketone, a polyacrylate, a polymethacrylate, a polyimide, acrylonitrile butadiene styrene (ABS), a polyphenylene sulfide, a vinyl polymer, a polyarylene ether, a polyarylene sulfide, a polysulfone, a polyether ketone, a polyamide-imide, a polyetherimide, a polyetherester, a copolymer comprising a polyether block and a polyamide block (PEBA or polyether block amide), a functionalized or nonfunctionalized ethylene/vinyl monomer polymer, a functionalized or nonfunctionalized ethylene/alkyl (meth)acrylate, a functionalized or nonfunctionalized (meth)acrylic acid polymer, a functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymer, an ethylene/vinyl monomer/carbonyl terpolymer, an ethylene/alkyl (meth)acrylate/carbonyl terpolymer, a methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymer, a polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymer, a chlorinated or chlorosulphonated polyethylene, polyvinylidene fluoride (PVDF), a phenolic resin, poly(ethylene/vinyl acetate), a polybutadiene, a polyisoprene, a styrenic block copolymer, a polyacrylonitrile, and a silicone. Clause 9. The method of Clause 1 or 2, wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyacetal, a polycarbonate, a polybutylene terephthalate (PBT), a polyethylene terephthalate (PET), a polyethylene naphthalate (PEN), a polytrimethylene terephthalate (PTT), a ethylene vinyl acetate copolymer (EVA), a polyhexamethylene terephthalate, and a polystyrene. Clause 10. The method of Clause 1 or 2, wherein the thermoplastic polymer comprises one or more substituted or unsubstituted C2to C40alpha olefins. Clause 11. The method of Clause 1 or 2, wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, decene, undecene, dodecene, norbornene, norbornadiene, dicyclopentadiene, cyclopentene, cycloheptene, cyclooctene, cyclooctadiene, cyclododecene, 7-oxanorbornene, 7-oxanorbornadiene, substituted derivatives thereof, and isomers thereof. Clause 12. The method of Clause 1 or 2, wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: polyethylene, polypropylene, ethylene-propylene copolymers, ethylene-propylene-diene copolymers, polybutene, polyisobutylene, polymethylpentene, poly (4-methyl-1-pentene), and any combination thereof. Clause 13. The method of Clause 1 or 2, wherein the nucleating agent comprises one or more selected from the group consisting of: a salt of benzoic acid, lithium benzoate, sodium benzoate, aluminum benzoate, potassium benzoate, sodium salts of organophosphates, finely divided organoclays, carbon nanotubes, silica, calcium carbonate, talc, benzene trisamides, nonitol derivatives, 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-sorbitol, sodium 2,4,8,10-tetra-tert-butyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin-6-olate-6-oxide, bicyclo[2.2.1]heptane-2,3-dicarboxylic acid disodium salt, 2,2′-methylene-bis-(4,6-di-tert-butylphenyl) phosphate, and any combination thereof. Clause 14. The method of Clause 1 or 2, wherein the nucleating agent is present in the polymer particle in an amount of at about 0.05 wt % to about 5 wt % of the thermoplastic polymer. Clause 15. The method of Clause 1 or 2, wherein the polymer particles have a circularity of about 0.90 to about 1.0. Clause 16. The method of Clause 1 or 2, wherein the polymer particles have an angle of repose of about 25° to about 45°. Clause 17. The method of Clause 1 or 2, wherein the polymer particles have a Hausner ratio of about 1.0 to about 1.5. Clause 18. The method of Clause 1 or 2, wherein the polymer particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90. Clause 19. The method of Clause 1 or 2, wherein the polymer particles have a diameter span of about 0.2 to about 10. Clause 20. The method of Clause 1 or 2, wherein the polymer particles have an aerated density of about 0.55 g/cm3to about 0.8 g/cm3. Clause 21. The method of Clause 1 or 2, wherein the polymer particles have a bulk density of about 0.3 g/cm3to about 0.8 g/cm3. Clause 22. The method of Clause 1 or 2, wherein the polymer particles have a tapped density of about 0.6 g/cm3to about 0.9 g/cm3. Clause 23. The method of Clause 1 or 2, wherein the polymer particles have a BET surface area of about 10 m2/g to about 500 m2/g. Clause 24. The method of Clause 1 or 2, wherein the polymer particles further comprise an emulsion stabilizer covering at least a portion of a surface of the polymer particles. Clause 25. The method of Clause 1 or 2, wherein the polymer particles further comprise a nanoparticle emulsion stabilizer embedded in a surface of the polymer particles. Clause 26. A method comprising: mixing a mixture comprising: a thermoplastic polymer, a nucleating agent, a carrier fluid, and optionally an emulsion stabilizer at a temperature at or greater than a melting point or softening temperature of the thermoplastic polymer to emulsify a thermoplastic polymer melt in the carrier fluid; cooling the mixture to below the melting point or softening temperature to form polymer particles; and separating the polymer particles from the carrier fluid, wherein the polymer particles comprise the thermoplastic polymer, the nucleating agent, the emulsion stabilizer, if included, and wherein the polymer particles have a crystallization temperature within about 10° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 27. A method comprising: mixing a mixture comprising: a thermoplastic polymer, a nucleating agent, a carrier fluid, and optionally an emulsion stabilizer at a temperature at or greater than a melting point or softening temperature of the thermoplastic polymer to emulsify a thermoplastic polymer melt in the carrier fluid, wherein the thermoplastic polymer prior to mixing has a crystallization temperature (Tc) and a melting temperature (Tm) that satisfies Tm≤Tc+60° C.; cooling the mixture to below the melting point or softening temperature to form polymer particles; and separating the polymer particles from the carrier fluid, wherein the polymer particles comprise the thermoplastic polymer, the nucleating agent, the emulsion stabilizer, if included, and wherein the polymer particles have a crystallization temperature within about 10° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 28. The method of Clause 26 or 27, wherein the thermoplastic polymer prior to mixing has a crystallization temperature (Tc) and a melting temperature (Tm) that satisfies Tm≤Tc+60° C. (or Tc+20° C.≤Tm≤Tc+60° C., or Tc+20° C.≤Tm≤Tc+50° C., or Tc+20° C.≤Tm≤Tc+40° C., or Tc+20° C.≤Tm≤Tc+30° C.). Clause 29. The method of Clause 26 or 27, wherein the polymer particles have a crystallization temperature within about 7° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 30. The method of Clause 26 or 27, wherein the polymer particles have a crystallization temperature within about 5° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 31. The method of Clause 26 or 27, wherein the polymer particles have a crystallization temperature within about 3° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 32. The method of Clause 26 or 27, wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyurethane, a polyacetal, a polycarbonate, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), ethylene vinyl acetate copolymer (EVA), polyhexamethylene terephthalate, a polystyrene, a polyvinyl chloride, a polytetrafluoroethene, a polyester (e.g., polylactic acid), a polyether, a polyether sulfone, a polyetherether ketone, a polyacrylate, a polymethacrylate, a polyimide, acrylonitrile butadiene styrene (ABS), a polyphenylene sulfide, a vinyl polymer, a polyarylene ether, a polyarylene sulfide, a polysulfone, a polyether ketone, a polyamide-imide, a polyetherimide, a polyetherester, a copolymer comprising a polyether block and a polyamide block (PEBA or polyether block amide), a functionalized or nonfunctionalized ethylene/vinyl monomer polymer, a functionalized or nonfunctionalized ethylene/alkyl (meth)acrylate, a functionalized or nonfunctionalized (meth)acrylic acid polymer, a functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymer, an ethylene/vinyl monomer/carbonyl terpolymer, an ethylene/alkyl (meth)acrylate/carbonyl terpolymer, a methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymer, a polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymer, a chlorinated or chlorosulphonated polyethylene, polyvinylidene fluoride (PVDF), a phenolic resin, poly(ethylene/vinyl acetate), a polybutadiene, a polyisoprene, a styrenic block copolymer, a polyacrylonitrile, and a silicone. Clause 33. The method of Clause 26 or 27, wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyacetal, a polycarbonate, a polybutylene terephthalate (PBT), a polyethylene terephthalate (PET), a polyethylene naphthalate (PEN), a polytrimethylene terephthalate (PTT), a ethylene vinyl acetate copolymer (EVA), a polyhexamethylene terephthalate, and a polystyrene. Clause 34. The method of Clause 26 or 27, wherein the thermoplastic polymer comprises one or more substituted or unsubstituted C2to C40alpha olefins. Clause 35. The method of Clause 26 or 27, wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, decene, undecene, dodecene, norbornene, norbornadiene, dicyclopentadiene, cyclopentene, cycloheptene, cyclooctene, cyclooctadiene, cyclododecene, 7-oxanorbornene, 7-oxanorbornadiene, substituted derivatives thereof, and isomers thereof. Clause 36. The method of Clause 26 or 27, wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: polyethylene, polypropylene, ethylene-propylene copolymers, ethylene-propylene-diene copolymers, polybutene, polyisobutylene, polymethylpentene, poly (4-methyl-1-pentene), and any combination thereof. Clause 37. The method of Clause 26 or 27, wherein the nucleating agent comprises one or more selected from the group consisting of: a salt of benzoic acid, lithium benzoate, sodium benzoate, aluminum benzoate, potassium benzoate, sodium salts of organophosphates, finely divided organoclays, carbon nanotubes, silica, calcium carbonate, talc, benzene trisamides, nonitol derivatives, 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-sorbitol, sodium 2,4,8,10-tetra-tert-butyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin-6-olate-6-oxide, bicyclo[2.2.1]heptane-2,3-dicarboxylic acid disodium salt, 2,2′-methylene-bis-(4,6-di-tert-butylphenyl) phosphate, and any combination thereof. Clause 38. The method of Clause 26 or 27, wherein the nucleating agent is present in the polymer particle in an amount of at about 0.05 wt % to about 5 wt % of the thermoplastic polymer. Clause 39. The method of Clause 26 or 27, wherein the polymer particles have a circularity of about 0.90 to about 1.0. Clause 40. The method of Clause 26 or 27, wherein the polymer particles have an angle of repose of about 25° to about 45°. Clause 41. The method of Clause 26 or 27, wherein the polymer particles have a Hausner ratio of about 1.0 to about 1.5. Clause 42. The method of Clause 26 or 27, wherein the polymer particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90. Clause 43. The method of Clause 26 or 27, wherein the polymer particles have a diameter span of about 0.2 to about 10. Clause 44. The method of Clause 26 or 27, wherein the polymer particles have an aerated density of about 0.55 g/cm3to about 0.8 g/cm3. Clause 45. The method of Clause 26 or 27, wherein the polymer particles have a bulk density of about 0.3 g/cm3to about 0.8 g/cm3. Clause 46. The method of Clause 26 or 27, wherein the polymer particles have a tapped density of about 0.6 g/cm3to about 0.9 g/cm3. Clause 47. The method of Clause 25, wherein the polymer particles have a BET surface area of about 10 m2/g to about 500 m2/g. Clause 48. The method of Clause 26 or 27, wherein the polymer particles further comprise an emulsion stabilizer covering at least a portion of a surface of the polymer particles. Clause 49. The method of Clause 26 or 27, wherein the polymer particles further comprise a nanoparticle emulsion stabilizer embedded in a surface of the polymer particles. Clause 50. A selective laser sintered article comprising before being laser sintering: polymer particles comprising: a thermoplastic polymer, and a nucleating agent, wherein the polymer particles have a crystallization temperature that is substantially the same as a crystallization temperature of the thermoplastic polymer prior to formation of the polymer particles Clause 51. The article of Clause 50, wherein the thermoplastic polymer prior to mixing has a crystallization temperature (Tc) and a melting temperature (Tm) that satisfies Tm≤Tc+60° C. (or Tc+20° C.≤Tm≤Tc+60° C., or Tc+20° C.≤Tm≤Tc+50° C., or Tc+20° C.≤Tm≤Tc+40° C., or Tc+20° C.≤Tm≤Tc+30° C.). Clause 52. The article of Clause 51, wherein the polymer particles have a crystallization temperature within about 10° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 53. The article of Clause 51, wherein the polymer particles have a crystallization temperature within about 7° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 54. The article of Clause 50, wherein the polymer particles have a crystallization temperature within about 5° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 55. The article of Clause 50, wherein the polymer particles have a crystallization temperature within about 3° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 56. The article of Clause 50, wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyurethane, a polyacetal, a polycarbonate, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), ethylene vinyl acetate copolymer (EVA), polyhexamethylene terephthalate, a polystyrene, a polyvinyl chloride, a polytetrafluoroethene, a polyester (e.g., polylactic acid), a polyether, a polyether sulfone, a polyetherether ketone, a polyacrylate, a polymethacrylate, a polyimide, acrylonitrile butadiene styrene (ABS), a polyphenylene sulfide, a vinyl polymer, a polyarylene ether, a polyarylene sulfide, a polysulfone, a polyether ketone, a polyamide-imide, a polyetherimide, a polyetherester, a copolymer comprising a polyether block and a polyamide block (PEBA or polyether block amide), a functionalized or nonfunctionalized ethylene/vinyl monomer polymer, a functionalized or nonfunctionalized ethylene/alkyl (meth)acrylate, a functionalized or nonfunctionalized (meth)acrylic acid polymer, a functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymer, an ethylene/vinyl monomer/carbonyl terpolymer, an ethylene/alkyl (meth)acrylate/carbonyl terpolymer, a methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymer, a polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymer, a chlorinated or chlorosulphonated polyethylene, polyvinylidene fluoride (PVDF), a phenolic resin, poly(ethylene/vinyl acetate), a polybutadiene, a polyisoprene, a styrenic block copolymer, a polyacrylonitrile, and a silicone. Clause 57. The article of Clause 50, wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyacetal, a polycarbonate, a polybutylene terephthalate (PBT), a polyethylene terephthalate (PET), a polyethylene naphthalate (PEN), a polytrimethylene terephthalate (PTT), a ethylene vinyl acetate copolymer (EVA), a polyhexamethylene terephthalate, and a polystyrene. Clause 58. The article of Clause 50, wherein the thermoplastic polymer comprises one or more substituted or unsubstituted C2to C40alpha olefins. Clause 59. The article of Clause 50, wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, decene, undecene, dodecene, norbornene, norbornadiene, dicyclopentadiene, cyclopentene, cycloheptene, cyclooctene, cyclooctadiene, cyclododecene, 7-oxanorbornene, 7-oxanorbornadiene, substituted derivatives thereof, and isomers thereof. Clause 60. The article of Clause 50, wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: polyethylene, polypropylene, ethylene-propylene copolymers, ethylene-propylene-diene copolymers, polybutene, polyisobutylene, polymethylpentene, poly (4-methyl-1-pentene), and any combination thereof. Clause 61. The article of Clause 50, wherein the nucleating agent comprises one or more selected from the group consisting of: a salt of benzoic acid, lithium benzoate, sodium benzoate, aluminum benzoate, potassium benzoate, sodium salts of organophosphates, finely divided organoclays, carbon nanotubes, silica, calcium carbonate, talc, benzene trisamides, nonitol derivatives, 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-sorbitol, sodium 2,4,8,10-tetra-tert-butyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin-6-olate-6-oxide, bicyclo[2.2.1]heptane-2,3-dicarboxylic acid disodium salt, 2,2′-methylene-bis-(4,6-di-tert-butylphenyl) phosphate, and any combination thereof. Clause 62. The article of Clause 50, wherein the nucleating agent is present in the polymer particle in an amount of at about 0.05 wt % to about 5 wt % of the thermoplastic polymer. Clause 63. The article of Clause 50, wherein the polymer particles have a circularity of about 0.90 to about 1.0. Clause 64. The article of Clause 50, wherein the polymer particles have an angle of repose of about 25° to about 45°. Clause 65. The article of Clause 50, wherein the polymer particles have a Hausner ratio of about 1.0 to about 1.5. Clause 66. The article of Clause 50, wherein the polymer particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90. Clause 67. The article of Clause 50, wherein the polymer particles have a diameter span of about 0.2 to about 10. Clause 68. The article of Clause 50, wherein the polymer particles have an aerated density of about 0.55 g/cm3to about 0.8 g/cm3. Clause 69. The article of Clause 50, wherein the polymer particles have a bulk density of about 0.3 g/cm3to about 0.8 g/cm3. Clause 70. The article of Clause 50, wherein the polymer particles have a tapped density of about 0.6 g/cm3to about 0.9 g/cm3. Clause 71. The article of Clause 50, wherein the polymer particles have a BET surface area of about 10 m2/g to about 500 m2/g. Clause 72. The article of Clause 50, wherein the polymer particles further comprise an emulsion stabilizer covering at least a portion of a surface of the polymer particles. Clause 73. The article of Clause 50, wherein the polymer particles further comprise a nanoparticle emulsion stabilizer embedded in a surface of the polymer particles. Clause 74. An article comprising: a consolidated body produced by selective laser sintering of polymer particles comprising melt emulsified polymer particles that comprise: a thermoplastic polymer, and a nucleating agent, wherein the melt emulsified polymer particles have a crystallization temperature is substantially the same as a crystallization temperature of the thermoplastic polymer prior to melt emulsification. Clause 75: The article of Clause 74, wherein the thermoplastic polymer prior to melt emulsification satisfies Tm≤Tc+60° C. (or Tc+20° C.≤Tm≤Tc+60° C., or Tc+20° C.≤Tm≤Tc+50° C., or Tc+20° C.≤Tm≤Tc+40° C., or Tc+20° C.≤Tm≤Tc+30° C.). Clause 76: The article of Clause 74, wherein melt emulsified polymer particles have a crystallization temperature within about 10° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 77: The article of Clause 74, wherein the melt emulsified polymer particles have a crystallization temperature within about 7° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 78: The article of Clause 74, wherein the melt emulsified polymer particles have a crystallization temperature within about 5° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 79: The article of Clause 74, wherein the melt emulsified polymer particles have a crystallization temperature within about 3° C. of a crystallization temperature of the thermoplastic polymer prior to mixing. Clause 80: The article of Clause 74, wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyurethane, a polyacetal, a polycarbonate, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), ethylene vinyl acetate copolymer (EVA), polyhexamethylene terephthalate, a polystyrene, a polyvinyl chloride, a polytetrafluoroethene, a polyester (e.g., polylactic acid), a polyether, a polyether sulfone, a polyetherether ketone, a polyacrylate, a polymethacrylate, a polyimide, acrylonitrile butadiene styrene (ABS), a polyphenylene sulfide, a vinyl polymer, a polyarylene ether, a polyarylene sulfide, a polysulfone, a polyether ketone, a polyamide-imide, a polyetherimide, a polyetherester, a copolymer comprising a polyether block and a polyamide block (PEBA or polyether block amide), a functionalized or nonfunctionalized ethylene/vinyl monomer polymer, a functionalized or nonfunctionalized ethylene/alkyl (meth)acrylate, a functionalized or nonfunctionalized (meth)acrylic acid polymer, a functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymer, an ethylene/vinyl monomer/carbonyl terpolymer, an ethylene/alkyl (meth)acrylate/carbonyl terpolymer, a methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymer, a polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymer, a chlorinated or chlorosulphonated polyethylene, polyvinylidene fluoride (PVDF), a phenolic resin, poly(ethylene/vinyl acetate), a polybutadiene, a polyisoprene, a styrenic block copolymer, a polyacrylonitrile, and a silicone. Clause 81: The article of Clause 74, wherein the thermoplastic polymer comprises one or more substituted or unsubstituted C2to C40alpha olefins. Clause 82: The article of Clause 74, wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, decene, undecene, dodecene, norbornene, norbornadiene, dicyclopentadiene, cyclopentene, cycloheptene, cyclooctene, cyclooctadiene, cyclododecene, 7-oxanorbornene, 7-oxanorbornadiene, substituted derivatives thereof, and isomers thereof. Clause 83: The article of Clause 74, wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: polyethylene, polypropylene, ethylene-propylene copolymers, ethylene-propylene-diene copolymers, polybutene, polyisobutylene, polymethylpentene, poly (4-methyl-1-pentene), and any combination thereof. Clause 84: The article of Clause 74, wherein the nucleating agent comprises one or more selected from the group consisting of: a salt of benzoic acid, lithium benzoate, sodium benzoate, aluminum benzoate, potassium benzoate, sodium salts of organophosphates, finely divided organoclays, carbon nanotubes, silica, calcium carbonate, talc, benzene trisamides, nonitol derivatives, 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-sorbitol, sodium 2,4,8,10-tetra-tert-butyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin-6-olate-6-oxide, bicyclo[2.2.1]heptane-2,3-dicarboxylic acid disodium salt, 2,2′-methylene-bis-(4,6-di-tert-butylphenyl) phosphate, and any combination thereof. Clause 85: The article of Clause 74, wherein the nucleating agent is present in the melt emulsified polymer particles in an amount of at about 0.05 wt % to about 5 wt % of the thermoplastic polymer. Clause 86: The article of Clause 74, wherein the melt emulsified polymer particles have a circularity of about 0.90 to about 1.0. Clause 87: The article of Clause 74, wherein the melt emulsified polymer particles have an angle of repose of about 25° to about 45°. Clause 88: The article of Clause 74, wherein the melt emulsified polymer particles have a Hausner ratio of about 1.0 to about 1.5. Clause 89: The article of Clause 74, wherein the melt emulsified polymer particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90. Clause 90: The article of Clause 74, wherein the melt emulsified polymer particles have a diameter span of about 0.2 to about 10. Clause 91: The article of Clause 74, wherein the melt emulsified polymer particles have an aerated density of about 0.55 g/cm3to about 0.8 g/cm3. Clause 92: The article of Clause 74, wherein the melt emulsified polymer particles have a bulk density of about 0.3 g/cm3to about 0.8 g/cm3. Clause 93: The article of Clause 74, wherein the melt emulsified polymer particles have a tapped density of about 0.6 g/cm3to about 0.9 g/cm3. Clause 94: The article of Clause 74, wherein the melt emulsified polymer particles have a BET surface area of about 10 m2/g to about 500 m2/g. Clause 95: The article of Clause 74, wherein the melt emulsified polymer particles further comprise an emulsion stabilizer covering at least a portion of a surface of the melt emulsified polymer particles. Clause 96: The article of Clause 74, wherein the melt emulsified polymer particles further comprise a nanoparticle emulsion stabilizer embedded in a surface of the melt emulsified polymer particles. Clause 97. A composition comprising: polymer particles comprising a thermoplastic polymer, a nucleating agent, optionally an emulsion stabilizer, and optionally a compatibilizer. Clause 98. The composition of Clause 97, wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyurethane, a polyacetal, a polycarbonate, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), ethylene vinyl acetate copolymer (EVA), polyhexamethylene terephthalate, a polystyrene, a polyvinyl chloride, a polytetrafluoroethene, a polyester (e.g., polylactic acid), a polyether, a polyether sulfone, a polyetherether ketone, a polyacrylate, a polymethacrylate, a polyimide, acrylonitrile butadiene styrene (ABS), a polyphenylene sulfide, a vinyl polymer, a polyarylene ether, a polyarylene sulfide, a polysulfone, a polyether ketone, a polyamide-imide, a polyetherimide, a polyetherester, a copolymer comprising a polyether block and a polyamide block (PEBA or polyether block amide), a functionalized or nonfunctionalized ethylene/vinyl monomer polymer, a functionalized or nonfunctionalized ethylene/alkyl (meth)acrylate, a functionalized or nonfunctionalized (meth)acrylic acid polymer, a functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymer, an ethylene/vinyl monomer/carbonyl terpolymer, an ethylene/alkyl (meth)acrylate/carbonyl terpolymer, a methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymer, a polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymer, a chlorinated or chlorosulphonated polyethylene, polyvinylidene fluoride (PVDF), a phenolic resin, poly(ethylene/vinyl acetate), a polybutadiene, a polyisoprene, a styrenic block copolymer, a polyacrylonitrile, and a silicone. Clause 99. The composition of Clause 97, wherein the thermoplastic polymer comprises one or more of: a thermoplastic polyolefin, a polyamide, a polyacetal, a polycarbonate, a polybutylene terephthalate (PBT), a polyethylene terephthalate (PET), a polyethylene naphthalate (PEN), a polytrimethylene terephthalate (PTT), a ethylene vinyl acetate copolymer (EVA), a polyhexamethylene terephthalate, and a polystyrene. Clause 100. The composition of Clause 97, wherein the thermoplastic polymer comprises one or more substituted or unsubstituted C2to C40alpha olefins. Clause 101. The composition of Clause 97, wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, decene, undecene, dodecene, norbornene, norbornadiene, dicyclopentadiene, cyclopentene, cycloheptene, cyclooctene, cyclooctadiene, cyclododecene, 7-oxanorbornene, 7-oxanorbornadiene, substituted derivatives thereof, and isomers thereof. Clause 102. The composition of Clause 97, wherein the thermoplastic polymer comprises a monomer selected from the group consisting of: polyethylene, polypropylene, ethylene-propylene copolymers, ethylene-propylene-diene copolymers, polybutene, polyisobutylene, polymethylpentene, poly (4-methyl-1-pentene), and any combination thereof. Clause 103. The composition of Clause 97, wherein the nucleating agent comprises one or more selected from the group consisting of: a salt of benzoic acid, lithium benzoate, sodium benzoate, aluminum benzoate, potassium benzoate, sodium salts of organophosphates, finely divided organoclays, carbon nanotubes, silica, calcium carbonate, talc, benzene trisamides, nonitol derivatives, 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-sorbitol, sodium 2,4,8,10-tetra-tert-butyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin-6-olate-6-oxide, bicyclo[2.2.1]heptane-2,3-dicarboxylic acid disodium salt, 2,2′-methylene-bis-(4,6-di-tert-butylphenyl) phosphate, and any combination thereof. Clause 104. The composition of Clause 97, wherein the nucleating agent is present in the polymer particle in an amount of at about 0.05 wt % to about 5 wt % of the thermoplastic polymer. Clause 105. The composition of Clause 97, wherein the polymer particles have a circularity of about 0.90 to about 1.0. Clause 106. The composition of Clause 97, wherein the polymer particles have an angle of repose of about 25° to about 45°. Clause 107. The composition of Clause 97, wherein the polymer particles have a Hausner ratio of about 1.0 to about 1.5. Clause 108. The composition of Clause 97, wherein the polymer particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90. Clause 109. The composition of Clause 97, wherein the polymer particles have a diameter span of about 0.2 to about 10. Clause 110. The composition of Clause 97, wherein the polymer particles have an aerated density of about 0.55 g/cm3to about 0.8 g/cm3 Clause 111. The composition of Clause 97, wherein the polymer particles have a bulk density of about 0.3 g/cm3to about 0.8 g/cm3. Clause 112. The composition of Clause 97, wherein the polymer particles have a tapped density of about 0.6 g/cm3to about 0.9 g/cm3. Clause 113. The composition of Clause 97, wherein the polymer particles have a BET surface area of about 10 m2/g to about 500 m2/g. Clause 114. The composition of Clause 97, wherein the polymer particles further comprise an emulsion stabilizer covering at least a portion of a surface of the polymer particles. Clause 115. The composition of Clause 97, wherein the polymer particles further comprise a nanoparticle emulsion stabilizer embedded in a surface of the polymer particles. Clause 116. A method comprising: depositing first polymer particles of any one of the compositions of Clauses 97-115 and optionally second polymer particles different than the first particles onto a surface; and once deposited, exposing at least a portion of the first and second, when included, particles to a laser to fuse the first and second, when included, particles and form a consolidated body. Clause 117. The method of Clause 116, wherein the consolidated body has a void percentage of about 5% or less. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the present specification and associated claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the incarnations of the present inventions. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claim, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. One or more illustrative incarnations incorporating one or more invention elements are presented herein. Not all features of a physical implementation are described or shown in this application for the sake of clarity. It is understood that in the development of a physical embodiment incorporating one or more elements of the present invention, numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related and other constraints, which vary by implementation and from time to time. While a developer's efforts might be time-consuming, such efforts would be, nevertheless, a routine undertaking for those of ordinary skill in the art and having benefit of this disclosure. While compositions and methods are described herein in terms of “comprising” various components or steps, the compositions and methods can also “consist essentially of” or “consist of” the various components and steps. To facilitate a better understanding of the embodiments of the present invention, the following examples of preferred or representative embodiments are given. In no way should the following examples be read to limit, or to define, the scope of the invention. EXAMPLES Sample 1 (comparative): A Haake twin-screw extruder was brought to a temperature around the melting point of the polymer (about 225° C.) and the rotors were started at 120 rpm. 65.1 g of polypropylene homopolymer pellets (Ultra HoPP 20, available from ResMart) were added to the heated extruder followed by 145 g poly(dimethylsiloxane) (PDMS) (PSF-30,000 from Clearco). The weight ratio of PDMS oil to polymer was 70:30 or 30 wt % polymer solids in 70 wt % oil. At temperature, the extruder was operated at 120 rpm for 10 min. The mixture was then discharged onto a metal tray and allowed to cool slowly. Once at room temperature, the PDMS was washed away from the polypropylene particles with three heptane washes and the polypropylene particles were isolated by vacuum filtration. The polypropylene particles were then dried overnight in a vacuum oven at room temperature to allow any residual heptane to evaporate. The dried polypropylene particles were then sieved through a 250 μm screen. Sample 2 (inventive): Sample 2 was prepared in the manner of Sample 1 with the exception that 0.20 wt % 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-sorbitol nucleating agent based on a total weight of polypropylene was added to the extruder after the addition of the polypropylene pellets. Sample 3 (inventive): Sample 3 was prepared in the manner of Sample 1 with the exception that 0.30 wt % 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-sorbitol nucleating agent based on a total weight of polypropylene was added to the extruder after the addition of the polypropylene pellets. Sample 4 (inventive): Sample 4 was prepared in the manner of Sample 1 with the exception that 0.20 wt % bicyclo[2.2.1]heptane-2,3-dicarboxylic acid disodium salt nucleating agent based on a total weight of polypropylene was added to the extruder after the addition of the polypropylene pellets. Sample 5 (inventive): Sample 5 was prepared in the manner of Sample 1 with the exception that 0.30 wt % bicyclo[2.2.1]heptane-2,3-dicarboxylic acid disodium salt nucleating agent based on a total weight of polypropylene was added to the extruder after the addition of the polypropylene pellets. Table 1 provides characteristics of the particles. A commercial example of ULTRASINT®PP powder (available from BASF) were also characterized as a comparative example. TABLE 1CommercialSampleSample 1Sample 2Sample 3Sample 4Sample 5nucleating agentnonenone0.2 wt %0.3 wt %0.2 wt %0.3 wt %sorbitolsorbitolbicyclobicycloparticle size62.247.968.675.370.966.9(microns)(after sieve)span (after sieve)1.051.121.211.241.00.96angle of repose (°)45.331.332.433.330.930.4melting temp. (° C.)135.0160.5162.0162.1159.5158.9crystallizationcould not116.7127.4107.4128.9127.7temp. (° C.)be measuredSEM ImageFIG. 2FIG. 3FIG. 4FIG. 5FIG. 6FIG. 7 With the exception of Sample 3, polypropylene particles made with nucleating agents showed an increase in crystallization temperature compared with Sample 1. Further, the melting temperature and a crystallization temperature for the polypropylene homopolymer (Ultra HoPP 20) of Samples 1-5 were measured to be about 166° C. and about 127.5° C., respectively. Sample 1 illustrates that melt emulsification methods may depress the crystallization temperature of the starting polymer. Samples 2, 4, and 5 illustrate that the inclusion of a nucleating agent may cause the crystallization temperature to be substantially maintained. At least some of the foregoing samples were processed by SLS methods using a Sharebot SnowWhite SLS Printer with a CO2laser. More specifically, a 30 mm×30 mm×0.1 mm square was printed as a preliminary screening object. The sieved particles were applied onto an aluminum plate using a bar coater (40 mil gap/approximately 1 mm thick layer of powder). The sample was placed in the SnowWhite chamber. The motors were disabled since a multilayer object was not printed. The environmental temperature control was enabled. The chamber temperature was set to 115° C. as determined by previous experimentation. Laser rate and laser power were varied to determine optimal print conditions. The chamber and powder bed were cooled to room temperature before the part was removed. Edge curl was visually identified by the naked eye and qualitatively analyzed. To quantify the amount of edge curl or warping, profile measurements of the sintered squares were taken. Warping was then calculated as the average of the left and right edge height minus the center height of the sintered square. Units are in millimeters. Table 2 shows that experimental samples made with nucleating agents had less warping than the comparative samples 1 and 2, which both contained no nucleating agents. Therefore, the addition of nucleating agents decreases the amount of warping in polypropylene single layer sintered objects. The nucleating agent does not interfere with good particle formation, size, span or shape. TABLE 2CommercialSampleSample 1Sample 2Sample 3Sample 4Sample 5nucleating agentnonenone0.2 wt %0.3 wt %0.2 wt %0.3 wt %sorbitolsorbitolbicyclobicyclowarping (mm)1.591.600.180.180.701.24(65% laser power)(40,000 rate) Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. The particular examples and configurations disclosed above are illustrative only, as the present invention may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. It is therefore evident that the particular illustrative examples disclosed above may be altered, combined, or modified and all such variations are considered within the scope and spirit of the present invention. The invention illustratively disclosed herein suitably may be practiced in the absence of any element that is not specifically disclosed herein and/or any optional element disclosed herein. While compositions and methods are described in terms of “comprising,” “containing,” or “including” various components or steps, the compositions and methods can also “consist essentially of” or “consist of” the various components and steps. All numbers and ranges disclosed above may vary by some amount. Whenever a numerical range with a lower limit and an upper limit is disclosed, any number and any included range falling within the range is specifically disclosed. In particular, every range of values (of the form, “from about a to about b,” or, equivalently, “from approximately a to b,” or, equivalently, “from approximately a-b”) disclosed herein is to be understood to set forth every number and range encompassed within the broader range of values. Also, the terms in the claims have their plain, ordinary meaning unless otherwise explicitly and clearly defined by the patentee. Moreover, the indefinite articles “a” or “an,” as used in the claims, are defined herein to mean one or more than one of the element that it introduces. | 126,767 |
11859104 | DETAILED DESCRIPTION OF THE INVENTION The identified advantages of the self-healing anti-corrosion coating compositions, as per the inventor's provided disclosure are as follows: Nanocontainers in the polymer coatings have more freedom of movement than those in the sol-gel coatings where they are tightly bound with the sol-gel. Since the cost of the raw materials (metal alkoxides) used the sol-gel coatings are high, this method is economically not feasible. Starting materials used for the nanocontainer-polymer coatings such as vinyl acrylate and polyvinyl butyral (PVB) are most cost effective. Curing of nanocontainer-polymer coatings at 30-70° C. is enough to get a uniform thick coating. Sol-gel coatings always require high temperature annealing to achieve a dense microstructure. Sintering at high temperatures introduces cracks and/or delamination of sol-gel coatings. Curing of nanocontainer-polymer coatings improve the chemical and physical interaction between the functional groups such as amine, hydroxyl etc. on the outer layer polyelectrolyte shell with the polymeric matrix, which enhances the nanocontainer compatibility and reduces the chances of coating damages. Successful commercially available sol-gel coatings are thin films. Thick films of (>1 μm) sol-gel coatings always have cracking problems. Uniform coatings with thickness greater than 1 μm could easily be obtained from nanocontainer-polymer formulation according to the invention. In the present invention so-called nanocontainers are described which are made from mesoporous carbon (Meso C). The use of mesoporous carbon as carrier of corrosion inhibitor is not well reported in the literature. Therefore, the present work has been conducted in the fabrication of corrosion inhibitor encapsulated mesoporous carbon based nanocontainer coatings for the corrosion protection of metal structures. Corrosion inhibitor was added directly into the pores of carbon matrix and then covered by polyelectrolyte layers to prevent the unwanted release. It was applied for coating preparation by mixing with vinyl acrylate primer. The advantage of mesoporous carbon nanocontainer over silica nanocontainer is that the coatings would be more hydrophobic and it can protect the UV radiation as well. Synthesis of Mesoporous Carbon/(BT)3/PAH Nanocontainer Mesoporous carbon (0.5 g) was dispersed in 15 ml of water by sonication. It was then degassed under vacuum to open up the pores. Benzotriazole (BT) (20 mg/ml) dissolved in water was added to this mixture with constant stirring and then again degassed under vacuum. The vacuum was adjusted at the point when the bubbling of air from the pores starts. This degassing process was continued till the bubbles from the mixture is completely disappeared. The excess of benzotriazole was removed by centrifugation and washing with water and then dried at 80° C. for 24 h. These steps were repeated three times to completely fill the pores of mesoporous carbon with benzotriazole. 10 mg/ml of poly (allyl amine) hydrochloride (PAH) was added as the covering layer for the benzotriazole encapsulated mesoporous carbon and a final structure of Meso Carbon/(BT)3/PAH was obtained. Mesoporous Carbon Nanocontainer Coatings on Carbon Steel The final mixtures of Meso Carbon/(BT)3/PAH nanocontainers were dried in oven at 80° C. for overnight. 0.5 g of mesoporous carbon nanocontainer was added slowly to a 30 g of solvent based vinyl acrylate with magnetic stirring and then was coated on the carbon steel substrate by dip coating. After drying at 80° C. for 2 h followed by 60° C. for 12 h all the sides were sealed with quick setting epoxy. A schematic representation of silica nanocontainer synthesis and coating on carbon steel substrate is given inFIG.12. Corrosion analysis of each coating was conducted by immersion test in 0.35M sodium chloride solution. In order to study the self-healing nature of the coatings a scratch was made before immersing in sodium chloride solution. Corrosion was monitored at periodic intervals using microscopic techniques and measured quantitatively by electrochemical techniques. Release of benzotriazole from the nanocontainers was monitored by UV-Visible-NIR spectrometer at different pH solutions. Results and Discussion FIG.1shows the release of benzotriazole from Meso carbon/(BT)3/PAH nanocontainer at different pH solutions measured by UV-Visible-NIR spectrometer. In the UV spectra, nanocontainers at pH 3 and 10 solutions showed a higher absorbance than those in pH 7 solution, indicating the more release of benzotriazole in acidic and alkaline media. It was due to its solubility that benzotriazole dissolves in acidic and alkaline solutions far better than neutral media. Additionally, UV spectra confirmed further the presence of benzotriazole inside the pores of carbon nanocontainer. In order to analyze the self-healing nature the scratched coatings of both vinyl acrylate and nanocontainer embedded vinyl acrylate were immersed in 0.35M sodium chloride solution. Sevier corrosion products on the scratches of vinyl acrylate alone coated substrate was clearly visible inFIGS.2&3. Optical and SEM images also showed the corrosion products on the scratches and EDAX results confirmed the formation of iron oxides as well (FIGS.4,5&6). Corrosion progressed with time and slowly it covered all the scratches. Immersion test conducted on the Meso carbon/(BT)3/PAH nanocontainer embedded coatings are given inFIGS.7&8. Compared with vinyl acrylate alone coated substrate nanocontainer coatings showed less corrosion products on the scratches (FIGS.7&8). Optical and SEM images of the corroded area, marked with circle1inFIG.8, was observed to be covered by a precipitate and the EDAX showed the presence of nitrogen in that area (FIGS.9,11&12). It indicated that benzotriazole released from the nanocontainer diminished the extent of corrosion that was apparent from the less intensity of corrosion products on the scratches of nanocontainer coating compared with vinyl acrylate coating (FIGS.3&8). The optical and SEM images of the non-corroded areas of the scratches given inFIG.10andFIG.13respectively showed clean surfaces. EDAX of this area also showed the presence of nitrogen and oxygen atoms along with iron (FIG.14). It indicated that benzotriazole released from the nanocontainer formed resistive layers with iron and oxygen on the metal surface which prevented corrosion. It can be suggested therefore that mesoporous carbon nanocontainers were effective carriers of benzotriazole which got released on demand at required areas of metal structures for corrosion protection. Self-healing performance of the nanocontainer embedded coatings were quantified by measuring impedance and open circuit potential (OCP) at different time of immersion in 0.35M sodium chloride solution. Electrochemical impedance (EIS) of vinyl acrylate coating and nanocontainer coating, measured using a three electrode set up, are given inFIGS.15&16respectively. Low frequency impedance (log Z) of nanocontainer coatings at initial time of immersion was at 5.5 Ωcm2which is slightly lower than the vinyl acrylate coating (6.2 Ωcm2). Low frequency impedance of vinyl acrylate coating decreased continuously and reached 4.6 Ωcm2by 4 d whereas for nanocontainer coating low frequency impedance reached a similar value by 13 d. This behavior of nanocontainer coating was due to the corrosion inhibitive nature of benzotriazole released from the Meso carbon/(BT)3/PAH nanocontainer. Open circuit potential (OCP) related to the corrosion potential, measured when no current or potential being applied to the cell, showed a decreasing trend to nanocontainer coating during the initial days of exposure and then started to increase after 50 hours (FIG.17). This type of OCP behavior clearly shows the self-healing nature of the coating. At the same time OCP of vinyl acrylate alone coated substrates didn't increase after the initial decrease and it continued to remain at the lower value during the further immersion in sodium chloride solution. Both immersion test and electrochemical analysis concluded that mesoporous carbon nanocontainers are effective in providing protection by inhibiting corrosion on the metal surface by the release of encapsulated corrosion inhibitor. | 8,335 |
11859105 | In the different figures, the same reference signs refer to the same or analogous elements. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The present invention will be described with respect to particular embodiments and with reference to certain drawings, but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. The dimensions and the relative dimensions do not correspond to actual reductions to practice of the invention. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequence, either temporally, spatially, in ranking or in any other manner. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. Moreover, the terms top, under and the like in the description and the claims are used for descriptive purposes and not necessarily for describing relative positions. It is to be understood that the terms so used are interchangeable with their antonyms under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other orientations than described or illustrated herein. It is to be noticed that the term “comprising”, used in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a device comprising means A and B” should not be limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination. Furthermore, some of the embodiments are described herein as a method or combination of elements of a method that can be implemented by a processor of a computer system or by other means of carrying out the function. Thus, a processor with the necessary instructions for carrying out such a method or element of a method forms a means for carrying out the method or element of a method. Furthermore, an element described herein of an apparatus embodiment is an example of a means for carrying out the function performed by the element for the purpose of carrying out the invention. In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practised without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description. In a first aspect, the present invention relates to a self-cleaning coating, comprising a photocatalytic matrix, and plasmonic nanoparticles embedded in the photocatalytic matrix. The photocatalytic matrix is typically a material which can embed the plasmonic nanoparticles and can photocatalytically degrade (e.g. decompose) an undesired pollutant. In embodiments, the self-cleaning coating may be suitable for degrading at least one pollutant. In embodiments, the pollutant may be an organic compound (e.g. a volatile organic compound), an inorganic compound (e.g. a nitrogen oxide) or a microorganism (e.g. a bacteria, fungus, virus or parasite). In embodiments, the pollutant may be present in a fluid, e.g. in a liquid or in a gas. The self-cleaning coating may be used in a gas environment (e.g. air) and/or submerged in a liquid (e.g. water, such as wastewater). The self-cleaning coating may be useful outside; e.g. to degrade an environmental pollutant, or to keep a window, wall or roof dirt free. Likewise, the self-cleaning coating may also be useful in-house; e.g. to keep a window of a fish tank free from dirt and algae, to degrade a tobacco smoke, or to provide an antimicrobial property to a wall, door, handle, etc. (e.g. in a hospital). Without being bound by theory, it is believed that photoexcitations in the photocatalytic matrix facilitate decomposition reactions involving the pollutant. These decomposition reactions may further comprise other reactants in the environment, such as oxygen or water. For example, an energy transfer may occur from a photoexcitation in the photocatalytic matrix to oxygen, resulting in the formation of a highly reactive oxygen species (e.g. an oxygen radical). These highly reactive oxygen species may then in turn react with the pollutant, or an intermediate degradation product, to form a further degradation product. In embodiments, degrading the at least one pollutant may comprise a series of oxidation steps. The degradation products of a fully degraded pollutant may, for example, comprise carbon dioxide and water. In embodiments, the self-cleaning coating may have a highly hydrophilic or highly hydrophobic surface. The highly hydrophilic surface may, for example, be characterized by a static water contact angle of 45° or lower, preferably 30° or lower, more preferably 20° or lower. The highly hydrophobic surface may, for example, be characterized by a static water contact angle of 135° or higher, preferably 150° or higher, more preferably 160° or higher. Typically in combination with water (e.g. rain), highly hydrophilic or highly hydrophobic surfaces provide an alternative form of self-cleaning by preventing the accumulation of pollutants (e.g. dirt) on the surface and having them easily washed them away (e.g. by rain). In the case of a highly hydrophilic surface, sheeting water may carry away the pollutants. Conversely, in the case of a highly hydrophobic surface, the pollutants may be carried away by rolling water droplets. This alternative form of self-cleaning is furthermore compatible with the photocatalytic self-cleaning and can thus be provided in addition thereto. In embodiments, the photocatalytic matrix may comprise TiO2, but any other photocatalyst like ZnO, WO3, CdS, etc. or their combination. TiO2is advantageously known to have good photocatalytic properties. Moreover, TiO2absorbs light in the UV range, thereby allowing it to function as a matrix in a self-cleaning coating which is advantageously transparent to visible light. This is a useful property in many applications, both when the self-cleaning coating is to be applied on a surface which is preferably transparent (such on a glass pane of a window or solar panel), as well as when a change in appearance of the surface (e.g. a color change) due to the coating is undesired. By introducing plasmonic nanoparticles, the coating thus remains substantially transparent, but it does absorb in the visible light region, which typically may give a haze of a certain colour. Additionally, TiO2is known to become highly hydrophilic (e.g. superhydrophilic) when exposed to light (e.g. sunlight). As such, a dual form of self-cleaning can be provided by the TiO2comprising self-cleaning coating, combining both the photocatalytic degradation of pollutants and the anti-sticking nature of a highly hydrophilic surface. It was surprisingly found within the present invention that the self-cleaning action of a photocatalytic matrix can be enhanced by embedding therein plasmonic nanoparticles. The plasmonic nanoparticles can for example advantageously extend the spectral range that can be exploited by the photocatalytic matrix (i.e. improve the spectral response), by absorbing light outside the absorption range of the photocatalytic matrix and subsequently transferring the energy associated with the excited state to the photocatalytic matrix (e.g. hot electron transfer). Alternatively, or additionally, the plasmonic nanoparticles may facilitate the spatial separation of excitons into distinct charge carriers (e.g. electrons and holes), due to the presence of a barrier (e.g. a Schottky barrier) near the matrix-nanoparticle interface. The spatial separation of charge carriers hinders their recombination rate; a recombination which would prevent them from contributing to the photocatalytic degradation. It should be noted that the concentration of nanoparticles need not be large to obtain a considerable effect. As such, even when the plasmonic nanoparticles absorb in the visible range of the electromagnetic spectrum, the self-cleaning coating can remain highly transparent and colourless. The concentration of nanoparticles may be within 0.01 weight % and 4 weight %, e.g. within 0.5 weight % and 4 weight %, e.g. between 1 weight % and 3 weight %. It is an advantage of embodiments of the present invention that the transmission of the film with embedded nanoparticles can be at least 50%, e.g. at least 60%, e.g. at least 70%, e.g. at least 75%, e.g. at least 80%. Moreover, embedding the plasmonic nanoparticles in the photocatalytic matrix brings additional advantages compared to e.g. providing the nanoparticles on top of the matrix. A first benefit is that a more even distribution of the nanoparticles can be obtained, while achieving a tighter integration between the nanoparticles and the matrix. This enables the spectral range of a larger portion of the matrix to be extended, e.g. to substantially the whole matrix, as opposed to only a top layer in contact with the nanoparticles. Simultaneously, the tighter integration lowers the energy transfer distance that has to be overcome. A second benefit is that the nanoparticles can be protected by the surrounding matrix from e.g. physical and/or chemical influences. Indeed, when the nanoparticles which are not embedded in but attached to a surface of the matrix, they are prone to detachment due to physical forces or prone to a change their nature due to chemical reactions with the environment. A third benefit is that the embedded nanoparticles do not take up valuable, reactive surface area from the photocatalytic matrix; thereby allowing a higher active surface area for the self-cleaning coating, compared to when the nanoparticles would cover the top of the matrix. In embodiments, the plasmonic nanoparticles may comprise a noble metal. In embodiments, the noble metal may be selected from the list of Ru, Rh, Pd, Ag, Os, Ir, Pt, Au, Hg, Re and Cu. In preferred embodiments, the noble metal may be selected from the list of Ru, Rh, Pd, Ag, Os, Ir, Pt and Au. In very preferred embodiments, the noble metal may be Au or Ag or alloys of both. In embodiments, the plasmonic nanoparticles may have a size of from 3 nm to 200 nm, e.g. from 5 nm to 200 nm, e.g. from 5 nm to 50 nm. In embodiments, the plasmonic nanoparticles may have an absorption band within the spectral region of 390 to 700 nm. In embodiments, the absorption band may substantially cover the entire spectral range of 390 nm to 700 nm. Noble metal plasmonic nanoparticles typically advantageously absorb light in the visible region, which may complement the absorption by the photocatalytic matrix (e.g. TiO2). Furthermore, the absorption characteristics typically depend on the size and shape of the nanoparticles; as such, these characteristics can be controlled through changes in the synthesis of the nanoparticles. In embodiments, the coating may have a thickness larger than 25 nm, advantageously larger than 40 nm. In embodiments, the coating may have a thickness up to 1 mm, or even higher. In embodiments, features of the first aspect and its embodiments may independently be as correspondingly described for any embodiment of any other aspect. In a second aspect, the present invention relates to an article with a self-cleaning surface, the article comprising at least one surface, and a layer of the self-cleaning coating, as defined in any embodiment of the first aspect, covering the surface. The self-cleaning coating can advantageously be provided on a variety of surfaces, thereby enabling a large variety of articles with self-cleaning properties. In embodiments, the article may comprise a glass pane, a construction material or a fabric. The glass pane may, for example, be comprised in a glass panel, e.g. for use in a fish tank or window, or in a solar panel, e.g. for use in a photovoltaic system. The construction material may, for example, be a brick, a tile, a plaster or a paint. The fabric may, for example, be used in clothing or in drapes. In embodiments, features of the second aspect and its embodiments may independently be as correspondingly described for any embodiment of any other aspect. In a third aspect, the present invention relates to a method for forming a self-cleaning coating, comprising:providing a first dispersion comprising plasmonic nanoparticles,providing a second dispersion (e.g. a solution) comprising a precursor of a photocatalytic matrix,forming a mixture of the first and second dispersion,coating the mixture on a surface, andcalcining the coated mixture. In this way, a self-cleaning coating can be obtained with advantageously well dispersed plasmonic nanoparticles, and this in a relatively simple and economical way. In embodiments, the plasmonic nanoparticles may comprise a noble metal. In embodiments, the precursor of the photocatalytic matrix may be a precursor of TiO2, e.g. titanium(IV)isopropoxide. In embodiments, the step of forming the mixture of the first and second dispersion may comprise forming a sol. The method is preferably based on the well-researched sol-gel process. This process is known to offer a good control of the synthesis, while being relatively economical. For example, using a sol-gel based method, the mixture can typically advantageously be sintered at a lower temperature, compared to other traditional synthesis methods. In embodiments, the step of calcining the coated mixture may comprise heating up the coated mixture to a temperature between 300° C. to 800° C., e.g. between 450 to 650° C., preferably between 500° C. to 600° C., such as 550° C. In embodiments, the first and/or second dispersion may further comprise an organic solvent, such as ethanol. In embodiments wherein the first dispersion further comprises an organic solvent, the plasmonic nanoparticles may be complexed with a stabilizing agent which is suitable for stabilizing the dispersion of the plasmonic nanoparticles in the organic solvent. In embodiments, the stabilizing agent may be polyvinylpyrrolidone (PVP). In embodiments, complexing the plasmonic nanoparticles with the stabilizing agent may comprise complexing the plasmonic nanoparticles with a first stabilizing agent (for example for stabilizing the plasmonic nanoparticles in an aqueous medium, e.g. sodium citrate) and subsequently exchanging the first stabilizing agent for a second stabilizing agent (for example for stabilizing the plasmonic nanoparticles in an organic medium, e.g. PVP). The method (e.g. sol-gel based method) may, in general, be catalysed either by an acid or base. An acid catalysed method may be preferred. In embodiments, the first solution may thus further comprise an acid, e.g. acetic acid. In embodiments, the step of coating the mixture on a surface may comprise a wet coating technique, such as spin coating or dip coating. This advantageously allows the coating to be formed on a variety of surfaces, using relatively economical techniques. In embodiments, features of the third aspect and its embodiments may independently be as correspondingly described for any embodiment of any other aspect. In a fourth aspect, the present invention relates to a use of plasmonic nanoparticles embedded in a photocatalytic matrix for enhancing a self-cleaning property of said photocatalytic matrix. In embodiments, features of the fourth aspect and its embodiments may independently be as correspondingly described for any embodiment of any other aspect. The invention will now be described by a detailed description of several embodiments of the invention. It is clear that other embodiments of the invention can be configured according to the knowledge of the person skilled in the art without departing from the true technical teaching of the invention, the invention being limited only by the terms of the appended claims. Example 1: Forming a Self-Cleaning Coating In a first example, a dispersion of Au plasmonic nanoparticles in ethanol (i.e. a solvent) was mixed with water and an acetic acid in a reaction vessel. Subsequently, a solution of a titanium(IV)isopropoxide photocatalytic matrix precursor ethanol (i.e. a solvent) was added dropwise to the reaction vessel under stirring; a sol was thereby obtained. The sol was then coated by spin or dip coating onto a substrate. The coated sol was calcined at 550° C. and a transparent self-cleaning coating was formed. Further by way of illustration, results will be discussed illustrating features and advantages of embodiments of the present invention. The results illustrate how embedded systems can be used as an economically feasible catalyst for self-cleaning applications. The photocatalytic self-cleaning activity was evaluated by means of a stearic acid degradation experiment, as this widely recognized model reaction is representative of the group of compounds that typically contaminates glass surfaces. First the substrate preparation used in this example is discussed. Silicon wafers (15 mm×30 mm) were cleaned ultrasonically in methanol and dried with compressed air. Glass substrates were obtained by cutting microscope slides (15 mm×25 mm) and cleaning them for 15 min at room temperature in fresh piranha solution (7:3 v/v sulfuric acid (Chem-Lab, 95-97%):hydrogen peroxide (Chem-Lab, 30%)) and rinsing them three times with distilled water. The cleaned glass slides were stored in distilled water and blown dry just before spin-coating with compressed air. Further, the synthesis of PVP stabilized gold nanoparticles used in the present example is discussed. Aqueous colloidal suspensions of Au nanoparticles were prepared using a modified Turkevich procedure but performed at higher concentrations (10 times more concentrated). In short, 10 mL of a 0.01 M HAuCl4.3H2O (Sigma-Aldrich, >99.9%) was diluted so a total metal concentration of 1 mM was obtained. The solution was stirred vigorously and brought to boil after which 10 mL of a freshly prepared 1 weight % sodium citrate (Sigma-Aldrich, 99%) solution was quickly added to the boiling solution. After exactly 30 minutes boiling the resulting colloidal Au suspension was immediately cooled to room temperature. The used stabilizing agent, sodium citrate, stabilizes the nanoparticles by charge repulsion and is only weakly bound to nanoparticle causing the as obtained Au nanoparticles to be unstable in organic media. The titanium dioxide precursor solution also contains organic solvents (e.g. ethanol, vide infra) thus necessitating a phase transfer of the nanoparticles from the aqueous phase to the organic phase. This is achieved by exchanging the sodium citrate with PVP (polyvinylpyrrolidone, Alfa Aesar, 10000 g mol−1). PVP was dissolved in water by ultrasonicating the solution for 15 minutes. An appropriate amount of the PVP solution (2.5 mM) was added to the colloidal Au suspension so approximately 60 PVP molecules were provided per nm2nanoparticle surface. The solution was stirred at 600 rpm for 24 h at room temperature to ensure complete exchange of stabilizing agent. The resulting PVP stabilized Au nanoparticles were finally centrifuged, washed and suspended in absolute ethanol (Emplura, 99.5%). UV-VIS absorption spectra of the colloidal Au nanoparticle solutions were measured with a Shimadzu UV-VIS 2600 double beam spectrometer. Further, the preparation of plasmon modified thin films is discussed. The sols were prepared by the hydrolysis of titanium(IV) isopropoxide (TTIP, Sigma-Aldrich, 97%) in the presence of acetic acid (Riedel-de Haën, 96%). A solution of TTIP and ethanol (0.05:1.64 molar ratio) (henceforth referred to as Mixture 1) was added dropwise to a solution containing water, ethanol and acetic acid (1.07:1.31:0.34 molar ratio) (referred to as Mixture 2) under vigorous stirring. In the case of Au/TiO2thin film preparation, the ethanol part of Mixture 2 was replaced by a concentrated dispersion containing appropriate amounts of gold nanoparticles in ethanol. This way sols were prepared with a final gold loading of 0.1-0.3-1 and 3 weight % (calculated relative to the total amount of TiO2formed assuming all TTIP is hydrolyzed). The viscosity change of the formed sol was monitored with a Brookfield LVDV-I prime Digital Viscosimeter to ensure all samples were spin-coated at the same viscosity. Film deposition was thus achieved by spin-coating both the glass and silicon substrates at 1000 rpm for one minute at room temperature. Finally, the samples were calcined at 823 K for three hours at a heating rate of 1 K min−1. A schematic overview of the synthesis procedure can be seen inFIG.1. In the following, the self-cleaning activity obtained with the exemplary system manufactured as described above is discussed. The photocatalytic self-cleaning test was conducted by means of a stearic acid degradation experiment, based on the method proposed by Paz et al. in J. Mater. Res. 10 (1995) 2842-2848. In short, a layer of stearic acid was applied on top of the prepared thin films on the silicon wafers by spin coating 100 μL of a 0.25 weight % solution of stearic acid (Sigma-Aldrich, >98.5%) in chloroform (Sigma-Aldrich, >99.8%) at 1000 rpm for one minute. The resulting sample was dried at 363 K and subsequently allowed to equilibrate in the test environment for one hour. For the photocatalytic experiments, the samples were illuminated with: combined simulated solar light (300 W Xe source (Oriel Instruments) equipped with an AM 1.5 solar simulator) and UVA light (λmax=350 nm, provided by a fluorescent lamp). The corresponding irradiance spectra and intensity outputs are given inFIG.2. Light intensity and photon fluxes were measured directly at sample distance with a calibrated intensity spectrometer (Avantes Avaspec 3648). The remaining surface coverage of stearic acid was measured using a Nicolet™ 380 (Thermo Fisher Scientific) spectrophotometer equipped with ZnSe windows. All spectra were recorded in the wavenumber range 400-4000 cm−1at resolution of 2 cm−1. For each measurement, eight spectra were averaged. The samples were placed at a fixed angel of 9° with the IR beam in order to minimize internal reflections. The stearic acid concentration is related to the integrated absorbance in the wavenumber range 2800-3000 cm−1so that one unit of integrated area (in a.u. cm−1) corresponds to 1.39×1016stearic acid molecules cm−2. The integrated intensity is 6.9 mW cm−2for the UVA LED source (300-400 nm, curve at the left of the spectrum) and 100.1 mW cm−2for the combined simulated solar light (AM 1.5, 300-800 nm, curve at the right of the spectrum). In the following, some characterisation results for PVP stabilized gold nanoparticles are further discussed. Concentrated gold suspensions were prepared according to the Turkevich method. The resulting colloidal solutions were dark red and showed a similar UV-VIS absorption spectrum as 100% Au suspensions, indicating that increasing the concentration has no effect on the final nanoparticle properties. The effect of replacing the stabilizing agent from sodium citrate to PVP can be seen inFIG.3. After ligand exchange a small red-shift of the plasmon peak was observed. This is in line with results obtained by Bastús et al., where a similar red-shift was observed after ligand exchange with various surfactants. This shift is ascribed to an increase in the hydrodynamic diameter, caused by capping the nanoparticles with a bigger, bulky molecule (like PVP). In the following, some characterization results for plasmon modified transparent thin films are presented. For obtaining these thin films, the PVP stabilized nanoparticles were dispersed in EtOH and added to Mixture 2 (FIG.1) to be incorporated into the TiO2matrix during the sol gel process. Thin films were deposited on both silicon and glass substrates by spin-coating the resulting sols at the same viscosity. Previous experiments on the effect of spin-coating speed and viscosity on the film thickness on silicon wafers have shown that changing the viscosity of the coating sol has limited effect on film thickness (as shown inFIG.5a). Altering the spinning speed on the other hand clearly influences film thickness, as is evidenced inFIG.5b. Based on these results, it was opted to use a spin-coating speed of 1000 rpm for the plasmon modified thin films, resulting in a layer thickness of ±94 nm (as shown inFIG.5b). Similar experiments performed on microscope glass slides have shown that the overall layer thickness is somewhat lower when this substrate is used (FIG.5a, open circles). The light transmittance of the coatings is evaluated (table 1). More particularly, table 1 shows the light transmittance of the coatings with varying gold loadings (calculated by measuring the light intensity coming through the glass slide and the coating in the wavelength range from 300 to 800 nm). Coating the glass slide with a thin, unmodified layer of TiO2reduces the amount of light passing through the sample by 17%. The coating is also visible as it has a slightly colored appearance as can be seen inFIG.4. It has however to be stressed that at this point, no actions have been undertaken to improve the transparency of the TiO2film. Lowering the film thickness will for instance already improve the transparency (keeping the tradeoff with photocatalytic activity in mind). Combining the TiO2with a low refractive index material like SiO2is another method that allows to control the optical properties of the resulting film. Progress can thus still be made. It can however be seen in table 1 that an additional loss of only 4% is observed when loading the films with 3 weight % Au (highest loading). This is a promising result as it indicates that adding gold nanoparticles only has a limited effect on the light transmitting properties of the resulting film. TABLE 1SampleTransmittance (%)Uncoated glass1000 weight % TiO2coating830.1 weight % Au/TiO2coating820.3 weight % Au/TiO2coating811 weight % Au/TiO2coating813 weight % Au/TiO2coating79 As indicated above, the photocatalytic activity of the films was evaluated by monitoring the stearic acid degradation, a widely accepted method for assessing the activity of self-cleaning materials as stearic acid is a good model compound for organic fouling on glass windows. The results of these experiments performed under both UVA and simulated solar light are shown inFIGS.6aand6b. As the added value of modifying the TiO2thin films with gold is our main interest, the formal quantum efficiencies of all samples are expressed relative to the unmodified sample. Under pure UVA illumination (FIG.6a) it can be seen that embedding gold nanoparticles into TiO2causes the photocatalytic activity to increase compared to the unmodified sample (up to 16% for the 3 weight % sample). This result differs from a system where surface modification with noble metals led to a reduced activity, likely due to the metal particles blocking the active sites and shielding part of the TiO2surface from incoming light. By altering the catalyst design, i.e. embedding the nanoparticles into the TiO2matrix, these issues are circumvented. In addition, the contact area between nanoparticle and TiO2is drastically increased, possibly leading to an improved electron transfer efficiency from the excited semiconductor to the passive Au nanoparticle electron sink. Under broadband solar light illumination (FIG.6b) a similar trend can be observed, where higher loadings lead to an increased activity relative to the unmodified sample. The relative improvement starts to saturate around 1 weight %, which thus can be considered to be a sufficient load. Relative improvements up to 29 and 40% (for the 1 weight % and 3 weight % sample respectively) are achieved. Interestingly, these improvements are far more than the ones observed for under pure UVA illumination (FIG.6aversusFIG.6b) and can therefore not be solely attributed to the UV part of the simulated sunlight. These results therefore hint at a synergistic effect of dually exciting the plasmonic photocatalyst under solar irradiation (semiconductor by UV light and plasmonic nanoparticles under visible light illumination). The efficiency improvement is also twice as much as observed for substrates with a surface modification with noble metals (i.e. substrates where the noble metals are not embedded) for a rainbow photocatalyst e.g. as defined in Verbruggen et al. Applied Catalysis B: Environmental 188 (2016) 147-153, which was optimized to respond to the entire solar spectrum by modifying the TiO2surface with gold-silver alloys of different sizes and compositions: 16% for the 1.5 weight % surface modified rainbow photocatalyst compared to 29% for the 1 weight % Au embedded photocatalyst under study. As in the current experiments only one nanoparticle composition is used which is not yet tailored to respond to most intense wavelengths of solar irradiation, let alone to the entire solar spectrum (solely pure gold embedded in TiO2), there seems to be ample room for further improving the activity. Further by way of illustration, embodiments of the present invention not being limited thereto, further test results regarding the activity of samples are provided below. Various samples have been tested towards their self-cleaning behavior, using stearic acid as a model compound for organic fouling on glass windows. The samples were in the present example tested under ambient conditions and using a 300 W Xe arc discharge lamp equipped with an AM1.5 filter as the light source, adjusted to an incident irradiance of 100 mW cm−2. The synthesized coatings were applied on Borofloat glass as the substrate. Pilkington Activ™ was used as the commercially available benchmark. The results are shown inFIG.7. The plain Borofloat glass showed no self-cleaning activity, as expected. The Pilkington Activ™ benchmark showed a modest activity that was outperformed by coatings according to embodiments of the present invention. Upon introduction of plasmonic gold nanoparticles, an increase in activity was observed with increasing gold fraction, leveling off at loadings around 3 weight %. When using Au0.3Ag0.7bimetallic plasmonic nanoparticles, with plasmon resonance in the wavelength range 490-500 nm, the absorbance of the coating became more adjusted to the peak intensity wavelengths of the solar simulator (and real solar light). This sample showed the highest activity, even at a metal loading as low as 0.3 weight %. For the practical application on glass surfaces, high transparency was proposed as one of the main physical properties. The Pilkington Activ™ benchmark glass shows a high transparency of just over 87%. When applying coatings according to embodiments of the present invention on Borofloat, a resulting transparency is achieved in the order of 86%, very close to the benchmark, as can be seen inFIG.8. The higher the gold loading, the lower the overall transparency. For the Au—Ag bimetallic nanoparticles integrated in the coating, a transparency of almost 87% was measured, which is as good as the commercial benchmark. Also by way of illustration, embodiments of the present invention not being limited thereto, an example is shown of characteristics of thin films according to embodiments of the present invention. The surface characteristics of a 0.5 weight % Au containing TiO2coating prepared according to methods according to embodiments of the present invention were compared with films prepared according to the method as described by Sonawane et al. in J. Molecular Catalysis A, 243 (2006) pages 68 to 76. Starting from a bare Borofloat glass substrate with an Arithmetical Mean Height Ra (average roughness factor) of 0.23 nm, applying the 0.5 weight % coating of Sonawane et al. resulted in an average roughness Ra of 1.92 nm. Coating methods according to embodiments of the present invention resulted in a smoother film with an Ra of 1.3 nm. The corresponding AFM images are shown inFIG.9. Further by way of illustration, the activity of a coating according to an embodiment of the present invention (being a coating on a silicon wafer), is tested by evaluating the degradation speed of stearic acid (degradation time scale tested is in the order of minutes, in air). Comparison is made with a coating obtained using the protocol of Sonawane et al. in J. Molecular Catalysis A, 243 (2006) pages 68 to 76. The degradation measurements obtained are shown inFIG.10, illustrating the degradation by coatings obtained with embodiments of the present invention and Au comprising coatings made according to the Sonawane et al. protocol (as a comparison). It can be seen that for the coatings made according to embodiments of the present invention, the degradation speed is similar as for the same coatings on glass, whereby addition of gold results in a significant improvement. Furthermore, it can be seen that for a similar coating but made using the Sonawane et al. protocol, the degradation reaches only 4% of the amount of degradation obtained with coatings of the present invention, and that the addition of Au does not result in an improvement. It is to be noted that the results given in the article by Sonawane et al. are results for long degradation times, substantially longer than the degradation times evaluated and required in the present examples. The degradation properties of coatings according to embodiments of the present invention therefore also have the advantages of being efficient, not only in degree of degradation but also in degradation time. The absolute measured degradation values for Sonawane et al. based coatings are 1.6×1011molecules/cm2/s, whereas based on coatings according to the present invention, the degradation values are 4.8×1012molecules/cm2/s. It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for devices according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and technical teachings of this invention. For example, any formulas given above are merely representative of procedures that may be used. Functionality may be added or deleted from the block diagrams and operations may be interchanged among functional blocks. Steps may be added or deleted to methods described within the scope of the present invention. | 37,092 |
11859106 | DETAILED DESCRIPTION The following detailed description is merely exemplary in nature and is not intended to limit the presently claimed invention or the application and uses of the presently claimed invention. Furthermore, there is no intention to be bound by any theory presented in the preceding technical field, background, summary or the following detailed description. The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. It will be appreciated that the terms “comprising”, “comprises” and “comprised of” as used herein comprise the terms “consisting of”, “consists” and “consists of”. Furthermore, the terms “(a)”, “(b)”, “(c)”, “(d)” etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the subject matter described herein are capable of operation in other sequences than described or illustrated herein. In case the terms “(A)”, “(B)” and “(C)” or “(a)”, “(b)”, “(c)”, “(d)”, “(i)”, “(ii)” etc. relate to steps of a method or use or assay there is no time or time interval coherence between the steps, that is, the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below. In the following passages, different aspects of the subject matter are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the presently claimed invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment but may refer. Furthermore, the features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some, but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the subject matter, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination. Furthermore, the ranges defined throughout the specification include the end values as well, i.e. a range of 1 to 10 implies that both 1 and 10 are included in the range. For the avoidance of doubt, the applicant shall be entitled to any equivalents according to applicable law. For the purposes of the presently claimed invention, a block polymer or a block copolymer is defined as a polymer or a copolymer formed, when two or more monomers cluster together and form ‘blocks’ of repeating units. For the purposes of the presently claimed invention, a random polymer or a random copolymer is defined as a polymer or a copolymer formed, when two or more monomers are added as repeating units in a completely random manner. For the purposes of the presently claimed invention, a graft polymer is a segmented copolymer with a linear backbone of one composite and randomly distributed branches of another composite. Reference throughout this specification to the term “copolymer” means that the copolymer comprises block or random copolymers obtainable by radical polymerization. For the purposes of the presently claimed invention, the mass-average (Mw) and number-average (Mn) molecular weight is determined by means of gel permeation chromatography at 40° C., using a high-performance liquid chromatography pump and a refractive index detector. The eluent used was tetrahydrofuran with an elution rate of 1 ml/min. The calibration is carried out by means of polystyrene standards. For the purposes of the presently claimed subject matter, a polar solvent is defined to be a solvent with large dipole moments and which contains bonds between atoms with very different electronegativities. For the purposes of the presently claimed invention, a dielectric constant value of a solvent indicates a measure of polarity of the solvent. Higher dielectric constant of a solvent is indicative of more polarity of the solvent. For the purposes of the presently claimed invention, a use of (meth) in a monomer or repeat unit indicates an optional methyl group. For the purposes of the presently claimed invention, transparent or transparency is defined as a property of a material to allow visible light completely or partially to pass through the material without being scattered. For the purposes of the presently claimed invention, a pigment is defined to be any substance that alters the colour of a material through selective absorption or any substance that scatters and reflects light. For the purposes of the presently claimed invention, effect pigments are defined as flake or platy structures that impart a directional light reflectance, scattering, absorption, or optically variable appearance to the substrate in or on which they are applied. For the purposes of the presently claimed invention, polydispersity or polydispersity index (PDI) is defined to be a measure of the distribution of molecular mass in a given polymer. For the purposes of the presently claimed invention, ‘% by weight’ or ‘wt. %’ as used in the presently claimed invention is with respect to the total weight of the coating composition. Further, sum of wt.-% of all the compounds, as described hereinbelow, in the respective component adds up to 100 wt.-%. The above-mentioned measurement techniques are well known to a person skilled in the art and therefore do not limit the presently claimed invention. Polymeric Pigment Dispersant An aspect of the presently claimed invention describes a polymeric pigment dispersant comprising a polymer backbone (P) and at least one moiety of the formula (I): wherein R1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl; R2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or a branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—O— group; R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—O— group; or R2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl, which are each substituted with one —C(═O)—O— group; and whereby the moiety of the formula (I) is bonded to the polymer backbone (P) via the —C(═O)—O— group. In an embodiment of the presently claimed invention, the R1 in formula (I) described hereinabove is selected from the group consisting of naphthyl, anthracenyl and phenanthrenyl which are unsubstituted or substituted with F, Cl, Br, I, —NO2, —CN, —OH, —O—C1-C6-alkyl, —C(═O)—C1-C6-alkyl, —C(═O)—O—C1-C6-alkyl, —C(═O)—O-phenyl, —CH2—C(═O)—C1-C6-alkyl, —C(═O)—NH(C1-C6)alkyl, —C(═O)—NH-phenyl, —C1-C6-alkyl; wherein —C1-C6-alkyl is itself unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents independently of each other selected from the group consisting of F, Cl, Br, I, —CN, —OH, —O—CF3, —O—CH3and —O—C2H5. In a preferred embodiment of the presently claimed invention, R1 in formula (I) described hereinabove is naphthyl which is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents independently of each other selected from the group consisting of F, Cl, Br, I, —NO2, —CN, —OH, —O—C1-C6-alkyl, —C(═O)—C1-C6-alkyl, —C(═O)—O—C1-C6-alkyl, —C(═O)—O-phenyl, —CH2—C(═O)—C1-C6-alkyl, —C(═O)—NH(C1-C6)alkyl, —C(═O)—NH-phenyl, —C1-C6-alkyl; wherein —C1-C6-alkyl is itself unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents independently of each other selected from the group consisting of F, Cl, Br, I, —CN, —OH, —O—CF3, —O—CH3and —O—C2H5. In a preferred embodiment of the presently claimed invention, R1 in formula (I) described hereinabove is selected from the group consisting of naphthyl, anthracenyl and phenanthrenyl which are each unsubstituted or substituted with 1, 2 or 3 —OH. In a preferred embodiment of the presently claimed invention, R2 and R3 in formula (I) described hereinabove together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—O— group. In another embodiment of the presently claimed invention, the at least one moiety of the formula (I) and the compound of general formula (IV), respectively, are obtained by reacting at least one compound of formula (II) wherein R2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; or R2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl which are each substituted with one —C(═O)—OH group; with at least one compound of formula (III) R1-NH2(III) wherein R1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl; optionally in the presence of at least one solvent (S1). In a preferred embodiment of the presently claimed invention, the at least one compound of formula (II) described hereinabove is selected from the group consisting of phthalic anhydride, hexahydrophthalic anhydride, dodecenyl succinic anhydride, octadecenyl succinic anhydride, tetrahydrophthalic anhydride, methyltetrahydrophthalic anhydride and endomethylene tetrahydrophthalic, which are each substituted with one —C(═O)—OH group. In a more preferred embodiment of the presently claimed invention, the at least one compound of formula (II) described hereinabove is selected from the group consisting of 1,2,4-benzenetricarboxylic anhydride and 1,2-cyclohexanecarboxylic anhydride. In a preferred embodiment of the presently claimed invention, the at least one compound of formula (III) described hereinabove is selected from the group consisting of 1-naphthylamine and 7-hydroxy 1-naphthylamine. In another embodiment of the presently claimed invention, the at least one solvent (S1) is a polar solvent having a boiling point in the range of ≥80° C. to ≤160° C. and a dielectric constant in the range of ≥11 to ≤30. In a preferred embodiment of the presently claimed invention, the at least one solvent (S1) is a polar solvent having a boiling point in the range of ≥80° C. to ≤130° C. and a dielectric constant in the range of ≥11 to ≤25. In a preferred embodiment of the presently claimed invention, the at least one solvent (S1) is selected from the group consisting of methyl N-amyl ketone, ethyl methyl ketone, methyl isoamyl ketone and isopropanol. For the purposes of the presently claimed invention, the compound of general formula (IV) can be more preferably synthesized by the reaction described hereinabove in the presence of less polar and lower boiling solvents like isopropanol, ethyl methyl ketone, methyl isoamyl ketone and methyl N-amyl ketone. In an embodiment of the presently claimed invention, the polymeric pigment dispersant has a number average molecular weight (Mn) in the range of from ≥1000 g/mol to ≤25000 g/mol, determined according to gel permeation chromatography against a polystyrene standard. In a preferred embodiment of the presently claimed invention, the polymeric pigment dispersant has a number average molecular weight (Mn) in the range of from ≥1000 g/mol to ≤15000 g/mol. In an embodiment of the presently claimed invention, the polymeric pigment dispersant has a polydispersity in the range of from ≥1.2 to ≤20, determined according to gel permeation chromatography against a polystyrene standard. In another embodiment of the presently claimed invention, the polymeric pigment dispersant has a polydispersity in the range of from ≥1.2 to ≤10, determined according to gel permeation chromatography against a polystyrene standard. In a preferred embodiment of the presently claimed invention, the polymeric pigment dispersant has a polydispersity in the range of from ≥1.2 to ≤3.5, determined according to gel permeation chromatography against a polystyrene standard. In a most preferred embodiment of the presently claimed invention, the polymeric pigment dispersant has a polydispersity in the range of from ≥1.2 to ≤2.2, determined according to gel permeation chromatography against a polystyrene standard. In an embodiment of the presently claimed invention, the total weight of the at least one moiety of the formula (I) is in the range of from ≥5 wt. % to ≤50 wt. %, based on the total weight of the polymeric pigment dispersant. In a preferred embodiment of the presently claimed invention, the total weight of the at least one moiety of the formula (I) is in the range of from ≥5 wt. % to ≤30 wt. %, based on the total weight of the polymeric pigment dispersant. Linear Di-Block Polymer In an embodiment of the presently claimed invention, the polymer backbone (P) described hereinabove is a linear di-block polymer. In another embodiment of the presently claimed invention, the linear di-block polymer is obtained by a living free radical polymerization. In an embodiment of the presently claimed invention, the linear di-block polymer is obtained by a living free radical polymerization referred to as atom transfer radical polymerization (ATRP). The ATRP process is described to provide highly uniform products having controlled structure and is also referred to as controlled radical polymerization (CRP). The ATRP process is described for preparation of copolymers which are useful in a wide variety of applications, including pigment dispersant in U.S. Pat. Nos. 6,365,666 B1 and 6,642,301 B2. The ATRP process description can be found in detail in U.S. Pat. Nos. 5,807,937 A, 5,763,548 A, 5,789,487 A and WO 1998/40415 A1. For purposes of the presently claimed invention, the linear di-block polymer can be obtained by other polymerization techniques like reversible addition-fragmentation chain transfer (RAFT) polymerization, single electron transfer living radical polymerization (SEL-LRP), nitroxide mediated radical polymerization (NMRP), living ring opening metathesis polymerization (ROMP), living anionic and living cationic polymerization. In a yet another embodiment of the presently claimed invention, the linear di-block polymer has a formula A-B, wherein A is a first polymer block which is obtained by reacting a first mixture comprising at least one glycidyl (meth)acrylate; and B is a second polymer block which is obtained by reacting a second mixture comprising at least one monomer selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate, polyethylene glycol (meth)acrylate and polyethylene glycol alkyl ether (meth)acrylate. In an embodiment of the presently claimed invention, the linear di-block polymer A-B is obtained by reacting the first polymer block A and the second polymer block B, optionally in the presence of at least one solvent and optionally in the presence of at least one catalyst. In another embodiment of the presently claimed invention, the linear di-block polymer has a formula A-B, wherein A is a first polymer block which is obtained by reacting a first mixture comprising at least one glycidyl (meth)acrylate; and B is a second polymer block which is obtained by reacting a second mixture comprising at least one monomer selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate, polyethylene glycol (meth)acrylate and polyethylene glycol alkyl ether (meth)acrylate; optionally in the presence of at least one solvent. In a preferred embodiment of the presently claimed invention, the first polymer block A described hereinabove is obtained by reacting a first mixture comprising at least one glycidyl (meth)acrylate. In a preferred embodiment of the presently claimed invention, the second polymer block B described hereinabove is obtained by reacting a second mixture comprising at least one monomer of alkyl (meth)acrylate, at least one monomer of hydroxyalkyl (meth)acrylate, at least one monomer of polyethylene glycol (meth)acrylate and at least one monomer of polyethylene glycol alkyl ether (meth)acrylate. In a preferred embodiment of the presently claimed invention, the second polymer block B described hereinabove is obtained by reacting a second mixture comprising at least one monomer of alkyl (meth)acrylate and at least one monomer of hydroxyalkyl (meth)acrylate. In an embodiment of the presently claimed invention, the alkyl (meth)acrylate described hereinabove is selected from the group consisting of methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, isopropyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, hexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, nonyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate and isodecyl (meth)acrylate. In a preferred embodiment of the presently claimed invention, the alkyl (meth)acrylate described hereinabove is selected from the group consisting of methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, isopropyl (meth)acrylate, butyl (meth)acrylate and isobutyl (meth)acrylate. In an embodiment of the presently claimed invention, the hydroxyalkyl (meth)acrylate described hereinabove is selected from the group consisting of 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate and 2-hydroxybutyl (meth)acrylate. In an embodiment of the presently claimed invention, the polyethylene glycol alkyl ether (meth)acrylate is selected from the group consisting of polyethylene glycol methylether acrylate, polyethylene glycol ethyl ether acrylate, polyethylene glycol propyl ether acrylate and polyethylene glycol butyl ether acrylate. In an embodiment of the presently claimed invention, the linear di-block polymer has a number average molecular weight (Mn) in the range of from ≥1000 g/mol to ≤25000 g/mol, determined according to gel permeation chromatography against a polystyrene standard. In a preferred embodiment of the presently claimed invention, the polymeric pigment dispersant has a number average molecular weight (Mn) in the range of from ≥1000 g/mol to ≤15000 g/mol. In an embodiment of the presently claimed invention, the linear di-block has a polydispersity in the range of from ≥1.2 to ≤20, determined according to gel permeation chromatography against a polystyrene standard. In another embodiment of the presently claimed invention, the linear di-block has a polydispersity in the range of from ≥1.2 to ≤10, determined according to gel permeation chromatography against a polystyrene standard. In a preferred embodiment of the presently claimed invention, the polymeric pigment dispersant has a polydispersity in the range of from ≥1.2 to ≤3.5, determined according to gel permeation chromatography against a polystyrene standard. In a most preferred embodiment of the presently claimed invention, the polymeric pigment dispersant has a polydispersity in the range of from ≥1.2 to ≤2.2, determined according to gel permeation chromatography against a polystyrene standard. In an embodiment of the presently claimed invention, the polymer backbone (P) described hereinabove is a block polymer with at least two blocks. Random Polymer In an embodiment of the presently claimed invention, the polymer backbone (P) described hereinabove is a random polymer. In a yet another embodiment of the presently claimed invention, the random polymer is obtained by a free radical polymerization. In an embodiment of the presently claimed invention, the random polymer is obtained by free radical polymerization referred to as atom transfer radical polymerization (ATRP). The ATRP process is described to provide highly uniform products having controlled structure and is also referred to as controlled radical polymerization (CRP). The ATRP process is described for preparation of copolymers which are useful in a wide variety of applications, including pigment dispersant in U.S. Pat. Nos. 6,365,666 B1 and 6,642,301 B2. The ATRP process description can be found in detail in U.S. Pat. Nos. 5,807,937 A, 5,763,548 A, 5,789,487 A and WO 1998/40415 A1. For the purposes of the presently claimed invention, the random polymer can be obtained by other polymerization techniques like reversible addition-fragmentation chain transfer (RAFT), ring-opening metathesis polymerization (ROMP), and anionic and cationic polymerizations. In an embodiment of the presently claimed invention, the random polymer is obtained by reacting a mixture (Mn) comprising: (a) glycidyl methacrylate and/or glycidyl acrylate; (b) at least one monomer selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate and cycloalkyl (meth)acrylate; (c) optionally at least one monomer of styrene; and (d) optionally at least one monomer selected from the group consisting of vinyl monomers, monoethylenically unsaturated monomers bearing urea or keto groups and benzyl (meth)acrylate, optionally in the presence of at least one solvent (S2). In a preferred embodiment of the presently claimed invention, the random polymer is obtained by reacting a mixture (Mn) comprising: (a) glycidyl methacrylate and/or glycidyl acrylate; and (b) at least one monomer selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate and cycloalkyl (meth)acrylate; In a preferred embodiment of the presently claimed invention, the random polymer is obtained by reacting a mixture (Mn) comprising: (a) glycidyl methacrylate and/or glycidyl acrylate; (b) at least one monomer selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate and cycloalkyl (meth)acrylate; and (c) at least one monomer of styrene. In an embodiment of the presently claimed invention, the random polymer is obtained by reacting a mixture (Mn) comprising: (a) glycidyl methacrylate and/or glycidyl acrylate; (b) at least one monomer selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate and cycloalkyl (meth)acrylate; (c) at least one monomer of styrene; and (d) at least one monomer selected from the group consisting of vinyl monomers, monoethylenically unsaturated monomers bearing urea or keto groups and benzyl (meth)acrylate, In a preferred embodiment of the presently claimed invention, the random polymer is obtained by reacting a mixture (Mn) comprising: (a) glycidyl methacrylate and/or glycidyl acrylate; (b) at least one monomer selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate and cycloalkyl (meth)acrylate; and (c) at least one monomer of styrene. in the presence of at least one solvent (S2). In a preferred embodiment of the presently claimed invention, the random polymer is obtained by reacting a mixture (Mn) comprising: (a) glycidyl methacrylate and/or glycidyl acrylate; and (b) at least one monomer selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate and cycloalkyl (meth)acrylate; in the presence of at least one solvent (S2). In an embodiment of the presently claimed invention, the alkyl (meth)acrylate described hereinabove is selected from the group consisting of methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, isopropyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, hexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, nonyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate and isodecyl (meth)acrylate). In a preferred embodiment of the presently claimed invention, the alkyl (meth)acrylate described hereinabove is selected from the group consisting of methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, isopropyl (meth)acrylate, butyl (meth)acrylate and isobutyl (meth)acrylate. In an embodiment of the presently claimed invention, the hydroxyalkyl (meth)acrylate described hereinabove is selected from the group consisting of 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate and 2-hydroxybutyl (meth)acrylate. In an embodiment of the presently claimed invention, the cycloalkyl (meth)acrylate described hereinabove is selected from the group consisting of cyclopentyl (meth)acrylate, cyclohexyl(meth)acrylate, dicyclopentadiene (meth)acrylate, dicyclopentanyl (meth)acrylate, tricyclodecanyl (meth)acrylate, isobornyl (meth)acrylate, 4-tert-butylcyclohexyl (meth)acrylate, norbornyl (meth)acrylate and bornyl (meth)acrylate. In an embodiment of the presently claimed invention, the at least one monomer of styrene described hereinabove is selected from the group consisting of 4-methyl styrene, 3-methyl styrene, 4-tert-butyl styrene, 4-tert-butoxy styrene, 2-bromostyrene, 3-bromostyrene, 4-bromostyrene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 4-chloro-ε-methylstyrene, 2,6-dichloro styrene, 2-flurostyrene, 3-fluorstyrene, 4-fluorostyrene, 2,6-difluorostyrene, 3-nitrostyrene and 4-acetoxy styrene. In an embodiment of the presently claimed invention, the at least one vinyl monomer described hereinabove is selected from the group consisting of 3-vinyl benzoic acid, 4-vinyl benzoic acid and 4-vinylbenzyl chloride. In an embodiment of the presently claimed invention, the monoethylenically unsaturated monomer bearing urea or keto groups described hereinabove is selected from the group consisting of 2-(2-oxo-imidazolidin-1-yl)ethyl (meth)acrylate, 2-ureido (meth)acrylate, N-[2-(2-oxooxazolidin-3-yl)ethyl]methacrylate, acetoacetoxyethyl acrylate, acetoacetoxypropyl methacrylate, acetoacetoxybutyl methacrylate, 2-(acetoacetoxy)ethyl methacrylate, diacetoneacrylamide (DAAM), diacetonemethacrylamide, N-(beta-ureido ethyl) acrylamide and N-(beta-ureido ethyl) methacrylamide. In an embodiment of the presently claimed invention, the solvent (S2) is selected from the group consisting of xylene, toluene, methanol, ethanol, n-propanol, isopropanol, butanol, butoxyethanol, acetone, butanone, pentanone, hexanone, methyl isobutyl ketone, ethyl acetate, butyl acetate, amyl acetate, methoxy propyl acetate, tetrahydrofuran, diethyl ether, ethylene glycol, polyethylene glycol and mixtures thereof. In a preferred embodiment of the presently claimed invention, the solvent (S2) is selected from the group consisting of toluene, n-propanol, isopropanol, methyl isobutyl ketone and mixtures thereof. In an embodiment of the presently claimed invention, the random polymer has a number average molecular weight (Mn) in the range of from ≥1000 g/mol to ≤25000 g/mol, determined according to gel permeation chromatography against a polystyrene standard. In a preferred embodiment of the presently claimed invention, the polymeric pigment dispersant has a number average molecular weight (Mn) in the range of from ≥1000 g/mol to ≤15000 g/mol. In an embodiment of the presently claimed invention, the random polymer has a polydispersity in the range of from ≥1.5 to ≤20, determined according to gel permeation chromatography against a polystyrene standard. In another embodiment of the presently claimed invention, the random polymer has a polydispersity in the range of from ≥1.5 to ≤10, determined according to gel permeation chromatography against a polystyrene standard. In a preferred embodiment of the presently claimed invention, the polymeric pigment dispersant has a polydispersity in the range of from ≥1.5 to ≤5, determined according to gel permeation chromatography against a polystyrene standard. In a most preferred embodiment of the presently claimed invention, the polymeric pigment dispersant has a polydispersity in the range of from ≥1.5 to ≤3, determined according to gel permeation chromatography against a polystyrene standard. Graft Polymer In an embodiment of the presently claimed invention, the polymeric pigment dispersant described hereinabove is a graft polymer. In an embodiment of the presently claimed invention, the graft polymer described hereinabove and hereinbelow comprises at least one polyester block. In a yet another embodiment of the presently claimed invention, the polyester block described hereinabove is obtained from monomeric units of a hydroxy-functional aliphatic acid or a hydroxy-functional aromatic acid or hydroxy-functional araliphatic acid. In a preferred embodiment of the presently claimed invention, the polyester block described hereinabove is obtained from monomeric units of a hydroxy-functional aliphatic acid. In an embodiment of the presently claimed invention, the hydroxy-functional aliphatic acid described hereinabove is selected from the group consisting of glycolic acid, lactic acid, 5-hydroxy valeric acid, 3-hydroxy-butyric acid, 4-hydroxy-valeric acid, 12-hydroxy stearic acid and 6-hydroxy caproic acid. In a preferred embodiment of the presently claimed invention, the polyester block described hereinabove is obtained in the presence of a saturated fatty acid or an unsaturated fatty acid. Representative examples of saturated or the unsaturated fatty acid is selected preferably from the group consisting of oleic acid, linolenic acid, palmitoleic acid and tall oil fatty acid. In another embodiment of the presently claimed invention, the polyester block described hereinabove is obtained from monomeric units of a lactone. In a yet another embodiment of the presently claimed invention, the lactone described hereinabove is selected from the group consisting of δ-valerolactone, ε-caprolactone, β-methyl-δ-valerolactone, 2-methyl-ε-caprolactone, 3-methyl-ε-caprolactone, 4-methyl-ε-caprolactone, 5-ter-butyl-ε-caprolactone, 7-methyl-ε-caprolactone, 4,4,6-trimethyl-ε-caprolactone and β-propiolactone. In an embodiment of the presently claimed invention, the total weight of the at least one polyester block described hereinabove is in the range of from ≥5 wt. % to ≤95 wt. %, based on the total weight of the polymeric pigment dispersant. In a preferred embodiment of the presently claimed invention, the total weight of the at least one polyester block described hereinabove is in the range of from ≥45 wt. % to ≤95 wt. %, based on the total weight of the polymeric pigment dispersant. In a most preferred embodiment of the presently claimed invention, the total weight of the at least one polyester block described hereinabove is in the range of from ≥45 wt. % to ≤80 wt. %, based on the total weight of the polymeric pigment dispersant In an embodiment of the presently claimed invention, the polyester block described hereinabove is bonded to the moiety of the formula (I) and/or the polymer backbone (P) via a —C(═O)—O— group. In another embodiment of the presently claimed invention, the graft polymer described hereinabove and hereinbelow comprises at least one polyether block. In another embodiment of the presently claimed invention, the at least one polyether block described hereinabove comprises a polyoxyethylene group comprising from 10 to 120 ethylene oxide units. In a preferred embodiment of the presently claimed invention, the at least one polyether block described hereinabove comprises a polyoxyethylene group comprising from 20 to 60 ethylene oxide units. In an embodiment of the presently claimed invention, the polyether block described hereinabove is bonded to the moiety of the formula (I) and/or the polymer backbone (P) via a —C(═O)—O— group. An aspect of the presently claimed invention is directed to a process for the preparation of at least one polymeric pigment dispersant comprising a linear di-block polymer backbone comprising at least the steps of: reacting a di-block polymer as described hereinabove with a compound of the formula (IV): wherein R1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl; R2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; or R2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl, which are each substituted with one —C(═O)—OH group; at a temperature from ≥80° C. to ≤150° C.; and wherein the linear di-block polymer comprises a first and a second block and is obtained by a living free radical polymerization described hereinabove, optionally in the presence of a solvent (S3). In an embodiment of the presently claimed invention, the solvent (S3) is selected from the group consisting of butyl acetate, methyl N-amyl ketone, methyl isoamyl ketone and isopropanol. In an embodiment, the presently claimed invention is directed to a process for the preparation of at least one polymeric pigment dispersant comprising a linear di-block polymer backbone comprising at least the steps of: reacting a di-block polymer as described hereinabove with a compound of the formula (IV): wherein R1 is selected from the group consisting of naphthyl, anthracenyl and phenanthrenyl which are each unsubstituted or substituted with 1, 2 or 3 —OH; and R2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—OH group; at a temperature from ≥100° C. to ≤130° C.; and wherein the linear di-block polymer comprises a first and a second block and is obtained by a living free radical polymerization described hereinabove, optionally in the presence of a solvent (S3). In a preferred embodiment, the presently claimed invention is directed to a process for the preparation of at least one polymeric pigment dispersant comprising a linear di-block polymer backbone comprising at least the steps of: reacting a di-block polymer as described hereinabove with a compound of the formula (IV): wherein R1 is naphthyl, which is unsubstituted or substituted with 1, 2 or 3 —OH; and R2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—OH group; at a temperature from ≥100° C. to ≤130° C.; and wherein the linear di-block polymer comprises a first and a second block and is obtained by a living free radical polymerization described hereinabove, optionally in the presence of a solvent (S3). An aspect of the presently claimed invention is directed to a process for the preparation of at least one polymeric pigment dispersant comprising a random polymer comprising at least the steps of: (a) reacting a random polymer as described hereinabove with a compound of the formula (IV): wherein R1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl; R2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; or R2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl, which are each substituted with one —C(═O)—OH group; and (b) reacting the compound obtained in step (a) with at least one monomer of a lactone at a temperature from ≥30° C. to ≤190° C. In an embodiment, the presently claimed invention is directed to a process for the preparation of at least one polymeric pigment dispersant comprising a random polymer comprising at least the steps of: (a) reacting a random polymer as described hereinabove with a compound of the formula (IV): wherein R1 is selected from the group consisting of naphthyl, anthracenyl and phenanthrenyl which are each unsubstituted or substituted with 1, 2 or 3 —OH; and R2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—OH group; and (b) reacting the compound obtained in step (a) with at least one monomer of a lactone at a temperature from ≥100° C. to ≤140° C. In a preferred embodiment, the presently claimed invention is directed to a process for the preparation of at least one polymeric pigment dispersant comprising a random polymer comprising at least the steps of: (a) reacting a random polymer as described hereinabove with a compound of the formula (IV): wherein R1 is naphthyl, which is unsubstituted or substituted with 1, 2 or 3 —OH; and R2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—OH group; and (b) reacting the compound obtained in step (a) with at least one monomer of a lactone at a temperature from ≥100° C. to ≤140° C. In an embodiment of the presently claimed invention, the at least one monomer of a lactone described hereinabove is selected from the group consisting of δ-valerolactone, ε-caprolactone, β-methyl-δ-valerolactone, 2-methyl-ε-caprolactone, 3-methyl-ε-caprolactone, 4-methyl-ε-caprolactone, 5-ter-butyl-ε-caprolactone, 7-methyl-ε-caprolactone, 4,4,6-trimethyl-ε-caprolactone and β-propiolactone. An aspect of the presently claimed invention is directed to a process for the preparation of at least one polymeric pigment dispersant comprising a random polymer comprising at least the steps of: (a) reacting at least one polyalkylene glycol monoalkyl ether and at least one carboxylic acid anhydride at a temperature in the range from ≥70° C. to ≤140° C. to obtain a mixture; and (b) reacting the mixture obtained in step (a) with a random polymer described hereinabove and a compound of the formula (IV): wherein R1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl; R2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; or R2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl, which are each substituted with one —C(═O)—OH group; at a temperature in the range from ≥70° C. to ≤140° C. In an embodiment, the presently claimed invention is directed to a process for the preparation of at least one polymeric pigment dispersant comprising a random polymer comprising at least the steps of: (a) reacting at least one polyalkylene glycol monoalkyl ether and at least one carboxylic acid anhydride at a temperature in the range from ≥100° C. to ≤140° C. to obtain a mixture; and (b) reacting the mixture obtained in step (a) with a random polymer described hereinabove and a compound of the formula (IV): wherein R1 is selected from the group consisting of naphthyl, anthracenyl and phenanthrenyl which are each unsubstituted or substituted with 1, 2 or 3 —OH; and R2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—OH group; at a temperature in the range from ≥100° C. to ≤140° C. In a preferred embodiment, the presently claimed invention is directed to a process for the preparation of at least one polymeric pigment dispersant comprising a random polymer comprising at least the steps of: (a) reacting at least one polyalkylene glycol monoalkyl ether and at least one carboxylic acid anhydride at a temperature in the range from ≥100° C. to ≤140° C. to obtain a mixture; and (b) reacting the mixture obtained in step (a) with a random polymer described hereinabove and a compound of the formula (IV): wherein R1 is naphthyl, which is unsubstituted or substituted with 1, 2 or 3 —OH; and R2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—OH group; at a temperature in the range from ≥100° C. to ≤140° C. Another aspect of the presently claimed invention is directed to a pigment dispersion comprising at least one polymeric pigment dispersant according to the presently claimed invention, at least one solvent (S5) and at least one pigment. For the purposes of the presently claimed invention, the at least one solvent (S5) is selected from the group consisting of organic solvents. Representative examples of classes of organic solvents include, but are not limited to, alcohols, ketones or ketoalcohols, ethers, esters and polyhydric alcohols. Representative examples of organic solvents include, but are not limited to, xylene, toluene, methanol, ethanol, n-propanol, isopropanol, acetone, methyl ethyl ketone, dimethyl ether, methyl ethyl ether, ethyl acetate, ethyl lactate, ethylene glycol, diethylene glycol and butyl-2-hydroxyethyl ether. For the purposes of the presently claimed invention, the at least one pigment is a virtually insoluble, finely dispersed, organic or inorganic colorant as per the definition in the German standard specification DIN 55944. Representative examples of organic pigments include but are not limited to, monoazo pigments, such as C.I. Pigment Brown 25; C.I. Pigment Orange 5, 13, 36 and 67; C.I. Pigment Red 1, 2, 3, 5, 8, 9, 12, 17, 22, 23, 31, 48:1, 48:2, 48:3, 48:4, 49, 49:1, 52:1, 52:2, 53, 53:1, 53:3, 57:1, 63, 112, 146, 170, 184, 210, 245 and 251; C.I. Pigment Yellow 1, 3, 73, 74, 65, 97, 151 and 183; disazo pigments, such as C.I. Pigment Orange 16, 34 and 44; C.I. Pigment Red 144, 166, 214 and 242; C.I. Pigment Yellow 12, 13, 14, 16, 17, 81, 83, 106, 113, 126, 127, 155, 174, 176 and 188; anthanthrone pigments, such as C.I. Pigment Red 168 (C.I. Vat Orange 3); anthraquinone pigments, such as C.I. Pigment Yellow 147 and 177; C.I. Pigment Violet 31; anthraquinone pigments, such as C.I. Pigment Yellow 147 and 177; C.I. Pigment Violet 31; anthrapyrimidine pigments: C.I. Pigment Yellow 108 (C.I. Vat Yellow 20); quinacridone pigments, such as C.I. Pigment Red 122, 202 and 206; C.I. Pigment Violet 19; quinophthalone pigments, such as C.I. Pigment Yellow 138; dioxazine pigments, such as C.I. Pigment Violet 23 and 37; flavanthrone pigments, such as C.I. Pigment Yellow 24 (C.I. Vat Yellow 1); indanthrone pigments, such as C.I. Pigment Blue 60 (C.I. Vat Blue 4) and 64 (C.I. Vat Blue 6); isoindoline pigments, such as C.I. Pigment Orange 69; C.I. Pigment Red 260; C.I. Pigment Yellow 139 and 185; isoindolinone pigments, such as C.I. Pigment Orange 61; C.I. Pigment Red 257 and 260; C.I. Pigment Yellow 109, 110, 173 and 185; isoviolanthrone pigments, such as C.I. Pigment Violet 31 (C.I. Vat Violet 1); metal complex pigments, such as C.I. Pigment Yellow 117, 150 and 153; C.I. Pigment Green 8; perinone pigments, such as C.I. Pigment Orange 43 (C.I. Vat Orange 7); C.I. Pigment Red 194 (C.I. Vat Red 15); perylene pigments, such as C.I. Pigment Black 31 and 32; C.I. Pigment Red 123, 149, 178, 179 (C.I. Vat Red 23), 190 (C.I. Vat Red 29) and 224; C.I. Pigment Violet 29; phthalocyanine pigments, such as C.I. Pigment Blue 15, 15:1, 15:2, 15:3, 15:4, 15:6 and 16; C.I. Pigment Green 7 and 36; pyranthrone pigments, such as C.I. Pigment Orange 51; C.I. Pigment Red 216 (C.I. Vat Orange 4); thioindigo pigments, such as C.I. Pigment Red 88 and 181 (C.I. Vat Red 1); C.I. Pigment Violet 38 (C.I. Vat Violet 3); triarylcarbonium pigments, such as C.I. Pigment Blue 1, 61 and 62; C.I. Pigment Green 1; C.I. Pigment Red 81, 81:1 and 169; C.I. Pigment Violet 1, 2, 3 and 27; C.I. Pigment Black 1 (aniline black); C.I. Pigment Yellow 101 (aldazine yellow), and C.I. Pigment Brown 22. Representative examples of inorganic pigments include, but are not limited to, white pigments such as titanium dioxide (C.I. Pigment White 6), zinc white, pigment grade zinc oxide; zinc sulfide, lithopone; lead white; furthermore white fillers such as barium sulfate and CaCO3, black pigments, such as iron oxide black (C.I. Pigment Black 11), iron manganese black, spinel black (C.I. Pigment Black 27), carbon black (C.I. Pigment Black 7); colour pigments, such as chromium oxide, chromium oxide hydrate green; chrome green (C.I. Pigment Green 48); cobalt green (C.I. Pigment Green 50); ultramarine green; cobalt blue (C.I. Pigment Blue 28 und 36); ultramarine blue, iron blue (C.I. Pigment Blue 27), manganese blue, ultramarine violet, cobalt violet, manganese violet, iron oxide read (C.I. Pigment Red 101); cadmium sulfoselenide (C.I. Pigment Red 108); molybdate read (C.I. Pigment Red 104); ultramarine read, iron oxide brown, mixed brown, spinel- and Korundum phases (C.I. Pigment Brown 24, 29 und 31), chrome orange; iron oxide yellow (C.I. Pigment Yellow 42); nickel titanium yellow (C.I. Pigment Yellow 53; C.I. Pigment Yellow 157 und 164); chrome titanium yellow; cadmium sulfide und cadmium zinc sulfide (C.I. Pigment Yellow 37 und 35); Chrome yellow (C.I. Pigment Yellow 34), zinc yellow, alkaline earth metal chromates; Naples yellow; bismuth vanadate (C.I. Pigment Yellow 184); interference pigments, such as metallic effect pigments based on coated metal platelets, pearl luster pigments based on mica platelets coated with metal oxide, and liquid crystal pigments. For the purposes of the presently claimed invention, the at least one pigment is selected from the group consisting of metallic pigments and effect pigments. Representative examples of effect pigments include but are not limited to red pearlescent mica, white pearlescent mica, green organic mica, yellow mica, blue base mica. For the purposes of the presently claimed invention, the at least one pigment can also comprise mixtures of two or more different pigments. For the purposes of the presently claimed invention, the at least one pigment is preferably selected from the group consisting of BASF Perrindo Maroon L3920, BASF Perrindo Maroon L 3990, Sun Chemical Perrindo Ma-roon 229-8801, Sun Chemical Perrindo Maroon 229-6438, Sun Chemical Perrindo Violet 29, Clariant Hostaperm Brown HFR01, Sun Chemical Palomar Blue 248-4816 and BASF Heliogen Blue 7081 D. In an embodiment of the presently claimed invention, the weight ratio of the polymeric pigment dispersant to the at least one pigment is in the range of from ≥0.1:1 to ≤3:1. In a preferred embodiment of the presently claimed invention, the weight ratio of the polymeric pigment dispersant to the at least one pigment is in the range of from ≥0.25:1 to ≤1.5:1. For the purposes of the presently claimed invention, the average particle size of the pigment particles is in the range of ≥10 nanometres to ≤10 microns, preferably in the range of ≥10 nanometres to ≤5 microns, more preferably in the range of ≥10 nanometres to ≤1 micron in diameter. For the purposes of the presently claimed invention, the pigment dispersion may be prepared by methods known to those of ordinary skill in the art. Representative examples of the methods for preparing pigment dispersions include, but are not limited to, the use of energy intensive mixing or grinding using ball mills or media mills. Another aspect of the presently claimed invention is directed to a coating composition comprising a pigment dispersion according to the presently claimed invention and at least one binder. For the purposes of the presently claimed invention, representative examples of binders include, but are not limited to, paints, fillers, and additives. The representative examples of additives include, but are not limited to, surfactants, light stabilizers, UV-absorbers, anti-foaming agents, dyes, plasticizers, levelling agents and anti-skinning agents. For the purposes of the presently claimed invention, the at least one binder is preferably selected from the group consisting of poly(meth)acrylates, polystyrenics, polyesters, alkyds, polysaccharides and polyurethanes. In an embodiment of the presently claimed invention, the coating composition is a solventborne composition. For the purposes of the presently claimed invention, the solventborne coating composition is a composition that comprises an organic solvent. Representative examples of organic solvents include, but are not limited to, xylene, toluene, methanol, ethanol, n-propanol, isopropanol, acetone, methyl ethyl ketone, dimethyl ether, methyl ethyl ether, ethyl acetate, ethyl lactate, ethylene glycol, diethylene glycol and butyl-2-hydroxyethyl ether. In an embodiment of the presently claimed invention, the coating composition is a waterborne composition. For the purposes of the presently claimed invention, the waterborne coating composition is a composition that comprises water as a main solvent. However, 0 wt. % to ≤10 wt. %, preferably 0 wt. % to ≤5 wt. %, and most preferably 0 wt. % to ≤1 wt. % of organic solvents may be present in the waterborne coating compositions. In an embodiment of the presently claimed invention, a clearcoat material comprises the coating composition described hereinabove. In an embodiment of the presently claimed invention, a basecoat material comprises the coating composition described hereinabove. An aspect of the presently claimed invention is directed to a use of a pigment dispersion according to the presently claimed invention in printing ink, automotive basecoat, automotive clearcoat, mill base, furniture coatings and wood coatings. In an embodiment of the presently claimed invention, the pigment dispersion described hereinabove is used as a clearcoat material for industrial coatings selected from the group consisting of automotive OEM finishing, the finishing of parts for installation in or on automobiles and/or utility vehicles and automotive refinish, topcoat material, and electrodepositable coating material. Another aspect of the presently claimed invention is directed to an article coated with at least one layer formed from the coating composition according to the presently claimed invention. For the purposes of the presently claimed invention, the coating composition can preferably be applied to the article by any of the customary application methods. Representative examples of the application methods include, but are not limited to, spraying, knife coating, spreading, pouring dipping, impregnating, trickling or rolling. With respect to such application, the substrate to be coated may itself be at rest, with the application unit or equipment being moved. Alternatively, the substrate to be coated, more particularly a coil, may be moved, with the application unit being at rest relative to the substrate or being moved appropriately. Pref-erable application methods are air spraying, airless spraying, high speed rotation, electro-static spray application, alone or in conjunction with hot spray application such as hot air spraying, for example. For the purposes of the presently claimed invention, the coating composition of the presently claimed invention can be applied to an uncoated or a precoated article. An aspect of the presently claimed invention is directed to a compound of formula (IV) wherein R1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl; R2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; or R2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl which are each substituted with one —C(═O)—OH group; whereby the following compound N-napthalenyl-4-carboxy-1,2-phthalimide is excluded. Another aspect of the presently claimed invention is directed to a compound of formula (IV) wherein R1 is selected from the group consisting of unsubstituted naphthyl or naphthyl substituted with 1, 2 or 3 —OH; and R2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—OH group. For the purposes of the presently claimed invention, the compound of formula (IV) described hereinabove and hereinbelow is also referred to as anchoring group or anchor or anchor(s). For the purposes of the presently claimed invention, the polymeric pigment dispersant described hereinabove and hereinbelow is also referred to as pigment dispersant or hyperdispersant or polymeric dispersant or dispersant. It is an advantage of the presently claimed invention, that the compounds of formula (IV) of the presently claimed invention were surprisingly found to provide colloidal stabilization against aggregation and/or agglomeration of the particulate or particles when functioning as a dispersant. The compounds of formula (IV) as described hereinabove and hereinbelow provide good interaction and strong adsorption with a pigment surface by even weak interactions like π-π and hydrogen bonding interaction. Further, it is an advantage of the presently claimed invention that the polymeric dispersants can be prepared in a simple and efficient method that is cost effective. The compounds of formula (IV) as described hereinabove and hereinbelow have increased solubility in low polarity solvents that makes the synthesis of the polymeric pigment dispersant easier than the conventionally known methods. The polymeric pigment dispersants of the presently claimed invention provide high chroma and transparent colour in comparison to traditional hyperdispersants or pigment dispersants. In a preferred embodiment, the presently claimed invention is directed to a polymeric pigment dispersant comprising a polymer backbone (P) and at least one moiety of the formula (I): wherein R1 is naphthyl, which is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents independently of each other selected from the group consisting of F, Cl, Br, I, —NO2, —CN, —OH, —C1-C6-alkyl, —C(═O)—C1-C6-alkyl, —C(═O)—O—C1-C6-alkyl, —C(═O)—O— phenyl, —CH2—C(═O)—C1-C6-alkyl, —C(═O)—NH(C1-C6)alkyl, —C(═O)—NH-phenyl, —C1-C6-alkyl; wherein —C1-C6-alkyl is itself unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents independently of each other selected from the group consisting of F, Cl, Br, I, —CN, —OH, —O—CF3, —O—CH3and —O—C2H5; R2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—O— group; and whereby the moiety of the formula (I) is bonded to the polymer backbone (P) via the —C(═O)—O— group. In a preferred embodiment, the presently claimed invention is directed to a polymeric pigment dispersant comprising a polymer backbone (P) and at least one moiety of the formula (I): wherein R1 is selected from the group consisting of naphthyl, anthracenyl and phenanthrenyl which are each unsubstituted or substituted with 1, 2 or 3 —OH; R2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—O— group; and whereby the moiety of the formula (I) is bonded to the polymer backbone (P) via the —C(═O)—O— group. In a preferred embodiment, the presently claimed invention is directed to a polymeric pigment dispersant comprising a polymer backbone (P) and at least one moiety of the formula (I): wherein R1 is naphthyl which is unsubstituted or substituted with 1, 2 or 3 —OH; R2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—O— group; and whereby the moiety of the formula (I) is bonded to the polymer backbone (P) via the —C(═O)—O— group. In a preferred embodiment of the presently claimed invention, wherein the at least one moiety of the formula (I) is obtained by reacting at least one compound of formula (II) wherein R2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl which are each substituted with one —C(═O)—OH group; with at least one compound of formula (III) R1—NH2(III) wherein R1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl; in the presence of at least one solvent. Embodiments In the following, there is provided a list of embodiments to further illustrate the present disclosure without intending to limit the disclosure to the specific embodiments listed below.1. A polymeric pigment dispersant comprising a polymer backbone (P) and a moiety of the formula (I): whereinR1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl;R2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or a branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—O— group;R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—O— group; orR2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl, which are each substituted with one —C(═O)—O— group; andwhereby the moiety of the formula (I) is bonded to the polymer backbone (P) via the —C(═O)—O— group.2. The polymeric pigment dispersant according to embodiment 1, wherein R1 is selected from the group consisting of naphthyl, anthracenyl and phenanthrenyl which are unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents independently of each other selected from the group consisting of F, Cl, Br, I, —NO2, —CN, —OH, —O—C1-C6-alkyl, —C(═O)—C1-C6-alkyl, —C(═O)—O—C1-C6-alkyl, —C(═O)—O-phenyl, —CH2—C(═O)—C1-C6-alkyl, —C(═O)—NH(C1-C6)alkyl, —C(═O)—NH-phenyl, —C1-C6-alkyl; wherein —C1-C6-alkyl is itself unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents independently of each other selected from the group consisting of F, Cl, Br, I, —CN, —OH, —O—CF3, —O—CH3and —O—C2H5.3. The polymeric pigment dispersant according to embodiment 1, wherein R1 is selected from the group consisting of naphthyl, anthracenyl and phenanthrenyl which are each unsubstituted or substituted with 1, 2 or 3 —OH.4. The polymeric pigment dispersant according to embodiment 1, wherein R2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with —C(═O)—O-group.5. The polymeric pigment dispersant according to embodiment 1, wherein the at least one moiety of the formula (I) is obtained by reacting at least one compound of formula (II) whereinR2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group;R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; orR2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl which are each substituted with one —C(═O)—OH group;with at least one compound of formula (III) R1—NH2(III)whereinR1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl;optionally in the presence of at least one solvent.6. The polymeric pigment dispersant according to embodiment 5, wherein the at least one compound of formula (II) is selected from the group consisting of phthalic anhydride, hexahydrophthalic anhydride, dodecenyl succinic anhydride, octadecenyl succinic anhydride, tetrahydrophthalic anhydride, methyltetrahydrophthalic anhydride and endomethylene tetrahydrophthalic anhydride, which are each substituted with at least one —C(═O)—OH group.7. The polymeric pigment dispersant according to embodiment 5, wherein the at least one compound of formula (III) is selected from the group consisting of 1-naphthylamine and 7-hydroxy 1-naphthylamine.8. The polymeric pigment dispersant according to embodiment 5, wherein the at least one solvent is a polar solvent having a boiling point in the range of ≥80° C. to ≤160° C. and a dielectric constant in the range of ≥11 to ≤30.9. The polymeric pigment dispersant according to embodiment 8, wherein the at least one solvent is selected from the group consisting of methyl N-amyl ketone, ethyl methyl ketone, methyl isoamyl ketone and isopropanol.10. The polymeric pigment dispersant according to embodiment 1, wherein the polymer backbone (P) is a linear di-block polymer.11. The polymeric pigment dispersant according to embodiment 10, wherein the linear di-block polymer is obtained by a living free radical polymerization.12. The polymeric pigment dispersant according to embodiment 10, wherein the linear di-block polymer has a formula A-B, whereinA is a first polymer block which is obtained by reacting a first mixture comprising at least one glycidyl (meth)acrylate; andB is a second polymer block which is obtained by reacting a second mixture comprising at least one monomer selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate, polyethylene glycol (meth)acrylate and polyethylene glycol alkyl ether (meth)acrylate.13. The polymeric pigment dispersant according to embodiment 12, wherein the alkyl (meth)acrylate is selected from the group consisting of methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, isopropyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, hexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, nonyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate and isodecyl (meth)acrylate.14. The polymeric pigment dispersant according to embodiment 12, wherein the hydroxyalkyl (meth)acrylate is selected from the group consisting of 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate and 2-hydroxybutyl (meth)acrylate.15. The polymeric pigment dispersant according to embodiment 12, wherein the polyethylene glycol alkyl ether (meth)acrylate is selected from the group consisting of polyethylene glycol methylether acrylate, polyethylene glycol ethyl ether acrylate, polyethylene glycol propyl ether acrylate and polyethylene glycol butyl ether acrylate.16. The polymeric pigment dispersant according to embodiment 1, wherein the polymeric pigment dispersant has a number average molecular weight (Mn) in the range of from ≥1000 g/mol to ≤25000 g/mol, determined according to gel permeation chromatography against a polystyrene standard.17. The polymeric pigment dispersant according to embodiment 1, wherein the polymeric pigment dispersant has a polydispersity in the range of from ≥1.2 to ≤20, determined according to gel permeation chromatography against a polystyrene standard.18. The polymeric pigment dispersant according to embodiment 1, wherein the total weight of the at least one moiety of formula (I) is in the range of from ≥5 wt. % to ≤50 wt. %, based on the total weight of the polymeric pigment dispersant.19. The polymeric pigment dispersant according to embodiment 1, wherein the polymer backbone (P) is a random polymer.20. The polymeric pigment dispersant according to embodiment 19, wherein the random polymer is obtained by free radical polymerization.21. The polymeric pigment dispersant according to embodiment 19, wherein the random polymer is obtained by reacting a mixture (Mn) comprising:(a) glycidyl methacrylate and/or glycidyl acrylate;(b) at least one monomer selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate and cycloalkyl (meth)acrylate;(c) optionally at least one monomer of styrene; and(d) optionally at least one monomer selected from the group consisting of vinyl monomers, monoethylenically unsaturated monomers bearing urea or keto groups and benzyl (meth)acrylate,optionally in the presence of at least one solvent.22. The polymeric pigment dispersant according to embodiment 21, wherein the alkyl (meth)acrylate is selected from the group consisting of methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, isopropyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, hexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, nonyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate and isodecyl (meth)acrylate).23. The polymeric pigment dispersant according to embodiment 21, wherein the hydroxyalkyl (meth)acrylate is selected from the group consisting of 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate and 2-hydroxybutyl (meth)acrylate.24. The polymeric pigment dispersant according to embodiment 21, wherein the cycloalkyl (meth)acrylate is selected from the group consisting of cyclopentyl (meth)acrylate, cy-clohexyl(meth)acrylate, dicyclopentadiene (meth)acrylate, dicyclopentanyl (meth)acrylate, tricyclodecanyl (meth)acrylate, isobornyl (meth)acrylate, 4-tert-butylcyclohexyl (meth)acrylate, norbornyl (meth)acrylate and bornyl (meth)acrylate.25. The polymeric pigment dispersant according to embodiment 21, wherein the at least one monomer of styrene is selected from the group consisting of 4-methyl styrene, 3-methyl styrene, 4-tert-butyl styrene, 4-tert-butoxy styrene, 2-bromostyrene, 3-bromostyrene, 4-bromostyrene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 4-chloro-α-methylstyrene, 2,6-dichloro styrene, 2-flurostyrene, 3-fluorstyrene, 4-fluorostyrene, 2,6-difluorostyrene, 3-nitrostyrene and 4-acetoxy styrene.26. The polymeric pigment dispersant according to embodiment 21, wherein the at least one vinyl monomer is selected from the group consisting of 3-vinyl benzoic acid, 4-vinyl benzoic acid and 4-vinylbenzyl chloride.27. The polymeric pigment dispersant according to embodiment 21, wherein the monoethylenically unsaturated monomer bearing urea or keto groups is selected from the group consisting of 2-(2-oxo-imidazolidin-1-yl)ethyl (meth)acrylate, 2-ureido (meth)acrylate, N-[2-(2-oxooxazolidin-3-yl)ethyl]methacrylate, acetoacetoxyethyl acrylate, acetoacetoxypropyl methacrylate, acetoacetoxybutyl methacrylate, 2-(aceto-acetoxy)ethyl methacrylate, diacetoneacrylamide (DAAM), diacetonemethacrylamide, N-(beta-ureido ethyl) acrylamide and N-(beta-ureido ethyl) methacrylamide.28. The polymeric pigment dispersant according to embodiment 21, wherein the solvent is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, butanol, butoxyethanol, acetone, butanone, pentanone, hexanone, methyl isobutyl ketone, ethyl acetate, butyl acetate, amyl acetate, methoxy propyl acetate, tetrahydrofuran, diethyl ether, ethylene glycol, polyethylene glycol and mixtures thereof.29. The polymeric pigment dispersant according to embodiment 19, wherein the random copolymer has a number average molecular weight (Mn) in the range of from ≥1000 g/mol to ≤25000 g/mol, determined according to gel permeation chromatography against a polystyrene standard.30. The polymeric pigment dispersant according to embodiment 19, wherein the random copolymer has a polydispersity in the range of from ≥1.5 to 20, determined according to gel permeation chromatography against a polystyrene standard.31. The polymeric pigment dispersant according to embodiment 19, wherein the polymeric pigment dispersant is a graft polymer.32. The polymeric pigment dispersant according to embodiment 31, wherein the graft polymer comprises at least one polyester block.33. The polymeric pigment dispersant according to embodiment 32, wherein the polyester block is obtained from monomeric units of a hydroxy-functional aliphatic acid or a hy-droxy-functional aromatic acid or a hydroxy-functional araliphatic acid.34. The polymeric pigment dispersant according to embodiment 32, wherein the polyester block is obtained in the presence of a saturated fatty acid or an unsaturated fatty acid.35. The polymeric pigment dispersant according to embodiment 34, wherein the saturated or the unsaturated fatty acid is selected from the group consisting of oleic acid, linolenic acid, palmitoleic acid and tall oil fatty acid.36. The polymeric pigment dispersant according to embodiment 33, wherein the hydroxy-functional aliphatic acid is selected from the group consisting of glycolic acid, lactic acid, 5-hydroxy valeric acid, 3-hydroxy-butyric acid, 4-hydroxy-valeric acid, 12-hydroxy stearic acid and 6-hydroxy caproic acid.37. The polymeric pigment dispersant according to embodiment 32, wherein the polyester block is obtained from monomeric units of a lactone.38. The polymeric pigment dispersant according to embodiment 37, wherein the lactone is selected from the group consisting of δ-valerolactone, ε-caprolactone, β-methyl-δ-valerolactone, 2-methyl-ε-caprolactone, 3-methyl-ε-caprolactone, 4-methyl-ε-caprolactone, 5-ter-butyl-ε-caprolactone, 7-methyl-ε-caprolactone, 4,4,6-trimethyl-ε-caprolactone and β-propiolactone.39. The polymeric pigment dispersant according to embodiment 32, wherein the total weight of the at least one polyester block is in the range of from ≥5 wt. % to ≤95 wt. %, based on the total weight of the polymeric pigment dispersant.40. The polymeric pigment dispersant according to embodiment 32, wherein the polyester block is bonded to the moiety of the formula (I) and/or the polymer backbone (P) via a —C(═O)—O— group.41. The polymeric pigment dispersant according to embodiment 31, wherein the graft polymer comprises at least one polyether block.42. The polymeric dispersant according to embodiment 41, wherein the at least one polyether block comprises a polyoxyethylene group comprising from 10 to 120 ethylene oxide units.43. The polymeric pigment dispersant according to embodiment 41 or 42, wherein the polyether block is bonded to the moiety of the formula (I) and/or the polymer backbone (P) via —C(═O)—O— group.44. A process for the preparation of at least one polymeric pigment dispersant according to embodiments 10 to 18 comprising at least the steps of:reacting a linear di-block polymer with a compound of the formula (IV): whereinR1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl;R2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group;R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; orR2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl, which are each substituted with one —C(═O)—OH group;at a temperature from ≥80° C. to ≤150° C.; andwherein the linear di-block polymer comprises a first and a second block and is obtained by a living free radical polymerization, optionally in the presence of a solvent.45. A process for the preparation of at least one polymeric pigment dispersant according to the embodiments 19 to 40 comprising at least the steps of:(a) reacting a random polymer as defined in embodiments 19 to 21 with a compound of the formula (IV): whereinR1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl;R2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group;R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; orR2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl, which are each substituted with one —C(═O)—OH group;and(b) reacting the compound obtained in step (a) with at least one monomer of a lactone at a temperature from 30° C. to ≤190° C.46. A process for the preparation of at least one polymeric pigment dispersant according to the embodiments 41 to 43 comprising at least the steps of:(a) reacting at least one polyalkylene glycol monoalkyl ether and at least one carboxylic acid anhydride at a temperature in the range from ≥70° C. to ≤140° C. to obtain a mixture; and(b) reacting the mixture obtained in step (a) with a random polymer as defined in embodiments 19 to 21 and a compound of the formula (IV): whereinR1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl;R2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group;R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; orR2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl, which are each substituted with one —C(═O)—OH group;at a temperature in the range from ≥70° C. to ≤140° C.47. A pigment dispersion comprising at least one polymeric pigment dispersant according to any one of the embodiments 1 to 43, at least one solvent and at least one pigment.48. The pigment dispersion according to the embodiment 47, wherein the weight ratio of the polymeric pigment dispersant to the at least one pigment is in the range of from ≥0.1:1 to 3:1.49. A coating composition comprising a pigment dispersion according to embodiment 47 or 48 and at least one binder.50. The coating composition according to embodiment 49, wherein the coating composition is a solventborne composition.51. The coating composition according to embodiment 49, wherein the coating composition is a waterborne composition.52. The use of a pigment dispersion according to embodiment 47 or 48 in printing ink, automotive basecoat, automotive clearcoat, mill base, furniture coatings and wood coatings.53. An article coated with at least one layer formed from the coating composition according to any one of the embodiments 49 to 51.54. A compound of formula (IV) whereinR1 is selected from the group consisting of unsubstituted or substituted naphthyl, unsubstituted or substituted anthracenyl and unsubstituted or substituted phenanthrenyl;R2 is selected from the group consisting of a hydrogen; a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group;R3 is selected from the group consisting of a linear or a branched, substituted C1-C14alkyl and a linear or branched, substituted C2-C14alkenyl, which are each substituted with one —C(═O)—OH group; orR2 and R3 together with the carbon atoms to which they are bonded form a substituted phenyl or a substituted C3-C10cycloalkyl or a substituted C4-C10cycloalkenyl which are each substituted with one —C(═O)—OH group;whereby the following compound N-napthalenyl-4-carboxy-1,2-phthalimide is excluded.55. A compound of formula (IV) whereinR1 is selected from the group consisting of unsubstituted naphthyl or naphthyl substituted with 1, 2 or 3 —OH; andR2 and R3 together with the carbon atoms to which they are bonded form a ring selected from the group consisting of phenyl and cyclohexyl which are each substituted with one —C(═O)—OH group. While the presently claimed invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the presently claimed invention Examples The presently claimed invention is illustrated in detail by non-restrictive working examples which follow. More particularly, the test methods specified hereinafter are part of the general disclosure of the application and are not restricted to the specific working examples. Preparation of the Compounds of Formula (IV) The preparation of the compounds of formula (IV) was carried out by reacting the anhydrides with an amine in a suitable solvent under reflux condition, followed by precipitation or vacuum drying under reduced pressure (Scheme 1: a to g). Synthesis of the Compounds of Formula (IV) (Anchors C to F) is Shown Below in Scheme 1: a-g Preparation of N-naphthalenyl-4-carboxy-1,2-phthalimide (Scheme 1a) A 500 mL round bottom flask was charged with 100 g (0.52 mol) of trimellitic anhydride (source: Sigma Aldrich), 81.98 g (0.57 mol) of 1-naphthylamine (source: Sigma Aldrich) and 65 g of DMF and refluxed for 2.5 h. Upon cooling to room temperature, the mixture was diluted in 300 mL of butyl acetate and precipitated in 500 mL of hexane. The precipitate was filtered off and dried under reduced pressure which resulted in 155 g of yellowish solid product (anchor C). Preparation of N-naphthalenyl-4-carboxy-1,2-phthalimide (Scheme 1b) In a 1 L round bottom flask attached with a dean-stark, 100 g (0.52 mol) of trimellitic anhydride (source: Sigma Aldrich), 81.98 g (0.57 mol) of 1-naphthylamine (source: Sigma Aldrich) and 200 g of isopropanol was refluxed for 15 h. The reaction was followed NMR spectroscopy. At the end of the reaction, ˜150 g of isopropanol was distilled off by heating at reflux condition. The remaining isopropanol was removed by vacuum to result in 212.94 g (85% solid) of product (anchor C). Preparation of N-naphthalenyl-4-carboxy-1,2-phthalimide (Scheme 1c) In a 1 L round bottom flask attached with a dean-stark, 100 g (0.52 mol) of trimellitic anhydride (source: Sigma Aldrich), 81.98 g (0.57 mol) of 1-naphthylamine (source: Sigma Aldrich) and 200 g of MIAK was refluxed for 2.5 h with distillation of water. The progress of the reaction was followed with the amount of water collected in the Dean-stark apparatus. At the end of the reaction, ˜150 g of MIAK was distilled off by heating at reflux condition. The remaining solvent was removed under reduced pressure to result in 200 g (91% solid) of product (anchor C). Preparation of N-(7-hydroxynaphthalenyl)-4-carboxy-1,2-phthalimide (Scheme 1d) A 250 mL round bottom flask was charged with 50 g (0.26 mol) of trimellitic anhydride (source: Sigma Aldrich), 46.0 g (0.29 mol) of 7-hydroxy 1-naphthylamine (source: Sigma Aldrich) and 60 g of DMF and refluxed for 3 h. Upon cooling to room temperature, the mixture was diluted in 300 mL of butyl acetate and precipitated in 500 mL of hexane. The precipitate was filtered off and dried under reduced pressure which resulted in 85 g (65% solid) of product (anchor D). Preparation of N-(7-hydroxynaphthalenyl)-4-carboxy-1,2-phthalimide (Scheme 1e) In a 500 mL round bottom flask attached with a dean-stark, 30.17 g (0.16 mol) of trimellitic anhydride (source: Sigma Aldrich), 25 g (0.16 mol) of 7-hydroxy 1-naphthylamine (source: Sigma Aldrich) and 150 g of MIAK was refluxed for 5 h with distillation of water. The reaction was followed with the amount of water collected in the Dean-stark apparatus. At the end of the reaction, ˜50 g of MIAK was distilled off by heating at reflux condition. The remaining MIAK was removed under reduced pressure 87.57 g (63% solid) of product (anchor D). Preparation of the Di-Block Polymeric Pigment Dispersants (I) Preparation of Solventborne Di-Block Prepolymer According to methods that are familiar to those skilled in the art, the di-block prepolymer for solventborne dispersants (prepolymer 1, prepolymer 2 and prepolymer 3) were synthesized out via controlled radical polymerization (CRP) in three major steps as described below. The raw material composition is provided in Tables 1-3. The characteristics of different backbones that are used in the synthesis of the random type of polymeric dispersants are shown in Table 4. Step A: A 5 L four neck round bottom flask fitted with a condenser, an agitator, and a ther-mocouple was charged with reagents 1-4 (Tables 1-2) and purged with nitrogen for 10 minutes. This was followed by addition of reagent 5 with further purging of 20 minutes. The dark brown mixture was heated to 70° C. and held at that temperature for 1.5 h. Nitrogen purging was continued until the temperature reached to 70° C. Step B: At the end of step A, the temperature of the reaction mixture was reduced to 60° C. and a mixture of reagents 6-9 (Tables 1-2) purged with nitrogen for 30 minutes, was trans-ferred to the reaction flask through a cannula under slight positive nitrogen pressure. The reaction temperature was increased to and held at 80° C. for 9.5 h. Step C: At the end of step B, the reaction mixture was exposed to air. A mixture of reagents 10 and 11 (Tables 1-2) was added directly to the flask and held at 80° C. for 5 h. Towards the end of this process the green colour of the resin disappeared and the initially yellow amberlyte-748 resin turned into bluish green. The solution was filtered through a solid filtration funnel to remove amberlyte resin beads. The acetic acid and some solvent was distilled off under reduced pressure until 10% of the volatile is removed. TABLE 1Synthesis of prepolymer 1Raw materialWeight (g)Mol1AButyl acetate1112.40009.57642Glycidyl methacrylate434.50003.05653TsCl57.20000.30004Bpy9.44900.06055Cu(0)3.84450.06056BButyl methacrylate406.12003.16867HPMA568.92003.94628n-butyl acetate500.00009Butyl acrylate962.50007.509610CAcetic acid27.43130.456811Amberlyte-748173.2500 where, glycidyl methacrylate and HPMA=2-hydroxypropyl methacrylate were obtained from Dow Chemical; butyl methacrylate, TsCl=p-toluene sulfonyl chloride, acetic acid and Bpy=bipyridyl were obtained from Sigma Aldrich; butyl acrylate was obtained from BASF and Amberlyte-748 resin was obtained from Alfa Aesar. TABLE 2Synthesis of prepolymer 2Raw materialWeight (g)Mol1AButyl acetate1100.00009.46972Glycidyl methacrylate470.00003.30633TsCl114.40000.60014Bpy7.08680.04545Cu (0)2.88340.04546BButyl methacrylate439.00003.42517HPMA681.300004.72538n-butyl acetate494.00009Butyl acrylate860.00006.709810CAcetic acid27.43130.456811Amberlyte-748173.2500 where, glycidyl methacrylate and HPMA=2-hydroxypropyl methacrylate were obtained from Dow Chemical; butyl methacrylate, TsCl=p-toluene sulfonyl chloride, acetic acid and Bpy=bipyridyl were obtained from Sigma Aldrich; butyl acrylate was obtained from BASF and Amberlyte-748 resin was obtained from Alfa Aesar. TABLE 3Synthesis of prepolymer 3Raw materialWeight (g)Mol1AMIBK1100.00009.46972Glycidyl methacrylate470.00003.30633TsCl114.40000.60014Bpy7.08680.04545Cu(0)2.88340.04546BPEGMEAcrylate480291.11000.60657HPMA115.65000.80218MIBK67.50000.58119CAcetic acid8.16000.135910AMBERLITE IRC748i51.970011MIBK30.00000.2583 where, MIBK=methyl isobutyl ketone; glycidyl methacrylate and HPMA=2-hydroxypropyl methacrylate were obtained from Dow Chemical; TsCl=p-toluene sulfonyl chloride, acetic acid, Bpy=bipyridyl and PEGMEAcrylate480=polyethylene glycol methylether acrylate, Mn480 were obtained from Sigma Aldrich; and Amberlyte-748 resin was obtained from Alfa Aesar. (II) Preparation of Solventborne Di-Block Polymeric Dispersants The di-block prepolymer, prepolymer 2 was reacted with anchors C and D (Scheme 1) using a catalytic amount of N,N-dimethyldodecylamine and butyl acetate under reflux condition (115° C.-124° C.) until the Weight per Epoxy (WPE) reached >15,000 to result in light brown transparent solutions dispersant 1 and dispersant 2 (Table 4) at about 50% non-volatile (NV). (III) Preparation of a Control Di-Block Polymeric Dispersant Using same procedure above for preparation of solventborne di-block polymeric dispersants, a comparative example of di-block copolymer was prepared by reacting prepolymer 2 with N-methylcarboxy-1,8-naphthalimide that is commercially available to result in dispersant 4 (Table 4). (IV) Preparation of Waterborne Di-Block Polymeric Dispersant The di-block prepolymer 3 was reacted with anchor C (Scheme 1) to result in dispersant 3 at 65% solid content. The mixture was heated under reflux condition (115° C.) until the WPE number reached 12,000. The final product was vacuum dried to result in the removal of MIBK. The resultant product was reduced in 50/50 (w/w) butyl cellosolve (source: Eastman Chemical Company) and DI water to result in a brown transparent solution at about 50% non-volatile (NV). TABLE 4Composition of final di-block dispersant resinsAnchorsDispersantwt %Molecularresin #Prepolymer#AnchorsSolvent/s(/solid)weightdispersant 1prepolymer 2CButyl23Mn=5,312acetateMw=8,286PDI =1.55dispersant 2prepolymer 2DButyl27Mn=5,517acetateMw=8,385PDI =1.52dispersant 3prepolymer 3CWater:butyl25Mn=4,393cellosolveMw=6,106(50:50)PDI =1.39dispersant 4*prepolymer 2N-methyl-carboxy-Butyl20Mn=5,1341,8-naphthalimideacetateMw=8,214PDI =1.60*not within the scope of the invention Preparation of Random Polymeric Pigment Dispersant (I) Preparation of Glycidyl Functional Polyacrylate Backbone (Acrylic Backbone BB-1 and BB-2) The glycidyl functional acrylic copolymers for the synthesis of the random type of polymeric dispersants were synthesized by random copolymerization of glycidyl methacrylate (GMA) with other vinyl and/or (meth)acrylate monomers via conventional state of the art free radical polymerization using solution polymerization technique. The important characteristics of these polyacrylates are described in Table 5. 2,2′-Azobis(2-methyl butyronitrile) AMBN was used as thermal initiator. The characteristics of different backbones used in the synthesis of comb type of hyper-dispersants are shown in Table 5. TABLE 5ComonomersAcrylicParts by weight (PbW)Solvent/s% NVEEWMolecularbackbonerespectively(PbW)(110° C./1 h)(g/eq)weightAcrylic-GMA//styrene/EHA/BzMAMIBK64.46%229.7Mn=2364BB-1(67.7/14.6/3.1/14.6)Mw=4548PDI =1.92Acrylic-GMA//styrene/EHAMIBK65.51%175.9Mn=2491BB-2(88.5/9.4/2.1)Mw=4599PDI =1.85 where, PbW=Parts by Weight GMA=glycidyl methacrylate (source: Mitsubishi Gas Chemical Company); MMA=methyl methacrylate; UMA=ureido methacrylate (used as 25% W/W solution in MMA) (source: BASF); EHA=ethyl hexyl acrylate (source: Sigma Aldrich); BzMA=benzyl methacrylate (source: Geo Specialty Chemical Company); MIBK=methyl isobutyl ketone (source: Sigma Aldrich). (II) Preparation of Solventborne Random Type Polymeric Dispersant (Dispersant 5) Step-1: Synthesis of the Anchor Grafted Intermediate: According to methods familiar to those skilled in the art, the glycidyl functional acrylic copolymer (Acrylic-BB-1) (36.3 g) was reacted with the anchor D (30.5 g) in the presence of a catalytic amount of zinc acetylacetonate at 110˜115° C. until almost all the epoxy groups were consumed as confirmed by FTIR spectroscopy. The reaction mass was cooled to ambient condition and diluted by adding ethyl methyl ketone (60 g) while cooling. Step-2: Grafting-from of Polyester Side Chains: According to methods familiar to those skilled in the art, linear polyester stabilization chains are ‘grafted from’ the anchor grafted intermediate in above step-1 by ring opening polymerization of lactone monomers. The intermediate is gradually heated to 125° C. while distilling out the solvent present in the intermediate. A mixture of ε-caprolactone (132.1 g) and 6-valerolactone (29.0 g) was run into the reactor along with tin(II) 2-ethylhexanoate (0.54 g) while maintaining the temperature between 120° C.-130° C. The reaction was further continued at 125° C. until the desired conversion of lactone was achieved as confirmed by measuring % NV as compared to the theoretical anticipated values. Upon achieving the desired conversion, the mass was cooled to 75° C. and diluted by n-butyl acetate and stirred until a homogeneous solution was observed. The final % NV of the dispersant was 60.8%. (III) Preparation of Solventborne Random Type Polymeric Dispersant (Dispersant 6) Step-1: Synthesis of the Anchor Grafted Intermediate: According to methods familiar to those skilled in the art, the glycidyl functional acrylic copolymer (Acrylic-BB-1) (36.9 g) was reacted with the anchor C (24.05 g) in the presence of a catalytic amount of zinc acetylacetonate at 115° C.-120° C. until almost all the epoxy groups were consumed. The reaction mass was cooled to ambient condition and diluted by adding ethyl methyl ketone (10 g) while cooling. Step-2: Grafting-from of Polyester Side Chains: According to methods familiar to those skilled in the art, linear polyester stabilization chains were ‘grafted from’ the anchor grafted intermediate in above step-1 by ring opening polymerization of lactone monomers. The intermediate was gradually heated to 125° C. while distilling out the solvent that was present in the intermediate. The mixture of ε-caprolactone (118.5 g) and δ-valerolactone (26.0 g) was run into the reactor along with tin(II) 2-ethylhexanoate (0.48 g) while maintaining the temperature between 105° C.-125° C. The reaction was further continued at 120° C. until the desired conversion of lactone was achieved as confirmed by measuring % NV as compared to the theoretical anticipated values. Upon achieving the desired conversion, the mass was cooled to 75° C. and diluted by n-butyl acetate and stirred until a homogeneous solution was observed. The final % NV of the dispersant was 61.7%. (IV) Preparation of Waterborne Random Type Polymeric Dispersant (Dispersant 7) The polyethylene glycol (Carbowax 2000 ®) (158.2 g) was charged to the reactor and heated to 120° C. under vacuum and maintained for 30 min. Vacuum was stopped and succinic anhydride (7.5 g) was added and reacted at 118° C.-120° C. for 3.5 h. The glycidyl functional acrylic copolymer (Acrylic-BB-2) (69.9 g) was added followed by the anchor C (48.4 g) in the presence of catalytic amounts of zinc acetylacetonate at 115° C. until almost all the epoxy groups were consumed (epoxy equivalent weight >15000 g/eq). The solvent present in the system was distilled out during the process by simple distillation. While cooling the reaction mass to ambient condition the mass was diluted by adding a mixture of ethyl methyl ketone (112 g) and 1-propoxy-2-propanol (28 g) while stirring. The final % NV of the dispersant was 66.5%. TABLE 6Composition of the final randompolymeric pigment dispersant resinsAnchorpolyacrylatewt. %Resin #backboneAnchorSolvent/s(/solid)Dispersant 5Acrylic-BB-1Dn-butyl acetate12.87%Dispersant 6Acrylic-BB-2Cn-butyl acetate12.57%Dispersant 7Acrylic-BB-2Cethyl methyl ketone:18.75%1-propoxy-2-propanol;(80:20) Evaluation and Observation of the Polymeric Dispersants In order to evaluate the polymeric dispersants synthesized according to the process described hereinabove, solventborne and waterborne samples were formulated and ground using a Lau Disperser. The sample grind, colour performance and the particle size distribution were measured. The evaluation was done with multiple high performance organic pigments for coatings that include, but are not limited to, BASF Perrindo Maroon L3920, BASF Perrindo Maroon L 39990, Sun Chemical Perrindo Maroon 229-8801, Sun Chemical Perrindo Maroon 229-6438, Sun Chemical Perrindo Violet 29, Clariant Hostaperm Brown HFR01, Sun Chemical Palomar Blue 248-4816, and BASF Heliogen Blue 7081 D. The pigments were obtained from Sun Chemical, New Jersey, USA and BASF Corporation, New Jersey, USA. 1) Formulation For solventborne trials, 10% pigment loading with various dispersant on pigment (DoP) concentrations was explored. DoP concentrations ranged from ˜30%-˜300%. The samples were evaluated in normal butyl acetate (Nexeo Solutions, Warren, Michigan, USA). Table 7 shows typical formulations of synthesized dispersants with ˜50% DoP. For waterborne trials, 10% pigment loading with various dispersant on pigment (DoP) concentrations was explored. DoP concentrations ranged from ˜25%-˜200%. The samples were evaluated in deionized water and deionized water/solvent mixtures, where the solvent component was at least 2.5 wt. % of the total formulation. Examples of the solvent used include, but are not limited to, propylene glycol n-propyl ether (Nexeo Solutions, Warren, Michigan, USA) and propylene glycol n-butyl ether (Dowanol PNB, The Dow Chemical Company, Midland, Michigan, USA). 2) Grinding To each formulation, 0.3 mm zirconium stabilized yttria beads (Fox Industries, Fairfield, New Jersey, USA) were added in order to grind the pigment. For solventborne systems, the beads were ˜100% of the total formulation weight, for example 100 g of formulation was added to 100 g of beads to make a total of 200 g. For waterborne systems, the beads were 200% of the total formulation weight, for example 100 g of formulation was added to 200 g of beads to make a total of 300 g. The prepped sample was then placed on the Lau Disperser—Model DAS H-TP 200-K with cooling system (LAU GmbH, Hemer, Germany) and shaken with the fan on for 540 minutes or 9 hours. Upon completion of the run, the samples were filtered to remove the beads and stored in aluminium paint cans. Filtered beads were washed with solvent and reused. TABLE 7Composition of the pigment dispersionsExam-Exam-Exam-Exam-Exam-Exam-Exam-Exam-Exam-ple 1ple 2ple 3ple 4ple 5ple 6ple 7*ple 8*ple 9*dispersant 19.869.869.86dispersant 210.110.110.1dispersant 4*9.849.849.84229-6438101010(maroon)L3920 (red)101010L3990 (maroon)101010n-butyl acetate80.1479.9080.1479.9080.1479.9080.1680.1680.16Total100100100100100100100100100*not within the scope of the invention Evaluation of Stability and Colour 1) Sample Stability After filtration, the fineness of the grind was evaluated using a Hegman gauge. Samples were considered to be passing if they showed a grind of <6 micron. 2) Colour Evaluation The solventborne pigment dispersions were evaluated for colour performance in R10CG0392D, a commercially available 1 component clear coat from BASF Corp. at 26701 Telegraph Rd. Southfield, Michigan 48033 using melinex drawdown sheets (Puetz GmbH+CO. Fo-lien KG, Taunusstein, Germany). The pigments (L3920, L3990, 229˜6438) were evaluated at 0.3% pigment to binder ratio by weight. The binder weight included only the 51% solids from the clearcoat. Pigment was added to R10CG0392D clearcoat under agitation. A 150 μm gap on the Byk drawdown bar was used (Byk-Chemie GmbH, Wesel, Germany) and allowed to flash for ˜20 minutes. The sample was subsequently baked for 20 minutes at 270° F. Samples were made in duplicate to ensure reproducibility. Once the sample cooled, the colour spectrum was measured using a Byk Mac i spectrophotometer (Byk-Chemie GmbH, Wesel, Germany). The melinex card with the tinted clearcoat drawdown was placed on top of a reflective mirror. The Byk Mac i is then placed on top of the melinex and mirror and colour data measured with d65 light at 15, 25, 45, 75, and 110 degrees off specular using GM CieLab weightings. Measurements were done five times per sample and replicate drawdowns of a given sample compared. This method is used for colour evaluation because higher particle size pigment agglomerates resulting in more scattered light which increases the measured lightness values of the film. As the 110° angle has the longest film path length, it is the most sensitive to detecting increases in scattering. Therefore, L* values (lightness) at the 110° angle were used for evaluation, whereby dispersions yielding lower L* values are more transparent and resemble therefore an improved distribution or stabilization or dispersion of the pigment particles. Table 8 provides typical L* values at the 110° angle for the formulated systems in Table 7. TABLE 8L* at 110°<dL> at 110°Example 110.22−0.88Example 210.01−1.16Example 311.57−1.84Example 411.59−1.82Example 511.95−1.41Example 612.41−0.84 where, L* is lightness value at 110°; <dL> is the weighted value of the difference in L* at 110° between a reference dispersion, i.e. dispersion composition with dispersant 4 (Examples 7*, 8* and 9*, with the anchor group as N-methylcarboxy-1,8-naphthalimide) and the dispersions with polymeric pigment dispersants of the presently claimed invention (Examples 1 to 6). Discussion of Results The results in the Table 8 shows that the Examples 1˜6 with polymeric pigment dispersants of the presently claimed invention show lower L* values. A negative value of <dL> indicates that the polymeric pigment dispersant has a lower L* value than the reference dispersion. This is also indicative that the dispersions with the polymeric pigment dispersants of the presently claimed invention are more transparent than the reference dispersion. Within each pigment type there is good correlation between smaller particle size and de-creased lightness values at 1100 related to scattering and opacity. A lower L* value generally correlates to smaller particle size. Advantages 1) The di-block polymeric dispersants of the presently claimed invention provide more efficient de-agglomeration over the dispersant made from the anchor molecule N-methylcarboxy-1,8-naphthalimide which was also prepared by the Controlled Radical Polymerization (CRP) method with di-block prepolymer. This indicates that more chromatic and transparent colour can be achieved via high energy micro milling process to result in pigment particles to less than 100 nm size range.2) The new anchors (formula IV) of the presently claimed invention are compatible with clearcoat coating compositions which contains organic acid catalyst. This is an advantage over the commercial dispersants that contain amines which reacts with the acid catalyst and loses the dispersing ability. Test Methods L* Value Determination The L* value was determined using Byk Mac i spectrophotometer (Byk-Chemie GmbH, Wesel, Germany). The colour data was measured with a D65 light source, and weighted dL or <dL> values were determined using GM CieLab weightings according to the standard DIN 6175˜2. Weight Per Epoxy (WPE) Determination The WPE was determined by titration with hydrogen bromide (HBr) according to ASTM D1652. Non-Volatile (NV) Determination The NV was determined in accordance with ASTM D2369 by removing the volatile component in a forced air draft oven set at 110° C. to 60 minutes. | 101,894 |
11859107 | DETAILED DESCRIPTION The disclosed resin removal systems will become better understood through review of the following detailed description in conjunction with the figures. The detailed description and figures provide merely examples of the various inventions described herein. Those skilled in the art will understand that the disclosed examples may be varied, modified, and altered without departing from the scope of the inventions described herein. Many variations are contemplated for different applications and design considerations; however, for the sake of brevity, each and every contemplated variation is not individually described in the following detailed description. Throughout the following detailed description, a variety of resin removal system examples are provided. Related features in the examples may be identical, similar, or dissimilar in different examples. For the sake of brevity, related features will not be redundantly explained in each example. Instead, the use of related feature names will cue the reader that the feature with a related feature name may be similar to the related feature in an example explained previously. Features specific to a given example will be described in that particular example. The reader should understand that a given feature need not be the same or similar to the specific portrayal of a related feature in any given figure or example. Botanicals are substances obtained or derived from natural materials and/or organic materials, such as plants, parts of plants, or native substances which are obtainable from organic or inorganic material. For example, the botanicals may be hydrocarbons, cannabidiols (CBD), tetrahydrocannabinols (THC), essential oils, and so forth that may be used in the food industry, the tobacco industry, the perfume industry, and the pharmaceutical industry for fragrances, flavoring mixtures, and medicinal remedies. To put the botanicals in a usable form, the botanicals are extracted from the natural materials and/or organic materials. Conventionally, to remove the botanicals from the natural material or the organic material an individual may use an ice water extraction also known as a water hash extraction. The ice water extraction may separate cannabinoids and trichomes from plant material which combined create a cannabis resin by using ice, water, and a screen to strain out resin glands which are inherently more dense than water. For example, an ice water hash machine may include a washing machine with a vertical or horizontal drum and plant matter and ice in a screen bag. The screen bag may be placed in the drum with the ice and water, then tumbled for a defined amount of time and using a motor to spin the washing machine drum before the liquid is drained through a tube into a bucket that contains bags that further separate particulates and resin. While the water hash extraction may separate the particulates and the resin from the plant material, the water hash extraction may be extremely labor-intensive and may be limited in scalability. For example, there may be physical limits as to the size that the vertical or horizontal drum of the washing machine may be scaled to due to the natural laws of physics. The physical limits of the size the drum may be scaled to may limit the ability to use the water hash extraction for mass production. Additionally, the conventional water hash extraction may require a relatively long amount of time to perform and may be time-intensive, where the process may require multiple runs to fully extract resin from the material. Furthermore, the conventional water hash extraction may not be automated as it requires manual labor to perform the process. The conventional water hash extraction may also use a relatively large amount of water and electricity, making the process not eco-friendly and costly. Implementations of the disclosure address the above-mentioned deficiencies and other deficiencies by providing methods, systems, devices, and/or apparatuses to extract botanicals from natural materials and/or organic materials. The resin removal system may include a water source, an air source, a washer, an internal filter, an external set of filters, and a processing tank. In one embodiment, a material may be inserted into the washer. Once the material is inserted into the washer, the water may be received from the water source and air may be received from the air source into the washer. As the high flow and high pressure of the water and the air are received into the washer, the water and the air may be circulated in the washer by spray patterns which create motion to expose the materials to forces which remove the resins from the material. As the resin-laden water and air are circulated in the washer, they flow through the internal filter and into the processing chamber. The internal filter may allow the water and the air to pass through to the external filters which may be suspended in the processing tank to collect the extracted resins. The external set of filters with the collected resins may be separate, or disconnected, from the washer and the resins may be removed for subsequent use. An advantage of the resin removal system may be that the resin removal system may be scaled up or down based on the desired use of the system, such as personal use or business use. Another advantage of the resin removal system may be to minimize the physical forces applied to the material to remove the resin, which may reduce the particularization and damage to the material during extraction. Another advantage of the resin removal system may be to reduce the time and labor required to extract resin from the material. Another advantage of the resin removal system may be to enable a user to automate the resin removal process and reduce or eliminate the amount of physical labor required to remove the resin from the material. FIG.1illustrates a resin removal system100to remove resin from a material124, according to an embodiment. In one embodiment, the resin removal system100may include a water source102and an air source104connected to a washer112. In one embodiment, the water source102may be connected to the washer112by a first conduit106, such as a first pipe, a first duct, or a first tube. In another embodiment, the air source104may be connected to the washer112by a second conduit108, such as a second pipe, a second duct, or a second tube. As discussed below, the first conduit106and the second conduit108may be connected to a manifold of the washer112. As the water and the air are received at the washer112, the water and/or the air may be mixed by the washer112to generate a water pattern to remove resins from a material124. The material124may be seeds, tubers, vegetables, fruits, cannabis, flowers, and so forth. In another embodiment, the second conduit108may be connected to the first conduit106via a connector110. In one example, the connector110may be a venturi valve that creates a constriction within the first conduit106(such as an hourglass shape) that varies the flow characteristics of water traveling through the first conduit106. As the water velocity in the throat of the venturi valve increases, the increase in velocity may create a natural pump or suction that may pull the air from the air source104and combine the air with the water. In another embodiment, the connector110may be a one-way valve, a globe valve, a gate valve, a ball valve, a butterfly valve, a diaphragm valve, a check valve, and so forth. In another embodiment, the water from the water source102may be pressurized by a first pump so that the water may be pressurized and directed from the water source102(via the first conduit106) to the washer112. The water source102may be a tank or container to store water and/or liquid. In one example, the water source102may be a 100-gallon to 300-gallon tank to store water and/or other liquids. In another example, the tank or container may store the water and/or other liquids at a cold or near freezing temperature, such as between 33 degrees Fahrenheit to 45 degrees Fahrenheit. In another example, the tank or container may store the water and/or other liquids at a heated temperature, such as between 75 degrees Fahrenheit and 120 degrees Fahrenheit. In another example, the water may include chemicals mixed in with the water. For example, to remove certain types of resins from materials, chemicals may also be required to remove the resin and those chemicals may be added to the water at the tank or container. In another example, the chemicals may be added in line with conduits for water or air or as a separate conduit connected to the washer112. In one embodiment, the washer112may be a metal material, a plastic material, a rubber material, a polyurethane material, a composite material, a foam material, and so forth. For example, the washer112may include a metal interior with a fiber composite housing to enclose a torus washer or toroidal washer, as discussed below. In another example, the interior of the torus washer or toroidal washer may include an interior with a stiff foam with food-grade coating and a housing that is a wood material or a plastic material. The washer112may be a material that can withstand high pressure, relatively hot and/or cold temperatures, and may have interior surfaces that are food grade surfaces. In another embodiment, the air from the air source104may be pressurized by a second pump so that the air may be pressurized and directed from the air source104(via the second conduit108) to the washer112. In another embodiment, the air from the air source104may be pressurized by a second pump so that the air may be pressurized and directed from the air source104(via the second conduit108, the connector110, and the first conduit106) to the washer112. In another embodiment, the air may be cooled at the air source104before providing the air to the washer112. In one example, the air source104may be air that is taken in or received from an area approximate a tube114. The tube114may be a coil that is within a container116that cools the coil to a defined temperature level. In one example, the tube114may be refrigerated. In another example, at least a portion of the tube114may be surrounded by a cold material, such as ice or a cooling gel. As the air is taken in by the cooled tube114, the temperature of the air may be cooled by the cooled tube114to decrease the temperature of the air below a threshold temperature level. In one embodiment, the threshold temperature level may be below 32 degrees (°) Fahrenheit (F) or 0° Celsius (C). In another embodiment, the threshold temperature level may be below 0° F. or −18° C. In another embodiment, the water from the water source102may be cooled to a threshold temperature. For example, the water may be cooled at the water source102by a refrigeration unit, a tube, and container similar to the tube114and the container116. In another example, the water may be cooled by ice added to the water at the water source. In another embodiment, the air from the air source104may be heated. For example, to heat the air, the air source104may include a forced air heater that may heat the air at the air source104or heat the air before the air being stored or sucked in by the air source104. In another example, the air source104may include the tube114where the air is stored and the tube114may be heated by a heating element to increase the temperature of the air. In another embodiment, the tube114may be copper because the copper tubing may transmute the cold or hot temperatures of the air. In another embodiment, the cold air or the heated air, when introduced into the washer112, may aid to quickly and efficiently separate and expose all sides of the material124in the chamber to extract the resin from the material124. In another embodiment, the first conduit106or the second conduit108may include a check valve118and a sprayer120that may be used to clean the resin removal system100. For example, when the check valve118is partially or fully opened, at least a portion of the water from the water source102may be diverted from the first conduit106or the second conduit108to the sprayer120. The sprayer120may then spray the water onto a part or all of the resin removal system100such as the water source102, the air source104, the washer112, a processing tank128, a filter134, a recycled water tank140, and so forth. In another embodiment, to clean part or all of the resin removal system100, air may be run through the resin removal system100. For example, to clean plant material in the resin removal system100, room temp air or heated air may be run through the resin removal system100to dry the resin removal system100. Once the resin removal system100has been dried, the material124that the resin was extracted from may be removed from the resin removal system100. In another example, room temperature or heated air may be run through the resin removal system100to screen rocks out of dirt and debris and/or to mix natural ingredients and compost into soil to produce organic planting soil. As the washer112receives the water and the air, the washer112may direct the water and the air to a material124held within the washer112to remove resins from the material124. As discussed below, the water and the air may be directed to form a defined pattern within the washer112to remove the resins from the material124. The resins may include hydrocarbons (such as terpenes and the oxygenated compounds), cannabidiols (CBD), tetrahydrocannabinols (THC), essential oils, and so forth. As discussed below, the water and/or the air may be injected or sprayed into the washer112in a directional flow to create a centrifugal or toroidal pattern that in turn generates a chaotic movement in the washer112to wash and clean the material124(such as cannabis, seeds, tubers, or other vegetables and fruits) and remove the resin from the material124. When the material124is seeds, the resin extracting process may separate and clean the seeds and then carry the seeds through the first filter122to the second filter126and separate the seeds via the sub-filters as discussed below. In one example, the washer112may include a housing with a first filter122. In one example, the washer112may include an opening in the bottom of the housing that the first filter122may be inserted into. To insert the material124into the washer112, the washer112may be detached and removed from the processing tank128and the material124may be inserted into an area enclosed by the first filter122. In one embodiment, the material124is loaded and unloaded in the washer112by disconnecting or removing the washer112from the processing tank128and then removing the first filter122from within the washer112. In one example, the washer112may be approximate to or rest on the processing tank128such that the washer112may be lifted off the processing tank128to disconnect the washer112from the processing tank128. In another example, the washer112may twist-lock onto the processing tank128such that the washer112may be rotated clockwise to unlock the washer112from the processing tank128and rotated counterclockwise to lock the washer112to the processing tank128, or vise versa. In another example, the first filter122may twist-lock into the opening of the washer112. As the water and the air are received into the washer112, the water and the air may be directed into the first filter122to remove the resins from the material124. In one example, the water and the air may be directed by the housing of the washer112toward the first filter122and the material124stored within the washer112. The water and the air may then be directed out the opening in the bottom of the housing of the washer112, and toward the processing tank128. As the water and/or the air are directed to the processing tank128, the force of the water and the air against the material124as the water and/or the air exit the opening in the washer112may wash or remove the resins from the material124and direct the resins toward the processing tank128. The processing tank128may be a plastic material, a metal material, a rubber material, a polyurethane material, and so forth. In one embodiment, the processing tank128may include a second filter126attached to a top of the processing tank128. As the water and/or the air exit the washer112and enter the processing tank128, the water and the air may pass through the second filter126. The second filter126may be porous to allow the water and the air to pass through the second filter126while the second filter126may remove the resins from the water and/or the air and trap or hold the resins within the second filter126. In one example, the water and/or the air may be stored in a housing of the processing tank128and the resins from the material124may be stored within the second filter126. A user of the resin removal system100may desire to use the resins removed from the material124for various purposes. For example, the resin removed from the material124may be hydrocarbons (such as terpenes and the oxygenated compounds), cannabidiols (CBD), tetrahydrocannabinols (THC), essential oils, and so forth. To remove the resin from the second filter126, the user may detach the washer112from above the processing tank128and then remove the second filter126from the processing tank128. Once the second filter126is removed from the processing tank128, the user may wash, scrape, skim, or otherwise extract the resin from the second filter126. In one embodiment, the water stored in the processing tank128may be discarded via an opening at the bottom of the processing tank128. For example, the opening at the bottom of the processing tank128may allow the water to flow onto the ground or the floor. In another example, the bottom of the processing tank128may be connected to a drain or a sewage connection to get rid of the water. In another embodiment, the resin removal system100may include a recycling system to clean and recycle the water from the processing tank128so that the water may be reused. When the resin removal system100uses recycled water, the resin removal system100may be a closed system that does not require new water to remove the resin from the material124. To recycle the water in the processing tank128, the opening at the bottom of the processing tank128may be connected to a third conduit132. In one embodiment, the third conduit132may be connected directly to the bottom of the processing tank128. In another embodiment, a check valve130may be connected to the bottom of the processing tank128and the third conduit132may be connected to the check valve130. The check valve130may allow a user to control when the water stored in the processing tank128is released. The third conduit132may be connected to a filter134. The filter134may remove particles or material from the water to clean the water for subsequent use. In one example, the water may be gravity fed to the filter134, where the processing tank128may be located at an elevation above the filter134to create natural pressure to force the water through the filter134. In another embodiment, the third conduit132may include a pump133, such as an inline pump, that may suck or pull the water from the processing tank128and direct the water through the filter134. In one embodiment, when the water has passed through the filter134, the water may be directed back to the first conduit106. In another embodiment, when the water has passed through the filter134, the water may be directed through a fourth conduit136to a water tank140. In one example, the water tank140may be used to store the recycled water. In one example, a pump may be connected to the water tank140such that when the pump is turned on, the pump may send the water stored in the water tank140through the resin removal system100several times before the water is to be heated or cooled again. In another example, the water tank140may store fresh water that is poured into the water tank140through an opening in the water tank140or another conduit connected to the water tank140. In another embodiment, the fourth conduit136may include a valve138, such as an inline check valve, that may control when the water may flow from the filter134, through the fourth conduit136, to the water tank140. A fifth conduit142may be connected to the water tank140, such as at the bottom of the water tank140. In one embodiment, the fourth conduit136may convey the water in the water tank140to the water source102. In another embodiment, the water tank140may be the water source102, where the fifth conduit142is connected to the first conduit106such that the water in the water tank140may be conveyed to the washer112via the fifth conduit142and the first conduit106. The connection of the first conduit106and the fifth conduit142may form the closed-loop system of the resin removal system100. In one example, the fifth conduit142may include a valve144, such as a ball valve or a check valve, to control when and how much water may flow out of the water tank140via the fifth conduit142. In addition to controlling the flow of the water and/or the air in the resin removal system100, the valves discussed herein for the resin removal system100may allow for replacement and/or cleaning of clogged areas of the resin removal system100without disrupting the entire water supply. In another example, the fifth conduit142may include a pump146, such as an inline pump, to suck the water from the water tank140and provide the water to the water source102or the first conduit106. In addition to controlling the flow and pressurizing the water and/or the air in the resin removal system100, the pumps discussed herein for the resin removal system100may allow for automating the resin removal system100with timers where the water and/or the air may automatically be provided to the resin removal system100at defined times of the day and/or for defined periods of time. FIG.2Aillustrates a top perspective view of a torus washer212with an octagonal shape, according to an embodiment. As discussed above, the resin removal system100inFIG.1may include a washer112to remove resin from material124. In one example, the washer112inFIG.1may be the torus washer212inFIG.2A. In one embodiment, the torus washer212may receive water and/or air from a source248. In one example, the source248may be the first conduit106and/or the second conduit108. In one embodiment, the torus washer212may receive the water and/or the air from the source248via a manifold structure. In one example, the manifold structure may receive the water and/or the air via a single inlet (such as one of256a-256d) connected to the source248. In another example, the manifold structure may receive the water and/or the air via multiple inlets256a-256d. In one example, the inlets256a-256dmay be plates or slots that direct low pressure or high-pressure water and/or air at a defined angle and/or direction. In one embodiment, the plates may be relatively stiff and/or rigid material (such as metal or plastic). In another embodiment, the plates may be a food-grade material or a material coated with a food-grade coating. The plates may include holes angled at a 45-degree angle to spray water into the torus washer212. The angle, size, and/or direction of the slots or holes of the inlets256a-256dmay generate the water and/or the airflow patterns as discussed herein. To receive the water and/or the air through the multiple inlets256a-256d, the manifold structure may include dividing the water and/or the air into multiple conduits. For example, when the manifold structure includes 4 inlets256a-256dat the torus washer212, the manifold structure may include a T-junction250to divide the water and/or the air from the source248so that a first portion of the water and/or the air flows into a first conduit252aand a second portion of the water and/or the air flows into a second conduit252b. The first conduit252amay be connected to a second T-junction253ato divide the water and/or the air in the first conduit252ainto a first portion that follows into a third conduit254aand a fourth conduit254b. The third conduit254amay be connected to the first inlet256aof the torus washer212and the fourth conduit254bmay be connected to the second inlet256bof the torus washer212. The second conduit252bmay be connected to a third T-junction253bto divide the water and/or the air in the second conduit252binto a first portion that follows into a fifth conduit254cand a sixth conduit254d. The fifth conduit254cmay be connected to the third inlet256cof the torus washer212and the sixth conduit254dmay be connected to the fourth inlet256dof the torus washer212. The number of inlets into the torus washer212and/or the manifold structure is not intended to be limiting and may vary. For example, the torus washer212may have two inlets on opposite sides of the torus washer212or inlets on each side of the torus washer212based on the desired amount of water and/or air a user may want to use to remove resin from the material, the type of material from which resin is removed, the size of the torus washer212, and so forth. In one embodiment, the torus washer212may be enclosed by a housing260. In one embodiment, the housing260may include handles to enable a user to more easily pick up the torus washer212. In another embodiment, the housing260may provide a protective cover to protect the torus washer from damage. The torus washer212may include a filter or screen258at approximately the center of the torus washer212. For example, at the center of the interior of the torus washer212may include a first filter258that is a barrel-shaped metal screen that extends 5-8 inches into the interior of the torus washer212and sits flush with the bottom of the torus washer212. As discussed above, the filter258may be secured to a housing of the torus washer212by a fastener or with a locking mechanism, such as a twist and lock mechanism. The filter258may allow liquids and particles smaller than the screen size to flow downward using gravity, into the processing tank128while keeping larger pieces inside the chamber for cleaning or disposal. In one example, the filter258may be a material so that the filter258may keep its shape in the face of the directional liquid and air flows. In one embodiment, the material may be a semi-rigid material such as a metal. In one embodiment, as the torus washer212receives the water and/or the air from the inlets256a-256d, the water and/or the air may circulate around an interior cavity of the torus washer212in a defined pattern, such as a circular pattern, a torus pattern, or a centrifugal pattern. The defined pattern may indicate a directional flow of the water and/or the air. As the water and/or the air circulates around the cavity in the defined pattern, the water and/or the air may chaotically or randomly move the material within the cavity of the torus washer212. The circular or torus pattern of the water and/or the air along with the chaotic or random movement of the material may dislodge or remove the resin from the material. As the water, the air, and the material circulate about the torus washer212, the water, the air, and the material may be pulled toward an opening at the bottom of the torus washer212below the filter258. As the water and/or the air exit through the opening, the resin that has been removed from the material may exit with the water and/or the air as the filter258restricts the material from exiting through the opening. FIG.2Billustrates a side exposed view of the torus washer212and the second filter126, according to an embodiment. Some of the features inFIG.2Bare the same or similar to some of the features inFIGS.1and2Aas noted by similar reference numbers, unless expressly described otherwise. As discussed above, the resin removal system100inFIG.1may include a washer112to remove resin from material124. As further discussed, as the water and/or the air may circulate around the cavity of the torus washer212in the defined pattern, the water and/or the air may also chaotically or randomly move the material within the cavity of the torus washer212to dislodge or remove the resin from the material. As the water, the air, and the material circulate about the torus washer212, the water, air, and material may be pulled toward an opening at the bottom of the torus washer below the filter258. In one example, the processing tank128may be located approximate to the torus washer212to receive the water, the air, and resin from the material from the torus washer212. In another example, the torus washer212may rest on top of the processing tank128with fasteners262that may extend from the bottom of the torus washer212to align the opening at the bottom of the torus washer212with an opening at the top of the second filter126. In one example, the torus washer212and the second filter126may interlock or interconnect. In another example, the torus washer212may freely sit on top of the processing tank128with the opening at the bottom of the torus washer212being aligned with the opening at the top of the second filter126when the fasteners262of the torus washer212are inserted into the top of the processing tank128. When the torus washer212is connected or aligned with the second filter126, the water, the air, and the resin, may flow out of the opening at the bottom of the torus washer212and into the second filter126. As the water, the air, and the resin flow through the second filter126, the resin may be caught or trapped by the second filter126while the water and/or the air pass through the second filter126to be stored in the processing tank128. In one embodiment, the second filter126may include multiple sub-filters that may catch or trap different portions of the resin with different particulate sizes by having different mesh sizes or mesh numbers for the sub-filters. In one embodiment, the sub-filters may be attached to a basket or tower structure263where the sub-filters are vertically stacked at defined distances along the basket or tower structure263and connected to the basket or tower structure263along the perimeter of the sub-filters and the basket or tower structure263. The basket or tower structure263may be connected to the processing tank128and the basket or tower structure263may hang directly below a bottom opening of the torus washer212, such as at a bottom center of the torus washer212. In another embodiment, the basket or tower structure263may be stiff and light material (such as flexible metal or plastic) and the sub-filters may be a flexible material (such as perforated metal or nylon material). In another embodiment, the sub-filters may be fastened to the basket or tower structure263with a fastener (such as a latch, a clasp, and so forth). For example, the filter258may include a filter to capture any materials larger than 177 microns and keep that material within the torus washer212. In this example, the sub-filters may include a first sub-filter264amay catch resin particulates with a first size (such as ranging between 176 microns to 145 microns) by having a first mesh size or a mesh number to catch the resin particulates with the first size, a second sub-filter264bto catch resin particulates with a second size (such as ranging between 144 microns to 90 microns) by having a second mesh size or a mesh number to catch the resin particulates with the second size, and a third sub-filter264cto catch resin particulates with a third size (such as ranging between 89 microns to 45 microns) by having a third mesh size or a mesh number to catch the resin particulates with the third size. In another example, the tower structure263may include a fourth sub-filter to catch resin particulates with a second size (such as ranging between 44 microns to 25 microns) by having a fourth mesh size or a mesh number to catch the resin particulates with the fourth size. The number of sub-filters, the mesh sizes, and particulate sizes is not intended to be limiting and may vary. The sub-filters264a-264cmay be configured to catch the different particulate sizes to separate different grades of the particulates. For example, the particulates caught by the first sub-filter264amay be the lowest grade of particulates, the particulates caught by the second sub-filter264bmay be a medium grade of particulates, and the particulates caught by the first sub-filter264amay be the highest grade of particulates. For example, recreational cannabis stores may sell 3-star cannabis concentrate, 4-star cannabis concentrate, and 5-star cannabis concentrate, where the 3-star cannabis concentrate may include resin that may be approximately 176 microns to 125 microns in size, the 4-star cannabis concentrate includes resin that may be approximately 124 microns to 45 microns in size, and 5-star cannabis concentrate may include resin that may be approximately 44 microns to 25 microns in size. The lower star or lower grade cannabis concentrate may be ranked lower because the cannabis concentrate includes less resin and more plant matter such that it includes a less concentrated resin as compared to higher ranked cannabis concentrate. In another example, 3-star cannabis concentrate may be the lowest grade product sold in the recreational and medical markets and 5-star cannabis concentrate may be the highest grade cannabis concentrate sold. In one embodiment, to remove the resin from the sub-filters264a-264c, the resin may be attached to or lay on top of the screens of the sub-filters264a-264c. When the sub-filters264a-264care removed from the basket or tower structure263, the resin may be removed and then used or stored for later use. In one example, once the resin has been removed from the sub-filters264a-264c, the resin may be frozen for later separation, drying, and packaging. In another example, once the resin has been removed from the sub-filters264a-264c, the resin may be prepared for drying and packaging. The number of sub-filters and the mesh size or mesh number of each sub-filter may vary based on the degree that a user may desire to separate resin particulates of different sizes. For example, the second filter126may include a single sub-filter when the second filter126is configured not to separate the different resin particulate sizes or the second filter126may include 10 sub-filters when the second filter126is configured to separate the different resin particulate sized to a high level of granularity. As discussed above, the torus washer212may be enclosed by a housing260. In one embodiment, the housing260may include handles260aand260bto enable a user to more easily pick up the torus washer212. The handles260aand260bmay be located on opposite sides of the housing260. FIG.3Aillustrates a top perspective view of a toroidal washer312with a donut shape, according to an embodiment. Some of the features inFIG.3Aare the same or similar to some of the features inFIGS.1-2Bas noted by similar reference numbers, unless expressly described otherwise. As discussed above, the resin removal system100inFIG.1may include a washer112to remove resin from the material124. In one example, the washer112inFIG.1may be the toroidal washer312inFIG.3A. In one embodiment, the toroidal washer312may receive water and/or air from a source348. In one example, the source348may be the first conduit106and/or the second conduit108inFIG.1A. In one embodiment, the toroidal washer312may receive the water and/or the air from the source348via an opening or inlet356at a top of the toroidal washer312. In one example, the inlet356may be located at the top center of the toroidal washer312. In another example, the source348may be a conduit fastened and sealed to the top of the toroidal washer312so that pressurized water and/or air may be sprayed into a cavity of the toroidal washer312. In another example, the source348may be a conduit located above the top opening of the toroidal washer312where the water and/or the air may naturally flow into the top opening via gravity pulling the water down into the top opening. In another example, the toroidal washer312may have inlets that are the same or similar to the inlets256a-256d(inFIG.2A) integrated into the sides of the toroidal washer312. The toroidal washer312may include a filter or screen358at approximately the center of the toroidal washer312. In one example, the filter358may be a barrel-shaped metal screen at the center of the toroidal washer312that may extend 5-8 inches into the interior of the toroidal washer312and may sit flush with a bottom of the toroidal washer312. The filter358may be fastened to the bottom of the toroidal washer312or may be attached to a housing of the toroidal washer312with a locking mechanism, such as a twist and lock mechanism. In one embodiment, as the toroidal washer312receives the water and/or the air from the inlet356, the water and/or the air may circulate around a cavity of the toroidal washer312in a defined pattern, such as a spherical pattern or a toroidal pattern. As the water and/or the air circulates around the cavity in the defined pattern, the water and/or the air may also chaotically or randomly move the material within the cavity of the toroidal washer312. The defined pattern of the water and/or the air along with the chaotic or random movement of the material may dislodge or remove the resin from the material124. As the water, the air, and the material circulate about the toroidal washer312, the water, air, and material may be pulled toward an opening at the bottom of the torus washer below the filter358. As the water and/or the air exit through the opening, the resin that has been removed from the material may exit with the water and/or the air as the filter358restricts the material from exiting through the opening. FIG.3Billustrates a side exposed view of the toroidal washer312and the second filter126, according to an embodiment. Some of the features inFIG.3Bare the same or similar to some of the features inFIGS.1-3Aas noted by the same reference numbers, unless expressly described otherwise. As discussed above, the resin removal system100inFIG.1may include a washer112to remove resin from material124. As further discussed, as the water and/or the air may circulate around the cavity of the toroidal washer312in the spherical or toroidal pattern, the water and/or the air may also chaotically or randomly move the material within the cavity of the toroidal washer312to dislodge or remove the resin from the material. As the water, the air, and the material circulate about the toroidal washer312, the water, air, and material may be pulled toward an opening at the bottom of the toroidal washer312below the filter358. In one example, the processing tank128may be located approximate to the toroidal washer312to receive the water, the air, and resin from the material from the toroidal washer312. In another example, the toroidal washer312may rest on top of the processing tank128with fasteners362that may extend from the bottom of the toroidal washer312to align the opening at the bottom of the toroidal washer312with an opening at the top of the second filter126. In one example, the toroidal washer312, and the second filter126may interlock or interconnect. In another example, the toroidal washer312may freely sit on top of the processing tank128with the opening at the bottom of the toroidal washer312being aligned with the opening at the top of the second filter126when the fasteners362of the toroidal washer312are inserted into the top of the processing tank128. When the toroidal washer312is connected or aligned with the second filter126, the water, the air, and the resin may flow out of the opening at the bottom of the toroidal washer312and into the second filter126. As the water, the air, and the resin flow through the second filter126, the resin may be caught or trapped by the second filter126while the water and/or the air pass through the second filter126to be stored in the processing tank128. As discussed above, the second filter126may include multiple sub-filters264a-264cthat may catch or trap different portions of the resin with different particulate sizes by having different mesh sizes or mesh numbers for the sub-filters. The disclosure above encompasses multiple distinct embodiments with independent utility. While these embodiments have been disclosed in a particular form, the specific embodiments disclosed and illustrated above are not to be considered in a limiting sense as numerous variations are possible. The subject matter of the embodiments includes the novel and non-obvious combinations and sub-combinations of the various elements, features, functions and/or properties disclosed above and inherent to those skilled in the art pertaining to such embodiments. Where the disclosure or subsequently filed claims recite “a” element, “a first” element, or any such equivalent term, the disclosure or claims is to be understood to incorporate one or more such elements, neither requiring nor excluding two or more such elements. Applicant(s) reserves the right to submit claims directed to combinations and sub-combinations of the disclosed embodiments that are believed to be novel and non-obvious. Embodiments embodied in other combinations and sub-combinations of features, functions, elements and/or properties may be claimed through amendment of those claims or presentation of new claims in the present application or a related application. Such amended or new claims, whether they are directed to the same embodiment or a different embodiment and whether they are different, broader, narrower or equal in scope to the original claims, are to be considered within the subject matter of the embodiments described herein. | 41,864 |
11859108 | DETAILED DESCRIPTION OF THE DISCLOSURE Although the invention will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of the invention. Various changes to the composition of a finishing medium or a finishing suspension and/or method of finishing an AMT Object may be made without departing from the scope of the invention. Ranges of values are disclosed herein. The ranges set out a lower limit value (“LLV”) and an upper limit value (“ULV”). Unless specified otherwise, the LLV, ULV, and all values between the LLV and ULV are part of the range. The present disclosure describes finishing mediums for removing support material and for surface finishing of AMT Objects. The finishing medium suspends media particles (forming a finishing suspension) and allows the media particles to flow through a machine that applies (e.g., sprays) the finishing suspension to the AMT Object being finished. The primary means for finishing using a finishing suspension that is in keeping with the invention is mechanical and chemical in nature. That mechanical aspect may be achieved by media particles impacting the outer surface of the AMT Object and the chemical aspect may be achieved through dissolution of dissolvable portions of the AMT Object. As used herein, unless otherwise indicated, the term “support material” refers to material that is operatively arranged to support portions of an AMT Object during an additive manufacturing process, but which is undesirable once the manufacturing process is complete. Support material can comprise the same material as the object that is being manufactured, or can be made of a different material. Materials that can be removed during finishing include, but are not limited to, materials used during Polyjet 3D-printing (e.g., SUP705, SUP706, SUP707, SUP708, and the like, and combinations thereof), fused deposition modeling (FDM) 3D-printing (e.g., SR20, SR30, SRT00, SRi 10, and the like, and combinations thereof), selective laser sintering (SLS) 3D-printing (e.g., nylon, polystyrene, steel, titanium, and the like), stereolithography (SLA) 3D-printing (e.g., photopolymers, light-activated resin, and the like), multi-jet fusion (MJF) 3D-printing (e.g., PA 12 (polyamide) and the like), DMLS, and binder jetting 3D-printing (e.g., steel, aluminum, titanium, copper, and the like, and combinations thereof), acrylonitrile butadiene styrene (ABS), and/or PLA (polylactic acid). A finishing medium that is in keeping with the invention may be used to carry particles for removing support material and/or removing a portion of the object being finished. The combination of a finishing medium and media particles is referred to herein as a finishing suspension. The finishing medium may comprise a polyol, an anti-corrosion agent, a hydrotrope, and water. Such a finishing medium may have a pH of 4 to 14 (e.g., 7 to 14 or 8). The finishing medium may comprise:(a) 1-20% by weight a polyol, including all 0.01% values and ranges therebetween;(b) optionally, 1-20% by weight an anti-corrosion agent, including all 0.01% values and ranges therebetween;(c) 0.001-10% by weight a hydrotrope, including all 0.0001% values and ranges therebetween; and(d) the remainder may be water. Such a finishing medium may have a pH of 4 to 14 (e.g., 7 to 14 or 8). A polyol, which may serve as a lubricating agent in a finishing medium that is in keeping with the invention, may aid in coating the object being finished. Such a coating may aid in dissolving undesired support material. Examples of polyols (e.g., glycols and glycol ethers) suitable for a finishing medium that is in keeping with the invention include, but are not limited to, propylene glycol, ethylene glycol, glycerol, methoxytriglycol, ethoxytriglycol, butoxytriglycol, diethylene glycol n-butyl 30 ether acetate, diethylene glycol monobutyl ether, ethylene glycol n-butyl ether acetate, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, ethylene glycol phenyl ether, diethylene glycol monohexyl ether, ethylene glycol monohexyl ether, diethylene glycol monomethyl ether, ethylene glycol monopropyl ether, di(propylene glycol) methyl ether, dipropylene glycol methyl ether acetate, dipropylene glycol n-butyl ether, propylene glycol diacetate, propylene glycol methyl ether, propylene glycol monomethyl ether acetate, propylene glycol n-butyl ether, propylene glycol phenyl ether, tripropylene glycol methyl ether, tripropylene glycol n-butyl ether, dipropylene glycol dimethyl ether, 2-butoxyethanol, and the like, and combinations thereof. For example, a finishing medium that is in keeping with the invention may be 1-50% by weight a polyol, including all 0.01% values and ranges therebetween, relative to the total weight of the finishing medium. In an example, a finishing medium that is in keeping with the invention may be 1-20% by weight a polyol, 1-10% by weight a polyol, 10-20% by weight a polyol, or 1-15% by weight a polyol. An anti-corrosion agent in a finishing medium that is in keeping with the invention may be included in order to keep metal components of the finishing machine and build plates from corroding. Anti-corrosion agents may be, for example, organooxygen compounds, organoamine compounds, organosulfur compounds, organophosphorus compounds, or a combination thereof. Examples of anti-corrosion agents suitable for a finishing medium that is in keeping with the invention include, but are not limited to, ethanolamine, diethanolamine, zinc dialkyldithiophosphate, benzotriazole, dibutylamine, combinations thereof, and the like. For example, a finishing medium that is in keeping with the invention may be 1-20% by weight an anti-corrosion agent, including all 0.01% values and ranges therebetween, relative to the total weight of the finishing medium. A hydrotrope may be included in a finishing medium that is in keeping with the invention in order to aid in solubilizing organic materials (e.g., the organic components of a finishing medium and resins from the object) in water. Examples of hydrotropes suitable for a finishing medium that is in keeping with the invention include, but are not limited to, SXS, xylene sulfonic acid, calcium xylene sulfonate, potassium xylene sulfonate, cumene sulfonic acid, sodium cumene sulfonate, toluene sulfonic acid, sodium toluene sulfonate, and combinations thereof. Such hydrotropes can be added to the finishing medium as a solid or can be added to the finishing medium as an aqueous solution comprising 1-50% hydrotrope by weight, including all 1% values and ranges therebetween. Such hydrotropes can be a mixture of isomers (e.g., stereoisomers, constitutional isomers, and the like). For example, a finishing medium that is in keeping with the invention may be 0.001-10% by weight a hydrotrope, including all 0.0001% values and ranges therebetween, relative to the total weight of the finishing medium. A finishing medium that is in keeping with the invention may be combined with one or more media particles. Herein, the combination of a finishing medium containing media particles is referred to as a finishing suspension. Media particles may aid in providing a mechanical force to remove support material and/or a portion of the object being finished. Examples of media particles include, but are not limited to, glass beads, steel (e.g., stainless steel) particles, ceramic materials/particles, aluminum oxide particles (e.g., alumina particles), silicon oxide particles (e.g., silica particles), zirconium oxide particles (e.g., zirconia particles), zirconium particles, silicon carbide particles, plastic particles, garnet particles, copper particles, corn cob particles, walnut shells, mica particles, feldspar particles, pumice particles, and the like. A finishing medium may be combined with a single type of media particles (e.g., solely alumina, stainless steel particles, ceramic particles (e.g., silica-based particles, silicon-based particles, and the like), or the like). Alternatively, a finishing suspension may have two or more types of media particles (e.g., alumina, stainless steel particles, ceramic particles (e.g., silica-based particles, silicon-based particles, and the like), and the like, and combinations thereof) and/or media particles of different sizes (e.g., alumina and stainless steel particles, where the alumina particles all have the same average size and the stainless steel particles have a different average size, or alumina where the alumina particles are various sizes). Such media particles can have a longest linear dimension (e.g., a diameter or radius) of 0.1-1000 μm, including all 0.1 μm values and ranges therebetween (e.g., 1-700 μm or 1-500 μm). The media particles may be of various shapes, such as, for example, round, oblong, irregular, jagged, angular, cubic, rectangular, and cylindrical. Media particles of one shape may be used with media particles of a different shape (e.g., round particles may be used with jagged particles). Media particles may be used to abrade and/or polish the AMT Object. The abrasion and/or polishing effect may be dependent on the shape and size of the media particles and the application pressure. For example, 1 to 100 pounds of media particles, including all 0.1 pound values and ranges therebetween, can be added for every 25 to 30 gallons of finishing medium, including every 0.1 gallon value and range therebetween. Thus, the ratio of pounds of media particles to gallons of finishing medium may be 1:30 to 4:1, including all integer ratio values and ranges therebetween. In various other examples, a finishing suspension comprises 1 to 2 pounds of media particles per gallon of finishing medium, including every 0.01 pound value and range therebetween. In a particular example of the invention, the finishing medium can comprise:(a) 1-20% by weight a propylene glycol, including all 0.010% values and ranges therebetween;(b) 1-20% by weight triethanolamine, including all 0.01% values and ranges therebetween;(c) 0.001-10% by weight SXS, including all 0.0001% values and ranges therebetween; and(d) the remainder is water. Such a finishing medium may have a pH of 4 to 14, including all 0.01 pH values and ranges therebetween (e.g., a pH of 7-14 or 4-9 or 8). In a particular example, a finishing suspension comprises a finishing medium comprising:(a) 10% by weight a propylene glycol;(b) 10% by weight triethanolamine;(c) 4% by weight a 40% by weight aqueous solution of SXS; and(d) the remainder is water. Such a finishing medium may have a pH of about 8. For example, the finishing suspension can comprise a finishing medium comprising:(a) 1-20% by weight a propylene glycol, including all 0.01% values and ranges therebetween;(b) 1-20% by weight triethanolamine, including all 0.01% values and ranges therebetween;(c) 0.001-10% by weight sodium xylene sulfonate, including all 0.0001% values and ranges therebetween; and(d) the remainder may be water, and media particles chosen from the group consisting of alumina particles, stainless steel particles, ceramic particles (e.g., silica-based particles, silicon-based particles, and the like), and combinations thereof. There may be 1 to 2 pounds of media particles for every gallon of finishing medium. Such a finishing medium/suspension may have a pH of 7 to 14, including all 0.01 pH values and ranges therebetween. In a particular example, the finishing suspension can comprise a finishing medium comprising:(a) 10% by weight a propylene glycol;(b) 10% by weight triethanolamine, including all 0.01% values and ranges therebetween;(c) 4% by weight a 40% by weight aqueous solution of SXS; and(d) the remainder is water, and media particles selected from the group consisting of alumina particles, stainless steel particles, ceramic particles, and combinations thereof. Such a finishing medium/suspension may have a pH of about 8 (e.g., a pH of 8±0.5). A finishing medium that is in keeping with the invention may help reduce media attrition. Media attrition occurs when the media particles inside the machine applying the finishing suspension break down (e.g., mechanically fracture) into a form that is less effective or ineffective. The media particles are less effective or ineffective when the particles are too small to carry enough momentum to impart an effective force on the AMT Object for purposes of removing material from the object. Particle size changes because large particles may fracture when impacting the object and/or as a result of being pumped. With time, the average size of the particles will become smaller and thus decrease in mass, and, as such, each particle becomes less effective at removing material from the object as they become smaller. The invention may be embodied as a method of using a finishing medium/suspension. The steps of such a method may be sufficient to remove undesirable material (e.g., support material, undesirable print material, undesirable metal, and the like) from an AMT Object. The method may comprise:(a) providing (301inFIG.3) a finishing medium/suspension that is in keeping with the invention; and(b) applying (303inFIG.3) (e.g., spraying) the finishing medium/suspension to an AMT Object, such that a portion of the object is removed. Applying the finishing medium to an AMT Object may involve spraying and/or otherwise coating the object, such that the finishing medium/suspension is applied to the object so that mechanical force is exerted on the object. To increase the speed and pressure of the finishing medium/suspension leaving the nozzle, air may be simultaneously forced through the same nozzle as the finishing medium/suspension to increase the velocity of the finishing medium/suspension leaving the nozzle. An AMT Object may be sprayed with the finishing medium/suspension at a pressure up to 60 psi (e.g., 35 psi), inclusive. Along with the finishing medium/suspension, air may be forced into the conduit carrying the finishing medium/suspension. A finishing medium or finishing suspension that is in keeping with the invention may be heated (302inFIG.3) prior to or during application (e.g., spraying). The finishing medium or finishing suspension may have a temperature of 50-140° F., (e.g., 70-100° F.), including all 0.01° F. values and ranges therebetween. Following application of the finishing medium/suspension, the object may be rinsed with, for example, water (304inFIG.3) in order to remove the finishing medium or suspension and the AMT Object may be dried. A finishing medium or finishing suspension that is in keeping with the invention may be applied by a machine (e.g., the PostProcess™ DECI DUO finishing unit). Such a machine may be the same or similar to the apparatuses disclosed in U.S. patent application Ser. No. 16/209,778. Such a machine may pump the finishing medium or finishing suspension through a nozzle that is directed at the object. The finishing medium or finishing suspension may be forced out of the nozzle at a high velocity. During such application (e.g., spraying), the object may be sprayed with a finishing medium or a finishing suspension (e.g., containing alumina particles, stainless steel particles, ceramic particles, or the like). The finishing medium (and media particles) impacts the outer surface of the object. The force of impact on the outer surface of the object assists in removing undesirable material (e.g., undesirable resin, undesirable print material, undesirable metal powder, undesirable support material, and the like). During application of the finishing medium or finishing suspension, the AMT Object may be rotated (e.g., rotated via a turntable) so that the AMT Object is thoroughly coated/impacted with the finishing medium and/or media particles. Generally, an initial finishing step can utilize a finishing suspension having larger media particles (e.g., larger media particles, such as, for example, aluminum oxide, silicon carbide, stainless steel, titanium) and then polished with a finishing suspension having smaller suspended particles (e.g., ceramic media particles, such as, for example, ZIRBLAST® and MICROBLAST© blasting media). In an example, larger and/or jagged particles may be used for abrading and spherical particles may be used for polishing. Roughness average (“Ra”) can be used to determine when an object is finished. A lower Ra indicates a smooth object, whereas a higher Ra indicates a rougher object. An object may be finished until the desired Ra is achieved. Ra is often measured in micro-inches. A lower roughness average indicates a smoother object. The following Statements describe non-limiting examples in keeping with the present invention.Statement 1. A finishing medium comprising: 1-20% by weight a polyol; optionally, 1-20% by weight an anti-corrosion agent; 0.001-10% by weight a hydrotrope; and water (e.g., the remainder of the finishing medium may be water).Statement 2. The finishing medium according to Statement 1, where the polyol is chosen from ethylene glycol, propylene glycol, glycerol, methoxytriglycol, ethoxytriglycol, butoxytriglycol, diethylene glycol n-butyl 30 ether acetate, diethylene glycol monobutyl ether, ethylene glycol n-butyl ether acetate, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, ethylene glycol phenyl ether, diethylene glycol monohexyl ether, ethylene glycol monohexyl ether, diethylene glycol monomethyl ether, ethylene glycol monopropyl ether, di(propylene glycol) methyl ether, dipropylene glycol methyl ether acetate, dipropylene glycol n-butyl ether, propylene glycol diacetate, propylene glycol methyl ether, propylene glycol monomethyl ether acetate, propylene glycol n-butyl ether, propylene glycol phenyl ether, tripropylene glycol methyl ether, tripropylene glycol n-butyl ether, dipropylene glycol dimethyl ether, 2-butoxyethanol, and combinations thereof.Statement 3. The finishing medium according to Statement 1 or Statement 2, where the anti-corrosion agent is chosen from one or more organooxygen compound, one or more organoamine compound, one or more organosulfur compound, one or more organosulfur compound, one or more organophosphorus compound, and combinations thereof, where non-limiting examples include ethanolamine, diethanolamine, zinc dialkyldithiophosphate, benzotriazole, dibutylamine, and combinations thereof.Statement 4. The finishing medium according to any one of the preceding Statements, where the hydrotrope is chosen from sodium xylene sulfonate, xylene sulfonic acid, calcium xylene sulfonate, potassium xylene sulfonate, cumene sulfonic acid, sodium cumene sulfonate, toluene sulfonic acid, sodium toluene sulfonate, and combinations thereof.Statement 5. The finishing medium according to Statement 4, where the hydrotrope is a mixture of hydrotrope isomers.Statement 6. The finishing medium according to Statement 4 or Statement 5, where the hydrotrope is provided via an aqueous solution comprising 1-50% by weight a hydrotrope.Statement 7. The finishing medium according to any one of the preceding Statements, where the finishing medium has a pH of 4 to 14 (e.g., 7 to 14 or 4 to 9 or 8).Statement 8. The finishing medium according to any one of the preceding Statements, where the polyol is propylene glycol; the anti-corrosion agent is triethanolamine; and the hydrotrope is an aqueous solution comprising 40% by weight sodium xylene sulfonate.Statement 9. The finishing medium according to any one of the preceding Statements, comprising: 10% by weight propylene glycol; 10% by weight triethanolamine; 4% by weight the aqueous solution having 40% by weight sodium xylene sulfonate; and 76% by weight water, where the finishing medium has pH of 4 to 14 (e.g., 7 to 14 or 4 to 9 or 8).Statement 10. A finishing suspension comprising media particles and a finishing medium according to any one of the preceding Statements.Statement 11. The finishing suspension according to Statement 10, where the media particles are chosen from glass beads, steel (e.g., stainless steel), ceramic materials, aluminum oxide/alumina, silica, zirconium, silicon carbide, plastic, garnet, copper, corn cob, walnut shells, mica, feldspar, pumice, and combinations thereof, where the media particles may be of various shapes, such as, for example, round, oblong, irregular, jagged, angular, cubic, rectangular, and cylindrical.Statement 12. The finishing suspension according to Statement 10 or Statement 11, where the media particles have a longest linear dimension of 0.1-1000 μm (e.g., 1-700 μm or 1-500 μm).Statement 13. A method of finishing an AMT Object comprising applying a finishing suspension according to any one of Statements 10-12 to an AMT Object such that a portion of the AMT Object is removed.Statement 14. The method according to Statement 13, where support material or a portion thereof is removed.Statement 15. The method according to Statement 13, where applying the finishing suspension comprises spraying the finishing suspension on the AMT Object.Statement 16. The method according to Statement 15, where the finishing suspension is sprayed at a pressure of 0-60 psi.Statement 17. The method according to any one of Statements 13-16, where the finishing suspension is at a temperature of 50-140° F.Statement 18. The method according to any one of Statements 13-17, where the object is made by fused deposition modeling, selective laser sintering, stereolithography, multi-jet fusion, direct metal laser sinter/binder jetting methods, or a combination thereof. The following example is presented to illustrate an embodiment of the invention. It is not intended to limit the scope of the invention. Example 1 This example describes a method of using a finishing medium/suspension that is in keeping with the invention. The AMT Object inFIG.1AandFIG.1Bwas printed by a DMLS process known in the art. The object, which was made from Ti6Al4V, was finished using a finishing medium having:(a) 10% by weight propylene glycol;(b) 10% by weight triethanolamine;(c) 4% by weight a 40% by weight aqueous solution of sodium xylene sulfonate; and(d) the remainder was water. Such a finishing medium had a pH of about 8. The finishing medium was combined with 36 grit (450 μm) aluminum oxide media particles. The resulting finishing suspension was initially heated to a temperature of about 80° F. and the AMT Object was abraded by spraying the finishing suspension at the object at a pressure of 35 psi. Air was supplied to the finishing suspension through an orifice located prior to the nozzle. Upstream of the orifice, the air pressure was 70 psi. The finishing suspension was applied (e.g., sprayed) to the object for 110 minutes while the object was on a turntable rotating at 10 rotations per minute (rpm). The object was then polished using the finishing medium combined with ZIRBLAST® (a combination of zirconia, silica, and alumina, the majority being zirconia), having a size of 300 grit (34 m). The finishing suspension was applied to (e.g., sprayed) the object for 40 minutes while the object was on a turntable rotating at 10 rpm. After measuring the Ra values at many locations on the unfinished AMT Object, it was determined that the high/low Ra values prior to finishing were 649/787. Following finishing, the high/low Ra values were determined to be 58/98. It will be appreciated that various aspects of the invention and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Various alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art, and these are also intended to be encompassed by the invention. Although embodiments of the invention have been described herein, the invention is not limited to such embodiments. Hence, the present invention is deemed only limited by the appended claims and the reasonable interpretation thereof. | 24,061 |
11859109 | DETAILED DESCRIPTION Disclosed is a polyurethane adhesive composition which includes (a) one or more urethane prepolymers having isocyanate moieties; (b) a catalytic amount of one or more catalysts; (c) one or more fillers; (d) one or more silane adhesion promoters; and (e) one or more monofunctional polyalkylene glycols. The term “one or more” as used herein shall be understood to mean that at least one, or more than one, of the recited components may be used. The one or more urethane prepolymers having isocyanate moieties component (a) of the polyurethane adhesive composition according to the present invention includes any conventional prepolymer used in polyurethane adhesive compositions. The urethane prepolymers for use in preparing the composition of the invention include any prepolymer having an average isocyanate functionality of at least 2.0 and a weight average molecular weight of at least 2,000. In one embodiment, the average isocyanate functionality of the prepolymer is at least 2.2, or at least 2.4. In one embodiment, the average isocyanate functionality is no greater than 4.0, or no greater than 3.5 or no greater than 3.0. In one embodiment, the weight average molecular weight of the prepolymer is at least 2,500 or at least 3,000, and no greater than 40,000, or no greater than 20,000, or no greater than 15,000 or no greater than 10,000. In general, the urethane prepolymer may be prepared by any suitable method, such as reacting one or more isocyanate compounds comprising a polyisocyanate with one or more isocyanate-reactive compounds. In one embodiment, the urethane prepolymer is obtained by reacting an isocyanate-reactive compound containing at least two isocyanate-reactive, active hydrogen containing groups with an excess stoichiometric amount of a polyisocyanate under reaction conditions sufficient to form the corresponding prepolymer. In one embodiment, the polyisocyanates have an average isocyanate functionality of at least 2.0 and an equivalent weight of at least 80. In one embodiment, the isocyanate functionality of the polyisocyanate is at least 2.0, or at least 2.2, or at least 2.4; and is no greater than 4.0, or no greater than 3.5, or no greater than 3.0. As one skilled in the art will understand, higher functionality may also be used, but may cause excessive cross-linking, and result in an adhesive which is too viscous to handle and apply, and can cause the cured adhesive to be too brittle. In one embodiment, the equivalent weight of the polyisocyanate is at least 80, or at least 110, or at least 120; and is no greater than 300, or no greater than 250, or no greater than 200. Suitable polyisocyanates include, for example, aromatic polyisocyanates, aliphatic polyisocyanates, cycloaliphatic polyisocyanates, araliphatic polyisocyanates, heterocyclic polyisocyanates, and mixtures thereof. Suitable aromatic polyisocyanates include, for example, m- and p-phenylene diisocyanate; toluene-2,4- and 2,6-diisocyanate (TDI); naphthylene-1,5-diisocyanate; methoxyphenyl-2,4-diisocyanate; diphenylmethane-4,4′, 2,4′-, and 2,2′-diisocyanate (MDI); 4,4′-biphenylene diisocyanate; 3,3′-dimethoxy-4,4′-biphenyl diisocyanate; 3,3′-dimethyl-4,4′-biphenyl diisocyanate; 3,3′-dimethyldiphenyl methane-4,4′-diisocyanate; 1,3-bis(isocyanatomethyl)benzene (xylylene diisocyante XDI); 4,4′,4″-triphenyl methane triisocyanate; polymethylene polyphenylisocyanate (PMDI); toluene-2,4,6-triisocyanate; 4,4′-dimethyldiphenylmethane-2,2′,5,5′-tetraisocyanate; diphenyletherdiisocyanate; 2,4,4′-triisocyanatodiphenylether; chlorophenylene-2,4-diisocyanate; blends thereof and polymeric and monomeric blends thereof. Suitable aliphatic polyisocyanates and cycloaliphatic polyisocyanates include, for example, cyclohexane diisocyanate; 1,3- and 1,4-bis(isocyanatomethyl)cyclohexane; 1-methyl-cyclohexane-2,4-diisocyanate; 1-methyl-cyclohexane-2,6-diisocyanate; methylene dicyclohexane diisocyanate; isophorone diisocyanate; ethylene diisocyanate; 1,6-hexamethylene diisocyanate; blends thereof and polymeric and monomeric blends thereof. Modified aromatic polyisocyanates that contain urethane, urea, biuret, carbodiimide, uretoneimine, allophonate or other groups formed by reaction of isocyanate groups are also useful. The aromatic polyisocyanates may be MDI or PMDI (or a mixture thereof that is commonly referred to as “polymeric MDI”), and so-called “liquid MDI” products that are mixtures of MDI and MDI derivatives that have biuret, carbodiimide, uretoneimine and/or allophonate linkages. In one embodiment, at least some of the polyisocyanates present in the isocyanate compounds may be aromatic polyisocyanates. If a mixture of aromatic and aliphatic polyisocyanates are present, 50% or more by number, or 75% or more by number, are aromatic polyisocyanates. In one embodiment, 80 to 98% by number of the polyisocyanates may be aromatic, and 2 to 20% by number may be aliphatic. All of the polyisocyanates used in forming the urethane prepolymers may be aromatic. In a further embodiment, the isocyanate compound includes MDI, e.g., 40 to 99 wt % of the 4,4′-isomer of MDI. Suitable isocyanate-reactive compounds include, for example, any organic compound having at least two isocyanate-reactive moieties, such as a compound containing an active hydrogen moiety, or an imino-functional compound. As used herein, an active hydrogen moiety refers to a moiety containing a hydrogen atom which, because of its position in the molecule, displays significant activity according to the Zerewitnoff test described by Wohler in the Journal of the American Chemical Society, Vol. 49, p. 3181 (1927). Representative examples of active hydrogen moieties include —COOH, —OH, —NH2, —NH—, —CONH2, —SH, and —CONH—. Suitable active hydrogen containing compounds include, for example, polyols, polyamines, polymercaptans and polyacids. Suitable imino-functional compounds include, for example, those which have at least one terminal imino group per molecule, such as those described in, for example, U.S. Pat. No. 4,910,279. In one embodiment, the isocyanate-reactive compound is a polyol. Suitable polyols include, for example, polyether polyols, polyester polyols, poly(alkylene carbonate)polyols, hydroxyl containing polythioethers, polymer polyols (dispersions of vinyl polymers in such polyols, commonly referred to as copolymer polyols) and mixtures thereof In one embodiment, the polyols are polyether polyols containing one or more alkylene oxide units in the backbone of the polyol. Suitable alkylene oxide units include, for example, ethylene oxide, propylene oxide, butylene oxide and mixtures thereof. The alkylene oxides can contain straight or branched chain alkylene units. In one embodiment, the polyols contain propylene oxide units, ethylene oxide units or a mixture thereof. In the embodiment where a mixture of alkylene oxide units is contained in a polyol, the different units can be randomly arranged or can be arranged in blocks of each alkylene oxide. In one embodiment, the polyol includes propylene oxide chains with ethylene oxide chains capping the polyol. In another embodiment, the polyols are a mixture of diols and triols. In one embodiment, the isocyanate-reactive compound can have a functionality of at least 1.5, or at least 1.8, or at least 2.0; and is no greater than 4.0, or no greater than 3.5, or no greater than 3.0. In one embodiment, the equivalent weight of the isocyanate-reactive compound is at least 200, or at least 500, or at least 1,000; and is no greater than 5,000, or no greater than 3,000, or no greater than 2,500. The urethane prepolymers will have a viscosity sufficient to allow the use of the prepolymers in adhesive formulations. In one embodiment, the prepolymers will have a viscosity of 6,000 centipoise (600 N-S/m2) or greater, or 8,000 centipoise (800 N-S/m2) or greater. In one embodiment, the prepolymers will have a viscosity of 30,000 centipoise (3,000 N-S/m2) or less, or 20,000 centipoise (2,000 N-S/m2) or less. As one skilled in the art will understand, above 30,000 centipoise (3,000 N-S/m2), the polyurethane compositions become too viscous to pump and therefore cannot be applied using conventional techniques. In addition, below 6,000 centipoise (600 N-S/m2), the prepolymers do not afford sufficient integrity to allow the compositions comprising the urethane prepolymers to be utilized in desired applications. As used herein, “viscosity” is measured by the Brookfield Viscometer, Model DV-E with a RV spindle #5 at a speed of 5 revolutions per second and at a temperature of 25° C. In general, the amount of isocyanate compounds used to prepare the urethane prepolymer is an amount that provides the desired properties, i.e., the appropriate free isocyanate content and viscosities as discussed above. In one embodiment, the amount of the isocyanate compound used to prepare the urethane prepolymer is an amount of 6.5 wt. % or greater, or 7.0 wt. % or greater or 7.5 wt. % or greater, based on the weight of the urethane prepolymer. In one embodiment, the amount of the isocyanate compound used to prepare the urethane prepolymer is an amount of 12 wt. % or less, or 10.5 wt. % or less or 10 wt. % or less, based on the weight of the urethane prepolymer. The amount of the isocyanate-reactive compound is an amount sufficient to react with most of the isocyanate groups of the isocyanate compound leaving enough isocyanate groups to give the desired free isocyanate content of the urethane prepolymer. In one embodiment, the isocyanate-reactive compound is present in an amount of 30 wt. % or greater, or 35 wt. % or greater or 40 wt. % or greater, based on the weight of the urethane prepolymer. In one embodiment, the isocyanate-reactive compound is present in an amount of 75 wt. % or less, or 65 wt. % or less or 60 wt. % or less, based on the weight of the urethane prepolymer. The urethane prepolymer may be prepared by any suitable method, such as bulk polymerization and solution polymerization. The reaction to prepare the prepolymer can be carried out under anhydrous conditions, or under an inert atmosphere such as a nitrogen blanket and to prevent crosslinking of the isocyanate groups by atmospheric moisture. The reaction can be carried out at a temperature between 0° C. and 150° C., or between 25° C. and until the residual isocyanate content determined by titration of a sample is very close to the desired theoretical value. In one embodiment, the isocyanate content in the prepolymers can be 0.1 wt. % or greater, or 1.5 wt. % or greater or 1.8 wt. % or greater. In one embodiment, the isocyanate content in the prepolymers can be 10 wt. % or less, or 5 wt. % or less or 3 wt. % or less. The term “isocyanate content” as used herein means the weight percentage of isocyanate moieties to the total weight of the prepolymer. The reactions to prepare the urethane prepolymer may be carried out in the presence of urethane catalysts. Suitable urethane catalysts include, for example, the stannous salts of carboxylic acids, such as stannous octoate, stannous oleate, stannous acetate, and stannous laurate; dialkyltin dicarboxylates, such as dibutyltin dilaurate and dibutyltin diacetate which are known in the art as urethane catalysts; as are tertiary amines and tin mercaptides. The amount of catalyst employed is generally between 0.005 and 5 wt. % of the mixture catalyzed, depending on the nature of the isocyanate. In general, the urethane prepolymer is present in the polyurethane adhesive composition according to the present invention in a sufficient amount such that the adhesive is capable of bonding substrates together. In one embodiment, the urethane prepolymer is present in an amount of 20 wt. % or greater, or 30 wt. % or greater, or 40 wt. % or greater, or wt. % or greater, based on the weight of the polyurethane adhesive composition. In one embodiment, the urethane prepolymer is present in an amount of 85 wt. % or less, or 80 wt. % or less, or 75 wt. % or less, or 70 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the urethane prepolymer is present in an amount of 30 wt. % to 65 wt. %, based on the weight of the polyurethane adhesive composition. In one embodiment, the prepolymer is present in an amount of 55 wt. % to 62 wt. %, based on the weight of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention further includes one or more catalysts which catalyze the reaction of isocyanate moieties with water or an active hydrogen containing compound. The catalyst can be any catalyst known to the skilled artisan for the reaction of isocyanate moieties with water or active hydrogen containing compounds. In one embodiment, one or more catalysts containing one or more tertiary amine groups, organotin catalysts, metal alkanoates catalysts, and mixtures thereof may be used. Suitable one or more catalysts containing one or more tertiary amine groups include, for example, dimorpholinodialkyl ethers, di((dialkylmorpholino)alkyl)ethers, substituted morpholine compounds, N-dialkyl amino alkyl ethers and alkyl substituted polyalkylene polyamines. In one embodiment, suitable one or more catalysts include, for example, bis-(2-dimethylaminoethyl)ether; triethylene diamine; pentamethyldiethylene triamine; N,N-dimethylcyclohexylamine; N,N-dimethyl piperazine 4-methoxyethyl morpholine; N-methylmorpholine; N-ethyl morpholine and mixtures thereof. In one embodiment, a class of catalyst is dimorpholino dialkyl ethers wherein the morpholine groups may be substituted with groups which do not interfere in the catalytic affect of the catalyst. Suitable dimorpholinodialkyl ether includes, for example, dimorpholinodiethyl ether. In one embodiment, the one or more catalysts containing one or more tertiary amine groups are present in an amount of 0.01 wt. % or greater, or 0.03 wt. % or greater, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more catalysts containing one or more tertiary amine groups are present in an amount of 2.0 wt. % or less, or 1.75 wt. % or less, or 1.0 wt. % or less, or 0.5 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more catalysts containing one or more tertiary amine groups are present in an amount of 0 wt. % to 1 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more catalysts containing one or more tertiary amine groups are present in an amount of 0.03 wt. % to 0.5 wt. % or less, based on the weight of the polyurethane adhesive composition. Suitable one or more organotin catalysts include, for example, alkyl tin oxides, stannous alkanoates, dialkyl tin carboxylates and tin mercaptides. Suitable stannous alkanoates include, for example, stannous octoate. Suitable alkyl tin oxides include, for example, dialkyl tin oxides, such as dibutyl tin oxide and its derivatives. In one embodiment, an organotin catalyst is a dialkyltin dicarboxylate or a dialkyltin dimercaptide. Suitable dialkyl dicarboxylates include, for example, 1,1-dimethyltin dilaurate; 1,1-dibutyltin diacetate and 1,1-dimethyl dimaleate. Suitable metal alkanoates include, for example, bismuth octoate and bismuth neodecanoate. In one embodiment, the organo tin compound or metal alkanoate is present in an amount of 60 parts per million or greater, or 120 parts by million or greater, based on the weight of the polyurethane adhesive composition. In one embodiment, the organo tin compound or metal alkanoate is present in an amount of 2.0 percent or less, or 1.5 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the organo tin compound or metal alkanoate is present in an amount of 0.1 wt. % to 1.6 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the organo tin compound or metal alkanoate is present in an amount of 0.6 wt. % to 1.3 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the catalytic amount of the one or more catalysts is an amount of 0.3 wt. % or greater, or 0.5 wt. % or greater, based on the weight of the polyurethane adhesive composition. In one embodiment, the catalytic amount of the one or more catalysts is an amount of 3.5 percent or less, or 3 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the catalytic amount of the one or more catalysts is an amount of 0.63 wt. % to 1.8 wt. %, based on the weight of the polyurethane adhesive composition. In one embodiment, the catalytic amount of the one or more catalysts is an amount of 0.1 wt. % to 2.6 wt. %, based on the weight of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention further includes one or more fillers and to improve the strength and rheology of the composition. Suitable fillers include, for example, an inorganic filler such as glass, silica, boron oxide, boron nitride, titanium oxide, titanium nitride, fly ash, calcium carbonate, various alumina-silicates including clays such as wollastonite and kaolin, metal particles such as iron, titanium, aluminum, copper, brass, bronze and the like; thermoset polymer particles such as polyurethane, cured particles of an epoxy, phenol-formaldehyde, or cresol-formaldehyde resin, crosslinked polystyrene and the like; thermo-plastics such as polystyrene, styrene-acrylonitrile copolymers, polyimide, polyamide-imide, polyether ketone, polyether-ether ketone, polyethyleneimine, poly(p-phenylene sulfide), polyoxymethylene, polycarbonate and the like; and various types of carbon such as activated carbon, graphite, carbon black and the like. When calcium carbonate is used as a filler, the calcium carbonate functions as a white pigment in the composition. Suitable calcium carbonates include, for example, any standard calcium carbonate. Suitable standard calcium carbonates are untreated, that is, they are not modified by treatment with other chemicals, such as organic acids or esters of organic acids. In one embodiment, the polyurethane adhesive composition according to the present invention includes calcium carbonate as the only white pigment. In general, the one or more calcium carbonates are present in a sufficient amount such that the desired adhesive properties of the polyurethane adhesive composition are achieved. In one embodiment, the one or more calcium carbonates are present in an amount of 5 wt. % or greater, or 8 wt. % or greater, or 12 wt. % or greater, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more calcium carbonates are present in an amount of 25 wt. % or less, or 20 wt. % or less, or 18 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more calcium carbonates are present in an amount of 8 wt. % to 20 wt. %, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more calcium carbonates are present in an amount of 12 wt. % to 18 wt. %, based on the weight of the polyurethane adhesive composition. When carbon black is used as a filler, the carbon black used may be a standard carbon black. Standard carbon black is carbon black which is not specifically surface treated or oxidized to render it nonconductive. Nonconductivity is generally understood to mean an impedance of the composition of at least 1010Ohm-cm. One or more nonconductive carbon blacks may be used in conjunction with the standard carbon black. The non-conductive carbon blacks may be high surface area carbon blacks, which exhibit an oil absorption of 110 cc/100 g or greater, or 115 cc/100 g or greater and/or an iodine number of 130 mg/g or greater, or 150 mg/g or greater. Suitable non-conductive carbon blacks include, for example, ELFTEX™ 57100 (available from Cabot), RAVEN™ 1040 and RAVEN™ 1060 carbon blacks (available from Birla Carbon). Suitable standard carbon blacks are well known in the art and include, for example, RAVEN™ 790, RAVEN™ 450, RAVEN™ 500, RAVEN™ 430, RAVEN™ 420 and RAVEN™ 410 carbon blacks (available from Birla Carbon) and CSX™ carbon blacks (available from Cabot), and PRINTEX™ carbon black (available from Degussa). In general, the one or more forms of carbon black are present in the polyurethane adhesive composition according to the present invention in a sufficient amount to reinforce the composition and to improve the rheology of the composition. In one embodiment, the one or more forms of carbon black are present in an amount such that the parts of the composition are nonconductive. In one embodiment, the one or more forms of carbon black are present in an amount of 10 wt. % or greater, or 14 wt. % or greater, or 18 wt. % or greater, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more forms of carbon black are present in an amount of 35 wt. % or less, or 30 wt. % or less, or 25 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more forms of carbon black are present in an amount of 15 wt. % to 23 wt. %, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more forms of carbon black are present in an amount of 19 wt. % to 23 wt. %, based on the weight of the polyurethane adhesive composition. In general, the one or more fillers are present in the polyurethane adhesive composition according to the present invention in a sufficient amount to reinforce the composition and to improve the rheology of the composition. In one embodiment, the one or more fillers may constitute 15 wt. % or greater, based on the total weight of the polyurethane adhesive composition. In one embodiment, the one or more fillers may constitute 20 wt. % or greater, based on the total weight of the polyurethane adhesive composition. In one embodiment, the one or more fillers may constitute 50 wt. % or less, based on the total weight of the polyurethane adhesive composition. In one embodiment, the one or more fillers may constitute 35 wt. % or less, based on the total weight of the polyurethane adhesive composition. In one embodiment, a polyurethane adhesive composition according to the present invention does not contain clay in any form as a filler. The polyurethane adhesive composition according to the present invention further includes one or more silane adhesion promoters in order to facilitate a durable bond between the polyurethane adhesive and, for example, a glass surface. In one embodiment, the one or more silane adhesion promoters are those which do not have a functional group which forms a salt with an acidic compound. In one embodiment, suitable one or more silane adhesion promoters include, for example, one or more alkoxysilanes. In one embodiment, suitable one or more alkoxysilanes are those react with isocyanate moieties. Suitable alkoxysilanes include, for example, mercaptosilanes, aminosilanes, isocyanato silanes, epoxy silanes, acrylic silanes and vinyl silanes. In one embodiment, suitable alkoxysilanes are trialkoxysilanes such as trimethoxy silanes. In one preferred embodiment, a class of alkoxysilanes is mercaptosilanes. “Mercaptosilanes” as used herein refer to any molecule having both a mercapto and a silane group which enhances the adhesion of polyurethane adhesive to a glass surface. Suitable mercaptosilanes include, for example, mercapto alkyl di- or tri-alkoxysilanes. In one embodiment, a suitable mercaptosilane can be of the general formula: wherein R is a hydrocarbylene group, R1is independently an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 20 carbon atoms, an aralkyl group having 7 to 20 carbon atoms or a triorganosiloxy group represented by (R2)3SiO—, wherein each of the R2groups independently represents a monovalent hydrocarbon group having 1 to 20 carbon atoms; X is independently a hydroxyl group or a hydrolyzable group; a is independently 0, 1 or 2; b is independently 0, 1, 2 or 3; and the sum of a and b is 3. The hydrolyzable group represented by X is not limited and can be any conventional hydrolyzable group. Suitable hydrolyzable groups include, for example, a hydrogen atom, a halogen atom, an alkoxy group, an acyloxy group, a ketoximate group, an amino group, an amido group, an acid amido group, an amino-oxy group, a mercaptosilane group, and an alkenyloxy group. In one embodiment, the one or more hydrolyzable groups include a hydrogen atom, an alkoxy group, an acyloxy group, a ketoximate group, an amino group, an amido group, an amino-oxy group, a mercaptosilane group, and an alkenyloxy group. In one embodiment, the one or more hydrolyzable groups are alkoxy groups such as, for example, a methoxy or ethoxy group, for ease in handling due to their mild hydrolyzability. Where two or more hydroxyl groups or hydrolyzable groups are present per reactive silicon group, they may be the same or different. In one embodiment, R1is an alkyl group, e.g., methyl or ethyl; a cycloalkyl group, e.g., cyclohexyl; an aryl group, e.g., phenyl; an aralkyl group, e.g., benzyl; or a triogansiloxy group of formula (R2)3Si— in which R2is methyl or phenyl. In another embodiment, R1and R2are a methyl group. In another embodiment, R is an arylene, alkarylene or an alkylene group such as a C2to C8alkylene group, or a C2to C4alkylene group or a C2to C3alkylene group. Representative examples of suitable one or more silane adhesion promoters include mercaptosilane propyl trimethoxysilane, mercaptosilane propyl methyl dimethoxysilane, bis-(trimethoxysilylpropyl)amine, isocyanato trimethoxysilane, N,N-bis[(3-triethoxysilyl)propyl]amine, N,N-bis[(3-tripropoxy-silyl)propyl]amine, N-(3-trimethoxysilyl)propyl-3-[N-(3-trimethoxysilyl)-propyl amino]prop ion-amide, N-(3-triethoxysilyl)propyl-3-[N-3-triethoxysilyl)-propyl-amino]propion amide, N-(3-trimethoxysilyl)propyl-3-[N-3-triethoxy silyl)-propylamino]propionamide, 3-trimeth-oxysilyl propyl 3-[N-(3-trimethoxysilyl)-propyl amino]-2-methyl propionate, 3-triethoxysilyl propyl 3-[N-(3-triethoxysilyl)-propylamino]-2-methyl propionate, and 3-trimethoxysilylpropyl 3-[N-(3-triethoxy silyl)-propylamino]-2-methyl propionate. In general, the one or more silane adhesion promoters are present in a sufficient amount to enhance the bonding of the isocyanate functional adhesive to the substrate, or glass or coated plastic surface. In one embodiment, the one or more silane adhesion promoters are present in an amount of 0.1 wt. % or more, or 0.4 wt. % or more, or 0.7 wt. % or more, or 1.0 wt. % or more, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more silane adhesion promoters are present in an amount of 5 wt. % or less, or 3 wt. % or less, or 2 wt. % or less, or 1.5 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more silane adhesion promoters are present in an amount of 0.7 wt. % to 3 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more silane adhesion promoters are present in an amount of 1.2 wt. % to 1.7 wt. % or less, based on the weight of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention further includes one or more monofunctional polyalkylene glycols. In general, the monofunctional polyalkylene glycol has a reactive hydrogen moiety on one end of its chain and an inert unreactive moiety on the other end. The reactive hydrogen moiety can be, for example, a primary or secondary amine or a hydroxyl moiety. In one embodiment, the reactive hydrogen moiety is a hydroxyl moiety. It is believed that when forming the polyurethane adhesive composition of the present invention, the reactive hydrogen moiety of the monofunctional polyalkylene glycols will react in situ with the isocyanate moieties of the urethane prepolymers, such that the residue of monofunctional polyalkylene glycols form the terminal groups of the urethane prepolymer, i.e., the urethane prepolymers are capped with the residue of the monofunctional polyalkylene glycols. In accordance with the present invention, within the polyurethane adhesive compositions, the one or more monofunctional polyalkylene glycols are incorporated in a sub-stoichiometric amount over the isocyanate moieties of the urethane prepolymers, such that the urethane prepolymers are partially capped with the residue of the monofunctional polyalkylene glycols. In one embodiment, about 0.1-50 mol %, or about 1-20 mol %, or about 2-10 mol % of the isocyanate moieties of the urethane prepolymers are reacted with the reactive hydrogen moieties of the one or more monofunctional polyalkylene glycols. The inert unreactive moiety can be any moiety which does not react with isocyanates such as, for example, a hydrocarbyloxy moiety. Suitable hydrocarbyloxy moieties include, for example, alkoxy, aryloxy, and alkylaryloxy. In one embodiment, the hydrocarbyloxy moiety is an alkoxy. Suitable alkoxy moieties include, for example, a C1to C20alkoxy, or a C1to C12alkoxy, or a C1to C6alkoxy or a C1to C4alkoxy. In one embodiment, a monofunctional polyalkylene glycol is a monofunctional hydroxyl substituted hydrocarbon initiated polyalkylene glycol which includes at one terminal end a hydrocarbyloxy moiety and at the other end a reactive hydrogen group. In between the terminal groups are a plurality of alkylene oxide moieties. Alternatively the compound can be referred to as a monofunctional hydrocarbyloxy polyalkyleneoxy glycol. In one embodiment, suitable alkylene oxide moieties can comprise ethylene oxide moieties, propylene oxide moieties, butylene oxide moieties or a mixture thereof. In one embodiment, the one or more monofunctional polyalkylene glycols are represented by general formula I and II: wherein R1and R3are independently a C1to C20hydrocarbyl group such an aliphatic or aromatic group with linear or branched structure and which may contain one or more unsaturated bonds, or hydrogen, with the proviso that one of R1and R3is hydrogen; each R2is independently hydrogen, methyl, or ethyl; and m is an integer of 1 to 20. In one embodiment, R1and R3are independently a C1to C12hydrocarbyl group, or a C1to C6hydrocarbyl group or a C1to C4hydrocarbyl group. In one embodiment, R1and R3are independently a C1to C20alkoxy, or a C1to C12alkoxy, or a C1to C6alkoxy or a C1to C4alkoxy. The one or more monofunctional polyalkylene glycols can be made by methods known in the art or are commercially available such as PAG-15 from The Dow Chemical Company. The one or more monofunctional polyalkylene glycols can have a number average molecular weight of 200 or greater, or 500 or greater. In one embodiment, the number average molecular weight of the monofunctional polyalkylene glycol is 2,000 or less, or from 1,000 or less. In general, the one or more monofunctional polyalkylene glycols are present in an amount of 0.1 wt. % or more, or 0.4 wt. % or more, or 0.8 wt. % or more, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more monofunctional polyalkylene glycols are present in an amount of 5 wt. % or less, or 3 wt. % or less, or 2 wt. % or less, or 1.5 wt. % or less, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more monofunctional polyalkylene glycols are present in an amount of 0.4 wt. % to 2 wt. %, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more monofunctional polyalkylene glycols are present in an amount of 0.6 wt. % to 1.3 wt. %, based on the weight of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention may further include one or more of the same or different dispersing aids, which wet the surface of the filler particles and help them disperse. The one or more dispersing aids may also have the effect of reducing viscosity. Suitable one or more dispersing aids include, for example, dispersing aids which are commercially available and sold by such sources as BYK Chemie under the BYK, DISPERBYK and ANTI-TERRA-U tradenames, such as alkylammonium salt of a low-molecular-weight polycarboxylic acid polymer and salts of unsaturated polyamine amides and low-molecular acidic polyesters, and fluorinated surfactants such as FC-4430, FC-4432 and FC-4434 from 3M Corporation. Such dispersing aids may constitute, for example, up to 2 wt. %, or up to 1 wt. %, of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention may further include one or more desiccants such as, for example, fumed silica, hydrophobically modified fumed silica, silica gel, aerogel, various zeolites and molecular sieves, and the like. One or more desiccants may constitute 1 wt. % or greater, or 5 wt. % or less, or 4 wt. % or less, based on the total weight of the polyurethane adhesive composition. In one embodiment, the polyurethane adhesive composition does not include a desiccant. The polyurethane adhesive composition according to the present invention may further include one or more plasticizers or solvents to modify rheological properties to a desired consistency. The one or more plasticizers or solvents should be free of water, inert to isocyanate groups and compatible with the prepolymer. The one or more plasticizers or solvents may be added to the reaction mixtures for preparing the prepolymer, or to the mixture for preparing the final adhesive composition. In one embodiment, the one or more plasticizers or solvents are added to the reaction mixtures for preparing the prepolymer and the adduct, so that such mixtures may be more easily mixed and handled. Suitable plasticizers and solvents are well known in the art and include, for example, straight and branched alkylphthalates, such as diisononyl phthalate, dioctyl phthalate and dibutyl phthalate, a partially hydrogenated terpene commercially available as “HB-40”, trioctyl phosphate, epoxy plasticizers, toluene-sulfamide, chloroparaffins, adipic acid esters, castor oil, xylene, 1-methyl-2-pyrrolidinone and toluene. The amount of plasticizer used is that amount sufficient to give the desired rheological properties and disperse the components in the composition of the invention. In one embodiment, the one or more plasticizers are present in an amount of 0 wt. % or greater, or 5 wt. % or greater, or 10 wt. % or greater, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more plasticizers are present in an amount of 35 wt. % or less, or 30 wt. % or less, or 25 wt. % or less, based on the weight of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention may further include one or more stabilizers, which function to protect the composition from moisture, thereby inhibiting advancement and preventing premature crosslinking of the isocyanates or silanol groups in the composition. Suitable one or more stabilizers include, for example, diethylmalonate, alkylphenol alkylates, paratoluene sulfonic isocyanates, benzoyl chloride, calcium oxide and orthoalkyl formates. In one embodiment, the one or more stabilizers are present in an amount of 0.1 wt. % or greater, or 0.5 wt. % or greater or wt. % or greater, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more stabilizers are present in an amount of 5.0 wt. % or less, or 2.0 wt. % or less, or 1.4 wt. % or less, based on the weight of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention may further include one or more curing agents. Suitable one or more curing agents include, for example, one or more chain extenders, crosslinking agents, polyols or polyamines. Polyols as described hereinabove can be utilized as curing agents. The one or more curing agents may include one or more low molecular weight compounds having two or more isocyanate reactive groups and a hydrocarbon backbone wherein the backbone may further include one or more heteroatoms. Suitable low molecular weight compounds may be compounds known in the art as chain extenders, difunctional compounds, or crosslinkers, having, on average, greater than two active hydrogen groups per compound. The heteroatoms in the backbone can be oxygen, sulfur, nitrogen or a mixture thereof. In one embodiment, the molecular weight of the low molecular weight compound is 250 or less, or 120 or less, or 100 or less. The low molecular weight compound includes one or more multifunctional alcohols, multifunctional alkanol amines, one or more adducts of multifunctional alcohol and an alkylene oxide, one or more adducts of a multifunctional alkanol amine and an alkylene oxide or a mixture thereof. Suitable multifunctional alcohols and multifunctional alkanol amines include, for example, ethane diol, propane diol, butane diol, hexane diol, heptane diol, octane diol, glycerine, trimethylol propane, pentaerythritol, neopentyl glycol, ethanol amines (diethanol amine, triethanol amine) and propanol amines (di-isopropanol amine, tri-isopropanol amine). In general, the one or more curing agents are used in a sufficient amount to obtain the desired G-Modulus (E-Modulus). In one embodiment, the one or more curing agents are present in an amount of 2 wt. % or greater, or 2.5 wt. % or greater, or 3.0 wt. % or greater, based on the weight of the polyurethane adhesive composition. In one embodiment, the one or more curing agents are present in an amount of 10 wt. % or less, or 8 wt. % or less, or 6 wt. % or less, based on the weight of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention may further include a polyoxyalkylene polyamine having 2 or more amines per polyamine. In one embodiment, the polyoxyalkylene polyamine can have 2 to 4 amines per polyamine or 2 to 3 amines per polyamine. In one embodiment, the polyoxyalkylene polyamine can have a weight average molecular weight of 200 or greater, or 400 or greater. In one embodiment, the polyoxyalkylene polyamine can have a weight average molecular weight of 5,000 or less or 3,000 or less. Suitable polyoxyalkylene polyamines include, for example, Jeffamine™ D-T-403 polypropylene oxide triamine having a molecular weight of 400 and Jeffamine™ D-400 polypropylene oxide diamine having a molecular weight of 400. In one embodiment, the polyoxyalkylene polyamines are present in an amount of 0.2 wt. % or greater, or 0.3 wt. % or greater, or 0.5 wt. % or greater, based on the total weight of the polyurethane adhesive composition. In one embodiment, the polyoxyalkylene polyamines are present in an amount of 6 wt. % or less, or 4 wt. % or less, or 2 wt. % or less, based on the total weight of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention may further include other durability stabilizers known in the art, including alkyl substituted phenols, phosphites, sebacates and cinnamates and preferably organophosphites. The durability stabilizers are present in a sufficient amount to enhance the durability of bond of the polyurethane adhesive composition to the substrate surface. Suitable phosphites include, for example, poly(dipropyleneglycol)phenyl phosphite (available from Dover Chemical Corporation under the trademark and designation DOVERPHOS 12), tetrakis isodecyl 4,4′isopropylidene diphosphite (available from Dover Chemical Corporation under the trademark and designation DOVERPHOS 675), and phenyl diisodecyl phosphite (available from Dover Chemical Corporation under the trademark and designation DOVERPHOS 7). In one embodiment, the one or more durability stabilizers are present in an amount of 0.1 wt. % or greater, or 0.2 wt. % or greater, based on the total weight of the polyurethane adhesive composition. In one embodiment, the one or more durability stabilizers are present in an amount of 1.0 wt. % or less, or 0.5 wt. % or less, based on the total weight of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention may further include one or more light stabilizers, which facilitates the system maintaining durable bond to the substrate for a significant portion of the life of the structure to which it is bonded. Suitable one or more light stabilizers include, for example, hindered amine light stabilizers, such as Tinuvin 1,2,3 bis-(1-octyloxy-2,2,6,6, tetramethyl-4-piperidinyl)sebacate and Tinuvin 765, bis(1,2,2,6,6,-pentamethyl-4-piperidinyOsebacate. In one embodiment, the one or more light stabilizers are present in an amount of 0.1 wt. % or greater, or 0.2 wt. % or greater, or 0.3 wt. % or greater, based on the total weight of the polyurethane adhesive composition. In one embodiment, the one or more light stabilizers are present in an amount of 3 wt. % or less, or 2 wt. % or less, or 1 wt. % or less, based on the total weight of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention may further include one or more ultraviolet (UV) light absorbers, which enhances the durability of the bond of the composition to a substrate. Suitable one or more ultraviolet light absorbers include, for example, benzophenones and benzotriazoles, such as Cyasorb UV-531 2-hydroxy-4-n-octoxybenzophenone and Tinuvin 571 2-(2H-benzotriazol-2-yl)-6-dodecyl-4-methylphenol, branched and linear. In one embodiment, the one or more UV light absorbers are present in an amount of 0.1 wt. % or greater, or 0.2 wt. % or greater, or 0.3 wt. % or greater, based on the total weight of the polyurethane adhesive composition. In one embodiment, the one or more UV light absorbers are present in an amount of 3 wt. % or less, or 2 wt. % or less, or 1 wt. % or less, based on the total weight of the polyurethane adhesive composition. The polyurethane adhesive composition according to the present invention can be formed by blending the components together by methods well known in the art. For example, the components can be blended in a suitable mixer. Such blending can be conducted, for example, in an inert atmosphere and in the absence of atmospheric moisture to prevent premature reaction. The mixing of the components can be done in any convenient way, depending on the particular application and available equipment. Mixing of the components can be done batchwise, mixing them by hand or by using various kinds of batch mixing devices, followed by application by brushing, pouring, applying a bead and/or in other suitable manner. In one embodiment, once the composition is formulated, it can be packaged in a suitable container such that it is protected from atmospheric moisture. Contact with atmospheric moisture could result in premature cross-linking of the urethane prepolymer utilized in the compositions of the invention. The polyurethane adhesive composition according to the present invention is used to bond porous and nonporous substrates together. For example, the polyurethane adhesive composition is applied to a first substrate and the polyurethane adhesive composition on the first substrate is then contacted with a second substrate. Thereafter, the polyurethane adhesive composition is exposed to curing conditions. In one embodiment, one substrate is glass or clear plastic coated with an abrasion resistant coating and the other substrate is a plastic, metal, fiberglass or composite substrate which may optionally be painted or coated. The plastic coated with an abrasion resistant coating can be any plastic which is clear, such as polycarbonate, acrylic, hydrogenated polystyrene or hydrogenated styrene conjugated diene block copolymers having greater than 50 percent styrene content. The coating can include any coating which is abrasion resistant such as a polysiloxane coating. In one embodiment, the coating has an ultraviolet pigmented light blocking additive. In one embodiment, the glass or coated plastic window has an opaque coating disposed in the region to be contacted with the adhesive to block UV light from reaching the adhesive. This is commonly referred to as a frit. In one embodiment, the opaque coating is an inorganic enamel or an organic coating. In one embodiment, the polyurethane adhesive composition according to the present invention can be applied to the surface of the glass or coated plastic, along the portion of the glass or coated plastic which is to be bonded to the structure. The polyurethane adhesive composition is thereafter contacted with the second substrate such that the polyurethane adhesive composition is disposed between the glass or coated plastic and the second substrate. The polyurethane adhesive composition is allowed to cure to form a durable bond between the glass or coated plastic and the substrate. Generally, the polyurethane adhesive composition according to the present invention can be applied at an ambient temperature in the presence of atmospheric moisture. Exposure to atmospheric moisture is sufficient to result in curing of the polyurethane adhesive composition. Curing may be further accelerated by applying heat to the curing composition by means of convection heat, or microwave heating. In another embodiment, the composition may be applied to the surface of the other substrate and then contacted with the glass or coated plastic as described. In one embodiment, the polyurethane adhesive composition according to the present invention can be applied to the surface in the absence of a pre-treatment step. In one embodiment, the polyurethane adhesive composition according to the present invention can be applied to fill gaps in structures and allowed to cure to seal about gaps in structures such as buildings or in vehicles. The polyurethane adhesive compositions can be applied as described hereinabove. In buildings, the polyurethane adhesive compositions can be used to seal gaps in structures. In vehicles, the polyurethane adhesive compositions can be utilized to seal gaps or seams between pans that may allow water to get in, for example, automobiles, buses, trucks, trailers, rail cars and specialty vehicles having such a gap or seal, such as about windows, door frames, trim, between body panels, and between door parts. Further handling may include, for example, transporting the assembly to a downstream work station, and further manufacturing steps which might include joining the assembly to one or more other components, various shaping and/or machining steps, the application of a coating, and the like. The completion of the cure can take place during and/or after such additional handling steps. Molecular weights as described herein are number average molecular weights which may be determined by Gel Permeation Chromatography (also referred to as GPC). The following examples are provided to illustrate the disclosed compositions, but are not intended to limit the scope thereof. All parts and percentages are by weight unless otherwise indicated. The following designations, symbols, terms and abbreviations are used in the examples below: Prepolymer 1 is a MDEPPO based Prepolymer. The prepolymer is a polyether polyurethane prepolymer prepared by mixing 22.571 g of a polyoxypropylene diol having an average molecular weight of 2000 g/mol commercially available under the trade name Voranol 2000L with 33.132 g of a polyoxypropylene triol having an average molecular weight of 4650 g/mol and commercially available under the trade name Arcol CP 4655. 33.779 g of plasticizer agent and 9.501 g diphenylmethane 4,4′-diisocyanate were added. Afterwards, 0.001 g of orthophsphoric acid in 0.009 g methyl ethyl ketone and 1 g of diethylmalonate were added. Then, the entire mixture was heated to 50° C. in a reactor and g of stannous octoate and was added. The reaction was carried out for 1 hour at 50° C. The resultant prepolymer is referred to herein as NCO-prepolymer. Prepolymer 2 is an Isocyanate Functional Polyester Prepolymer. The prepolymer was prepared by mixing 46.7 g of plasticizer agent (branched plasticizer), 30.15 g of a iscocyanate (Diphenylmethane 4,4′-diisocyanate) commercially available under the trade name Isonate M125U with 190.0 g of a polyester polyol commercially available under the trade name DYNACOL 7381. Then, the entire mixture was stirred for 8 hours. Vestinol 9 is Diisononylphtalate having a molecular weight: 418.6 g/mol, available from Evonik. Aerosil R208 is pyrogenic silica having a BET surface: ca. 80 to 140 m2/g, available from Evonik. Printex 30 is carbon black, available from Orion Carbons. Carbital 120 is uncoated calcium carbonate having a surface area BET: 2 m2/g, available from Imerys. DEM is diethyl malonate. Desmodure N3300 is a HDI-trimerisat, hexamethylene diisocyanate trimer having an NCO content: 21.8±0.3% and a viscosity at 23° C.: 3.000±750 mPa*s, available from Covestro. VORANATE M600 is a polymeric MDI (polymeric methylene diphenyl diisocyanate) having an isocyanate equiv. of 137 to 139, and isocyanate content of 30.2 to 31.1 and a viscosity at 25° C. of 520 to 680 mPa*s, available from DOW. SILQUEST A189 is gamma-mercaptopropyltrimethoxysilane, available from Momentive. SILQUEST A1170 is bis-(trimethoxysilylpropyl)amine, available from Momentive. DMDEE is 2,2′-dimorpholinodiethylether, available from BASF. UL28+Vestinol is a dimethyl-tin-dilaureate/Vestinol mixture. The mixture is prepared by adding 0.24 g catalyst UL28 to 9.76 g Vestinol 9 plasticizer. After stirring the solution became filled into a flask under dry nitrogen to exclude moisture. PAG-15 is a monofunctional polyalkylene glycol, available from DOW, having a chain length of C12to C15, plus units of propylene oxide resulting in a molecular weight of 1100 g/mol. Preparation of Adhesive Compositions The polyurethane adhesive compositions set forth below in Table 1 were prepared as follows. A planetary mixture was charged with the stated amounts of PPO based prepolymer 1 as well as with all liquid additives (DEM, silanes, Vestinol 9, Voranate M600, Desmodur N3300 and PAG-15). The mixture was stirred for 35 minutes under vacuum at room temperature. Then the appropriate amounts of carbon black, calcium carbonate and Aerosil R208 were added. The mixture was then stirred and heated until 60° C. to 70° C. under an atmosphere of nitrogen and subsequently 35 minutes under vacuum. When the temperature exceeded 60° C., the appropriate amount of polyester prepolymer 2 was added into the planetary mixer and stirred for another 10 minutes. Then the appropriate amounts of diisononylphtalate added as Vestinol 9, the UL28+Vestinol catalyst and the DMDEE catalyst were added and the mixture was stirred 15 minutes under vacuum or until a homogeneous pasteous black mixture was observed. Testing and Analytical Procedures Substrates: The following ceramic frit types were used SGS Ferro 14305, SGS Ferro 14502 and PLK Johnson Matthey C 24-8708 IR-9872-L. Substrate Preparation. Adhesive application with nozzle and applicator. The polyurethane adhesive compositions were applied to the unprimed ceramic fit types discussed above using the applicator. The adhesive bead was flattened to rectangular shape with a spatula. The test specimens were then stored for the desired cure time and environmental conditions listed at Table 1. Peel testing of cured bead on glass substrate was then carried out after the following climate conditions listed below in Table 1. TABLE 1TestingTem-ConditioningConditionperatureDetailsbefore testingDuration7 D RTRT7 days atna7 daysCycle23° C./50% r.h.7 DRT7 days at 90° C.+1 hour at7 days +90° C.23° C.1 h7 DRT4x (16 hours atat 23° C. for 727 daysClimate38° C./98% r.h. +hours beforeChange4 hours at −40° C. +testingCycle4 hours at 80° C.)14 DRT14 days at−20° C. for 214 daysCata-70° C./100% r.h.hours and 2plasmahours at 23° C. The ingredients and amounts used in the tested adhesives are listed in the following Table 2 along with the test results. All amounts listed are in weight percent. TABLE 2CompEx. 1Ex. 1CompositionPrepolymer 158.2557.25Prepolymer 21.21.2Voranate M6000.60.6Desmodur N33000.80.8Printex 302121Carbital 1201414Aerosil 2080.80.8DEM0.050.05Vestinol 9 DINP0.80.8Silquest A 1890.990.99Dynasilan 11700.110.11PAG-151DMDEE0.40.4UL 28 (2.4%) + vestinol11Total100.00100.00Peel Strength7 D RT100% CF100% CF7 D 90° C.100% CF100% CF7 D Climate Change Cycle100% CF100% CF14 D Cataplasma100% AF100% CF The data from Table 2 show that the polyurethane adhesive compositions within the scope of the present invention resulted in significantly improved glass adhesion duration after being subjected to the 14-day cataplasm conditioning when using the monofunctional polyalkylene glycol in combination with calcium carbonate and the silane adhesion promoters. | 53,318 |
11859110 | DESCRIPTION OF EMBODIMENTS Herein, a numeral value range represented by “from . . . to . . . ” means a range including the numeral values represented before and after “to” as a lower limit value and an upper limit value, respectively. An upper limit or a lower limit described in one numeral value range, among numeral value ranges stepwisely described herein, may be replaced with an upper limit or a lower limit of other numeral value range stepwisely described. An upper limit or a lower limit disclosed herein may be replaced with any value shown in Examples. [Stacked Substrates Body] Hereinafter, one embodiment of the body including stacked substrates (stacked substrates body) of the invention will be described. A stacked substrates body of the present embodiment has a configuration where a first substrate, an adhesion layer including a reaction product of a compound (A) which has a cationic functional group containing at least one of a primary nitrogen atom or a secondary nitrogen atom and which has a weight average molecular weight of from 90 to 400000 and a crosslinking agent (B) which has three or more —C(═O)OX groups (X is a hydrogen atom or an alkyl group having from 1 to 6 carbon atoms) in a molecule, in which from one to six of the three or more —C(═O)OX groups are —C(═O)OH groups and which has a weight average molecular weight of from 200 to 600, and a second substrate are layered in the listed order. The compound (A) preferably includes at least one selected from the group consisting of an aliphatic amine having a weight average molecular weight of from 10000 to 400000 and a compound having a siloxane bond (Si—O bond) and an amino group and having a weight average molecular weight of from 130 to 10000. In the stacked substrates body of the embodiment, the first substrate and the second substrate are bonded by the adhesion layer including the reaction product of the compound (A) and the crosslinking agent (B). The adhesion layer, which is formed on a surface of the first substrate, has a surface excellent in smoothness, and thus can have a uniform thickness and is also excellent in bonding strength between the substrates. The adhesion layer, which is also excellent in bonding strength between the substrates even in the case of having a reduced thickness, is thus advantageous in a case in which a multilayer three-dimensional structure is formed with a decrease in size being achieved. The adhesion layer, which can be reduced in thickness, thus allows a polar solvent (D) to be easily volatilized, resulting in suppression of the occurrence of a void, in manufacturing of the stacked substrates body. Such suppression of the occurrence of a void enables an adhesion area to be hardly small, resulting in suppression of unintended releasing of the substrates. [Adhesion Layer] The stacked substrates body of the embodiment includes an adhesion layer including a reaction product of a compound (A) which has a cationic functional group containing at least one of a primary nitrogen atom or a secondary nitrogen atom and which has a weight average molecular weight of from 90 to 400000 and a crosslinking agent (B) which has three or more —C(═O)OX groups (X is a hydrogen atom or an alkyl group having from 1 to 6 carbon atoms) in a molecule, in which from one to six of the three or more —C(═O)OX groups are —C(═O)OF groups and which has a weight average molecular weight of from 200 to 600. The adhesion layer is excellent in bonding strength between the substrates, and can suppress releasing of the substrates. The thickness of the adhesion layer is preferably from 0.1 nm to 5000 nm, more preferably from 0.5 nm to 3000 nm, still more preferably from 0.5 nm to 2000 nm, particularly preferably from 0.5 nm to 1000 nm, much more preferably from 0.5 nm to 500 nm. The stacked substrates body of the embodiment can ensure a high bonding strength between the substrates even at a relatively thin thickness of the adhesion layer, of from 0.1 nm to 5000 nm. The thickness of the adhesion layer may be determined by releasing at least one of the substrates from the stacked substrates body and subjecting the resultant to measurement with an ellipsometer. In the case of a thickness of 10 nm or more, fitting may be performed with an optical model of air/(Cauchy+Lorenz oscillator model)/natural oxide film/silicon substrate. In the case of a thickness of less than 10 nm, fitting may be performed with an optical model of air/SiO2/natural oxide film/silicon substrate. In a case in which such releasing is difficult, such measurement may be made by cutting the stacked substrates body and observing the resulting cut surface with a scanning electron microscope (SEM) or a transmission electron microscope (TEM). SEM or TEM may be selected depending on the thickness of the stacked substrates body. The reaction product in the adhesion layer preferably includes at least one of an amide bond or an imide bond from the viewpoint of imparting excellent heat resistance. (Compound (A)) The compound (A) is a compound which has a cationic functional group containing at least one of a primary nitrogen atom or a secondary nitrogen atom and which has a weight average molecular weight of from 90 to 400000. The cationic functional group is not particularly limited as long as the functional group is a functional group which can be positively charged and which contains at least one of a primary nitrogen atom or a secondary nitrogen atom. The compound (A) may contain a tertiary nitrogen atom, in addition to the primary nitrogen atom and the secondary nitrogen atom. Herein, the “primary nitrogen atom” refers to a nitrogen atom (for example, a nitrogen atom contained in a primary amino group (—NH2group)) bonded to only two hydrogen atoms and one atom other than a hydrogen atom, or a nitrogen atom (cation) bonded to only three hydrogen atoms and one atom other than a hydrogen atom. The “secondary nitrogen atom” refers to a nitrogen atom (namely, a nitrogen atom contained in a functional group represented by the following Formula (a)) bonded to only one hydrogen atom and two atoms other than a hydrogen atom, or a nitrogen atom (cation) bonded to only two hydrogen atoms and two atoms other than a hydrogen atom. The “tertiary nitrogen atom” refers to a nitrogen atom (namely, a nitrogen atom contained in a functional group represented by the following Formula (b)) bonded to only three atoms other than a hydrogen atom, or a nitrogen atom (cation) bonded to only one hydrogen atom and three atoms other than a hydrogen atom. In Formula (a) and Formula (b), * represents a bonding position with an atom other than a hydrogen atom. The functional group represented by Formula (a) may be a functional group constituting a part of a secondary amino group (—NHRagroup; wherein RR represents an alkyl group), or may be a divalent linking group contained in the skeleton of the polymer. The functional group represented by Formula (b) (namely, a tertiary nitrogen atom) may be a functional group constituting a part of a tertiary amino group (—NRbRcgroup; wherein Rband Rceach independently represent an alkyl group), or may be a trivalent linking group contained in the skeleton of the polymer. The weight average molecular weight of the compound (A) is from 90 to 400000. Examples of the compound (A) include an aliphatic amine and a compound having a siloxane bond (Si—O bond) and an amino group. In a case in which the compound (A) is an aliphatic amine, the weight average molecular weight is preferably from 10000 to 200000. In a case in which the compound (A) is a compound having a siloxane bond (Si—O bond) and an amino group, the weight average molecular weight is preferably from 130 to 10000, more preferably from 130 to 5000, further preferably from 130 to 2000. The weight average molecular weight herein refers to a weight average molecular weight of any other than a monomer, in terms of polyethylene glycol, as measured by a GPC (Gel Permeation Chromatography) method. Specifically, the weight average molecular weight is calculated in analysis software (EMPOWER 3 manufactured by Waters Corporation) with polyethylene glycol/polyethylene oxide as a standard by detecting the refractive index at a flow rate of 1.0 mL/min with an aqueous solution having a concentration of sodium nitrate of 0.1 mol/L as a developing solvent, by use of an analyzer Shodex DET RI-101 and two analytical columns (TSKgel G6000 PWXL-CP and TSKgel G3000 PWXL-CP manufactured by TOSOH CORPORATION). The compound (A) may further have, if necessary, an anionic functional group, a nonionic functional group, or the like. The nonionic functional group may be a hydrogen bond receptor or a hydrogen bond doner. Examples of the nonionic functional group can include a hydroxy group, a carbonyl group, and an ether group (—O—). The anionic functional group is not particularly limited as long as the functional group is a functional group which can be negatively charged. Examples of the anionic functional group can include a carboxylic acid group, a sulfonic acid group, and a sulfuric acid group. Examples of the compound (A) include an aliphatic amine, more specifically, a polyalkyleneimine which is a polymer of an alkyleneimine such as ethylene imine, propyleneimine, butylene imine, pentylene imine, hexylene imine, heptylene imine, octylene imine, trimethylene imine, tetramethylene imine, pentamethylene imine, hexamethylene imine, or octamethylene imine: polyallylamine; and polyacrylamide. Polyethyleneimine (PEI) can be produced by a known method described in Japanese Patent Publication (JP-B) No. S43-8828. JP-B No. S49-33120, JP-A No. 2001-213958, WO 2010/137711, or the like. Polyalkyleneimine other than polyethyleneimine can also be produced by a similar method as in polyethyleneimine. It is also preferable that the compound (A) is a derivative of the above polyalkyleneimine (polyalkyleneimine derivative; particularly preferably polyethyleneimine derivative). The polyalkyleneimine derivative is not particularly limited as long as the derivative is a compound which can be produced using the polyalkyleneimine. Specific examples can include a polyalkyleneimine derivative obtained by introducing an alkyl group (preferably an alkyl group having from 1 to 10 carbon atoms), an aryl group, or the like into the polyalkyleneimine, and a polyalkyleneimine derivative obtained by introducing a crosslinkable group such as a hydroxyl group into the polyalkyleneimine. Such polyalkyleneimine derivatives can be produced by a method commonly performed using the polyalkyleneimine. Specifically, such derivatives can be produced according to the method described in, for example, JP-A No. H06-016809. A preferable polyalkyleneimine derivative is also a highly branched type polyalkyleneimine obtained by allowing a cationic functional group-containing monomer to react with the polyalkyleneimine to result in an enhancement in the branching degree of the polyalkyleneimine. Examples of the method of obtaining such a highly branched type polyalkyleneimine include a method in which a cationic functional group-containing monomer is allowed to react with a polyalkyleneimine having a plurality of secondary nitrogen atoms in the backbone, thereby replacing at least one of the plurality of secondary nitrogen atoms with the cationic functional group-containing monomer, and a method in which a cationic functional group-containing monomer is allowed to react with a polyalkyleneimine having a plurality of primary nitrogen atoms at a terminal, thereby replacing at least one of the plurality of primary nitrogen atoms with the cationic functional group-containing monomer. Examples of the cationic functional group introduced for an enhancement in the branching degree can include an aminoethyl group, an aminopropyl group, a diaminopropyl group, an aminobutyl group, a diaminobutyl group, and a triaminobutyl group, and an aminoethyl group is preferable from the viewpoint of decreasing the equivalent of the cationic functional group and increasing the density of the cationic functional group. The polyethyleneimine and the derivative thereof may be each a commercially available product. For example, the polyethyleneimine and the derivative thereof may be each appropriately selected from any polyethyleneimines and derivatives thereof which are commercially available from NIPPON SHOKUBAI CO., LTD., BASF SE, MP-Biomedicals, LLC., and the like. Examples of the compound (A) include a compound having a Si—O bond and an amino group, in addition to the above aliphatic amine. Examples of the compound having a Si—O bond and an amino group include siloxane diamine, a silane coupling agent having an amino group, and a siloxane polymer of a silane coupling agent having an amino group. Examples of the silane coupling agent having an amino group include a compound represented by the following Formula (A-3). R1in Formula (A-3) represents an optionally substituted alkyl group having from 1 to 4 carbon atoms. R2and R3each independently represent an optionally substituted alkylene group having from 1 to 12 carbon atoms (the group optionally containing a carbonyl group, an ether group, or the like in the skeleton), an ether group, or a carbonyl group. R4and R5each independently represent an optionally substituted alkylene group having from 1 to 4 carbon atoms, or a single bond. Ar represents a divalent or trivalent aromatic ring. X1represents hydrogen, or an optionally substituted alkyl group having from 1 to 5 carbon atoms. X2represents hydrogen, a cycloalkyl group, a heterocyclic group, an aryl group, or an optionally substituted alkyl group having from 1 to 5 carbon atoms (the group optionally containing a carbonyl group, an ether group, or the like in the skeleton). A plurality of R3's, R2's, R3's, R4's, R5's, and X1's may be each the same as or different from each other. Examples of each substituent of the alkyl groups and the alkylene groups in R1, R2, R3, R4, R5, X1, and X2independently include an amino group, a hydroxy group, an alkoxy group, a cyano group, a carboxylic acid group, a sulfonic acid group, and halogen. Examples of the divalent or trivalent aromatic ring in Ar include a divalent or trivalent benzene ring. Examples of the aryl group in X2include a phenyl group, a methylbenzyl group, and a vinylbenzyl group. Specific examples of the silane coupling agent represented by Formula (A-3) include N-(2-aminoethyl)-3-aminopropylmethyldiethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminoisobutyldimethylmethoxysilane, N-(2-aminoethyl)-3-aminoisobutylmethyldimethoxysilane, N-(2-aminoethyl)-11-aminoundecyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, N-phenyl-3-aminopropyltrimethoxysilane, (aminoethylaminoethyl)phenyltriethoxysilane, methylbenzylaminoethylaminopropyltrimethoxysilane, benzylaminoethylaminopropyltriethoxysilane, 3-ureidopropyltriethoxysilane, (aminoethylaminoethyl)phenethyltrimethoxysilane, (aminoethylaminomethyl)phenethyltrimethoxysilane, N-[2-[3-(trimethoxysilyl)propylamino]ethyl]ethylenediamine, 3-aminopropyldiethoxymethylsilane, 3-aminopropyldimethoxymethylsilane, 3-aminopropyldimethylethoxysilane, 3-aminopropyldimethylmethoxysilane, trimethoxy[2-(2-aminoethyl)-3-aminopropyl]silane, diaminomethylmethyldiethoxysilane, methylaminomethylmethyldiethoxysilane, p-aminophenyltrimethoxysilane, N-methylaminopropyltriethoxysilane, N-methylaminopropylmethyldiethoxysilane, (phenylaminomethyl)methyldiethoxysilane, acetamidopropyltrimethoxysilane, and hydrolysates thereof. Examples of other silane coupling agent having an amino group, than that represented by Formula (A-3), include N,N-bis[3-(trimethoxysilyl)propyl]ethylenediamine, N,N′-bis[3-(trimethoxysilyl)propyl]ethylenediamine, bis[(3-triethoxysilyl)propyl]amine, piperazinylpropylmethyldimethoxysilane, bis[3-(triethoxysilyl)propyl]urea, bis(methyldiethoxysilylpropyl)amine, 2,2-dimethoxy-1,6-diaza-2-silacyclooctane, 3,5-diamino-N-(4-(methoxydimethylsilyl)phenyl)benzamide, 3,5-diamino-N-(4-(triethoxysilyl)phenyl)benzamide, 5-(ethoxydimethylsilyl)benzene-1,3-diamine, and hydrolysates thereof. The silane coupling agent having an amino group may be used singly, or in combination of two or more kinds thereof. The silane coupling agent having an amino group may be used in combination with a silane coupling agent having no amino group. For example, a silane coupling agent having a mercapto group may be used in order to improve adhesiveness to a metal. A polymer (siloxane polymer) formed from such a silane coupling agent via a siloxane bond (Si—O—Si) may also be used. For example, a polymer having a linear siloxane structure, a polymer having a branched siloxane structure, a polymer having a cyclic siloxane structure, a polymer having a cage-like siloxane structure, or the like can be obtained from a hydrolysate of 3-aminopropyltrimethoxysilane. Such a cage-like siloxane structure is represented by, for example, the following Formula (A-1). Examples of the siloxane diamine include a compound represented by the following Formula (A-2). In Formula (A-2), i is an integer of from 0 to 4, j is an integer of from 1 to 3, and Me is a methyl group. Examples of the siloxane diamine include 1,3-bis(3-aminopropyl)tetramethyldisiloxane (i=0 and j=1 in Formula (A-2)) and 1,3-bis(2-aminoethylamino)propyltetramethyldisiloxane (i=1 and j=1 in Formula (A-2)). Examples of the compound (A) include an amine compound having not any Si—O bond, but a ring structure in the molecule, in addition to the aliphatic amine and the compound having a Si—O bond and an amino group. The compound (A) may contain not only at least one selected from the group consisting of the aliphatic amine and the compound having a Si—O bond and an amino group, but also an amine compound having not any Si—O bond, but a ring structure in the molecule, in particular, an amine compound having not any Si—O bond, but a ring structure in the molecule, and having a weight average molecular weight of from 90 to 600. Examples of the amine compound having not any Si—O bond, but a ring structure in the molecule, and having a weight average molecular weight of from 90 to 600 include an alicyclic amine, an aromatic ring amine, and a heterocyclic (aminocyclic) amine. The amine compound may have a plurality of ring structures in the molecule, and the plurality of ring structures may be the same as or different from each other. Such an amine compound having a ring structure is more preferably a compound having an aromatic ring because a thermally more stable compound is easily obtained. The amine compound having not any Si—O bond, but a ring structure in the molecule, and having a weight average molecular weight of from 90 to 600 is preferably a compound having a primary amino group from the viewpoint of being capable of easily forming a thermally crosslinked structure such as amide, amide-imide, or imide with the crosslinking agent (B) and enhancing heat resistance. The above amine compound is preferably a diamine compound having two primary amino groups, a triamine compound having three primary amino groups, or the like from the viewpoint of being capable of easily increasing the number of thermally crosslinked structures such as amide, amide-imide, or imide with the crosslinking agent (B) and more enhancing heat resistance. Examples of the alicyclic amine include cyclohexylamine and dimethylaminocyclohexane. Examples of the aromatic ring amine include diaminodiphenyl ether, xylenediamine (preferably p-xylenediamine), diaminobenzene, diaminotoluene, methylenedianiline, dimethyldiaminobiphenyl, bis (trifluoromethyl) diaminobiphenyl, diaminobenzophenone, diaminobenzanilide, bis(aminophenyl)fluorene, bis(aminophenoxy)benzene, bis(aminophenoxy)biphenyl, dicarboxydiaminodiphenylmethane, diaminoresorcin, dihydroxybenzidine, diaminobenzidine, 1,3,5-triaminophenoxybenzene, 2,2′-dimethylbenzidine, tris(4-aminophenyl)amine, 2,7-diaminofluorene, 1,9-diaminofluorene, and dibenzylamine. Examples of the heterocyclic ring of a heterocyclic amine include a heterocyclic ring containing a sulfur atom as a heteroatom (for example, a thiophene ring), or a heterocyclic ring containing a nitrogen atom as a heteroatom (for example, a 5-membered ring such as a pyrrole ring, a pyrrolidine ring, a pyrazole ring, an imidazole ring, or a triazole ring; a 6-membered ring such as an isocyanuryl ring, a pyridine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, a piperidine ring, a piperazine ring, or a triazine ring; or a condensed ring such as an indole ring, an indoline ring, a quinoline ring, an acridine ring, a naphthyridine ring, a quinazoline ring, a purine ring, or a quinoxaline ring). Examples of a heterocyclic amine having a nitrogen-containing heterocyclic ring include melamine, ammeline, melam, melem, and tris(4-aminophenyl)amine. Examples of an amine compound having both a heterocyclic ring and an aromatic ring include N2,N4,N6-tris(4-aminophenyl)-1,3,5-triazine-2,4,6-triamine. The compound (A) has a primary or secondary amino group, and thus can electrostatically interact with a functional group such as a hydroxyl group, an epoxy group, a carboxy group, an amino group, or a mercapto group which can be present on surfaces of the first substrate and the second substrate, or can tightly form a covalent bond with the functional group, thereby allowing the substrates to strongly adhere to each other. The compound (A) has a primary or secondary amino group, and thus is easily dissolved in a polar solvent (D) described below. Such a compound (A) to be easily dissolved in the polar solvent (D) is used, thereby increasing affinity with a hydrophilic surface of a substrate such as a silicon substrate, whereby a smooth film can be easily formed and the thickness of the adhesion layer can be decreased. The compound (A) is preferably the aliphatic amine or the compound having a Si—O bond and an amino group from the viewpoint of formation of a smooth thin film, and is more preferably the compound having a Si—O bond and an amino group from the viewpoint of heat resistance. The compound (A) is preferably a compound having a Si—O bond and a primary amino group from the viewpoint of formation of a thermally crosslinked structure such as amide, amide-imide, or imide for a more enhancement in heat resistance. In a case in which the compound (A) contains the compound having a Si—O bond and an amino group, the ratio of the total number of primary nitrogen atoms and secondary nitrogen atoms to the number of silicon atoms (total number of primary nitrogen atoms and secondary nitrogen atoms/number of silicon atoms) in the compound (A) is preferably from 0.2 to 5 from the viewpoint of formation of a smooth thin film. In a case in which the compound (A) contains the compound having a Si—O bond and an amino group, the molar ratio of a non-crosslinkable group such as a methyl group bound to a Si element to a Si element in the compound having a Si—O bond and an amino group preferably satisfies a relationship of (non-crosslinkable group)/Si<2 from the viewpoint of adhesion ability of the substrates. It is presumed that when this relationship can be satisfied, the density of crosslinking (crosslinking of a Si—O—Si bond with an amide bond, an imide bond, or the like) in the resultant film is increased, the substrates are bonded with a sufficient adhesion force, and releasing of the substrates is suppressed. As described above, the compound (A) has a cationic functional group containing at least one of a primary nitrogen atom or a secondary nitrogen atom. In a case in which the compound (A) contains a primary nitrogen atom, the proportion of the primary nitrogen atom in all the nitrogen atoms in the compound (A) is preferably 20% by mol or more, more preferably 25% by mol or more, further preferably 30% by mol or more. The compound (A) may have a cationic functional group containing a primary nitrogen atom and containing no nitrogen atom other than the primary nitrogen atom (for example, a secondary nitrogen atom or a tertiary nitrogen atom). In a case in which the compound (A) contains a secondary nitrogen atom, the proportion of the secondary nitrogen atom in all the nitrogen atoms in the compound (A) is preferably from 5% by mol to 50% by mol, more preferably from 10% by mol to 45% by mol. The compound (A) may contain a tertiary nitrogen atom, in addition to the primary nitrogen atom and the secondary nitrogen atom. In a case in which the compound (A) contains a tertiary nitrogen atom, the proportion of the tertiary nitrogen atom in all the nitrogen atoms in the compound (A) is preferably from 20% by mol to 50% by mol, more preferably from 25% by mol to 45% by mol. In the embodiment, the content of the component derived from the compound (A) in the adhesion layer is not particularly limited, and can be, for example, from 1% by mass to 82% by mass or less and is preferably from 5% by mass to 82% by mass, more preferably from 13% by mass to 82% by mass, with respect to the entire adhesion layer. (Crosslinking Agent (B)) The crosslinking agent (B) is a compound which has three or more —C(═O)OX groups (X is a hydrogen atom or an alkyl group having from 1 to 6 carbon atoms) in the molecule, in which from one to six of three or more —C(═O)OX groups (hereinafter, also referred to as “COOX”.) are —C(═O)OH groups (hereinafter, also referred to as “COOH”.) and which has a weight average molecular weight of from 200 to 600. The crosslinking agent (B) is a compound having three or more —C(═O)OX groups in the molecule (X is a hydrogen atom or an alkyl group having from 1 to 6 carbon atoms), and is preferably a compound having from three to six —C(═O)OX groups in the molecule, more preferably a compound having three or four —C(═O)OX groups in the molecule. Examples of each X in the —C(═O)OX groups in the crosslinking agent (B) include a hydrogen atom, or an alkyl group having from 1 to 6 carbon atoms, and in particular, a hydrogen atom, a methyl group, an ethyl group, and a propyl group are preferable. X's in the —C(═O)OX groups may be the same as or different from each other. The crosslinking agent (B) is a compound having from one to six —C(═O)OH groups, which corresponds to a case in which X is hydrogen atom, in the molecule, and is preferably a compound having from one to four —C(═O)OH groups in the molecule, more preferably a compound having from two to four —C(═O)OH groups in the molecule, still more preferably a compound having two or three —C(═O)OH groups in the molecule. The crosslinking agent (B) is a compound having a weight average molecular weight of from 200 to 600, and is preferably a compound having a weight average molecular weight of from 200 to 400. The crosslinking agent (B) preferably have a ring structure in the molecule. Examples of the ring structure include an alicyclic structure and an aromatic ring structure. The crosslinking agent (B) may have a plurality of ring structures in the molecule, and the plurality of ring structures may be the same as or different from each other. Examples of the alicyclic structure include an alicyclic structure having from 3 to 8 carbon atoms, preferably include an alicyclic structure having from 4 to 6, and the ring structure may be a saturated or unsaturated ring structure. More specific examples of the alicyclic structure include a saturated alicyclic structure such as a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, or a cyclooctane ring; and an unsaturated alicyclic structure such as a cyclopropene ring, a cyclobutene ring, a cyclopentene ring, a cyclohexene ring, a cycloheptene ring, or a cyclooctene ring. The aromatic ring structure is not particularly limited as long as the structure is a ring structure exhibiting aromaticity, and examples include a benzene aromatic ring such as a benzene ring, a naphthalene ring, an anthracene ring, or a perylene ring, an aromatic heterocyclic ring such as a pyridine ring or a thiophene ring, and a nonbenzene aromatic ring such as an indene ring or an azulene ring. The ring structure of the crosslinking agent (B) in the molecule is preferably, for example, at least one selected from the group consisting of a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a benzene ring, and a naphthalene ring, and is more preferably at least one of a benzene ring or a naphthalene ring from the viewpoint of further enhancing heat resistance of the adhesion layer. As described above, the crosslinking agent (B) may have a plurality of ring structures in the molecule, and may have a biphenyl structure, a benzophenone structure, a diphenyl ether structure, or the like in a case in which the ring structure is benzene. The ring structure of the crosslinking agent (B) in the molecule is preferably a ring structure having two or more —C(═O)OX groups. The crosslinking agent (B) preferably has a fluorine atom in the molecule, more preferably has from one to six fluorine atoms in the molecule, further preferably has from three to six fluorine atoms in the molecule. For example, the crosslinking agent (B) may have a fluoroalkyl group in the molecule, specifically, may have a trifluoroalkyl group or a hexafluoroisopropyl group. Examples of the crosslinking agent (B) further include a carboxylic acid compound such as alicyclic carboxylic acid, benzenecarboxylic acid, naphthalenecarboxylic acid, diphthalic acid, or fluorinated aromatic ring carboxylic acid; and a carboxylic acid ester compound such as an alicyclic carboxylic acid ester, a benzenecarboxylic acid ester, a naphthalenecarboxylic acid ester, a diphthalic acid ester, or a fluorinated aromatic ring carboxylic acid ester. The carboxylic acid ester compound is a compound which has a carboxy group (—C(═O)OH group) in the molecule and in which at least one X in three or more —C(═O)OX groups is an alkyl group having from 1 to 6 carbon atoms (in other words, an ester bond is contained). In the embodiment, the crosslinking agent (B) is such a carboxylic acid ester compound, whereby aggregation due to association of the compound (A) with the crosslinking agent (B) is inhibited, aggregates and pits are decreased, and the film thickness is easily adjusted. The carboxylic acid compound is preferably a tetravalent or lower carboxylic acid compound containing four or less —C(═O)OH groups, more preferably a trivalent or tetravalent carboxylic acid compound containing three or four —C(═O)OH groups. The carboxylic acid ester compound is preferably a compound containing three or less carboxy groups (—C(═O)OH groups) and three or less ester bonds in the molecule, more preferably a compound containing two or less carboxy groups and two or less ester bonds in the molecule. In a case in which each X in three or more —C(═O)OX groups in the carboxylic acid ester compound is an alkyl group having from 1 to 6 carbon atoms, such X is preferably a methyl group, an ethyl group, a propyl group, a butyl group, or the like and is preferably an ethyl group or a propyl group from the viewpoint that aggregation due to association of the compound (A) with the crosslinking agent (B) is inhibited. Specific examples of the carboxylic acid compound include, without any limitation, an alicyclic carboxylic acid such as 1,2,3,4-cyclobutanctetracarboxylic acid, 1,2,3,4-cyclopentanetetracarboxylic acid, 1,3,5-cyclohexanetricarboxylic acid, 1,2,4-cyclohexanetricarboxylic acid, 1,2,4,5-cyclohexanetetracarboxylic acid, or 1,2,3,4,5,6-cyclohexanehexacarboxylic acid; a benzenecarboxylic acid such as 1,2,4-benzenetricarboxylic acid, 1,3,5-benzenetricarboxylic acid, pyromellitic acid, benzenepentacarboxylic acid, or mellitic acid; a naphthalenecarboxylic acid such as 1,4,5,8-naphthalenetetracarboxylic acid or 2,3,6,7-naphthalenetetracarboxylic acid; a diphthalic acid such as 3,3′,5,5′-tetracarboxy diphenylmethane, biphenyl-3,3′,5,5′-tetracarboxylic acid, biphenyl-3,4′,5-tricarboxylic acid, biphenyl-3,3′,4,4′-tetracarboxylic acid, benzophenone-3,3′,4,4′-tetracarboxylic acid, 4,4′-oxydiphthalic acid, 3,4′-oxydiphthalic acid, 1,3-bis(phthalic acid)tetramethyldisiloxane, 4,4′-(ethyne-1,2-diyl)diphthalic acid(4,4′-(Ethyne-1,2-diyl)diphthalic acid), 4,4′-(1,4-phenylenebis(oxy))diphthalic acid (4,4′-(1,4-phenylenebis(oxy))diphthalic acid), 4,4′-([1,1′-biphenyl]-4,4′-diylbis(oxy))diphthalic acid, (4,4′-([1,1′-biphenyl]-4,4′-diylbis(oxy))diphthalic acid), 4,4′-((oxybis(4,1-phenylene))bis(oxy))diphthalic acid, or (4,4′-((oxybis(4,1-phenylene))bis(oxy))diphthalic acid); a perylene carboxylic acid such as perylene-3,4,9,10-tetracarboxylic acid; an anthracene carboxylic acid such as anthracene-2,3,6,7-tetracarboxylic acid; and a fluorinated aromatic ring carboxylic acid such as 4,4′-(bexafluoroisopropylidene)diphthalic acid, 9,9-bis(trifluoromethyl)-9H-xanthene-2,3,6,7-tetracarboxylic acid, or 1,4-ditrifluoromethylpyromellitic acid. Specific examples of the carboxylic acid ester compound include any compound in the above specific examples of the carboxylic acid compound, in which at least one carboxy group is substituted with an ester group. Examples of the carboxylic acid ester compound include half esterified compounds represented by the following Formulae (B-1) to (B-6). Each R in Formulae (B-1) to (B-6) is independently an alkyl group having from 1 to 6 carbon atoms, and, in particular, is preferably a methyl group, an ethyl group, a propyl group, or a butyl group, more preferably an ethyl group or a propyl group. Such a half-esterified compound can be produced by, for example, mixing carboxylic acid anhydride as an anhydride of the above carboxylic acid compound, with an alcohol solvent, and opening the carboxylic acid anhydride. In the embodiment, the content of the component derived from the crosslinking agent (B) in the adhesion layer is not particularly limited, and, for example, the ratio ((—(C═O)—Y)/N) of the number of carbonyl groups (—(C═O)—Y) in the substance derived from the crosslinking agent (B) to the number of all the nitrogen atoms in the substance derived from the compound (A) is preferably from 0.1 to 3.0, more preferably from 0.3 to 2.5, further preferably from 0.4 to 2.2. Y in —(C═O)—Y represents a nitrogen atom imide-crosslinked or amide-crosslinked, OH, or an ester group. The ratio (—(C═O)—Y)/N is from 0.1 to 3.0, whereby the adhesion layer suitably has a thermally crosslinked structure such as amide, amide-imide, or imide, and is more excellent in heat resistance. The compound (A) has an uncrosslinked cationic functional group, and it is thus considered that the adhesion layer is low in crosslinking density and is not sufficient in heat resistance in the case of including the compound (A) and not including the crosslinking agent (B) as the component thereof. The adhesion layer is increased in crosslinking density and has high heat resistance due to formation of a covalent bond by a reaction of the cationic functional group of the compound (A) with the carboxy group of the crosslinking agent (B). (Polar Solvent (D)) The solution for formation of the adhesion layer in the stacked substrates body of the embodiment, for example, the solution containing the compound (A), the solution containing the crosslinking agent (B), or the solution containing the compound (A) and the crosslinking agent (B), for use in the method of manufacturing the stacked substrates body described below, preferably contains a polar solvent (D). The polar solvent (D) here refers to a solvent having a relative dielectric constant of 5 or more at room temperature. Specific examples of the polar solvent (D) include a protonic inorganic compound such as water or heavy water; an alcohol such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, isopentyl alcohol, cyclohexanol, ethylene glycol, propylene glycol, 2-methoxyethanol, 2-ethoxyethanol, benzyl alcohol, diethylene glycol, triethylene glycol, or glycerin; an ether such as tetrahydrofuran or dimethoxyethane; an aldehyde or ketone such as furfural, acetone, ethyl methyl ketone, or cyclohexane; an acid derivative such as acetic anhydride, ethyl acetate, butyl acetate, ethylene carbonate, propylene carbonate, formaldehyde, N-methylformamide, N,N-dimethylformamide, N-methylacetamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, or hexamethylphosphoramide; a nitrile such as acetonitrile or propionitrile; a nitro compound such as nitromethane or nitrobenzene; and a sulfur compound such as dimethylsulfoxide. The polar solvent (D) preferably contains a protonic solvent, more preferably water, further preferably ultrapure water. The content of the polar solvent (D) in the solution for formation of the adhesion layer is not particularly limited, and is, for example, from 1.0% by mass to 99.99896% by mass, preferably from 40% by mass to 99.99896% by mass, with respect to the total solution. The boiling point of the polar solvent (D) is preferably 150° C. or less, more preferably 120° C. or less, from the viewpoint that the polar solvent (D) is volatilized by heating in formation of the adhesion layer and thus the amount of the remaining solvent in the adhesion layer is reduced. (Additive (C)) The solution for formation of the adhesion layer in the stacked substrates body of the embodiment may contain an additive (C), in addition to the above compound (A), the crosslinking agent (B) and the polar solvent (D). Examples of the additive (C) include an acid (C-1) having a carboxy group and having a weight average molecular weight of from 46 to 195, and a base (C-2) having a nitrogen atom, having a weight average molecular weight of from 17 to 120, and not having any ring structure. While the additive (C) is volatilized by heating in formation of the adhesion layer, the adhesion layer in the stacked substrates body of the embodiment may contain such an additive (C). The acid (C-1) is an acid having a carboxy group and having a weight average molecular weight of from 46 to 195. It is presumed that the acid (C-1) is contained in the additive (C), whereby ionic bonding formed from an amino group in the compound (A) and a carboxy group in the acid (C-1) inhibits aggregation due to association of the compound (A) with the crosslinking agent (B). More specifically, it is presumed that the interaction (for example, electrostatic interaction) between an ammonium ion derived from an amino group in the compound (A) and a carboxylate ion derived from a carboxy group in the acid (C-1) is stronger than the interaction between an ammonium ion derived from an amino group in the compound (A) and a carboxylate ion derived from a carboxy group in the crosslinking agent (B), whereby such aggregation is inhibited. The invention is not limited to such presumptions at all. The acid (C-1) is not particularly limited as long as the acid is a compound which has a carboxy group and which has a weight average molecular weight of from 46 to 195, and examples thereof include a monocarboxylic acid compound, a dicarboxylic acid compound, and an oxydicarboxylic acid compound. More specific examples of the acid (C-1) include formic acid, acetic acid, malonic acid, oxalic acid, citric acid, benzoic acid, lactic acid, glycolic acid, glyceric acid, butyric acid, methoxyacetic acid, ethoxyacetic acid, phthalic acid, terephthalic acid, picolinic acid, salicylic acid, and 3,4,5-trihydroxybenzoic acid. In the embodiment, the content of the acid (C-1) in the solution for formation of the adhesion layer in the stacked substrates body is not particularly limited, and, for example, the ratio (COOH/N) of the number of carboxy groups in the acid (C-1) to the total number of nitrogen atoms in the compound (A) is preferably from 0.01 to 10, more preferably from 0.02 to 6, further preferably from 0.5 to 3. The base (C-2) is a base having a nitrogen atom and having a weight average molecular weight of from 17 to 120. It is presumed that the solution for formation of the adhesion layer in the stacked substrates body of the embodiment contains the base (C-2) in the additive (C), whereby ionic bonding formed from a carboxy group in the crosslinking agent (B) and an amino group in the base (C-2) inhibits aggregation due to association of the compound (A) with the crosslinking agent (B). More specifically, it is presumed that the interaction between a carboxylate ion derived from a carboxy group in the crosslinking agent (B) and an ammonium ion derived from an amino group in the base (C-2) is stronger than the interaction between an ammonium ion derived from an amino group in the compound (A) and a carboxylate ion derived from a carboxy group in the crosslinking agent (B), whereby such aggregation is inhibited. The invention is not limited to such presumptions at all. The base (C-2) is not particularly limited as long as the base is a compound having a nitrogen atom and having a weight average molecular weight of from 17 to 120, and not having any ring structure, and examples thereof include a monoamine compound and a diamine compound. More specific examples of the base (C-2) include ammonia, ethylamine, ethanolamine, diethylamine, triethylamine, ethylenediamine, N-acetylethylenediamine, N-(2-aminoethyl)ethanolamine, and N-(2-aminoethyl)glycine. In the embodiment, the content of the base (C-2) in the solution for formation of the adhesion layer in the stacked substrates body is not particularly limited, and, for example, the ratio (N/COOH) of the number of nitrogen atoms in the base (C-2) to the number of carboxy groups in the crosslinking agent (B) is preferably from 0.5 to 5, more preferably from 0.9 to 3. (Other Components) The respective contents of sodium and potassium in the adhesion layer in the stacked substrates body of the embodiment are preferably 10 mass ppb or less on an element basis. In a case in which the respective contents of sodium and potassium are 10 mass ppb or less on an element basis, the occurrence of inconvenience in electrical characteristics of a semiconductor device, such as malfunction of a transistor, can be suppressed. In a case in which the adhesion layer of the stacked substrates body of the embodiment is required to have insulation, tetraethoxysilane, tetramethoxysilane, bistriethoxysilylethane, bistriethoxysilylmethane, bis(methyldiethoxysilyl)ethane, 1,1,3,3,5,5-hexaethoxy-1,3,5-trisilacyclohexane, 1,3,5,7-tetramethyl-1,3,5,7-tetrahydroxylcyclosiloxane, 1,1,4,4-tetramethyl-1,4-diethoxydisylethylene, 1,3,5-triethoxy-1,3,5-trimethyl-1,3,5-trisilacyclohexane, or a siloxane polymer thereof may be mixed therewith, in order to improve insulation or mechanical strength. Furthermore, methyltriethoxysilane, dimethyldiethoxysilane, trimethylethoxysilane, or the like may be mixed therewith in order to improve hydrophobicity of an adhesion layer having insulation. Such a compound may also be mixed for control of etching selectivity. The solution for formation of the adhesion layer in the stacked substrates body may contain any solvent other than the polar solvent (D), and examples of such any other solvent include normal hexane. The solution for formation of the adhesion layer in the stacked substrates body may contain phthalic acid, benzoic acid or the like, or a derivative thereof, for example, in order to improve electrical characteristics. The solution for formation of the adhesion layer in the stacked substrates body may contain benzotriazole or a derivative thereof, for example, in order to suppress corrosion of copper. The pH of the solution for formation of the adhesion layer in the stacked substrates body is not particularly limited, and is preferably from 2.0 to 12.0. The adhesion layer in the stacked substrates body may a crosslinkable compound such as an epoxy compound, an isocyanate compound, and a polyvalent acrylate compound. The content of such a crosslinkable compound is preferably 5% by mass or less, more preferably 1% by mass or less, further preferably 0.1% by mass or less, with respect to the total mass of the adhesion layer, and particularly preferably, such a compound is not contained, from the viewpoint that the occurrence of any outgas is suppressed and heat resistance is enhanced. [First Substrate and Second Substrate] The stacked substrates body of the embodiment is obtained by layering the first substrate, the adhesion layer, and the second substrate in the listed order. The respective materials of the first substrate and the second substrate are not particularly limited, and may be any materials commonly used. The respective materials of the first substrate and the second substrate may be the same as or different from each other. Each of the first substrate and the second substrate preferably includes at least one element selected from the group consisting of Si, Al, Ti, Zr, Hf, Fe, Ni, Cu, Ag, Au, Ga, Ge, Sn, Pd, As, Pt, Mg, In, Ta, and Nb, more preferably includes at least one element selected from the group consisting of Si, Ga, Ge, and As, and is further preferably semiconductor substrate including at least one element selected from the group consisting of Si, Ga, Ge, and As. Examples of the respective materials of the first substrate and the second substrate include a semiconductor: Si, InP, GaN, GaAs, InGaAs, InGaAlAs, SiGe, SiC, oxide, carbide, or nitride: borosilicate glass (PYREX (registered trade mark)), quartz glass (SiO2), sapphire (Al2O3), ZrO2, Si3N4, AlN, MgAl2O4, a piezoelectric body, a dielectric body: BaTiO3, LiNbO3, SrTiO3, LiTaO3, diamond, and metal: Al, Ti, Fe, Cu, Ag, Au, Pt, Pd, Ta, or Nb. Other respective materials of the first substrate and the second substrate may be a resin: polydimethylsiloxane (PDMS), an epoxy resin, a phenol resin, polyimide, a benzocyclobutene resin, polybenzoxazole, or the like. Such respective materials are used in the followings as main applications. Si is used in a semiconductor memory. LSI layering, a CMOS image sensor, MEMS sealing, an optical device, a LED, or the like; SiO2is used in a semiconductor memory, LSI layering, MEMS sealing, microfluidics, a CMOS image sensor, an optical device, LED, or the like; BaTiO3. LiNbO3, SrTiO3, and LiTaO3are used in a surface acoustic wave device; PDMS is used in microfluidics; InGaAlAs, InGaAs, and InP are used in an optical device; InGaAlAs, GaAs, and GaN are used in LED, or the like. A surface of at least one of the first substrate or the second substrate for use in manufacturing of the stacked substrates body on which the adhesion layer is to be formed, preferably, surfaces of the first substrate and the second substrate, on which the adhesion layer is to be formed, preferably have at least one selected from the group consisting of a hydroxyl group, an epoxy group, a carboxy group, an amino group, and a mercapto group. Thus, bonding strength between the substrates can be enhanced. A hydroxyl group can be provided on such each surface of the first substrate and the second substrate by performing a surface treatment such as a plasma treatment, a chemical treatment, or an ozone treatment. An epoxy group can be provided on such each surface of the first substrate and the second substrate by performing a surface treatment such as silane coupling with epoxysilane. A carboxy group can be provided on such each surface of the first substrate and the second substrate by performing a surface treatment such as silane coupling with carboxysilane. An amino group can be provided on such each surface of the first substrate and the second substrate by performing a surface treatment such as silane coupling with aminosilane. At least one selected from the group consisting of a hydroxyl group, an epoxy group, a carboxy group, an amino group, and a mercapto group is preferably present in the state of being bonded to at least one element selected from the group consisting of Si, Al, Ti, Zr, Hf, Fe, Ni, Cu, Ag, Au, Ga, Ge, Sn, Pd, As, Pt, Mg, In, Ta, and Nb contained in the first substrate or the second substrate. In particular, a surface of at least one of the first substrate or the second substrate, on which the adhesion layer is to be formed, more preferably has a silanol group (Si—OH group) containing a hydroxyl group. At least one of the first substrate or the second substrate may be provided with an electrode on a surface thereof, the surface being closer to the adhesion layer. The thicknesses of the first substrate and the second substrate are each independently preferably from 1 μm to 1 mm, more preferably from 2 μm to 900 μm. The respective shapes of the first substrate and the second substrate are not particularly limited. For example, in a case in which the first substrate and the second substrate are each a silicon substrate, such a silicon substrate may be a silicon substrate on which an interlayer insulating layer (Low-k film) is formed, or a silicon substrate on which fine grooves (recesses), fine through holes, or the like are formed. In the stacked substrates body of the embodiment, still another substrate may be layered on a surface of at least one of the first substrate or the second substrate, the surface being located opposite to the surface closer to the adhesion layer. A preferable material of such another substrate is the same as preferable respective materials of the first substrate and the second substrate. The material of such another substrate may be the same as or different from that of at least one of the first substrate or the second substrate. (Examples of the Stacked Structure of the Stacked Substrates Body) Hereinafter, examples of the stacked structure of the stacked substrates body in each application are shown. For MEMS packaging: Si/adhesion layer/Si, SiO2/adhesion layer/Si, SiO2/adhesion layer/SiO2, Cu/adhesion layer/Cu, For microfluidics; PDMS/adhesion layer/PDMS, PDMS/adhesion layer/SiO2, For CMOS image sensor; SiO2/adhesion layer/SiO2, Si/adhesion layer/Si, SiO2/adhesion layer/Si, For silicon through via (TSV); SiO2(provided with Cu electrode)/adhesion layer/SiO2(provided with Cu electrode), For memory and LSI; SiO2/adhesion layer/SiO2, For optical device; (InGaAlAs, InGaAs, InP, GaAs)/adhesion layer/Si, For LED; (InGaAlAs, GaAs, GaN)/adhesion layer/Si, (InGaAlAs, GaAs, GaN)/adhesion layer/SiO2, (InGaAlAs, GaAs, GaN)/adhesion layer/(Au, Ag, Al), InGaAlAs, GaAs, GaN)/adhesion layer/sapphire For surface acoustic wave device; (BaTiO3, LiNbO3, SrTiO3, LiTaO3)/adhesion layer/(MgAl2O4, SiO2, Si, Al2O3)). A higher tensile bonding strength of the stacked substrates body in the embodiment is more preferable from the viewpoint of suppression of unintended releasing in a semiconductor process and from the viewpoint of reliability. Specifically, the tensile bonding strength of the stacked substrates body is preferably 5 MPa or more, more preferably 10 MPa or more. The tensile bonding strength of the stacked substrates body can be determined from a yield point obtained in measurement with a tensile tester. The tensile bonding strength may be 200 MPa or less, or may be 100 MPa or less. The stacked substrates body of the embodiment preferably has a tensile bonding strength of 5 MPa or more and a thickness of the adhesion layer of from 0.1 nm to 5000 nm, more preferably a tensile bonding strength of 5 MPa or more and a thickness of the adhesion layer of from 0.5 nm to 3000 nm, further preferably a tensile bonding strength of 10 MPa or more and a thickness of the adhesion layer of from 5 nm to 2000 nm, particularly preferably a tensile bonding strength of 10 MPa or more and a thickness of the adhesion layer of from 5 nm to 500 nm. The temperature at which the pressure of outgas reaches 2×10−6Pa in the stacked substrates body of the embodiment is preferably 400° C. or more, more preferably 420° C. or more, further preferably 440° C. or more from the viewpoint of suppression of a reduction in bonding strength of the stacked substrates body due to outgas. The temperature at which the pressure of outgas reaches 2×10−6Pa is a value obtained by measurement under a reduced pressure environment. Such a reduced pressure environment is at 10−7Pa. The temperature at which the pressure of outgas reaches 2×10−6Pa may be 600° C. or less or may be 550° C. or less. The rate of the total void area in the stacked substrates body of the embodiment (void area ratio) is preferably 30% or less, more preferably 20% or less, further preferably 10% or less. The void area ratio is a value calculated by dividing the total void area by the total area where transmitted light is observable, and multiplying the quotient by 100, in infrared light transmission observation. In a case where such infrared light transmission observation is difficult to perform, such a value can be determined in the same procedure by use of reflected wave in an ultrasonic microscope, transmitted wave in an ultrasonic microscope, or reflected light of infrared light, preferably by use of reflected wave in an ultrasonic microscope. (Method of Manufacturing Stacked Substrates Body) Hereinafter, a method of manufacturing a stacked substrates body of one embodiment of the invention will be described. Examples of the method of manufacturing a stacked substrates body of the embodiment include a first manufacturing method and a second manufacturing method, described below. The method of manufacturing a stacked substrates body of the invention is not limited to such methods. (First Manufacturing Method) A first method of manufacturing a stacked substrates body includes a first step of forming a film including a compound (A) which has a cationic functional group containing at least one of a primary nitrogen atom or a secondary nitrogen atom and which has a weight average molecular weight of from 90 to 400000, on a first substrate, a second step of providing a crosslinking agent (B) which has three or more —C(═O)OX groups (X is a hydrogen atom or an alkyl group having from 1 to 6 carbon atoms) in a molecule, in which from one to six of the three or more —C(═O)OX groups are —C(═O)OH groups and which has a weight average molecular weight of from 200 to 600, onto the film, a third step of layering a second substrate on a surface on which a film including the compound (A) and the crosslinking agent (B) is formed, and a heating step of heating the film including the compound (A) and the crosslinking agent (B) to a temperature of from 70° C. to 450° C., thereby forming an adhesion layer including a reaction product of the compound (A) and the crosslinking agent (B). The compound (A) preferably includes at least one selected from the group consisting of an aliphatic amine having a weight average molecular weight of from 10000 to 400000 and a compound having a siloxane bond (Si—O bond) and an amino group and having a weight average molecular weight of from 130 to 10000. Hereinafter, each of the steps of the first method of manufacturing a stacked substrates body will be described. <First Step> Examples of the method of forming the film including the compound (A) on the first substrate include a method of forming the film including the compound (A) on the substrate by use of a solution containing the compound (A). The method of forming the film is not particularly limited, and a method commonly used may be adopted. Examples of such a method commonly used include a dipping method, a spraying method, a spin coating method, and a bar coating method. For example, a bar coating method is preferably used in the case of formation of a film having a micrometer-sized thickness, and a spraying method is preferably used in the case of a film having a nanometer-sized thickness (from several nm to several hundred nm). For example, the method of forming the film including the compound (A) by a spin coating method is not particularly limited, and, for example, a method can be used which includes dropping the solution containing the compound (A) onto a surface of the first substrate with rotation of the substrate by a spin coater, and thereafter increasing the number of revolutions of the first substrate for drying. Various conditions of the method of forming the film including the compound (A) by a spin coating method, for example, the number of revolutions of the substrate, the amount and the time of dropping of the solution containing the compound (A), and the number of revolutions of the substrate in drying, are not particularly limited, and may be appropriately adjusted in consideration of the thickness of a film to be formed. <Drying Step> The first method of manufacturing a stacked substrates body may include a drying step of drying the first substrate on which the film including the compound (A) is formed, in a condition of a temperature of from 70° C. to 250° C. The temperature here refers to the temperature of a surface of the first substrate, on which the film including the compound (A) is formed. In particular, in a case in which Cu and SiO2are present in a surface of the first substrate, the surface being closer to the adhesion layer, the method can include the drying step, thereby allowing both the difficulty of remaining of a polymer on Cu and the ease of remaining of a polymer on SiO2to be more effectively satisfied. Specifically, the temperature is 70° C. or more, whereby the remaining ability of a polymer provided to SiO2is suitably maintained. The temperature is 250° C. or less, whereby a polymer can further hardly remain on Cu. The temperature is more preferably from 80° C. to 200° C., more preferably from 85° C. to 170° C., further preferably from 90° C. to 150° C. The drying in the drying step can be performed by a common method, and can be performed using, for example, a hot plate. The atmosphere where the drying is performed is not particularly limited, and may be performed, for example, under an air atmosphere or under an atmosphere of an inert gas (nitrogen gas, argon gas, helium gas, or the like). The drying time is not particularly limited, and is preferably 300 seconds or less, more preferably 200 seconds or less, further preferably 120 seconds or less, particularly preferably 80 seconds or less. The lower limit of the drying time is not particularly limited, and the lower limit can be, for example, 10 seconds, preferably 20 seconds, more preferably 30 seconds. <Washing Step> The first method of manufacturing a stacked substrates body may include a washing step of washing the first substrate on which the film including the compound (A) is formed, with a polar solvent or the like in order to remove an excess of the compound (A) provided to the first substrate. In a case in which the first method of manufacturing a stacked substrates body includes the drying step, it is preferable that the washing step is performed after the drying step, and it is more preferable that the washing step is performed after the drying step and after the second step. <Second Step> Examples of the method of providing the crosslinking agent (B) onto the film including the compound (A) include a method of providing the crosslinking agent (B) onto the film including the compound (A) by use of a solution containing the crosslinking agent (B). In a case in which the solution containing the crosslinking agent (B) is used, the crosslinking agent (B) can be provided onto the film including the compound (A) in the same manner as the method described in the first step. The first step, the drying step, the second step, and the washing step may be, if necessary, further repeated after the second step, depending on the thickness of the adhesion layer to be formed. <Third Step> A second substrate is layered on a surface on which the film including the compound (A) and the crosslinking agent (B) is formed, after the second step or after a heating step described below. Thus, a stacked substrates body where the first substrate, the film including the compound (A) and the crosslinking agent (B), and the second substrate are sequentially layered is obtained. The film including the compound (A) and the crosslinking agent (B) may be formed in advance on a surface of the second substrate, the surface being closer to the first substrate, before the third step, from the viewpoint of a more enhancement in bonding strength. The pressure at which the layering in the third step is performed is not particularly limited, and the layering is preferably performed at an absolute pressure of higher than 10−4Pa and equal to or lower than the atmospheric pressure. The absolute pressure is more preferably from 10−3Pa to the atmospheric pressure, further preferably from 100 Pa to the atmospheric pressure, particularly preferably from 1000 Pa to the atmospheric pressure. The layering in such a layering step may be performed under an air atmosphere or under an atmosphere of an inert gas (nitrogen gas, argon gas, helium gas, or the like). <Heating Step> The first method of manufacturing a stacked substrates body includes a heating step of heating the film including the compound (A) and the crosslinking agent (B) at a temperature of from 70° C. to 450° C., after the second step. The temperature here refers to the temperature of a surface of the first substrate or the second substrate, on which the film including the compound (A) and the crosslinking agent (B) is formed. Since the method includes a heating step, the solvent contained in the film including the compound (A) and the crosslinking agent (B) is removed, and the compound (A) and the crosslinking agent (B) react with each other due to heating, and form a reaction product, whereby a film including the reaction product is formed. The temperature is preferably from 100° C. to 450° C., more preferably from 100° C. to 430° C., further preferably from 150° C. to 420° C. The temperature may be from 70° C. to 250° C., may be from 80° C. to 200° C., may be from 85° C. to 170° C., or may be from 90° C. to 150° C. The pressure in heating to be performed in the heating step is not particularly limited, and is preferably an absolute pressure of higher than 17 Pa and equal to or lower than the atmospheric pressure. The absolute pressure is more preferably from 1000 Pa to the atmospheric pressure, further preferably from 5000 Pa to the atmospheric pressure, particularly preferably from 10000 Pa to the atmospheric pressure. The heating in the heating step can be performed by a common method using a furnace or a hot plate. Such a furnace that can be used is, for example, SPX-1120 manufactured by APPEX CORPORATION or VF-1000 LP manufactured by Koyo Thermo Systems Co., Ltd. The heating in the heating step may be performed under an air atmosphere or under an atmosphere of an inert gas (nitrogen gas, argon gas, helium gas, or the like). The heating time in the heating step is not particularly limited, and is, for example, 3 hours or less, preferably 1 hour or less. The lower limit of the heating time is not particularly limited, and, for example, can be 5 minutes, can be 3 minutes, or can be 30 seconds. In a case in which the film including the compound (A) and the crosslinking agent (B) is heated at from 70° C. to 250° C., the heating time may be 300 seconds or less, may be 200 seconds or less, may be 120 seconds or less, or may be 80 seconds or less. The lower limit of the heating time here can be, for example, 10 seconds, preferably 20 seconds, more preferably 30 seconds. The heating step may include a step of heating the film including the compound (A) and the crosslinking agent (B) at from 70° C. to 250° C. as described above (low-temperature heating step) and a step of heating the film at from 100° C. to 450° C. (high-temperature heating step where heating is made at a higher temperature than that in the low-temperature heating step). A surface of the first substrate, on which the film including the compound (A) and the crosslinking agent (B) is formed, may be irradiated with ultraviolet light for the purpose of a decrease in the time of the heating step. Such ultraviolet light is preferably ultraviolet light having a wavelength of from 170 nm to 230 nm, excimer light having a wavelength of 222 nm, excimer light having a wavelength of 172 nm, or the like. Such irradiation with ultraviolet light is preferably performed under an inert gas atmosphere. The heating step may be any step to be performed after the second step, or may be performed after the drying step or the washing step, if necessary, performed. The heating step may be performed before the third step, may be performed after the third step, or may be performed both before and after the third step. A stacked substrates body may be pressed at the same time as the heating in the heating step performed after the third step. Here, a heating step of performing the heating and the pressing may be performed after the third step, or the heating step and the third step may be performed in any order after the second step and thereafter such a heating step of performing the heating and the pressing may be further performed. The pressing pressure in the heating step of performing the heating and the pressing is preferably from 0.1 MPa to 50 MPa, more preferably from 0.1 MPa to 10 MPa, further preferably from 0.1 MPa to 5 MPa. The pressing apparatus that may be here used is, for example, TEST MINI PRESS manufactured by Toyo Seiki Seisaku-sho, Ltd. The heating temperature in the heating step of performing the heating and the pressing is preferably from 100° C. to 450° C., more preferably from 100° C. to 400° C., further preferably from 150° C. to 350° C. Thus, in a case in which a semiconductor circuit is formed on a substrate, any damage on the semiconductor circuit tends to be suppressed. <Pressurizing Step> The first method of manufacturing a stacked substrates body may include a pressurizing step of pressing a stacked substrates body, after the third step, preferably after the third step and after the heating step. The pressing pressure in the pressurizing step is preferably from 0.1 MPa to 50 MPa, more preferably from 0.1 MPa to 10 MPa. The pressing apparatus that may be here used is, for example TEST MINI PRESS manufactured by Toyo Seiki Seisaku-sho, Ltd. The pressurizing time is not particularly limited, and can be, for example, from 0.5 seconds to 1 hour. The temperature in the pressurizing step is preferably 10° C. or more but less than 100° C., more preferably from 10° C. to 70° C., further preferably from 15° C. to 50° C., particularly preferably from 20° C. to 30° C. The temperature here refers to the temperature of a surface of the first substrate or the second substrate, on which the film including the compound (A) and the crosslinking agent (B) is formed. It is preferable to press a stacked substrates body in at least one of the heating step or the pressurizing step in the first method of manufacturing a stacked substrates body. Such pressing of a stacked substrates body tends to allow for an increase in adhesion area and impart a more excellent bonding strength. The conditions in the pressing are as described above. <Post-Heating Step> The first method of manufacturing a stacked substrates body may include a post-heating step of heating the stacked substrates body, after the pressurizing step. The method includes the pressurizing step and the post-heating step, whereby bonding strength tends to be more excellent. Hereinafter, preferable conditions in the post-heating step will be described. Herein, description of items common to the heating step is omitted. The heating temperature in the post-heating step is preferably from 100° C. to 450° C., more preferably from 150° C. to 420° C., further preferably from 150° C. to 400° C. The heating in the post-heating step is preferably performed at an absolute pressure of higher than 17 Pa and equal to or lower than the atmospheric pressure. The absolute pressure is more preferably from 1000 Pa to the atmospheric pressure, further preferably from 5000 Pa to the atmospheric pressure, particularly preferably from 10000 Pa to the atmospheric pressure. It is preferable not to perform any pressing of the stacked substrates body in the post-heating step. (Second Manufacturing Method) A second method of manufacturing a stacked substrates body includes a fifth step of forming a film including a compound (A) which has a cationic functional group containing at least one of a primary nitrogen atom or a secondary nitrogen atom and which has a weight average molecular weight of from 90 to 400000 and a crosslinking agent (B) which has three or more —C(═O)OX groups (X is a hydrogen atom or an alkyl group having from 1 to 6 carbon atoms) in a molecule, in which from one to six of the three or more —C(═O)OX groups are —C(═O)OH groups and which has a weight average molecular weight of from 200 to 600, on a first substrate, a sixth step of layering a second substrate on a surface where the film including the compound (A) and the crosslinking agent (B) is formed, and a heating step of heating the film including the compound (A) and the crosslinking agent (B) to a temperature of from 70° C. to 450° C., thereby forming an adhesion layer including a reaction product of the compound (A) and the crosslinking agent (B). The compound (A) preferably includes at least one selected from the group consisting of an aliphatic amine having a weight average molecular weight of from 10000 to 400000 and a compound having a siloxane bond (Si—O bond) and an amino group and having a weight average molecular weight of from 130 to 10000. <Fifth Step> Examples of the method of forming the film including the compound (A) and the crosslinking agent (B), on the first substrate, include a method of forming the film including the compound (A) and the crosslinking agent (B), on the first substrate, by use of a solution containing the compound (A) and the crosslinking agent (B). The solution containing the compound (A) and the crosslinking agent (B) may be prepared by mixing the compound (A) and the crosslinking agent (B). The film including the compound (A) and the crosslinking agent (B) may be formed on the first substrate by the same method as the method described in the first step. The second manufacturing method may, if necessary, undergo the drying step and the washing step after the fifth step, as in the first manufacturing method. <Sixth Step> A second substrate is layered on a surface on which the film including the compound (A) and the crosslinking agent (B) is provided. The sixth step may be performed by the same method as that in the third step. <Heating Step> The second method of manufacturing a stacked substrates body includes a heating step of heating the film including the compound (A) and the crosslinking agent (B) at from 70° C. to 450° C., after the fifth step. The method includes the heating step, thereby allowing the compound (A) and the crosslinking agent (B) to react with each other due to heating, thereby providing a reaction product and then forming an adhesion layer as a film including the reaction product. The heating step in the second manufacturing method may be performed by the same method as that in the heating step in the first manufacturing method. The second manufacturing method may include a pressurizing step of pressing a stacked substrates body, after the sixth step, preferably after the sixth step and after the heating step. The pressurizing step in the second manufacturing method may be performed by the same method as that in the pressurizing step in the first manufacturing method. The second manufacturing method may include a post-heating step of heating the stacked substrates body, after the pressurizing step. The post-heating step in the second manufacturing method may be performed by the same method as that in the post-heating step in the first manufacturing method. It is preferable in the first manufacturing method and the second manufacturing method that the solution for formation of the adhesion layer, namely, the solution containing the compound (A), the solution containing the crosslinking agent (B), or the solution containing the compound (A) and the crosslinking agent (B), preferably contains a polar solvent (D). The polar solvent (D) may be added to the compound (A), the crosslinking agent (B), or a mixture of the compound (A) and the crosslinking agent (B) at any timing in production of the solution for formation of the adhesion layer. The timing at which other components are added is also not particularly limited. The method includes a step of, for example, providing the solution containing the compound (A) on a surface of the first substrate, thereby forming the film including the compound (A) (first step), or a step of, for example, providing the solution containing the compound (A) and the crosslinking agent (B) on a surface of the first substrate, thereby forming the film including the compound (A) and the crosslinking agent (B) (fifth step). In a case in which the solution containing the compound (A), or the solution containing the compound (A) and the crosslinking agent (B) is provided on the first substrate, the content of the compound (A) in the solution containing the compound (A), or the content of the compound (A) in the solution containing the compound (A) and the crosslinking agent (B) is not particularly limited, and can be, for example, from 0.001% by mass to 30% by mass or less and is preferably from 0.01% by mass to 20% by mass or less, more preferably from 0.04% by mass to 20% by mass or less, with respect to the entire solution. The method includes a step of, for example, forming the film including the compound (A) on a surface of the first substrate and thereafter providing the solution containing the crosslinking agent (B) on the film, thereby providing the crosslinking agent (B) on the film (second step), or a step of, for example, providing the solution containing the compound (A) and the crosslinking agent (B) on a surface of the first substrate, thereby forming the film including the compound (A) and the crosslinking agent (B) (fifth step). In a case in which the solution containing the crosslinking agent (B), or the solution containing the compound (A) and the crosslinking agent (B) is provided on the first substrate, the content of the crosslinking agent (B) in the solution containing the crosslinking agent (B), or the content of the crosslinking agent (B) in the solution containing the compound (A) and the crosslinking agent (B) is not particularly limited, and, for example, the ratio (COOH/N) of the number of carboxy groups in the crosslinking agent (B) to the number of all the nitrogen atoms in the compound (A) is preferably from 0.1 to 3.0, more preferably from 0.3 to 2.5, further preferably from 0.4 to 2.2. A ratio COOH/N of from 0.1 to 3.0 enables a thermally crosslinked structure such as amide, amide-imide, or imide to be easily formed in the heating step in the first manufacturing method and the second manufacturing method, and thus enables a film more excellent in heat resistance to be manufactured. In the first manufacturing method and the second manufacturing method, at least one additive (C) selected from the group consisting of an acid (C-1) having a carboxy group and having a weight average molecular weight of from 46 to 195 and a base (C-2) having a nitrogen atom, having a weight average molecular weight of from 17 to 120, and not having any ring structure may be added to the compound (A) or the crosslinking agent (B). The timing at which the additive (C) is added is not particularly limited. In a case in which the acid (C-1) is added as the additive (C) in the first manufacturing method, it is preferable to form a film including the acid (C-1) and the compound (A) in the first step and thereafter provide the crosslinking agent (B) onto the film in the second step. Thus, whitening and gelation of a composition can be suitably suppressed in mixing of the compound (A) and the crosslinking agent (B). It is herein preferable to suppress gelation from the viewpoint of making the thickness of the adhesion layer uniform. In a case in which the acid (C-1) is added as the additive (C) in the second manufacturing method, it is preferable to mix a mixture of the acid (C-1) and the compound (A), with the crosslinking agent (B). In other words, it is preferable to mix the compound (A) and the acid (C-1) in advance before mixing of the compound (A) and the crosslinking agent (B). Thus, whitening and gelation of a composition (gelation is not preferable because any time may be taken for making the composition transparent) can be suitably suppressed in mixing of the compound (A) and the crosslinking agent (B). In a case in which the base (C-2) is added as the additive (C) in the first manufacturing method, it is preferable to form a film including the compound (A) in the first step and thereafter provide a mixture of the crosslinking agent (B) and the base (C-2) onto the film in the second step. Thus, whitening and gelation of a composition can be suitably suppressed in mixing of the compound (A) and the crosslinking agent (B). In a case in which the base (C-2) is added as the additive (C) in the second manufacturing method, it is preferable to mix a mixture of the base (C-2) and the crosslinking agent (B) with the compound (A). In other words, it is preferable to mix the crosslinking agent (B) and the base (C-2) in advance before mixing of the compound (A) and the crosslinking agent (B). Thus, whitening and gelation of a composition (gelation is not preferable because any time may be taken for making the composition transparent) can be suitably suppressed in mixing of the compound (A) and the crosslinking agent (B). The first manufacturing method and the second manufacturing method tend to allow the thickness of the adhesion layer to be thinner and allow for a more enhancement in bonding strength as compared with, for example, a case in which the adhesion layer is formed by coating a substrate with polyamic acid. EXAMPLES Hereinafter, the invention will be more specifically described with reference to Examples, but the invention is not intended to be limited to these Examples. Hereinafter, water was used in the case of no indication of any solvent. Hereinafter, ultrapure water (MILLI-Q water manufactured by Millipore Corporation, resistance of 18 MΩ·cm (25° C.) or less) was used as “water”. Each solution for formation of the adhesion layer was prepared in Example 1 to Example 1 and Comparative Example 1 to Comparative Example 5. The details are as indicated below. A solution of the compound (A), a solution of the crosslinking agent (B), a solution where the base (C-2) was added to the crosslinking agent (B), and other solution were each mixed after checking of no precipitate present in each of the solutions. Example 1 A solution containing the compound (A) was obtained by adding 4.0 g of 3-aminopropyldiethoxymethylsilane (3APDES; (3-Aminopropyl)diethoxymethylsilane) prepared as the compound (A), to 56.0 g of 1-propanol (1PrOH), further adding 20.0 g of an aqueous 8.8% by mass formic acid (FA) solution for dissolution so that the concentration of 3APDES was 5% by mass, and stirring the resultant at room temperature for 1 hour and then warming it in a water bath at 60° C. for 1 hour, 3APDES was here present as a hydrolysate in the solution. The hydrolysate of 3APDES had a structure where one methyl group being a non-crosslinkable group, two hydroxyl groups being crosslinkable groups, and one aminopropyl group being a crosslinkable group were bound to Si. In other words, the (non-crosslinkable group)/Si was 1. 1-Propyl half ester trimellitic acid (1PrheTMA; 1-propyl half ester TMA) was prepared as the crosslinking agent (B), 1 PrheTMA was produced by adding trimellitic anhydride to 1-propanol, and completely dissolving a trimellitic anhydride powder. Next, a solution 1 containing the compound (A), the crosslinking agent (B) and the acid (C-1) was prepared by mixing the solution containing the compound (A) and the solution containing the crosslinking agent (B) so that the concentration shown in Table 1 was achieved. In Table 1, the concentration in parentheses with respect to 3APDES (2% by mass) represents the concentration of 3APDES in the solution containing the compound (A) and the crosslinking agent (B). The numerical value in parentheses with respect to 1PrheTMA [1.03] represents the ratio (COOH/N) of the number of carboxy groups in 1PrheTMA as the crosslinking agent (B) to the number of all the nitrogen atoms in 3APDES as the compound (A). The numerical value in parentheses with respect to FA, 1.83, represents the ratio (COOH/N) of the number of carboxy groups in FA as the acid (C-1) to the number of all the nitrogen atoms in 3APDES as the compound (A). The concentration in parentheses with respect to 1 PrOH (86.6% by mass) represents the concentration of 1PrOH in the solution containing the compound (A) and the crosslinking agent (B). Example 2 A solution 2 containing the compound (A), the crosslinking agent (B), and the acid (C-1) was prepared by preparing 2.0 g of the solution 1 produced in Example 1, and adding the solution to 12.0 g of 1PrOH and further adding 6.0 g of water. In Table 1, the concentration in parentheses with respect to 3APDES (0.2% by mass) represents the concentration of 3APDES in the solution containing the compound (A) and the crosslinking agent (B). The numerical value in parentheses with respect to 1PrheTMA [1.03] represents the ratio (COOH/N) of the number of carboxy groups in 1PrheTMA as the crosslinking agent (B) to the number of all the nitrogen atoms in 3APDES as the compound (A). The numerical value in parentheses with respect to FA, 1.83, represents the ratio (COOH/N) of the number of carboxy groups in FA as the acid (C-1) to the number of all the nitrogen atoms in 3APDES as the compound (A). The concentration in parentheses with respect to 1PrOH (68.6% by mass) represents the concentration of 1PrOH in the solution containing the compound (A) and the crosslinking agent (B). Example 3 Polyethyleneimine (Mw=70,000, primary nitrogen atom/secondary nitrogen atom/tertiary nitrogen atom=31/40/29) being a branched polyethyleneimine (BPEI), manufactured by BASF SE, was prepared as the compound (A). Production was made by adding 12.67 g of water to 7.0 g of trimellitic acid (TMA) as the crosslinking agent (B), and further adding 30.33 g of an aqueous 8.4% by mass ammonia (NH3) solution for complete dissolution of TMA. Next, the solution containing the compound (A) and the solution containing the crosslinking agent (B) were mixed so that the concentration shown in Table 1 was achieved, and a solution 3 containing the compound (A) and the crosslinking agent (B) was prepared. In Table 1, the concentration in parentheses with respect to BPEI (0.15% by mass) represents the concentration of BPEI in the solution containing the compound (A) and the crosslinking agent (B). The numerical value in parentheses with respect to TMA [1.5] represents the ratio (COOH/N) of the number of carboxy groups in TMA as the crosslinking agent (B) to the number of all the nitrogen atoms in BPEI as the compound (A). The numerical value in parentheses with respect to NH3, 1.5, represents the ratio (N/COOH) of the number of all the nitrogen atoms in NH3as the base (C-2) to the number of carboxy groups in TMA as the crosslinking agent (B). Example 4 to Example 11 Each of solutions 1 and 4 to 8 was prepared in the same manner as in Example 1 except that the components and the amounts thereof were changed as described in Table 1 in each of Examples 4 to 11. In Table 1, the concentrations in parentheses of 3APDES, 3APTES, and BPEI, each serving as the compound (A), represent the concentrations of 3APDES, 3APTES, and BPEI in the solution containing the compound (A) and the crosslinking agent (B), respectively. The numerical values in parentheses with respect to 1PrheTMA, TMA, ehePMA, PMA, and TMSA, each serving as the crosslinking agent (B), each represent the ratio (COOH/N) of the number of carboxy groups in the crosslinking agent (B) to the number of all the nitrogen atoms in the compound (A). The numerical value in parentheses with respect to FA represents the ratio (COOH/N) of the number of carboxy groups in FA as the acid (C-1) to the number of all the nitrogen atoms in the compound (A). The numerical value in parentheses with respect to NH3represents the ratio (N/COOH) of the number of all the nitrogen atoms in NH3as the base (C-2) to the number of carboxy groups in the crosslinking agent (B). The concentrations in parentheses with respect to 1 PrOH and EtOH represent the concentrations of 1PrOH and EtOH in the solution containing the compound (A) and the crosslinking agent (B), respectively. Abbreviations of the compound (A), the crosslinking agent (B), the acid (C-1), the base (C-2), and the polar solvent (D) in Table 1 and Table 2 are as follows. 3APDES was hydrolyzed in the solution as described above. 3APTES was also hydrolyzed in the solution, and the hydrolysate of 3APTES had a structure where no non-crosslinkable group was present on Si, and three hydroxyl groups being crosslinkable groups and one aminopropyl group being a crosslinkable group were bound to Si. In other words, the (non-crosslinkable group)/Si was 0. <Compound (A)> 3 APDES: 3-aminopropyldiethoxymethylsilane (hydrolyzed in solution) 3APTES: 3-aminopropyltriethoxysilane (hydrolyzed in solution) BPEI: branched polyethyleneimine <Crosslinking Agent (B)> 1 PrheTMA: 1-propyl half ester trimellitic acid TMA: trimellitic acid ehePMA: ethyl half ester pyromellitic acid PMA: pyromellitic acid TMSA: 1,3,5-benzenetricarboxylic acid <Acid (C-1)> FA: formic acid <Base (C-2)> NH3: ammonia <Polar Solvent (DP) 1PrOH: 1-propanol EtOH: ethanol Comparative Example 1 to Comparative Example 5 Solutions 9 to 13 were prepared in the same manner as in Example 1 except that the components and the amounts thereof were changed as described in Table 2 in Comparative Examples 1 to 5, respectively. In Comparative Example 1, biphenyltetracarboxylic acid dianhydride (BPDA) and para-phenylenediamine (pDA) were allowed to react with each other in an N-methyl-2-pyrrolidone (NMP) solvent (97.5% by mass), thereby preparing a solution 9 containing polyamic acid (2.5% by mass) made of BPDA and pDA. In Comparative Example 2, p-xylenediamine (pXDA) was dissolved in a mixed solvent of water and 1-propanol (1 PrOH) and thereafter left to still stand overnight, thereby providing a pXDA solution 1. Ammonia (NH3) and water were mixed with 1,3,5-benzenetricarboxylic acid (TMSA), thereby providing a mixed solution 1 of TMSA and NH3. Next, the pXDA solution 1, the mixed solution 1 of TMSA and NH3, and water were mixed so that the concentration shown in Table 2 was achieved, thereby preparing a solution 10. In Comparative Example 3 and Comparative Example 4, BPEI and 3APDES were used for mixing with water so that the concentrations in parentheses were achieved, and MA was mixed with each of the resulting solutions so that the ratios (COOH/N) of the numbers of carboxy groups in malonic acid (MA) to the numbers of all the nitrogen atoms in BPEI and 3APDES corresponded to a numerical value in parentheses of 1.0, thereby preparing a solution 11 and a solution 12, respectively. In Comparative Example 5, a solution containing a hydrolysate of tetraethoxysilane (TEOS) and a siloxane polymer in a mixture of ethanol, water and nitric acid was obtained according to the A2** method described in THE JOURNAL OF PHYSICAL CHEMISTRY C (2011), vol. 115, pages 12981-12989, and thereafter water, ethanol, and 1-propanol were added so as to be in amounts of numerical numbers in parentheses, thereby preparing a solution 13. The concentrations in parentheses with respect to 1 PrOH, EtOH, and nitric acid represent the concentrations of 1PrOH, EtOH, and nitric acid in the solution, respectively. <Formation of Adhesion Layer> A silicon substrate having a diameter of 4 inches (silicon wafer) was prepared as a substrate to be coated with the resulting solution containing the compound (A) and the crosslinking agent (B). After the silicon substrate was treated with UV (ultraviolet) ozone for 5 minutes, the silicon substrate was placed on a spin coater, 2.0 mL of the composition prepared in each of Examples and each of Comparative Examples was dropped thereon at a constant speed for 10 seconds and held for 13 seconds, and thereafter the resultant was rotated at 2000 rpm (rpm meaning the rotational speed) for 1 second and at 600 rpm for 30 seconds, and then rotated at 2000 rpm for 10 seconds for drying. Thus, an adhesion layer was formed on the silicon substrate. Next, the adhesion layer was heated and dried at 125° C. for 1 minute in the heating step (low-temperature heating step). Each of the resulting adhesion layers of Examples and Comparative Examples was evaluated with respect to the thickness of each of the adhesion layers, the crosslinked structure, the surface smoothness, and the thickness uniformity in wafer. (Measurement of Thickness of Adhesion Layer) The thickness of each of the adhesion layers was measured with an ellipsometer (optical porosimeter (PS-1100) manufactured by manufactured by SEMILAB JAPAN K.K.). In the case of a thickness of 10 nm or more, fitting was performed with an optical model of air/(Cauchy+Lorenz oscillator model)/natural oxide film/silicon substrate. In the case of a thickness of less than 10 nm, fitting was performed with an optical model of air/SiO2/natural oxide film/silicon substrate. The results are shown in Table 1 and Table 2. (Confirmation of Crosslinked Structure) The crosslinked structure of each of the adhesion layers was measured by FT-IR (Fourier transform infrared spectroscopy). The analyzer used was as follows. —FT-IR Analyzer— Infrared absorption analyzer (DIGILAB Excalibur (manufactured by Digilab Inc.)) —Measurement Conditions— IR source: air-cooled ceramic. Beam splitter: wide range KBr, Detector: Peltier cooling DTGS, Measurement wavenumber range: from 7500 cm−1to 400 cm−1, Resolution: 4 cm−1, Integration times: 256, Background: use of Si bare wafer, Measurement atmosphere: N2(10 L/min), Incident angle of IR (infrared): 72° (=Brewster angle of Si) —Determination Conditions— An imide bond was determined by the presence of vibration peaks at 1770 cm−1and 1720 cm−1. An amide bond was determined by the presence of vibration peaks at 1650 cm−1and 1550 cm−1. A siloxane bond was determined by the presence of vibration peaks at from 1000 to 1100 cm−1. The results are shown in Table 1 and Table 2. A sample after heating under a nitrogen atmosphere at 250° C. for 1 hour was used in FT-IR measurement. (Confirmation of Surface Smoothness) —SPM Morphology Observation— The surface smoothness of the adhesion layer was evaluated by morphology observation with SPM. Measurement was carried out in an area of 3 microns×3 microns square in a dynamic force microscope mode by use of SPA 400 (manufactured by Hitachi High-Technologies Corporation) as a scanning probe microscope (SPM). In a case in which the root mean square surface roughness (RMS) measured with SPM was 0.5 nm or less, the surface was determined to be “smooth”. The results are shown in Table 1 and Table 2. Any film after heating at 400° C. for 10 minutes was an objective of SPM morphology observation. (Thickness Uniformity in Wafer) The surface of each of the adhesion layers was visually observed, and any adhesion layer in which any of unevenness such as an interference pattern, a particle (an aggregate of any component in the adhesion layer), or cissing (a portion where the adhesion layer was not partially formed) was observed was rated as “C” with no uniformity in wafer. Each adhesion layer other than those rated as “C” was subjected to thickness distribution measurement with an ellipsometer (optical porosimeter (PS-1100) manufactured by manufactured by SEMILAB JAPAN K.K.). Specifically, a silicon wafer having a diameter of 4 inches (before bonding) on which the composition was formed into a film was cut to a size of 1 cm×1 cm square, and the film thickness in the wafer was measured with respect to 1 cm. Any sample where the difference in film thickness between the maximum film thickness and the minimum film thickness was 10% or less of average film thickness was determined to be excellent in thickness uniformity in wafer and was rated as “A”. The results are shown in Table 1 and Table 2. <Formation of Stacked Substrates Body> Example 1 to Example 3 and Comparative Example 1 to Comparative Example 4 A silicon bare wafer having a diameter of 4 inches (second substrate), treated with UV ozone for 5 minutes, was attached onto a silicon wafer (first substrate), on which the adhesion layer was formed. Such an operation corresponded to the sixth step. Next, the resultant was subjected to thermal compression bonding in a pressing apparatus at 250° C. and at 1 MPa for 1 hour (60 minutes), thereby providing a stacked substrates body. Such an operation corresponded to the heating step. <Formation of Stacked Substrates Body> Example 4 A silicon wafer (first substrate) on which the adhesion layer was formed was heated under a nitrogen atmosphere at 400° C. for 10 minutes. Next, a silicon bare wafer having a diameter of 4 inches (second substrate), treated with UV ozone for 5 minutes, was attached onto the silicon wafer (first substrate) on which the adhesion layer was formed. The resultant was subjected to thermal compression bonding in a pressing apparatus at 250° C. and at 1 MPa for 1 hour (60 minutes), thereby providing a stacked substrates body. <Formation of Stacked Substrates Body> Example 5 and Example 7 to Example 11 A silicon wafer (first substrate) on which the adhesion layer was formed was heated under a nitrogen atmosphere at 400° C. for 10 minutes. Next, a silicon bare wafer having a diameter of 4 inches (second substrate), treated with UV ozone for 5 minutes, was attached onto the silicon wafer (first substrate) on which the adhesion layer was formed. The resultant was subjected to compression bonding in a pressing apparatus at 23° C. and at 1 MPa for 1 minute, thereby providing a stacked substrates body. Such an operation corresponded to the pressurizing step. <Formation of Stacked Substrates Body> Example 6 and Comparative Example 5 A silicon wafer (first substrate) on which the adhesion layer was formed was heated under a nitrogen atmosphere at 400° C. for 10 minutes. Next, a silicon bare wafer having a diameter of 4 inches (second substrate), treated with UV ozone for 5 minutes, was attached onto the silicon wafer (first substrate) on which the adhesion layer was formed. The resultant was subjected to compression bonding in a pressing apparatus at 23° C. and at 1 MPa for 1 minute. Next, the resultant was heated under a nitrogen atmosphere at 400° C. for 30 minutes, thereby providing a stacked substrates body. Each of the resulting substrate laminated bodies of Examples and Comparative Examples was evaluated with respect to the tensile bonding strength, the outgas, and the void. (Tensile Bonding Strength) Each of the substrate laminated bodies was cut to a size of 1 cm×1 cm square by use of a dicer (DAD 3240 manufactured by DISCO). Subsequently, a metal pin having a diameter of 7 mm, with an epoxy resin, was allowed to adhere to both the upper and lower surfaces of each of the stacked substrates body cut (cured by epoxy at room temperature), thereby forming a sample for tensile bonding strength measurement. The sample for tensile bonding strength measurement was used to perform measurement of a yield point with a tensile tester. Each of the tensile bonding strengths determined from such a yield point is shown in Table 1 and Table 2. (Measurement of Outgas) Each of the substrate laminated bodies was cut to a size of 7 mm×7 mm square by use of a dicer (DAD 3240 manufactured by DISCO), thereby producing a sample for outgas measurement. The sample for outgas measurement was used for measurement of the amount of outgas due to heating, with EMD-WA 1000S manufactured by ESCO Co., Ltd. The atmosphere pressure (base pressure) was 10−7Pa and the rate of temperature rise was 30° C./min. The surface temperature of the silicon substrate was determined as the temperature of a thermocouple under a stage, calibrated by use of a peak derived from the outgas from a standard specimen (H+-injected silicon, CaC2O4-dropped and Ar+-injected silicon wafer). The temperature at which the pressure of outgas reached 2×10−6Pa was determined with temperature rise. The results are shown in Table 1 and Table 2. A higher temperature means less occurrence of outgas. (Void Measurement) Each of the substrate laminated bodies was disposed on a stage on an IR lamp of IR 200 manufactured by SUESS MICROTEC SE. Next, void observation was performed by an IR camera disposed on the stage, with the second substrate being interposed. The total void area was divided by the total area where transmitted light could be observed, and thus the void area ratio was calculated. A void area ratio of 30% or less was rated as “favorable”, and a void area ratio of more than 30% was rated as “poor”. The results are shown in Table 1 and Table 2. TABLE 1Manufacturing conditionsHeating stepEvaluation resultsLow-High-ThicknessTemp. attemp.temp.High-temp.Post-ofTensileoutgasheatingheatingheating stepPressurizingheatingadhesionThicknessCross-bondingpressurestepstep(pressing)stepsteplayeruniformitylinkedstrengthof 2 × 10−6PaSampleTe*Ti*Te*Ti*Te*Pr*Ti*Te*Pr*Ti*Te*Ti*(nm)Smoothnessin waferstructure(mPa)Void(° C.)Ex. 1Solution1251——250160—————85.4SmoothAAmide->25.8—4501:3APDES (2% by mass) +imide + siloxane1prheTMA[1.03] + FA{1.83} +1PrOH (86.6% by mass)Ex. 2Solution1251——250160—————8.5SmoothAAmide->5——2:3APDES (0.2%imide + siloxaneby mass) +1prheTMA[1.03] +FA{1.83} +1PrOH (68.6%by mass)Ex. 3Solution 3:BPEI1251——250160—————6.9SmoothAAmide->5——(0.15% by mass) +imideTMA [1.5] +NH3<1.5>Ex. 4Solution125140010250160—————104SmoothAImide + siloxane>10.7Favorable—4:3APDES (2% by mass) +ehePMA[1] +1PrOH (33.3% by mass) +EtOH (31.7% by mass)Ex. 5Solution125140010———2311104SmoothAImide + siloxane>18.5Favorable5154:3APDES (2% bymass) +ehePMA[1] +1PrOH (33.3%by mass) +EtOH (31.7% bymass)Ex. 6Solution125140010———231140030104SmoothAImide + siloxane>30.9Favorable5314:3APDES (2% bymass) +ehePMA[1] +1PrOH (33.3%by mass) +EtOH (31.7% bymass)Ex. 7Solution125140010———231185.9SmoothAAmide->17.1Favorable4871:3APDES (2% byimide + siloxanemass) +1prheTMA[1.03] +FA{1.83} +1PrOH (86.6%by mass)Ex. 8Solution125140010———231132SmoothAAmide->16.0Favorable4885:3APTES(2% byimide + siloxanemass) +PMA[1] +NH3<1>Ex. 9Solution125140010———231128.7SmoothAAmide +>13.7Favorable4906:3APTES(2% bysiloxanemass) +TMSA[0.5] +NH3<1.5>Ex. 10Solution125140010———2311720SmoothAImide + siloxane>14.5Favorable4447:3APDES (10%by mass) +ehePMA[1] +1PrOH (33.3%by mass) +EtOH (25.2% bymass)Ex. 11Solution125140010———23112536SmoothAImide + siloxane>16.8Favorable4288:3APDES (20%by mass) +ehePMA[1] +1PrOH (2.5% bymass) +EtOH (41.3% bymass)“Ex.” represents “Example”,“Temp.” represents “Temperature”,“Te*” represents “Temperature (° C.)”,“Ti*” represents “Time (min)”, and“Pr*” represents “Pressure (MPa)”. TABLE 2Manufacturing conditionsHeating stepLow-High-High-temp.temperaturetemperaturePost-heatingheatingheating stepPressurizingheatingstepstep(pressing)stepstepSampleTe*Ti*Te*Ti*Te*Pr*Ti*Te*Pr*Ti*Te*Ti*Comp.Solution1251——250160—————Ex. 19:BPDA-pDApolyamicacid(2.5% bymass) +NMP(97.5% bymass)Comp.Solution1251——250160—————Ex. 210:pxDA(3% bymass) +TMSA[1] +NH3<1.5> +1PrOH (12% bymass)Comp.Solution 11:BPEI1251——250160—————Ex. 3(0.15% by mass) +MA[1.0]Comp.Solution1251——250160—————Ex. 412:3APDES (2%by mass) +MA[1.0]Comp.Solution125140010———231140030Ex. 513:TEOS(8.4%by mass) +EtOH (12.9%by mass) +1PrOH (75% bymass) +HNO3(0.075%by mass)Evaluation resultsTemp. atThick-outgasnessThick-pressureofnessTensileofadhesionuni-Cross-bonding2 × 10−6layerSmooth-formitylinkedstrengthPaSample(nm)nessin waferstructure(mpa)Void(° C.)Comp.Solution2000—CImideNG——Ex. 19:BPDA-pDA(uneven-(releasedpolyamicness)inacid(2.5% bydicing)mass) +NMP(97.5% bymass)Comp.Solution236—C—NGPoor370Ex. 210:pxDA(particle)(released(3% byinmass) +dicing)TMSA[1] +NH3<1.5> +1PrOH(12% bymass)Comp.Solution 11:1.2———0——Ex. 3BPEI(0.15% bymass) +MA[1.0]Comp.Solution——C——Poor—Ex. 412:3APDES(partial(2%cissing)by mass) +MA[1.0]Comp.Solution73.5SmoothASiloxaneNGPoor354Ex. 513:TEOS(released(8.4%inby mass) +dicing)EtOH(12.9%by mass) +1PrOH(75% bymass) +HNO3(0.075%by mass)“Comp. Ex.” represents “Comparative Example”,“Temp.” represents “Temperature”,“Te*” represents “Temperature(° C.)”,“Ti*” represents “Time (min)”, and“Pr*” represents “Pressure (MPa)”. It was found that each of the substrate laminated bodies of Examples had a bonding strength of 5 MPa or more and a high bonding strength was obtained by forming the reaction product of the compound (A) and the crosslinking agent (B) into the adhesion layer. It was also found that a thin film of from 6.9 nm to 2536 nm was formed on each of the adhesion layers used in Examples 1 to 11. The stacked substrates body of Comparative Example 4 was not suitable as a stacked substrates body because a plurality of portions having a size of several mm, with cissing of the adhesion layer, was caused on the silicon wafer. The substrate laminated bodies of Examples 4 to 11 where void evaluation was performed were suppressed in the occurrence of void as compared with those of Comparative Example 2, Comparative Example 4 and Comparative Example 5 where void evaluation was performed. The substrate laminated bodies of Example 1 and Examples 5 to 11 where outgas evaluation was performed exhibited a high temperature at which the pressure of outgas reached 2×10−6Pa and were suppressed in the occurrence of outgas as compared with those of Comparative Example 2 and Comparative Example 5 where outgas evaluation was performed. The stacked substrates body of each of Examples is presumed based on the above to hardly cause unintended releasing to occur as compared with the stacked substrates body of each of Comparative Examples. The disclosure of Japanese Patent Application No. 2017-090591 filed on Apr. 28, 2017 is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described in this specification are incorporated herein by reference to the same extent as if each individual document, patent application, and technical specification were specifically and individually indicated to be incorporated by reference. | 103,384 |
11859111 | DETAILED DESCRIPTION The silicone pressure sensitive adhesive composition (composition) herein comprises (A) a polydiorganosiloxane gum, (B) a polyorganosilicate resin, (C) a polyorganohydrogensiloxane, (D) a polydialkylcyclosiloxane, (E) a hydrosilylation reaction catalyst, (F) a hydrosilylation reaction catalyst inhibitor, and (G) an organic solvent. (A) Alkenyl-Functional Polydiorganosiloxane Gum Starting material (A) in the composition is a polydiorganosiloxane gum (gum). The gum may have unit formula (R13SiO1/2)a(R12R2SiO1/2)b(R12SiO2/2)c(R1R2SiO2/2)d, where each R1is an independently selected alkyl group of 1 to 10 carbon atoms; each R2is an independently selected alkenyl group with 2 to 10 carbon atoms; subscript a is 0, 1, or 2; subscript b is 0, 1, or 2; a quantity (a+b)=2, subscript c≥0; subscript d≥0; a quantity (c+d) has a value sufficient to provide the gum with a molecular weight ≥400,000 Da; and a quantity (b+d) is sufficient to provide a silicon bonded alkenyl content of at least 0.01 weight % based on weight of the gum. Alternatively, the quantity (b+d) may have a value sufficient to provide the gum with an alkenyl content of at least 0.05%, and alternatively at least 0.06 weight %. At the same time, the quantity (b+d) may have a value sufficient to provide the gum with an alkenyl content up to 0.1%, alternatively up to 0.07% based on the weight of the gum. Alternatively, the quantity (c+d) may have a value sufficient to provide the gum with a molecular weight of 400,000 Da to 1,000,000 Da; alternatively 500,000 Da to 900,000 Da; and alternatively 600,000 Da to 800,000 Da. The alkyl groups for R1may be methyl, ethyl, propyl (including n-propyl and/or isopropyl), butyl (including n-butyl, t-butyl, sec-butyl, and/or isobutyl), pentyl (including cyclopentyl, n-pentyl, and branched isomeric species with 5 carbon atoms), hexyl (including cyclohexyl, n-hexyl, and branched isomeric species with 6 carbon atoms), heptyl (including cycloheptyl, n-heptyl, and branched isomeric species with 7 carbon atoms), octyl (including cyclooctyl, n-octyl, and branched isomeric species with 8 carbon atoms), nonyl (including cyclononyl, n-nonyl, and branched isomeric species with 9 carbon atoms), and decyl (including cyclodecyl, n-decyl, and branched isomeric species with 10 carbon atoms). Alternatively, each R1may be methyl, ethyl, propyl or butyl; alternatively methyl or ethyl; and alternatively methyl. The alkenyl groups for R2are capable of undergoing hydrosilylation reaction with the silicon bonded hydrogen atoms (SiH) of starting material (C). Suitable alkenyl groups for R2may be selected from the group consisting of vinyl, allyl, and hexenyl; alternatively vinyl and hexenyl; and alternatively vinyl. Examples of suitable gums include a trimethylsiloxy-terminated poly(dimethylsiloxane/methylvinylsiloxane) copolymer, a dimethylvinylsiloxy-terminated polydimethylsiloxane homopolymer, a dimethylvinylsiloxy-terminated poly(dimethylsiloxane/methylvinylsiloxane) copolymer, a dimethylhexenylsiloxy-terminated poly(dimethylsiloxane/methylhexenylsiloxane) copolymer, a dimethylhexenylsiloxy-terminated polydimethylsiloxane homopolymer, a dimethylhexenylsiloxy-terminated poly(dimethylsiloxane/methylhexenylsiloxane) copolymer, and a combination of two or more thereof. Gums can be made by known methods, such as those described in U.S. Pat. No. 3,983,298 to Hahn, et al.; U.S. Pat. No. 7,728,080 to Aoki; and U.S. Pat. No. 8,754,174 to Aoki; e.g., by catalytically polymerizing octamethylcyclotetrasiloxane or similar oligomer with a silane compound or a siloxane compound having dimethylvinylsiloxane units and a silane compound or a siloxane compound having dimethylsiloxane units and/or methylvinylsiloxane units. One skilled in the art would recognize that the thus polymerized reaction product contains low molecular weight cyclic siloxanes, and therefore, the low molecular cyclic siloxanes may be fully or partially removed, e.g., by stripping or distilling at elevated temperature and/or under reduced pressure by introducing an inert gas. The gum selected for use in the silicone pressure sensitive adhesive composition may be one gum or may comprise more than one gum, where the gums differ in at least one property such as molecular weight, selection of alkyl and/or alkenyl groups, type of terminal siloxane units, and sequence of the siloxane units in the polymer backbone. (B) Polyorganosilicate Resin The composition further comprises (B) a polyorganosilicate resin (resin). The resin may comprise unit formula (R13SiO1/2)e(R12R2SiO1/2)f(SiO4/2)g(ZO1/2)h, where R1and R2are as described above, Z is a hydrogen atom or an alkyl group of 1 to 6 carbon atoms, subscripts e, f, g, and h represent mole fractions of each unit, e≥0, f≥0, g>0, h≥0, and a quantity (e+f+g)=1. Subscripts e, f, and g may have values such that a molar ratio of monofunctional units, i.e., the units (R13SiO1/2) and (R12R2SiO1/2) to quadrifunctional units, i.e., units of formula (SiO4/2) are present in a ratio of 0.6:1 to 1.1:1, alternatively 0.9:1 to 1.1:1. Subscript h may have a value sufficient to give the resin a (ZO1/2) group content up to 5.0 weight %, alternatively 0 to 4.5%, alternatively 0 to 4.0%, alternatively 0 to 3.5%, alternatively 0 to 3.0%, alternatively 0 to 2.5%, alternatively 0 to 2.0%, alternatively 0.5 to 1.5%, and alternatively 0 to 1.1% based on weight of the resin. Alternatively, Z may be hydrogen, and the resin may have a hydroxyl group content of 0 to 5.0%, alternatively 0 to 4.5%, alternatively 0 to 4.0%, alternatively 0 to 3.5%, alternatively 0 to 3.0%, alternatively 0 to 2.5%, alternatively 0 to 2.0%, alternatively 0.5 to 1.5%, and alternatively 0.9% to 1.1% based on weight of the resin. The resin may optionally further comprise up to 20% of difunctional units, e.g., of formula (R12SiO2/2) and/or trifunctional units of formula (R1SiO3/2). The resin may have a number average molecular weight (Mn) measured by GPC of 3,000 Da to 10,000 Da, alternatively 4,000 Da to 10,000 Da, alternatively 5,000 Da to 8,000 Da, alternatively 6,000 Da to 7,000 Da. Alternatively, the resin may have a weight average molecular weight (Mw) of 5,000 Da to 50,000 Da, alternatively 8,000 Da to 40,000 Da, alternatively 10,000 Da to 30,000 Da, alternatively 13,000 Da to 20,000 Da. The polyorganosilicate resin may be prepared by known methods, such as those disclosed in U.S. Pat. No. 8,017,712 to Berry, et al. and the references cited therein and U.S. Pat. No. 10,351,742 to Brown, et al. and the references cited therein. Polyorganosilicate resins are also commercially available from various sources such as Dow Silicones Corporation of Midland, Michigan, USA; Momentive Performance Materials of Albany, New York, USA, and Bluestar Silicones USA Corp. of East Brunswick, New Jersey, USA. For example, DOWSIL™ MQ-1600 Solid Resin, DOWSIL™ MQ-1601 Solid Resin, and DOWSIL™ MQ-1640 Flake Resin are available from Dow Silicones Corporation. The resin is present in the composition in an amount sufficient to provide a molar ratio of amount of (B) resin to amount of (A) gum (i.e., Resin:Gum ratio) of 1.5:1 to 0.5:1; alternatively 1.25:1 to 0.6:1; alternatively 1:1 to 0.7:1; and alternatively 0.9:1 to 0.8:1. The resin selected for use in the silicone pressure sensitive adhesive composition may be one resin or may comprise more than one resin, where the resins differ in at least one property such as molecular weight, selection of alkyl and/or alkenyl groups, type of terminal siloxane units, and molecular weight. (C) Polyorganohydrogensiloxane Crosslinker Starting material (C) is a polyorganohydrogensiloxane that may have unit formula (c1): (R13SiO1/2)k(R12HSiO1/2)m(R1HSiO2/2)i(R12SiO2/2)j, where R1is an alkyl group as described above, and subscripts k, m, i, and j represent numbers of each unit in the formula and have values such that k is 0, 1, or 2; m is 0, 1, or 2; a quantity (k+m)=2; i>0; and j≥0. A quantity (i+j+k+m) is sufficient to provide the polyorganohydrogensiloxane with a degree of polymerization of 5 to 2000, alternatively 10 to 1000, alternatively 30 to 100; alternatively 50 to 80. Alternatively, when k=2, m=0, and j=0, the polyorganohydrogensiloxane may have unit formula (c2): (R13SiO1/2)2(HR1SiO2/2)i, where R1is an alkyl group as described above, subscript i is sufficient to give the polyorganohydrogensiloxane a viscosity of 10 to 50 cSt, alternatively 20 to 40 cSt, measured at 25° C. and 0.1 to 50 RPM on a Brookfield DV-III cone & plate viscometer with #CP-52 spindle. One skilled in the art would recognize that rotation rate decreases as viscosity increases and would be able to select the appropriate rotation rate when using this test method to measure viscosity. Alternatively, subscript i can be 30 to 100, alternatively 50 to 80, alternatively 60 to 70, and alternatively 70 to 80. Suitable polyorganohydrogensiloxanes for use in the composition are exemplified by:(c3) α,ω-dimethylhydrogensiloxy-terminated poly(dimethylsiloxane/methylhydrogensiloxane),(c4) α,ω-dimethylhydrogensiloxy-terminated polymethylhydrogensiloxane,(c5) α,ω-trimethylsiloxy-terminated poly(dimethylsiloxane/methylhydrogensiloxane),(c6) α,ω-trimethylsiloxy-terminated polymethylhydrogensiloxane,(c7) α-dimethylhydrogensiloxy-ω-trimethylsiloxy-terminated poly(dimethylsiloxane/methylhydrogensiloxane),(c8) α-dimethylhydrogensiloxy-ω-trimethylsiloxy-terminated polymethylhydrogensiloxane, and(c9) a combination of two or more thereof. Methods of preparing polyorganohydrogensiloxanes, such as hydrolysis and condensation of organohalosilanes, are well known in the art, for example, in U.S. Pat. No. 2,823,218 to Speier, et al.; U.S. Pat. No. 3,957,713 to Jeram et al. and U.S. Pat. No. 4,329,273 to Hardman, et al. Polyorganohydrogensiloxanes are also commercially available, such as those available from Gelest, Inc. of Morrisville, Pennsylvania, USA, for example, HMS-H271, HMS-071, HMS-993, HMS-301, HMS-301 R, HMS-031, HMS-991, HMS-992, HMS-993, HMS-082, HMS-151, HMS-013, HMS-053, HAM-301, and HMS-HM271. The polyorganohydrogensiloxane selected for use in the silicone pressure sensitive adhesive composition may be one polyorganohydrogensiloxane or may comprise more than one polyorganohydrogensiloxane, where the polyorganohydrogensiloxanes differ in at least one property such as molecular weight, selection of alkyl groups, type of terminal siloxane units, and sequence of the siloxane units in the polymer backbone. Starting materials (A), (B), and (C) are present in amounts sufficient to provide a molar ratio of silicon bonded hydrogen atoms in starting material (C) to alkenyl groups in starting materials (A) and (B) of 10:1 to 50:1, alternatively 15:1 to 45:1, alternatively 20:1 to 40:1, and alternatively 25:1 to 35:1. (D) Polydialkylcyclosiloxane Starting material (D) is a low molecular weight cyclic siloxane. More specifically, starting material (D) is a polydialkylcyclosiloxane selected from the group consisting of: (d1) an octaalkylcyclotetrasiloxane of unit formula (R12SiO2/2)4, (d2) a decaaalkylcyclopentasiloxane of unit formula (R12SiO2/2)5, (d3) a dodecaalkylcyclohexasiloxane of unit formula (R12SiO2/2)6, and (d4) combinations of two or more of (d1), (d2), and (d3), where R1is an alkyl group as described above. The amount of starting material (d1) in the composition may be 0.18 to 8.0 weight parts, alternatively 0.27 to 8.0 weight parts, and alternatively 0.18 weight parts to 4.01 weight parts, per 100 weight parts of starting material (A). Alternatively, the amount of starting material (d1) may be at least 0.18 weight part, alternatively at least 0.27 weight part, alternatively at least 0.30 weight part, alternatively at least 0.33 weight part, and alternatively at least 0.35 weight part, per 100 parts by weight of starting material (A). At the same time, the amount of (d1) may be up to 8.0 weight parts, alternatively up to 7.5 weight parts, alternatively up to 7.0 weight parts, alternatively up to 6.5 weight parts, alternatively up to 6.0 weight parts, alternatively up to 5.5 weight parts, alternatively up to 5.0 weight parts, alternatively up to 4.5 weight parts, alternatively up to 4.1 weight parts, alternatively up to 4.01 weight parts, and alternatively up to 4.0 weight parts, per 100 parts by weight of starting material (A). The amount of starting material (d2) in the composition may be 0.17 to 8.8 weight parts, alternatively 0.17 to 4.43 weight parts, and alternatively 0.39 weight part to 8.8 weight parts, per 100 weight parts of starting material (A). Alternatively, the amount of starting material (d2) may be at least 0.17 weight part, alternatively at least 0.39 weight part, alternatively at least 0.41 weight part, alternatively at least 0.43 weight part, alternatively at least 0.45 weight part, alternatively at least 0.47 weight part, alternatively at least 0.49 weight part, and alternatively at least 0.51 weight part, per 100 weight parts of starting material (A). At the same time, the amount of (d2) may be up to 8.8 weight parts, alternatively up to 8.5 weight parts, alternatively up to 8.0 weight parts, alternatively up to 7.5 weight parts, alternatively up to 7.0 weight parts, alternatively up to 6.5 weight parts, alternatively up to 6.0 weight parts, alternatively up to 5.5 weight parts, alternatively up to 5.0 weight parts, alternatively up to 4.5 weight parts, alternatively up to 4.43 weight parts, and alternatively up to 4.4 weight parts, per 100 weight parts of starting material (A). The amount of starting material (d3) in the composition may be 0.16 to 5.0 weight parts, alternatively 0.26 weight part to 5.0 weight parts, and alternatively 0.16 to 1.02 weight parts, per 100 weight parts of starting material (A). Alternatively, the amount of starting material (d3) may be at least 0.16 weight part, alternatively at least 0.26 weight parts, alternatively at least 0.28 weight parts, alternatively at least 0.30 weight parts, alternatively at least 0.32 weight parts, alternatively at least 0.33 weight parts, and alternatively at least 0.34 weight parts, per 100 weight parts of starting material (A). At the same time, the amount of (d3) may be up to 5.0 weight parts, alternatively up to 4.5 weight parts, alternatively up to 4.4 weight parts, alternatively up to 4.0 weight parts, alternatively up to 3.5 weight parts, alternatively up to 3.0 weight parts, alternatively up to 2.5 weight parts, alternatively up to 2.4 weight parts, alternatively up to 2.3 weight parts, and alternatively 1.02 weight parts, per 100 weight parts of starting material (A). The total amount of starting material (D) the polydialkylcyclosiloxane, i.e., the combined amounts of (d1), (d2) and/or (d3) in the composition is 0.91 to 17.2 parts by weight, per 100 parts by weight of starting material (A). Alternatively, the total amount of starting material (D) may be at least 0.91 weight part, alternatively at least 0.93 weight part, alternatively at least 0.95 weight part, alternatively at least 0.97 weight part, alternatively at least 0.99 weight part, alternatively at least 1.0 weight part, alternatively at least 1.1 weight parts, and alternatively at least 1.2 weight parts, per 100 weight parts of starting material (A). At the same time, the total amount of starting material (D) may be up to 17.2 weight parts, alternatively up to 17 weight parts, alternatively up to 16 weight parts, alternatively up to 15 weight parts, alternatively up to 14 weight parts, alternatively up to 13 weight parts, alternatively up to 12 weight parts, alternatively up to 11 weight parts, alternatively up to 10 weight parts, alternatively up to 9 weight parts, alternatively up to 8.9 weight parts, alternatively up to 8.8 weight parts, alternatively up to 8.5 weight parts, and alternatively up to 8 weight parts, per 100 weight parts of starting material (A). Polydialkylcyclosiloxanes are known in the art and are commercially available. For example, octaalkylcyclotetrasiloxanes such as 2,2,4,4,6,6,8,8,-octamethylcyclotetrasiloxane; decaaalkylcyclopentasiloxanes such as 2,2,4,4,6,6,8,8,-decamethylcyclopentasiloxane; and dodecaalkylcyclohexasiloxanes such as 2,2,4,4,6,6,8,8,10,10-dodecamethylcyclohexasiloxane are known in the art and are commercially available from various sources such as Dow Silicones Corporation of Midland, Michigan, USA; Gelest, Inc. of Morrisville, Pennsylvania, USA; and Millipore Sigma of St. Louis, Missouri, USA. The inventors surprisingly found that curing the silicone pressure sensitive adhesive composition described herein, which includes (D) the polydialkylcyclosiloxane described above, formed a silicone pressure sensitive adhesive with reduced stain visible to human eyes after removal of the silicone pressure sensitive adhesive from various adherends, as described below in the Examples herein. This was particularly surprising in view of previous disclosures, e.g., in U.S. Pat. Nos. 3,983,298; 7,728,080; and 8,754,174; all of which disclose removing low molecular weight cyclic siloxanes from the starting materials used to make pressure sensitive adhesive compositions. (E) Hydrosilylation Reaction Catalyst Hydrosilylation reaction catalysts are known in the art and are commercially available. Hydrosilylation reaction catalysts include platinum group metal catalysts. Such hydrosilylation reaction catalysts can be E1) a metal selected from platinum, rhodium, ruthenium, palladium, osmium, and iridium. Alternatively, the hydrosilylation reaction catalyst may be E2) a compound of such a metal, for example, chloridotris(triphenylphosphane)rhodium(I) (Wilkinson's Catalyst), a rhodium diphosphine chelate such as [1,2-bis(diphenylphosphino)ethane]dichlorodirhodium or [1,2-bis(diethylphospino)ethane]dichlorodirhodium, chloroplatinic acid (Speier's Catalyst), chloroplatinic acid hexahydrate, platinum dichloride, and E3) a complex of a compound, E2), with a low molecular weight organopolysiloxane, or E4) a platinum group metal compound microencapsulated in a matrix or coreshell type structure. Complexes of platinum with low molecular weight organopolysiloxanes include 1,3-diethenyl-1,1,3,3-tetramethyldisiloxane complexes with platinum (Karstedt's Catalyst) and Pt(0) complex in tetramethyltetravinylcyclotetrasiloxane (Ashby's Catalyst). Alternatively, the hydrosilylation reaction catalyst may be E5) a compound or complex, as described above, microencapsulated in a resin matrix. Specific examples of platinum-containing catalysts include chloroplatinic acid, either in hexahydrate form or anhydrous form, or a platinum-containing catalyst which is obtained by a method comprising reacting chloroplatinic acid with an aliphatically unsaturated organosilicon compound such as divinyltetramethyldisiloxane, or alkene-platinum-silyl complexes as described in U.S. Pat. No. 6,605,734 to Roy. These alkene-platinum-silyl complexes may be prepared, for example by mixing 0.015 mole (COD)PtCl2with 0.045 mole COD and 0.0612 moles HMeSiCl2, where COD represents cyclooctadiene and Me represents methyl. Other exemplary hydrosilylation reaction catalysts are described in U.S. Pat. No. 2,823,218 to Speier; U.S. Pat. No. 3,159,601 to Ashby; U.S. Pat. No. 3,220,972 to Lamoreaux; U.S. Pat. No. 3,296,291 to Chalk, et al.; U.S. Pat. No. 3,419,593 to Willing; U.S. Pat. No. 3,516,946 to Modic; U.S. Pat. No. 3,814,730 to Karstedt; U.S. Pat. No. 3,928,629 to Chandra; U.S. Pat. No. 3,989,668 to Lee, et al.; U.S. Pat. No. 4,766,176 to Lee, et al.; U.S. Pat. No. 4,784,879 to Lee, et al.; U.S. Pat. No. 5,017,654 to Togashi; U.S. Pat. No. 5,036,117 to Chung, et al.; and U.S. Pat. No. 5,175,325 to Brown; and EP 0 347 895 A to Togashi, et al. Hydrosilylation reaction catalysts are commercially available, for example, SYL-OFF™ 4000 Catalyst and SYL-OFF™ 2700 are available from Dow Silicones Corporation. The amount of catalyst in the composition will depend on various factors including the selection of starting materials A), B), and C) and their respective contents of alkenyl groups and silicon bonded hydrogen atoms, and the amount of F) hydrosilylation reaction inhibitor present in the composition, however, the amount of catalyst is sufficient to catalyze hydrosilylation reaction of SiH and alkenyl groups, alternatively the amount of catalyst is sufficient to provide 10 ppm to 75000 ppm of the platinum group metal based on combined weights of starting materials A), B), C), D), E), and F). (F) Hydrosilylation Reaction Catalyst Inhibitor Starting material (F) is a hydrosilylation reaction inhibitor (inhibitor) that can be used for altering rate of reaction of the silicon bonded hydrogen atoms of starting material (C) and the alkenyl groups of starting materials (A) and (B), as compared to reaction rate of the same starting materials but with the inhibitor omitted. Inhibitors are exemplified by acetylenic alcohols such as methyl butynol, ethynyl cyclohexanol, dimethyl hexynol, and 3,5-dimethyl-1-hexyn-3-ol, 1-butyn-3-ol, 1-propyn-3-ol, 2-methyl-3-butyn-2-ol, 3-methyl-1-butyn-3-ol, 3-methyl-1-pentyn-3-ol, 3-phenyl-1-butyn-3-ol, 4-ethyl-1-octyn-3-ol, 3,5-dimethyl-1-hexyn-3-ol, and 1-ethynyl-1-cyclohexanol, and a combination thereof; olefinic siloxanes such as cycloalkenylsiloxanes exemplified by methylvinylcyclosiloxanes exemplified by 1,3,5,7-tetramethyl-1,3,5,7-tetravinylcyclotetrasiloxane, 1,3,5,7-tetramethyl-1,3,5,7-tetrahexenylcyclotetrasiloxane, and a combination thereof; ene-yne compounds such as 3-methyl-3-penten-1-yne, 3,5-dimethyl-3-hexen-1-yne, and a combination thereof; triazoles such as benzotriazole; phosphines; mercaptans; hydrazines; amines, such as tetramethyl ethylenediamine, 3-dimethylamino-1-propyne, n-methylpropargylamine, propargylamine, and 1-ethynylcyclohexylamine; dialkyl fumarates such as diethyl fumarate, dialkenyl fumarates such as diallyl fumarate, dialkoxyalkyl fumarates, maleates such as diallyl maleate and diethyl maleate; nitriles; ethers; carbon monoxide; alkenes such as cyclooctadiene, divinyltetramethyldisiloxane; alcohols such as benzyl alcohol; and a combination thereof. Exemplary olefinic siloxanes are disclosed, for example, in U.S. Pat. No. 3,989,667. Exemplary acetylenic alcohols are disclosed, for example, in U.S. Pat. No. 3,445,420. Alternatively, the inhibitor may be a silylated acetylenic compound. Without wishing to be bound by theory, it is thought that adding a silylated acetylenic compound reduces yellowing of the reaction product prepared from hydrosilylation reaction as compared to a reaction product from hydrosilylation of starting materials that do not include a silylated acetylenic compound or that include an organic acetylenic alcohol inhibitor, such as those described above. The silylated acetylenic compound is exemplified by (3-methyl-1-butyn-3-oxy)trimethylsilane, ((1,1-dimethyl-2-propynyl)oxy)trimethylsilane, bis(3-methyl-1-butyn-3-oxy)dimethylsilane, bis(3-methyl-1-butyn-3-oxy)silanemethylvinylsilane, bis((1,1-dimethyl-2-propynyl)oxy)dimethylsilane, methyl(tris(1,1-dimethyl-2-propynyloxy))silane, methyl(tris(3-methyl-1-butyn-3-oxy))silane, (3-methyl-1-butyn-3-oxy)dimethylphenylsilane, (3-methyl-1-butyn-3-oxy)dimethylhexenylsilane, (3-methyl-1-butyn-3-oxy)triethylsilane, bis(3-methyl-1-butyn-3-oxy)methyltrifluoropropylsilane, (3,5-dimethyl-1-hexyn-3-oxy)trimethylsilane, (3-phenyl-1-butyn-3-oxy)diphenylmethylsilane, (3-phenyl-1-butyn-3-oxy)dimethylphenylsilane, (3-phenyl-1-butyn-3-oxy)dimethylvinylsilane, (3-phenyl-1-butyn-3-oxy)dimethylhexenylsilane, (cyclohexyl-1-ethyn-1-oxy)dimethylhexenylsilane, (cyclohexyl-1-ethyn-1-oxy)dimethylvinylsilane, (cyclohexyl-1-ethyn-1-oxy)diphenylmethylsilane, (cyclohexyl-1-ethyn-1-oxy)trimethylsilane, and combinations thereof. Alternatively, the silylated acetylenic compound is exemplified by methyl(tris(1,1-dimethyl-2-propynyloxy))silane, ((1,1-dimethyl-2-propynyl)oxy)trimethylsilane, or a combination thereof. The silylated acetylenic compound useful as the inhibitor herein may be prepared by methods known in the art, for example, U.S. Pat. No. 6,677,407 to Bilgrien, et al. discloses silylating an acetylenic alcohol described above by reacting it with a chlorosilane in the presence of an acid receptor. The amount of inhibitor in the composition will depend on various factors including the desired reaction rate, the particular inhibitor used, and the selection and amount of starting materials (A), (B), and (C). However, when present, the amount of inhibitor may be 0.1 to 5 parts by weight, per 100 parts by weight of starting material (A). (G) Organic Solvent Starting material (G) in the composition is an organic solvent. The organic solvent can be an alcohol such as methanol, ethanol, isopropanol, butanol, or n-propanol; a ketone such as acetone, methylethyl ketone, or methyl isobutyl ketone; an aromatic hydrocarbon such as benzene, toluene, or xylene; an aliphatic hydrocarbon such as hexane, heptane, or octane; a glycol ether such as propylene glycol methyl ether, dipropylene glycol methyl ether, propylene glycol n-butyl ether, propylene glycol n-propyl ether, or ethylene glycol n-butyl ether, tetrahydrofuran; mineral spirits; naphtha; ethyl acetate; or a combination thereof. The amount of solvent will depend on various factors including the type of solvent selected and the amount and type of other starting materials selected for the composition. However, the amount of solvent may be 10% to 90%, alternatively 20% to 80%, alternatively 30% to 70%, and alternatively 40% to 60%, based on combined weights of all starting materials in the composition. The solvent may be added during preparation of the composition, for example, to aid mixing and delivery of one or more starting materials. For example, the gum, the resin and/or the catalyst may be delivered in a solvent. The solvent may be one solvent or a combination of two or more different solvents. For example, certain catalysts (e.g., chloroplatinic acid) may be dissolved in an alcohol solvent and the gum and/or the resin may be dissolved in a different solvent, such as toluene. The composition may be free of filler or contain only a limited amount of filler, such as 0 to 30% by weight of the composition. Without wishing to be bound by theory, it is thought that fillers can agglomerate or otherwise stick to the coater equipment used to apply the composition to a substrate. Fillers can also hinder optical properties by creating haze of the pressure sensitive adhesive and protective film formed therewith. Furthermore, the composition may be free of other components which would hinder optical properties, such as by increasing haze. Without wishing to be bound by theory, it is thought that hydroxyl-functional polydiorganosiloxane gum may contribute to haze and/or migration of the pressure sensitive adhesive. It is further thought that certain low molecular weight side-products formed inherently during the manufacture of polyorganosilicate resins, such as neopentamer, may also contribute to haze and/or migration of the pressure sensitive adhesive. Therefore, the composition may also be free of neopentamer. Method for Making the Silicone Pressure Sensitive Adhesive Composition The pressure sensitive adhesive composition can be prepared by a method comprising combining all starting materials by any convenient means such as mixing at ambient or elevated temperature. The inhibitor may be added before the hydrosilylation reaction catalyst, for example, when the pressure sensitive adhesive composition will be prepared at elevated temperature and/or the composition will be prepared as a one part composition. One or more of the starting materials may optionally be devolatilized before their addition to the pressure sensitive adhesive composition. For example, (A) the gum and/or (B) the resin may be stripped (if not previously devolatilized), optionally with vacuum and/or inert gas flow, to remove any volatile by-products of manufacture before their use in the pressure sensitive adhesive composition. The (B) resin and all or a portion of the (A) gum may be combined, e.g., using a devolatilizing extruder, before adding the resulting resin/gum blend to the remaining starting materials of the composition. The method may further comprise delivering one or more starting materials in a solvent. For example, the gum, the resin and/or the hydrosilylation reaction catalyst may be dissolved in a solvent when combined with one or more of the other starting materials in the composition. Alternatively, the resin may be prepared as a solid flake or powder, optionally combined with a portion of the gum, and thereafter the resin or the resin/gum combination may be dissolved in solvent before combining with the other starting materials of the composition. Alternatively, the pressure sensitive adhesive composition may be prepared as a multiple part composition, for example, when the pressure sensitive adhesive composition will be stored for a long period of time before use, e.g., up to 6 hours before coating the pressure sensitive adhesive composition on a substrate. In the multiple part composition, the hydrosilylation reaction catalyst is stored in a separate part from any starting material having a silicon bonded hydrogen atom, for example the polyorganohydrogensiloxane, and the parts are combined shortly before use of the pressure sensitive adhesive composition. For example, a multiple part composition may be prepared by combining starting materials comprising at least some of the gum, the polyorganohydrogensiloxane, at least some of the solvent, and optionally one or more other additional starting materials described above to form a base part, by any convenient means such as mixing, with the proviso that the hydrosilylation reaction catalyst is not included in the base part. A curing agent may be prepared by combining starting materials comprising at least some of the gum, the hydrosilylation reaction catalyst, solvent and optionally one or more other additional starting materials described above by any convenient means such as mixing, with the proviso that any starting material with silicon bonded hydrogen atoms (such as the polyorganohydrogensiloxane) is not included in the curing agent part. The starting materials may be combined at ambient or elevated temperature. The hydrosilylation reaction inhibitor may be included in one or more of the base part, the curing agent part, or a separate additional part. Alternatively, the polyorganosilicate resin may be added to the base part, the curing agent part, or a separate additional part. The polydialkylcyclosiloxane may be added to the base part, the curing agent part, or a separate additional part. When a two part composition is used, the weight ratio of amounts of base part to curing agent part may range from 1:1 to 10:1. The pressure sensitive adhesive composition will cure via hydrosilylation reaction to form a pressure sensitive adhesive. Method of Use The method described above may further comprise one or more additional steps. The pressure sensitive adhesive composition prepared as described above may be used to form an adhesive article, e.g., a pressure sensitive adhesive (prepared by curing the pressure sensitive adhesive composition described above) on a substrate. The method may, therefore, further comprise applying the pressure sensitive adhesive composition to a substrate. Applying the pressure sensitive adhesive composition to the substrate can be performed by any convenient means. For example, the pressure sensitive adhesive curable composition may be applied onto a substrate by gravure coater, comma coater, offset coater, offset-gravure coater, roller coater, reverse-roller coater, air-knife coater, or curtain coater. The substrate can be any material that can withstand the curing conditions (described below) used to cure the pressure sensitive adhesive composition to form the pressure sensitive adhesive on the substrate. For example, any substrate that can withstand heat treatment at a temperature equal to or greater than 70° C., alternatively, 70° C. to 150° C. is suitable. Examples of materials suitable for such substrates including plastic films such as polyimide (PI), polyetheretherketone (PEEK), polyethylene naphthalate (PEN), liquid-crystal polyarylate, polyamideimide (PAI), polyether sulfide (PES), polyethylene terephthalate (PET), polyethylene (PE), or polypropylene (PP). Alternatively, the substrate may be a metal foil such as aluminum foil or copper foil. The thickness of the substrate is not critical, however, the thickness may be 5 micrometers to 300 micrometers. To improve bonding of the pressure sensitive adhesive to the substrate, the method for forming the adhesive article may optionally further comprise treating the substrate before applying the pressure sensitive adhesive composition. Treating the substrate may be performed by any convenient means, such as applying a primer, or subjecting the substrate to corona-discharge treatment, etching, or plasma treatment before applying the pressure sensitive adhesive composition to the substrate. An adhesive article such as a film or tape may be prepared by applying the pressure sensitive adhesive curable composition described above onto the substrate described above. The method may optionally further comprise removing all, or a portion, of the solvent before and/or during curing. Removing solvent may be performed by any convenient means, such as heating at a temperature that vaporizes the solvent without fully curing the pressure sensitive adhesive composition, e.g., heating at a temperature of 70° C. to 120° C., alternatively 50° C. to 100° C., and alternatively 70° C. to 80° C. for a time sufficient to remove all or a portion of the solvent (e.g., 30 seconds to 1 hour, alternatively 1 minute to 5 minutes). Curing the pressure sensitive adhesive composition may be performed by heating at a temperature of 70° C. to 200° C., alternatively 80° C. to 180° C., and alternatively 90° C. to 160° C., and alternatively 100° C. to 150° C. for a time sufficient to cure the pressure sensitive adhesive composition (e.g., for 30 seconds to an hour, alternatively 1 to 5 minutes). If cure speed needs to be increased or the process oven temperatures lowered, the catalyst level can be increased. This forms a pressure sensitive adhesive on the substrate. Curing may be performed by placing the substrate in an oven. The amount of the pressure sensitive adhesive composition to be applied to the substrate depends on the specific application, however, the amount may be sufficient such that after curing thickness of the pressure sensitive adhesive may be 1 micrometers to 100 micrometers, and for protective film the thickness may be 3 micrometers to 80 micrometers, and alternatively 4 micrometers to 50 micrometers. The method described herein may optionally further comprise applying a removable release liner to the pressure sensitive adhesive opposite the substrate, e.g., to protect the pressure sensitive adhesive before use of the adhesive article. The release liner may be applied before, during or after curing the pressure sensitive adhesive composition; alternatively after curing. The adhesive article prepared as described above is suitable for use as a protective film, e.g., for protection of exteriors of appliances, which may comprise plastic, metal such as copper or stainless steel, and/or glass. For example, a method for protecting an appliance, exemplified by a frame and back plate of a home appliance, may comprise applying a protective film (i.e., an adhesive article comprising the pressure sensitive adhesive prepared as described herein) to a surface of the appliance. The method further comprises removing the protective film after storage and shipping the device. The protective film may be removed with no or minimal stain on the surface of the appliance. In an alternative embodiment, the adhesive article prepared as described above may be used for surface protection of an electronic device, such as screen or other surface protection during assembly of the device, storage of the device, shipment of the device, such as a smartphone or tablet, or at an end user for screen protection of such a device. The pressure sensitive adhesive and substrate selected are typically transparent for screen protection applications. The adhesive article may be removed without damaging the electronic device after storage and shipping. EXAMPLES These examples are intended to illustrate the invention to one skilled in the art and should not be interpreted to limit the scope of the invention set forth in the claims. Starting materials used in these examples are described below in Table 1. TABLE 1Starting MaterialsStartingMaterialChemical DescriptionSourceA-1Dimethylvinylsiloxy-terminated, poly(dimethyl/DSCmethylvinyl)siloxane with a vinyl content of0.0669 weight % and Mn of 702,000 DaA-2Dimethylvinylsiloxy-terminated, poly(dimethyl/DSCmethylvinyl)siloxane with a vinyl content of0.0654 weight % and Mn of 700,000 DaB-1Polymethylsilicate resin comprising M units ofDSCformula (Me3SiO1/2), Q units of formula (SiO4/2)and hydroxyl content (HO1/2), where molar ratioof the M:Q units was 0.9:1 to 1.1:1, Mw was17,000 Da, Mn was 6,200 to 6,900, and thehydroxyl content was 0.9 to 1.1 weight % basedon weight of the resin.C-1Trimethylsiloxy-terminatedDSCpolymethylhydrogensiloxane homopolymer with aDP of 36D-11,1,1,3,3,5,5,7,7,7-dodecamethylpentasiloxaneDSCwith viscosity of 2 cStD-2Trimethylsiloxy terminated polydimethylsiloxaneDSCwith viscosity of 5 cStD-3Trimethylsiloxy terminated polydimethylsiloxaneDSCwith viscosity of 10 cStD42,2,4,4,6,6-octamethylcyclotetrasiloxaneDSCD52,2,4,4,6,6,8,8-decamethylcyclopentasiloxaneDSCD62,2,4,4,6,6,8,8,10,10-DSCdodecamethylcyclohexasiloxaneD-7Trimethylsiloxy terminatedDSCpoly(dimethylsiloxane-co-methylphenylsiloxane)with viscosity of 50 cStD-81,1,1,3,3,3-hexamethyldisiloxaneDSCD-91,1,1,3,3,5,5,5-octamethyltrisiloxaneDSCE-1Platinum - vinylmethylpolysiloxane complexSYL-OFF ™4000 catalystfrom DSCF-11-ethynyl-1-cyclohexanolBASFG-1TolueneSK Chemical In Table 1, DSC refers to Dow Silicones Corporation of Midland, Michigan, USA. Comparative Example 0 (Run 0) A silicone pressure sensitive adhesive composition, which included a hydroxyl-functional polydiorganosiloxane gum, was prepared according to U.S. Pat. No. 8,178,207. This composition was coated on a substrate and cured according to Reference Example 2, described below. The resulting pressure sensitive adhesive sheet was evaluated according to Reference Examples 3-7 below. The pressure sensitive adhesive in this comparative example failed the discoloration test in Reference Example 7. Reference Example 1—Sample Preparation 100 parts by weight of a dimethylvinylsiloxy-terminated, poly(dimethyl/methylvinyl)siloxane gum (A-1 and/or A-2 in Table 1), was mixed with polymethylsilicate resin (B-1) and (G-1) toluene to form a 41% solution. To the solution were added a polydimethylsiloxane additive (one or more of D-1 to D-9 in Table 1). Inhibitor (ETCH) (F-1) was added, and the resulting combination was mixed well. The catalyst (E-1) was then added in an amount targeting 115 ppm of platinum in the mixture. The composition of each sample (Run) is shown below in Tables 2 and 3. TABLE 2Silicone Pressure Sensitive Adhesive CompositionsStarting Material fromTable 1 or CalculatedRun-Run-Run-Run-Run-Run-Run-Run-Parameter1 (C)2 (W)3 (C)4 (C)5 (C)6 (C)7 (C)8 (C)A-2100.00100.00100.00100.00100.00100.00100.00100.00B-188.9688.9688.9688.9688.9688.9688.9688.96D40.0330.6670.0330.0330.0330.0330.0330.033D50.0710.7540.0710.0710.0710.0710.0710.071D60.0632.3540.0630.0630.0630.0630.0630.063D-8003.4400000D-90003.440000D-100003.44000D-2000003.4400D-30000003.440D-700000003.44C-14.614.614.614.614.614.614.614.61F-10.740.740.740.740.740.740.740.74E-14.614.614.614.614.614.614.614.61G-1272.11272.11272.11272.11272.11272.11272.11272.11Resin/Gum ratio0.890.890.890.890.890.890.890.89SiH/Vi ratio (excluding Pt)30.4530.4530.4530.4530.4530.4530.4530.45Pt content (ppm)115.82115.82115.82115.82115.82115.82115.82115.82D4/D5/D6 content (g)0.1673.7750.1670.1670.1670.1670.1670.167 TABLE 3Silicone Pressure Sensitive Adhesive Compositions (continued)Starting Material orRunRunRunRunRunRunRunRunRunCalculated Parameter9 (C)10 (W)11 (W)12 (W)13 (C)14 (W)15 (W)16 (W)17 (C)A-147.2966.3995.25000000A-252.7133.614.75100.00100.00100.00100.00100.00100.00B-189.5989.8490.2288.9688.9688.9688.9688.9688.96D40.2620.3540.4940.6670.6671.0352.0364.0408.046D50.3860.5130.7050.7540.7541.1732.2754.4798.886D60.2580.3370.4560.3335.0710.3330.3330.3330.333C-14.644.664.684.614.614.614.614.614.61F-10.740.740.750.740.740.740.740.740.74E-14.644.664.684.614.614.614.614.614.61G-1274.02274.79275.96272.11272.11272.11272.11272.11272.11Resin/Gum ratio0.890.890.890.890.890.890.890.890.89SiH/Vi ratio (excluding Pt)30.1229.9929.8030.4530.4530.4530.4530.4530.45Pt content (ppm)115.82115.82115.82115.82115.82115.82115.82115.82115.82D4/D5/D6 content (g)0.9061.2041.6551.7546.4922.5414.6458.85217.266 In Tables 2 and 3, (C) denotes a comparative example, and (W) denotes a working example. Reference Example 2—Si PSA Coating and Cure The silicone-based pressure-sensitive adhesive compositions prepared as described above were applied onto a 25 um PET film sold under the trade name of SK Chemical (SKC). A pressure-sensitive adhesive layer which after curing had a thickness of 30 um was formed by heating the coated film for 2 min at 100° C. with 1000 rpm of wind speed. A pressure sensitive adhesive sheet was formed. Reference Example 3—Initial Adhesion Measurement Pressure sensitive adhesive strips were formed by cutting the pressure sensitive adhesive sheets prepared according to Reference Example 2 into 1 inch-wide strips. Each strip was adhered under pressure of 2 kgf developed by rubber rollers to an adherend in the form of a mirror-surface stainless steel plate (SUS304) to form test specimens. Adhesion strength of the strips to the stainless-steel plate (SUS304) was measured according to a 180-degree peel test specified in ASTM D1000. Reference Example 4—Adhesion Measurement for Aged Condition at 85° C./85% of Humidity (Adhesion Stability) Samples were prepared as described above in Reference Example 3, except the test specimens were aged for 1 day (24 hr) in an oven at 85° C./85% moisture. After cooling to room temperature, the adhesion force was measured according to the same 180-degree peel test specified in ASTM D1000 as in Reference Example 3. Reference Example 5—Probe Tack Test An adhesive layer prepared as described in Reference Example 2 showed tackiness to a surface of stainless column measured according to probe tack test specified in ASTM D 2979. The pressure sensitive adhesive sheets were cut to obtain sheet into 1 inch-wide strips. A probe in the shape of a cylinder was brought into contact with a test specimen to which a strip was attached, and the tack property (instantaneous adhesion) that occurred when it was peeled at 0.5 cm/sec of speed with 1 sec of dwell time was evaluated. Reference Example 6—Migration Check Under Aged Condition at 60° C./90% of Humidity (Stain Test) Stain of the pressure sensitive adhesives prepared as described in Reference Example 2 were evaluated as follows. Pressure sensitive adhesive sheets were cut to obtain sheet into 2 inch-wide strips, and then each strip was attached on a surface of a target adherend (polycarbonate) to create a test specimen. The test specimen was kept at 60° C./90% of humidity condition for 3 days and then cooled down to room temperature condition. The strips were peeled off from the adherends, and then the position where the pressure sensitive adhesive contacted the adherend was visually inspected by naked eyes to determine whether the pressure sensitive adhesive created a stain remaining on the adherend. If a strong stain remained on the surface, it was scored to 5. If a faint or no stain remained on the surface, it was scored to 1. Results for each sample (Run) are also shown below in Tables 4. ‘Fail’ in Table 4 denotes worse stain and is undesirable for certain applications such as the protective film application described herein. Reference Example 7—Migration Check Under Post Cure Condition at 200° C. (Discoloration Test) Stain called as ‘Discoloration’ of the pressure sensitive adhesives prepared as described in reference Example 2 was evaluated follows and the test method is referred to ‘A CIELAB color space’ specified in ASTM D2244. Pressure sensitive adhesive tapes were obtained by cutting a sheet prepared as described in Reference Example 2 into 2 inch-wide strips, and then the strips were attached on a surface of a copper film to form test specimens. The test specimens were kept at 200° C. for 10 mins and then cooled to room temperature. The strips were peeled off from the copper film, and then the residue of silicone was measured by Spectro-meter called as hazy meter on reflective mode. Once silicone residue called as migration remained on the surface of Cu film, the surface color of Cu film was changed to be getting low brightness and red brown. This discoloration is correlated with silicone migration strength how much silicone residue remained on the surface of Cu film and the variation of color is correlated to each value of ΔL*, Δa*, Δb* and delta E. ΔL* means a variation of lightness between Cu film having silicone residue and Cu film as reference. If the value is getting negative, it correlated with low brightness caused by silicone migration. Δa* represents the variation of red color between Cu film having silicone residue and Cu film as reference. If the value if getting positive, it is correlated with strong red color of Cu film changed by silicone migration. Δb* represents the variation of yellow color between Cu film having silicone residue and Cu film as reference. If the value is getting positive, the color of Cu film is changed to more yellow due to strong silicone migration. ΔE is the deviation of ΔL*, Δa* and Δb* from reference to determine how much stain remained on surface of Cu film is through comparing each optical index, L*, a* and b*. If ΔE is getting positive, the Cu film having strong stain or discoloration caused by silicone migration. The value of each index is calculated following below formula and the result for each sample are shown in Table 4. ΔL*=L*example−L*Cu filmFormula (1): Δa*=a*example−a*Cu filmFormula (2): Δb*=b*example−b*Cu filmFormula (3): ΔE*=√{square root over ((ΔL*)2+(Δa*)2+(Δb*)2)} Formula (4): TABLE 4Test Results of Reference Examples 3 to 7InitialAdhesionTheAdhesionBuild up -StainVisualdeviation ofStrength24 hProbe(Pass/StaindiscolorationRun(gf/in)(gf/in)TackFail)Score(delta E)0 (C)173.7382.2131.1Pass1.54.651 (C)169.7364.3120.0Fail4.31.692 (W)164.0364.7131.4Pass1.51.963 (C)154.4320.0111.0Fail3.8N/D4 (C)141.9317.7128.8Fail4.0N/D5 (C)162.7344.3114.5Fail4.0N/D6 (C)157.0330.0111.0Fail3.8N/D7 (C)143.6311.5121.0Fail4.0N/D8 (C)153.3322.0126.0Fail3.8N/D9 (C)157.8319.397.0Fail3.02.0310 (W)162.2330.3121.0Pass1.52.2411 (W)155.8340.4101.5Pass1.42.5512 (W)156.7332.7112.2Pass1.01.8513 (C)151.1315.0117.8Fail3.31.8814 (W)164.4347.0122.3Pass1.02.0315 (W)149.8324.0118.6Pass1.52.1516 (W)161.1355.5132.8Pass1.01.7417 (C)142.2311.0135.5Fail4.02.03 In Table 4, ‘N/D’ means ‘Not detected’ due to strong stain with high visual stain score and stain marked as ‘fail’. The silicone pressure sensitive adhesives prepared by curing the compositions described in Table 2 and Table 3 have <200 gf/in of initial adhesion, <150 g of tack property, and <400 gf/in of adhesion change after aging for 24 hrs at 85° C./85% of humidity condition. The most important thing is to provide minimal or no stain called as ‘a kind of silicone migration’ which remains ‘discoloration’ on surface of adherend where a silicone pressure sensitive adhesive is attached. ‘Stain’ of a silicone pressure sensitive adhesive is measured by both test methods, which is checked with naked eyes and is detected by spectrometer. For example, Run 0 prepared as described in Comparative Example 0, and evaluated described above in Reference Example 6, had visual stain score ‘1.5’ with ‘Pass’ but Run 0 evaluated as described above in Reference Example 7, showed high deviation of discoloration with delta E ‘4.65’. So, Run 0 failed because the sample provide minimal or no stain measured by both test conditions. Run 1, Run 3 to Run 9, Run 13 and Run 17 also failed one or both stain test conditions. It is desirable for the silicone pressure sensitive adhesive according to this invention to provide minimal or no stain under both test conditions. Runs 1 to 7 screened various compounds as migration reducing additives for the silicone pressure sensitive adhesive composition. Run 2 showed that certain polydialkylcyclosiloxanes were useful to reduce stain of a silicone pressure sensitive adhesive made from the composition. Runs 1 and 3 to 9 were comparative and showed that when no additive was used, or when a linear polydiorganosiloxane was used instead of a cyclic, stain performance was poor under the conditions tested. Runs 9-17 showed that when pressure sensitive adhesive compositions containing the amounts of each polydialkylcyclosiloxane within the ranges described herein were cured, improved stain properties were achieved, however, when too little or too much of one or more of the polydialkylcyclosiloxane species was present, more staining was observed on one or more adherends. INDUSTRIAL APPLICABILITY Without wishing to be bound by theory, it is thought that migration that can result in staining is caused by unreacted silicone species in a silicone pressure sensitive adhesive. Previous attempts to minimize or eliminate migration included increasing crosslink density of the cured silicone pressure sensitive adhesive, e.g., by controlling the type and amount of crosslinker, changing the functional group and amount of the polydiorganosiloxane gum, and elimination of volatile unreacted species, such as linear and cyclic polydiorganosiloxanes. The inventors surprisingly found that adding certain volatile cyclic siloxanes of the types and in the amounts described herein actually reduced (rather than increased) staining. The protective film prepared using the pressure sensitive adhesive composition and methods described above may be used to protect a surface of an appliance from damage such as scratching during storage and shipping or poisoning such as attracting dust or foreign material during assembly process in device. Definitions and Usage of Terms All amounts, ratios, and percentages are by weight unless otherwise indicated. The amounts of all starting materials in a composition total 100% by weight. The Summary and the Abstract are hereby incorporated by reference. The articles ‘a’, ‘an’, and ‘the’ each refer to one or more, unless otherwise indicated by the context of specification. The singular includes the plural unless otherwise indicated. The term “comprising” and derivatives thereof, such as “comprise” and “comprises” are used herein in their broadest sense to mean and encompass the notions of “including,” “include,” “consist(ing) essentially of,” and “consist(ing) of. The use of “for example,” “e.g.,” “such as,” and “including” to list illustrative examples does not limit to only the listed examples. Thus, “for example” or “such as” means “for example, but not limited to” or “such as, but not limited to” and encompasses other similar or equivalent examples. It is to be understood that the appended claims are not limited to express and particular compounds, compositions, or methods described in the detailed description, which may vary between particular embodiments which fall within the scope of the appended claims. With respect to any Markush groups relied upon herein for describing particular features or aspects of various embodiments, different, special, and/or unexpected results may be obtained from each member of the respective Markush group independent from all other Markush members. Each member of a Markush group may be relied upon individually and or in combination and provides adequate support for specific embodiments within the scope of the appended claims. Any ranges and subranges relied upon in describing various embodiments of the present invention independently and collectively fall within the scope of the appended claims and are understood to describe and contemplate all ranges including whole and/or fractional values therein, even if such values are not expressly written herein. One of skill in the art readily recognizes that the enumerated ranges and subranges sufficiently describe and enable various embodiments of the present invention, and such ranges and subranges may be further delineated into relevant halves, thirds, quarters, fifths, and so on. As just one example, a range “1.0 to 17” may be further delineated into a lower third, i.e., 1 to 6.3, a middle third, i.e., 6.4 to 11.6, and an upper third, i.e., 11.7 to 17, which individually and collectively are within the scope of the appended claims and may be relied upon individually and/or collectively and provide adequate support for specific embodiments within the scope of the appended claims. In addition, with respect to the language which defines or modifies a range, such as “at least,” “greater than,” “less than,” “no more than,” and the like, it is to be understood that such language includes subranges and/or an upper or lower limit. Furthermore, an individual number within a disclosed range may be relied upon and provides adequate support for specific embodiments within the scope of the appended claims. For example, a range “1.0 to 17” includes various individual integers, such as 1.0, 3.0, 5.0, 8.0, and 17 as well as individual numbers including a decimal point (or fraction), such as 1.2, 4.1, 4.8, 8.7, and 8.8 which may be relied upon and provide adequate support for specific embodiments within the scope of the appended claims. Abbreviations used herein are defined in Table 5. TABLE 5AbbreviationsAbbreviationDefinition° C.Degrees CelsiusCODcyclooctadienecStcentistokesDaDaltonsDPDegree of polymerizationETCH1-ethynyl-1-cyclohexanolgGramsgfGrams forceGPCGel Permeation ChromatographyhrhoursininchkgfKilograms forceMemethylminminutesMnNumber average molecular weight, which can be evaluatedby GPC as described in U.S. Pat. No. 9,593,209 inReference Example 1MwWeight average molecular weight, which can be evaluatedby GPC as described in U.S. Pat. No. 9,593,209 inReference Example 1ummicrometerVivinyl Embodiments of the Invention In a first embodiment, a silicone pressure sensitive adhesive composition comprises:100 parts by weight of (A) a polydiorganosiloxane gum of unit formula (R13SiO1/2)a(R12R2SiO1/2)b(R12SiO2/2)c(R1R2SiO2/2)d, where each R1is an independently selected alkyl group of 1 to 10 carbon atoms; each R2is an independently selected alkenyl group with 2 to 10 carbon atoms; subscript a is 0, 1, or 2; subscript b is 0, 1, or 2; subscript c≥0; subscript d≥0; (c+d) has a value sufficient to provide the gum with a molecular weight ≥400,000 Da; a quantity (b+d) is sufficient to provide a silicon bonded alkenyl content of at least 0.06 weight % based on weight of the polydiorganosiloxane gum;(B) a polyorganosilicate resin with unit formula (R13SiO1/2)e(R12R2SiO1/2)f(SiO4/2)g(ZO1/2)h, where R1and R2are as described above, Z is a hydrogen atom or an alkyl group of 1 to 6 carbon atoms, subscripts e, f, g, and h represent mole fractions of each unit, e≥0, f>0, g>0, h≥0, and a quantity (e+f+g)=1; where starting materials (A) and (B) are present in amounts sufficient to provide a molar ratio of amount of (B) to amount of (A) (Resin:Gum ratio) of 1.5:1 to 0.5:1;(C) a polyorganohydrogensiloxane of unit formula (R13SiO1/2)2(HR1SiO2/2)i, where R1is as described above, subscript i is sufficient to give the polyorganohydrogensiloxane, a viscosity of 10 to 30 cSt;where starting materials (A), (B), and (C) are present in amounts sufficient to provide a molar ratio of silicon bonded hydrogen atoms in starting material (C) to alkenyl groups in starting materials (A) and (B) of 10:1 to 50:1(D) a polydialkylcyclosiloxane selected from the group consisting of0.35 to 4.1 parts by weight of (d1) an octaalkylcyclotetrasiloxane of unit formula (R12SiO2/2)4,0.51 to 4.5 parts by weight of (d2) a decaalkylcyclopentasiloxane of unit formula (R12SiO2/2)5,0.33 to 2.4 parts by weight of (d3) a dodecaalkylcyclohexasiloxane of unit formula (R12SiO2/2)6, and(d4) combinations of two or more of (d1), (d2), and (d3),with the proviso that combined amounts of (d1), (d2) and (d3) total 1.2 to 8.9 parts by weight, per 100 parts by weight of starting material (A);(E) a hydrosilylation reaction catalyst in an amount sufficient to provide 10 ppm to 7,500 ppm of platinum metal by weight based on combined weights of starting materials (A), (B), (C), (D), (E), and (F); and0.1 to 5 parts by weight of (F) a hydrosilylation reaction catalyst inhibitor; and(G) an organic solvent, in an amount sufficient to provide 10% to 90 weight % solvent based on combined weights of starting materials (A), (B), (C), (D), (E), (F), and (G); with the proviso that the silicone pressure sensitive adhesive composition is free of hydroxyl-functional polydiorganosiloxane gum. In a second embodiment, in the composition of the first embodiment, each R1is methyl and each R2is vinyl. In a third embodiment, in the composition of the first embodiment (or the second embodiment), the gum has a number average molecular weight of 600,000 Da to 800,000 Da and an alkenyl content of at least 0.06 weight %. In a fourth embodiment, in the composition of the first embodiment (or any one of the first to third embodiments), the resin has a number average molecular weight of 5,000 Da to 8,000 Da and a ratio of monofunctional units to quadrifunctional units of 0.9:1 to 1.1:1. In a fifth embodiment, in the composition of the first embodiment (or any one of the first to fourth embodiments), starting material (D) comprises 0.36 weight part to 4.0 weight parts of (d1) the octaalkylcyclotetrasiloxane, 0.52 weight part to 4.4 weight parts of (d2) the decaaalkylcyclopentasiloxane, and 0.34 weight part to 2.3 weight parts of (d3) the dodecaalkylcyclohexasiloxane. In a sixth embodiment, in the composition of the first embodiment (or any one of the first to fifth embodiments), the combined amounts of (d1), (d2) and (d3) total 1.3 to 8.8 parts by weight, per 100 parts by weight of starting material (A). In a seventh embodiment, a method comprises: optionally 1) treating a surface of a substrate, 2) applying a silicone pressure sensitive adhesive composition to the surface of the substrate, where the silicone pressure sensitive adhesive composition comprises 100 parts by weight of (A) a polydiorganosiloxane gum of unit formula (R13SiO1/2)a(R12R2SiO1/2)b(R12SiO2/2)c(R1R2SiO2/2)d, where each R1is an independently selected alkyl group of 1 to 10 carbon atoms; each R2is an independently selected alkenyl group with 2 to 10 carbon atoms; subscript a is 0, 1, or 2; subscript b is 0, 1, or 2; subscript c≥0; subscript d≥0; (c+d) has a value sufficient to provide the gum with a molecular weight ≥400,000 Da; a quantity (b+d) is sufficient to provide a silicon bonded alkenyl content of at least 0.06 weight % based on weight of the polydiorganosiloxane gum;(B) a polyorganosilicate resin with unit formula (R13SiO1/2)e(R12R2SiO1/2)f(SiO4/2)g(ZO1/2)h, where R1and R2are as described above, Z is a hydrogen atom or an alkyl group of 1 to 6 carbon atoms, subscripts e, f, g, and h represent mole fractions of each unit, e≥0, f>0, g>0, h≥0, and a quantity (e+f+g)=1; where starting materials (A) and (B) are present in amounts sufficient to provide a molar ratio of amount of (B) to amount of (A) (Resin:Gum ratio) of 1.5:1 to 0.5:1;(C) a polyorganohydrogensiloxane of unit formula (R13SiO1/2)2(HR1SiO2/2)i, where R1is as described above, subscript i is sufficient to give the polyorganohydrogensiloxane, a viscosity of 10 to 30 cSt;where starting materials (A), (B), and (C) are present in amounts sufficient to provide a molar ratio of silicon bonded hydrogen atoms in starting material (C) to alkenyl groups in starting materials (A) and (B) of 10:1 to 50:1(D) a polydialkylcyclosiloxane selected from the group consisting of0.27 to 8.0 parts by weight of (d1) an octaalkylcyclotetrasiloxane of unit formula (R12SiO2/2)4,0.39 to 8.8 parts by weight of (d2) a decaalkylcyclopentasiloxane of unit formula (R12SiO2/2)5,0.26 to 5.0 parts by weight of (d3) a dodecaalkylcyclohexasiloxane of unit formula (R12SiO2/2)6, and(d4) combinations of two or more of (d1), (d2), and (d3),with the proviso that combined amounts of (d1), (d2) and (d3) is 0.91 to 17.2 parts by weight, per 100 parts by weight of starting material (A);(E) a hydrosilylation reaction catalyst in an amount sufficient to provide 10 ppm to 7,500 ppm of platinum metal by weight based on combined weights of starting materials (A), (B), (C), (D), (E), and (F); and0.1 to 5 parts by weight of (F) a hydrosilylation reaction catalyst inhibitor; and(G) an organic solvent, in an amount sufficient to provide 10% to 90 weight % solvent based on combined weights of starting materials (A), (B), (C), (D), (E), (F), and (G); with the proviso that the silicone pressure sensitive adhesive composition is free of hydroxyl-functional polydiorganosiloxane gum, 3) removing all or a portion of the solvent, and 4) curing the composition, thereby forming an adhesive article. In an eighth embodiment, in the method of the seventh embodiment, starting material (D) is selected from the group consisting of0.35 to 4.1 parts by weight of (d1) the octaalkylcyclotetrasiloxane of unit formula (R12SiO2/2)4,0.51 to 4.5 parts by weight of (d2) the decaalkylcyclopentasiloxane of unit formula (R12SiO2/2)5,0.33 to 2.4 parts by weight of (d3) the dodecaalkylcyclohexasiloxane of unit formula (R12SiO2/2)6, and(d4) combinations of two or more of (d1), (d2), and (d3),with the proviso that combined amounts of (d1), (d2) and (d3) total 1.2 to 8.9 parts by weight, per 100 parts by weight of starting material (A). In a ninth embodiment, in the seventh (or eighth) embodiment, the substrate is a plastic film and the adhesive article is a protective film. In a tenth embodiment, the method of the ninth embodiment further comprises applying the protective film to an adherend, protecting the adherend, and thereafter removing the protective film. In an eleventh embodiment, in the method of the tenth embodiment, the adherend is a surface of an appliance or electronic device. In a twelfth embodiment, in the method of the seventh embodiment (or any one of the seventh to eleventh embodiments), starting material (D) comprises 0.36 weight part to 4.0 weight parts of (d1) the octaalkylcyclotetrasiloxane, 0.52 weight part to 4.4 weight parts of (d2) the decaaalkylcyclopentasiloxane, and 0.34 weight part to 2.3 weight parts of (d3) the dodecaalkylcyclohexasiloxane. In a thirteenth embodiment, in the method of the seventh embodiment (or any one of the seventh to twelfth embodiments), the combined amounts of (d1), (d2) and (d3) total 1.3 to 8.8 parts by weight, per 100 parts by weight of starting material (A). In a fourteenth embodiment, a protective film is prepared by the method of the seventh embodiment (alternatively any one of the seventh to the ninth embodiments). In a fifteenth embodiment, the protective film of the fourteenth embodiment is used for protecting an adherend during assembly, storage, shipping, or a combination of two or more thereof. In a sixteenth embodiment, a silicone pressure sensitive adhesive composition comprises:100 parts by weight of (A) a polydiorganosiloxane gum of unit formula (R13SiO1/2)a(R12R2SiO1/2)b(R12SiO2/2)c(R1R2SiO2/2)d, where each R1is an independently selected alkyl group of 1 to 10 carbon atoms; each R2is an independently selected alkenyl group with 2 to 10 carbon atoms; subscript a is 0, 1, or 2; subscript b is 0, 1, or 2; subscript c≥0; subscript d≥0; (c+d) has a value sufficient to provide the gum with a molecular weight ≥400,000 Da; a quantity (b+d) is sufficient to provide a silicon bonded alkenyl content of at least 0.06 weight % based on weight of the polydiorganosiloxane gum;(B) a polyorganosilicate resin with unit formula (R13SiO1/2)e(R12R2SiO1/2)f(SiO4/2)g(ZO1/2)h, where R1and R2are as described above, Z is a hydrogen atom or an alkyl group of 1 to 6 carbon atoms, subscripts e, f, g, and h represent mole fractions of each unit, e≥0, f>0, g>0, h≥0, and a quantity (e+f+g)=1; where starting materials (A) and (B) are present in amounts sufficient to provide a molar ratio of amount of (B) to amount of (A) (Resin:Gum ratio) of 1.5:1 to 0.5:1;(C) a polyorganohydrogensiloxane of unit formula (R13SiO1/2)2(HR1SiO2/2)i, where R1is as described above, subscript i is sufficient to give the polyorganohydrogensiloxane, a viscosity of 10 to 30 cSt;where starting materials (A), (B), and (C) are present in amounts sufficient to provide a molar ratio of silicon bonded hydrogen atoms in starting material (C) to alkenyl groups in starting materials (A) and (B) of 10:1 to 50:1(D) a polydialkylcyclosiloxane selected from the group consisting of0.18 to 8.0 parts by weight of (d1) an octaalkylcyclotetrasiloxane of unit formula (R12SiO2/2)4,0.17 to 8.8 parts by weight of (d2) a decaalkylcyclopentasiloxane of unit formula (R12SiO2/2)5,0.16 to 5.0 parts by weight of (d3) a dodecaalkylcyclohexasiloxane of unit formula (R12SiO2/2)6, and(d4) combinations of two or more of (d1), (d2), and (d3),with the proviso that combined amounts of (d1), (d2) and (d3) total 1.2 to 8.9 parts by weight, per 100 parts by weight of starting material (A);(E) a hydrosilylation reaction catalyst in an amount sufficient to provide 10 ppm to 7,500 ppm of platinum metal by weight based on combined weights of starting materials (A), (B), (C), (D), (E), and (F); and0.1 to 5 parts by weight of (F) a hydrosilylation reaction catalyst inhibitor; and(G) an organic solvent, in an amount sufficient to provide 10% to 90 weight % solvent based on combined weights of starting materials (A), (B), (C), (D), (E), (F), and (G); with the proviso that the silicone pressure sensitive adhesive composition is free of hydroxyl-functional polydiorganosiloxane gum. In a seventeenth embodiment, in the composition of the sixteenth embodiment, each R1is methyl and each R2is vinyl. In an eighteenth embodiment, in the composition of the sixteenth embodiment (or the seventeenth embodiment), the gum has a number average molecular weight of 600,000 Da to 800,000 Da and an alkenyl content of at least 0.06 weight %. In a nineteenth embodiment, in the composition of the sixteenth embodiment (or any one of the sixteenth to eighteenth embodiments), the resin has a number average molecular weight of 5,000 Da to 8,000 Da and a ratio of monofunctional units to quadrifunctional units of 0.9:1 to 1.1:1. In a twentieth embodiment, in the composition of the sixteenth embodiment (or any one of the sixteenth to nineteenth embodiments), starting material (D) comprises 0.18 weight part to 4.01 weight parts of (d1) the octaalkylcyclotetrasiloxane, 0.17 weight part to 4.43 weight parts of (d2) the decaaalkylcyclopentasiloxane, and 0.16 weight part to 1.02 weight parts of (d3) the dodecaalkylcyclohexasiloxane. In a twenty-first embodiment, in the composition of the sixteenth embodiment (or any one of the sixteenth to twentieth embodiments), the combined amounts of (d1), (d2) and (d3) total 1.3 to 8.8 parts by weight, per 100 parts by weight of starting material (A). In a twenty-second embodiment, a method comprises: optionally 1) treating a surface of a substrate, 2) applying a silicone pressure sensitive adhesive composition to the surface of the substrate, where the silicone pressure sensitive adhesive composition comprises 100 parts by weight of (A) a polydiorganosiloxane gum of unit formula (R13SiO1/2)a(R12R2SiO1/2)b(R12SiO2/2)c(R1R2SiO2/2)d, where each R1is an independently selected alkyl group of 1 to 10 carbon atoms; each R2is an independently selected alkenyl group with 2 to 10 carbon atoms; subscript a is 0, 1, or 2; subscript b is 0, 1, or 2; subscript c≥0; subscript d≥0; (c+d) has a value sufficient to provide the gum with a molecular weight ≥400,000 Da; a quantity (b+d) is sufficient to provide a silicon bonded alkenyl content of at least 0.06 weight % based on weight of the polydiorganosiloxane gum;(B) a polyorganosilicate resin with unit formula (R13SiO1/2)e(R12R2SiO1/2)f(SiO4/2)g(ZO1/2)h, where R1and R2are as described above, Z is a hydrogen atom or an alkyl group of 1 to 6 carbon atoms, subscripts e, f, g, and h represent mole fractions of each unit, e≥0, f>0, g>0, h≥0, and a quantity (e+f+g)=1; where starting materials (A) and (B) are present in amounts sufficient to provide a molar ratio of amount of (B) to amount of (A) (Resin:Gum ratio) of 1.5:1 to 0.5:1;(C) a polyorganohydrogensiloxane of unit formula (R13SiO1/2)2(HR1SiO2/2)i, where R1is as described above, subscript i is sufficient to give the polyorganohydrogensiloxane, a viscosity of 10 to 30 cSt;where starting materials (A), (B), and (C) are present in amounts sufficient to provide a molar ratio of silicon bonded hydrogen atoms in starting material (C) to alkenyl groups in starting materials (A) and (B) of 10:1 to 50:1(D) a polydialkylcyclosiloxane selected from the group consisting of0.18 to 8.0 parts by weight of (d1) an octaalkylcyclotetrasiloxane of unit formula (R12SiO2/2)4,0.17 to 8.8 parts by weight of (d2) a decaalkylcyclopentasiloxane of unit formula (R12SiO2/2)5,0.16 to 5.0 parts by weight of (d3) a dodecaalkylcyclohexasiloxane of unit formula (R12SiO2/2)6, and(d4) combinations of two or more of (d1), (d2), and (d3),with the proviso that combined amounts of (d1), (d2) and (d3) is 0.91 to 17.2 parts by weight, per 100 parts by weight of starting material (A);(E) a hydrosilylation reaction catalyst in an amount sufficient to provide 10 ppm to 7,500 ppm of platinum metal by weight based on combined weights of starting materials (A), (B), (C), (D), (E), and (F); and0.1 to 5 parts by weight of (F) a hydrosilylation reaction catalyst inhibitor; and(G) an organic solvent, in an amount sufficient to provide 10% to 90 weight % solvent based on combined weights of starting materials (A), (B), (C), (D), (E), (F), and (G); with the proviso that the silicone pressure sensitive adhesive composition is free of hydroxyl-functional polydiorganosiloxane gum, 3) removing all or a portion of the solvent, and 4) curing the composition, thereby forming an adhesive article. In a twenty-third embodiment, in the method of the twenty-second embodiment, starting material (D) is selected from the group consisting of0.18 to 4.01 parts by weight of (d1) the octaalkylcyclotetrasiloxane of unit formula (R12SiO2/2)4,0.17 to 4.43 parts by weight of (d2) the decaalkylcyclopentasiloxane of unit formula (R12SiO2/2)5,0.16 to 1.02 parts by weight of (d3) the dodecaalkylcyclohexasiloxane of unit formula (R12SiO2/2)6, and(d4) combinations of two or more of (d1), (d2), and (d3),with the proviso that combined amounts of (d1), (d2) and (d3) total 1.2 to 8.9 parts by weight, per 100 parts by weight of starting material (A). In a twenty-fourth embodiment, in the method of the twenty-second (or twenty-third) embodiment, the substrate is a plastic film and the adhesive article is a protective film. In a twenty-fifth embodiment, the method of the twenty-fourth embodiment further comprises applying the protective film to an adherend, protecting the adherend, and thereafter removing the protective film. In a twenty-sixth embodiment, in the method of the twenty-fifth embodiment, the adherend is a surface of an appliance or electronic device. In a twenty-seventh embodiment, a protective film is prepared by the method of the twenty-second embodiment (alternatively any one of the twenty-second to the twenty-fourth embodiments). In a twenty-eighth embodiment, the protective film of the twenty-seventh embodiment is used for protecting an adherend during assembly, storage, shipping, or a combination of two or more thereof. | 72,591 |
11859112 | DETAILED DESCRIPTION The present disclosure will be hereinafter described. <Paste Composition> A paste composition of this embodiment contains: (A) copper fine particles having a thickness or minor axis of 10 to 500 nm and coated with amino alcohol having a predetermined structure; and (B) an organic solvent. [(A) Copper Fine Particles] In this embodiment, (A) the copper fine particles have a thickness or minor axis of 10 to 500 nm and are coated with amino alcohol represented by the chemical formula (1). Such copper fine particles can be produced as follows, for example. Note that, in this specification, coat means that the aforesaid amino alcohol adheres to the whole or part of the surfaces of the copper fine particles. (Method of Producing (A) the Copper Fine Particles) It is possible to obtain the copper fine particles used in this embodiment by mixing a copper-containing compound, the amino alcohol, and a reducing compound in the organic solvent and heating the resultant mixture to a temperature at which the copper-containing compound is pyrolyzed, to produce the copper fine particles. The copper fine particles obtained by this production method have surfaces coated with the amino alcohol, and their oxidation is lessened owing to the amino alcohol coating the surfaces, so that copper fine particles with desired properties and qualities are obtained. The following describes the raw materials used in the production of the copper fine particles of this embodiment. <Copper-Containing Compound> The copper-containing compound used here is a material for precipitating metallic copper to produce the copper fine particles. The copper-containing compound is decomposed by the heating to release copper ions. The copper-containing compound may be one that releases these copper ions which are then reduced to the metallic copper. This copper-containing compound may also be one that is decomposed by the heating to release the copper ions and organic matter ions derived from the copper-containing compound. Examples of such a copper-containing compound include copper carboxylate in which carboxylic acid such as formic acid, oxalic acid, malonic acid, benzoic acid, or phthalic acid is combined with copper. Other examples of the copper-containing compound include cuprous oxide, copper nitrate, and copper sulfate. <Amino Alcohol> The amino alcohol used here is alcohol having an amino group represented by the following chemical formula (1). (In the formula, R1s may be identical or different, and each independently represent a hydrogen atom, an alkyl group with a carbon number of 1 to 4, a hydroxy group, or a methoxy group, n and m each represent an integer of 0 to 10, and m+n is 10 or less.) Specific examples include aminoethanol, heptaminol, propanolamine, 1-amino-2-propanol, 2-aminodibutanol, 2-diethylaminoethanol, 3-diethylamino-1,2-propanediol, 3-dimethylamino-1,2-propanediol, 3-methylamino-1,2-propanediol, and 3-amino-1,2-propanediol. These may have a boiling point of 70 to 300° C. from the viewpoint of sinterability. Further, from the viewpoint of workability, the amino alcohol may also be liquid at room temperature. <Reducing Compound> The reducing compound used here is not particularly limited as long as it has reducing power to reduce the copper ions generated as a result of the decomposition of the copper-containing compound and liberate the metallic copper. Further, the boiling point of the reducing compound may be 70° C. or higher. The boiling point of the reducing compound may be higher than or equal to a heating temperature in the heating process. In addition, the reducing compound may be a compound dissolved in (B) the later-described organic solvent formed from carbon, hydrogen, and oxygen. Such a reducing compound is typically a hydrazine derivative. Examples of the hydrazine derivative include hydrazine monohydrate, methylhydrazine, ethylhydrazine, n-propylhydrazine, i-propylhydrazine, n-butylhydrazine, i-butylhydrazine, sec-butylhydrazine, t-butylhydrazine, n-pentylhydrazine, i-pentylhydrazine, neo-pentylhydrazine, t-pentylhydrazine, n-hexylhydrazine, i-hexylhydrazine, n-heptylhydrazine, n-octylhydrazine, n-nonylhydrazine, n-decylhydrazine, n-undecylhydrazine, n-dodecylhydrazine, cyclohexylhydrazine, phenylhydrazine, 4-methylphenylhydrazine, benzylhydrazine, 2-phenylethylhydrazine, 2-hydrazinoethanol, and acetohydrazine. <Organic Solvent> In the production of the copper fine particles used in this embodiment, the above-described copper-containing compound, amino alcohol, and reducing compound may be mixed in the organic solvent. As the organic solvent used here, one that can be used as a reaction solvent not impairing the natures of a complex and so on generated from the mixture obtained as a result of the above mixing is usable without any particular limitation. The organic solvent may also be alcohol that has compatibility with the above-described reducing compound. Further, since the reduction reaction of the copper ions which is caused by the reducing compound is an exothermic reaction, an organic solvent that does not volatilize during the reduction reaction is also acceptable. If the organic solvent volatilizes, it is difficult to control the generation of the copper ions resulting from the decomposition of the copper-containing compound-amine compound complex and the precipitation of the metallic copper due to the reduction of the generated copper ions, and as a result, shape stability may deteriorate. Therefore, the organic solvent may have a boiling point of 70° C. or higher and may be formed from carbon, hydrogen, and oxygen. If the boiling point of the organic solvent is 70° C. or higher, it is easy to control the generation of the copper ions resulting from the decomposition of the copper-containing compound-alcohol amine compound complex and the precipitation of the metallic copper due to the reduction of the generated copper ions, leading to the stability of the shape of the copper fine particles. Examples of the aforesaid alcohol used as the organic solvent include 1-propanol, 2-propanol, butanol, pentanol, hexanol, heptanol, octanol, ethylene glycol, 1,3-propanediol, 1,2-propanediol, butyl carbitol, butyl carbitol acetate, ethyl carbitol, ethyl carbitol acetate, diethylene glycol diethyl ether, and butyl cellosolve. Note that this organic solvent does not include the aforesaid amino alcohol or reducing compound. <Another Amine Compound> Another amine compound may be further added when the copper fine particles used in this embodiment are produced. Examples of the other amine compound include a compound containing at least one selected from the following alkylamines and alkoxyamines. This amine compound is not particularly limited as long as it forms a complex with the copper-containing compound. Those appropriately selected from these amine compounds can be used according to the condition of the pyrolysis of the copper-containing compound to be used, the properties expected of the copper fine particles to be produced, and so on. These amine compounds have a function of lessening the oxidation of the copper fine particles by adhering to the surfaces of the copper fine particles obtained as a result of the pyrolysis of the copper-containing compound. By controlling a growth direction of the metallic copper in this way, it is possible to obtain copper fine particles with a specific shape such as a polyhedral shape or a plate shape. The structure of the alkylamine is not particularly limited as long as it is an amine compound in which an aliphatic hydrocarbon group such as an alkyl group is bonded to an amino group. It is, for example, alkyl monoamine having one amino group or alkyldiamine having two amino groups. Note that the above alkyl group may further have a substituent. Specific examples of the alkyl monoamine include dipropylamine, butylamine, dibutylamine, hexylamine, cyclohexylamine, heptylamine, octylamine, nonylamine, decylamine, 3-aminopropyltriethoxysilane, dodecylamine, and oleylamine. Examples of the alkyldiamine include ethylenediamine, N,N-dimethylethylenediamine, N,N′-dimethylethylenediamine, N,N-diethylethylenediamine, N,N′-diethylethylenediamine, 1,3-propanediamine, 2,2-dimethyl-1,3-propanediamine, N,N-dimethyl-1,3-diaminopropane, N,N′-dimethyl-1,3-diaminopropane, N,N-diethyl-1,3-diaminopropane, 1,4-diaminobutane, 1,5-diamino-2-methylpentane, 1,6-diaminohexane, N,N′-dimethyl-1,6-diaminohexane, 1,7-diaminoheptane, and 1,8-diaminooctane. Note that the alkylamine does not include the alkoxyamine described below. The structure of the alkoxyamine is not particularly limited as long as it is an amine compound having an alkoxyl group, and it is, for example, alkoxy monoamine having one amino group or alkoxydiamine having two amino groups. Specifically, examples of the alkoxy monoamine include methoxyethylamine, 2-ethoxyethylamine, and 3-butoxypropylamine, and examples of the alkoxydiamine include N-methoxy-1,3-propanediamine and N-methoxy-1,4-butanediamine. In consideration of coordination force to the copper generated as a result of the reduction, the alkoxyamine may be alkoxy monoamine such as primary amine (R2ONH2) or secondary amine (R3(R4O)NH). Here, the substituent R2of the primary amine mentioned in the aforesaid alkylamine and alkoxyamine represents an alkyl group and may be an alkyl group with a carbon number of 4 to 18. Further, the substituents R3and R4of the secondary amine each represent an alkyl group, and both may be alkyl groups with a carbon number of 4 to 18. The substituents R3and R4may be identical or different. Further, these alkyl groups may have a substituent such as a silyl group or a glycidyl group. The boiling point of this amine compound may be not lower than 70° C. nor higher than 200° C., or may be not lower than 120° C. nor higher than 200° C. If the boiling point of the amine compound is 70° C. or higher, the amine volatilizes less in the heating process. If the boiling point of the amine compound is 200° C. or lower, the amine compound volatilizes during the sintering of the copper fine particles to be removed to the outside of the system, resulting in good low-temperature sinterability. The copper fine particles can be produced as follows using the above-described copper-containing compound, amino alcohol, and reducing compound, and further the organic solvent and the amine compound which are added as required. <Forming of the Mixture> In the method of producing the copper fine particles of the present disclosure, the organic solvent is first put in a reaction vessel, and the above-described copper-containing compound, amino alcohol, and reducing compound, and as required, the organic solvent and the amine compound are mixed in the organic solvent. As for the mixing order, the aforesaid compounds may be mixed in any order. In this mixing, the copper-containing compound and the reducing compound may be put into a reaction solution multiple times at predetermined time intervals. By making the copper-containing compound and the amino alcohol react multiple times in this way, it is possible to generate the copper fine particles having a desired shape or particle size. In forming the mixture, the copper-containing compound and the amino alcohol may be first mixed and kept mixed at 0 to 50° C. for about five to thirty minutes, followed by the addition of the reducing compound and mixing. This results in the efficient formation of the complex of the copper-containing compound and the amino alcohol in the mixture. In this mixing, as for the amount of each of the compounds used relative to 1 mol of the copper-containing compound, that of the amino alcohol may be 0.5 to 10 mol and that of the reducing compound may be 0.5 to 5 mol. The amount of the organic solvent used only needs to be large enough to cause the sufficient reaction of the components, and may be, for example, about 50 to 2000 mL. <Heating of the Mixture> In the next step, by the sufficient heating of the mixture formed and obtained above, a pyrolysis reaction of the copper-containing compound is made to progress. By this heating, the copper-containing compound forming the complex is decomposed into the organic matter ions derived from the copper-containing compound and the copper ions. The copper ions resulting from the decomposition are reduced by the reducing compound, and the metallic copper precipitates and grows into the copper fine particles. Then, the organic matter ions derived from the copper-containing compound, which are generated at the same time when the metallic copper precipitates at this time, tend to be coordinated on a specific crystal face of the precipitated metallic copper. This enables to control the growth direction of the copper fine particles to be generated, and also enables to efficiently obtain the polyhedral or plate-shaped copper fine particles. Further, the later-described amine compound has a function of controlling the grown of the copper fine particles by adhering to their surfaces, thereby preventing the particles from becoming coarse. The heating temperature of the mixture is a temperature at which the copper-containing compound can be pyrolyzed and reduced and the polyhedral or plate-shaped copper fine particles can be generated. For example, the heating temperature only needs to be 70° C. to 150° C. or may be 80 to 120° C. Further, the heating temperature is preferably lower than the boiling points of the raw material components and the organic solvent. The heating temperature within the above range enables the efficient generation of the copper fine particles and also leads to a decrease in the volatilization of the amine compound. The heating temperature of 70° C. or higher causes the progress of the quantitative pyrolysis of the copper-containing compound. Further, the heating temperature of 150° C. or lower leads to a decrease in the volatilization amount of the amine, resulting in the stable progress of the pyrolysis. The precipitated copper fine particles are separated from the organic solvent and so on by centrifugation or the like. A solid of the precipitated copper fine particles may be dried under reduced pressure. The copper fine particles of this embodiment can be obtained by such an operation. <Shape and Size of the Copper Fine Particles> The copper fine particles of this embodiment are in the state in which the amino alcohol forms a coordination bond with the copper atoms which are generated when the complex formed from the copper-containing compound and the reducing agent are pyrolyzed in the amino alcohol, as described above. The copper fine particles coated with the amino alcohol and the organic matter ion species derived from the copper-containing compound are considered to be formed because these copper atoms aggregate. Therefore, it is possible to obtain any shape and size of the copper fine particles by appropriately selecting the types of the copper-containing compound, the amino alcohol, and the reducing agent that are to be used and the reaction temperature. When the other amine compound is further added in the mixture of the copper-containing compound, the amino alcohol, and the reducing agent, the amine compound adheres to the surfaces of the copper fine particles generated by the aforesaid pyrolysis to lessen the oxidation and control the growth direction of the metallic copper. By thus controlling the growth direction of the metallic copper, it is possible to obtain the copper fine particles with a specific shape such as the polyhedral or plate shape. In this embodiment, the plate shape refers to a particle that has a uniform thickness and whose long side in a direction perpendicular to the thickness direction is three times the thickness or more. The polyhedral shape refers to a particle whose shape is similar to the plate shape described above but whose long side in a direction perpendicular to the thickness direction is less than three times the thickness. The copper fine particles obtained by the above-described copper fine particle production method can be fired at low temperature. A conductive paste using the copper fine particles does not require a reducing atmosphere during the firing. Therefore, the resistance of the copper fine particles of this embodiment can be low even if they are fired at low temperature. In addition, a fine sintered film can be obtained because the amount of outgassing, which can cause voids, is small. According to the above-described copper fine particle production method, it is possible to efficiently produce the copper fine particles that can be fired at low temperature in the atmosphere with a simple operation. It is possible to confirm the shape of the obtained copper fine particles by observing them with a scanning electron microscope (product name: JSM-7600F; SEM, manufactured by JEOL Ltd.). Further, the dimensions (thickness, minor axis, and major axis) of the copper fine particles in this specification are each calculated as an average value of those of 10 copper fine particles (n=10) randomly selected based on an observation image of the above SEM. The average value is an arithmetic mean value, which may be calculated using ten or more copper fine particles. Regarding the above-described copper fine particles, the higher a ratio of copper oxide to the whole copper, the lower the activity of the surfaces of the copper fine particles and the more difficult they are to sinter. This ratio of the copper oxide can be expressed as the degree of oxidation found by the following formula (I). The lower the degree of oxidation, the better, and it may be less than 3%. The degree of oxidation of less than 3% results in a paste composition with good sinterability and low resistance. degree of oxidation (%)=([CuO]+[Cu2O])/([Cu]+[CuO]+[Cu2O])×100 (I) In the formula, [Cu] represents the content (mass %) of copper (Cu) in the copper fine particles, [CuO] represents the content (mass %) of copper (II) oxide in the copper fine particles, and [Cu2O] represents the content (mass %) of copper (I) oxide in the copper fine particles. The degree of oxidation can be calculated from the component contents measured using X-ray diffraction (XRD). The contents of the components can be obtained through the quantification by the RIR (reference intensity ratio) method from an integral intensity ratio of the strongest line peaks of the aforesaid Cu, CuO, and Cu2O components obtained by XRD. The paste composition of this embodiment is made with (A) the copper fine particles, which are obtained by the above production method, having a thickness or minor axis of 10 to 500 nm and coated with the amino alcohol with a carbon number of 3 to 10. As (B) the organic solvent of this embodiment, a known organic solvent is usable. Examples of (B) the organic solvent include alcohol (hydroxy compound) that functions as a reducing agent. (B) the organic solvent above is increased in the reducing power by being increased in temperature by a heating process during paste curing (sintering). Consequently, the paste composition can have a dense sintered structure and thus is highly conductive and highly adherent to a substrate such as a lead frame. This mechanism is considered to occur as follows. Since a joint part is sandwiched between a semiconductor element and a substrate, the heating during the sintering brings part of the organic solvent into a reflux state. Accordingly, the organic solvent does not volatilize immediately but remains at the joint part for a while. At this time, the copper oxide partly present in the copper fine particles of the paste composition and metal oxide (for example, copper oxide) present on the surface of the substrate to be joined are reduced to metals (for example, coppers) by the organic solvent (for example, the alcohol that functions as the reducing agent). The sintering of the copper particles with the reduced metals (for example, coppers) then progresses. Consequently, the paste composition at the joint part forms a metallic bond that is highly conductive and highly adherent to the substrate. Specifically, the boiling point of (B) the organic solvent only needs to be 100 to 300° C., and may be 150 to 290° C. If the boiling point is 100° C. or higher, stable adhesion strength can be obtained without reducing ability being lowered by the volatilization of a dispersion medium. Further, if the boiling point is 300° C. or lower, the solvent is prevented from remaining in the film without volatilizing, leading to good sinterability. Specific examples of (B) the organic solvent include 1-propanol, 2-propanol, butanol, pentanol, hexanol, heptanol, octanol, ethylene glycol, diethylene glycol, 1,3-propanediol, 1,2-propanediol, butyl carbitol, butyl carbitol acetate, ethyl carbitol, ethyl carbitol acetate, diethylene glycol diethyl ether, and butyl cellosolve. These solvents each can be used alone or two kinds or more of these can be used in combination. Further, in the paste composition of this embodiment, an organic solvent other than the above-mentioned ones may be added according to its use. The compounding amount of (B) the organic solvent relative to 100 parts by mass of (A) the copper fine particles only needs to be 2 to 20 parts by mass and may be 5 to 15 parts by mass. The compounding amount within this range enables to produce the paste composition having good workability. Further, in the paste composition of this embodiment, (C) carboxylic acid and (D) a thermosetting resin such as an epoxy compound, a phenolic compound, an acrylic compound, or a maleimide compound may be mixed according to its use. Further, in the paste composition of this embodiment, a curing agent, a curing accelerator, a dispersant, metal powder of Cu, Ag, Al, Ni, or the like, and metal oxide powder of silica, alumina, or the like may be mixed as required. (C) the carboxylic acid may be any of aliphatic carboxylic acid, aromatic carboxylic acid, and an anhydride of any of these carboxylic acids. Adding the carboxylic acid improves the dispersibility and low-temperature sinterability of the copper fine particles, making it possible to obtain stable adhesion strength. In the paste composition of this embodiment, the carboxylic acid is blended to not only remove an oxide film of a base material to be joined but also remove a coating layer of (A) the copper fine particles which is generated by a ligand (protective group) exchange reaction at the time of the heating for joining, and remove an oxide film and copper oxide which are contained therein. In addition, since the carboxylic acid decomposes or transpires during the heating for joining, it does not impede the progress of the subsequent sintering of the coppers. Because of this, in the paste composition of this embodiment, the sintering of the coppers is promoted at lower temperature than before the addition. The decomposition temperature of (C) the carboxylic acid may be 100 to 300° C. or may be 150 to 290° C. The decomposition temperature of (C) the carboxylic acid within this range is effective for removing the oxide film of the base material to be joined. If the decomposition temperature of (C) the carboxylic acid is 100° C. or higher, sinterability becomes good owing to the reducing operation of the carboxylic acid, making it possible to obtain a fine sintered film. Further, if the decomposition temperature of (C) the carboxylic acid is 300° C. or lower, the dispersion medium does not remain in the joining member after the sintering. Examples of the aliphatic carboxylic acid include malonic acid, methylmalonic acid, dimethylmalonic acid, ethylmalonic acid, arylmalonic acid, 2,2′-thiodiacetic acid, 3,3′-thiodipropionic acid, 2,2′-(ethylenedithio)diacetic acid, 3,3′-dithiodipropionic acid, 2-ethyl-2-hydroxybutyric acid, dithiodiglycolic acid, diglycolic acid, acetylenedicarboxylic acid, maleic acid, malic acid, 2-isopropylmalic acid, tartaric acid, itaconic acid, 1,3-acetonedicarboxylic acid, tricarballylic acid, muconic acid, β-hydromuconic acid, succinic acid, methylsuccinic acid, dimethylsuccinic acid, glutaric acid, α-ketoglutaric acid, 2-methylglutaric acid, 3-methylglutaric acid, 2,2-dimethylglutaric acid, 3,3-dimethylglutaric acid, 2,2-bis(hydroxymethyl)propionic acid, citric acid, adipic acid, 3-tert-butyladipic acid, pimelic acid, phenyloxalic acid, phenylacetic acid, nitrophenylacetic acid, phenoxyacetic acid, nitrophenoxyacetic acid, phenylthioacetic acid, hydroxyphenylacetic acid, dihydroxyphenylacetic acid, mandelic acid, hydroxymandelic acid, dihydroxymandelic acid, 1,2,3,4-butanetetracarboxylic acid, suberic acid, 4,4′-dithiodibutyric acid, cinnamic acid, nitrocinnamic acid, hydroxycinnamic acid, dihydroxycinnamic acid, coumaric acid, phenylpyruvic acid, hydroxyphenylpyruvic acid, caffeic acid, homophthalic acid, tolylacetic acid, phenoxypropionic acid, hydroxyphenylpropionic acid, benzyloxyacetic acid, phenyllactic acid, tropic acid, 3-(phenylsulfonyl)propionic acid, 3,3-tetramethyleneglutaric acid, 5-oxoazelaic acid, azelaic acid, phenylsuccinic acid, 1,2-phenylenediacetic acid, 1,3-phenylenediacetic acid, 1,4-phenylenediacetic acid, benzylmalonic acid, sebacic acid, dodecanedioic acid, undecanedioic acid, diphenylacetic acid, benzilic acid, dicyclohexylacetic acid, tetradecanedioic acid, 2,2-diphenylpropionic acid, 3,3-diphenylpropionic acid, 4,4-bis(4-hydroxyphenyl)valeric acid, pimaric acid, palustric acid, isopimaric acid, abietic acid, dehydroabietic acid, neoabietic acid, and agathic acid. Examples of the aromatic carboxylic acid include benzoic acid, 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2, 5-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid, 1,2,3-benzenetricarboxylic acid, 1,2,4-benzenetricarboxylic acid, 1,3,5-benzenetricarboxylic acid, 2-[bis(4-hydroxyphenyl)methyl]benzoic acid, 1-naphthoic acid, 2-naphthoic acid, 1-hydroxy-2-naphthoic acid, 2-hydroxy-1-naphthoic acid, 3-hydroxy-2-naphthoic acid, 6-hydroxy-2-naphthoic acid, 1,4-dihydroxy-2-naphthoic acid, 3,5-dihydroxy-2-naphthoic acid, 3,7-dihydroxy-2-naphthoic acid, 2,3-naphthalenedicarboxylic acid, 2,6-naphthalenedicarboxylic acid, 2-phenoxybenzoic acid, biphenyl-4-carboxylic acid, biphenyl-2-carboxylic acid, and 2-benzoylbenzoic acid. Among these, from the viewpoint of storage stability and easy availability, usable are succinic acid, malic acid, itaconic acid, 2,2-bis(hydroxymethyl)propionic acid, adipic acid, 3,3′-thiodipropionic acid, 3,3′-dithiodipropionic acid, 1,2,3,4-butanetetracarboxylic acid, suberic acid, sebacic acid, phenylsuccinic acid, dodecanedioic acid, diphenylacetic acid, benzilic acid, 4,4-bis(4-hydroxyphenyl)valeric acid, abietic acid, 2,5-dihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid, 1,2,4-benzenetricarboxylic acid, 1,3,5-benzenetricarboxylic acid, 2-[bis(4-hydroxyphenyl)methyl]benzoic acid, acetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride, valeric anhydride, trimethylacetic anhydride, hexanoic anhydride, heptanoic anhydride, decanoic anhydride, lauric anhydride, myristic anhydride, palmitic anhydride, stearic anhydride, docosanoic anhydride, crotonic anhydride, methacrylic anhydride, oleic anhydride, linoleic anhydride, chloroacetic anhydride, iodoacetic anhydride, dichloroacetic anhydride, trifluoroacetic anhydride, chlorodifluoroacetic anhydride, trichloroacetic anhydride, pentafluoropropionic anhydride, heptafluorobutyric anhydride, succinic anhydride, methylsuccinic anhydride, 2,2-dimethylsuccinic anhydride, itaconic anhydride, maleic anhydride, glutaric anhydride, diglycolic anhydride, benzoic anhydride, phenylsuccinic anhydride, phenylmaleic anhydride, homophthalic anhydride, isatoic anhydride, phthalic anhydride, tetrafluorophthalic anhydride, and tetrabromophthalic anhydride. These compounds each may be used alone or two or more of them may be used in combination. (C) the carboxylic acid may also be carboxylic anhydride. In particular, having a high coordination ability on the surfaces of the copper fine particles, the carboxylic anhydride substitutes for the protective groups on the surfaces of the copper fine particles, so that the carboxylic anhydride is coordinated on the surfaces of the copper fine particles. The copper fine particles on whose surfaces the carboxylic anhydride is coordinated exhibit good dispersibility, and also exhibit good low-temperature sinterability owing to the excellent volatility of the carboxylic anhydride. The content of (C) the carboxylic acid relative to 100 parts by mass of the component (A) is 0 to 5 parts by mass and may be 0.01 to 5 parts by mass. The paste composition in which this content is 5 parts by mass or less can have good reliability without any void. As (D) the thermosetting resin, any thermosetting resin ordinarily used as an adhesive is usable without limitation. The thermosetting resin may be a liquid resin, and a resin that is in a liquid state at room temperature (25° C.) is usable. Examples of (D) the thermosetting resin include an epoxy resin, a phenolic resin, a radical polymerizable acrylic resin, and a maleimide resin. The paste composition of this embodiment becomes an adhesive material with moderate viscosity by containing (D) the thermosetting resin. In addition, since the paste composition of this embodiment contains (D) the thermosetting resin, the reaction heat at the time of its curing raises the temperature of the resin composition to promote the sinterability of the copper fine particles. Here, the compounding amount of (D) the thermosetting resin relative to 100 parts by mass of (A) the copper fine particles is 0 to 10 parts by mass and may be 1 to 10 parts by mass. If the compounding amount of the component (D) is 10 parts by mass or less, the obtained paste composition exhibits good thermal conductivity and has an excellent heat dissipating property. In addition, the obtained paste composition is not degraded much by the influences of light and heat, and thus can be used as a joining material for long-life light-emitting devices. As described above, the paste composition of this embodiment contains: (A) the copper fine particles having a thickness or minor axis of 10 to 500 nm and coated with the amino alcohol represented by the chemical formula (1); and (B) the organic solvent. Further, in the method of producing the paste composition of this embodiment, after (C) the carboxylic acid, (D) the thermosetting resin, and the other components which are added as required are mixed, a kneading process with a disperser, a kneader, a 3-roll mill, a planetary mixer, or the like is further performed. Next, the obtained resin composition is defoamed, whereby the paste composition is obtained. Note that, in this specification, the paste composition includes those with a low viscosity such as slurry and ink. The viscosity of the paste composition of this embodiment is, for example, 40 to 300 Pa·s and may be 60 to 200 Pa·s. [Semiconductor Device and Electrical/Electronic Component] In a semiconductor device of this embodiment, a semiconductor element is bonded on a substrate that serves as an element support member, using the above-described paste composition. That is, the paste composition is used here as a die attach paste. The semiconductor element used here may be any known semiconductor element, for example, a transistor, a diode, or the like. Further, this semiconductor element includes a light-emitting element such as a LED. Further, the type of the light-emitting element is not particularly limited. Examples thereof include those in which a nitride semiconductor such as InN, AlN, GaN, InGaN, AlGaN, or InGaAlN is formed as a light-emitting layer on a substrate by the MOCVD method or the like. Further, the element support member can be a support member formed of a material such as copper, silver-plated copper, PPF (preplated lead frame), glass epoxy, or ceramic. The semiconductor device using the paste composition of this embodiment has advantages that its electrical resistance value is sufficiently small, and it undergoes only a little change over time and thus has a long life with little decrease in output over time even after a long drive time. Further, in an electrical/electronic component of this embodiment, a heat generating member and a heat dissipating member are bonded through the above-described paste composition. That is, the paste composition is used here as a heat dissipating member bonding material. Here, the heat generating member may be the aforesaid semiconductor element, a member having the semiconductor element, or any other heat generating member. Examples of the heat generating member other than the semiconductor element include an optical pickup and a power transistor. Further, examples of the heat dissipating member include a heat sink and a heat spreader. In the electrical/electronic component in which the heat dissipating member is thus bonded to the heat generating member using the above-described paste composition, the heat dissipating member is capable of efficiently releasing the heat generated by the heat generating member to the outside to reduce a temperature increase of the heat generating member. Note that the heat generating member and the heat dissipating member may be directly bonded with the paste composition, or may be indirectly bonded with another member high in thermal conductivity therebetween. [Substrate Having a Conductive Pattern] A substrate used as this substrate having the conductive pattern is not particularly limited. For example, an organic substrate, a ceramic substrate, a glass substrate, or the like is usable. In particular, from the viewpoint of flexibility, the substrate used may be a film made of polyimide, polyethylene terephthalate (PET), or polyethylene naphthalate (PEN). Here, the paste composition containing the copper fine particles is used as a material for forming conductive wiring. The above-described paste composition can be low in resistance at 150° C. Therefore, it is possible to form the conductive pattern by directly drawing the conductive pattern with a desired shape by applying the above-described paste composition on the substrate where to form the wiring, and by heating the paste composition to fuse the copper fine particles in the paste composition drawn on the substrate. Using the paste composition of this embodiment makes it possible to replace an electronic circuit and electronic element production process which employs a subtractive process by photolithography, a vacuum process such as sputtering, or a wet process such as etching or plating, with a printing method under atmospheric pressure. This can make an electronic circuit manufacturing method resource-saving and highly productive. EXAMPLES Next, the present disclosure will be described in more detail using Examples and Comparative Example. Production of the Copper Fine Particles Reference Example 1 Copper citrate (5 mmol), citric acid (3.75 mmol), and butyl cellosolve (3 ml) were put in a 50 mL sample bottle and mixed at 90° C. for five minutes in an aluminum block type thermostirrer. To this, 1-amino-2-propanol (60 mmol) was added, followed by heating for another five minutes, whereby a copper precursor solution was prepared. This solution was cooled to room temperature, and then hydrazinoethanol (20 mmol) dissolved in 3 mL 1-propanol was added to the copper precursor solution in the sample bottle, followed by five-minute stirring. This was heated and stirred again for two hours in the aluminum block type thermostirrer at 90° C. Five minutes later, 2 mL ethanol (Kanto Chemical, special grade) was added, and a solid was obtained by centrifugation (4000 rpm (1 minute)). When the centrifuged solid was dried under reduced pressure, powdery copper fine particles 1 (0.66 g yield, 97.2% yield ratio) with a copper gloss were obtained. The copper fine particles 1 had surfaces coated with the 1-amino-2-propanol. Reference Example 2 A solid was obtained by the same operation using the same base materials as those in Reference Example 1 except that the 1-amino-2-propanol in Reference Example 1 was replaced with 4-amino-1-butanol (30 mmol) and octylamine (30 mmol) was further added. When the centrifuged solid was dried under reduced pressure, powdery copper fine particles 2 (0.62 g yield, 94.5% yield ratio) having a copper luster were obtained. The copper fine particles 2 had surfaces coated with the 4-amino-1-butanol. Reference Example 3 Cuprous oxide (8.75 mmol) and 1-propanol (5 mL) were put in a 50 mL sample bottle and mixed at 90° C. for five minutes in an aluminum block type thermostirrer. To this, 4-amino-1-butanol (30 mmol) and octylamine (30 mmol) were added, followed by heating for another five minutes, whereby a copper precursor solution was prepared. This solution was cooled to room temperature, and then hydrazine monohydrate (20 mmol) dissolved in 3 mL 1-propanol was added to the copper precursor solution in the sample bottle, followed by five-minute stirring. This was heated and stirred again for two hours in the aluminum block type thermostirrer at 90° C. Five minutes later, 2 mL ethanol (Kanto Chemical, special grade) was added, and a solid was obtained by centrifugation (4000 rpm (one minute)). When the centrifuged solid was dried under reduced pressure, powdery copper fine particles 3 (1.0 g yield, 98.5% yield ratio) with a copper gloss were obtained. The copper fine particles 3 had surfaces coated with the 4-amino-1-butanol. Reference Example 4 Copper oxalate (3.33 mmol) was put in a mixture solution of hydrazine monohydrate (13.2 mmol) and 5 mL methanol being a reaction medium, which were mixed in advance at room temperature, and then the mixture was stirred for ten minutes, whereby a copper oxalate-hydrazine complex (composite compound) was generated. To the obtained copper oxalate-hydrazine complex, oleylamine (16.6 mmol) was added, followed by ten-minute stirring at room temperature, whereby a suspension was prepared. After the stirring, a container containing the mixture solution was heated in a 150° C. oil bath. As a result of the heating, the mixture solution foamed and reddened, and then it was heated and stirred for one hour, whereby a suspension with a copper luster was obtained. After it was cooled to room temperature, ethanol (Kanto Chemical, special grade) (2 mL) was added, and a solid was obtained by centrifugation (4000 rpm (one minute)). When the centrifuged solid was dried under reduced pressure, powdery copper fine particles 4 (0.62 g yield, 61.5% yield ratio) with a copper luster were obtained. The copper fine particles 4 had surfaces coated with the oleylamine. The obtained copper fine particles of Reference Examples 1 to 4 were observed with a scanning electron microscope (product name: JSM-7600F; SEM, manufactured by JEOL Ltd.), and particle size and particle shape were observed to be evaluated.FIGS.1to4are electron micrographs of these copper fine particles. Further, regarding the above copper fine particles, the degree of oxidation, the amount of outgassing, and the yield ratio were also examined as follows. These properties are summarized in Table 1. TABLE 1ReferenceReferenceReferenceReferenceExample 1Example 2Example 3Example 4(copper(copper(copper(copperfinefinefinefineparticles 1)particles 2)particles 3)particles 4)Degree of1.51.00.05.6oxidation(%)ParticleMinor axis:Thickness:Minor axis:20size (nm)22120123Thickness:Minor axis:50100ParticleMixture ofPlate shapedPolyhedralSphericalshapepolyhedralandplate shapesCoating1-amino-2-Aminobutanol,Aminobutanol,OleylamineaminepropanoloctylamineoctylamineAmount of0.72.80.511.6outgassing(%)Yield ratio97.294.598.561.5 <Method of Evaluating the Copper Fine Particles> [Degree of Oxidation] Based on X-ray diffraction (XRD), the contents of Cu, CuO, and Cu2O components were determined from an integral intensity ratio of their strongest line peaks by the RIR (reference intensity ratio) method, and the degree of oxidation of the copper fine particles was calculated by the following formula (I). degree of oxidation (%)=([CuO]+[Cu2O])/([Cu]+[CuO]+[Cu2O])×100 (I) In the formula, [Cu] represents the content (mass %) of copper (Cu) in the copper fine particles, [CuO] represents the content (mass %) of copper (II) oxide in the copper fine particles, and [Cu2O] represents the content (mass %) of copper (I) oxide in the copper fine particles. [Particle Size] As the particle size of the copper fine particles, an average value of those of 10 copper fine particles (n=10) randomly selected based on an image of the obtained solid product observed with a scanning electron microscope (product name: JSM-7600F: SEM, manufactured by JOEL Ltd.) was calculated. At this time, the major axis, the minor axis, and the thickness can also be calculated by the same method. [Particle Shape] The particle shape of the copper fine particles was observed with a scanning electron microscope (JSM-7600F; SEM, manufactured by JEOL Ltd.). [Amount of Outgassing] The amount of outgassing of the copper fine particles was measured using dry powder of the obtained copper fine particles by simultaneous differential thermal and thermogravimetric analysis (TG-DTA) while they were heated from 40 to 500° C. at a temperature increase rate of 10° C./min., and an amount of mass by which the mass after the measurement decreased from that before the measurement was calculated as the amount of outgassing (%). Examples 1 to 4, Comparative Example 1 To produce paste compositions, the components were mixed according to the formulas (part by mass) in Table 2 and the mixtures were kneaded with a roll. The obtained paste compositions were evaluated by the following methods. Table 2 also shows the results of the evaluation. As the materials used in Examples 1 to 4 and Comparative Example 1, commercially available products were used, except for the copper fine particles. [(A) Copper Fine Particles](A1): The copper fine particles 1 obtained in Reference Example 1(A2): The copper fine particles 2 obtained in Reference Example 2(A3): The copper fine particles 3 obtained in Reference Example 3 [Other Copper Fine Particles](CA1): The copper fine particles 4 obtained in Reference Example 4 [(B) Organic solvent](B1): Diethylene glycol (manufactured by Tokyo Chemical Industry Co., Ltd.) [(C) Carboxylic acid](C1): Glutaric anhydride (manufactured by Wako Pure Chemical Corporation) [(D) Thermosetting resin](D1): Bisphenol A-type epoxy resin (product name: jER828 manufactured by Mitsubishi Chemical Corporation) Curing accelerator: imidazole (product name: 2E4MZ manufactured by Shikoku Chemicals Corporation) TABLE 2Ex-Ex-Ex-Ex-Compar-ampleampleampleampleative1234Example 1Composition(A) Copper fine particles(A1) Copper fine particles 1100100———(part(A2) Copper fine particles 2——100——by mass)(A3) Copper fine particles 3———100—Other copper fine particles(CA1) Copper fine particles 4————100(B) Organic solventDiethylene glycol1010101010(C) Carboxylic acidGlutaric anhydride0.3—0.30.30.3(D) Thermosetting resinBisphenol A-type epoxy resin1010101010Curing acceleratorImidazole11111PropertiesViscosity [Pa · s]3234323338Pot life [days]>7>7>7>7>7Thermal conductivity [W/m · K]12010612512880Electrical resistance [Ω]175° C.9 × 10−69 × 10−69 × 10−68 × 10−6Unableto measure200° C.8 × 10−68 × 10−68 × 10−67 × 10−65 × 10−6225° C.6 × 10−66 × 10−66 × 10−64 × 10−64 × 10−6Thermal-time adhesion strengthNormal state353132365[N/chip] to copper frameAfter moisture absorption process353132355Thermal-time adhesion strengthNormal state302829334[N/chip] to PPFAfter moisture absorption process302729324Thermal-time adhesionAfter 100-hour heating process302829324strength after high-After 1000-hour heating process302828322temperature processAfter 100 thermal cycles302828324[N/chip] to PPFAfter 1000 thermal cycles302728322Thermal shock resistanceAfter IR reflow0/50/50/50/55/5[NG number/5] on copper frameAfter 1000 thermal cycles0/50/50/50/55/5Thermal shock resistanceAfter IR reflow0/50/50/50/55/5[NG number/5] on PPFAfter 1000 thermal cycles0/50/50/50/55/5Void ratioGoodGoodGoodGoodUn-acceptable <Method of Evaluating the Paste Compositions> [Viscosity] The viscosity of each of the paste compositions was measured at 25° C. at 5 rpm using an E-type viscometer (3° cone). [Pot Life] The number of days it took for the viscosity after the resin paste was left in a 25° C. thermostatic bath to increase to 1.5 times or more of the initial viscosity was measured. [Thermal Conductivity] The thermal conductivity of each of the paste compositions cured at 175° C. for thirty minutes was measured by a laser flash method according to JIS R 1611-1997. [Electrical Resistance] Test pieces were each fabricated by applying the paste composition on a glass substrate (1 mm thickness) up to a 200 μm by a screen-printing method, followed by curing at 175° C., 200° C., and 225° C. for sixty minutes. The electrical resistance of each of the cured paste compositions was measured by a four-terminal method using a high-precision high-performance resistivity meter “MCP-T600” (product name, manufactured by Mitsubishi Chemical Corporation). <Method of Evaluating Semiconductor Devices> [Thermal-Time Adhesion Strength] Test pieces were each fabricated by mounting a gold-backside chip whose 4 mm×4 mm faying surface was provided with a gold deposition layer, on a pure copper frame and PPF (copper frame plated with Ni—Pd/Au) using the paste composition, followed by curing at 200° C. for sixty minutes. The test pieces each having the chip mounted on the frame were subjected to a moisture absorption process under the condition of 85° C., 85% relative humidity, and 72 hours. The thermal-time adhesion strength of each of the paste compositions was determined by measuring thermal-time die shear strength at 260° C. between the chip and the frame using a mount strength measuring device. [Thermal-Time Adhesion Strength after a High-Temperature Heating Process] Test pieces were each fabricated by mounting a gold-backside chip whose 4 mm×4 mm faying surface was provided with a gold deposition layer, on PPF (copper frame plated with Ni—Pd/Au) using the paste composition for semiconductors and joining them by curing at 200° C. for sixty minutes. As the thermal-time adhesion strength of each of the paste compositions after a high-temperature heating process, thermal-time die shear strength at 260° C. was measured using a mount strength measuring device after the heating process was performed at 250° C. for 100 hours and 1000 hours. As the thermal-time adhesion strength of each of the paste compositions after a high-temperature heating process by a thermal cycle process, thermal-time die shear strength at 260° C. was measured using a mount strength measuring device after the paste composition was subjected to 100-cycle and 1000-cycle processes, with one cycle consisting of the operation of heating from −40° C. to 250° C. and cooling to −40° C. [Thermal Shock Resistance] Test pieces were each fabricated by mounting a gold-backside silicon chip whose 6 mm×6 mm faying surface was provided with a gold deposition layer, on a copper frame and PPF using the paste composition. The curing condition of the paste in joining the above silicon chip to the copper frame and the PPF was such that the paste was cured on a hotplate by 200° C., sixty-second heating (HP curing) or was cured by 200° C., sixty-minute heating using an oven (OV curing). The silicon chips mounted on the above frames were each resin-sealed with an epoxy encapsulant (product name: KE-G3000D) manufactured by KYOCERA Corporation under the following conditions, whereby packages were obtained. In a thermal shock resistance test, the above resin-sealed packages were subjected to a moisture absorption process under the condition of 85° C., 85% relative humidity, and 168 hours, followed by an IR reflow process (260° C., ten seconds), and a thermal cycle process (1000 cycles, with one cycle consisting of the operation of raising the temperature from −55° C. to 150° C. and cooling to −55° C.) was performed. For the evaluation, the number of cracks occurring inside each of the packages after each of the processes was observed using an ultrasonic microscope. In the evaluation result of the thermal shock resistance, the number of samples where cracks occurred among five samples was indicated. Test pieces and curing conditions of the epoxy encapsulantPackage type: 80 pQFP (14 mm×20 mm×2 mm thickness)Chip overview: silicon chip and gold-plated-backside chipLead frame: PPF and copperMolding with the epoxy encapsulant: 175°, two minutesPost-mold curing: 175° C., eight hours [Void Ratio] The void ratio of each of the paste compositions was found by the observation using a microfocus X-ray inspection device (SMX-1000, manufactured by Shimadzu Corporation). The evaluation criteria of the void ratio were as follows: the incidence rate of less than 5% was determined as good, 5% or more and less than 8% was determined as acceptable, and 8% or more was determined as unacceptable. The above void ratio was calculated by the following formula by observing a solder-joint part from a direction perpendicular to a faying surface with an X-ray transmission device and finding the area of voids and the area of the joint part. void ratio (%)=area of voids÷(area of voids+area of joint part)×100 The above results led to the findings that the paste composition containing the copper fine particles of the present disclosure is excellent in sinterability at low temperature of about 175° C. It has also been found out that the paste composition has good thermal conductivity since it can be in a good sintered state owing to the contained carboxylic anhydride. In addition, the copper fine particles obtained in Examples have a particle thickness or minor axis of about 10 to 500 nm, can be sintered at low temperature, and have a small amount of outgassing. Therefore, the copper fine particles obtained in Examples can also be used as an element bonding die attach paste, a heat dissipating member bonding material, and a conductive wiring material of wiring boards. The use of this conductive paste allows the low-temperature sintering and makes it possible to obtain highly reliable semiconductor devices, electrical/electronic devices, and substrates with conductive patterns. | 50,908 |
11859113 | DETAILED DESCRIPTION OF THE INVENTION A first aspect of the present invention relates to a metal-free ECA composition, in the form of a curable paste or ink, comprising an adhesive polymer component and an electrically conductive carbon-based component, characterized by components having different topological morphologies. In a preferred embodiment, the ECA composition is a paste which is processable at low temperature, lower than 100° C. including room temperature, where the term “processable” indicates the possibility of application by means of doctor blading or spin coating. As the adhesive polymer component, a polymer may be used, which is known as a solar cell encapsulant. The polymer component can be selected from polyethylene-vinyl acetate (EVA), polyolefin elastomers (POEs), polyvinyl butyral (PVB), poly(acrylic acid) (PAA), (methyl methacrylate)(PMMA) and polyacrylates or commercial polyacrylate mixtures (e.g. Hydrolac 610L, polyacrylate water dispersion). The polymer component is preferably an EVA copolymer, obtained by the copolymerization of ethylene with vinyl acetate, with a vinyl acetate content of 5 to 50% by weight, preferably 15 to 45% by weight. Commercial copolymers such as for example ELVAX® from DuPont, whose commercial grades have a vinyl acetate content generally of 9 to 40% by weight, can be used. The POEs used within the scope of the invention comprise ethylene copolymers with various monomers such as propylene, butene, hexane and octene. In practice, ethylene-octene and ethylene-butene are commercial products that exhibit excellent elasticity, dielectric properties and easy processability. POEs can be combined with different polymers, including polyethylene, polypropylene and polyamide in order to modulate the material properties. Examples of commercial POEs comprise PHOTOCAP® 35521P HLT (STR), ENGAGE™ (Dow Chemical) and TAFMER™ (Mitsui Chemicals). EVA and POE polymers can be specifically optimized with additives (for example metal peroxides) so as to adjust their melting point and/or their cross-linking temperature. Adhesion of EVA and POE polymers to silicon and metal (including Ag and Cu) surfaces, mechanical elasticity and excellent mechanical and thermal fatigue resistance, are well known in the art and make these materials a technological standard as encapsulants in the PV field. The carbon-based electro-conductive component is a mixture comprising at least 0D acetylene black (or carbon black)nanoparticles, 1D carbon nanotubes and 2D flakes or plates (platelets) of graphene or graphene derivatives. Graphene flakes are preferably obtained by means of the wet-jet milling exfoliation process in solvents described in WO2017089987 to the Applicant. Graphene derivatives comprise reduced graphene oxide. The invention is based on the experimental acknowledgement that the use in the adhesive composition object of the invention of a mixture of the three above mentioned carbon-based fillers involves a substantial reduction in volumetric resistivity, reaching values which allow the use of the adhesive composition instead of conventional metal filler-based electrically conductive adhesive compositions. According to the invention, the electro-conductive component comprises:acetylene black or carbon black particles from 15 to 45% by weight, preferably from 35 to 45% by weight,carbon nanotubes from 5 to 25% by weight, preferably from 10% to 20% by weight,flakes or plates of graphene or graphene derivatives from 35 to 70% by weight, preferably graphene flakes from 35 to 45% by weight, the above mentioned percentages being referred to 100 parts by weight of the conductive component. The combination of carbon nanomaterials with different topological morphologies significantly increases electrical performance compared to the use of single carbon nanomaterials; thus, the electrical conductivity of the ECAs object of the invention can be adjusted by varying the weight ratio of the carbon nanomaterials. Although the explanation of the mechanism is not binding for the scope of the invention, flakes of graphene/graphene derivatives are believed to provide excellent conductivity as far as flakes of graphene/graphene derivatives are concerned. Acetylene black nanoparticles fill the voids between the flakes of graphene/graphene derivatives that are electrically connected. Carbon nanotubes create highly conductive pathways that connect compact conductive domains formed by acetylene black nanoparticles and graphene flakes. In the paste adhesive, the percentage by weight of the polymer component and of the conductive component as compared to the solid content can vary according to the final use of the ECAs, in accordance with the following data:polymer adhesive component 5-40% by weight,electrically conductive component 60-95% by weight. Experimentally, the mechanical properties, such as tensile strength and elongation at break, improve as the percentage by weight of the adhesive component increases. However, an excess content of the adhesive component results in low electrical conductivity (conductivity <10 S m−1). The preferred content of adhesive component for the formulation of a paste with high electrical conductivity ranges from 20 to 30% by weight, more preferably 25% by weight. This value results in an excellent electrical connection of carbon nanomaterials. With reference to mechanical properties, the specific mechanical properties of flakes of graphene/graphene derivatives and carbon nanotubes allow the ECA to be mechanically strengthened. In addition, the excellent thermal conductivity of graphene/graphene derivatives and carbon nanotubes allows effective heat dissipation, improving the reliability of ECAs in electrical and thermal durability tests. In particular, the synergistic combination of nanomaterials allows to obtain greater electrical performance (volumetric resistivity lower than 10−1Ωcm) than that obtained with the individual carbon components (volumetric resistivity greater than 10 Ωcm for graphene- and acetylene black-based ECAs; volumetric resistivity >10−1cm for single-walled carbon nanotubes). The compositions object of the invention thus allow to avoid the use of precious metals such as Ag and Au as conductive material, reducing the overall cost of the ECA. Another aspect of the invention relates to the process for the preparation of the above described ECAs comprising the following steps:i) providing an adhesive component by melting, at a temperature preferably in the range from 120 to 180° C., a polymer selected from EVA, POE, PVB, PAA, PMMA, polyacrylates and dissolving or dispersing said polymer (or mixture of polymers) in a compatible solvent, preferably selected from chlorobenzene, chloroform, xylene, isopranol and their mixtures;ii) providing an electrically conductive component by mixing the powders of carbon nanomaterials comprising acetylene or carbon black nanoparticles, carbon nanotubes and flakes or plates of graphene or graphene derivatives, in the above mentioned proportions;iii) homogeneously mixing the adhesive and electrically conductive components by mechanical stirring at a temperature ranging from 40 to 60° C., to obtain a paste (slurry);iv) depositing the obtained paste, by means of process techniques in solution, optionally compatible with low temperatures (preferably <100° C., including room temperature), thus obtaining the ECA composition. “Compatible solvent” herein means a solvent capable of dissolving or dispersing the polymer component, without causing aggregation phenomena. In step i), the adhesive component is advantageously formulated in highly volatile organic solvents (i.e. having a high vapor pressure, preferably greater than about 0.8 kPa, and preferably with low process temperatures (<100° C., including room temperature (25° C.)), particularly chlorobenzene, xylene and isopranol, whose vapor pressures at 25° C. are: ˜1.6 kPa for chlorobenzene, 1.1 kPa for m-xylene, ˜0.88 for o-xylene, ˜1.16 kPa for p-xylene, 5.8 kPa for isopranol. Further solvents that may be used within the scope of the invention are reported in the claims and in the following experimental section. The solvents cited are also intended to include water solutions of such solvents, when compatible with the polymer component. Water can be used as a solvent or dispersant, or as a component of a solvent mixture, for example with polymer alcohols having sufficient water solubility. Such polymers mainly belong to the class of acrylates. The amount of solvent in the paste adhesive generally is comprised between 50 and 90% by weight referred to 100 parts by weight of the ECA (solvent included). Thanks to the use of highly volatile solvents in the preparation process, the herein described ECAs can be processed (i.e. deposited/applied) and cured at low temperature (<100° C.), including room temperature (25° C.). This avoids the high temperature treatment of pastes which is a pre-requisite for the applicability of traditional welding (welding temperature greater than 180° C. for Sn—Pb welding) and for commercially available ECA pastes (curing temperature typically >100° C.). The ECAs object of the invention can be advantageously applied to thermally sensitive substrates including various plastic materials and semiconductors of solar cells. It is also an object of the invention the use of the ECAs object of the invention for the connection of Cu ribbon, conventionally coated with Sn, to Ag busbars in HTJ-Si, whose ribbon application process is not compatible with traditional welding. In these applications, mechanical adhesion and the quality of the electrical contact between the Ag busbar/ECA/Cu ribbon was assessed before and after mechanical, thermal and electrical stress, based on standard resistance tests reported in the IS/IEC 61730.2 and IEC 61215 standards. The performance of the ECAs object of the invention for the process for applying the conductive ribbon in HJT-Si cells was comparable to that obtained by conventional Ag-filled ECAs. Another object of the invention is the use of the ECA pastes described herein for the production of carbon-based back-electrodes with a surface resistance of less than 200Ω sq−1for a thickness of less than 10 μm. The ECAs were deposited on PSC perovskite-based films using liquid-phase process techniques (e.g. spin-coating) at room temperature. Multiple deposition cycles were effective for the production of back-electrodes with a surface resistance of less than 500Ω sq−1, exceeding the values exhibited by TCO-based back-electrodes (used for example in HJT-Si or double-side PSC technologies). Example 1: ECA with EVA Adhesive Component or Elastomer Polyolefines In the tests that follow and in the following examples, the following materials were used for the conductive component:acetylene black nanoparticles (Sigma Aldrich), particle size: 24 nm,carbon nanotubes purchased from Cheap Tubes (single-walled carbon tubes, outer diameter: 1-4 nm, inner diameter: 0.8-1.6 nm, length: 5-30 μm);graphene flake powder isolated by drying a flake dispersion obtained by means of graphite wet-jet milling exfoliation in N-methyl-2-pyrrolidone in accordance with WO2017/089987;commercial graphene nanoplates purchased from Sigma Aldrich and reduced graphene oxide powders (Sigma Aldrich). In example 1, the following polymers were used:EVA: commercial product ELVAX®, Du Pont (40% by weight of vinyl acetate) elastomer polyolefins: commercial product PHOTOCAP® 35521P HLT, STR. In all the prepared samples, a percentage of 25% by weight of the adhesive component was used. The adhesive components were previously melted at 150° C. for EVA or 180° C. for polyolefin and dissolved in chlorobenzene or in a mixture of xylene isomers having the above mentioned vapor pressures; 6 mL of solvent were used for 1 g of solid polymer component The exemplified ECA compositions have a content of polymer component of 25% by weight referred to 100 parts of polymer component and conductive component. The ECAs were obtained by depositing the corresponding paste (slurry) by means of a doctor blade and subsequent drying of such pastes at 50° C. for 10 minutes. The thickness of the resulting ECAs is between 25 and 45 μm depending on the ECA formulation, measured by means of an optical profilometer. Table 1 shows the percentages by weight of each carbon nanomaterial, the average volumetric resistivity and the error (standard deviation) for each ECA tested. The EVA- and POE-based ECAs are respectively called C-EVA-ECA-X and C-polyolefin-ECA-X, where X indicates different compositions of electro-conductive component and/or solvent. The compositions indicated with an asterisk are shown by way of comparison. TABLE 1Composition of the electro-conductive component and volumetric resistivity for representativeECA samples (percentage by weight of the adhesive component = 25%).ReducedAverageAcetyleneCarbonGrapheneGraphenegraphenevolumetricblacknanotubesflakesnanoplatesoxideresistivityErrorECA(% wt)(% wt)(% wt)(% wt)(% wt)Solvent(Ω cm)(Ω cm)*C-EVA-1000000Chlorobenzene5.1431.061ECA-1*C-EVA-0010000Chlorobenzene8.52512.838ECA-2*C-EVA-2008000Chlorobenzene0.7940.090ECA-3C-EVA-42.51542.500Chlorobenzene0.0680.007ECA-4C-EVA-42.51542.500Xylene0.1460.038ECA-4BC-polyolefin-42.51542.500Chlorobenzene1.3970.504ECA-4C-EVA-17156800Chlorobenzene0.0980.015ECA-5C-EVA-17156800Xylene0.3790.046ECA-5BC-polyolefin-17156800Chlorobenzene1.2320.262ECA-5C-EVA-42.515042.50Chlorobenzene0.3690.061ECA-6C-EVA-17150680Chlorobenzene0.1290.013ECA-7C-EVA-42.5150042.5Chlorobenzene0.2680.039ECA-8C-EVA-17150068Chlorobenzene0.1910.050ECA-9*comparative The combination of acetylene black, carbon nanotubes and graphene flakes (produced by wet-jet milling) in the electro-conductive component, in the compositions according to the invention, is effective in reducing the volumetric resistivity to values lower than 10−1Ωcm for the preferred compositions C-ECA-4 and C-ECA-5 which use EVA polymer and a chlorobenzene solvent as compared to the corresponding comparative compositions. The volumetric resistivity of C-EVA-ECA-4 is comparable with that measured for commercially available Ag-based ECAs from Henkel (i.e. 0.055±0.007 Ωcm). The polyolefin-based ECAs as the adhesive component show greater volumetric resistivity compared to EVA-based ones having the same composition of the electrically conductive component; however, the experimental tests have also shown for these compositions a reduction in resistivity compared to corresponding compositions having a single carbon-filler morphology or including two filler morphologies. Perspectively, the optimization of the C-polyolefin-ECA composition can further reduce the volume resistivities obtained. The experimental data confirm the use of chlorobenzene as the preferred solvent for the exemplified polymer components. SEM analysis (FIG.1) shows that the C-ECA-EVA-4 composition, which is the sample with the best electrical conductivity among those reported, consists of a lattice structure of graphene flakes filled with acetylene black nanoparticles. The conductive lattice structure is electrically connected by filling the voids among the highly conductive graphene flakes. Carbon nanotubes are not resolved due to their nanometric size. However, they are expected to provide electrical connections among multiple carbon nanomaterials. The EVA polymer acts as a bonding component to mechanically bond the entire film structure. Example 2: Product Reliability In order to test the reliability of the C-EVA-ECAs object of the invention, their electrical resistances were measured depending on the deformation applied. The results shown inFIG.2indicate that the C-EVA-ECA compositions have a behavior similar to that of commercially available Ag-based ECAs (Henkel), upon a tensile deformation of up to 20%. At tensile deformation values greater than 20%, the C-EVA-ECA compositions with greater percentage by weight of graphene (C-EVA-ECA-5) show an optimal retention of the initial electrical resistance, which is also greater than that obtained for Ag-based ECAs. The reliability of C-EVA-ECAs was also tested by thermal stress.FIG.3illustrates the electrical resistances of the same compositions illustrated inFIG.2as a function of temperature. The temperature is varied from 20° C. (room temperature) to 250° C. For each resistance sampling point as a function of temperature, the temperature is kept constant for 5 minutes. The increase time between different temperatures tested is 5 minutes. After reaching 250° C., the temperature is allowed to cool down to room temperature. The results indicate that the C-EVA-ECA compositions maintain their initial electrical resistance up to a temperature of 120° C. Above this temperature, the electrical resistance decreases as the temperature increases. This effect should be deemed caused by the cross-linking of EVA at temperatures greater than 160° C. In fact, the changes in electrical resistance are then partially maintained when the samples are cooled to room temperature. The standard lamination temperature of PV modules (between 145 and 160° C.) improves the final electrical performance. Overall, the C-EVA-ECA compositions show reliable electrical performance under mechanical and thermal stress which can also reach values greater than those of the practical operating condition of C-EVA-ECA in electrical devices, including solar cells. Example 3: Validation of HJT-Si Solar Cells The C-EVA-ECA-5 composition was validated as a composition suitable for the application process of metal ribbons to metal contact grids (ribbon tabbing) for the serial connection of HJT-Si solar cells. Tests were carried out with the use of C-EVA-ECA-5 due to its excellent mechanical and electrical properties (see examples 1-3).FIG.4shows the HJT-Si structure used for the tests. The table alongside the figure also shows the thicknesses of the layers of the HUT-Si structure. The metal contact grids are deposited by silkscreen printing on the front and rear of the HJT-Si. Such grids consist of rectangular strips (busbars) perpendicular to “super-thin” grid fingers. Sn-coated Cu strips are used as ribbons. These ribbons are connected to the busbar by C-EVA-ECA-5.FIG.5illustrates a diagram of the ribbon/busbar connection by means of the aforementioned adhesive composition. The quality of the electrical contact is assessed by measuring the electrical resistance between the non-contacting part of the busbar and the floating part of the ribbon (contact resistance). The busbar/ribbon contact area is 0.3 cm×1 cm. The contact resistance obtained using C-EVA-ECA-5 is 0.219Ω. This value is better than that obtained using commercially available Ag-based ECAs (0.295Ω). In order to determine the mechanical and electrical reliability of the contact, the same measurement is performed after encapsulation of the busbar/ribbon contact area with EVA. Following traditional encapsulation with EVA, the contact resistance obtained with C-EVA-ECA-5 is 0.293Ω. Again, this value is lower than that of the contact resistance measured using Ag-based ECAs (0.351Ω). Busbar/ribbon contact resistance tests are carried out by measuring the contact resistance at different applied currents. FIG.6illustrates the normalized contact resistances obtained using Ag-based ECAs and C-EVA-ECA-5 as a function of the applied current, starting from 0.01 A and increasing up to 1.5 A. The insert illustrates the results obtained after two hours at an applied current of 1.5 A, starting from such applied current and reducing the applied current to 0.01 A. It should be noted that the maximum normalized applied current on the contact area is comparable or greater than those used for the hot-spot resistance test of solar cells reported in IEC 61730.2 (MQT 09) (minimum current tested 1.25 times greater than the short-circuit current of the entire solar cells). The purpose of this test is to determine the module's capability of withstanding hot-spot heating effects, i.e. melting of solder or encapsulation deterioration. This defect could be caused by defective cells, misaligned cells, shadowing or fouling. Since the absolute temperature and the relative performance losses are not criteria for this test, the most severe hot-spot conditions are used (corresponding to a minimum current 1.25 times greater than that of the short-circuit current of the entire solar cells), to ensure the reliability of the project. In fact, hot-spot heating takes place in a module when its operating current exceeds the reduced short-circuit current of a shaded or defective cell or groups of cells. When this condition occurs, the cell (or group of cells) affected thereby is forced with reverse polarization and must dissipate energy, causing overheating. If the energy dissipation is sufficiently high or sufficiently localized, the cell with reverse polarization can overheat, resulting, depending on the technology, in melting of solder, deterioration of the encapsulant of the front and/or back cover, breakage of the substrate superstrate and/or glass cover. Herein, ideal ECAs must show reliable mechanical and electrical contact by providing both thermal fatigue resistance and suitable heat dissipation. The results ofFIG.6indicate that, similarly to the contact resistance obtained using Ag-based ECAs, the contact resistance obtained using C-EVA-ECA according to the invention has excellent holding as the applied current increases. After two hours, at an applied current of 1.5 A, the contact resistance obtained using Ag-based ECAs increases by about 20%, while the contact resistance obtained using C-EVA-ECA-5 is maintained. After reducing again the applied current to 0.01 A, both contact resistances show values similar to those measured at the beginning of the test. The contact reliability in withstanding thermal variance, fatigue and other stresses caused by temperature changes is determined by measuring the contact resistance at representative temperatures. In more detail, the temperature is varied from 20° C. (room) to 100° C. After reaching 250° C., the temperature is allowed to drop to room temperature. The contact resistance is therefore measured at the temperature of −70° C. It should be noted that the upper and lower temperature limits are greater and lower than those used during the thermal cycling test (MQT 11) reported in IEC 61215, whose purpose is to determine the module's capability of withstanding thermal variance, fatigue and other stresses caused by repeated temperature changes. The contact resistance obtained using C-EVA-ECA drops from 0.293Ω to 0.257Ω. By cooling the contact to room temperature, the contact resistance is 0.2477Ω. After cooling the contact to −70° C., the electrical resistance decreases from 0.2477Ω to 0.1933Ω. After the contact returns to room temperature, the contact resistance increases to 0.2441Ω. This value is comparable to that measured at room temperature in the initial stage of the tests. Overall, resistance tests indicate that the C-EVA-ECA composition maintains its electrical performance under electrical or thermal stress. Example 4: Validation of PSCs The C-EVA-ECA-4 composition is deposited on active films of mesoscopic PSCs to provide cost-effective carbon-based back-electrodes obtained through a liquid-phase process at room temperature. According to previous reports (Najafi et al. in ACS Nano, 2018, 12(11), pages 10736-10754) architectures made from fluoride-doped tin oxide (FTO)/compact TiO2(cTiO2)/mesoporous TiO2(mTiO2)/CH3NH3PbI3/2,2′,7,7′-tetrakis(N,N-di-4-methoxyphenylamino)-9,9′-spirobifluorene (spiro-OMeTAD) are used as benchmarking PSCs not completed by Au-based back-electrodes. The TiO2layers perform a selective extraction and transport function of the negative charge photo-generated by perovskite (in our case CH3NH3PbI3), and form the so-called electron transporting layer (ETL). The spiro-OMeTAD performs a selective extraction and transport function of the positive charge photo-generated by perovskite, and forms the so-called hole transporting layer (HTL). The concentration of the active materials (electrically conductive and adhesive components) of C-EVA-ECA-4 is adjusted to 111 mg m−1in order to provide suitable viscosity for the spin-coating process. The deposition of C-EVA-ECA-4 on CH3NH3PbI3/spiro-OMeTAD is carried out by deposition via dynamic spin-coating at room temperature with a two-stage protocol (stage 1: 1000 rpm, 3 min.; stage 2: 4000 rpm, 3 min.). As illustrated by the cross-sectional SEM image ofFIG.7, the deposition of C-EVA-ECA-4 does not significantly deteriorate the underlying layered structure. However, an interpenetration of C-EVA-ECA within the spiro-OMeTAD is to be expected, as the C-EVA-ECA solvent can dissolve the spiro-OMeTAD. Operationally, the efficiencies of C-EVA-ECA-based PSCs can be increased by subsequent deposition of spiro-OMeTAD over the C-EVA-ECA. In addition, mixtures of spiro-OMeTAD and C-EVA-ECA can be used to deposit an ECA with a dual HTL and electrode function. Finally, materials with the function of HTL alternative to the spiro-OMeTAD and not dissolvable in the C-EVA-ECA solvent can be used to avoid interpenetration of the C-EVA-ECA in the underlying structure of the solar cell. Alternatively, other ECAs object of the invention and discussed in example 7 can be used to avoid the use of solvents dissolving the materials with the HTL function. No heat treatment is applied to the C-EVA-ECA-4-based PSC. The resulting C-EVA-ECA-4-based back-electrodes have a surface resistance of 155±20 ΩSQ−1for a thickness of less than 10 μm. Multiple deposition cycles are effective for the production of C-EVA-ECA-4-based back-electrodes with surface resistances of less than 50Ω sq−1, exceeding the values often obtained by TCO-based back-electrodes used for example in HJT-Si or double-side PSC technologies. Example 5: Validation of Batteries and Supercapacitors The C-EVA-ECA-4 and C-EVA-ECA-5 compositions were used for the mechanical and electrical connection of electrodes in series of battery cells and supercapacitors, ensuring total reliability of the electrical contact of electrodes in series with electrical resistances lower than 0.1Ω on contact areas equal to or greater than 1 cm×1 cm and C-EVA-ECA thicknesses between 1 and 400 μm. The reliability of the mechanical and electrical contacts of electrodes in series is ensured by an even distribution of the compression forces acting on the electrodes themselves, deriving from the elastic properties of C-EVA-ECAs. Example 6: ECAs with an EVA Adhesive Component with Different Vinyl Acetate Content According to the procedure of example 1, ECA compositions were prepared using EVA polymers with different vinyl acetate content. In particular, the following EVA copolymers were used:EVA with 40% by weight vinyl acetate (Sigma Aldrich) called EVA-B;EVA with 25% by weight vinyl acetate (Sigma Aldrich) called EVA-C;EVA with 18% by weight vinyl acetate (Sigma Aldrich) called EVA-D;EVA with 12% by weight vinyl acetate (Sigma Aldrich) called EVA-E. The main features of some of the ECAs thus prepared (composition of the electro-conductive component, solvent, volumetric resistivity) are reported in table 2 below. TABLE 2Composition of the electro-conductive component and volumetric resistivityfor representative EVA-based ECA samples with different vinyl acetatecontent (percentage by weight of the adhesive component = 25%).AcetyleneCarbonGrapheneVolumetricblacknanotubesflakesresistivityErrorECA(% wt)(% wt)(% wt)Solvent(Ω cm)(Ω cm)C-EVA-42.51542.5Chlorobenzene0.1240.012B-ECAC-EVA-42.51542.5Chlorobenzene0.1440.017C-ECAC-EVA-42.51542.5Chlorobenzene0.2550.020D-ECA Example 7: ECAs with Different Polymer Components and Solvents In addition to the polymers used in the tests of example 1, other polymers were used, reported as encapsulating materials, following the procedure described in example 1. Specifically, polyvinyl butyral (PVB), poly(acrylic acid) (PAA), poly(methyl methacrylate) (PMMA) and commercial acrylate mixtures (Hydrolac 610L, material supplied in the form of an aqueous dispersion) were used. Herein, other solvents were also used in addition to chlorobenzene and xylene, capable of properly dissolving or dispersing the polymers used. In particular, the following solvents were used:for PVB, acetic acid and other solvents with lower volatility such as cyclohexanone, butanol, dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dimethylsulfoxide (DMSO). For these tests, the ECAs produced by doctor-blade deposition were dried at 50° C. for 60 minutes;for PMMA, nitroethane, toluene, chloroform, ethyl acetate, chlorobenzene and cyclohexanone (solvent with less volatility); the ECAs produced by doctor-blade deposition were dried at 50° C. for 60 minutes;for PAA, isopropanol (IPA) and ethanol;for commercial acrylate mixtures, water. Table 3 below shows the main features of the above mentioned ECAs, wherein the electrically conductive component composition corresponding to that of the C-EVA-ECA-4 (or C-EVA-ECA-4B or C-polyolefin-ECA-4) products was used. TABLE 3Composition of electrically conductive component and volumetric resistivity forECAs using different adhesive polymer components and solvents in addition tochlorobenzene (percentage by weight of adhesive polymer component = 25%).AcetyleneCarbonGrapheneVolumetricblacknanotubesflakesresistivityErrorECA(% wt)(% wt)(% wt)Solvent(Ω cm)(Ω cm)C-PVB-42.51542.5Acetic acid0.1740.023ECA-1C-PVB-42.51542.5Butanol0.3850.041ECA-2C-PVB-42.51542.5Cyclohexanone0.3380.055ECA-3C-PVB-42.51542.5DMSO0.2300.046ECA-4C-PVB-42.51542.5DMF0.0910.009ECA-5C-PVB-42.51542.5NMP0.0740.013ECA-6C-PMMA-42.51542.5Ethyl acetate0.1320.031ECA-1C-PMMA-42.51542.5Nitroethane0.0920.009ECA-2C-PMMA-42.51542.5Toluene0.2530.032ECA-3C-PMMA-42.51542.5Chloroform0.2010.030ECA-4C-PMMA-42.51542.5Chlorobenzene0.6570.091ECA-5C-PMMA-42.51542.5Cyclohexanone0.4290.0656C-PAA-42.51542.5IPA0.0610.007ECA-1C-PAA-42.51542.5Ethanol0.0150.004ECA-2C-acrylate42.51542.5Water0.0450.009mixture-ECA Example 8: Adhesive Compositions with Different Solvents and Process Restrictions Although the solvents used for the ECAs shown in example 1, table and for example 7, table 3, can generally be used for the formulation of the ECAs according to the procedures given in example 1, they can impose limitations in relation to the methods used for processing (i.e. depositing/applying) the ECAs. Depending on the properties of the solvents used, it is suggested that the resulting ECAs are processed at low temperature (<100° C.) with the techniques shown in table 4 below. Furthermore, other techniques such as gravure and flexographic printing can be used within the scope of the invention; the use of other deposition parameters, such as substrate temperature and paste temperature, other than those indicated in the notes relating to table 4 also falls within the scope of the invention. TABLE 4Preferred process methods for ECAs depending on the solventMethod of process (i.e., deposition/application))DoctorSpinSprayScreenECA's solventbladingacoatingbcoatingcprintingd,eChlorobenzeneyesyesyesyesChloroformyesyesyesyesTolueneyesyesyesyesIPAyesyesyesyesEthanolyesyesyesyesWateryesyesyesyesAcetic acidyesyesyesyesEthyl acetateyesyesyesyesNitroethaneyesyesyesyesXyleneyesnonoyes(mixture ofisomers)NMPyesnonoyesDMFyesnonoyesDMSOyesnonoyesCyclohexanoneyesnonoyesButanolyesnonoyesasubstrate maintained at temperature <100° C.bpaste and substrate maintained at room temperaturecas a function of the solid component content of the composition, the amount of solvent can be increased to values above 90% referred to 100 parts of ECA (solvent included)das a function of the materials of the ECA, the solvent and the concentration of the solid components, silkscreens are selected depending on material, mesh number and mesh tensionesubstrate maintained at temperature <100° C. Example 9 (Comparative) By way of comparison, the formulation described in example 3 of CN 109320893 was reproduced. The following table 5 shows the materials and the corresponding amounts used for the reproduction of such example. TABLE 5Composition of the product reported in example 3 of CN 108384103Specifications(production andMaterialsupply method)Amount (mg)grapheneWet-jet milling180exfoliationcarbon nanotubesCheap tubes30Carbon blackAlfa Aesar130Epoxy resinSigma Aldrich250Hydroxyl acrylic acidVecom Srl200resinCuring agent (1)Sigma Aldrich180AnilineSigma Aldrich60Silicon nanopowderAlfa Aesar40FeSO4•7H2OSigma Aldrich300FeCl3•6H2OSigma Aldrich150EVA (EVA-B)Sigma Aldrich600White oilSigma Aldrich60Zirconium hydrogenSigma Aldrich10phosphateButyl acrylate emulsionSigma Aldrich80NMPSigma Aldrich400PolytetrafluoroethyleneSigma Aldrich80PolyvinylideneSigma Aldrich130chloride emulsionSodium dodecyl sulfateSigma Aldrich30AuxiliarySigma Aldrich20(1): curing agent: polyether amine The material obtained is in a wet solid form formed by separate lumps, so that the material cannot be processed by the deposition methods previously described such as, in particular, by doctor blading, spin coating, spray coating and silkscreen printing.FIG.8shows a comparison of the film obtained by doctor-blade deposition of the C-EVA-ECA-4 product according to the invention (on the left) with the product obtained in this comparative example (on the right). In order to measure the resistivity of the composite obtained, a lump of the material was pressed in the form of a film. The measured volumetric resistivity is 3.99 Ωcm which is about two orders of magnitude greater than that shown by the products according to the invention, using the same adhesive polymer component (EVA). | 33,892 |
11859115 | DETAILED DESCRIPTION It is a task of the present Disclosure to avoid the disadvantages mentioned in connection with the prior art and to provide a method for activating and deactivating the phosphorescence of a structure, a method for producing a phosphorescent structure, and a phosphorescent structure which is inexpensive, readily available and simple to produce. The problem may be solved at least by a method according to Example 1 and by the method of Example 2. The task of the present Disclosure may be further solved by a method for activating and deactivating the phosphorescence of a structure, wherein for activation in a first activation step for photochemically deactivating oxygen in the structure, the structure may be illuminated with light of a first characteristic, and in a second activation step for initiating the phosphorescence, the structure may be illuminated with light of a second characteristic, wherein for deactivation in a deactivation step, oxygen is introduced into the structure. This enables the activation of the phosphorescence of a structure with phosphorescent organic materials, which was produced under normal atmospheric pressure, in which oxygen is thus present in the region of the phosphorescent organic materials. The oxygen may stop the phosphorescence. Only the photochemical deactivation of the oxygen in the first activation step may make possible the initiation of phosphorescence in the second activation step by pumping electrons into a long-lived higher-energy state by the light of the second characteristic and the long-term residence of the electrons there until excitation with emission of a photon. The deactivation of the phosphorescence of the structure may be achieved in the deactivation step by introducing oxygen. Furthermore, it is advantageously possible with the method according to the Disclosure to activate the phosphorescence of the structure and to deactivate it again by the deactivation step. It is widely possible to reactivate the phosphorescence of the structure after deactivation. For the purposes of the present Disclosure, the terms irradiating light, irradiating light, illuminating with light, and illuminating with light, as well as the terms irradiating, irradiating and illuminating should be understood as being synonymous. It is conceivable to perform the activation and/or the deactivation of the phosphorescence only on parts of the structure. Thus, it is advantageously possible to design areas of the structure with activated phosphorescence and areas of the structure with deactivated phosphorescence. The partial activation and/or deactivation of phosphorescence enables geometric patterns on the structure to be activated and/or deactivated. It is conceivable that information can be stored by the geometric patterns. To this end, it is conceivable, for example, to execute the patterns as a font, logo, image, or machine-readable code, such as a bar code or a QR code. Due to the possibility of activating phosphorescence and deactivating phosphorescence, the geometric design of phosphorescent and non-phosphorescent areas on the structure may be reversible. That is, a designed phosphorescent pattern may be producible and erasable. The structure may then be suitable for producing a pattern again. That is, the structure may be rewritable using a method of the Disclosure. In particular, the first material may comprise a first organic material and/or the second material may comprise a second organic material. Preferably, the first material may be a first organic material and/or the second material may be a second organic material. As a result, the use of metals or rear earth elements that are expensive and/or are harmful to health and the environment can be dispensed with. In a preferred aspect of the Disclosure, the first material includes a first organic material that is oxidizable by singlet oxygen. Preferably, the first material may be a first organic material. Preferably, the first organic material may be capable of forming a chemical bond with singlet oxygen. For example, the first material may comprise a polymer, preferably an organic polymer. Preferably, the first material may be a polymer, especially an organic polymer. Preferably, the first material may be transparent and can be processed by wet processing. For example, the first material may comprise polymethyl methacrylate (PMMA), polystyrene (PS) and/or cycloolefin copolymers (COC). In a preferred aspect of the Disclosure, the second material may comprise a second organic material, preferably an organic polymer. The second material may preferably be an organic polymer. The second material may preferably have a second organic material that is impermeable to oxygen at ambient temperature. Preferably, the second material may be transparent and can be processed, for example, by wet processing. Particularly preferably, the second material may be a second organic material that can be processed in combination with the first material. The second material may comprise, for example, polyvinyl alcohol (PVA) and/or ethylene vinyl alcohol (EVOH) copolymers. The phosphor may be mixed with the first, in particular organic, material, for example the phosphor and the first, in particular organic, material form a host-guest complex, with the first, in particular organic, material forming the host and the phosphor acting as the guest. Oxygen, in particular molecular oxygen, i.e. O2, is present in the region of the phosphor. Preferably, the oxygen, in particular the molecular oxygen, is not bound to the phosphor and/or to the first, in particular organic, material in the non-irradiated and/or non-heated state, i.e., for example, at an ambient temperature. In a preferred aspect of the Disclosure, the phosphor is an organic phosphor that is particularly preferably capable of being excited to phosphorescence at an ambient temperature. Preferably, the phosphor is an organic phosphor whose phosphorescence is inhibited by oxygen. Particularly preferably, the phosphor is an organic phosphor that can be processed by wet processing. For the purposes of the present Disclosure, the ambient temperature refers to the temperature of the medium surrounding the structure during activation and/or deactivation, for example air, at which the second material, in particular an organic material, is impermeable to oxygen. Preferably, the ambient temperature is room temperature, i.e. 293 K. In the oxygen-impermeable state, the second, in particular organic, material advantageously prevents oxygen from penetrating to the first, in particular organic, material and/or the phosphor admixed thereto. By introducing heat and/or light, i.e. thermally and/or photochemically, oxygen, in particular molecular oxygen, may be introduced into the structure in the deactivation step. Preferably, the oxygen may penetrate to the first, in particular organic, material and/or the phosphor and may stop the phosphorescence. Advantageous aspects of the Disclosure can be taken from the dependent claims, as well as from the description with reference to the drawings. According to a preferred aspect of the Disclosure, in the deactivation step, the heat and/or the light of a third characteristic transforms the second, in particular organic, material from an oxygen-impermeable state to an oxygen-permeable state, so that oxygen penetrates to the first, in particular organic, material and/or the phosphor and inhibits phosphorescence. Preferably, molecular oxygen diffuses through the oxygen permeable second, in particular organic, material. For example, infrared light (IR light) may be used as the light of the third characteristic. This advantageously favors the introduction of oxygen to deactivate the phosphorescence. Preferably, in the deactivation step, heat is introduced into the structure by the light of the third characteristic, the heat converting the second, in particular organic, material from an oxygen-impermeable state to an oxygen-permeable state. Alternatively or additionally, the second, in particular organic, material may be photochemically converted from an oxygen-impermeable state to an oxygen-permeable state. According to a preferred aspect of the present Disclosure, the introduction of oxygen is deferred in the deactivation step. Deferment is advantageously the simplest method for deactivation. Due to imperfections of the structure, oxygen is introduced over time by diffusion processes. Heating the structure produces a much faster introduction of oxygen. Illuminating the structure with light of the third characteristic is an advantageously elegant and simple technical realization of heating the structure. According to a further preferred aspect of the present Disclosure, it is provided that light of the first characteristic is used as light with a first intensity, wherein the light of the first characteristic has a wavelength of less than 700 nm, preferably less than 550 nm, particularly preferably less than 460 nm, wherein preferably as light of the second characteristic the light of the first characteristic with a second intensity is used. The light of the first characteristic thus has a first intensity and the light of the second characteristic has a second intensity, the second intensity preferably being different from the first intensity. Particularly preferably, the light of the first characteristic and the light of the second characteristic differ only in intensity. This advantageously allows for deactivation of oxygen in a photochemical reaction. The light of the first characteristic is preferably UV light, for example UV light with a wavelength of approximately 365 nm. Furthermore, it is advantageously possible to use the same light source for the light of the first characteristic and the light of the second characteristic if the light of the first characteristic and the light of the second characteristic do not differ in wavelength but differ in intensity, i.e. the light of the second characteristic is the light of the first characteristic with a second intensity. The light of the first characteristic then has a first intensity. In this case, the first intensity is higher than the second intensity. It is conceivable that the first intensity is 10 times to 100 times greater, preferably 20 times to 90 times greater, particularly preferably 50 times to 80 times greater and in particular circa 70 times greater than the second intensity. It is conceivable that the first intensity is between 1 mWcm−2and 20 mWcm−2, preferably between 3 mWcm−2and 15 mWcm−2, particularly preferably between 5 mWcm−2and 10 mWcm−2and in particular at circa 7 mWcm−2. Furthermore, it is conceivable that the second intensity is between 0.01 mWcm−2and 1 mWcm−2, preferably between 0.05 mWcm−2and 0.5 mWcm−2and in particular at circa 0.1 mWcm−2. It is conceivable that one or more filters and/or two polarizers and/or one or more beam splitters are used to generate the difference of the first intensity and the second intensity, i.e. to generate the attenuation of the light intensity. According to a preferred further aspect of the present Disclosure, it is provided that in the first activation step the oxygen is bound to a first, in particular organic, material in a binding step, wherein prior to the binding step the oxygen is preferably converted from a triplet ground state of the oxygen to an excited singlet state of the oxygen in a triplet-triplet interaction with a phosphor admixed to the first, in particular organic, material. This advantageously enables photochemical deactivation of the oxygen, thus making phosphorescence possible. The oxygen is usually present in a triplet ground state of oxygen. The oxygen is preferably converted from the triplet ground state of oxygen to an excited singlet state of oxygen in a triplet-triplet interaction with the phosphor. This excited singlet state of oxygen is highly reactive. Thus, the oxygen can be bound in the binding step by oxidation of the first, especially organic, material. Conceivably, the phosphor is a doping of the first, in particular organic, material. According to a preferred further aspect of the present Disclosure, it is provided that prior to the triplet-triplet interaction of the light of the first characteristic, the phosphor is transferred from a singlet state of the phosphor to an excited singlet state of the phosphor and subsequently by intercombination from the excited singlet state of the phosphor to a triplet state of the phosphor, wherein preferably in the second activation step the phosphor is transferred from a singlet state of the phosphor to an excited triplet state of the phosphor. Preferably, the phosphor is organic. The phosphor is typically in an unexcited singlet state, preferably the singlet ground state. The light of the first characteristic converts the phosphor to an excited singlet state of the phosphor from which the phosphor can transition by intercombination to an excited triplet state of the phosphor, which is then available for triplet-triplet interaction with oxygen. According to a preferred further aspect of the present Disclosure, heat is introduced into the structure in the deactivation step by the light of the third characteristic, wherein preferably a second, in particular organic, material is converted from an oxygen impermeable state to an oxygen permeable state by the heat. The second, in particular organic, material may form an oxygen barrier in the non-irradiated and/or non-heated state, which keeps oxygen away from the first, in particular organic, material and thus enables a reduction of the unbound oxygen by oxidation of the first, in particular organic, material carried out in the first activation step. Conceivably, by irradiating the third light, i.e., the light of the third characteristic, preferably IR light, the first, in particular organic, material is heated and this heat is transported to the second, in particular organic, material. The heat transfers the second, in particular organic, material from an oxygen-impermeable state to an oxygen-permeable state. Thus, oxygen penetrates to the first, in particular organic, material and stops phosphorescence. Conceivably, the second, particularly organic, material includes ethylene vinyl alcohol (EVOH) copolymers and/or polyvinyl alcohol (PVA). Preferably, the second, in particular organic, material exclusively includes EVOH or PVA, apart from any impurities that may be due to production technology. According to a preferred further aspect of the present Disclosure, it is provided that a long-chain organic polymer, preferably polymethyl methacrylate (PMMA), polystyrene (PS) and/or cycloolefin copolymers (COC), is used as the first, in particular organic, material, wherein the first, in particular organic, material preferably has the phosphor as a dopant and/or as a side chain. That is, the first material includes an organic material. Preferably, the first material includes a long-chain organic polymer. Particularly preferably, the first material includes PMMA, PS and/or COC. Particularly preferably, the first material is an organic material and exclusively includes a long-chain polymer, in particular PMMA, PS or COC. Preferably, PMMA, PS or COC forms a host-guest complex with the phosphor. PMMA, PS and COC are inexpensive, robust and very easy to process. In this context, PMMA, PS and COC are optically transparent, essentially non-toxic, and advantageously suitable for binding oxygen in the first activation step and thus deactivating it. According to a preferred further aspect of the present Disclosure, it is provided that as phosphor N,N′-di(1-naphthyl)-N,N′-diphenyl-(1,1′-biphenyl)-4,4′-diamines (NPB), tetra-N-phenylbensidines (TPB), PhenDPA, PhenTPA, thianthrenes (TA), benzophenone-thianthrenes (BP-TA), Bromo-benzophenone-thianthrenes (Br-BP-TA), benzophenone-2-thianthrenes (BP-2TA), diphenylsulfone-thianthrenes (DPS-TA), diphenylsulfone-2-thianthrenes (DPS-2TA), Bromodiphenylsulfone-thianthrenes (Br-DPS-TA), difluoroborone-9-hydroxyphenalenones (BF2(HPhN)), and/or difluoroborone-6-hydroxybenz[de]anthracene-7-one (BF2(HBAN)) are used. NPB is widely used in the semiconductor industry, for example in the production of OLEDs. It is easy to process and inexpensive. Preferably, the phosphor features NPB, PhenDPA, PhenTPA, TA, BP-TA, Br-BP-TA, BP-2TA, DPS-TA, DPS-2TA, Br-DPS-TA, BF2(HPhN) and/or BF2(HBAN). Particularly preferably, the phosphor exclusively includes NPB, PhenDPA, PhenTPA, TA, BP-TA, Br-BP-TA, BP-2TA, DPS-TA, DPS-2TA, Br-DPS-TA, BF2(HPhN) or BF2(HBAN). The phosphor is preferably selected from the group of the following compounds: wherein R1, R2, and R3are identical or different from each other. Furthermore:R1may be a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl or a substituted or unsubstituted alkyl or a substituted or unsubstituted heteroalkyl or hydrogen;R2may be a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl or a substituted or unsubstituted alkyl or a substituted or unsubstituted heteroalkyl or hydrogen;R3may be a substituted or unsubstituted alkyl or a substituted or unsubstituted heteroalkyl or hydrogen or nitro group. R3may be selected from the group H, OR4or NO2; R4may be H or a (C1-C8) alkyl; R5may be either H, a halogen or a thianthrene; X is P or N; Y1, Y2, Y3and Y4are each independently selected from C or N, where either two or four of Y1, Y2, Y3and Y4are N. Z1and Z2are preferably independently selected from each other. Z1is either an enol or sulfoxide. Z2is absent or a heteroatom or selected from the group consisting of —NR4. Z3is selected from the group consisting of —NR4or —CR4R4. According to a preferred further aspect of the present Disclosure, it is provided that the structure is partially covered with a mask in the first activation step and/or the structure is only partially illuminated in the first activation step with the first light from a light beam meandering locally or scanning line by line and/or the structure is only partially illuminated in the first activation step by illuminating the structure with a light beam having a steel profile. Steel profile of a light beam in the sense of the present Disclosure means that the intensity of the light beam incident on the surface of the structure varies locally to such an extent that the intensity of the light beam is above a threshold value for carrying out a reaction at points of high intensity and below this threshold value at points of low intensity. The reaction can be, for example, the transfer of the phosphor from the singlet state of the phosphor to the excited singlet state of the phosphor or the heating up to the transfer of the second, in particular organic, material from the oxygen impermeable state to the oxygen permeable state. This enables the targeted partial activation of the phosphorescence of the structure and thus the creation of geometric phosphorescent patterns. This makes it possible, for example, to deposit information in the form of writing, images, logos, codes, pictograms, machine-readable writing, bar codes, QR codes, or the like on the structure. Due to the possibility of repeated activation, deactivation, and reactivation of the phosphorescence, the structure can be rewritten several times. It is also conceivable that the structure in the deactivation step is partially covered with a mask and/or the structure in the deactivation step is only partially illuminated with the light of the third characteristic by a locally meandering or line-scanning light beam and/or the structure in the deactivation step is only partially illuminated by illuminating the structure with a light beam having a steel profile. Another object of the present Disclosure is a structure for use in a method according to any one of Examples 1 to 12, wherein the structure includes a first and a second material, wherein a phosphor is admixed with the first material and oxygen is present in the region of the phosphor in the non-irradiated and/or non-heated state, and wherein the second material is oxygen impermeable at an ambient temperature and acts as an oxygen barrier between the first material and an environment of the structure in the oxygen impermeable state. Preferably, the first material includes a first organic material and/or the second material includes a second organic material. Particularly preferably, the first material is a first organic material and the second material is a second organic material. The first, in particular organic, material is preferably different from the second, in particular organic, material. The first, in particular organic, material forms a guest-host complex with the phosphor, for example, wherein the first, in particular organic, material forms the host and the phosphor forms the guest. The second, in particular organic, material acts as an oxygen barrier at an ambient temperature, for example room temperature, i.e. 293 K, and prevents oxygen from penetrating to the first, in particular organic, material, and thus also to the phosphor. Thus, oxygen is prevented from inhibiting phosphorescence. In a preferred aspect of the Disclosure, the first material includes a first organic material that is oxidizable by singlet oxygen. Particularly preferably, the first material is a first organic material. Preferably, the first organic material is capable of forming a chemical bond with singlet oxygen. For example, the first material includes a polymer, preferably an organic polymer. Particularly preferably, the first material is a polymer, especially an organic polymer. Preferably, the first material is transparent and can be processed by wet processing. For example, the first material includes polymethyl methacrylate (PMMA), polystyrene (PS) and/or cycloolefin copolymers (COC). In a preferred aspect of the Disclosure, the second material includes a second organic material, preferably an organic polymer. The second material is preferably an organic polymer. The second material preferably has a second organic material that is impermeable to oxygen at ambient temperature. Preferably, the second material is transparent and can be processed, for example, by wet processing. Particularly preferably, the second material is a second organic material that can be processed in combination with the first material. For example, the second material has polyvinyl alcohol (PVA) and/or ethylene vinyl alcohol (EVOH) copolymers. In a preferred aspect of the Disclosure, the phosphor is an organic phosphor that is particularly preferably capable of being excited to phosphorescence at an ambient temperature. Preferably, the phosphor may be an organic phosphor whose phosphorescence is inhibited by oxygen. Particularly preferably, the phosphor may be an organic phosphor that can be processed by wet processing. Preferably, the phosphor has NPB, PhenDPA, PhenTPA, TA, BP-TA, Br-BP-TA, BP-2TA, DPS-TA, DPS-2TA, Br-DPS-TA, BF2(HPhN), and/or BF2(HBAN). Particularly preferably, the phosphor exclusively features NPB, PhenDPA, PhenTPA, TA, BP-TA, Br-BP-TA, BP-2TA, DPS-TA, DPS-2TA, Br-DPS-TA, BF2(HPhN), or BF2(HBAN). Particularly preferably, the phosphor has at least one of the following compounds: R1, R2and R3being identical or different from each other. Furthermore:R1is a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl or a substituted or unsubstituted alkyl or a substituted or unsubstituted heteroalkyl or hydrogen;R2is a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl or a substituted or unsubstituted alkyl or a substituted or unsubstituted heteroalkyl or hydrogen;R3is a substituted or unsubstituted alkyl or a substituted or unsubstituted heteroalkyl or hydrogen or nitro group. R3is selected from the group H, OR4or NO2; R4is H or a (C1-C8) alkyl; R5is either H, a halogen or a thianthrene; X is P or N; Y1, Y2, Y3and Y4are each independently selected from C or N, where either two or four of Y1, Y2, Y3and Y4are N. Z1and Z2are preferably independently selected from each other. Z1is either an enol or sulfoxide. Z2is absent or a heteroatom or selected from the group consisting of —NR4. Z3is selected from the group consisting of —NR4or —CR4R4. According to a preferred aspect of the Disclosure, the second material, in particular organic material, can be converted from an oxygen impermeable state to an oxygen permeable state by supplying heat and/or light. Preferably, the conversion from the oxygen-impermeable to the oxygen-permeable state is carried out thermally and/or photochemically. According to another preferred aspect of the Disclosure, the structure includes a substrate. The substrate includes, for example, the second, in particular organic, material. Preferably, the substrate is transparent. For example, the substrate includes a film, in particular a transparent film, or a glass plate. The substrate is preferably provided with an adhesive and/or magnetic underside. For example, the substrate has a self-adhesive and/or magnetic film. Thus, the structure can be applied easily and, in the case of the magnetic foil, reversibly. Conceivably, the substrate is a preferably transparent sheet. It is conceivable, for example, that the substrate is a glass or plastic sheet. However, it is also conceivable that the substrate is flexible. It is further conceivable that the substrate is a film, a rubberized structure or a rubber. According to a preferred aspect of the Disclosure, the structure includes a first layer with a first layer thickness of the first, in particular organic, material and/or at least one second layer with a second layer thickness of the second, in particular organic, material, wherein the first layer is arranged between the substrate and the at least second layer. Preferably, the structure includes exactly one second layer. Alternatively, the structure has a plurality of, for example two, second layers. The number of layers can determine the quality of the oxygen barrier. In particular, the oxygen impermeability of the oxygen barrier formed at ambient temperature by the second, in particular organic, material increases with the number of second layers of the structure. For example, the first layer thickness is 200 nm to 2000 nm, preferably 900 nm. The at least second layer thickness is, for example, between 800 nm and 30 μm or between 500 nm and 50 μm. Preferably, the sum of the thicknesses of the second layers is between 800 nm and 30 μm or between 500 nm and 50 μm. According to a preferred aspect of the Disclosure, the first, in particular organic, material and the second, in particular organic, material are applied to the substrate as a mixture. The first, in particular organic, material preferably includes PMMA, PS and/or COC, in particular the first material is an organic material and exclusively includes PMMA, PS or COC. The second, in particular organic, material preferably includes EVOH and/or PVA, in particular the second material is an organic material and exclusively includes EVOH or PVA. A further object of the present Disclosure is a method for manufacturing a structure according to any one of Examples 14 to 27, wherein a first, in particular organic, material and a second, in particular organic, material are applied to a substrate, wherein a phosphor is admixed to the first, in particular organic, material. The method of manufacture may be suitable for producing a structure in which phosphorescence can be activated and deactivated. In particular, the phosphorescence of the structure can also be activated and deactivated in partial areas. The production process is very simple to carry out and advantageously does not require the absence of oxygen. Thus, the process for fabrication can be carried out outside vacuum chambers or chambers flooded with inert gas. It is conceivable that the second, in particular organic, material is used as substrate. However, it is also conceivable that glass, metal or plastic is used as the substrate. According to a preferred aspect of the present Disclosure, it is provided that the first, in particular organic, material is applied to the substrate by means of rotary coating and/or a line application method and/or pipetting and/or a printing method and/or a spraying method, in particular as a first layer, and/or the second, in particular organic, material is applied by means of rotary coating and/or line application method and/or pipetting and/or printing method and/or spraying method, in particular as at least a second layer. Preferably, the first, in particular organic, material and the second, in particular organic, material each form a layer. It is conceivable that the first, in particular organic, material is PMMA doped with NPB. Alternatively, PMMA and NPB form a guest-host complex, with PMMA acting as host and NPB as guest. It is further conceivable that the first, particularly organic, material is dissolved in an organic solvent for application. Conceivably, the first, in particular organic, material is dissolved in anisole, chlorobenzene, water, or ethyl lactate. Conceivably, the second, in particular organic, material is also dissolved in an organic solvent for application. Conceivably, the second, in particular organic, material is dissolved in anisole, chlorobenzene, water, or ethyl lactate. Preferably, the first, in particular organic, material and/or the second, in particular organic, material are dried after application. This allows the solvent to be evaporated in a controlled manner. It is conceivable that this is done in an oven or on a hotplate. Furthermore, it is conceivable that the first, in particular organic, material and the second, in particular organic, material are applied as a mixture. Thus, no oxygen-impermeable substrate is necessary. According to a further preferred aspect of the present Disclosure, it is envisaged that a solid substrate or a film is used as the substrate, preferably a solid substrate with a self-adhesive rear side facing away from the first, in particular organic, material or a film with a self-adhesive rear side facing away from the first, in particular organic, material. All of the foregoing statements in the Disclosure apply equally to the method for activation according to the Disclosure, the method for deactivation and the method for activation and deactivation of phosphorescence, the structure according to the Disclosure and the method for production thereof according to the Disclosure. Another object of the Disclosure is a label including a functional layer, wherein the functional layer includes a structure according to any one of Examples 13 to 29. The label according to the Disclosure can be written and erased several times. The writing is preferably performed by irradiating with light of the first characteristic. By irradiating with light of the first characteristic, oxygen present in the region of the first, in particular organic, material, in particular in the region of the phosphor, is preferably converted into an excited singlet state and reacts with the second, in particular organic, material. The oxygen can thus no longer prevent phosphorescence. Preferably, the label can be read by irradiating it with light of a second characteristic. In particular, the light of the second characteristic excites the phosphor to phosphorescence. The label can be erased by means of the introduction of heat and/or the irradiation of light of a third characteristic, in particular IR light. The introduction of heat and/or the irradiation of light of the third characteristic transforms the second, in particular organic, material from an oxygen-impermeable to an oxygen-permeable state. Oxygen can penetrate from the environment to the first, in particular organic, material and in particular to the phosphor and prevent phosphorescence. Conceivably, the label includes a substrate. In addition, it is conceivable that the functional layer is arranged on the substrate. It is further conceivable that the substrate is arranged in a plane. However, it is also conceivable that the substrate is arranged in a non-planar surface. For this purpose, the substrate has a non-constant geometric profile. Conceivably, the functional structure has the same geometric profile as the substrate. It is further conceivable that the substrate is impermeable to oxygen. According to a preferred aspect of the present Disclosure, it is provided that the functional layer includes a first, in particular organic, material and a second, in particular organic, material, wherein a phosphor for phosphorescence is admixed with the first, in particular organic, material and wherein the second, in particular organic, material is in an oxygen-impermeable state at room temperature. According to a preferred further aspect of the present Disclosure, it is provided that the first, in particular organic, material is arranged in a lower layer and the second, in particular organic, material is arranged in an upper layer, wherein the lower layer is arranged between a substrate and the upper layer. Preferably, the lower layer has a layer thickness of between 200 nm and 2000 nm, preferably between 500 nm and 1500 nm, in particular of circa 900 nm and/or the upper layer has a layer thickness of between 500 nm and 50 μm. It is conceivable that the substrate is made of the second, in particular organic, material. Thus, advantageously, a layer structure consisting of two outer layers of the second, in particular organic, material and a layer of the first, in particular organic, material enclosed by the two outer layers is realized. Furthermore, however, it is also conceivable that the first, in particular organic, material and the second, in particular organic, material are a mixture. According to a preferred further aspect of the present Disclosure, it is provided that the functional layer can be transferred from the non-phosphorescent state to the phosphorescent state by the incidence of light of a first characteristic on the first, in particular organic, material and/or can be transferred from the phosphorescent state to the non-phosphorescent state by the incidence of light of a second characteristic on the functional layer and/or can be transferred from the phosphorescent state to the non-phosphorescent state by the introduction of heat into the functional layer. Conceivably, the light of the first characteristic is also suitable for exciting phosphorescence. The light of the first characteristic may have a wavelength of less than 700 nm, preferably less than 550 nm, particularly preferably less than 460 nm. Preferably, the light of the second characteristic is IR light. Furthermore, it is advantageously possible to use the same light source for the light of the first characteristic and the light for exciting phosphorescence, if the light of the first characteristic and the light for exciting phosphorescence do not differ in wavelength but in intensity, i.e. the light for exciting phosphorescence is the light of the first characteristic with a second intensity. The light of the first characteristic then has a first intensity. In this case, the first intensity is higher than the second intensity. It is conceivable that the first intensity is 10 times to 100 times greater, preferably 20 times to 90 times greater, particularly preferably 50 times to 80 times greater and in particular circa 70 times greater than the second intensity. It is conceivable that the first intensity is between 1 mWcm−2and 20 mWcm−2, preferably between 3 mWcm−2and 15 mWcm−2, particularly preferably between 5 mWcm−2and 10 mWcm−2and in particular at circa 7 mWcm−2. Furthermore, it is conceivable that the second intensity is between 0.01 mWcm−2and 1 mWcm−2, preferably between 0.05 mWcm−2and 0.5 mWcm−2and in particular at circa 0.1 mWcm−2. According to a preferred further aspect of the present Disclosure, it is provided that the first material, in particular organic material, is configured for binding oxygen by the incidence of light of the first characteristic. This allows oxygen to be removed from the functional layer, thus enabling phosphorescence. According to a preferred further aspect of the present Disclosure, it is provided that the second, in particular organic, material is convertible to an oxygen permeable state by the incidence of light of the second characteristic and/or the application of heat. Thus, it is advantageously possible to prevent phosphorescence in the functional layer by the introduction of oxygen. It is conceivable that the functional layer is configured in such a way that when light of the second characteristic is incident, the first, in particular organic, material is heated and the heat is transported to the second, in particular organic, material. It is further conceivable that this heating allows the second, in particular organic, material to be converted into an oxygen-permeable state. According to a preferred further aspect of the present Disclosure, it is provided that the first, in particular organic, material is PMMA, PS and/or COC and/or the second, in particular organic, material includes EVOH and/or PVA and/or the phosphor includes NPB, PhenDPA, PhenTPA, TA, BP-TA, Br-BP-TA, BP-2TA, DPS-TA, DPS-2TA, Br-DPS-TA, BF2(HPhN) and/or BF2(HBAN). Particularly preferably, the phosphor exclusively features NPB, PhenDPA, PhenTPA, TA, BP-TA, Br-BP-TA, BP-2TA, DPS-TA, DPS-2TA, Br-DPS-TA, BF2(HPhN), or BF2(HBAN). These materials are widely used, easy to process and inexpensive. Preferably, two weight percent NPB is admixed to the first, in particular organic, material. According to a preferred further aspect of the present Disclosure, it is provided that the substrate is a film, preferably the side of the substrate facing away from the functional layer is self-adhesive or magnetic. This advantageously enables the label to be easily applied to objects to be labeled. A film is flexible in this case. Thus, the label can also be applied to uneven objects. According to a preferred further aspect of the present Disclosure, it is provided that the film is transparent. This enables a completely unobtrusive appearance of the label. The label can thus be applied to windowpanes or screens, for example, without affecting their function. This is particularly advantageous in the case of objects to be labeled which offer hardly any surface, which is not visually functional. According to a preferred further aspect of the present Disclosure, it is provided that the substrate is a plastic plate, preferably a transparent plastic plate, or a metal plate, wherein preferably the side of the substrate facing away from the functional layer is self-adhesive or magnetic. This advantageously enables mechanical protection of the functional layer. A further object of the present Disclosure is a method for writing on a label according to Example 30, wherein for writing on the label in a writing process, dots of the functional layer are selectively transferred locally from the non-phosphorescent state to the phosphorescent state in a non-contact manner, wherein a phosphorescent region is formed by the dots, wherein during the writing process the phosphorescent region is irradiated with light of a first characteristic, wherein in a binding step the oxygen present in the phosphorescent region is bound to the first, in particular organic, material. Dot, in the sense of the present Disclosure, means a locally extended location in the functional layer. The sum of all dots is the functional layer. Selectively locally transferable means in the sense of the present Disclosure that the dots are individually selectively transferable. That is, one point can be transferred without transferring another point. In the sense of the present Disclosure, the phosphorescent region means a region suitable for phosphorescence. In particular, oxygen present in the region of the phosphor or the first material, in particular organic material, is transferred to the excited singlet state only in the phosphorescent region. By selectively irradiating partial areas of the label, oxygen is prevented from inhibiting phosphorescence only in these areas, and thus a structure that can be excited to phosphorescence by irradiation with light is made available only in these areas. Preferably, during the writing process, the phosphorescent region is irradiated with light of the first characteristic, the functional layer being partially covered with a mask in such a way that only the phosphorescent area is illuminated and/or the functional layer is illuminated with light of the first characteristic by a locally meandering or line-by-line scanning light beam only in the phosphorescent area and/or the functional layer is illuminated only in the phosphorescent area, in that the functional layer is illuminated with a light beam having a beam profile, the beam profile on the functional layer corresponding to the phosphorescent area. The label can thus be quickly and reliably written with a code or other characters. Advantageously, the character used to write on the label is not visible to the naked eye. In particular, when a transparent substrate is used, the label is thus barely visible to the naked eye. Preferably, the label described is transparent. Advantageously, a label is thus provided which is suitable for use even in exposed locations, for example also on transparent objects such as a glass bottle or a window. It is therefore not necessary to attach labels to the inside or other places of a product that are not directly visible. The label can be attached to the outside of the product in an easily readable location and still remain virtually invisible to traffic. The visual appearance of the product may not be compromised by the label. This simplifies picking, packing and logistics of goods, for example. The label is preferably read by irradiating the label with light of a second characteristic. Preferably, the light of the second characteristic differs from the light of the first characteristic only in intensity. By irradiating with light of the second characteristic, the phosphor is excited to phosphorescence in the areas previously irradiated with light of the first characteristic. Another object of the present Disclosure is a method for erasing a label according to Example 30, wherein for erasing the label in an erasing process the functional layer is substantially completely converted into the non-phosphorescent state, wherein during the erasing process heat is introduced into the functional layer and/or the functional layer is irradiated with light of a second characteristic, wherein the second material is converted from an oxygen-impermeable state into an oxygen-permeable state by the heat and/or by the irradiation with the light of the second characteristic. Oxygen present in the environment can thus penetrate to the phosphor and inhibit phosphorescence. Preferably, heat is introduced by irradiation with light of the second characteristic, in particular IR light. This provides a simple, fast and inexpensive method of extinguishing the label. Another object of the present Disclosure is a method for writing and erasing a label according to Example 30, wherein the label is written in a writing process according to any one of Examples 31 to 33 and erased in a subsequent erasing process according to any one of Examples 34 to 36. Preferably, the label is written in a writing process according to any one of Examples 31 to 33, erased in a subsequent erasing process according to any one of Examples 34 to 36, and rewritten in a writing process following the erasing process according to any one of Examples 31 to 33. Repeated writing and erasing of the label eliminates the need to apply new labels. According to a preferred aspect of the Disclosure, for writing the label in a writing process, points of the functional layer are selectively transferred locally from the non-phosphorescent state to the phosphorescent state without contact, wherein a phosphorescent region is formed by the points, for erasing the label in an erasing process, the functional layer is transferred substantially completely to the non-phosphorescent state, and for rewriting the label, the writing process is carried out. According to a preferred aspect of the present Disclosure, it is provided that during the writing operation the phosphorescent region is illuminated with light of the first characteristic, preferably the functional layer is partially covered with a mask such, that only the phosphorescent area is illuminated and/or the functional layer is illuminated with light of the first characteristic by a locally meandering or line-by-line scanning light beam only in the phosphorescent area and/or the functional layer is illuminated only in the phosphorescent area by illuminating the functional layer with a light beam having a steel profile, the beam profile on the functional layer corresponding to the phosphorescent area. According to a preferred aspect of the present Disclosure, it is provided that UV light is used as the light of the first characteristic, wherein preferably oxygen is bound to the first, in particular organic, material in a binding step, wherein prior to the binding step preferably the oxygen is converted from a triplet ground state of oxygen to an excited singlet state of oxygen in a triplet-triplet interaction with the phosphor, wherein the phosphor is transferred from a singlet state of the phosphor to an excited singlet state of the phosphor prior to the triplet-triplet interaction by the light of the first characteristic, and subsequently by intercombination from the excited singlet state of the phosphor to an excited triplet state of the phosphor. According to a preferred further aspect of the present Disclosure, it is provided that heat is introduced into the functional layer during the quenching process, preferably the heat converting the second, in particular organic, material from an oxygen-impermeable state to an oxygen-permeable state. However, it is also conceivable that oxygen is introduced into the functional layer during the quenching process by waiting. Due to imperfections of the functional layer, the oxygen barrier formed by the second, in particular organic, material is not perfect, so that oxygen can diffuse in over a longer period of time. According to a preferred further aspect of the present Disclosure, it is provided that the heat is introduced by irradiation with light of the second characteristic, wherein the light of the second characteristic is preferably IR light. This enables contactless erasure of the information on the label. It is conceivable that in this case the functional layer is partially covered with a mask in such a way that an area to be erased is illuminated and/or the functional layer is illuminated with light of the second characteristic by a light beam meandering locally or scanning line by line only in the area to be erased and/or the functional layer is illuminated only in the area to be erased by illuminating the functional layer with a light beam having a steel profile, the beam profile on the functional layer corresponding to the area to be erased. Another object of the present Disclosure is a sensor for determining the dose of ultraviolet light, including a structure according to any one of Examples 13 to 29. In particular, the sensor determines the energy absorbed by the sensor per unit area upon irradiation with ultraviolet light. The sensor according to the Disclosure thus provides a measuring device for determining the dose of ultraviolet radiation incident on an object. In particular, the sensor according to the Disclosure provides information on the absolute value of the dose of ultraviolet radiation (UV dose). The sensor does not require any electronics and is thus independent of a power source. Preferably, the sensor is designed as a foil. This allows flexible and easy mounting. Thus, an electronics-free, large-area executable and flexible sensor for the determination of the UV dose is provided. According to a preferred aspect of the Disclosure, the sensor has a dose threshold value, whereby phosphorescence sets in upon irradiation with ultraviolet light at a dose that corresponds to or exceeds the dose threshold value. The dose threshold may be varied, for example, by the material composition of the structure and by the first and second layer thicknesses of the structure. According to a preferred aspect of the Disclosure, the sensor has a main extension plane and the dose threshold is homogeneous in the main extension plane. This advantageously provides a sensor for spatially resolved determination of the UV dose. The main extension plane of the sensor runs in particular parallel to the substrate and/or to the first and second layers of the structure. When the main extension plane of the sensor is irradiated with UV light, phosphorescence is triggered in the areas in which the dose threshold is reached or exceeded. In the remaining areas, phosphorescence is inhibited by molecular oxygen present in the structure. In particular, the sensor can be used to determine when and where a specific UV dose defined by the dose threshold has been exceeded. A two-dimensional UV dose threshold measurement is thus advantageously enabled. According to a preferred aspect of the Disclosure, the sensor has a neutral density filter. With the aid of the neutral density filter, the dose threshold can be set and easily changed. Preferably, the neutral density filter is applied as a foil on the sensor surface. Alternatively, the dose threshold can be set via material parameters of the structure. According to a preferred aspect of the Disclosure, the sensor has a principal extension plane and the dose threshold has a gradient or gradation of transparency in the principal extension plane. For example, the sensor has a neutral density filter, wherein the neutral density filter has a gradient or gradation of transparency. For example, the neutral density filter is designed as a gradient gray filter or graduated neutral density filter (GND filter). Alternatively or additionally, the material composition of the structure exhibits a gradient or gradation in composition. According to a preferred aspect of the Disclosure, the dose threshold increases along at least one axis in the principal extension plane. Thus, the UV dose required to trigger phosphorescence changes along at least one axis in the main extension plane of the sensor. For example, the at least one axis is parallel to an edge of the sensor. Preferably, the dose threshold increases along the at least one axis. For example, when irradiated with a low UV dose, the sensor illuminates only in the lower region of the at least one axis due to phosphorescence, while at a higher dose the illuminated region extends along the at least one axis. Preferably, the sensor has a scale along the at least one axis, the scale indicating the respective dose threshold. Thus, the irradiated UV dose can be read directly. The use of reading devices is advantageously avoided. This enables one-dimensional but absolute UV dose value determinations. A further object of the present Disclosure is a method for spatially resolved determination of a dose of ultraviolet radiation, in particular incident on an object, with a sensor according to one of Examples 39 to 42, wherein in an irradiation step the sensor, preferably with the object, is irradiated with ultraviolet light of a dose to be determined and wherein in a determination step in the regions of the main extension plane of the sensor, in which the irradiated dose reaches or exceeds the dose threshold value, molecular oxygen is bound in the sensor and phosphorescence is triggered, and in the regions of the main extension plane of the sensor in which the irradiated dose falls below the dose threshold value, molecular oxygen in the sensor prevents the occurrence of phosphorescence. In particular, molecular oxygen present in the region of the first, in particular organic, material and/or in the region of the phosphor prevents phosphorescence in the regions of the main extension plane of the sensor in which the irradiated dose falls below the dose threshold. In the regions of the main extension plane of the sensor in which the irradiated dose reaches or exceeds the dose threshold, molecular oxygen present in the region of the first, in particular organic, material and/or in the region of the phosphor is photochemically deactivated. In particular, the oxygen is bound to the first, in particular organic, material. The oxygen can thus no longer prevent phosphorescence. Further irradiation with UV light stimulates the phosphor to phosphoresce. Advantageously, this provides a method for two-dimensional UV dose threshold determination. In particular, the method according to the Disclosure can be used to check the homogeneity of an irradiation with UV light. If the phosphorescence appears everywhere and simultaneously in the main extension plane of the sensor, the UV dose is homogeneous. A further object of the present Disclosure is a method for absolute value determination of a dose of ultraviolet radiation, in particular incident on an object, with a sensor according to any one of Examples 43 to 46, wherein in an irradiation step the sensor, preferably with the object, is irradiated with ultraviolet light of a dose to be determined and wherein in a determination step in the regions of the main extension plane of the sensor, in which the irradiated dose in each case reaches or exceeds the dose threshold value which is variable in the main extension plane, molecular oxygen is bound in the sensor and phosphorescence is triggered, and in the regions of the main extension plane of the sensor in which the irradiated dose in each case falls below the dose threshold value which is variable in the main extension plane, molecular oxygen in the sensor prevents the occurrence of the phosphorescence. This advantageously provides a method, in particular for the one-dimensional determination of absolute UV dose values. In particular, molecular oxygen present in the region of the first, in particular organic, material and/or in the region of the phosphor prevents phosphorescence in the regions of the main extension plane of the sensor in which the irradiated dose falls below the dose threshold value. In the regions of the main extension plane of the sensor in which the irradiated dose reaches or exceeds the dose threshold, molecular oxygen present in the region of the first, in particular organic, material and/or in the region of the phosphor is photochemically deactivated. In particular, the oxygen is bound to the first, in particular organic, material. The oxygen can thus no longer prevent phosphorescence. Further irradiation with UV light stimulates the phosphor to phosphoresce. According to a preferred aspect of the Disclosure, the sensor is irradiated with light and/or heated in a neutralization step, whereby the irradiation and/or heating causes oxygen to penetrate the sensor and prevent phosphorescence. The sensor is thus advantageously neutralized and can be reused for further UV dose determinations. According to a preferred aspect of the Disclosure, the neutralization step is followed by at least one irradiation step and at least one determination step according to one of Examples 47 or 48. The UV dose determination is preferably repeated several times for the same or for different objects and/or for the same or for different UV sources. According to a preferred aspect of the Disclosure, the sensor is arranged on a roller of a production line. This makes it advantageous to determine the UV dose with which objects on the production line are actually irradiated. Depending on the aspect of the Disclosure, information about the spatial resolution of the UV dose and/or the absolute value of the UV dose can thus be obtained. Preferably, several sensors are arranged on the production line so that both a two-dimensional UV dose threshold measurement and a one-dimensional absolute UV dose measurement are possible. Since the sensor is free of electronics and can be designed as a film, especially a self-adhesive film, it can be easily and cost-effectively integrated into existing production lines. There is no need for costly installation. The sensor can also be easily adapted to the individual application in terms of size, shape, and sensitivity. Alternatively, the sensor is arranged directly on the object whose UV dose is to be determined. Here, it is particularly advantageous that the sensor is free of electronics and can be operated independently of a power source. The sensor can be applied directly to the object, for example, as a self-adhesive film. Further details, features and advantages of the Disclosure will be apparent from the drawings, and from the following description of preferred aspects of the Disclosure based on the drawings. In this connection, the drawings merely illustrate exemplary aspects of the Disclosure, which do not restrict the essential idea of the Disclosure. In the various figures, identical parts are always provided with the same reference signs and are therefore generally also named or mentioned only once in each case.FIG.1Ashows a schematic view of the structure1according to an exemplary aspect of the present Disclosure. The structure1has the substrate2. The substrate2is a transparent film. However, it is also conceivable that the substrate2includes the second organic material. The first organic material3is deposited on the substrate2in a 900 nm thick layer. The first organic material3consists, for example, of polymethyl methacrylate (PMMA), to which approximately two mass percent N,N′-di(1-naphthyl)-N,N′-diphenyl-(1,T-biphenyl)-4,4′-diamine is admixed. Over the layer of the first organic material3, the second organic material4is applied in another layer and contains ethylene-vinyl alcohol copolymers. In a normal state at room temperature, the second organic material4is impermeable to oxygen and serves as an oxygen barrier between the first organic material3and the environment of the structure1. The first material may, for example, alternatively or additionally contain PS and/or COC. In addition to NPB, suitable phosphors include, for example, PhenDPA, PhenTPA, TA, BP-TA, Br-BP-TA, BP-2TA, DPS-TA, DPS-2TA, Br-DPS-TA, BF2(HPhN) and/or BF2(HBAN). FIG.1Bschematically shows the process of activating the phosphorescence30of the structure1, more specifically the first activation step, according to an exemplary aspect of the Disclosure is shown. The structure1is partially illuminated by the light of the first characteristic8. For this purpose, the structure1is partially covered with a mask7with respect to the light source of the light of the first characteristic8. Preferably, the mask has a resolution of up to 700 dpi. The light of the first characteristic8thus irradiates the first area5of the structure. The second area6of the structure is not irradiated by the light of the first characteristic8. FIG.2shows a schematic of the first activation step according to an exemplary aspect of the Disclosure. The light of the first characteristic8(not shown) with a wavelength of approximately 365 nm induces a transition of the phosphor mixed with the first organic material3in the first region of the structure8(seeFIG.1B) from the singlet state of the phosphorSO to an excited singlet state of the phosphor S1. From this excited singlet state of phosphor S1, a part of the phosphor transitions to an excited triplet state of phosphor T1via intercombination10. Oxygen9is present in the layer of the first organic material3, which prevents phosphorescence30. The oxygen9is in a triplet ground state of oxygen T0. In a triplet-triplet interaction11, the phosphor passes from the excited triplet state of phosphor T1to the singlet state of phosphor SO and the oxygen9passes from the triplet ground state of oxygen T0to an excited singlet state of oxygen ST. The oxygen9is highly reactive in its excited singlet state ST, oxidizing the first organic material3and becoming bound (not shown). Thus, the oxygen9present in the layer of the first organic material3is effectively deactivated. The second organic material4acts as an oxygen barrier to prevent additional oxygen from the outside from entering the layer of the first organic material3. The second region6is not illuminated by the light of the first characteristic8and consequently is not activated. In this region, the oxygen9is not bound to the first organic material3and thus is not deactivated. FIG.3shows a schematic of the second activation step according to an exemplary aspect of the present Disclosure. For the second activation step, the mask7is removed and the light of the first characteristic8is still used for irradiation at a significantly reduced intensity (not shown here). In the first region5, a transition of the phosphor from the singlet state of the phosphor SO to the excited singlet state of the phosphor S1is then further induced. From this excited singlet state of phosphor S1, the phosphor can transition to the excited triplet state of dopant T1via intercombination10. The transition from the excited triplet state of phosphor T1to the singlet state of phosphor T0is quantum mechanically “forbidden” and thus the excited triplet state of phosphor T1has long lifetimes. Nevertheless, transitions from the excited triplet state of phosphor T1to the singlet state of phosphor SO occur, distributed over a long period of time. Even after the source of light of the first characteristic8is switched off, phosphorescence30is produced. Since oxygen9is not deactivated in the second region6, it prevents phosphorescence30here (seeFIG.1B). Thus, the structure1phosphoresces only in the first region5. FIG.4Ashows a schematic view of the label12according to an exemplary aspect of the present Disclosure. The label12has the substrate2. The substrate2is a transparent film. On the substrate2, the first organic material3is deposited in a 900 nm thick lower layer. The first organic material3consists, for example, of polymethyl methacrylate (PMMA), which is admixed with approximately two mass percent N,N′-di(1-naphthyl)-N,N′-diphenyl-(1,1′-biphenyl)-4,4′-diamine. The first material may, for example, alternatively or additionally contain PS and/or COC. In addition to NPB, suitable phosphors include, for example, PhenDPA, PhenTPA, TA, BP-TA, Br-BP-TA, BP-2TA, DPS-TA, DPS-2TA, Br-DPS-TA, BF2(HPhN) and/or BF2(HBAN) as appropriate. Above the layer of the first organic material3, the second organic material4is applied in an upper layer and contains, for example, ethylene-vinyl alcohol copolymers and/or PVA. In a normal state at room temperature, the second organic material4is impermeable to oxygen and serves as an oxygen barrier between the first organic material3and the environment of the label12. On the side of the substrate2facing away from the first organic material3, the transparent film is preferably adhesive or magnetic. Thus, it can be easily, and in the case of the magnetic film also reversibly, applied to goods, for example. FIG.4Bschematically shows the writing of the label12according to an exemplary aspect of the present Disclosure. The label12is partially illuminated by the light of the first characteristic8. For this purpose, the label12is positioned opposite the light source of the light of the first characteristic8partially covered with a mask7the mask is preferably a negative of the character with which the label12is to be described. Possible characters are for example, one-dimensional, two-dimensional and three-dimensional codes such as barcodes, QR codes or others. It is also conceivable to include an indication of origin on the label. This can be in the form of an image, for example the brand, the manufacturer or supplier. The light of the first characteristic8thus irradiates the phosphorescent area5of the label. The non-phosphorescent area6of the label12is not irradiated by the light of the first characteristic8. The first phosphorescent region5can thus be excited to phosphoresce30by irradiation of light of the second characteristic. The label12can thus advantageously be written with a resolution of up to 700 dpi. The label12is usually readable by the naked eye in the written state. Rather, the irradiation of light of the second characteristic is necessary to excite phosphorescence in the area5. Excitation to phosphorescence in region6is not possible because molecular oxygen is present here, which prevents it. Advantageously, therefore, a label12is provided whose contents are not visible to the naked eye. The label12is erased by the introduction of heat and/or light of a third characteristic. For example, the label12will be erased by irradiation with IR light having a wavelength of about 4 μm for a period of 1 min. The radiation is preferably absorbed by PMMA, PS and/or COC, which is heated as a result. The second organic material4, which acts as an oxygen barrier at ambient temperature, is thus converted to an oxygen-permeable state. The first organic material3is again filled with oxygen, phosphorescence is prevented. Write and erase cycles can be repeated several times. For example, if the write and erase procedures are repeated 40 times, the emission still reaches 40% of its initial value.FIG.5Aschematically shows the structure of the sensor13for determining the dose of ultraviolet light17according to an exemplary aspect of the present Disclosure. The sensor13has a structure1. The structure1has a first and a second material3,4, wherein a phosphor is admixed with the first material3and oxygen9is present in the region of the phosphor, and wherein the second material4is impermeable to oxygen at an ambient temperature and, in the oxygen-impermeable state, acts as an oxygen barrier between the first material3and an environment of the structure1. Preferably, the second material4is convertible from the oxygen-impermeable state to the oxygen-permeable state by supplying heat and/or light. Preferably, the structure1further includes a substrate2. For example, the substrate2includes the second material4. The substrate2preferably includes a film, particularly preferably a self-adhesive or magnetic film. This gives the sensor13a high degree of flexibility. Furthermore, the sensor13can thus be attached easily, if necessary even reversibly. The structure1has, for example, a first layer with a first layer thickness of the first material3and/or at least one second layer with a second layer thickness of the second material4. The first layer is preferably arranged between the substrate2and the at least second layer. The first material3is preferably an organic material. The second material4is also preferably an organic material. The first material3and the phosphor preferably form a guest-host complex. For example, the first material3includes PMMA, PS and/or COC and the second material4includes, for example, EVOH and/or PVA. For example, the phosphor includes NPB, PhenDPA, PhenTPA, TA, BP-TA, Br-BP-TA, BP-2TA, DPS-TA, DPS-2TA, Br-DPS-TA, BFz (HPhN) and/or BFz (HBAN). The sensor13preferably has a dose threshold, wherein when the sensor13is irradiated with ultraviolet light17at a dose equal to or greater than the dose threshold, phosphorescence30begins. The dose threshold is dependent on, and can be varied via, the material composition of the structure1, among other factors. Irradiation of ultraviolet light17causes molecular oxygen9present in the region of the first material3and/or in the region of the phosphor to be bound by UV energy input. The molecular oxygen9can no longer influence the no longer suppress phosphorescence30. There is a sudden increase in phosphorescence30due to further irradiation with UV light. This occurs, depending on the irradiated intensity of the UV radiation, after a defined period of time, seeFIG.5B. The sudden, significant increase in phosphorescence30after a defined irradiation time is the basis for dose determination. The irradiation time required for the sudden increase in phosphorescence30depends on the material composition, the first and second layer thicknesses, the number of second layers and the intensity of the UV radiation17. The higher the intensity of the UV radiation17, the shorter the necessary irradiation time. In this case, the molecular oxygen9is only bound in the areas of the sensor13in which the dose threshold value is reached or exceeded. This allows a spatially resolved representation of the UV radiation17in real time as an on-off state of the phosphorescence30. FIG.6Aschematically shows the sensor13and the method for determining the UV dose according to an exemplary aspect of the present Disclosure. The sensor13has the features described in the description ofFIGS.5A and5B. The sensor13preferably has a main extension plane14. The main extension plane14is parallel to the substrate2. In the main extension plane14, the dose threshold is homogeneous. That is, the same UV dose is required at each point in the main extension plane14to excite phosphorescence30. For spatially resolved determination of a dose of ultraviolet radiation, the sensor13is irradiated with UV light17in an irradiation step at a dose to be determined. In a determination step, in the regions5of the main extension plane14in which the irradiated dose reaches or exceeds the dose threshold, molecular oxygen9is bound in the sensor13and phosphorescence30is triggered. In the regions6in which the irradiated dose falls below the dose threshold, the molecular oxygen9present in particular in the region of the first, in particular organic, material3and/or in the region of the phosphor prevents phosphorescence30. In the regions5in which the UV dose reaches or exceeds the dose threshold, phosphorescence30occurs. In the regions6in which the UV dose remains below the dose threshold, no phosphorescence30occurs. Thus, the appearance of phosphorescence30can be used to determine when and where the UV dose has reached or exceeded the dose threshold. Thus, advantageously, a method for spatially resolved determination of a UV dose is provided. This can be used, for example, to check irradiation homogeneity. Homogeneous irradiation, i.e. irradiation with a spatially homogeneous UV dose, has occurred if the phosphorescence30occurs everywhere simultaneously in the main extension plane14. The dose threshold can be adjusted via material parameters or a neutral density filter preferably arranged on the sensor surface. Preferably, the neutral density filter is designed as a foil. With the sensor13according to the described aspect of the Disclosure, two-dimensional UV dose threshold determinations can thus be carried out. FIG.6Bschematically shows the sensor13and the method for determining the UV dose according to an alternative exemplary aspect of the present Disclosure. The sensor13has the features described in the description ofFIGS.5A and5B. Preferably, the sensor13has a main extension plane14. The main extension plane14is parallel to the substrate2. In the main extension plane14, the dose threshold exhibits a gradient or gradation along an axis15. Along the axis15, the dose threshold increases, for example linearly. To realize such a gradient or gradation of the dose threshold, the sensor13has, for example, a gray gradient filter or GND filter. Alternatively, the material composition of the structure1exhibits a gradient or gradation in composition. In the initial region of axis15, a lower UV dose is sufficient to excite phosphorescence30. In the end region of axis15, a higher UV dose is necessary to produce phosphorescence30. In an irradiation step, the sensor13is irradiated with UV light17of a dose to be determined. In a determination step, in the regions5of the main extension plane14of the sensor13in which the irradiated dose reaches or exceeds the dose threshold value variable in the main extension plane14, respectively, molecular oxygen9is bound in the sensor13and phosphorescence30is triggered. In the areas6of the main extension plane14in which the irradiated dose in each case falls below the dose threshold value variable in the main extension plane14, molecular oxygen9present in the sensor13, in particular in the area of the first, in particular organic, material3and/or in the area of the phosphor, prevents phosphorescence. At a very low UV dose, the sensor13illuminates only in the initial area of the axis15; at a higher UV dose, the illuminated area5grows upward. Preferably, the sensor13has a scale16along the at least one axis15, which indicates the respective dose threshold. This allows a direct reading of the irradiated UV dose without additional reading devices. The sensor13can thus be used to determine one-dimensional absolute values of the UV dose. The methods described with reference toFIGS.6A and6Bare preferably followed by a neutralization step in which the sensor13is irradiated with light and/or heated. As a result of the irradiation and/or heating, oxygen penetrates the sensor13and stops the phosphorescence30. In particular, the second, in particular organic, material4changes from the oxygen-impermeable to an oxygen-permeable state. The second, in particular organic, material4no longer acts as an oxygen barrier. Oxygen can penetrate into the structure1from the area surrounding the sensor13. In particular, the oxygen penetrates into the region of the first, in particular organic, material3and/or into the region of the phosphor and here prevents phosphorescence30. The sensor13is thus advantageously neutralized and can be used for a further determination of a UV dose. Preferably, the irradiation, determination and neutralization steps are repeated several times, for example for different UV sources or different objects. FIG.7schematically shows the sensor13according to an exemplary aspect of the present Disclosure. The sensor13has the features described with reference to the precedingFIG.6B. The sensor13is arranged on a roller18of a production line. The sensor13is preferably bonded to the roll18. For this purpose, the sensor13preferably has a self-adhesive film. The sensor13is preferably placed like the objects transported on the roll18in ordinary operation. Thus, the measured values determined by means of the sensor13provide information about the UV dose to which the objects are exposed. Alternatively, the sensor13is placed directly on the object. For example, the axis15runs along the transport direction of the roller18. The sensor13preferably extends perpendicular to the axis15over the used width of the roller18. The roller18transports objects, for example, for UV curing under a UV source. The sensor13can be used to determine the absolute value of the UV dose over the entire used width of the roller18. Deviations from the nominal value can thus be identified and corrected if necessary. Additional aspects of the disclosure will be described by example: In Example 1, a method for activating the phosphorescence (30) of a structure (1) is disclosed: wherein the structure (1) includes a first and a second material (3,4); wherein a phosphor is admixed with the first material (3) and oxygen (9) is present in the region of the phosphor; and wherein the second material (4) is oxygen-impermeable at a temperature and, in the oxygen-impermeable state, acts as an oxygen barrier between the first material (3) and an environment of the structure (1); wherein, in order to activate the phosphorescence (30), oxygen (9) present in the structure (1) is photochemically deactivated in a first activation step by irradiating the structure (1) with light of a first characteristic (8), and wherein, in a second activation step, the phosphorescence (30) is initiated by irradiating the structure (1) with light of a second characteristic. In Example 2, a method for deactivating the phosphorescence (30) of a structure (1) is disclosed, wherein the structure (1) includes a first and a second material (3,4); wherein a phosphor is admixed with the first material (3); wherein the first material (3) is capable of containing molecular oxygen (9); wherein the second material (4) is oxygen-impermeable at an ambient temperature and in the oxygen-impermeable state, acts as an oxygen barrier between the first material (3) and an environment of the structure (1); wherein oxygen (9) is introduced into the structure (1) in a deactivation step to deactivate the phosphorescence (30) by heating the structure (1) in the deactivation step and/or irradiating it with light of a third characteristic. In Example 3, a method for activating and deactivating the phosphorescence (30) of a structure (1) is disclosed, wherein the structure (1) includes a first and a second material (3,4), wherein a phosphor is admixed with the first material (3) and oxygen (9) is present in the region of the phosphor; wherein the second material (4) is oxygen-impermeable at an ambient temperature and, in the oxygen-impermeable state, acts as an oxygen barrier between the first material (3) and an environment of the structure (1); wherein, in order to activate the phosphorescence (30), in a first activation step oxygen (9) present in the structure is photochemically deactivated by irradiating the structure (1) with light of a first characteristic (8); and wherein, in a second activation step, the phosphorescence (30) is activated by irradiating the structure (1) with light of a second characteristic (8); wherein, in order to deactivate the phosphorescence (30), oxygen (9) is introduced into the structure (1) in a deactivation step by heating the structure (1) in the deactivation step and/or by irradiating the structure with light of a third characteristic. In Example 4, the method of any one of Examples 2 and 3 is disclosed, wherein in the deactivation step the second material (4) is converted from an oxygen impermeable state to an oxygen permeable state by the heat and/or the light of a third characteristic, so that oxygen (9) penetrates to the first material (3) and/or the phosphor and inhibits the phosphorescence (30). In Example 5, the method of any one of Examples 2 to 4 is disclosed, wherein IR light is used as light of the third characteristic. In Example 6, the method of any one of Examples 2 to 5 is disclosed, wherein in the deactivation step heat is introduced into the structure (1) from the light of the third characteristic, the heat converting the second material (4) from an oxygen-impermeable state to an oxygen-permeable state. In Example 7, the method of any one of the preceding Examples 1 and 3 to 6 is disclosed, wherein light with a first intensity is used as light of the first characteristic (8), wherein the light of the first characteristic has a wavelength of less than 700 nm, preferably less than 550 nm, particularly preferably less than 460 nm, wherein preferably the light of the first characteristic (8) with a second intensity is used as light of the second characteristic. In Example 8, the method of any one of the preceding Examples 1 and 3 to 7 is disclosed, wherein in the first activation step the oxygen (9) is bound to a first material (3) in a binding step, wherein prior to the binding step the oxygen (9) is preferably converted from a triplet ground state of the oxygen (T0) into an excited singlet state of the oxygen (ST) in a triplet-triplet interaction (11) with a phosphor admixed with the first material (3). In Example 9, the method of Example 8 is disclosed, wherein the phosphor is transferred from a singlet state of the phosphor (SO) to an excited singlet state of the phosphor (S1) by the light of the first characteristic (8) prior to the triplet Triplet interaction (11), and subsequently from the excited singlet state of the phosphor (S1) to an excited triplet state of the phosphor (T1) by intercombination (10), wherein preferably in the second activation step the phosphor is transferred from a singlet state of the phosphor (SO) to an excited triplet state of the phosphor (T1). In Example 10, the method of any one of Examples 1 to 9 is disclosed, wherein a long-chain organic polymer, preferably polymethyl methacrylate, polystyrene and/or cycloolefin copolymers, is used as the first material (3), wherein the first material (3) preferably has the phosphor as a dopant and/or as a side chain. In Example 11, the method of any one of Examples 1 to 10 is disclosed, wherein the phosphor used is N,N′-di(1-naphthyl)-N,N′-diphenyl-(1,1′-biphenyl)-4,4′-diamines, tetra-N-phenylbenzidines, PhenDPA, PhenTPA, thianthrenes, benzophenone-thianthrenes, bromo-benzophenone-thianthrenes, benzophenone-2-thianthrenes, diphenylsulfone-thianthrenes, diphenylsulfone-2-thianthrenes, bromo-diphenylsulfone-thianthrenes, difluoroborone-9-hydroxyphenalenones and/or difluoroborone-6-hydroxybenz[de]anthracene-7-one. In Example 12, the method of any one of the preceding Examples is disclosed, wherein the structure (1) is partially covered with a mask (7) in the first activation step and/or the structure (1) is only partially illuminated in the first activation step with the light of the first characteristic (8) from a locally meandering or line-by-line scanning light beam and/or the structure (1) is only partially illuminated in the first activation step by illuminating the structure (1) with a light beam having a steel profile. In Example 13, a structure (1) for use in the method of any one of Examples 1 to 12 is disclosed, wherein the structure (1) includes a first and a second material (3,4), wherein a phosphor is admixed with the first material (3) and oxygen (9) is present in the region of the phosphor in the non-irradiated and/or non-heated state, and wherein the second material (4) is oxygen-impermeable at an ambient temperature and acts as an oxygen barrier between the first material (3) and an environment of the structure (1) in the oxygen-impermeable state. In Example 14, the structure (1) of Example 13 is disclosed, wherein the second material (4) can be converted from the oxygen-impermeable state to an oxygen-permeable state by supplying heat and/or light. In Example 15, the structure (1) of Example 13 and 14 is disclosed, wherein the structure (1) includes a substrate (2). In Example 16, the structure (1) of Example 15 is disclosed, wherein the substrate (2) includes the second material (4). In Example 17, the structure (1) of any one of Examples 15 and 16 is disclosed, wherein the substrate (2) is transparent. In Example 18, the structure (1) of any one of Examples 15 to 17 is disclosed, wherein the substrate (2) includes a self-adhesive film. In Example 19, the structure (1) of any one of Examples 15 to 18 is disclosed, wherein the substrate (2) includes a glass plate. In Example 20, the structure (1) of any one of Examples 15 to 19 is disclosed, wherein the structure (1) includes a first layer having a first layer thickness of the first material (3) and/or includes at least one second layer having a second layer thickness of the second material (4), wherein the first layer is arranged between the substrate (2) and the at least second layer. In Example 21, the structure (1) of Example 20 is disclosed, wherein the first layer thickness is between 200 nm and 2000 nm, preferably 900 nm. In Example 22, the structure (1) of any one of Examples 20 and 21 is disclosed, wherein the second layer thickness is between 800 nm and 30 μm. In Example 23, the structure (1) of any one of Examples 15 to 22 is disclosed, wherein the first material (3) and the second material (4) are applied as a mixture on the substrate (2). In Example 24, the structure (1) of any one of Examples 13 to 23 is disclosed, wherein the first material (3) is an organic material and/or the second material (4) is an organic material. In Example 25, the structure (1) of any one of Examples 13 to 24 is disclosed, wherein the first material (3) includes polymethyl methacrylate, polystyrene and/or cycloolefin copolymers. In Example 26, the structure (1) of any one of Examples 13 to 25 is disclosed, wherein the second material (4) includes ethylene vinyl alcohol copolymers and/or polyvinyl alcohol. In Example 27, the structure (1) of any one of Examples 13 to 26 is disclosed, wherein the phosphor includes N,N′-di(1-naphthyl)-N,N′-diphenyl-(1,1′-biphenyl)-4,4′-diamine, tetra-N-phenylbenzidines, PhenDPA, PhenTPA, thianthrenes, benzophenone-thianthrenes, bromo-benzophenone-thianthrenes, benzophenone-2-thianthrenes, diphenylsulfone-thianthrenes, diphenylsulfone-2-thianthrenes, bromo-diphenylsulfone-thianthrenes, difluoroborone-9-hydroxyphenalenones, and/or difluoroborone-6-hydroxybenz[de]anthracene-7-one. In Example 28, a method of manufacturing a structure (1) of any one of Examples 14 to 27 is disclosed, wherein a first material (3) and a second material (4) are applied to a substrate (2), wherein a phosphor is admixed with the first material (3). In Example 29, the method of Example 28 is disclosed, wherein the first material (3) is applied as a first layer to the substrate (2) by rotary coating and/or a line application method and/or pipetting and/or a printing method and/or a spraying method, and/or the second material (4) is applied as at least a second layer by rotary coating or a line application method or pipetting. In Example 30, a label (12) is disclosed including a functional layer, wherein the functional layer includes the structure (1) of any one of Examples 13 to 29. In Example 31, a method for writing the label (12) of Example 30 is disclosed, wherein for writing the label (12) in a writing process, dots of the functional layer are selectively transferred locally from the non-phosphorescent state to the phosphorescent state in a contactless manner, wherein a phosphorescent region (5) is formed by the dots, wherein during the writing process the phosphorescent region (5) is irradiated with light of a first characteristic (8), wherein in a binding step the oxygen (9) present in the phosphorescent region is bound to the first material (3). In Example 32, a method for writing on the label (12) of Example 31 is disclosed, wherein during the writing process the phosphorescent region (5) is irradiated with light of the first characteristic (8), wherein the functional layer is partially covered with a mask (15) such, that only the phosphorescent area (5) is illuminated and/or the functional layer is illuminated with light of the first characteristic (8) by a locally meandering or line-by-line scanning light beam only in the phosphorescent area (5) and/or the functional layer is illuminated only in the phosphorescent area (5), in that the functional layer is illuminated with a light beam having a beam profile, the beam profile on the functional layer corresponding to the phosphorescent area (5). In Example 33, the method of writing the label (12) of any one of Examples 31 and 32 is disclosed, wherein the oxygen (9) is converted from a triplet ground state (T0) of the oxygen (9) to an excited singlet state (ST) of the oxygen (9) by a Triplet-Triplet interaction (11) with the phosphor prior to the binding step, wherein the phosphor is excited from a singlet state (SO) of the phosphor to an excited singlet state (S1) of the phosphor before the triplet-triplet interaction (11) by the light of the first characteristic (8), and subsequently is transferred from the excited singlet state (S1) of the phosphor to an excited triplet state (T1) of the phosphor by intercombination (10). In Example 34, a method for erasing the label (12) of Example 30 is disclosed, wherein for erasing the label (12) in an erasing process the functional layer is substantially completely converted to the non-phosphorescent state, wherein during the erasing process heat is introduced into the functional layer and/or the functional layer is irradiated with light of a second characteristic, wherein the second material (4) is converted from an oxygen-impermeable state to an oxygen-permeable state by the heat and/or by the irradiation with the light of the second characteristic. In Example 35, a method for erasing the label (12) of Example 34 is disclosed, wherein the heat is introduced by irradiation with light of the second characteristic. In Example 36, the method of erasing the label (12) of any one of Examples 34 and 35 is disclosed, wherein the light of the second characteristic is infrared light. In Example 37, a method of writing and erasing the label (12) of Example 30 is disclosed, wherein the label (12) is written in the writing process of any one of Examples 31 to 33 and erased in the subsequent erasing process of any one of Examples 34 to 36. In Example 38, a method for writing and erasing the label (12) of Example 30 is disclosed, wherein the label (12) is written in the writing method of any one of Examples 31 to 33, is erased in the subsequent erasing method of any one of Examples 34 to 36, and is rewritten in the writing method following the erasing method of any one of Examples 31 to 33. In Example 39, a sensor (13) for determining a dose of ultraviolet light (17) is disclosed, including the structure (1) of any one of Examples 13 to 29. In Example 40, the sensor (13) of Example 39 is disclosed, wherein the sensor (13) includes a dose threshold, wherein upon irradiation with ultraviolet light (17) at a dose equal to or exceeding the dose threshold, phosphorescence (30) begins. In Example 41, the sensor (13) of Example 40 is disclosed, wherein the sensor (13) includes a main extension plane (14) and the dose threshold is homogeneous in the main extension plane (14). In Example 42, the sensor (13) of Example 41 is disclosed, wherein the sensor (13) includes a neutral density filter. In Example 43, the sensor (13) of Example 40 is disclosed, wherein the sensor (13) includes a main extension plane (14) and the dose threshold in the main extension plane (14) includes a gradient or a gradation. In Example 44, the sensor (13) of Example 43 is disclosed, wherein the sensor (13) includes a neutral density filter, wherein the neutral density filter includes a gradient or gradation of transparency. In Example 45, the sensor (13) of Example 43 is disclosed, wherein the material composition of the structure (1) includes a gradient or gradation in composition. In Example 46, the sensor (13) of any one of Examples 43 to 45 is disclosed, wherein the dose threshold increases along at least one axis (15) in the main extension plane (14), and wherein the sensor (13) includes a scale (16) along the at least one axis (15), the scale (16) indicating the respective dose threshold. In Example 47, a method for spatially resolved determination of a dose of ultraviolet radiation with the sensor (13) of any one of Examples 39 to 42 is disclosed, wherein in an irradiation step the sensor (13) is irradiated with ultraviolet light (17) of a dose to be determined and wherein in a determination step in the regions of the main extension plane (14) of the sensor (13), in which the irradiated dose reaches or exceeds the dose threshold value, molecular oxygen is bound in the sensor (13) and phosphorescence (30) is triggered, and in the regions of the main extension plane (14) of the sensor (13) in which the irradiated dose falls below the dose threshold value, molecular oxygen in the sensor (13) prevents the occurrence of phosphorescence (30). In Example 48, a method for absolute value determination of a dose of ultraviolet radiation with the sensor (13) of any one of Examples 43 to 46 is disclosed, wherein in an irradiation step the sensor (13) is irradiated with ultraviolet light (17) of a dose to be determined and wherein in a determination step in the regions of the main extension plane (14) of the sensor (13) in which the irradiated dose in each case reaches or exceeds the dose threshold value variable in the main extension plane (14), molecular oxygen is bound in the sensor (13) and phosphorescence (30) is triggered, and in the regions of the main extension plane (14) of the sensor (13) in which the irradiated dose falls below the dose threshold value variable in the main extension plane (14) in each case, molecular oxygen in the sensor (13) prevents the occurrence of the phosphorescence (30). In Example 49, the method of any one of Examples 47 and 48 is disclosed, wherein the sensor (13) is irradiated with light and/or heated in a neutralization step, wherein the irradiation and/or the heating causes oxygen to enter the sensor (13) and suppress the phosphorescence (30). In Example 50, the method of Example 49 is disclosed, wherein the neutralization step is followed by at least one irradiation step and at least one determination step of one of Examples 47 or 48, respectively. In Example 51, the method of any one of Examples 47 to 50 is disclosed, wherein the sensor (13) is arranged on a roller (18) of a production line. LIST OF REFERENCE SIGNS 1Structure2Substrate3First organic material4Second organic material5First region6Second area7Mask8Light of first characteristic9Oxygen10Intercombination11Triplet-triplet interaction12Label13Sensor14Main extension plane15Axis16Scale17UV light18Roll of a production line30PhosphorescenceS0Singlet state of the phosphorS1Excited singlet state of the phosphorS1′ Excited singlet state of oxygen T0Triplet ground state of oxygenT1Excited Triplet state of the phosphor | 93,977 |
11859116 | DETAILED DESCRIPTION Reference will now be made in more detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Throughout the disclosure, the expression “at least one of a, b or c” indicates only a, only b, only c, both a and b, both a and c, both b and c, all of a, b, and c, or variations thereof. Expressions such as “at least one selected from,” “one of,” and “selected from,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list. Further, the use of “may” when describing embodiments of the present disclosure refers to “one or more embodiments of the present disclosure.” Because the disclosure may have diverse modified embodiments, embodiments are illustrated in the drawings and are described in the detailed description. An effect and a characteristic of the disclosure, and a method of accomplishing these will be apparent when referring to embodiments described with reference to the drawings. The disclosure may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. One or more embodiments of the disclosure will be described below in more detail with reference to the accompanying drawings. Those components that are the same or are in correspondence with each other are rendered the same reference numeral regardless of the figure number, and redundant explanations are not provided. An expression used in the singular encompasses the expression of the plural, unless it has a clearly different meaning in the context. It will be further understood that the terms “comprises” and/or “comprising” used herein specify the presence of stated features or elements, but do not preclude the presence or addition of one or more other features or elements. It will be understood that when a layer, region, or component is referred to as being “on” or “onto” another layer, region, or component, it may be directly or indirectly formed on the other layer, region, or component. That is, for example, intervening layers, regions, or components may be present. Sizes of elements in the drawings may be exaggerated for convenience of explanation. In other words, because sizes and thicknesses of components in the drawings are arbitrarily illustrated for convenience of explanation, the following embodiments are not limited thereto. As used herein, the terms “use,” “using,” and “used” may be considered synonymous with the terms “utilize,” “utilizing,” and “utilized,” respectively. In addition, the terms “substantially,” “about,” and similar terms are used as terms of approximation and not as terms of degree, and are intended to account for the inherent deviations in measured or calculated values that would be recognized by those of ordinary skill in the art. “About” or “approximately,” as used herein, is inclusive of the stated value and means within an acceptable range of deviation for the particular value as determined by one of ordinary skill in the art, considering the measurement in question and the error associated with measurement of the particular quantity (i.e., the limitations of the measurement system). For example, “about” may mean within one or more standard deviations, or within ±30%, 20%, 10%, 5% of the stated value. Also, any numerical range recited herein is intended to include all subranges of the same numerical precision subsumed within the recited range. For example, a range of “1.0 to 10.0” is intended to include all subranges between (and including) the recited minimum value of 1.0 and the recited maximum value of 10.0, that is, having a minimum value equal to or greater than 1.0 and a maximum value equal to or less than 10.0, such as, for example, 2.4 to 7.6. Any maximum numerical limitation recited herein is intended to include all lower numerical limitations subsumed therein and any minimum numerical limitation recited in this specification is intended to include all higher numerical limitations subsumed therein. Accordingly, Applicant reserves the right to amend this specification, including the claims, to expressly recite any sub-range subsumed within the ranges expressly recited herein. In the present specification, the term “room temperature” refers to about 25° C. The term “interlayer” as used herein refers to a single layer and/or all layers between a first electrode and a second electrode of a light-emitting device. A material included in the “interlayer” may be an organic material and/or an inorganic material. The expression “(an interlayer) includes at least one compound represented by Formula 1” as used herein may include a case in which “(an interlayer) includes one or more identical compounds represented by Formula 1” and a case in which “(an organic layer) includes two or more different compounds represented by Formula 1”. In the present specification, the term “quantum dot” refers to a crystal of a semiconductor compound, and may include any material capable of emitting light of various emission wavelengths according to the size of the crystal. In the present specification, the term “miscible” refers to ability of one or more components, such as liquids, solids, and/or gases, to mix with each other in a single and homogeneous shape. For example, two liquids are referred to be miscible when different components therein can be mixed to a single and homogeneous liquid that is only distinguished at a molecular level. In the present specification, the term “immiscible” refers to ability of two or more components, such as liquids, solids, and/or gases, to mix with each other in two or more shapes (e.g., layers). For example, when an organic solvent is immiscible with a water-soluble solvent (e.g., hexane and water), the organic solvent may be seen as a separate layer that does not mix with the water-soluble solvent. Quantum Dot Composition One or more embodiments of the present disclosure provide a quantum dot composition including: a first solvent; a second solvent different from the first solvent; first quantum dots including a hole-transporting ligand; and second quantum dots including an electron-transporting ligand, wherein the first solvent and the second solvent may be miscible solvents having different boiling points from each other, a degree of dispersion of the first quantum dots is greater in the first solvent than in the second solvent, and a degree of dispersion of the second quantum dots is greater in the second solvent than in the first solvent. Quantum Dots The first quantum dot and the second quantum dot may each include crystals of a semiconductor compound. The hole-transporting ligand may be coordinated on the surface of the first quantum dot, and the electron-transporting ligand may be coordinated on the surface of the second quantum dot. In one or more embodiments, the first quantum dot and the second quantum dot may each independently include: a Group III-VI semiconductor compound; a Group II-VI semiconductor compound; a Group III-V semiconductor compound; a Group I-III-VI semiconductor compound; a Group IV-VI semiconductor compound; a Group IV element or compound; or any combination thereof. Non-limiting examples of the Group III-VI semiconductor compound are: a binary compound, such as GaS, GaSe, Ga2Se3, GaTe, InS, InSe, In2S3, In2Se3, and/or InTe; a ternary compound, such as InGaS3, and/or InGaSe3; or any combination thereof. Non-limiting examples of the Group II-VI semiconductor compound are: a binary compound, such as CdSe, CdTe, ZnS, ZnSe, ZnTe, ZnO, HgS, HgSe, HgTe, MgSe, MgS, and/or the like; a ternary compound, such as CdSeS, CdSeTe, CdSTe, ZnSeS, ZnSeTe, ZnSTe, HgSeS, HgSeTe, HgSTe, CdZnS, CdZnSe, CdZnTe, CdHgS, CdHgSe, CdHgTe, HgZnS, HgZnSe, HgZnTe, MgZnSe, MgZnS, and/or the like; a quaternary compound, such as CdZnSeS, CdZnSeTe, CdZnSTe, CdHgSeS, CdHgSeTe, CdHgSTe, HgZnSeS, HgZnSeTe, HgZnSTe, and/or the like; or any combination thereof. Non-limiting examples of the Group III-V semiconductor compound are: a binary compound, such as GaN, GaP, GaAs, GaSb, AlN, AlP, AlAs, AlSb, InN, InP, InAs, InSb, and/or the like; a ternary compound, such as GaNP, GaNAs, GaNSb, GaPAs, GaPSb, AlNP, AlNAs, AlNSb, AlPAs, AlPSb, InGaP, InNP, InAlP, InNAs, InNSb, InPAs, InPSb, GaAlNP, and/or the like; a quaternary compound, such as GaAlNAs, GaAlNSb, GaAlPAs, GaAlPSb, GaInNP, GaInNAs, GaInNSb, GaInPAs, GaInPSb, InAlNP, InAlNAs, InAlNSb, InAlPAs, InAlPSb, and/or the like; or any combination thereof. In one or more embodiments, the Group III-V semiconductor compound may further include a Group II element. Non-limiting examples of the Group III-V semiconductor compound further including a Group II element are InZnP, InGaZnP, InAlZnP, and/or the like. Non-limiting examples of the Group semiconductor compound are: a ternary compound, such as AgInS, AgInS2, CuInS, CuInS2, CuGaO2, AgGaO2, AgAlO2and/or the like; or any combination thereof. Non-limiting examples of the Group IV-VI semiconductor compound are: a binary compound, such as SnS, SnSe, SnTe, PbS, PbSe, PbTe, and/or the like; a ternary compound, such as SnSeS, SnSeTe, SnSTe, PbSeS, PbSeTe, PbSTe, SnPbS, SnPbSe, SnPbTe, and/or the like; a quaternary compound, such as SnPbSSe, SnPbSeTe, SnPbSTe, and/or the like; or any combination thereof. The Group IV element or compound may include: a single element compound, such as Si and/or Ge; a binary compound, such as SiC and/or SiGe; or any combination thereof, without limitation. Each element included in a multi-element compound, such as the binary compound, the ternary compound, and/or the quaternary compound, may exist in a particle with a uniform concentration or non-uniform concentration. In one or more embodiments, the first quantum dot and the second quantum dot may each have a single structure or a dual core-shell structure. In the case of the single structure, the concentration of each element included in the corresponding quantum dots is uniform. For example, a material included in the core may be different from a material included in the shell. The shell of each of the first quantum dot and the second quantum dot may serve as a protective layer for maintaining semiconductor characteristics by preventing (or reducing) chemical modification of the core of each of the first quantum dot and second quantum dot, and/or may serve as a charging layer for imparting electrophoretic characteristics to the first quantum dot and second quantum dot. The shell may have a single-layered structure or a multi-layered structure. The interface between the core and the shell may have a concentration gradient in which concentration of the element present in the shell decreases toward the center. Non-limiting examples of the shell of each of the first quantum dot and second quantum dot are an oxide of metal or non-metal, a semiconductor compound, or any combination thereof. Non-limiting examples of the oxide of metal or non-metal are: a binary compound, such as SiO2, Al2O3, TiO2, ZnO, MnO, Mn2O3, Mn3O4, CuO, FeO, Fe2O3, Fe3O4, CoO, Co3O4, and/or NiO; a ternary compound, such as MgAl2O4, CoFe2O4, NiFe2O4, and/or CoMn2O4; or any combination thereof. Non-limiting examples of the semiconductor compound are: as described herein, a Group III-VI semiconductor compound; a Group II-VI semiconductor compound; a Group III-V semiconductor compound; a Group III-VI semiconductor compound; a Group I-III-VI semiconductor compound; a Group IV-VI semiconductor compound; or any combination thereof. For example, the semiconductor compound may include CdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, ZnSeS, ZnTeS, GaAs, GaP, GaSb, HgS, HgSe, HgTe, InAs, InP, InGaP, InSb, AlAs, AlP, AlSb, or any combination thereof. The first quantum dot and second quantum dot may each have a full width at half maximum (FWHM) of an emission wavelength spectrum of equal to or less than about 45 nm, for example, equal to or less than about 40 nm, and for example, equal to or less than about 30 nm. Within these ranges, a formed light-emitting device using the quantum dot composition may have improved color purity or color reproducibility. In addition, because light emitted through these quantum dots may be emitted in all directions, the wide viewing angle of the formed light-emitting device using the quantum dot composition may be improved. In one or more embodiments, the first quantum dot and second quantum dot may each be, for example, a spherical, a pyramidal, a multi-arm, and/or cubic nanoparticle, a nanotube, a nanowire, a nanofiber, and/or a nanoplate particle. Because an energy band gap may be adjusted by controlling the size of the quantum dots, light having various wavelength bands may be obtained from a quantum dot emission layer. Therefore, by using quantum dots of different sizes, a light-emitting device that emits light of various wavelengths may be implemented. In detail, the size of the quantum dots may be selected to emit red, green, and/or blue light. In one or more embodiments, the size of the quantum dots may be configured to emit white light by combining light of various colors. The first quantum dot and second quantum dot may each independently have a diameter of, for example, about 1 nm to about 15 nm, and for example, about 5 nm to about 15 nm. The first quantum dot and second quantum dot may each independently be synthesized by a wet chemical process, an organometallic chemical vapor deposition process, a molecular beam epitaxy process, and/or any process similar thereto. According to the wet chemical process, a precursor material is mixed with an organic solvent to grow quantum dot particle crystals. When the crystals grow, an organic solvent naturally may act as a dispersant coordinated on the surface of the quantum dot crystals and control (or manage) the growth of the crystals, so that the growth of quantum dot particles may be controlled (or managed) through a process which is more easily performed than vapor deposition methods, such as metal organic chemical vapor deposition (MOCVD) and/or molecular beam epitaxy (MBE), and which can be performed at low costs. The first quantum dot may include a hole-transporting ligand. For example, the hole-transporting ligand may be coordinated on the surface of the first quantum dot. In one or more embodiments, the hole-transporting ligand may include an aromatic hydrocarbon group-containing compound. For example, the hole-transporting ligand may be an aromatic hydrocarbon group-containing carboxylic acid, an aromatic hydrocarbon group-containing amine, an aromatic hydrocarbon group-containing alcohol, an aromatic hydrocarbon group-containing thiol, an aromatic hydrocarbon group-containing phosphine oxide, an aromatic hydrocarbon group-containing phosphine, an aromatic hydrocarbon group-containing phosphonic acid, an aromatic hydrocarbon group-containing ester, an aromatic hydrocarbon group-containing acid anhydride, or any combination thereof. In one or more embodiments, the aromatic hydrocarbon group may be a C6-C60aryl group unsubstituted or substituted with at least one R10a, a C1-C60heteroaryl group unsubstituted or substituted with at least one R10a, or any combination thereof. In one or more embodiments, the aromatic hydrocarbon group may be a phenyl group, a biphenyl group, a naphthyl group, a fluorenyl group, a spiro-bifluorenyl group, a benzofluorenyl group, a dibenzofluorenyl group, a phenanthrenyl group, an anthracenyl group, a fluoranthenyl group, an acenaphthenyl group, a triphenylenyl group, a pyrenyl group, a chrysenyl group, a perylenyl group, a pentaphenyl group, a hexacenyl group, a pentacenyl group, a thiophenyl group, a furanyl group, a carbazolyl group, an indolyl group, an isoindolyl group, a benzofuranyl group, a benzothiophenyl group, a dibenzofuranyl group, a dibenzothiophenyl group, a benzocarbazolyl group, a dibenzocarbazolyl group, a dibenzosilolyl group, a pyridinyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, a thiadiazolyl group, an oxadiazolyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, a triazinyl group, a quinolinyl group, an isoquinolinyl group, a benzoquinolinyl group, a phthalazinyl group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group, a phenanthridinyl group, an acridinyl group, a phenanthrolinyl group, a phenazinyl group, a benzimidazolyl group, an isobenzothiazolyl group, a benzoxazolyl group, an isobenzoxazolyl group, a triazolyl group, a tetrazolyl group, an imidazopyridinyl group, an imidazopyrimidinyl group, an azacarbazolyl group, or any combination thereof, each unsubstituted or substituted with at least one R10a. R10amay be: deuterium, —F, —Cl, —Br, —I, a hydroxyl group, a cyano group, or a nitro group; a C1-C60alkyl group, a C2-C60alkenyl group, a C2-C60alkynyl group, or a C1-C60alkoxy group, each unsubstituted or substituted with deuterium, —F, —Cl, —Br, —I, a hydroxyl group, a cyano group, a nitro group, a C3-C60carbocyclic group, a C1-C60heterocyclic group, a C6-C60aryloxy group, a C6-C60arylthio group, —Si(Q11)(Q12)(Q13), —N(Q11)(Q12), —B(Q11)(Q12), —C(═O)(Q11), —S(═O)2(Q11), —P(═O)(Q11)(Q12), or any combination thereof; a C3-C60carbocyclic group, a C1-C60heterocyclic group, a C6-C60aryloxy group, or a C6-C60arylthio group, unsubstituted or substituted with deuterium, —F, —Cl, —Br, —I, a hydroxyl group, a cyano group, a nitro group, a C1-C60alkyl group, a C2-C60alkenyl group, a C2-C60alkynyl group, a C1-C60alkoxy group, a C3-C60carbocyclic group, a C1-C60heterocyclic group, a C6-C60aryloxy group, a C6-C60arylthio group, —Si(Q21)(Q22)(Q23), —N(Q21)(Q22), —B(Q21)(Q22), —C(═O)(Q21), —S(═O)2(Q21), —P(═O)(Q21)(Q22), or any combination thereof; or —Si(Q31)(Q32)(Q33), —N(Q31)(Q32), —B(Q31)(Q32), —C(═O)(Q31), —S(═O)2(Q31), or —P(═O)(Q31)(Q32), wherein Q1to Q3, Q11to Q13, Q21to Q23, and Q31to Q33may each independently be: hydrogen; deuterium; —F; —Cl; —Br; —I; a hydroxyl group; a cyano group; a nitro group; a C1-C60alkyl group; a C2-C60alkenyl group; a C2-C60alkynyl group; a C1-C60alkoxy group; or a C3-C60carbocyclic group or a C1-C60heterocyclic group, each unsubstituted or substituted with deuterium, —F, a cyano group, a C1-C60alkyl group, a C1-C60alkoxy group, a phenyl group, a biphenyl group, or any combination thereof. In one or more embodiments, the aromatic hydrocarbon group may be a phenyl group, a biphenyl group, a naphthyl group, a fluorenyl group, a spiro-bifluorenyl group, a benzofluorenyl group, a dibenzofluorenyl group, a phenanthrenyl group, an anthracenyl group, a fluoranthenyl group, an acenaphthenyl group, a triphenylenyl group, a pyrenyl group, a chrysenyl group, a perylenyl group, a pentaphenyl group, a hexacenyl group, a pentacenyl group, a thiophenyl group, a furanyl group, a carbazolyl group, an indolyl group, an isoindolyl group, a benzofuranyl group, a benzothiophenyl group, a dibenzofuranyl group, a dibenzothiophenyl group, a benzocarbazolyl group, a dibenzocarbazolyl group, a dibenzosilolyl group, a pyridinyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, a thiadiazolyl group, an oxadiazolyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, a triazinyl group, a quinolinyl group, an isoquinolinyl group, a benzoquinolinyl group, a phthalazinyl group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group, a phenanthridinyl group, an acridinyl group, a phenanthrolinyl group, a phenazinyl group, a benzoimidazolyl group, an isobenzothiazolyl group, a benzoxazolyl group, an isobenzoxazolyl group, a triazolyl group, a tetrazolyl group, an imidazopyridinyl group, an imidazopyrimidinyl group, an azacarbazolyl group, or any combination thereof, each unsubstituted or substituted with deuterium, —F, —Cl, —Br, —I, a hydroxyl group, a cyano group, a nitro group, an amidino group, a hydrazino group, a hydrazono group, a C1-C20alkyl group, a C1-C20alkoxy group, a phenyl group, a biphenyl group, a naphthyl group, a fluorenyl group, a spiro-bifluorenyl group, a benzofluorenyl group, a dibenzofluorenyl group, a phenanthrenyl group, an anthracenyl group, a fluoranthenyl group, an acenaphthenyl group, a triphenylenyl group, a pyrenyl group, a chrysenyl group, a perylenyl group, a pentaphenyl group, a hexacenyl group, a pentacenyl group, a thiophenyl group, a furanyl group, a carbazolyl group, an indolyl group, an isoindolyl group, a benzofuranyl group, a benzothiophenyl group, a dibenzofuranyl group, a dibenzothiophenyl group, a benzocarbazolyl group, a dibenzocarbazolyl group, a dibenzosilolyl group, a pyridinyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, a thiadiazolyl group, an oxadiazolyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, a triazinyl group, a quinolinyl group, an isoquinolinyl group, a benzoquinolinyl group, a phthalazinyl group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group, a phenanthridinyl group, an acridinyl group, a phenanthrolinyl group, a phenazinyl group, a benzoimidazolyl group, an isobenzothiazolyl group, a benzoxazolyl group, an isobenzoxazolyl group, a triazolyl group, a tetrazolyl group, an imidazopyridinyl group, an imidazopyrimidinyl group, an azacarbazolyl group, —Si(Q31)(Q32)(Q33), —N(Q31)(Q32), —B(Q31)(Q32), —C(═O)(Q31), —S(═O)2(Q31), —P(═O)(Q31)(Q32), or any combination thereof, wherein Q31to Q33may each independently be a C1-C10alkyl group, a C1-C10alkoxy group, a phenyl group, a biphenyl group, a terphenyl group, or a naphthyl group. For example, the hole-transporting ligand may be represented by Formula 1: wherein, in Formula 1, Ar may be a phenyl group, a naphthyl group, a fluorenyl group, a spiro-bifluorenyl group, a phenanthrenyl group, an anthracenyl group, a fluoranthenyl group, an acenaphthenyl group, a triphenylenyl group, a pyrenyl group, a chrysenyl group, a perylenyl group, a thiophenyl group, a furanyl group, an indolyl group, an isoindolyl group, a benzofuranyl group, a benzothiophenyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, a thiadiazolyl group, an oxadiazolyl group, a pyridinyl group, a pyrimidinyl group, a pyrazinyl group, a pyridazinyl group, a triazinyl group, a quinolinyl group, an isoquinolinyl group, a benzoquinolinyl group, a phthalazinyl group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group, a phenanthridinyl group, an acridinyl group, a phenanthrolinyl group, a phenazinyl group, a benzoimidazolyl group, an isobenzothiazolyl group, a benzoxazolyl group, an isobenzoxazolyl group, a triazolyl group, a tetrazolyl group, a carbazolyl group, a benzofuranyl group, a benzothiophenyl group, a dibenzofuranyl group, a dibenzothiophenyl group, or a dibenzosilolyl group, each unsubstituted or substituted with at least one R10a, Li may be a single bond, a C1-C10alkylene group unsubstituted or substituted with at least one R10a, a C1-C10alkenylene group unsubstituted or substituted with at least one R10a, a C1-C10alkynylene group unsubstituted or substituted with at least one R10a, a C3-C60carbocyclic group unsubstituted or substituted with at least one R10a, or a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, m may be an integer from 1 to 5, n may be an integer from 1 to 10, and R10amay be the same as described herein. In one or more embodiments, Ar in Formula 1 may be a phenyl group, a naphthyl group, a fluorenyl group, a spiro-bifluorenyl group, a phenanthrenyl group, an anthracenyl group, a fluoranthenyl group, an acenaphthenyl group, a triphenylenyl group, a pyrenyl group, a chrysenyl group, a perylenyl group, a thiophenyl group, a furanyl group, an indolyl group, an isoindolyl group, a benzofuranyl group, a benzothiophenyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, a thiadiazolyl group, an oxadiazolyl group, a pyridinyl group, a pyrimidinyl group, a pyrazinyl group, a pyridazinyl group, a triazinyl group, a quinolinyl group, an isoquinolinyl group, a benzoquinolinyl group, a phthalazinyl group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group, a phenanthridinyl group, an acridinyl group, a phenanthrolinyl group, a phenazinyl group, a benzoimidazolyl group, an isobenzothiazolyl group, a benzoxazolyl group, an isobenzoxazolyl group, a triazolyl group, a tetrazolyl group, a carbazolyl group, a benzofuranyl group, a benzothiophenyl group, a dibenzofuranyl group, a dibenzothiophenyl group, or a dibenzosilolyl group, each substituted with deuterium, —F, —Cl, —Br, —I, a hydroxyl group, a cyano group, a nitro group, an amidino group, a hydrazino group, a hydrazono group, a C1-C20alkyl group, a C1-C20alkoxy group, a phenyl group, a naphthyl group, a fluorenyl group, a spiro-bifluorenyl group, a phenanthrenyl group, an anthracenyl group, a fluoranthenyl group, an acenaphthenyl group, a triphenylenyl group, a pyrenyl group, a chrysenyl group, a perylenyl group, a thiophenyl group, a furanyl group, an indolyl group, an isoindolyl group, a benzofuranyl group, a benzothiophenyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, a thiadiazolyl group, an oxadiazolyl group, a pyridinyl group, a pyrimidinyl group, a pyrazinyl group, a pyridazinyl group, a triazinyl group, a quinolinyl group, an isoquinolinyl group, a benzoquinolinyl group, a phthalazinyl group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group, a phenanthridinyl group, an acridinyl group, a phenanthrolinyl group, a phenazinyl group, a benzoimidazolyl group, an isobenzothiazolyl group, a benzoxazolyl group, an isobenzoxazolyl group, a triazolyl group, a tetrazolyl group, a carbazolyl group, a benzofuranyl group, a benzothiophenyl group, a dibenzofuranyl group, a dibenzothiophenyl group, a dibenzosilolyl group, or any combination thereof. In one or more embodiments, Ar in Formula 1 may be one of groups represented by Formulae 2-1 to 2-8: wherein, in Formulae 2-1 to 2-8, R10b, R10c, and R10dmay each be the same as described in connection with R10a, c3 may be an integer from 0 to 3, c4 may be an integer from 0 to 4, c5 may be an integer from 0 to 5, and * indicates a binding site to a neighboring atom. When m in Formula 1 is 2 or more, two or more of Li(s) may be identical to or different from each other. In Formula 1, *—SH may be an anchoring group binding to the surface of the quantum dots. Here, the anchoring group refers to a linking group that allows a ligand to be attached to a quantum dot when the ligand is coordinated on the quantum dots. In one or more embodiments, the hole-transporting ligand may be at least one selected from Ligands A to D, but embodiments of the present disclosure are not limited thereto: As the hole-transporting ligand includes the aromatic hydrocarbon group according to the present embodiments, a highest occupied molecular orbital (HOMO) level may be improved, thereby facilitating injection of holes into an emission layer. The hole-transporting ligand may be reacted by mixing a hole-transporting ligand together with an organic solvent and a precursor material for forming the first quantum dots, or may be attached to the surface of the first quantum dots through a ligand exchange reaction after the hole-transporting ligand is added to a mixture of an organic solvent and the first quantum dots to which a random ligand (e.g., any suitable ligand) is attached. However, embodiments of the present disclosure are not limited thereto. The second quantum dot may include an electron-transporting ligand. For example, the electron-transporting ligand may be coordinated on the surface of the second quantum dot. In one or more embodiments, the electron-transporting ligand may include an aliphatic hydrocarbon group-containing compound, a halogen ion, BF4−, or any combination thereof. In one or more embodiments, the aliphatic hydrocarbon group may include a C1-C60alkyl group unsubstituted or substituted with at least one R10a, a C2-C60alkenyl group unsubstituted or substituted with at least one R10a, a C2-C60alkynyl group unsubstituted or substituted with at least one R10a, a C3-C10cycloalkyl group unsubstituted or substituted with at least one R10a, a C3-C10cycloalkenyl group unsubstituted or substituted with at least one R10a, or any combination thereof. In one or more embodiments, the electron-transporting ligand may be an aliphatic hydrocarbon group-containing carboxylic acid, an aliphatic hydrocarbon group-containing amine, an aliphatic hydrocarbon group-containing alcohol, an aliphatic hydrocarbon group-containing thiol, an aliphatic hydrocarbon group-containing phosphine oxide, an aliphatic hydrocarbon group-containing phosphine, an aliphatic hydrocarbon group-containing phosphonic acid, an aliphatic hydrocarbon group-containing ester, an aliphatic hydrocarbon group-containing acid anhydride, an aliphatic hydrocarbon group-containing halide, an aliphatic hydrocarbon group-containing acyl halide, a halogen ion, BF4−, or any combination thereof. In one or more embodiments, the electron-transporting ligand may be RCOOH, RNH2, R2NH, R3N, ROH, RSH, R3PO, R3P, RPO(OH)2, RCOOR′, RCOOCOR′, R—X, RCOX, a halogen ion, BF4−, or any combination thereof, wherein R and R′ may each independently be a C1-C60alkyl group unsubstituted or substituted with at least one R10a, a C2-C60alkenyl group unsubstituted or substituted with at least one R10a, a C2-C60alkynyl group unsubstituted or substituted with at least one R10a, a C3-C10cycloalkyl group unsubstituted or substituted with at least one R10a, or a C3-C10cycloalkenyl group unsubstituted or substituted with at least one R10a, and X may be Cl, Br, or I. In one or more embodiments, the electron-transporting ligand may be formic acid, acetic acid, propanoic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, dodecanoic acid, hexadecanoic acid, octadecanoic acid, oleic acid, stearic acid, palmitic acid, methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, octylamine, dodecylamine, hexadecylamine, octadecylamine, oleylamine, dimethylamine, diethylamine, dipropylamine, methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, dodecanol, hexadecanol, octadecanol, methanethiol, ethanethiol, propanethiol, butanethiol, pentanethiol, hexanethiol, heptanethiol, octanethiol, dodecanthiol, hexadecanthiol, octadecanthiol, trimethylphosphine oxide, triethylphosphine oxide, tripropylphosphine oxide, tributylphosphine oxide, trioctylphosphine oxide, F−, Cl−, Br−, I−, BF4−, or any combination thereof, but embodiments of the present disclosure are not limited thereto. In one or more embodiments, the electron-transporting ligand may be dodecanthiol, but embodiments of the present disclosure are not limited thereto. As the electron-transporting ligand includes the aliphatic hydrocarbon group of the present embodiments, injection of electrons into an emission layer may be facilitated. The electron-transporting ligand may be reacted by mixing an electron-transporting ligand together with an organic solvent and a precursor material for forming the second quantum dot, or may be attached to the surface of the second quantum dot through a ligand exchange reaction after the electron-transporting ligand is added to a mixture of an organic solvent and the second quantum dots to which a random ligand (e.g., any suitable ligand) is attached. However, embodiments of the present disclosure are not limited thereto. In one or more embodiments, both the hole-transporting ligand and the electron-transporting ligand may be thiol compounds. For example, the hole-transporting ligand may be an aromatic hydrocarbon group-containing thiol, and the electron-transporting ligand may be an aliphatic hydrocarbon group-containing thiol. In one or more embodiments, a total amount of the first quantum dots and the second quantum dots may be, based on the total weight of the quantum dot composition, in a range of about 0.1 wt % to about 20 wt %, for example, about 1 wt % to about 20 wt %, and for example, about 3 wt % to about 15 wt %, but embodiments of the present disclosure are not limited thereto. When the total amount is satisfied within these ranges, the quantum dot composition may have a suitable solid content concentration for a soluble process. Solvent A degree of dispersion of quantum dots in a solvent may be observed with a naked eye, or may be measured by transmittance comparison using an optical device, such as Turbiscan™, UV-Vis spectrometer, and/or dynamic light scattering (DLS), or may be measured using a particle size analyzer, and/or atom probe tomography (APT). The degree of dispersion of the first quantum dots may be greater in the first solvent than in the second solvent, and the degree of dispersion of the second quantum dots may be greater in the second solvent than in the first solvent. In this regard, the first solvent may improve the dispersibility of the first quantum dots, and the second solvent may improve the dispersibility of the second quantum dots, and accordingly, the quantum dot composition may have excellent dispersibility. In one or more embodiments, the first solvent may include an aromatic hydrocarbon solvent. For example, when the first quantum dot includes an aromatic hydrocarbon group-containing compound coordinated on the surface thereof, the first solvent including an aromatic hydrocarbon solvent may improve the dispersibility of the first quantum dots. For example, the first solvent may include toluene, xylene, ethylbenzene, diethylbenzene, mesitylene, propylbenzene, cyclohexylbenzene, dimethoxybenzene, anisole, ethoxytoluene, phenoxytoluene, isopropylbiphenyl, dimethylanisole, propylanisole, 1-ethylnaphthalene, 2-ethylnaphthalene, 2-ethylbiphenyl, octylbenzene, or any combination thereof. In one or more embodiments, the second solvent may include an aliphatic hydrocarbon solvent, an aromatic hydrocarbon solvent, or any combination thereof. For example, when the second quantum dot includes an aliphatic hydrocarbon group-containing compound coordinated on the surface thereof, the second solvent including an aliphatic hydrocarbon solvent, an aromatic hydrocarbon solvent, or any combination thereof may improve the dispersibility of the second quantum dots. For example, the second solvent may include n-octane, n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, 2-methylheptane, 3-methylheptane, 4-methylheptane, 2,2-dimethylhexane, 2,3-dimethylhexane, 2,4-dimethylhexane, 2,5-dimethylhexane, 3,3-dimethylhexane, 3-ethylhexane, 2,2,4-trimethylpentane, 2-methyloctane, 2-methylnonane, 2-methyldecane, 2-methylundecane, 2-methyldodecane, 2-methyltridecane, methylcyclohexane, ethylcyclohexane, 1,1-dimethylcyclohexane, 1,2-dimethylcyclohexane, cycloheptane, methylcycloheptane, bicyclohexyl, decaline, toluene, xylene, ethylbenzene, diethylbenzene, mesitylene, propylbenzene, cyclohexylbenzene, dimethoxybenzene, anisole, ethoxytoluene, phenoxytoluene, isopropylbiphenyl, dimethylanisole, propylanisole, 1-ethylnaphthalene, 2-ethylnaphthalene, 2-ethylbiphenyl, octylbenzene, or any combination thereof. The first solvent and the second solvent may be miscible solvents having different boiling points from each other. In a related art, a quantum dot composition including quantum dots (such as both the first quantum dots and the second quantum dots) including charge-transporting ligands with different characteristics may be prepared by a method of mixing two dispersion solutions, after preparing a dispersion solution for each of dispersing the first quantum dots and the second quantum dots by using immiscible solvents (such as, for example, a hydrophilic solvent and a hydrophobic solvent). However, in this case, the quantum dot composition may have poor dispersibility because the quantum dot(s) having selectivity to a hydrophilic solvent or a hydrophobic solvent may be precipitated. The quantum dot composition according to one or more embodiments, by using the solvents that are miscible with each other, may reduce a precipitation of the quantum dots during the preparation and storage of the quantum dot composition, thereby significantly improving the dispersibility of the quantum dot composition. In this regard, the quantum dot composition may be suitable for manufacturing a quantum dot emission layer of a light-emitting device by a soluble process. In addition, because the first solvent and the second solvent have different boiling points from each other, when the quantum dot composition is used in the manufacture of a light-emitting device to be described hereinbelow, there is an advantage in that a double-layered emission layer may be formed by a single process of forming an emission layer by sequentially removing the solvents. In one or more embodiments, a total amount of the first solvent and second solvent may be, based on the total weight of the quantum dot composition, in a range of about 80 wt % to about 99.9 wt %, for example about 85 wt % to about 97 wt %, but embodiments of the present disclosure are not limited thereto. Within these ranges, the quantum dots may be appropriately (or suitably) dispersed in the quantum dot composition and may have a suitable solid content composition for a soluble process. In one or more embodiments, a volume ratio of the first solvent to the second solvent may be in a range of about 1:9 to about 9:1, for example, about 2:8 to about 8:2, and for example, about 3:7 to about 7:3, but embodiments of the present disclosure are not limited thereto. In one or more embodiments, viscosity of the quantum dot composition may be, in the storage state, in a range of about 1 cP to about 10 cP, for example, about 2 cP to about 7 cP, but embodiments of the present disclosure are not limited thereto. The quantum dot composition having the viscosity within these ranges may be suitable for manufacturing a quantum dot emission layer of a light-emitting device by a soluble process. Any suitable method of measuring the viscosity in the art may be used, and for example, a rheometer (for example, a Brookfield DV-I Prime rheometer) may be used for the measurement. In one or more embodiments, a surface tension of the quantum dot composition may be, at a temperature of 25° C., in a range of about 10 dynes/cm to about 40 dynes/cm, for example, about 25 dynes/cm to about 35 dynes/cm, but embodiments of the present disclosure are not limited thereto. The quantum dot composition having the surface tension within these ranges may be suitable for manufacturing a quantum dot emission layer of a light-emitting device by a soluble process. Any suitable method of measuring the surface tension in the art may be used, and for example, a tensiometer (for example, a bubble pressure tensiometer from SITA Process Solutions) may be used for the measurement. In one or more embodiments, vapor pressure of the quantum dot composition may be, at a temperature of 25° C., in a range of about 10−5mmHg to about 10−2mmHg, but embodiments of the present disclosure are not limited thereto. The quantum dot composition having the vapor pressure within these ranges may be suitable for manufacturing a quantum dot emission layer of a light-emitting device by a soluble process. In one or more embodiments, the quantum dot composition may further include a hole-transporting compound and/or an electron-transporting compound. The hole-transporting compound may be the same as described in connection with a compound included in a hole transport region to be described hereinbelow, and the electron-transporting compound may be the same as described in connection with a compound included in an electron transport region to be described hereinbelow. In the quantum dot composition, the amount of the hole-transporting compound or the electron-transporting compound may be, based on the total weight of the quantum dot composition, in a range of about 0.5 wt % to about 20 wt %, for example, about 0.5 wt % to about 15 wt %, but embodiments of the present disclosure are not limited thereto. Additives The quantum dot composition may further include an additive for the purpose of controlling an energy band level, controlling charge mobility, and/or improving coating uniformity. The additive may include a dispersant, an adhesion promoter, a leveling agent, an antioxidant, an ultraviolet absorber, or any combination thereof. For example, the quantum dot composition may further include a dispersant to improve the degree of dispersion of the first quantum dots and the second quantum dots. The dispersant may be used to prevent or reduce the agglomeration of the quantum dots in the quantum dot composition, and to impart the role of a protective layer of the quantum dots during a soluble process. The dispersant may include an anion-based polymer material, a cation-based polymer material, and/or a nonionic-based polymer material. An amount of the dispersant may be, per 100 parts by weight of the quantum dots, in a range of about 10 parts by weight to about 50 parts by weight, for example, about 15 parts by weight to about 30 parts by weight. When the amount of the dispersant is satisfied within these ranges, the agglomeration of the quantum dots may be substantially prevented or reduced, and the dispersant may serve as a protective layer for the quantum dots. The adhesion promoter may include a silane coupling agent having a reactive substituent selected from a carboxyl group, a methacryloyl group, an isocyanate group, an epoxy group, and a combination thereof, which may each independently be added to increase adhesion to a substrate. However, embodiments of the present disclosure are not limited thereto. For example, the silane coupling agent may include trimethoxysilylbenzoic acid, γ-methacryloxypropyltrimethoxysilane, vinyltriacetoxysilane, vinyltrimethoxysilane, γ-isocyanatepropyltriethoxysilane, γ-glysidoxypropyltrimethoxysilane, β-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, or any combination thereof. The leveling agent may be added to improve coating properties of the quantum dot composition. The leveling agent may include, for example, a silicon-based compound, a fluorine-based compound, a siloxane-based compound, a nonionic surfactant, an ionic surfactant, a titanate coupling agent, and/or the like, but embodiments of the present disclosure are not limited thereto. For example, the leveling agent may include a silicon-based compound, a fluorine-based compound, or any combination thereof. The silicon-based compound may be, although not particularly limited, dimethyl silicon, methyl silicon, phenyl silicon, methyl phenyl silicon, alkyl-modified silicon, alkoxy-modified silicon, polyether-modified silicon, and/or the like. For example, the silicon-based compound may be dimethyl silicon, methylphenyl silicon, and/or the like. The fluorine-based compound may be, although not particularly limited, polytetrafluoroethylene, polyvinylidenfluoride, fluoroalkylmethacrylate, perfluoropolyether, perfluoroalkylethylene oxide, and/or the like. For example, the fluorine-based compound may be polytetrafluoroethylene. The siloxane-based compound may be, although not particularly limited, a dimethyl siloxane compound (product name: KF96L-1, KF96L-5, KF96L-10, and KF96L-100 of Shin-Etsu Chemical Co., Ltd.). The leveling agent may be used alone or in combination of two or more materials. An amount of the leveling agent may vary depending on the desired performance, but may be, based on the total weight of the quantum dot composition, in a range of about 0.001 wt % to about 5 wt %, for example, about 0.001 wt % to about 1 wt %. When the amount of the leveling agent is satisfied within these ranges, the fluidity of the quantum dot composition and the film uniformity may be improved. The quantum dot composition may be used to manufacture a light-emitting apparatus. Considering excellent inkjet ejection stability of the quantum dot composition, the quantum dot composition may be, for example, used for inkjet printing, but embodiments of the present disclosure are not limited thereto. Method of Manufacturing Light-Emitting Device One or more embodiments of the present disclosure provide a method of manufacturing a light-emitting device, the method including: providing a quantum dot composition on a first electrode, wherein the quantum dot composition includes a first solvent, a second solvent different from the first solvent, first quantum dots including a hole-transporting ligand, and second quantum dots including an electron-transporting ligand, wherein the first solvent and the second solvent are miscible solvents having different boiling points from each other, a degree of dispersion of the first quantum dots is greater in the first solvent than in the second solvent, and a degree of dispersion of the second quantum dots is greater in the second solvent than in the first solvent; forming a first emission layer by removing a solvent having a lower boiling point among the first solvent and the second solvent; forming a second emission layer by removing a solvent having a higher boiling point among the first solvent and the second solvent; and forming a second electrode on the second emission layer. The quantum dot composition may be the same as described herein. In the forming of the first emission layer and the second emission layer, the removing of the solvent may be performed by vacuum or heat, but embodiments of the present disclosure are not limited thereto. In one or more embodiments, the boiling point of the first solvent may be lower than that of the second solvent. For example, at the same temperature, the vapor pressure of the first solvent may be greater than that of the second solvent. In this case, because the first solvent is removed before the second solvent, the first quantum dots having a greater degree of dispersion in the first solvent than in the second solvent may be precipitated first on a first electrode. Accordingly, a first emission layer including the first quantum dots (hereinafter also referred to as a first quantum dot-containing first emission layer) may be formed first on the first electrode. Next, the second solvent having a higher boiling point than the first solvent is removed to form a second emission layer including the second quantum dots (hereinafter also referred to as a second quantum dot-containing second emission layer). Therefore, on the first electrode, the first quantum dot-containing first emission layer and the second quantum dot-containing second emission layer may be sequentially formed in this stated order. Here, the first quantum dot-containing first emission layer may have a maximum concentration of the first quantum dots, and the second quantum dot-containing second emission layer may have a maximum concentration of the second quantum dots. Although the first solvent and the second solvent are sequentially removed, the first and second emission layers may be prepared by a single process using one composition, rather than separate compositions. In this regard, the first quantum dot-containing first emission layer may further include a small amount of the second quantum dots and the second quantum dot-containing second emission layer may further include a small amount of the first quantum dots. In one or more embodiments, the first electrode may be an anode, and the second electrode may be a cathode. In one or more embodiments, the method of manufacturing the light-emitting device may further include: before the providing of the quantum dot composition on the first electrode, forming a hole transport region on the first electrode; and before the forming of the second electrode, forming an electron transport region on the second emission layer, wherein the first electrode may be an anode, and the second electrode may be a cathode. The light-emitting device thus manufactured may have a structure in which the first electrode (which is a hole injection electrode), the hole transport region, the first emission layer, the second emission layer, the electron transport region, and the second electrode (which is an electron injection electrode), are sequentially stacked in this stated order. For example, when the boiling point of the first solvent is lower than that of the second solvent, the first emission layer may be a first quantum dot-containing first emission layer, and the second emission layer may be a second quantum dot-containing second emission layer. In one or more embodiments, the boiling point of the second solvent may be lower than that of the first solvent. For example, at the same temperature, the vapor pressure of the second solvent may be greater than that of the first solvent. In this case, because the second solvent is removed before the first solvent, the second quantum dots having a greater dispersion degree in the second solvent than in the first solvent may be precipitated first on a first electrode. Accordingly, a first emission layer including the second quantum dots (hereinafter also referred to as a second quantum dot-containing first emission layer) may be formed first on the first electrode. Next, the first solvent having a higher boiling point than the second solvent is removed to form a second emission layer including the first quantum dots (hereinafter also referred to as a first quantum dot-containing second emission layer). Therefore, the second quantum dot-containing first emission layer and the first quantum dot-containing second emission layer may be sequentially formed in this stated order on the first electrode. Here, the second quantum dot-containing first emission layer may have a maximum concentration of the second quantum dots, and the first quantum dot-containing second emission layer may have a maximum concentration of the first quantum dots. Although the first solvent and the second solvent are sequentially removed, the first emission layer and the second emission layer are prepared in a single process using one composition, rather than separate compositions. In this regard, the second quantum dot-containing first emission layer may further include a small amount of the first quantum dots and the first quantum dot-containing second emission layer may further include a small amount of the second quantum dots. In one or more embodiments, the first electrode may be a cathode, and the second electrode may be an anode. In one or more embodiments, the method of manufacturing the light-emitting device may further include: before the providing of the quantum dot composition on the first electrode, forming an electron transport region on the first electrode; and before the forming of the second electrode, forming a hole transport region on the second emission layer, wherein the first electrode may be a cathode, and the second electrode may be an anode. The light-emitting device thus manufactured may have a structure in which the first electrode (which is an electron injection electrode), the electron transport region, the first emission layer, the second emission layer, the hole transport region, and the second electrode (which is a hole injection electrode), are sequentially stacked in this stated order. For example, when the boiling point of the second solvent is lower than that of the first solvent, the first emission layer may be a second quantum dot-containing first emission layer, and the second emission layer may be a first quantum dot-containing second emission layer. As such, when the first electrode is an anode (which is a hole injection electrode), and the second electrode is a cathode (which is an electron injection electrode), a solvent having a lower boiling point than that of the second solvent may be selected as the first solvent so that the first quantum dots are arranged on a bottom side (i.e., a side closer to the first electrode). However, when the first electrode is a cathode (which is an electron injection electrode), and the second electrode is an anode (which is a hole injection electrode), a solvent having a lower boiling point than that of the first solvent may be selected as the second solvent so that the second quantum dots are arranged on a bottom side (i.e., a side closer to the first electrode). As described above, according to one or more embodiments of the present disclosure, by selecting the first solvent and the second solvent having different boiling points from each other, a light-emitting device having a conventional structure or an inverted structure may be manufactured. The quantum dot composition may be provided on the first electrode by a soluble process, but embodiments of the present disclosure are not limited thereto. The soluble process may include an inkjet printing process, a spin coating process, a slit coating process, a drop casting process, a casting process, a gravure coating process, a bar coating process, a roll coating process, a dip coating process, a spray coating process, a screen coating process, a flexographic printing process, an offset printing process, and/or a nozzle printing process, but embodiments of the present disclosure are not limited thereto. In one or more embodiments, the soluble process may be performed by an inkjet printing method, but embodiments of the present disclosure are not limited thereto. For example, the quantum dot composition may be provided in the form of microdroplets on the first electrode by an inkjet printing method. The quantum dot composition has excellent (or suitable) inkjet ejection stability, and thus may be suitably used for an inkjet printing method. For the inkjet printing method, an inkjet printer having an inkjet head equipped with a piezo-type (or kind) nozzle applying pressure according to a voltage may be used. For example, the quantum dot composition may be ejected from a nozzle of an inkjet head. Here, an ejection amount of the quantum dot from a nozzle of an inkjet head composition may be in a range of about 1 pL per once to about 50 pL per once, for example, about 1 pL per once to about 30 pL per once, and for example, about 1 pL per once to about 20 pL per once. To minimize or reduce clogging of the nozzle and improve ejection precision, an aperture diameter of the inkjet head may be in a range of about 5 μm to about 50 μm, for example, about 10 μm to about 30 μm, but embodiments of the present disclosure are not limited thereto. An ejection pressure of the inkjet head may be, based on the shear rate, in a range of about 1,000 s−1to about 10,000 s−1, but embodiments of the present disclosure are not limited thereto. The temperature at the time of forming a coating film is not particularly limited. However, in consideration of suppression or reduction of crystallization of materials included in the quantum dot composition, the temperature may be in a range of about 10° C. to about 50° C., for example, about 15° C. to about 40° C., for example, about 15° C. to about 30° C., and for example, about 20° C. to about 25° C. Light-Emitting Device The light-emitting device manufactured using the quantum dot composition according to one or more of the present embodiments may include: the first electrode; the second electrode facing the first electrode; and the first emission layer and the second emission layer that are arranged between the first electrode and the second electrode. One of the first emission layer and the second emission layer may include the first quantum dots, and the other of the first emission layer and the second emission layer may include the second quantum dots. For example, one of the first emission layer and the second emission layer may have a maximum concentration of the first quantum dots, and the other of the first emission layer and the second emission layer may have a maximum concentration of the second quantum dots. Because the first emission layer and the second emission layer are prepared by a single process using the quantum dot composition, even when the first emission layer has a maximum concentration of the first quantum dots, the first emission layer may further include a small amount of the second quantum dots. Likewise, even when the second emission layer has a maximum concentration of the second quantum dots, the second emission layer may further include a small amount of the first quantum dots. In one or more embodiments, the second emission layer may be arranged between the first emission layer and the second electrode. In one or more embodiments, the first emission layer may be in direct contact with the second emission layer. For example, one surface of the first emission layer and one surface of the second emission layer may be in contact with each other. In one or more embodiments, the first emission layer and the second emission layer may have different electrical characteristics from each other. In one or more embodiments, the first emission layer may have hole transport characteristics, and the second emission layer may have electron transport characteristics. In one or more embodiments, the first emission layer may have strong electron transport characteristics, and the second emission layer may have strong hole transport characteristics. A total thickness of the emission layers may be in a range of about 7 nm to about 100 nm, for example, about 10 nm to about 30 nm. Within these ranges, the light-emitting device may have excellent (or improved) emission efficiency and/or lifespan properties due to the control (or substantial control) of pores that may be generated by quantum dot particle arrangement. In one or more embodiments, both the first emission layer and the second emission layer may emit first light belonging to a predetermined (or set) wavelength region. For example, the first light may belong to one of a first wavelength region between 430 nm and 480 nm, a second wavelength region between 520 nm and 570 nm, or a third wavelength region between 600 nm and 650 nm. In one or more embodiments, the first emission layer and the second emission layer may emit light having different wavelengths from each other. In this case, the light-emitting device may emit light obtained by mixing light emitted from the first emission layer and light emitted from the second emission layer. In one or more embodiments, in the light-emitting device, the first electrode may be an anode, and the second electrode may be a cathode. The light-emitting device may further include a hole transport region between the first electrode and the emission layer, and an electron transport region between the emission layer and the second electrode, wherein the hole transport region may include a hole injection layer, a hole transport layer, an emission auxiliary layer, an electron blocking layer, or any combination thereof, and the electron transport region may include a hole blocking layer, an electron transport layer, an electron injection layer, or any combination thereof. When the light-emitting device is a full color light-emitting device, the emission layer may include an emission layer that is to emit light of different color for each subpixel. In one or more embodiments, the emission layer may be patterned, for each subpixel, as a first color emission layer, a second color emission layer, and a third color emission layer. Here, at least one emission layer of the first color emission layer, the second color emission layer, or the third color emission layer may essentially include the first and second emission layers including the quantum dots. For example, the first color emission layer may include the first and second emission layers including the quantum dots, and the second color emission layer and the third color emission layer may each be an organic emission layer including an organic compound. Here, the first color through the third color are different colors, and more particularly, the first color through the third color (e.g., the first color, the second color, and the third color) may have different maximum emission wavelengths from each other. The first color through the third color may be white when combined with each other. In one or more embodiments, the emission layer may further include a fourth color emission layer, and at least one emission layer among the first color emission layer through the fourth color emission layer may be the first and second emission layers including the quantum dots, and the other emission layers may be organic emission layers each including an organic compound. As such, the emission layer may have various suitable modifications. In this regard, the first color through the fourth color (e.g., the first color, the second color, the third color, and the fourth color) may be different colors, and for example, the first color through the fourth color may have different maximum emission wavelengths from each other. The first color through the fourth color may be white when combined with each other. The emission layer may further include, in addition to the quantum dots, at least one of an organic compound or a semiconductor compound, but compounds to be included in the emission layer are not limited thereto. In one or more embodiments, the organic compound may include a host and a dopant. The host and the dopant may each be understood by referring to the related description to be presented hereinbelow. Description ofFIG.1 FIG.1is a schematic view of a light-emitting device10manufactured using a quantum dot composition according to one or more embodiments. The light-emitting device10includes a first electrode110, an interlayer150, and a second electrode190, and the interlayer150includes a first emission layer130and a second emission layer140. Hereinafter, a structure of the light-emitting device10and a manufacturing method thereof will be described with reference toFIG.1. First Electrode110 InFIG.1, a substrate may be additionally arranged under the first electrode110or above the second electrode190. As the substrate, a glass substrate and/or a plastic substrate may be used. In one or more embodiments, the substrate may be a flexible substrate, and may include plastics with excellent (or suitable) heat resistance and/or durability, such as polyimide, polyethylene terephthalate (PET), polycarbonate, polyethylene naphthalate, polyarylate (PAR), polyetherimide, or any combination thereof. The first electrode110may be formed by, for example, depositing or sputtering a material for forming the first electrode110on the substrate. When the first electrode110is an anode, the material for forming the first electrode110may be a high work function material that facilitates injection of holes. The first electrode110may be a reflective electrode, a semi-transmissive electrode, or a transmissive electrode. In one or more embodiments, when the first electrode110is a transmissive electrode, the material for forming the first electrode110may include indium tin oxide (ITO), indium zinc oxide (IZO), tin oxide (SnO2), zinc oxide (ZnO), or any combination thereof. In one or more embodiments, when the first electrode110is a semi-transmissive electrode or a reflective electrode, the material for forming the first electrode110may include magnesium (Mg), silver (Ag), aluminum (Al), aluminum-lithium (Al—Li), calcium (Ca), magnesium-indium (Mg—In), magnesium-silver (Mg—Ag), or any combination thereof. The first electrode110may have a single-layered structure including or consisting of a single layer or a multi-layered structure including a plurality of layers. For example, the first electrode110may have a three-layered structure of ITO/Ag/ITO. Interlayer150 The interlayer150is arranged on the first electrode110. The interlayer150includes a first emission layer130and a second emission layer140. The interlayer150may further include a hole transport region between the first electrode110and the first emission layer130and an electron transport region between the second emission layer140and the second electrode190. Hole Transport Region in Interlayer150 The hole transport region may have: i) a single-layered structure including or consisting of a single layer including or consisting of a single material, ii) a single-layered structure including or consisting of a single layer including or consisting of a plurality of different materials, or iii) a multi-layered structure including a plurality of layers including different materials. The hole transport region may include a hole injection layer, a hole transport layer, an emission auxiliary layer, an electron blocking layer, or any combination thereof. For example, the hole transport region may have a multi-layered structure including a hole injection layer/hole transport layer structure, a hole injection layer/hole transport layer/emission auxiliary layer structure, a hole injection layer/emission auxiliary layer structure, a hole transport layer/emission auxiliary layer structure, or a hole injection layer/hole transport layer/electron blocking layer structure, wherein, in each structure, layers are stacked sequentially on the first electrode110. The hole transport region may include a compound represented by Formula 201, a compound represented by Formula 202, or any combination thereof: wherein, in Formulae 201 and 202, L201to L204may each independently be a C3-C60carbocyclic group unsubstituted or substituted with at least one R10aor a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, L205may be *—O—*′, *—N(Q201)-*′, a C1-C20alkylene group unsubstituted or substituted with at least one R10a, a C2-C20alkenylene group unsubstituted or substituted with at least one R10a, a C3-C60carbocyclic group unsubstituted or substituted with at least one R10a, or a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, xa1 to xa4 may each independently be an integer from 0 to 5, xa5 may be an integer from 1 to 10, R201to R204and Q201may each independently be a C3-C60carbocyclic group unsubstituted or substituted with at least one R10aor a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, R201and R202may optionally be linked to each other via a single bond, a C1-C5alkylene group unsubstituted or substituted with at least one R10a, or a C2-C5alkenylene group unsubstituted or substituted with at least one R10a, to form a C8-C60polycyclic group (for example, a carbazole group and/or the like) unsubstituted or substituted with at least one R10a(see e.g., Compound HT16 and/or the like), R203and R204may optionally be linked to each other via a single bond, a C1-C5alkylene group unsubstituted or substituted with at least one R10a, or a C2-C5alkenylene group unsubstituted or substituted with at least one R10a, to form a C8-C60polycyclic group unsubstituted or substituted with at least one R10a, and na1 may be an integer from 1 to 4. For example, each of Formulae 201 and 202 may include at least one of groups represented by Formulae CY201 to CY217: wherein, in Formulae CY201 to CY217, R10band R10cmay each be the same as described in connection with R10a, ring CY201 to ring CY204 may each independently be a C3-C20carbocyclic group or a C1-C20heterocyclic group, and at least one hydrogen in Formulae CY201 to CY217 may be unsubstituted or substituted with at least one R10a. In one or more embodiments, ring CY201 to ring CY204 in Formulae CY201 to CY217 may each independently be a benzene group, a naphthalene group, a phenanthrene group, or an anthracene group. In one or more embodiments, each of Formulae 201 and 202 may include at least one of groups represented by Formulae CY201 to CY203. In one or more embodiments, Formula 201 may include at least one of the groups represented by Formulae CY201 to CY203 and at least one of groups represented by Formulae CY204 to CY217. In one or more embodiments, xa1 in Formula 201 may be 1, R201may be a group represented by one of Formulae CY201 to CY203, xa2 may be 0, and R202may be a group represented by one of Formulae CY204 to CY207. In one or more embodiments, each of Formulae 201 and 202 may not include a group represented by one of Formulae CY201 to CY203. In one or more embodiments, each of Formulae 201 and 202 may not include a group represented by one of Formulae CY201 to CY203, and may include at least one of groups represented by Formulae CY204 to CY217. In one or more embodiments, each of Formulae 201 and 202 may not include a group represented by one of Formulae CY201 to CY217. For example, the hole transport region may be selected from Compounds HT1 to HT44, m-MTDATA, TDATA, 2-TNATA, NPB(NPD), β-NPB, TPD, Spiro-TPD, Spiro-NPB, methylated-NPB, TAPC, HMTPD, 4,4′,4″-tris(N-carbazolyl)triphenylamine (TCTA), polyaniline/dodecylbenzenesulfonic acid (PANI/DBSA), poly(3,4-ethylenedioxythiophene)/poly(4-styrenesulfonate) (PEDOT/PSS), polyaniline/camphor sulfonic acid (PANI/CSA), polyaniline/poly(4-styrenesulfonate) (PANI/PSS), and any combinations thereof: A thickness of the hole transport region may be in a range of about 50 Å to about 10,000 Å, for example, about 100 Å to about 4,000 Å. When the hole transport region includes a hole injection layer, a hole transport layer, or any combination thereof, a thickness of the hole injection layer may be in a range of about 100 Å to about 9,000 Å, for example, about 100 Å to about 1,000 Å, and a thickness of the hole transport layer may be in a range of about 50 Å to about 2,000 Å, for example, about 100 Å to about 1,500 Å. When the thicknesses of the hole transport region, the hole injection layer, and the hole transport layer are within these ranges, satisfactory (or suitable) hole transporting characteristics may be obtained without a substantial increase in driving voltage. The emission auxiliary layer may increase light-emission efficiency by compensating for an optical resonance distance according to the wavelength of light emitted by the emission layers130and140, and the electron blocking layer may block or reduce the flow of electrons from the electron transport region. The emission auxiliary layer and the electron blocking layer may include any of the materials described above. p-Dopant The hole transport region may further include, in addition to the materials described above, a charge-generation material for the improvement of conductive properties. The charge-generation material may be uniformly or non-uniformly dispersed in the hole transport region (for example, in the form of a single layer including or consisting of a charge-generation material). The charge-generation material may be, for example, a p-dopant. For example, a lowest unoccupied molecular orbital (LUMO) energy level of the p-dopant may be equal to or less than about −3.5 eV. In one or more embodiments, the p-dopant may include a quinone derivative, a cyano group-containing compound, a compound containing element EL1 and element EL2, or any combination thereof. Non-limiting examples of the quinone derivative are TCNQ, F4-TCNQ, and/or the like. Non-limiting examples of the cyano group-containing compound are HAT-CN, a compound represented by Formula 221, and/or the like: wherein, in Formula 221, R221to R223may each independently be a C3-C60carbocyclic group unsubstituted or substituted with at least one R10aor a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, and at least one selected from R221to R223may each independently be a C3-C60carbocyclic group or a C1-C60heterocyclic group, each substituted with: a cyano group; —F; —Cl; —Br; —I; a C1-C20alkyl group substituted with a cyano group, —F, —Cl, —Br, —I, or any combination thereof; or any combination thereof. In the compound containing element EL1 and element EL2, element EL1 may be metal, metalloid, or a combination thereof, and element EL2 may be non-metal, metalloid, or a combination thereof. Examples of the metal are: alkali metal (for example, lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), etc.); alkaline earth metal (for example, beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), etc.); transition metal (for example, titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb), tantalum (Ta), chromium (Cr), molybdenum (Mo), tungsten (W), manganese (Mn), technetium (Tc), rhenium (Re), iron (Fe), ruthenium (Ru), osmium (Os), cobalt (Co), rhodium (Rh), iridium (Ir), nickel (Ni), palladium (Pd), platinum (Pt), copper (Cu), silver (Ag), gold (Au), etc.); post-transition metal (for example, zinc (Zn), indium (In), tin (Sn), etc.); and/or lanthanide metal (for example, lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), etc.). Examples of the metalloid are silicon (Si), antimony (Sb), and/or tellurium (Te). Examples of the non-metal are oxygen (O) and/or halogen (for example, F, Cl, Br, I, etc.). Examples of the compound containing element EL1 and element EL2 are metal oxide, metal halide (for example, metal fluoride, metal chloride, metal bromide, and/or metal iodide), metalloid halide (for example, metalloid fluoride, metalloid chloride, metalloid bromide, and/or metalloid iodide), metal telluride, or any combination thereof. Examples of the metal oxide are tungsten oxide (for example, WO, W2O3, WO2, WO3, W2O5, etc.), vanadium oxide (for example, VO, V2O3, VO2, V2O5, etc.), molybdenum oxide (MoO, Mo2O3, MoO2, MoO3, Mo2O5, etc.), and/or rhenium oxide (for example, ReO3, etc.). Examples of the metal halide are alkali metal halide, alkaline earth metal halide, transition metal halide, post-transition metal halide, and/or lanthanide metal halide. Examples of the alkali metal halide are LiF, NaF, KF, RbF, CsF, LiCl, NaCl, KCl, RbCl, CsCl, LiBr, NaBr, KBr, RbBr, CsBr, LiI, NaI, KI, RbI, and/or CsI. Examples of the alkaline earth metal halide are BeF2, MgF2, CaF2, SrF2, BaF2, BeCl2, MgCl2, CaCl2), SrCl2, BaCl2, BeBr2, MgBr2, CaBr2, SrBr2, BaBr2, BeI2, MgI2, CaI2, SrI2, and/or BaI2. Examples of the transition metal halide are titanium halide (for example, TiF4, TiCl4, TiBr4, TiI4, etc.), zirconium halide (for example, ZrF4, ZrCl4, ZrBr4, ZrI4, etc.), hafnium halide (for example, HfF4, HfCl4, HfBr4, HfI4, etc.), vanadium halide (for example, VF3, VCl3, VBr3, VI3, etc.), niobium halide (for example, NbF3, NbC13, NbBr3, NbI3, etc.), tantalum halide (for example, TaF3, TaCl3, TaBr3, TaI3, etc.), chromium halide (for example, CrF3, CrC13, CrBr3, CrI3, etc.), molybdenum halide (for example, MoF3, MoCl3, MoBr3, MoI3, etc.), tungsten halide (for example, WF3, WCl3, WBr3, WI3, etc.), manganese halide (for example, MnF2, MnCl2, MnBr2, MnI2, etc.), technetium halide (for example, TcF2, TcCl2, TcBr2, TcI2, etc.), rhenium halide (for example, ReF2, ReCl2, ReBr2, ReI2, etc.), iron halide (for example, FeF2, FeCl2, FeBr2, FeI2, etc.), ruthenium halide (for example, RuF2, RuCl2, RuBr2, RuI2, etc.), osmium halide (for example, OsF2, OsCl2, OsBr2, OsI2, etc.), cobalt halide (for example, CoF2, CoCl2, CoBr2, CoI2, etc.), rhodium halide (for example, RhF2, RhCl2, RhBr2, RhI2, etc.), iridium halide (for example, IrF2, IrCl2, IrBr2, IrI2, etc.), nickel halide (for example, NiF2, NiCl2, NiBr2, NiI2, etc.), palladium halide (for example, PdF2, PdCl2, PdBr2, PdI2, etc.), platinum halide (for example, PtF2, PtCl2, PtBr2, PtI2, etc.), copper halide (for example, CuF, CuCl, CuBr, CuI, etc.), silver halide (for example, AgF, AgCl, AgBr, AgI, etc.), and/or gold halide (for example, AuF, AuCl, AuBr, AuI, etc.). Examples of the post-transition metal halide are zinc halide (for example, ZnF2, ZnCl2, ZnBr2, ZnI2, etc.), indium halide (for example, InI3, etc.), and/or tin halide (for example, SnI2, etc.). Examples of the lanthanide metal halide are YbF, YbF2, YbF3, SmF3, YbCl, YbCl2, YbCl3SmCl3, YbBr, YbBr2, YbBr3SmBr3, YbI, YbI2, YbI3, and/or SmI3. An example of the metalloid halide is antimony halide (for example, SbCl5, etc.). Examples of the metal telluride are alkali metal telluride (for example, Li2Te, Na2Te, K2Te, Rb2Te, Cs2Te, etc.), alkaline earth metal telluride (for example, BeTe, MgTe, CaTe, SrTe, BaTe, etc.), transition metal telluride (for example, TiTe2, ZrTe2, HfTe2, V2Te3, Nb2Te3, Ta2Te3, Cr2Te3, Mo2Te3, W2Te3, MnTe, TcTe, ReTe, FeTe, RuTe, OsTe, CoTe, RhTe, IrTe, NiTe, PdTe, PtTe, Cu2Te, CuTe, Ag2Te, AgTe, Au2Te, etc.), post-transition metal telluride (for example, ZnTe, etc.), and/or lanthanide metal telluride (for example, LaTe, CeTe, PrTe, NdTe, PmTe, EuTe, GdTe, TbTe, DyTe, HoTe, ErTe, TmTe, YbTe, LuTe, etc.). Emission Layers130and140in Interlayer150 The emission layer150may include the first emission layer130and the second emission layer140. The first emission layer130and the second emission layer140may each be the same as described herein. The first emission layer130may include first quantum dots131, and the second emission layer140may include second quantum dots141. The first quantum dots131and the second quantum dots141may each be the same as described herein. The first emission layer130may be in direct contact with the second emission layer140. Host In one or more embodiments, the host may include a compound represented by Formula 301: [Ar301]xb11-[(L301)xb1-R301]xb21, Formula 301 wherein, in Formula 301, Ar301and L301may each independently be a C3-C60carbocyclic group unsubstituted or substituted with at least one R10aor a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, xb11 may be 1, 2, or 3, xb1 may be an integer from 0 to 5, R301may be hydrogen, deuterium, —F, —Cl, —Br, —I, a hydroxyl group, a cyano group, a nitro group, a C1-C60alkyl group unsubstituted or substituted with at least one R10a, a C2-C60alkenyl group unsubstituted or substituted with at least one R10a, a C2-C60alkynyl group unsubstituted or substituted with at least one R10a, a C1-C60alkoxy group unsubstituted or substituted with at least one R10a, a C3-C60carbocyclic group unsubstituted or substituted with at least one R10a, a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, —Si(Q301)(Q302)(Q303), —N(Q301)(Q302), —B(Q301)(Q302), —C(═O)(Q301), —S(═O)2(Q301), or —P(═O)(Q301)(Q302), xb21 may be an integer from 1 to 5, and Q301to Q303may each be the same as described in connection with Q1. For example, when xb11 in Formula 301 is 2 or more, two or more of Ar301(s) may be linked to each other via a single bond. In one or more embodiments, the host may include a compound represented by Formula 301-1, a compound represented by Formula 301-2, or any combination thereof: wherein, in Formulae 301-1 and 301-2, ring A301to ring A304may each independently be a C3-C60carbocyclic group unsubstituted or substituted with at least one R10aor a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, X301may be O, S, N-[(L304)xb4-R304], C(R304)(R305), or Si(R304)(R305), xb22 and xb23 may each independently be 0, 1, or 2, L301, xb1, and R301may each be the same as described herein, L302to L304may each independently be the same as described in connection with L301, xb2 to xb4 may each independently be the same as described in connection with xb1, and R302to R305and R311to R314may each be the same as described in connection with R301. In one or more embodiments, the host may include an alkaline earth-metal complex. In one or more embodiments, the host may include a Be complex (for example, Compound H55), an Mg complex, a Zn complex, or any combination thereof. In one or more embodiments, the host may include any of Compounds H1 to H124, 9,10-di(2-naphthyl)anthracene (ADN), 2-methyl-9,10-bis(naphthalen-2-yl)anthracene (MADN), 9,10-di-(2-naphthyl)-2-t-butyl-anthracene (TBADN), 4,4′-bis(N-carbazolyl)-1,1′-biphenyl (CBP), 1,3-di-9-carbazolylbenzene (mCP), 1,3,5-tri(carbazol-9-yl)benzene (TCP), or any combination thereof: Phosphorescent Dopant The phosphorescent dopant may include at least one transition metal as a central metal. The phosphorescent dopant may include a monodentate ligand, a bidentate ligand, a tridentate ligand, a tetradentate ligand, a pentadentate ligand, a hexadentate ligand, or any combination thereof. The phosphorescent dopant may be electrically neutral. For example, the phosphorescent dopant may include an organometallic compound represented by Formula 401: M(L401)xc1(L402)xc2Formula 401 wherein, in Formulae 401 and 402, M may be transition metal (for example, iridium (Ir), platinum (Pt), palladium (Pd), osmium (Os), titanium (Ti), gold (Au), hafnium (Hf), europium (Eu), terbium (Tb), rhodium (Rh), rhenium (Re), or thulium (Tm)), L401may be a ligand represented by Formula 402, and xc1 may be 1, 2, or 3, wherein, when xc1 is 3 or more, two or more of L401(s) may be identical to or different from each other, L402may be an organic ligand, and xc2 may be 0, 1, 2, 3, or 4, wherein, when xc2 is 2 or more, two or more of L402(s) may be identical to or different from each other, X401and X402may each independently be nitrogen or carbon, ring A401and ring A402may each independently be a C3-C60carbocyclic group or a C1-C60heterocyclic group, T401may be a single bond, *—O—*′, *—S—*′, *—C(═O)—*′, *—N(Q411)-*′, *—C(Q411)(Q412)-*′, *—C(Q411)=C(Q412)-*′, *—C(Q411)=*′, or *═C(Q411)=*′, X403and X404may each independently be a chemical bond (for example, a covalent bond or a coordinate bond), O, S, N(Q413), B(Q413), P(Q413), C(Q413)(Q414), or Si(Q413)(Q414), Q411to Q414may each be the same as described in connection with Q1, R401and R402may each independently be hydrogen, deuterium, —F, —Cl, —Br, —I, a hydroxyl group, a cyano group, a nitro group, a C1-C20alkyl group unsubstituted or substituted with at least one R10a, a C1-C20alkoxy group unsubstituted or substituted with at least one R10a, a C3-C60carbocyclic group unsubstituted or substituted with at least one R10a, a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, —Si(Q401)(Q402)(Q403), —N(Q401)(Q402), —B(Q401)(Q402), —C(═O)(Q401), —S(═O)2(Q401), or —P(═O)(Q401)(Q402), Q401to Q403may each be the same as described in connection with Q1, xc11 and xc12 may each independently be an integer from 0 to 10, and * and *′ in Formula 402 each indicate a binding site to M in Formula 401. For example, in Formula 402, i) X401may be nitrogen and X402may be carbon, or ii) each of X401and X402may be nitrogen. In one or more embodiments, in Formula 401, when xc1 is 2 or more, two rings A401(s) in two or more of L401(s) may be optionally linked to each other via T402, which is a linking group, and two rings A402(s) may optionally be linked to each other via T403, which is a linking group (see e.g., Compounds PD1 to PD4 and PD7), wherein T402and T403may each be the same as described in connection with T401. In Formula 401, L402may be an organic ligand. For example, L402may include a halogen group, a diketone group (for example, an acetylacetonate group), a carboxylic acid group (for example, a picolinate group), —C(═O), an isonitrile group, —CN group, a phosphorus group (for example, a phosphine group, a phosphite group, etc.), or any combination thereof. The phosphorescent dopant may include, for example, one of compounds PD1 to PD25, or any combination thereof: Fluorescent Dopant The fluorescent dopant may include an amine group-containing compound, a styryl group-containing compound, or any combination thereof. In one or more embodiments, the fluorescent dopant may include a compound represented by Formula 501: wherein, in Formula 501, Ar501, L501to L503, R501, and R502may each independently be a C3-C60carbocyclic group unsubstituted or substituted with at least one R10aor a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, xd1 to xd3 may each independently be 0, 1, 2, or 3, and xd4 may be 1, 2, 3, 4, 5, or 6. In one or more embodiments, Ar501in Formula 501 may be a condensed cyclic group (for example, an anthracene group, a chrysene group, or a pyrene group) in which three or more monocyclic groups are condensed together. In one or more embodiments, xd4 in Formula 501 may be 2. For example, the fluorescent dopant may include any of Compounds FD1 to FD36, DPVBi, DPAVBi, or any combination thereof: Delayed Fluorescence Material The emission layer may include a delayed fluorescence material. In the present specification, the delayed fluorescence material may be selected from compounds capable of emitting delayed fluorescence based on a delayed fluorescence emission mechanism. The delayed fluorescent material included in the emission layer may act as a host or a dopant depending on the type (or kind) of other materials included in the emission layer. In one or more embodiments, a difference between a triplet energy level (eV) of the delayed fluorescence material and a singlet energy level (eV) of the delayed fluorescence material may be equal to or greater than 0 eV and equal to or less than about 0.5 eV. When the difference between the triplet energy level (eV) of the delayed fluorescence material and the singlet energy level (eV) of the delayed fluorescence material is satisfied within the range above, up-conversion from the triplet state to the singlet state of the delayed fluorescence materials may effectively (or suitably) occur, and thus, the light-emitting device10may have improved luminescence efficiency. In one or more embodiments, the delayed fluorescence material may include i) a material including at least one electron donor (for example, a π electron-rich C3-C60cyclic group, such as a carbazole group) and at least one electron acceptor (for example, a sulfoxide group, a cyano group, and/or a π electron-deficient nitrogen-containing C1-C60cyclic group), and ii) a material including a C8-C60polycyclic group in which two or more cyclic groups are condensed while sharing boron (B). In one or more embodiments, the delayed fluorescence material may include at least one of Compounds DF1 to DF9: Electron Transport Region in Interlayer150 The electron transport region may have: i) a single-layered structure including or consisting of a single layer including or consisting of a single material, ii) a single-layered structure including or consisting of a single layer including or consisting of a plurality of different materials, or iii) a multi-layered structure including a plurality of layers including different materials. The electron transport region may include a buffer layer, a hole blocking layer, an electron control layer, an electron transport layer, an electron injection layer, or any combination thereof. For example, the electron transport region may have an electron transport layer/electron injection layer structure, a hole blocking layer/electron transport layer/electron injection layer structure, an electron control layer/electron transport layer/electron injection layer structure, or a buffer layer/electron transport layer/electron injection layer structure, wherein, in each structure, layers are sequentially stacked on the emission layers130and140. The electron transport region (for example, the buffer layer, the hole blocking layer, the electron control layer, and/or the electron transport layer in the electron transport region) may include a metal-free compound including at least one π electron-deficient nitrogen-containing C1-C60cyclic group. In one or more embodiments, the electron transport region may include a compound represented by Formula 601: [Ar601]xe11-[(L601)xe1-R601]xe21, Formula 601 wherein, in Formula 601, Ar601and L601may each independently be a C3-C60carbocyclic group unsubstituted or substituted with at least one R10aor a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, xe11 may be 1, 2, or 3, xe1 may be 0, 1, 2, 3, 4, or 5, R601may be a C3-C60carbocyclic group unsubstituted or substituted with at least one R10a, a C1-C60heterocyclic group unsubstituted or substituted with at least one R10a, —Si(Q601)(Q602)(Q603), —C(═O)(Q601), —S(═O)2(Q601), or —P(═O)(Q601)(Q602), Q601to Q603may each be the same as described in connection with Q1, xe21 may be 1, 2, 3, 4, or 5, and at least one of Ar601, L601, or R601may each independently be a π electron-deficient nitrogen-containing C1-C60cyclic group unsubstituted or substituted with at least one R10a. In one or more embodiments, when xe11 in Formula 601 is 2 or more, two or more of Ar601(s) may be linked together via a single bond. In one or more embodiments, Ar601in Formula 601 may be a substituted or unsubstituted anthracene group. In one or more embodiments, the electron transport region may include a compound represented by Formula 601-1: wherein, in Formula 601-1, X614may be N or C(R614), X615may be N or C(R615), and X616may be N or C(R616), wherein at least one of X614to X616may be N, L611to L613may each be the same as described in connection with L601, xe611 to xe613 may each be the same as described in connection with xe1, R611to R613may each be the same as described in connection with R601, and R614to R616may each independently be hydrogen, deuterium, —F, —Cl, —Br, —I, a hydroxyl group, a cyano group, a nitro group, a C1-C20alkyl group, a C1-C20alkoxy group, a C3-C60carbocyclic group unsubstituted or substituted with at least one R10a, or a C1-C60heterocyclic group substituted or unsubstituted at least one R10a. For example, xe1 and xe611 to xe613 in Formulae 601 and 601-1 may each independently be 0, 1, or 2. The electron transport region may include any of Compounds ET1 to ET45, 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline (BCP), 4,7-diphenyl-1,10-phenanthroline (Bphen), Alq3, BAlq, TAZ, NTAZ, or any combination thereof: A thickness of the electron transport region may be in a range about 50 Å to about 5,000 Å, for example, about 100 Å to about 4,000 Å. When the electron transport region includes a buffer layer, a hole blocking layer, an electron control layer, an electron transport layer, or any combination thereof, a thickness of the buffer layer, the hole blocking layer, or the electron control layer may each independently be in a range of about 20 Å to about 1000 Å, for example, about 30 Å to about 300 Å, and a thickness of the electron transport layer may be in a range of about 100 Å to about 1000 Å, for example, about 150 Å to about 500 Å. When the thicknesses of the buffer layer, the hole blocking layer, the electron control layer, the electron transport layer, and/or the electron transport layer are within these ranges, satisfactory (or suitable) hole transporting characteristics may be obtained without a substantial increase in driving voltage. The electron transport region (for example, the electron transport layer in the electron transport region) may further include, in addition to the materials described above, a metal-containing material. The metal-containing material may include an alkali metal complex, alkaline earth metal complex, or any combination thereof. A metal ion of the alkali metal complex may be a Li ion, a Na ion, a K ion, a Rb ion, or a Cs ion; and a metal ion of the alkaline earth metal complex may be a Be ion, a Mg ion, a Ca ion, a Sr ion, or a Ba ion. A ligand coordinated with the metal ion of the alkali metal complex or the alkaline earth-metal complex may include a hydroxyquinoline, a hydroxyisoquinoline, a hydroxybenzoquinoline, a hydroxyacridine, a hydroxyphenanthridine, a hydroxyphenyloxazole, a hydroxyphenylthiazole, a hydroxyphenyloxadiazole, a hydroxyphenylthiadiazole, a hydroxyphenylpyridine, a hydroxyphenylbenzimidazole, a hydroxyphenylbenzothiazole, a bipyridine, a phenanthroline, a cyclopentadiene, or any combination thereof. For example, the metal-containing material may include a Li complex. The Li complex may include, for example, Compound ET-D1 (LiQ) and/or Compound ET-D2: The electron transport region may include an electron injection layer that facilitates the injection of electrons from the second electrode190. The electron injection layer may be in direct contact with the second electrode190. The electron injection layer may have: i) a single-layered structure including or consisting of a single layer including or consisting of a single material, ii) a single-layered structure including or consisting of a single layer including or consisting of a plurality of different materials, or iii) a multi-layered structure including a plurality of layers including different materials. The electron injection layer may include an alkali metal, alkaline earth metal, a rare earth metal, an alkali metal-containing compound, alkaline earth metal-containing compound, a rare earth metal-containing compound, an alkali metal complex, alkaline earth metal complex, a rare earth metal complex, or any combination thereof. The alkali metal may include Li, Na, K, Rb, Cs, or any combination thereof. The alkaline earth metal may include Mg, Ca, Sr, Ba, or any combination thereof. The rare earth metal may include Sc, Y, Ce, Tb, Yb, Gd, or any combination thereof. The alkali metal-containing compound, the alkaline earth metal-containing compound, and the rare earth metal-containing compound may be one or more oxides, halides (for example, fluorides, chlorides, bromides, and/or iodides), tellurides, or any combination thereof, of the alkali metal, the alkaline earth metal, and the rare earth metal, respectively. The alkali metal-containing compound may include one or more alkali metal oxides (such as Li2O, Cs2O, and/or K2O), alkali metal halides (such as LiF, NaF, CsF, KF, LiI, NaI, CsI, and/or KI), or any combination thereof. The alkaline earth metal-containing compound may include an alkaline earth metal compound, such as BaO, SrO, CaO, BaxSr1-xO (where x is a real number satisfying the condition of 0<x<1), BaxCa1-xO (where x is a real number satisfying the condition of 0<x<1), and/or the like. The rare earth metal-containing compound may include YbF3, ScF3, Sc2O3, Y2O3, Ce2O3, GdF3, TbF3, Yb13, ScI3, Tb13, or any combination thereof. For example, the rare earth metal-containing compound may include lanthanide metal telluride. Non-limiting examples of the lanthanide metal telluride are LaTe, CeTe, PrTe, NdTe, PmTe, SmTe, EuTe, GdTe, TbTe, DyTe, HoTe, ErTe, TmTe, YbTe, LuTe, La2Te3, Ce2Te3, Pr2Te3, Nd2Te3, Pm2Te3, Sm2Te3, Eu2Te3, Gd2Te3, Tb2Te3, Dy2Te3, Ho2Te3, Er2Te3, Tm2Te3, Yb2Te3, and/or Lu2Te3. The alkali metal complex, the alkaline earth-metal complex, and the rare earth metal complex may include i) one of ions of the alkali metal, the alkaline earth metal, or the rare earth metal and ii), as a ligand bonded to the metal ion, for example, a hydroxyquinoline, a hydroxyisoquinoline, a hydroxybenzoquinoline, a hydroxyacridine, a hydroxyphenanthridine, a hydroxyphenyloxazole, a hydroxyphenylthiazole, a hydroxyphenyloxadiazole, a hydroxyphenylthiadiazole, a hydroxyphenylpyridine, a hydroxyphenyl benzimidazole, a hydroxyphenylbenzothiazole, a bipyridine, a phenanthroline, a cyclopentadiene, or any combination thereof. The electron injection layer may include or consist of an alkali metal, an alkaline earth metal, a rare earth metal, an alkali metal-containing compound, an alkaline earth metal-containing compound, a rare earth metal-containing compound, an alkali metal complex, an alkaline earth metal complex, a rare earth metal complex, or any combination thereof, as described above. For example, the electron injection layer may further include an organic material (for example, a compound represented by Formula 601). In one or more embodiments, the electron injection layer may include or consist of i) an alkali metal-containing compound (for example, an alkali metal halide), or ii) a) an alkali metal-containing compound (for example, an alkali metal halide); and b) an alkali metal, an alkaline earth metal, a rare earth metal, or any combination thereof. For example, the electron injection layer may be a KI:Yb co-deposited layer, an RbI:Yb co-deposited layer, and/or the like. When the electron injection layer further includes an organic material, the alkali metal, the alkaline earth metal, the rare earth metal, the alkali metal-containing compound, the alkaline earth metal-containing compound, the rare earth metal-containing compound, the alkali metal complex, the alkaline earth-metal complex, the rare earth metal complex, or any combination thereof may be homogeneously or non-homogeneously dispersed in a matrix including the organic material. A thickness of the electron injection layer may be in a range of about 1 Å to about 100 Å, for example, about 3 Å to about 90 Å. When the thickness of the electron injection layer is within these ranges, satisfactory (or suitable) electron injection characteristics may be obtained without a substantial increase in driving voltage. Second Electrode190 The second electrode190is arranged on the interlayer150having the above-described structure. The second electrode190may be a cathode, which is an electron injection electrode, and in this regard, as a material for forming the second electrode190, a metal, an alloy, a suitable electrically conductive compound, or any combination thereof, each having a low work function, may be used. The second electrode150may include at least one of lithium (Li), silver (Ag), magnesium (Mg), aluminum (Al), aluminum-lithium (Al—Li), calcium (Ca), magnesium-indium (Mg—In), magnesium-silver (Mg—Ag), ytterbium (Yb), silver-ytterbium (Ag—Yb), ITO, IZO, or any combination thereof. The second electrode190may be a transmissive electrode, a semi-transmissive electrode, or a reflective electrode. The second electrode190may have a single-layered structure or a multi-layered structure including two or more layers. Capping Layer A first capping layer may be arranged outside the first electrode110, and/or a second capping layer may be arranged outside the second electrode190. For example, the light-emitting device100may have a structure in which the first capping layer, the first electrode110, the interlayer150, and the second electrode190are sequentially stacked in this stated order; a structure in which the first electrode110, the interlayer150, the second electrode190, and the second capping layer are sequentially stacked in this stated order; or a structure in which the first capping layer, the first electrode110, the interlayer150, the second electrode190, and the second capping layer are sequentially stacked in this stated order. Light generated in the emission layer130or140included in the interlayer150of the light-emitting device10may be extracted toward the outside through the first electrode110, which is a semi-transmissive electrode or a transmissive electrode, and the first capping layer, and/or light generated in the emission layer130or140included in the interlayer150of the light-emitting device10may be extracted toward the outside through the second electrode150, which is a semi-transmissive electrode or a transmissive electrode, and the second capping layer. The first capping layer and the second capping layer may increase external luminescence efficiency according to the principle of constructive interference. Accordingly, light extraction efficiency of the light-emitting device10may be increased, thereby improving luminescence efficiency of the light-emitting device10. Each of the first capping layer and second capping layer may include a material having a refractive index (at 589 nm) of equal to or more than about 1.6. The first capping layer and the second capping layer may each independently be an organic capping layer including an organic material, an inorganic capping layer including an inorganic material, or a composite capping layer including an organic material and an inorganic material. At least one of the first capping layer or the second capping layer may each independently include a carbocyclic compound, a heterocyclic compound, an amine group-containing compound, a porphyrin derivative, a phthalocyanine derivative, a naphthalocyanine derivative, an alkali metal complex, an alkaline earth-based complex, or any combination thereof. The carbocyclic compound, the heterocyclic compound, and the amine group-containing compound may each independently be optionally be substituted with a substituent containing O, N, S, Se, Si, F, Cl, Br, I, or any combination thereof. In one or more embodiments, at least one of the first capping layer or the second capping layer may each independently include the amine group-containing compound. In one or more embodiments, at least one of the first capping layer or the second capping layer may each independently include a compound represented by Formula 201, a compound represented by Formula 202, or any combination thereof. In one or more embodiments, at least one of the first capping layer or the second capping layer may each independently include any of Compounds HT28 to HT33, any of Compounds CP1 to CP6, β-NPB, or any combination thereof: Electronic Apparatus The light-emitting device may be included in various suitable electronic apparatuses. For example, an electronic apparatus including the light-emitting device may be a light-emitting apparatus, an authentication apparatus, and/or the like. Such an electronic apparatus (for example, light-emitting apparatus) may further include, in addition to the light-emitting device, i) a color filter, ii) a color conversion layer, or iii) both a color filter and a color conversion layer. The color filter and/or the color conversion layer may be arranged in at least one traveling direction of light emitted from the light-emitting device. For example, light emitted from the light-emitting device may be blue light or white light. The light-emitting device may be the same as described above. In one or more embodiments, the color conversion layer may include quantum dots. The quantum dots may be, for example, the same as described herein. The electronic apparatus may include a first substrate. The first substrate may include a plurality of subpixel areas, the color filter may include a plurality of color filter areas respectively corresponding to the subpixel areas, and the color conversion layer may include a plurality of color conversion areas respectively corresponding to the subpixel areas. A pixel-defining film may be arranged among the subpixel areas to define each of the subpixel areas. The color filter may further include a plurality of color filter areas, and light-blocking patterns located among the color filter areas, and the color conversion layer may include a plurality of color conversion areas, and light-blocking patterns located among the color conversion areas. The color filter areas (and/or the color conversion areas) may include a first area to emit first color light, a second area to emit second color light, and/or a third area to emit third color light, and the first color light, the second color light, and/or the third color light may have different maximum emission wavelengths from one another. For example, the first color light may be red light, the second color light may be green light, and the third color light may be blue light. For example, the color filter areas (and/or the color conversion areas) may include quantum dots. In one or more embodiments, the first area may include red quantum dots, the second area may include green quantum dots, and the third area may not include quantum dots. The quantum dots may be the same as described herein. The first area, the second area, and/or the third area may each include a scatter. For example, the light-emitting device may emit first light, the first area may absorb the first light to emit first first-color light, the second area may absorb the first light to emit second first-color light, and the third area may absorb the first light to emit third first-color light. Here, the first first-color light, the second first-color light, and the third-first light may have different maximum emission wavelengths from one another. For example, the first light may be blue light, the first first-color light may be red light, the second first-color light may be green light, and the third first-color light may be blue light. The electronic apparatus may further include a thin-film transistor, in addition to the light-emitting device as described above. The thin-film transistor may include a source electrode, a drain electrode, and an active layer, wherein any one of the source electrode and the drain electrode may be electrically connected to any one of the first electrode and the second electrode of the light-emitting device. The thin-film transistor may include a gate electrode, a gate insulating film, and/or the like. The active layer may include crystalline silicon, amorphous silicon, organic semiconductor, oxide semiconductor, and/or the like. The electronic apparatus may further include a sealing portion for sealing the light-emitting device. The sealing portion may be arranged between the color filter and the light-emitting device and/or between the color conversion layer and the light-emitting device. The sealing portion allows light from the light-emitting device to be extracted to the outside, while simultaneously (or concurrently) preventing or reducing the penetration of ambient air and/or moisture into the light-emitting device. The sealing portion may be a sealing substrate including a transparent glass substrate and/or a plastic substrate. The sealing portion may be a thin-film encapsulation layer including at least one layer of an organic layer and/or an inorganic layer. When the sealing portion is a thin-film encapsulation layer, the electronic apparatus may be flexible. Various suitable functional layers may be additionally arranged on the sealing portion, in addition to the color filter and/or the color conversion layer, according to the use of the electronic apparatus. The functional layers may include a touch screen layer, a polarizing layer, and/or the like. The touch screen layer may be a pressure-sensitive touch screen layer, a capacitive touch screen layer, or an infra-red touch screen layer. The authentication apparatus may be, for example, a biometric authentication apparatus that authenticates an individual by using biometric information of a living body (for example, fingertips, pupils, etc.). The authentication apparatus may further include, in addition to the light-emitting device, a biometric information collector. The electronic apparatus may be applied to various suitable displays, light sources, lighting, personal computers (for example, a mobile personal computer), mobile phones, digital cameras, electronic organizers, electronic dictionaries, electronic game machines, medical instruments (for example, electronic thermometers, sphygmomanometers, blood glucose meters, pulse measurement devices, pulse wave measurement devices, electrocardiogram displays, ultrasonic diagnostic devices, and/or endoscope displays), fish finders, various measuring instruments, meters (for example, meters for a vehicle, an aircraft, and/or a vessel), projectors, and/or the like. Respective layers included in the hole transport region, the emission layer, and respective layers included in the electron transport region may be formed in a certain region by using one or more suitable methods including vacuum deposition, spin coating, casting, Langmuir-Blodgett (LB) deposition, ink-jet printing, laser-printing, laser-induced thermal imaging, and/or the like. When respective layers included in the hole transport region, the emission layer, and respective layers included in the electron transport region are formed by vacuum deposition, the deposition conditions may include, for example, a deposition temperature in a range of about 100° C. to about 500° C., a vacuum degree of about 10−8torr to about 10−3torr, and a deposition speed in a range of about 0.01 Å/sec to about 100 Å/sec, depending on a material to be included in a layer to be formed and a structure of a layer to be formed. Definition of Terms The term “C3-C60carbocyclic group” as used herein refers to a cyclic group consisting of carbon atoms only and having three to sixty carbon atoms, and the term “C1-C60heterocyclic group” as used herein refers to a cyclic group that further includes a heteroatom in addition to 1 to 60 carbon atoms. The C3-C60carbocyclic group and the C1-C60heterocyclic group may each independently be a monocyclic group consisting of one ring, or a polycyclic group in which two or more rings are condensed with each other. For example, the number of ring-forming atoms of the C1-C60heterocyclic group may be from 3 to 61. The “cyclic group” as used herein may include both the C3-C60carbocyclic group and the C1-C60heterocyclic group. The term “T1electron-rich C3-C60cyclic group” as used herein refers to a cyclic group that includes 3 to 60 carbon atoms and does not include *—N═*′ as a ring-forming moiety, and the term “T1electron-deficient nitrogen-containing C1-C60cyclic group” as used herein refers to a heterocyclic group that includes 1 to 60 carbon atoms and includes *—N═*′ as a ring-forming moiety. For example, the C3-C60carbocyclic group may be i) group T1 or ii) a condensed cyclic group in which two or more groups T1are condensed with each other (for example, the C3-C60carbocyclic group may be a cyclopentadiene group, an adamantane group, a norbornane group, a benzene group, a pentalene group, a naphthalene group, an azulene group, an indacene group, an acenaphthylene group, a phenalene group, a phenanthrene group, an anthracene group, a fluoranthene group, a triphenylene group, a pyrene group, a chrysene group, a perylene group, a pentaphene group, a heptalene group, a naphthacene group, a picene group, a hexacene group, a pentacene group, a rubicene group, a coronene group, an ovalene group, an indene group, a fluorene group, a spiro-bifluorene group, a benzofluorene group, an indenophenanthrene group, and/or an indenoanthracene group), the C1-C60heterocyclic group may be i) group T2, ii) a condensed cyclic group in which two or more groups T2are condensed with each other, or iii) a condensed cyclic group in which at least one group T2 and at least one group T1 are condensed with each other (for example, the C1-C60heterocyclic group may be a pyrrole group, a thiophene group, a furan group, an indole group, a benzoindole group, a naphtho indole group, an isoindole group, a benzoisoindole group, a naphtho isoindole group, a benzosilole group, a benzothiophene group, a benzofuran group, a carbazole group, a dibenzosilole group, a dibenzothiophene group, a dibenzofuran group, an indenocarbazole group, an indolocarbazole group, a benzofurocarbazole group, a benzothienocarbazole group, a benzosilolocarbazole group, a benzoindolocarbazole group, a benzocarbazole group, a benzonaphthofuran group, a benzonaphthothiophene group, a benzonaphthosilole group, a benzofurodibenzofuran group, a benzofurodibenzothiophene group, a benzothienodibenzothiophene group, a pyrazole group, an imidazole group, a triazole group, an oxazole group, an isoxazole group, an oxadiazole group, a thiazole group, an isothiazole group, a thiadiazole group, a benzopyrazole group, a benzimidazole group, a benzoxazole group, a benzoisoxazole group, a benzothiazole group, a benzoisothiazole group, a pyridine group, a pyrimidine group, a pyrazine group, a pyridazine group, a triazine group, a quinoline group, an isoquinoline group, a benzoquinoline group, a benzoisoquinoline group, a quinoxaline group, a benzoquinoxaline group, a quinazoline group, a benzoquinazoline group, a phenanthroline group, a cinnoline group, a phthalazine group, a naphthyridine group, an imidazopyridine group, an imidazopyrimidine group, an imidazotriazine group, an imidazopyrazine group, an imidazopyridazine group, an azacarbazole group, an azafluorene group, an azadibenzosilole group, an azadibenzothiophene group, an azadibenzofuran group, etc.), the π electron-rich C3-C60cyclic group may be i) group T1, ii) a condensed cyclic group in which two or more groups T1are condensed with each other, iii) group T3, iv) a condensed cyclic group in which two or more groups T3are condensed with each other, or v) a condensed cyclic group in which at least one group T3 and at least one group T1 are condensed with each other (for example, the π electron-rich C3-C60cyclic group may be the C3-C60carbocyclic group, a pyrrole group, a thiophene group, a furan group, an indole group, a benzoindole group, a naphtho indole group, an isoindole group, a benzoisoindole group, a naphtho isoindole group, a benzosilole group, a benzothiophene group, a benzofuran group, a carbazole group, a dibenzosilole group, a dibenzothiophene group, a dibenzofuran group, an indenocarbazole group, an indolocarbazole group, a benzofurocarbazole group, a benzothienocarbazole group, a benzosilolocarbazole group, a benzoindolocarbazole group, a benzocarbazole group, a benzonaphthofuran group, a benzonaphthothiophene group, a benzonaphthosilole group, a benzofurodibenzofuran group, a benzofurodibenzothiophene group, a benzothienodibenzothiophene group, etc.), the π electron-deficient nitrogen-containing C1-C60cyclic group may be i) group T4, ii) a condensed cyclic group in which two or more groups T4are condensed with each other, iii) a condensed cyclic group in which at least one group T4 and at least one group T1 are condensed with each other, iv) a condensed cyclic group in which at least one group T4 and at least one group T3 are condensed with each other, or v) a condensed cyclic group in which at least one group T4, at least one group T1, and at least one group T3 are condensed with one another (for example, the π electron-deficient nitrogen-containing C1-C60cyclic group may be a pyrazole group, an imidazole group, a triazole group, an oxazole group, an isoxazole group, an oxadiazole group, a thiazole group, an isothiazole group, a thiadiazole group, a benzopyrazole group, a benzimidazole group, a benzoxazole group, a benzoisoxazole group, a benzothiazole group, a benzoisothiazole group, a pyridine group, a pyrimidine group, a pyrazine group, a pyridazine group, a triazine group, a quinoline group, an isoquinoline group, a benzoquinoline group, a benzoisoquinoline group, a quinoxaline group, a benzoquinoxaline group, a quinazoline group, a benzoquinazoline group, a phenanthroline group, a cinnoline group, a phthalazine group, a naphthyridine group, an imidazopyridine group, an imidazopyrimidine group, an imidazotriazine group, an imidazopyrazine group, an imidazopyridazine group, an azacarbazole group, an azafluorene group, an azadibenzosilole group, an azadibenzothiophene group, an azadibenzofuran group, etc.), group T1 may be a cyclopropane group, a cyclobutane group, a cyclopentane group, a cyclohexane group, a cycloheptane group, a cyclooctane group, a cyclobutene group, a cyclopentene group, a cyclopentadiene group, a cyclohexene group, a cyclohexadiene group, a cycloheptene group, an adamantane group, a norbornane (or a bicyclo[2.2.1]heptane) group, a norbornene group, a bicyclo[1.1.1]pentane group, a bicyclo[2.1.1]hexane group, a bicyclo[2.2.2]octane group, and/or a benzene group, group T2 may be a furan group, a thiophene group, a 1H-pyrrole group, a silole group, a borole group, a 2H-pyrrole group, a 3H-pyrrole group, an imidazole group, a pyrazole group, a triazole group, a tetrazole group, an oxazole group, an isoxazole group, an oxadiazole group, a thiazole group, an isothiazole group, a thiadiazole group, an azasilole group, an azaborole group, a pyridine group, a pyrimidine group, a pyrazine group, a pyridazine group, a triazine group, and/or a tetrazine group, group T3 may be a furan group, a thiophene group, a 1H-pyrrole group, a silole group, and/or a borole group, and group T4 may be a 2H-pyrrole group, a 3H-pyrrole group, an imidazole group, a pyrazole group, a triazole group, a tetrazole group, an oxazole group, an isoxazole group, an oxadiazole group, a thiazole group, an isothiazole group, a thiadiazole group, an azasilole group, an azaborole group, a pyridine group, a pyrimidine group, a pyrazine group, a pyridazine group, a triazine group, and/or a tetrazine group. The terms “the cyclic group, the C3-C60carbocyclic group, the C1-C60heterocyclic group, the π electron-rich C3-C60cyclic group, and/or the π electron-deficient nitrogen-containing C1-C60cyclic group” as used herein refer to a group condensed to any cyclic group or a polyvalent group (for example, a divalent group, a trivalent group, a tetravalent group, etc.), depending on the structure of a formula in connection with which the terms are used. For example, “a benzene group” may be a benzo group, a phenyl group, a phenylene group, and/or the like, which should be easily understood by one of ordinary skill in the art according to the structure of a formula including the “benzene group”. Non-limiting examples of the monovalent C3-C60carbocyclic group and the monovalent C1-C60heterocyclic group are a C3-C10cycloalkyl group, a C1-C10heterocycloalkyl group, a C3-C10cycloalkenyl group, a C1-C10heterocycloalkenyl group, a C6-C60aryl group, a C1-C60heteroaryl group, a monovalent non-aromatic condensed polycyclic group, and/or a monovalent non-aromatic condensed heteropolycyclic group, and non-limiting examples of the divalent C3-C60carbocyclic group and the monovalent C1-C60heterocyclic group are a C3-C10cycloalkylene group, a C1-C10heterocycloalkylene group, a C3-C10cycloalkenylene group, a C1-C10heterocycloalkenylene group, a C6-C60arylene group, a C1-C60heteroarylene group, a divalent non-aromatic condensed polycyclic group, and/or a substituted or unsubstituted divalent non-aromatic condensed heteropolycyclic group. The term “C1-C60alkyl group” as used herein refers to a linear or branched aliphatic hydrocarbon monovalent group that includes 1 to 60 carbon atoms, and non-limiting examples thereof are a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, an n-pentyl group, a tert-pentyl group, a neopentyl group, an isopentyl group, a sec-pentyl group, a 3-pentyl group, a sec-isopentyl group, an n-hexyl group, an isohexyl group, a sec-hexyl group, a tert-hexyl group, an n-heptyl group, an isoheptyl group, a sec-heptyl group, a tert-heptyl group, an n-octyl group, an isooctyl group, a sec-octyl group, a tert-octyl group, an n-nonyl group, an isononyl group, a sec-nonyl group, a tert-nonyl group, an n-decyl group, an isodecyl group, a sec-decyl group, and/or a tert-decyl group. The term “C1-C60alkylene group” as used herein refers to a divalent group having the same structure as the C1-C60alkyl group. The term “C2-C60alkenyl group” as used herein refers to a monovalent hydrocarbon group having at least one carbon-carbon double bond in the middle and/or at either terminus of the C2-C60alkyl group, and non-limiting examples thereof are an ethenyl group, a propenyl group, and/or a butenyl group. The term “C2-C60alkenylene group” as used herein refers to a divalent group having the same structure as the C2-C60alkenyl group. The term “C2-C60alkynyl group” as used herein refers to a monovalent hydrocarbon group having at least one carbon-carbon triple bond in the middle and/or at either terminus of the C2-C60alkyl group, and non-limiting examples thereof include an ethynyl group, and/or a propynyl group. The term “C1-C60alkynylene group” as used herein refers to a divalent group having the same structure as the C1-C60alkynyl group. The term “C1-C60alkoxy group” as used herein refers to a monovalent group represented by —OA101(wherein Ani is the C1-C60alkyl group), and non-limiting examples thereof include a methoxy group, an ethoxy group, and/or an isopropyloxy group. The term “C3-C10cycloalkyl group” as used herein refers to a monovalent saturated hydrocarbon cyclic group having 3 to 10 carbon atoms, and non-limiting examples thereof are a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, an adamantanyl group, a norbornanyl group (or bicyclo[2.2.1]heptyl group), a bicyclo[1.1.1]pentyl group, a bicyclo[2.1.1]hexyl group, and/or a bicyclo[2.2.2]octyl group. The term “C3-C10cycloalkylene group” as used herein refers to a divalent group having the same structure as the C3-C10cycloalkyl group. The term “C1-C10heterocycloalkyl group” as used herein refers to a monovalent cyclic group that has 1 to 10 carbon atoms and includes at least one heteroatom as a ring-forming atom, in addition to ring-forming carbon atoms, and non-limiting examples thereof are a 1,2,3,4-oxatriazolidinyl group, a tetrahydrofuranyl group, and/or a tetrahydrothiophenyl group. The term “C1-C10heterocycloalkylene group” as used herein refers to a divalent group having the same structure as the C1-C10heterocycloalkyl group. The term “C3-C10cycloalkenyl group” as used herein refers to a monovalent cyclic group that includes 3 to 10 carbon atoms and at least one carbon-carbon double bond in the ring thereof and has no aromaticity, and non-limiting examples thereof are a cyclopentenyl group, a cyclohexenyl group, and/or a cycloheptenyl group. The term “C3-C10cycloalkenylene group” as used herein refers to a divalent group having the same structure as the C3-C10cycloalkenyl group. The term “C1-C10heterocycloalkenyl group” as used herein refers to a monovalent cyclic group that has 1 to 10 carbon atoms and at least one heteroatom as a ring-forming atom, in addition to ring-forming carbon atoms, and at least one carbon-carbon double bond in the cyclic structure thereof. Non-limiting examples of the C1-C10heterocycloalkenyl group include a 4,5-dihydro-1,2,3,4-oxatriazolyl group, a 2,3-dihydrofuranyl group, and/or a 2,3-dihydrothiophenyl group. The term “C1-C10heterocycloalkenylene group” as used herein refers to a divalent group having the same structure as the C1-C10heterocycloalkenyl group. The term “C6-C60aryl group” as used herein refers to a monovalent group having a carbocyclic aromatic system including 6 to 60 carbon atoms. Non-limiting examples of the C6-C60aryl group are a phenyl group, a pentalenyl group, a naphthyl group, an azulenyl group, an indacenyl group, an acenaphthyl group, a phenalenyl group, a phenanthrenyl group, an anthracenyl group, a fluoranthenyl group, a triphenylenyl group, a pyrenyl group, a chrysenyl group, a perylenyl group, a pentaphenyl group, a heptalenyl group, a naphthacenyl group, a picenyl group, a hexacenyl group, a pentacenyl group, a rubicenyl group, a coronenyl group, and/or an ovalenyl group. The term “C6-C60arylene group” as used herein refers to a divalent group having the same structure as the C6-C60aryl group. When the C6-C60aryl group and the C6-C60arylene group each independently include two or more rings, the respective two or more rings may be condensed with each other. The term “C1-C60heteroaryl group” as used herein refers to a monovalent group having a heterocyclic aromatic system that has 1 to 60 carbon atoms and at least one heteroatom as a ring-forming atom, in addition to ring-forming carbon atoms. The term “C1-C60heteroarylene group” as used herein refers to a divalent group having a heterocyclic aromatic system that has the same structure as the C1-C60heteroaryl group. Non-limiting examples of the C1-C60heteroaryl group are a pyridinyl group, a pyrimidinyl group, a pyrazinyl group, a pyridazinyl group, a triazinyl group, a quinolinyl group, a benzoquinolinyl group, an isoquinolinyl group, a benzoisoquinolinyl group, a quinoxalinyl group, a benzoquinoxalinyl group, a quinazolinyl group, a benzoquinazolinyl group, a cinnolinyl group, a phenanthrolinyl group, a phthalazinyl group, and/or a naphthyridinyl group. When the C1-C60heteroaryl group and the C1-C60heteroarylene group each independently include two or more rings, the respective two or more rings may be condensed with each other. The term “monovalent non-aromatic condensed polycyclic group” as used herein refers to a monovalent group having two or more rings condensed to each other, only carbon atoms (for example, having 8 to 60 carbon atoms) as ring-forming atoms, and no aromaticity in its molecular structure when considered as a whole. Non-limiting examples of the monovalent non-aromatic condensed polycyclic group are an indenyl group, a fluorenyl group, a spiro-bifluorenyl group, a benzofluorenyl group, an indenophenanthrenyl group, and/or an indenoanthracenyl group. The term “divalent non-aromatic condensed polycyclic group” as used herein refers to a divalent group having the same structure as a monovalent non-aromatic condensed polycyclic group. The term “monovalent non-aromatic condensed heteropolycyclic group” as used herein refers to a monovalent group having two or more rings condensed to each other, at least one heteroatom other than carbon atoms (for example, having 1 to 60 carbon atoms), as a ring-forming atom, and no aromaticity in its molecular structure when considered as a whole. Non-limiting examples of the monovalent non-aromatic condensed heteropolycyclic group are a 9,10-dihydroacridinyl group and/or 9H-xanthenyl group. The term “divalent non-aromatic condensed heteropolycyclic group” as used herein refers to a divalent group having the same structure as a monovalent non-aromatic condensed heteropolycyclic group. The term “C6-C60aryloxy group” as used herein refers to a monovalent group represented by —OA102(wherein A102is the C6-C60aryl group), and the term “C6-C60arylthio group” as used herein refers to a monovalent group represented by —SA103(wherein A103is the C6-C60aryl group). The term “R10a” as used herein refers to: deuterium, —F, —Cl, —Br, —I, a hydroxyl group, a cyano group, or a nitro group; a C1-C60alkyl group, a C2-C60alkenyl group, a C2-C60alkynyl group, or a C1-C60alkoxy group, each unsubstituted or substituted with deuterium, —F, —Cl, —Br, —I, a hydroxyl group, a cyano group, a nitro group, a C3-C60carbocyclic group, a C1-C60heterocyclic group, a C6-C60aryloxy group, a C6-C60arylthio group, —Si(Q11)(Q12)(Q13), —N(Q11)(Q12), —B(Q11)(Q12), —C(═O)(Q11), —S(═O)2(Q11), —P(═O)(Q11)(Q12), or any combination thereof; a C3-C60carbocyclic group, a C1-C60heterocyclic group, a C6-C60aryloxy group, or a C6-C60arylthio group, each unsubstituted or substituted with deuterium, —F, —Cl, —Br, —I, a hydroxyl group, a cyano group, a nitro group, a C1-C60alkyl group, a C2-C60alkenyl group, a C2-C60alkynyl group, a C1-C60alkoxy group, a C3-C60carbocyclic group, a C1-C60heterocyclic group, a C6-C60aryloxy group, a C6-C60arylthio group, —Si(Q21)(Q22)(Q23), —N(Q21)(Q22), —B(Q21)(Q22), —C(═O)(Q21), —S(═O)2(Q21), —P(═O)(Q21)(Q22), or any combination thereof; or —Si(Q31)(Q32)(Q33), —N(Q31)(Q32), —B(Q31)(Q32), —C(═O)(Q31), —S(═O)2(Q31), or —P(═O)(Q31)(Q32), wherein Q11to Q13, Q21to Q23, and Q31to Q33may each independently be: hydrogen; deuterium; —F; —Cl; —Br; —I; a hydroxyl group; a cyano group; a nitro group; C1-C60alkyl group; C2-C60alkenyl group; C2-C60alkynyl group; C1-C60alkoxy group; or a C3-C60carbocyclic group or a C1-C60heterocyclic group, each unsubstituted or substituted with deuterium, —F, a cyano group, a C1-C60alkyl group, a C1-C60alkoxy group, a phenyl group, a biphenyl group, or any combination thereof. The term “heteroatom” as used herein refers to any atom other than a carbon atom. Non-limiting examples of the heteroatom are O, S, N, P, Si, B, Ge, Se, and/or any combination thereof. The term “Ph” as used herein refers to a phenyl group, the term “Me” as used herein refers to a methyl group, the term “Et” as used herein refers to an ethyl group, the term “ter-Bu” or “But” as used herein refers to a tert-butyl group, and the term “OMe” as used herein refers to a methoxy group. The term “biphenyl group” as used herein refers to “a phenyl group substituted with a phenyl group”. For example, the “biphenyl group” may be a substituted phenyl group having a C6-C60aryl group as a substituent. The term “terphenyl group” as used herein refers to “a phenyl group substituted with a biphenyl group”. For example, the “terphenyl group” may be a substituted phenyl group having, as a substituent, a C6-C60aryl group substituted with a C6-C60aryl group. * and *′ as used herein, unless defined otherwise, each refer to a binding site to a neighboring atom in a corresponding formula. Hereinafter, a quantum dot composition and a light-emitting device according to embodiments will be described in more detail with reference to Examples. EXAMPLES Preparation Example 1: Preparation of Surface-Modified Quantum Dot Dispersion Solution 0.5 g of quantum dots having core/shell compositions (core: InP/shell: ZnSe/ZnS) were dissolved in 10 g of 1-octadecene, 0.2 g of Ligand A was added thereto. Then, the reaction was allowed to proceed for 0.5 hours, while raising and maintaining the temperature at 100° C. in the nitrogen atmosphere, thereby preparing surface-modified first quantum dots. Surface-modified second quantum dots were prepared in substantially the same manner as described above, except that Ligand A was changed to dodecanthiol. Subsequently, 1.5 g of the surface-modified first quantum dots and 1.5 g of the surface-modified second quantum dots were dispersed in 100 g of a mixed solvent of cyclohexylbenzene and n-hexadecane (at a weight ratio 1:1), thereby eventually preparing a surface-modified quantum dot dispersion solution having a solid content concentration of 3 wt %. Preparation Example 2: Preparation of Quantum Dot Dispersion Solution 3.0 g of the surface-modified first quantum dots was dispersed in 100 g of a solvent of cyclohexylbenzene, thereby preparing a quantum dot dispersion solution having a solid content concentration of 3 wt %. Evaluation Example 1: Evaluation of Electron and Hole Concentrations A light-emitting device having the following composition and thickness was used as a light-emitting device of Example 1 for simulation evaluation: ITO anode (125 nm)/PEDOT:PSS hole injection layer (140 nm)/TFB hole transport layer (40 nm)/first emission layer (first quantum dots) (10 nm)/second emission layer (second quantum dots) (10 nm)/ZnO electron transport layer (70 nm)/LiQ:Ag cathode (5 nm:100 nm). A light-emitting device having the following composition and thickness was used as a light-emitting device of Comparative Example 1 for simulation evaluation: ITO anode (125 nm)/PEDOT:PSS hole injection layer (140 nm)/TFB hole transport layer (40 nm)/emission layer (first quantum dots) (20 nm)/ZnO electron transport layer (70 nm)/LiQ:Ag cathode (5 nm:100 nm). To evaluate electron concentration and hole concentration according to the distance from the anode in the light-emitting devices of Example 1 and Comparative Example 1, a simulation program to which an interface model was applied was used as a method of confirming a charge concentration level in the emission layer. Results thereof are each shown inFIGS.2and3. Referring toFIGS.2and3, it was confirmed that the light-emitting device of Example 1 had, compared to the light-emitting device of Comparative Example 1, an increased electron concentration in a region of the emission layer close to the electron transport layer and an increased hole concentration in a region of the emission layer close to the hole transport layer. That is, it can be understood that, by forming an emission layer as a double-layered emission layer according to the present embodiments, the holes and electrons can be smoothly injected into the emission layer based on the appropriately (or suitably) adjusted HOMO and LUMO energy levels of the emission layer. However, the mechanism of the present disclosure is not limited thereto. Evaluation Example 2: J-V Curve Evaluation As a light-emitting device of Example 1, a light-emitting device having the following composition and thickness was prepared using the quantum dot dispersion solution of Preparation Example 1, and was used for evaluation: ITO (125 nm)/PEDOT:PSS (140 nm)/TFB (40 nm)/first emission layer (first quantum dots) (10 nm)/second emission layer (second quantum dots) (10 nm)/ZnO (70 nm)/LiQ:Ag (5 nm:100 nm). As a light-emitting device of Comparative Example 1, a light-emitting device having the following composition and thickness was prepared using the quantum dot dispersion solution of Preparation Example 2, and was used for evaluation: ITO (125 nm)/PEDOT:PSS (140 nm)/TFB (40 nm)/emission layer (first quantum dots) (20 nm)/ZnO (70 nm)/LiQ:Ag (5 nm:100 nm). The current density according to the voltage of each device was evaluated, and results are shown in J-V curves inFIG.4. As shown inFIG.4, it was confirmed that the current density of the light-emitting device of Example 1 increased when driving at the same voltage, compared to the light-emitting device of Comparative Example 1. That is, it can be understood that, by forming an emission layer as a double-layered emission layer according to the present embodiments, using, for example, the quantum dot dispersion solution of Preparation Example 1, the holes and electrons can be smoothly injected in to the emission layer based on the appropriately (or suitably) adjusted HOMO and LUMO energy levels of the emission layer. However, the mechanism of the present disclosure is not limited thereto. According to the one or more embodiments, a quantum dot composition may have improved dispersibility of quantum dots in a solvent. When manufacturing a light-emitting device using the quantum dot composition, a double-layered emission layer may be formed by a single process, thereby simplifying a process step. It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present disclosure as defined by the following claims and their equivalents. | 136,138 |
11859117 | EMBODIMENTS OF THE PRESENT DISCLOSURE In order to make the purpose, technical solutions, and advantages of the present application more clear, the following describes the present application in further detail with reference to the drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the application, and are not used to limit the application. In the description of the present application, it should be understood that the terms “first” and “second” are used for description purposes only, and cannot be understood as indicating or implying relative importance or implicitly specifying the number of the indicated technical features. Thus, the features defined as “first” and “second” may explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of “plurality” is two or more, unless otherwise specifically limited. In the first aspect, as shown inFIG.1, the embodiments of the present application provide a method for preparing core-shell structure QDs, including the following steps:S01, providing initial QD cores, mixing the initial QD cores with an organic carboxylic acid, so that the organic carboxylic acid is bonded to the surface of the initial QD cores;S02, preparing a shell layer on the surface of the initial QD cores, where the step of preparing the shell layer on the surface of the initial QD cores is performed in a shell-growth reaction system containing the organic carboxylic acid;S03, mixing and heating the solution system, obtained after the completion of the shell-layer growth reaction, with an organic amine;Or, mixing and heating the solution system, obtained after the completion of the shell-layer growth reaction, with an organic phosphine;Or, mixing and heating the solution system, obtained after the completion of the shell-layer growth reaction, with a mixed solution of an organic amine and an organic phosphine. According to the method for preparing QDs provided in the examples of the present application, the initial QD cores are mixed with an organic carboxylic acid, and the organic carboxylic acid tends to bind to the surface of the cations of the initial QD cores, such that the organic carboxylic acid is bonded to the surface of the initial QD cores to fill the cationic vacancies of the QD cores, thereby reducing the defect states at the interface between the core and the shell, and providing a desired epitaxial interface for the growth of the shell layer. At the same time, the organic carboxylic acid can also have the effect of passivating the surface of the QD cores, so that the QD cores will not self-mature in the stage of heating up to the shell-growth temperature, and thus QDs with uniform particle size are obtained. In the subsequent shell-growth process, the organic ligands obtained after pyrolysis of the shell-source anionic precursor and the shell-source cationic precursor, together with the organic carboxylic acid in the shell-growth reaction system, are bonded to the surface of the shell layer, making the prepared core-shell structure QDs have desired monodispersity. After the growth of the shell layer is completed, the system obtained after the completion of the shell-layer growth reaction is further mixed with at least one of an organic phosphine and/or an organic amine for subsequent treatment. Here, when the system obtained after the completion of the shell-layer growth reaction is mixed with an organic phosphine for subsequent treatment, the organic phosphine is bonded to the non-metallic elements on the surface of the nanocrystalline shell layer to passivate the anionic vacancies, and thus reduce the defect states on the surface of the core-shell nanocrystals and further increase the fluorescence intensity of the core-shell structure QDs; when the system obtained after the completion of the shell-layer growth reaction is mixed with an organic amine for subsequent treatment, the organic amine can complex with the residual cationic precursor in the mixed solution of the core-shell structure QDs, thereby reducing the freezing point of the cationic precursor, and thus further conducive to the subsequent cleaning of the QDs and the improvement of the purity. Therefore, when used to form a device film layer, the prepared QDs can effectively avoid the influence of the residual cationic precursor impurities in the solution of the core-shell structure QD on the stability of the device, and improve the film-forming quality of the QD solid films. For example, in an embodiment of step S01, the initial QD cores may be at least one selected from but not limited to group II/VI QD cores, group III/V QD cores, group III/VI QD cores, and group II/III/VI QD cores. As an example, the group II/VI QD cores may be selected from but not limited to CdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, HgS, HgSe, HgTe, CdZnSe, CdSSe, ZnSSe, ZnCdS, ZnCdSe, ZnSeS, ZnCdTe, ZnCdSSe, ZnCdSeS, and ZnCdTeS; the group III/V QD cores may be selected from InAs, InP, GaAs, GaP, GaSb, InSb, AlAs, AlP, AlSb, InGaAs, GaAsP and InAsP, but not limited to thereto; as an example, the group III/VI QD cores may be selected from but not limited to InS, In2S3, InSe, In2Se3, In4Se3, In2Se3, InTe, In2Se3, GaS, Ga2Se3, GaSe, Ga2Se3, GaTe, Ga2Te3; the group QD cores may be selected from but not limited to CuInS, CuInZnS, and CuInSeS. In some embodiments, the initial QD cores may be selected from group II/VI QD cores. In some embodiments, the initial QD cores are initial QD cores containing surface ligands. The surface ligand may be at least one selected from an organic carboxylic acid ligand, an organic phosphonic acid ligand, an organic phosphine ligand, and a phosphine oxide ligand. For example, the organic carboxylic acid ligand may be selected from but not limited to at least one of oleic acid, tetradecanoic acid, and dodecanoic acid; the organic phosphonic acid ligand may be selected from but not limited to at least one of octadecylphosphonic acid, tetradecylphosphonic acid, and dodecylphosphonic acid; the organic phosphine ligand may be selected from but not limited to at least one of trioctylphosphine and tributylphosphine; and the phosphine oxide ligand may be selected from but not limited to at least one of trioctylphosphine oxide and tributylphosphine oxide. In step S01, the initial QD cores are mixed with an organic carboxylic acid, and the organic carboxylic acid tends to bind to the surface of the cations of the initial QD cores, such that the organic carboxylic acid is bonded to the surface of the initial QD cores to fill the cationic vacancies of the QD cores, thereby reducing the defect states at the interface between the core and the shell, and providing a desired epitaxial interface for the growth of the shell layer. At the same time, the organic carboxylic acid can also have the effect of passivating the surface of the QD cores, so that the QD cores will not self-mature in the stage of heating up to the shell-growth temperature, and thus QDs with uniform particle size are obtained. In the subsequent shell-growth process, the organic ligands obtained after pyrolysis of the shell-source anionic precursor and the shell-source cationic precursor, together with the organic carboxylic acid in the shell-growth reaction system, are bonded to the surface of the shell layer, making the prepared core-shell structure QDs have desired monodispersity. In some embodiments, the organic carboxylic acid may be selected from organic carboxylic acids having 8 to 18 carbon atoms. At this time, it has a relatively small steric hindrance, which facilitates the binding of the organic carboxylic acid to the surface of the initial QD cores. Further, the organic carboxylic acid may be selected from linear organic carboxylic acids containing a single carboxyl group. The linear organic carboxylic acids are beneficial to reducing steric hindrance and promoting the occurrence of passivation. For example, the organic carboxylic acid may be at least one selected from oleic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, and octadecanoic acid. In step S01, in some embodiments, in order to facilitate the organic carboxylic acid to be sufficiently and stably bonded to the surface of the initial QD cores, in the step of mixing the initial QD cores with the organic carboxylic acid, the mixing conditions are as follows: the initial QD cores are mixed with the organic carboxylic acid, and heated at a temperature condition of 80˜150° C. for 20˜60 minutes to bond the organic carboxylic acid to the surface of the initial QD cores. In step S01, in some embodiments, the initial QD cores may be formulated into a solution and mixed with the organic carboxylic acid. In some embodiments, in order to make the initial QD cores uniformly dispersed in the solvent (the initial QD cores are individually present in the solution and maintain a proper distance from each other), provide a desired condition for the growth of the shell layer on the surface of the QD cores, and obtain a shell layer with desired dispersion and a uniform thickness, in the initial QD core solution, the ratio of the mass of the initial QD cores to the volume of the solvent may be 10 mg:(5˜15) ml. In step S01, in some embodiments, in the step of mixing the initial QD cores with the organic carboxylic acid, according to a mass-molar ratio of 10 mg:(3˜10) mmol between the QD cores and the organic carboxylic acid, the initial QD cores may be dispersed into a solution containing the organic carboxylic acid for surface modification of the initial QD cores. In order to ensure that the organic carboxylic acid is fully bonded to the initial QD cores and reduce the defect states on the surface of the initial QD cores, the organic carboxylic acid reagent may have a certain amount of excess. However, the excessive amount of the organic carboxylic acid reagent may not be too much, otherwise the viscosity may be too large, which may affect the subsequent shell-layer growth rate, and may be detrimental to the formation of the shell layer. In one embodiment, in step S02, the step of preparing the shell layer on the surface of the initial QD cores may be performed in a shell-growth reaction system containing the organic carboxylic acid. For example, in one embodiment, when the organic carboxylic acid added in step S01is excessive, the organic carboxylic acid in the shell-growth reaction system may come from step S01, that is, the initial QD cores may be mixed with the organic carboxylic acid such that the remaining materials after the step of bonding the organic carboxylic acid to the surface of the initial QD cores may include the organic carboxylic acid; when the organic carboxylic acid added in step S01is not excessive, or although the organic carboxylic acid added in step S01is excessive, the organic carboxylic acid becomes insufficient as the growth of the shell layer proceeds, adding an appropriate amount of an organic carboxylic acid to the shell-growth reaction system may also be possible during the process of preparing the shell layer on the surface of the initial QD cores, such that a sufficient amount of carboxylic acid may be bonded to the surface of the growing shell layer, making the prepared QDs have desired monodispersity. Of course, it should be noted that a specific kind of shell-source anionic precursor (e.g., a complex precursor formed by a non-metallic element. such as Te, Se, S, P, etc. and an oleic acid) and shell-source cationic precursor (zinc oleate, cadmium oleate, etc.), which may produce organic carboxylic acid ligands after pyrolysis, may be selected. It is precisely because this part of the organic ligands produced after pyrolysis are insufficient for fully modifying (especially as the thickness of the shell layer increases) the surface of the growing shell layer, the growth of the shell layer may need to be performed in a shell-growth reaction system that contains an organic carboxylic acid. In the shell-growth reaction system that contains the organic carboxylic acid, the organic carboxylic acid may come from the organic carboxylic acid remained after the step of mixing the initial QD cores with the organic carboxylic acid to bond the organic carboxylic acid to the surface of the initial QD cores, and/or an appropriate amount of the organic carboxylic acid added into the shell-growth reaction system during the shell-growth process. In one embodiment, in step S02, the shell-growth reaction system may refer to a reaction material system applied in the process of growing a shell layer on the surface of the initial QD cores. In one embodiment of the present application, the shell-source precursor may be injected once into the solution containing the initial QD cores for the growth of a shell layer. In another embodiment of the present application, the shell-source precursor may be injected multiple times into the solution containing the initial QD cores or the shell-growth solution system for the growth of multiple shell layers. For example, a shell-source precursor may be added to the initial QD cores for a first shell growth to prepare a first shell layer; further, on the basis of the first shell layer, a shell-source precursor may be added for a second shell growth to prepare a second shell layer on the surface of the first shell layer; and in this way, after N times of shell growth, an Nthshell layer may be prepared. In this embodiment, the surface of each shell layer is combined with the organic carboxylic acid in the shell-growth reaction system and the organic ligands after the pyrolysis of the shell-source anionic precursor and the shell-source cationic precursor, such that after preparing and obtaining each shell layer, the material may have desired monodispersity, which may be conducive to the subsequent growth of the shell layer or having desired dispersion performance as a product application. In the embodiments of the present application, the shell-source precursor may include a shell-source cationic precursor and a shell-source anionic precursor. Here, the shell-source cationic precursor may be at least one of organic metal carboxylates formed from oxides or metal salts of metals, such as Cd, Zn, Pb, Ag, Hg, Fe, In, Al, etc., and an organic carboxylic acid. Further, the shell-source cationic precursor may be at least one selected from zinc oleate, lead oleate, silver oleate, mercury oleate, indium oleate, copper oleate, iron oleate, manganese oleate, aluminum oleate, zinc stearate, lead stearate, silver stearate, mercury stearate, indium stearate, copper stearate, iron stearate, manganese stearate, aluminum stearate, zinc tetradecanoate, lead tetradecanoate, silver tetradecanoate, mercury tetradecanoate, indium tetradecanoate, copper tetradecanoate, iron tetradecanoate, manganese tetradecanoate, aluminum tetradecanoate, zinc hexadecanoate, lead hexadecanoate, silver hexadecanoate, mercury hexadecanoate, indium hexadecanoate, copper hexadecanoate, iron hexadecanoate, manganese hexadecanoate, aluminum hexadecanoate, zinc dodecanoate, lead dodecanoate, silver dodecanoate, mercury dodecanoate, indium dodecanoate, copper dodecanoate, iron dodecanoate, manganese dodecanoate, aluminum dodecanoate, zinc octadecanoate, lead octadecanoate, silver octadecanoate, mercury octadecanoate, indium octadecanoate, copper octadecanoate, iron octadecanoate, manganese octadecanoate, and aluminum octadecanoate, but not limited thereto. In the embodiments of the present application, after dispersing non-metallic elements such as Te, Se, S, P, etc. into organic molecules to form an anionic complex, the shell-source anionic precursor may be prepared. When the shell-source anionic precursor is an anionic complex formed by non-metallic elements such as Te, Se, S, P, etc. and organic molecules, the organic molecules may be at least one selected from trioctylphosphine, tributylphosphine, oleic acid, and octadecene, but not limited thereto. In the embodiments of the present application, when the anionic precursor is a mercaptan, the organic molecule of the non-metal atom may be an organic molecule containing a single functional group, e.g., the thiol (—HS) functional group (such as octadecanethiol, heptadecanethiol, hexadecanethiol, pentadecanethiol, tetradecanethiol, tridecanethiol, dodecanethiol, octanethiol, etc. but not limited to thereto). In the embodiments of the present application, the selection of the shell source is not limited. In some embodiments, the band gap of the obtained shell layer may be greater than the band gap of the initial QD cores. In some embodiments of the present application, the shell-source cationic precursor may be at least one selected from organometallic carboxylates of Cd, Zn, and Pb, and the shell-source anionic precursor may be selected from anionic complexes or thiols formed by dispersing the elements of Te, Se and S into organic molecules. In the embodiments of the present application, each time the shell source is injected for shell growth, the order of adding the shell-source cationic precursor and the shell-source anionic precursor is not strictly limited. For example, the shell source is a mixed precursor solution in which a shell-source cationic precursor and a shell-source anionic precursor are dispersed; the method of adding the shell source may include: injecting the cationic precursor and the anionic precursor into solvents to respectively prepare a cationic precursor solution and an anionic precursor solution, and injecting the shell-source cationic precursor solution first and then injecting the shell-source anionic precursor solution; or, injecting the cationic precursor and the anionic precursor into solvents to respectively prepare a cationic precursor solution and an anionic precursor solution, and injecting the shell-source anionic precursor solution first and then injecting the shell-source cationic precursor solution; or, injecting the cationic precursor and the anionic precursor into a solvent to prepare a mixed solution containing the cationic precursor and the anionic precursor, and injecting the mixed solution into the solution containing the initial QD cores or the shell-growth solution system. In some embodiments, the concentration range of the shell-source cationic precursor solution may be (0.5˜1.5) mmol/ml; the concentration range of the shell-source anionic precursor solution may be (0.5˜1.5) mmol/ml. Proper concentrations may be conducive to the uniform bonding of shell-source cationic precursor and shell-source anionic precursor on the surface of the initial QD cores to form a uniform and stable shell layer through crystallization. In some embodiments, according to a mass ratio of (1˜1.5) mmol:10 mg between the shell-source cationic precursor and the initial QD cores, and/or a mass ratio of (1 1.5) mmol:10 mg between the shell-source anionic precursor and the initial QD cores, the shell-source precursors may be injected into the solution containing the initial QD cores or the shell-growth solution system. The method is conducive to uniform and stable bonding of the anionic precursor and the cationic precursor on the surface of the initial QD cores, and obtaining a shell layer with an appropriate thickness. Further, the temperature for preparing the shell layer on the surface of the initial QD cores after the modification treatment may be 150˜320° C. The temperature range is conducive to crystallization of the anionic and cationic precursors into shells, and does not affect the stability of the QDs. In step S03, in one embodiment, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with an organic phosphine, such that the organic phosphine may be bonded to the non-metal atoms on the surface of the shell layer of the QDs to reduce the defect states on the surface of the core-shell nanocrystals and further increase the fluorescence intensity of the core-shell structure QDs. In step S03, in one embodiment, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with an organic phosphine, and heated at a temperature condition of 100˜320° C. for 10˜60 minutes. Under the condition, the organic phosphine may be bonded to the non-metal atoms on the surface of the shell layer of the QDs to reduce the defect states on the surface of the core-shell nanocrystals and further increase the fluorescence intensity of the core-shell structure QDs. To mix the organic phosphine with the solution system obtained after the completion of the shell-layer growth reaction, when the treatment temperature is too low and/or the time is too short, the organic phosphine may not have a significant effect on passivating the anionic vacancies, and may even not play any passivating role at all, and thus may not be able to increase the fluorescence intensity of the core-shell structure nanocrystals; To mix the organic phosphine with the solution system obtained after the completion of the shell-layer growth reaction, when the treatment temperature is too high, not only the organic phosphine may be easily volatilized, thereby affecting the modification treatment effect, but high temperature condition may affect the structural stability of the core-shell nanocrystals. In step S03, in the step of mixing and heating the system obtained after the completion of the shell-layer growth reaction with the organic phosphine, in some embodiments, according to a molar-mass ratio of (2˜5) mmol:10 mg between the organic phosphine and the initial QD cores, the core-shell structure QDs may be dispersed into a solution containing the organic phosphine. When the content of the organic phosphine is too low, the effect of passivating anionic vacancies may not be significant, and thus it may be difficult to significantly increase the fluorescence intensity of the core-shell structure QDs. When the content of the organic phosphine is too high, it may affect the film-forming performance of the core-shell structure nanocrystals when preparing the film layer. In the step S03, in one embodiment, the system obtained after the completion of the shell-layer growth reaction may be mixed with an organic amine, such that the organic amine can be complexed with the shell-source cationic precursor that remains in the solution system after the completion of the shell-layer growth reaction, thereby reducing the freezing point of the shell-source cationic precursor remaining in the solution system, which is beneficial to the subsequent cleaning of the QD mixture and the improvement of the purity. Therefore, when used to prepare device film layers, the prepared QDs may effectively avoid the influence of the residual cationic precursor impurities in the core-shell structure QD solution on the device stability, and thus improve the film-forming quality of the QD solid films. In step S03, in one embodiment, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with an organic amine, and heated at a temperature condition of 80˜320° C. for 10˜60 minutes. Under the condition, the organic amine may be bonded to the non-metal atoms on the surface of the shell layer of the QDs to reduce the freezing point of the shell-source cationic precursor remaining in the solution system, thereby improving the purity of the core-shell structure QDs. To mix the solution system obtained after the completion of the shell-layer growth reaction and the organic amine, when the temperature is too low and/or the time is too short, the effect of the organic amine to complex the remaining cationic precursor may not be significant, and thus the purity of the core-shell structure QDs may not be improved; when the temperature is too high and/or the time is too long, the high temperature condition may affect the structural stability of the core-shell structure QDs, causing phenomena such as ligand shedding, etc. In step S03, in the step of mixing the system obtained after the completion of the shell-layer growth reaction with the organic amine to bond the organic amine to the surface of the shell layer, in some embodiments, according to a molar-mass ratio of (5˜10) mmol:10 mg between the organic amine and the initial QD cores, the core-shell structure QDs may be dispersed into a solution containing the organic amine. When the content of the organic amine is too low, the effect of improving the purity of the core-shell structure QDs may not be significant. When the content of the organic amine is too high, the remaining organic amine after complexing with the residual cationic precursor in the mixed liquid with the core-shell structure QDs may exchange with the ligands on the surface of the core-shell structure QDs. The organic amine ligands are unstable (the organic amine ligands that are exchanged may be removed during the cleaning process), and easy to fall off. Therefore, defects may be introduced to the fall-off positions and thus reduce the photo-thermal stability, fluorescence intensity, and solubility of the core-shell structure QDs. For example, in some embodiments, the organic amine used as a post-treatment reagent may be an organic amine having 8 to 18 carbon atoms. Further, the organic amine reagent may be selected from linear organic amines containing a single amino group. The linear organic amines are beneficial to reducing steric hindrance and promoting organic amine to be bonded to the surface of the shell layer. For example, the organic amine reagent may be at least one selected from oleylamine, trioctylamine, dodecylamine, tetradecylamine, hexadecylamine, and octadecylamine. In step S03, in one embodiment, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with a mixed solution of an organic amine and an organic phosphine, so that the organic phosphine and the organic amine are bonded to the non-metal atoms on the surface of the shell layer of the QDs to increase the fluorescence intensity and purity of the core-shell structure QDs. In step S03, in one embodiment, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with a mixed solution of an organic amine and an organic phosphine, and heated at a temperature condition of 80˜320° C. for 10˜90 minutes. Under the condition, the organic amine and the organic phosphine may be bonded to the non-metal atoms on the surface of the shell layer of the QDs to improve the purity and fluorescence intensity of the core-shell structure QDs. To mix the solution system obtained after the completion of the shell-layer growth reaction with the mixed solution of the organic amine and the organic phosphine, when the temperature is too low and/or the time is too short, the effect of the organic amine and the organic phosphine to complex the remaining cationic precursor may not be significant, and thus the purity and fluorescence intensity of core-shell structure QDs may not be improved; when the temperature is too high and/or the time is too long, the high temperature condition may affect the structural stability of the core-shell structure QDs, causing phenomena such as ligand shedding, etc. In step S03, in the step of mixing and heating the solution system obtained after the completion of the shell-layer growth reaction with the mixed solution of the organic amine and the organic phosphine, according to a molar-mass ratio of (5˜10) mmol:10 mg between the organic amine and the initial QD cores and a molar-mass ratio of (2˜5) mmol:10 mg between the organic phosphine and the initial QD cores, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with the mixed solution of the organic amine and the organic phosphine. The influence of the content of the organic phosphine and the organic amine may be referred to the description provided above. In the second aspect, as shown inFIG.2, the embodiments of the present application provide a method for preparing QD core-shell structure nanocrystals, including the following steps:E01, providing initial QD cores, mixing the initial QD cores with an organic amine, so that the organic amine is bonded to the surface of the initial QD cores;E02, performing a shell-layer growth reaction on the surface of the initial QD cores to prepare a shell layer;E03, mixing and heating the solution system, obtained after the completion of the shell-layer growth reaction, with an organic carboxylic acid;Or, mixing and heating the solution system, obtained after the completion of the shell-layer growth reaction, with an organic phosphine;Or, mixing and heating the solution system, obtained after the completion of the shell-layer growth reaction, with a mixed solution of an organic carboxylic acid and an organic phosphine. According to the method for preparing QDs provided in the examples of the present application, the initial QD cores are mixed with an organic amine, and the organic amine is bonded to the surface of the initial QD cores to fill the cationic vacancies of the initial QD cores, thereby reducing the defect states at the interface between the core and the shell, and providing a desired epitaxial interface for the growth of the shell layer. Further, because the binding force between the organic amine and the metal atoms on the surface of the QD cores is relatively weak, the organic amine requires less energy to be desorbed from the metal atoms on the surface of the previous shell layer. During the subsequent shell-growth process, the anions in the shell-source precursor are easier to be bonded to the metal ions on the surface of the cores for epitaxial growth, which may avoid the large lattice stress between the atoms at the interface between the QD cores and the shell layer, thereby reducing the presence of lattice defects on the surface of the epicrystalline shell layer. In addition, due to the dipole effect of the amino functional group of the organic amine, the shell layer is driven to grow according to the crystal orientations of the QD cores during epitaxial crystallization, such that the shell layer obtained by the shell growth is consistent with the crystal form of the QD cores, which further reduces the lattice defects between the atoms on the surface of the QD cores and the shell layer. After the growth of the shell layer is completed, the system obtained after the completion of the shell-layer growth reaction is further mixed with at least one of an organic phosphine and/or an organic carboxylic acid for subsequent treatment. Here, when the system obtained after the completion of the shell-layer growth reaction is mixed with an organic phosphine for subsequent treatment, the organic phosphine is bonded to the non-metallic elements on the surface of the nanocrystalline shell layer to passivate the anionic vacancies, and thus reduce the defect states on the surface of the core-shell nanocrystals and further increase the fluorescence intensity of the core-shell structure QDs; when the system obtained after the completion of the shell-layer growth reaction is mixed with an organic carboxylic acid for subsequent treatment, the organic carboxylic acid can effectively eliminate the protonated organic amine connected to the surface of the core-shell structure nanocrystalline shell layer (in the process of modifying the QD cores with an organic amine, a portion of the organic amine falls off and, in the subsequent shell-growth process, binds to the surface of the growing metal atoms. Although most of the organic amine is removed during the shell-growth process, a portion of the organic amine is still bonded to the surface of the metal atoms of the shell layer without being removed from the surface; the portion of the organic amine that has not fallen off will eventually form a protonated organic amine), thereby reducing the charged organic amine ligands on the surface of the core-shell structure nanocrystals, which further reduces the excitons (electrons) generated by the core-shell structure nanocrystals when emitting light being trapped by the charged organic amine ligands on the surface. Therefore, the effect of this post-treatment is to further improve the transient fluorescence lifetime of the core-shell structure nanocrystals. For example, in an embodiment of step E01, the initial QD cores may be at least one selected from but not limited to group II/VI QD cores, group III/V QD cores, group III/VI QD cores, and group II/III/VI QD cores. As an example, the group II/VI QD cores may be selected from but not limited to CdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, HgS, HgSe, HgTe, CdZnSe, CdSSe, ZnSSe, ZnCdS, ZnCdSe, ZnSeS, ZnCdTe, ZnCdSSe, ZnCdSeS, and ZnCdTeS; the group III/V QD cores may be selected from InAs, InP, GaAs, GaP, GaSb, InSb, AlAs, AlP, AlSb, InGaAs, GaAsP and InAsP, but not limited to thereto; as an example, the group III/VI QD cores may be selected from but not limited to InS, In2S3, InSe, In2Se3, In4Se3, In2Se3, InTe, In2Se3, GaS, Ga2Se3, GaSe, Ga2Se3, GaTe, Ga2Te3; the group IUIIUVI QD cores may be selected from but not limited to CuInS, CuInZnS, and CuInSeS. In some embodiments, the initial QD cores may be selected from group II/VI QD cores. In some embodiments, the initial QD cores are initial QD cores containing surface ligands. The surface ligand may be at least one selected from an organic carboxylic acid ligand, an organic phosphonic acid ligand, an organic phosphine ligand, and a phosphine oxide ligand. For example, the organic carboxylic acid ligand may be selected from but not limited to at least one of oleic acid, tetradecanoic acid, and dodecanoic acid; the organic phosphonic acid ligand may be selected from but not limited to at least one of octadecylphosphonic acid, tetradecylphosphonic acid, and dodecylphosphonic acid; the organic phosphine ligand may be selected from but not limited to at least one of trioctylphosphine and tributylphosphine; and the phosphine oxide ligand may be selected from but not limited to at least one of trioctylphosphine oxide and tributylphosphine oxide. In step E01, the initial QD cores are mixed with an organic amine, and the organic amine tends to bind to the surface of the cations of the initial QD cores, such that the organic amine is bonded to the surface of the initial QD cores to fill the cationic vacancies of the initial QD cores, thereby reducing the defect states at the interface between the core and the shell, and reducing the presence of lattice defects on the surface of the epicrystalline shell layer. In some embodiments, the organic amine may be selected from organic amines having 8 to 18 carbon atoms. In this case, the organic amine may have a relatively small steric hindrance, which facilitates the bonding of the organic amine to the surface of the initial QD cores. In some embodiments, the organic amine may be selected from linear organic amines containing a single amino group. The linear organic amines are beneficial to reducing steric hindrance and promoting the occurrence of modification. For example, the organic amine reagent may be at least one selected from oleylamine, trioctylamine, dodecylamine, tetradecylamine, hexadecylamine, and octadecylamine. In step E01, in some embodiments, in order to facilitate the organic amine to be sufficiently and stably bonded to the surface of the initial QD cores, in the step of mixing the initial QD cores with the organic amine, the mixing conditions are as follows: the initial QD cores are mixed with the organic amine, heated at a temperature condition of 80˜150° C. for 20˜60 minutes to bond the organic amine to the surface of the initial QD cores. In step E01, in some embodiments, the initial QD cores may be formulated into a solution and mixed with the organic amine. In some embodiments, in order to make the initial QD cores uniformly dispersed in the solvent (the initial QD cores are individually present in the solution and maintain a proper distance from each other), provide a desired condition for the growth of the shell layer on the surface of the QD cores, and obtain a shell layer with desired dispersion and a uniform thickness, in the initial QD core solution, the ratio of the mass of the initial QD cores to the volume of the solvent may be 10 mg:(5˜15) ml. In step E01, in some embodiments, in the step of mixing the initial QD cores with the organic amine, according to a mass-molar ratio of 10 mg:(3˜10) mmol between the QD cores and the organic amine, the initial QD cores may be dispersed into a solution containing the organic amine for surface modification of the initial QD cores. In order to ensure that the organic amine is fully bonded to the initial QD cores and reduce the defect states on the surface of the initial QD cores, the organic amine may have a certain amount of excess. However, the excessive amount of the organic amine may not be too much, otherwise the viscosity may be too large, which may affect the subsequent shell-layer growth rate, and may be detrimental to the formation of the shell layer. In step E02described above, the shell-layer growth reaction may be performed on the surface of the initial QD cores for the preparation of the shell layer. In one embodiment of the present application, the shell-source precursor may be injected once into the solution containing the initial QD cores for the growth of a shell layer. In another embodiment of the present application, the shell-source precursor may be injected multiple times into the solution containing the initial QD cores or the shell-growth solution system for the growth of multiple shell layers. For example, a shell-source precursor may be added to the initial QD cores for a first shell growth to prepare a first shell layer; further, on the basis of the first shell layer, a shell-source precursor may be added for a second shell growth to prepare a second shell layer on the surface of the first shell layer; and in this way, after N times of shell growth, an Nthshell layer may be prepared. In this embodiment, the surface of each shell layer is combined with the organic carboxylic acid in the shell-growth reaction system and the organic ligands after the pyrolysis of the shell-source anionic precursor and the shell-source cationic precursor, such that after preparing and obtaining each shell layer, the material may have desired monodispersity, which may be conducive to the subsequent growth of the shell layer or having desired dispersion performance as a product application. In the embodiments of the present application, the shell-source precursor may include a shell-source cationic precursor and a shell-source anionic precursor. Here, the shell-source cationic precursor may be at least one of organic metal carboxylates formed from oxides or metal salts of metals, such as Cd, Zn, Pb, Ag, Hg, Fe, In, Al, etc., and an organic carboxylic acid. Further, the shell-source cationic precursor may be at least one selected from zinc oleate, lead oleate, silver oleate, mercury oleate, indium oleate, copper oleate, iron oleate, manganese oleate, aluminum oleate, zinc stearate, lead stearate, silver stearate, mercury stearate, indium stearate, copper stearate, iron stearate, manganese stearate, aluminum stearate, zinc tetradecanoate, lead tetradecanoate, silver tetradecanoate, mercury tetradecanoate, indium tetradecanoate, copper tetradecanoate, iron tetradecanoate, manganese tetradecanoate, aluminum tetradecanoate, zinc hexadecanoate, lead hexadecanoate, silver hexadecanoate, mercury hexadecanoate, indium hexadecanoate, copper hexadecanoate, iron hexadecanoate, manganese hexadecanoate, aluminum hexadecanoate, zinc dodecanoate, lead dodecanoate, silver dodecanoate, mercury dodecanoate, indium dodecanoate, copper dodecanoate, iron dodecanoate, manganese dodecanoate, aluminum dodecanoate, zinc octadecanoate, lead octadecanoate, silver octadecanoate, mercury octadecanoate, indium octadecanoate, copper octadecanoate, iron octadecanoate, manganese octadecanoate, and aluminum octadecanoate, but not limited thereto. In the embodiments of the present application, after dispersing non-metallic elements such as Te, Se, S, P, etc. into organic molecules to form an anionic complex, the shell-source anionic precursor may be prepared. When the shell-source anionic precursor is an anionic complex formed by non-metallic elements such as Te, Se, S, P, etc. and organic molecules, the organic molecules may be at least one selected from trioctylphosphine, tributylphosphine, oleic acid, and octadecene, but not limited thereto. In the embodiments of the present application, when the anionic precursor is a mercaptan, the organic molecule of the non-metal atom may be an organic molecule containing a single functional group, e.g., the thiol (—HS) functional group (such as octadecanethiol, heptadecanethiol, hexadecanethiol, pentadecanethiol, tetradecanethiol, tridecanethiol, dodecanethiol, octanethiol, etc. but not limited to thereto). In the embodiments of the present application, the selection of the shell source is not limited. In some embodiments, the band gap of the obtained shell layer may be greater than the band gap of the initial QD cores. In some embodiments of the present application, the shell-source cationic precursor may be at least one selected from organometallic carboxylates of Cd, Zn, and Pb, and the shell-source anionic precursor may be selected from anionic complexes or thiols formed by dispersing the elements of Te, Se and S into organic molecules. In the embodiments of the present application, each time the shell source is injected for shell growth, the order of adding the shell-source cationic precursor and the shell-source anionic precursor is not strictly limited. For example, the shell source is a mixed precursor solution in which a shell-source cationic precursor and a shell-source anionic precursor are dispersed; the method of adding the shell source may include: injecting the cationic precursor and the anionic precursor into solvents to respectively prepare a cationic precursor solution and an anionic precursor solution, and injecting the shell-source cationic precursor solution first and then injecting the shell-source anionic precursor solution; or, injecting the cationic precursor and the anionic precursor into solvents to respectively prepare a cationic precursor solution and an anionic precursor solution, and injecting the shell-source anionic precursor solution first and then injecting the shell-source cationic precursor solution; or, injecting the cationic precursor and the anionic precursor into a solvent to prepare a mixed solution containing the cationic precursor and the anionic precursor, and injecting the mixed solution into the solution containing the initial QD cores or the shell-growth solution system. In some embodiments, the concentration range of the shell-source cationic precursor solution may be (0.5˜1.5) mmol/ml; the concentration range of the shell-source anionic precursor solution may be (0.5˜1.5) mmol/ml. Proper concentrations may be conducive to the uniform bonding of shell-source cationic precursor and shell-source anionic precursor on the surface of the initial QD cores to form a uniform and stable shell layer through crystallization. In some embodiments, according to a mass ratio of (1˜1.5) mmol:10 mg between the shell-source cationic precursor and the initial QD cores, and/or a mass ratio of (1 1.5) mmol:10 mg between the shell-source anionic precursor and the initial QD cores, the shell-source precursors may be injected into the solution containing the initial QD cores or the shell-growth solution system. The method is conducive to uniform and stable bonding of the anionic precursor and the cationic precursor on the surface of the initial QD cores, and obtaining a shell layer with an appropriate thickness. In some embodiments, the temperature for preparing the shell layer on the surface of the initial QD cores after the modification treatment may be 150˜320° C. The temperature range is conducive to crystallization of the anionic and cationic precursors into shells, and does not affect the stability of the QDs. In step E03, in one embodiment, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with an organic phosphine, such that the organic phosphine may be bonded to the non-metal atoms on the surface of the shell layer of the QDs to reduce the defect states on the surface of the core-shell nanocrystals and further increase the fluorescence intensity of the core-shell structure QDs. In step E03, in one embodiment, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with an organic phosphine, and heated at a temperature condition of 100˜320° C. for 10˜60 minutes. Under the condition, the organic phosphine may be bonded to the non-metal atoms on the surface of the shell layer of the QDs to reduce the defect states on the surface of the core-shell nanocrystals and further increase the fluorescence intensity of the core-shell structure QDs. To mix the organic phosphine with the solution system obtained after the completion of the shell-layer growth reaction, when the treatment temperature is too low and/or the time is too short, the organic phosphine may not have a significant effect on passivating the anionic vacancies, and may even not play any passivating role at all, and thus may not be able to increase the fluorescence intensity of the core-shell structure nanocrystals; To mix the organic phosphine with the solution system obtained after the completion of the shell-layer growth reaction, when the treatment temperature is too high, not only the organic phosphine may be easily volatilized, thereby affecting the modification treatment effect, but high temperature condition may affect the structural stability of the core-shell nanocrystals. In step E03, in the step of mixing and heating the system obtained after the completion of the shell-layer growth reaction with the organic phosphine, in some embodiments, according to a molar-mass ratio of (2˜5) mmol:10 mg between the organic phosphine and the initial QD cores, the core-shell structure QDs may be dispersed into a solution containing the organic phosphine. When the content of the organic phosphine is too low, the effect of passivating anionic vacancies may not be significant, and thus it may be difficult to significantly increase the fluorescence intensity of the core-shell structure QDs. When the content of the organic phosphine is too high, it may affect the film-forming performance of the core-shell structure nanocrystals when preparing the film layer. In step E03, in one embodiment, the system obtained after the completion of the shell-layer growth reaction may be mixed with an organic carboxylic acid, such that the organic carboxylic acid may be complexed with the shell-source cationic precursor that remains in the solution system after the completion of the shell-layer growth reaction, thereby eliminating the protonated organic amine on the surface of the shell layer of the core-shell structure nanocrystals, and improving the transient fluorescence lifetime of the core-shell structure nanocrystals. In step E03, in one embodiment, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with an organic carboxylic acid, and heated at a temperature condition of 240˜320° C. for 30˜90 minutes. Under the condition, the organic carboxylic acid may be bonded to the non-metal atoms on the surface of the shell layer of the QDs to improve the transient fluorescence lifetime of the core-shell structure nanocrystals. To mix the solution system obtained after the completion of the shell-layer growth reaction with the organic carboxylic acid, when the temperature is too low and/or the time is too short, the effect of the organic carboxylic acid to complex the remaining cationic precursor may not be significant, and thus the transient fluorescence lifetime of the core-shell structure nanocrystals may not be improved; when the temperature is too high and/or the time is too long, the high temperature condition may affect the structural stability of the core-shell structure QDs, causing phenomena such as ligand shedding, etc. In step E03, in the step of mixing the system obtained after the completion of the shell-layer growth reaction with the organic carboxylic acid to bond the organic carboxylic acid to the surface of the shell layer, in some embodiments, according to a molar-mass ratio of (5˜10) mmol:10 mg between the organic carboxylic acid and the initial QD cores, the core-shell structure QDs may be dispersed into a solution containing the organic carboxylic acid. When the content of the organic carboxylic acid is too low, the effect of eliminating protonated organic amine bonded to the surface of the shell layer of the core-shell nanocrystals may not be significant, making it difficult to significantly improve the transient fluorescence lifetime of the nanocrystals. When the content of the organic carboxylic acid is too high, in a case where the obtained QDs are used as a device functional layer such as a QD light-emitting layer, the film-forming performance of the film layer may be degraded, which further affects the light-emitting performance of the device. In some embodiments, the organic acid may be selected from organic acids having 8 to 18 carbon atoms. In some examples, the organic acid reagent may be selected from linear organic acids containing a single carboxylic group. The linear amines are beneficial to reducing steric hindrance and promoting the occurrence of complexation. For example, the organic acid reagent may be at least one selected from oleic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, and octadecanoic acid. In step E03, in one embodiment, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with a mixed solution of an organic carboxylic acid and an organic phosphine, such that the organic phosphine and the organic carboxylic acid may be bonded to the non-metal atoms on the surface of the shell layer of the QDs. The organic phosphine may be coordinately bonded to the non-metal atoms on the surface of the shell layer of the nanocrystals to further passivate the anionic vacancies, thereby reducing the defect states on the surface of the core-shell structure nanocrystals and improving the fluorescence intensity of the core-shell structure nanocrystals. When the organic acid post-processes the core-shell structure nanocrystals, the protonated organic amines bonded to the surface of the shell layer of the core-shell structure nanocrystals may be effectively eliminated, thereby improving the transient fluorescence lifetime of the nanocrystals. At the same time, the organic acid and the organic phosphine may form interlaced ligands on the surface of the core-shell structure nanocrystals and maybe bonded to the metal and non-metal atoms on the surface of the nanocrystals. The interlaced ligands may further improve the solubility and the stability of the nanocrystals. In addition, when the solution system obtained after the completion of the shell-layer growth reaction is mixed with a mixed solution of an organic carboxylic acid and an organic phosphine, the organic carboxylic acid may promote the decomposition of a part of the shell that is unstable in crystallization on the surface of the core-shell QDs. The metal atoms obtained after the decomposition and the organic carboxylic acid may again form a metal cationic precursor, and the anions obtained after the decomposition and the organic phosphine may again form an anionic precursor. Further, the re-formed anionic and cationic precursors in the post-processing process may undergo shell-layer growth again on the surface of the core-shell QDs. When the re-formed shell layer grows, the core-shell QDs with small particles may preferentially grow again due to the relatively large body surface and the fast growth rate, and thus the final effect is that the size of the core-shell QDs may be relatively uniform. In step E03, in one embodiment, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with a mixed solution of an organic carboxylic acid and an organic phosphine, and heated at a temperature condition of 100˜320° C. for 10˜60 minutes. Under the condition, the organic carboxylic acid and the organic phosphine may be bonded to the non-metal atoms on the surface of the shell layer of the QDs to improve the transient fluorescence lifetime and the fluorescence intensity of the core-shell structure QDs. To mix the solution system obtained after the completion of the shell-layer growth reaction with the mixed solution of the organic carboxylic acid and the organic phosphine, when the temperature is too low and/or the time is too short, the effect of the organic carboxylic acid and the organic phosphine to complex the remaining cationic precursor may not be significant, and thus the transient fluorescence lifetime and the fluorescence intensity of core-shell structure QDs may not be improved; when the temperature is too high and/or the time is too long, the high temperature condition may affect the structural stability of the core-shell structure QDs, causing phenomena such as ligand shedding, etc. In step E03, in the step of mixing and heating the solution system obtained after the completion of the shell-layer growth reaction with the mixed solution of the organic carboxylic acid and the organic phosphine, according to a molar-mass ratio of (5˜10) mmol:10 mg between the organic carboxylic acid and the initial QD cores and a molar-mass ratio of (2˜5) mmol:10 mg between the organic phosphine and the initial QD cores, the solution system obtained after the completion of the shell-layer growth reaction may be mixed with the mixed solution of the organic carboxylic acid and the organic phosphine. The influence of the content of the organic phosphine and the organic carboxylic acid may be referred to the description provided above. The embodiments of the present application also provide a core-shell structure QD prepared by the above method. In the embodiments of the present application, applications of the core-shell structure QDs in the fields of optical devices, optical films, core-shell structure QD inks, glue, biological probes, etc. are provided. In some embodiments, the optical device may include, but are not limited to, QD light-emitting diode (LED), and QD sensitized battery. In some embodiments, the optical film may include, but is not limited to, QD light-emitting barrier film, QD light-emitting tube, etc. In some embodiments, the core-shell structure QD ink may include, but is not limited to, an ink formed by combining QDs with other different chemical solvents in different ratios. In some embodiments, the glue may include, but is not limited to, glue composed of core-shell structure QDs and other different chemical reagents according to different viscosity ratios. In some embodiments, the biological probe may be made of QDs modified with specific substances. The following is a description with reference to specific embodiments. Embodiment 1 A preparation method for QDs includes the following steps:1. Preparing cadmium selenide (CdSe) initial QD cores,11) Preparing a cadmium precursor: adding 0.25 mmol of CdO, 0.5 mmol of octadecylphosphonic acid, and 3 g of trioctylphosphine oxide together in a 50 ml three-necked flask, dissolving the mixture by heating to 380° C., the mixture becoming a clear and transparent solution, and keeping the mixture at this temperature;12) Preparing of a Se precursor: taking and stirring 0.5 mmol of a Se source solution and 1 ml of trioctylphosphine at room temperature until the mixture becomes clear, keeping the mixture for later use;13) Preparing CdSe initial QDs: prior to injecting the Se precursor, injecting 1 ml of a trioctylphosphine solution into 11), and when the temperature of the solution returns to 380° C., injecting the Se precursor for 30 seconds, and then injecting 10 ml of octadecene to quench the reaction and cool to room temperature before cleaning;14) Cleaning and purifying CdSe initial QDs: adding 30 ml of acetone to the QD mixture to centrifuge the QDs, and dispersing the centrifuged CdSe initial QDs in 10 ml of n-hexane for later use.2. Treating cadmium selenide (CdSe) initial QD cores, Taking 2 ml of the solution prepared in step 1) with CdSe initial QDs dispersed in n-hexane, adding it to a solution containing 1 ml of oleic acid and 10 ml of octadecene, heating the mixture to 150° C. and venting for 20 minutes, and then raising the temperature of the CdSe solution to 300° C.3. Preparing CdSe/ZnS core-shell QDs,31) Preparaing a ZnS shell source: taking and dispersing 1 mmol of zinc oleate precursor and 1.5 mmol of 1-octadecanethiol together in 10 ml of octadecene solution, stirring and heating at 80° C. to make the turbid liquid clear, and then cooling to room temperature for later use;32) Growing a ZnS shell layer: injecting the ZnS shell source prepared in step 31) into the CdSe initial QD core solution prepared in step 2) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes;33) After the cyclic reaction is completed, cooling the prepared CdSe/ZnS QD solution to room temperature without any post-treatment.4. Purifying the CdSe/ZnS core-shell QDs. Adding an appropriate amount of ethyl acetate and ethanol to the QD mixture prepared in step 3) to centrifuge the CdSe/ZnS QD solution, dispersing the centrifuged CdSe/ZnS QD solution again in an appropriate amount of chloroform solution, adding acetone and methanol to the solution for precipitation and centrifugal separation, and repeating this step once; and then vacuum drying the resulting CdSe/ZnS QDs. The solubility of the CdSe/ZnS QDs prepared according to the method of this embodiment is improved, and the corresponding effect is that the monodispersity of CdSe/ZnS core-shell QDs can be improved; the absorbance of the CdSe/ZnS solution (a concentration of 0.05 mg/ml) is tested by a UV-visible fluorescence spectrometer, where the absorbance value ranges from 0.86 to 1.53. Embodiment 2 A preparation method for core-shell structure QDs includes the following steps:1. Preparing CdS initial QD cores,11) Preparing a {Cd(OA)2} precursor,Adding 1 mmol of CdO, 4 mmol of oleic acid (OA), and 10 ml of octadecene (ODE) in a three-necked flask, evacuating at room temperature for 30 minutes first, heating to 180° C. for 60 minutes for argon evacuation, maintaining at 180° C. and evacuating for 30 minutes, and then cooling to room temperature for later use;12) Preparing a selenium (Se) precursor: weighing 10 mmol of Se and adding it into 10 ml of trioctylphosphine oxide (TOP), heating to 170° C. for 30 minutes, and then lowering the temperature to 140° C.;13) Preparing a sulfur (S-TOP) precursor: weighing 20 mmol of S and adding it into 10 ml of trioctylphosphine oxide (TOP), heating to 170° C. for 30 minutes, and then lowering the temperature to 140° C.;14) Preparing a sulfur (S-ODE) precursor: weighing 5 mmol of S and adding it into 10 ml of octadecene (ODE), heating to 110° C. for 60 minutes, and then keeping the temperature at 110° C.;15) Heating the cadmium oleate {Cd(OA)2} precursor prepared in step 11) to 250° C., extracting 2 ml of S-ODE precursor prepared in step 14) into a three-necked flask and reacting for 10 minutes to prepare the CdS initial QD cores, dispersing the prepared CdS initial QD cores in n-hexane through centrifugal drying.2. Preparing CdS/CdSe core-shell QDs as follows:21) Preparing a CdSe shell source: taking 1 mmol of cadmium oleate precursor and 1.5 mmol of Se-TOP and dispersing them in 10 ml of octadecene solution, and then stirring for later use.22) Taking and dispersing 10 mg of CdS initial QD cores in 1 ml of OA and 10 ml of ODE, venting at room temperature for 20 minutes, and then heating to 300° C.,23) Growing a CdS shell layer: dropping the CdS shell source prepared in step 21) into the CdSe initial QD core solution prepared in step 1) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes,24) Adding a precipitant to the CdS/CdSe core-shell QD mixture prepared in step 23), and centrifuging to separate the prepared CdS/CdSe core-shell QDs in n-hexane.3. Preparing oil-soluble red CdS/CdSe/CdS as follows:31) Preparing a CdS shell source: taking and dispersing 1 mmol of cadmium oleate precursor and 1.5 mmol of 1-dodecanethiol together in 10 ml of octadecene solution, stirring and heating at 80° C. to make the turbid liquid clear, and then cooling to room temperature for later use;32) Taking and dispersing 10 mg of CdS/CdSe shell-core structure QDs in 1 ml of OA and 10 ml of ODE, venting at room temperature for 20 minutes, and then heating to 300° C.,33) Growing a CdS shell layer: dropping the CdS shell source prepared in step 31) into the CdS/CdSe shell-core structure QD solution prepared in step 2) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes.34) After the cyclic reaction is completed, cooling the prepared CdS/CdSe/CdS QD solution to room temperature without any post-treatment.4. Purifying the oil-soluble red CdS/CdSe/CdS.41) Adding an appropriate amount of ethyl acetate and ethanol to the QD mixture prepared in step 3) to centrifuge the CdS/CdSe/CdS QD solution, dispersing the centrifuged CdS/CdSe/CdS QD solution again in an appropriate amount of chloroform solution, adding acetone and methanol to the solution for precipitation and centrifugal separation, and repeating this step once; and then vacuum drying the resulting CdS/CdSe/CdS QDs. The solubility of the CdS/CdSe/CdS QDs prepared according to the method of this embodiment is improved, and the corresponding effect is that the monodispersity of CdS/CdSe/CdS core-shell QDs can be improved; the absorbance of the CdS/CdSe/CdS solution (a concentration of 0.05 mg/ml) is tested by a UV-visible fluorescence spectrometer, where the absorbance value ranges from 0.85 to 1.62. Embodiment 3 A preparation method for core-shell structure QDs includes the following steps:1. Preparing cadmium selenide (CdSe) initial QD cores,11) Preparing a cadmium precursor: adding 0.25 mmol of CdO, 0.5 mmol of octadecylphosphonic acid, and 3 g of trioctylphosphine oxide together in a 50 ml three-necked flask, dissolving the mixture by heating to 380° C., the mixture becoming a clear and transparent solution, and keeping the mixture at this temperature;12) Preparing of a Se precursor: taking and stirring 0.5 mmol of a Se source solution and 1 ml of trioctylphosphine at room temperature until the mixture becomes clear, keeping the mixture for later use;13) Preparing CdSe QDs: prior to injecting the Se precursor prepared in step 12), injecting 1 ml of a trioctylphosphine solution into the solution prepared in step 11), and when the temperature of the solution returns to 380° C., injecting the Se precursor for 30 seconds, and then injecting 10 ml of octadecene to quench the reaction and cool to room temperature before cleaning;14) Cleaning and purifying CdSe initial QDs: adding 30 ml of acetone to the QD mixture to centrifuge the QDs, and dispersing the centrifuged CdSe initial QDs in 10 ml of n-hexane for later use.2. Treating cadmium selenide (CdSe) initial QD cores, Dispersing CdSe initial QD cores: Taking 2 ml of the solution prepared in step 1) with CdSe initial QDs dispersed in n-hexane, adding it to a solution containing 1 ml of oleic acid and 10 ml of octadecene, heating the CdSe initial QD solution to 150° C. and venting for 20 minutes to remove the excessive n-hexane, and then raising the temperature of the CdSe solution to 300° C.3. Preparing CdSe/CdS core-shell QDs,31) Preparing a CdS shell source: taking and dispersing 1 mmol of cadmium oleate precursor and 1.5 mmol of 1-dodecanethiol together in 10 ml of octadecene solution, stirring and heating at 80° C. to make the turbid liquid clear, and then cooling to room temperature for later use;32) Growing a CdS shell layer: dropping the CdS shell source prepared in step 31) into the CdSe initial QD core solution prepared in step 2) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes;33) After the cyclic shell-layer growth is completed, adding 5 mmol of oleylamine to the mixture, and performing an aging treatment at 300° C. for 60 minutes;34) After the cyclic reaction is completed, cooling the prepared CdSe/CdS QD solution to room temperature without any post-treatment.4. Purifying CdSe/CdS core-shell QDs,41) Adding an appropriate amount of ethyl acetate and ethanol to the QD mixture prepared in step 3) to centrifuge the CdSe/CdS QD solution, dispersing the centrifuged CdSe/CdS QD solution again in an appropriate amount of chloroform solution, adding acetone and methanol to the solution for precipitation and centrifugal separation, and repeating this step once; and then vacuum drying the resulting CdSe/CdS QDs. The fluorescence intensity of the CdSe/CdS QDs prepared according to the method of this example is somewhat reduced, but the stability after being prepared as a device is improved. The quantum yield (QY) of the CdSe/CdS solution at room temperature is tested by the integrating sphere of a fluorescence spectrometer (Edinburgh-FS5), where the QY value ranges from 70% to 79%; the external quantum efficiency (EQE) of the QLED device is reduced by 1%˜5% after 30 days of testing. Embodiment 4 A preparation method for core-shell structure QDs includes the following steps:1. Preparing cadmium selenide (CdSe) initial QD cores,11) Preparing a cadmium precursor: adding 0.25 mmol of CdO, 0.5 mmol of octadecylphosphonic acid, and 3 g of trioctylphosphine oxide together in a 50 ml three-necked flask, dissolving the mixture by heating to 380° C., the mixture becoming a clear and transparent solution, and keeping the mixture at this temperature;12) Preparing of a Se precursor: taking and stirring 0.5 mmol of a Se source solution and 1 ml of trioctylphosphine at room temperature until the mixture becomes clear, keeping the mixture for later use;13) Preparing CdSe QDs: prior to injecting the Se precursor prepared in step 12), injecting 1 ml of a trioctylphosphine solution into the solution prepared in step 11), and when the temperature of the solution returns to 380° C., injecting the Se precursor for 30 seconds, and then injecting 10 ml of octadecene to quench the reaction and cool to room temperature before cleaning;14) Cleaning and purifying CdSe initial QDs: adding 30 ml of acetone to the QD mixture to centrifuge the QDs, and dispersing the centrifuged CdSe initial QDs in 10 ml of n-hexane for later use.2. Treating cadmium selenide (CdSe) initial QD cores, Dispersing CdSe initial QD cores: Taking 2 ml of the solution prepared in step 1) with CdSe initial QDs dispersed in n-hexane, adding it to a solution containing 1 ml of oleic acid and 10 ml of octadecene, heating the CdSe initial QD solution to 150° C. and venting for 20 minutes to remove the excessive n-hexane, and then raising the temperature of the CdSe solution to 300° C.3. Preparing CdSe/CdS core-shell QDs,31) Preparing a CdS shell source: taking and dispersing 1 mmol of cadmium oleate precursor and 1.5 mmol of 1-dodecanethiol together in 10 ml of octadecene solution, stirring and heating at 80° C. to make the turbid liquid clear, and then cooling to room temperature for later use;32) Growing a CdS shell layer: dropping the CdS shell source prepared in step 31) into the CdSe initial QD core solution prepared in step 2) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes.33) After the cyclic shell-layer growth is completed, adding 5 mmol of trioctylphosphine to the mixture, and performing an aging treatment at 300° C. for 60 minutes;34) After the cyclic reaction is completed, cooling the prepared CdSe/CdS QD solution to room temperature without any post-treatment.4. Purifying CdSe/CdS core-shell QDs, Adding an appropriate amount of ethyl acetate and ethanol to the QD mixture prepared in step 3) to centrifuge the CdSe/CdS QD solution, dispersing the centrifuged CdSe/CdS QD solution again in an appropriate amount of chloroform solution, adding acetone and methanol to the solution for precipitation and centrifugal separation, and repeating this step once; and then vacuum drying the resulting CdSe/CdS QDs. The CdSe/CdS QDs prepared according to the method of this example can further improve the fluorescence intensity of the QDs. The quantum yield (QY) of the CdSe/CdS solution at room temperature is tested by the integrating sphere of a fluorescence spectrometer (Edinburgh-FS5), where the QY value ranges from 78% to 89%. Embodiment 5 A preparation method for core-shell structure QDs includes the following steps:1. Preparing cadmium selenide (CdSe) initial QD cores,11) Preparing a cadmium precursor: adding 0.25 mmol of CdO, 0.5 mmol of octadecylphosphonic acid, and 3 g of trioctylphosphine oxide together in a 50 ml three-necked flask, dissolving the mixture by heating to 380° C., the mixture becoming a clear and transparent solution, and keeping the mixture at this temperature;12) Preparing of a Se precursor: taking and stirring 0.5 mmol of a Se source solution and 1 ml of trioctylphosphine at room temperature until the mixture becomes clear, keeping the mixture for later use;13) Preparing CdSe QDs: prior to injecting the Se precursor prepared in step 12), injecting 1 ml of a trioctylphosphine solution into the solution prepared in step 11), and when the temperature of the solution returns to 380° C., injecting the Se precursor for 30 seconds, and then injecting 10 ml of octadecene to quench the reaction and cool to room temperature before cleaning;14) Cleaning and purifying CdSe QDs: adding 30 ml of acetone to the QD mixture to centrifuge the QDs, and dispersing the centrifuged CdSe QDs in 10 ml of n-hexane for later use.2. Treating cadmium selenide (CdSe) initial QD cores, Dispersing CdSe initial QD cores: Taking 2 ml of the solution prepared in step 1) with CdSe initial QDs dispersed in n-hexane, adding it to a solution containing 1 ml of oleic acid and 10 ml of octadecene, heating the CdSe initial QD solution to 150° C. and venting for 20 minutes to remove the excessive n-hexane, and then raising the temperature of the CdSe solution to 300° C.3. Preparing CdSe/CdS core-shell QDs,31) Preparing a CdS shell source: taking and dispersing 1 mmol of cadmium oleate precursor and 1.5 mmol of 1-octadecanethiol together in 10 ml of octadecene solution, stirring and heating at 80° C. to make the turbid liquid clear, and then cooling to room temperature for later use;32) Growing a CdS shell layer: dropping the CdS shell source prepared in step 31) into the CdSe initial QD core solution prepared in step 2) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes;33) After the cyclic shell-layer growth is completed, adding a mixture of 1 ml of oleylamine and 2 mmol of tributylphosphine to the mixture, and performing an aging treatment at 300° C. for 60 minutes;34) After the cyclic reaction is completed, cooling the prepared CdSe/CdS initial QD solution to room temperature without any post-treatment.4. Purifying CdSe/CdS core-shell QDs, Adding an appropriate amount of ethyl acetate and ethanol to the QD mixture prepared in step 3) to centrifuge the CdSe/CdS QD solution, dispersing the centrifuged CdSe/CdS QD solution again in an appropriate amount of chloroform solution, adding acetone and methanol to the solution for precipitation and centrifugal separation, and repeating this step once; and then vacuum drying the resulting CdSe/CdS QDs. The CdSe/CdS QDs prepared according to the method of this example can improve the stability. The quantum yield (QY) of the solution at room temperature is tested after 30 days by the integrating sphere of a fluorescence spectrometer (Edinburgh-FS5), where the QY value ranged from 83 to 91%. Embodiment 6 A preparation method for core-shell structure nanocrystals including the following steps:1. Preparing cadmium selenide (CdSe) initial QD cores,11) Preparing a cadmium precursor: adding 0.25 mmol of CdO, 0.5 mmol of octadecylphosphonic acid, and 3 g of trioctylphosphine oxide together in a 50 ml three-necked flask, dissolving the mixture by heating to 380° C., the mixture becoming a clear and transparent solution, and keeping the mixture at this temperature;12) Preparing of a Se precursor: taking and stirring 0.5 mmol of a Se source solution and 1 ml of trioctylphosphine at room temperature until the mixture becomes clear, keeping the mixture for later use;13) Preparing CdSe QDs: injecting 1 ml of a trioctylphosphine solution into the solution prepared in step 11), and when the temperature of the solution returns to 380° C., injecting the Se precursor prepared in step 12) for 30 seconds, and then injecting 10 ml of octadecene to quench the reaction and cool to room temperature before cleaning;14) Cleaning and purifying CdSe initial QDs: adding 30 ml of acetone to the initial QD mixture to centrifuge the QDs, and dispersing the centrifuged CdSe initial QDs in 10 ml of n-hexane for later use.2. Treating cadmium selenide (CdSe) initial QD cores, Dispersing CdSe initial QD cores: Taking 2 ml of the solution prepared in step 1) with CdSe initial QDs dispersed in n-hexane, adding it to a solution containing 1 ml of oleylamine and 10 ml of octadecene, heating the CdSe initial QD solution to 150° C. and venting for 20 minutes to remove the excessive n-hexane, and then raising the temperature of the CdSe solution to 300° C.3. Preparing CdSe/ZnS core-shell QDs,31) Preparaing a ZnS shell source: taking and dispersing 1 mmol of zinc oleate precursor and 1.5 mmol of 1-octadecanethiol together in 10 ml of octadecene solution, stirring and heating at 80° C. to make the turbid liquid clear, and then cooling to room temperature for later use;32) Growing a ZnS shell layer: injecting the ZnS shell source prepared in step 31) into the CdSe initial QD core solution prepared in step 2) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes;33) After the cyclic reaction is completed, cooling the prepared CdSe/ZnS QD solution to room temperature without any post-treatment.4. Purifying the CdSe/ZnS core-shell QDs. Adding an appropriate amount of ethyl acetate and ethanol to the QD mixture prepared in step 3) to centrifuge the CdSe/ZnS QD solution, dispersing the centrifuged CdSe/ZnS QD solution again in an appropriate amount of chloroform solution, adding acetone and methanol to the solution for precipitation and centrifugal separation, and repeating this step once; and then vacuum drying the resulting CdSe/ZnS QDs. The CdSe/ZnS QDs prepared according to the method of this example can reduce the generation of shell-layer defects, and the corresponding effect is that the fluorescence intensity of CdSe/ZnS core-shell QDs can be improved. The quantum yield (QY) of the solution at room temperature is tested by the integrating sphere of a fluorescence spectrometer (Edinburgh-FS5), where the QY value ranges from 78% to 83%. Embodiment 7 A preparation method for core-shell structure nanocrystals including the following steps:1. Preparing CdSe initial QDs as the following:11) Preparing a {Cd(OA)2}precursor, Adding 1 mmol of CdO, 4 mmol of oleic acid (OA), and 10 ml of octadecene (ODE) in a three-necked flask, evacuating at room temperature for 30 minutes first, heating to 180° C. for 60 minutes for argon evacuation, maintaining at 180° C. and evacuating for 30 minutes, and then cooling to room temperature for later use;12) Preparing a selenium (Se) precursor, Weighing 10 mmol of Se and adding it into 10 ml of trioctylphosphine oxide (TOP), heating to 170° C. for 30 minutes, and then lowering the temperature to 140° C.;13) Preparing a sulfur (S-TOP) precursor, Weighing 20 mmol of S and adding it into 10 ml of trioctylphosphine oxide (TOP), heating to 170° C. for 30 minutes, and then lowering the temperature to 140° C.;14) Preparing a sulfur (S-ODE) precursor, Weighing 5 mmol of S and adding it into 10 ml of octadecene (ODE), heating to 110° C. for 60 minutes, and then keeping the temperature at 110° C.;15) Heating the cadmium oleate {Cd(OA)2} precursor prepared in step 11) to 250° C., extracting 2 ml of S-ODE precursor prepared in step 14) into a three-necked flask and reacting for 10 minutes to prepare the CdS initial QD cores, dispersing the prepared CdS initial QD cores in n-hexane through centrifugal drying.2. Preparing CdS/CdSe core-shell QDs as follows:21) Preparing a CdSe shell source: taking 1 mmol of cadmium oleate precursor and 1.5 mmol of Se-TOP and dispersing them in 10 ml of octadecene solution, and then stirring for later use.22) Taking and dispersing 10 mg of CdS initial QD cores in 1 ml of OA and 10 ml of ODE, venting at room temperature for 20 minutes, and then heating to 300° C.,23) Growing a CdS shell layer: dropping the CdS shell source prepared in step 21) into the CdSe initial QD core solution prepared in step 1) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes,24) Adding a precipitant to the CdS/CdSe core-shell QD mixture prepared in step 23), and centrifuging to separate the prepared CdS/CdSe core-shell QDs in n-hexane.3. Preparing CdS/CdSe/CdS shell-core QDs as follows:31) Preparing a CdS shell source: taking and dispersing 1 mmol of cadmium oleate precursor and 1.5 mmol of 1-dodecanethiol together in 10 ml of octadecene solution, stirring and heating at 80° C. to make the turbid liquid clear, and then cooling to room temperature for later use;32) Taking and dispersing 10 mg of CdS/CdSe shell-core structure QDs in 1 ml of OA and 10 ml of ODE, venting at room temperature for 20 minutes, and then heating to 300° C.,33) Growing a CdS shell layer: dropping the CdS shell source prepared in step 31) into the CdS/CdSe QD solution prepared in step 2) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes.34) After the cyclic reaction is completed, cooling the prepared CdS/CdSe/CdS QD solution to room temperature without any post-treatment.4. Purifying the CdS/CdSe/CdS QDs. Adding an appropriate amount of ethyl acetate and ethanol to the QD mixture prepared in step 3) to centrifuge the CdS/CdSe/CdS QD solution, dispersing the centrifuged CdS/CdSe/CdS QD solution again in an appropriate amount of chloroform solution, adding acetone and methanol to the solution for precipitation and centrifugal separation, and repeating this step once; and then vacuum drying the resulting CdS/CdSe/CdS QDs. The CdS/CdSe/CdS QDs prepared according to the method of this example can reduce the generation of shell-layer defects, and the corresponding effect is that the fluorescence intensity of CdS/CdSe/CdS core-shell QDs can be improved. The quantum yield (QY) of the solution at room temperature is tested by the integrating sphere of a fluorescence spectrometer (Edinburgh-FS5), where the QY value ranges from 75% to 85%. Embodiment 8 A preparation method for core-shell structure nanocrystals including the following steps:1. Preparing cadmium selenide (CdSe) QD cores,11) Preparing a cadmium precursor: adding 0.25 mmol of CdO, 0.5 mmol of octadecylphosphonic acid, and 3 g of trioctylphosphine oxide together in a 50 ml three-necked flask, dissolving the mixture by heating to 380° C., the mixture becoming a clear and transparent solution, and keeping the mixture at this temperature;12) Preparing of a Se precursor: taking and stirring 0.5 mmol of a Se source solution and 1 ml of trioctylphosphine at room temperature until the mixture becomes clear, keeping the mixture for later use;13) Preparing CdSe initial QDs: injecting 1 ml of a trioctylphosphine solution into the solution prepared in step 11), and when the temperature of the solution returns to 380° C., injecting the Se precursor prepared in step 12) for 30 seconds, and then injecting 10 ml of octadecene to quench the reaction and cool to room temperature before cleaning;14) Cleaning and purifying CdSe initial QDs: adding 30 ml of acetone to the initial QD mixture to centrifuge the QDs, and dispersing the centrifuged CdSe initial QDs in 10 ml of n-hexane for later use.2. Treating cadmium selenide (CdSe) initial QD cores, Dispersing CdSe initial QD cores: Taking 2 ml of the solution prepared in step 1) with CdSe initial QDs dispersed in n-hexane, adding it to a solution containing 1 ml of oleylamine and 10 ml of octadecene, heating the CdSe initial QD solution to 150° C. and venting for 20 minutes to remove the excessive n-hexane, and then raising the temperature of the CdSe solution to 300° C.3. Preparing CdSe/CdS core-shell QDs,31) Preparing a CdS shell source: taking and dispersing 1 mmol of cadmium oleate precursor and 1.5 mmol of 1-dodecanethiol together in 10 ml of octadecene solution, stirring and heating at 80° C. to make the turbid liquid clear, and then cooling to room temperature for later use;32) Growing a CdS shell layer: dropping the CdS shell source prepared in step 31) into the CdSe initial QD core solution prepared in step 2) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes;33) After the cyclic shell-layer growth is completed, adding 5 mmol of oleic acid to the mixture, and performing an aging treatment at 300° C. for 60 minutes;34) After the cyclic reaction is completed, cooling the prepared CdSe/CdS QD solution to room temperature without any post-treatment.4. Purifying CdSe/CdS core-shell QDs, Adding an appropriate amount of ethyl acetate and ethanol to the QD mixture prepared in step 3) to centrifuge the CdSe/CdS QD solution, dispersing the centrifuged CdSe/CdS QD solution again in an appropriate amount of chloroform solution, adding acetone and methanol to the solution for precipitation and centrifugal separation, and repeating this step once; and then vacuum drying the resulting CdSe/CdS QDs. The CdSe/CdS QDs prepared according to the method of this example not only reduce the generation of shell defects during shell-layer growth but also reduce the defect states on the surface of the CdSe/CdS core-shell QDs. Further, the corresponding effect is that not only the fluorescence intensity of the CdSe/CdS core-shell QDs is improved but, at the same time, the transient fluorescence lifetime of the CdSe/CdS core-shell QDs is also extended; the quantum yield (QY) of the solution at room temperature is tested by the integrating sphere of a fluorescence spectrometer (Edinburgh-FS5) and the transient fluorescence lifetime of the CdSe/CdS core-shell QDs is tested by transient fluorescence spectroscopy, where the QY value ranges from 80% to 89%, and the lifetime value ranges from 25 ns to 30 ns. Embodiment 9 A preparation method for core-shell structure nanocrystals including the following steps:1. Preparing cadmium selenide (CdSe) QD cores,11) Preparing a cadmium precursor: adding 0.25 mmol of CdO, 0.5 mmol of octadecylphosphonic acid, and 3 g of trioctylphosphine oxide together in a 50 ml three-necked flask, dissolving the mixture by heating to 380° C., the mixture becoming a clear and transparent solution, and keeping the mixture at this temperature;12) Preparing of a Se precursor: taking and stirring 0.5 mmol of a Se source solution and 1 ml of trioctylphosphine at room temperature until the mixture becomes clear, keeping the mixture for later use;13) Preparing CdSe QDs: injecting 1 ml of a trioctylphosphine solution into the solution prepared in step 11), and when the temperature of the solution returns to 380° C., injecting the Se precursor prepared in step 12) for 30 seconds, and then injecting 10 ml of octadecene to quench the reaction and cool to room temperature before cleaning;14) Cleaning and purifying CdSe initial QDs: adding 30 ml of acetone to the initial QD mixture to centrifuge the QDs, and dispersing the centrifuged CdSe initial QDs in 10 ml of n-hexane for later use.2. Treating cadmium selenide (CdSe) initial QD cores, Dispersing CdSe initial QD cores: Taking 2 ml of the solution prepared in step 1) with CdSe initial QDs dispersed in n-hexane, adding it to a solution containing 1 ml of oleylamine and 10 ml of octadecene, heating the CdSe initial QD solution to 150° C. and venting for 20 minutes to remove the excessive n-hexane, and then raising the temperature of the CdSe solution to 300° C.3. Preparing CdSe/CdS core-shell QDs,31) Preparing a CdS shell source: taking and dispersing 1 mmol of cadmium oleate precursor and 1.5 mmol of 1-dodecanethiol together in 10 ml of octadecene solution, stirring and heating at 80° C. to make the turbid liquid clear, and then cooling to room temperature for later use;32) Growing a CdS shell layer: dropping the CdS shell source prepared in step 31) into the CdSe QD core solution prepared in step 2) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes;33) After the cyclic shell-layer growth is completed, adding 5 mmol of trioctylphosphine to the mixture, and performing an aging treatment at 300° C. for 60 minutes;34) After the cyclic reaction is completed, cooling the prepared CdSe/CdS QD solution to room temperature without any post-treatment.4. Purifying CdSe/CdS core-shell QDs, Adding an appropriate amount of ethyl acetate and ethanol to the QD mixture prepared in step 3) to centrifuge the CdSe/CdS QD solution, dispersing the centrifuged CdSe/CdS QD solution again in an appropriate amount of chloroform solution, adding acetone and methanol to the solution for precipitation and centrifugal separation, and repeating this step once; and then vacuum drying the resulting CdSe/CdS QDs. The CdSe/CdS QDs prepared according to the method of this example can further improve the fluorescence intensity of the QDs. The quantum yield (QY) of the CdSe/CdS solution at room temperature is tested by the integrating sphere of a fluorescence spectrometer (Edinburgh-FS5), where the QY value ranges from 78% to 88%. Embodiment 10 A preparation method for core-shell structure nanocrystals including the following steps:1. Preparing cadmium selenide (CdSe) QD cores,11) Preparing a cadmium precursor: adding 0.25 mmol of CdO, 0.5 mmol of octadecylphosphonic acid, and 3 g of trioctylphosphine oxide together in a 50 ml three-necked flask, dissolving the mixture by heating to 380° C., the mixture becoming a clear and transparent solution, and keeping the mixture at this temperature;12) Preparing of a Se precursor: taking and stirring 0.5 mmol of a Se source solution and 1 ml of trioctylphosphine at room temperature until the mixture becomes clear, keeping the mixture for later use;13) Preparing CdSe QDs: injecting 1 ml of a trioctylphosphine solution into the solution prepared in step 11), and when the temperature of the solution returns to 380° C., injecting the Se precursor prepared in step 12) for 30 seconds, and then injecting 10 ml of octadecene to quench the reaction and cool to room temperature before cleaning;14) Cleaning and purifying CdSe initial QDs: adding 30 ml of acetone to the QD mixture to centrifuge the QDs, and dispersing the centrifuged CdSe initial QDs in 10 ml of n-hexane for later use.2. Treating cadmium selenide (CdSe) initial QD cores, Dispersing CdSe initial QD cores: Taking 2 ml of the solution prepared in step 1) with CdSe initial QDs dispersed in n-hexane, adding it to a solution containing 1 ml of oleylamine and 10 ml of octadecene, heating the CdSe initial QD solution to 150° C. and venting for 20 minutes to remove the excessive n-hexane, and then raising the temperature of the CdSe solution to 300° C.3. Preparing CdSe/CdS core-shell QDs,31) Preparing a CdS shell source: taking and dispersing 1 mmol of cadmium oleate precursor and 1.5 mmol of 1-octadecanethiol together in 10 ml of octadecene solution, stirring and heating at 80° C. to make the turbid liquid clear, and then cooling to room temperature for later use;32) Growing a CdS shell layer: dropping the CdS shell source prepared in step 31) into the CdSe QD core solution prepared in step 2) at a dropping rate of 6 ml/h for shell-layer growth, where the injection time is 80 minutes;33) After the cyclic shell-layer growth is completed, adding a mixture of 1 ml of oleic acid and 2 mmol of tributylphosphine to the mixture, and performing an aging treatment at 300° C. for 60 minutes.34) After the cyclic reaction is completed, cooling the prepared CdSe/CdS QD solution to room temperature without any post-treatment.4. Purifying CdSe/CdS core-shell QDs, Adding an appropriate amount of ethyl acetate and ethanol to the QD mixture prepared in step 3) to centrifuge the CdSe/CdS QD solution, dispersing the centrifuged CdSe/CdS QD solution again in an appropriate amount of chloroform solution, adding acetone and methanol to the solution for precipitation and centrifugal separation, and repeating this step once; and then vacuum drying the resulting CdSe/CdS QDs. The CdSe/CdS QDs prepared according to the method of this example can improve the stability. The quantum yield (QY) of the solution at room temperature is tested after 30 days by the integrating sphere of a fluorescence spectrometer (Edinburgh-FS5), where the QY value ranges from 83% to 91%. The absorbance of the CdSe/CdS solution (a concentration of 0.05 mg/ml) is tested by a UV-visible fluorescence spectrometer, where the absorbance value ranges from 0.9 to 1.5. The above are only the preferred embodiments of this application and are not intended to limit this application. Any modification, equivalent replacement, and improvement made within the spirit and principle of this application should be included in the protection scope of the present application. | 90,578 |
11859118 | DETAILED DESCRIPTION Applicants have now discovered that by tightly bonding a polymer to the outer surface of the quantum dot, stability of the quantum dot can be maintained even in a variety of harsh manufacturing conditions, such as, but not limited to, extrusion molding, injection molding, and other techniques. As described further below, in particular embodiments, the polymer is chosen such that it cross-links with the passivation layer (e.g. Al2O3) of the quantum dot such that the bond dissociation energy associated with the polymer/passivation layer is greater than the energy needed to melt the cross-linked polymer. In other words, the bond between the polymer and the passivation layer is not broken at extrusion (or other manufacturing) temperatures. This tight bond essentially protects the quantum dot during melting operations such as extrusion and injection molding. Previously, quantum dots exposed to such temperatures simply went dark, their optoelectronic properties extinguished by the processing conditions. Described herein are methods for making quantum dots, quantum dot-containing polymer resins and the polymer resins themselves. These methods are applicable to various types of quantum dots provided the polymer can tightly bond to the surface of the quantum dot. By tightly bonding a polymer to the outer surface of the quantum dot, particularly a passivated quantum dot, stability of the quantum dot can be maintained even in a variety of harsh manufacturing conditions, such as, but not limited to, extrusion molding, injection molding, cast molding, solvent casting, and other techniques. As described further below, in particular embodiments, the polymer is chosen such that it cross-links with the passivation layer (e.g. Al2O3) of the quantum dot such that the bond dissociation energy associated with the bonds between the polymer and the passivation layer is greater than the energy needed to melt the cross-linked polymer. In other words, the bond between the polymer and the passivation layer is not broken at melt temperatures incurred, for example during extrusion (or other manufacturing) processes. This tight bond essentially protects the quantum dot during melting operations such as extrusion and injection molding. Previously, quantum dots exposed to such temperatures simply went dark, their optoelectronic properties were extinguished by the processing conditions. Described herein are methods for making quantum dot-containing polymer resins and the polymer resins themselves. These methods are applicable to various types of quantum dots provided the polymer can tightly bond to the surface of the quantum dot. As noted above, although improved stability can be had by using the polymers and methods disclosed herein with any quantum dot, be it homogenous or alloy-gradient, size-tuned or stoichiometrically tuned, capped or uncapped, passivated or unpassivated, so long as the polymer can tightly bind to the outer surface of the quantum dot, achieving efficient and stable quantum dot (QD) photoluminescence, over the visible range of light, under the combined conditions of high photon flux and chemically adverse external environments benefits from a multi-tiered approach. First, the QD cores should have a similar surface area across the visible range. Additionally, it is specifically contemplated that cadmium-free (Cd-free) quantum dots may also be used in the methods and polymers described herein. Any Cd-free quantum dot may be used, but those described in U.S. Provisional Patent Application No. 62/338,915 entitled Cadmium-Free Quantum Dots, the disclosure of which is incorporated by reference, and set forth below, are well-suited for use with the methods and polymers disclosed herein. Second, core passivation should provide both confinement of the exciton wavefunction to the core and a physical barrier to water and oxygen. Third, the dispersive matrix that provides separation in space for the individual QDs must also provide a stable electronic configuration outside the QD volume that is conducive to photoluminescence, while itself being stable against photodegradation. The embodiment of these three elements into usable materials for the thermoplastic, thermoset and solvent cast production of optical components would accelerate the acceptance of quantum dot based components for display and lighting applications. 1. The Core It is a basic property of metal and semiconductor materials that their propensity for chemical reactions increases with an increase in surface area to mass. Thus, a 1 cm cube of metal will simply heat up when exposed to flame while that same mass will ignite if ground to a micron-sized powder. The same is true of QD cores with respect to environmental degradation and photodegradation. QDs tuned by core size will differentially degrade due to the increased reactivity of smaller cores (blue-green emitters versus larger cores (yellow-red emitters) because of a higher surface area to mass ratio. This is true in both situations of environmental attack by water and oxygen and under conditions of high photon flux where destructive free radicals are created on the QD surface. At the surface of QDs, there is a population of atoms that are incompletely part of the periodic 3D crystal lattice of the interior. These atoms have vacant or lone-pair electron orbitals. These dangling bonds are the source of undesired chemical reactions both with the external environment and in non-radiative carrier relaxation processes during the photoluminescent emission cycle in which electrons pool at these sites instead of recombining with a hole. This effect is magnified with smaller QDs that have a higher surface area/mass ratio than larger QDs. Thus, in an optical device composed of multi-colored size-tuned QDs, it is likely that faster degradation of the QDs emitting at the blue end of the visible spectrum will be observed over time, especially under conditions of exposure to water and oxygen combined with high photon flux. It is desirable to have all QD cores in an optoelectronic device be of similar size. This desired core configurations can be achieved by using QDs synthesized by the methods of Nie (U.S. Pat. Nos. 7,981,667 and 84,201,550 and Qu (U.S. Pat. No. 8,454,927). These QDs are tuned by composition and not by size. While same color size-tunable dots could be used, when considering the entire visible range, stoichiometrically-tuned quantum dots advantageously have the same size regardless of emission wavelength. Stoichiometrically-tuned quantum dots can be made in accordance with the Nie and Qu patents discussed above or other available methods. An improved method, involving the use of a pH controller to fine tune the emission wavelength is disclosed in U.S. Provisional Patent Application No. 62/338,888 entitled Tunable Semiconductor Nanocrystals And Films And 3-D Structures Containing Them the disclosure of which is incorporated by reference and set forth below herein. Quantum dots made by the methods disclosed therein result in core/shell quantum dots having substantially the same size regardless of emission wavelength. Capping (i.e. First Passivation Layer) There are two methods to passivate the dangling bonds on the surface of QDs for higher quantum efficiency (QE) and improved photo/chemical stability: 1) passivating with low MW organic ligands or 2) passivating with inorganic shells. Passivation with organic ligands is simple and straightforward but the surface metal-organic ligand bond is relatively unstable and can be broken and displaced by chemical and/or photochemical reactions. Passivation with inorganic shells is embodied by the well-known core-shell type of QD, and is often referred to as “capping” such as with a ZnS shell. The surface passivation of QD cores with inorganic shells is more stable and has the additional desired effect of providing better confinement of the exciton wavefunction to the core, thus increasing QE. If a QD core is located within a shell material with a larger bandgap energy, the electron and hole wavefunctions are better confined to the core. The recombination probability of the two wavefunctions (electron and hole) increases while the non-radiative decay process via interaction with dangling bonds on the surface decreases. Bandgap and electronic energy levels for common group II-VI, III-V and II-VI semiconductors are shown inFIG.1. These core-shell structures are improved with respect to QE and photostability (PS) but are still susceptible to chemical attack by water and oxygen from the environment. This capping is present in traditional core-shell quantum dots, and can be applied to a number of quantum dots, including the Cd-Free quantum dots and the stoichiometrically/pH controller tuned quantum dots disclosed herein, as well as other quantum dots. 2. Passivation (Second Layer): It is desirable to provide a second shell of an even wider bandgap material over the first shell that would further confine the exciton wavefunction, passivate the dangling bonds on the outer surface of the first shell material and provide a physical barrier to the diffusion of water and oxygen. This can be realized by adding a second shell, a passivation layer, of Al2O3 as described in U.S. Pat. No. 9,425,253 (Qu and Miller) hereby incorporated by reference. The bandgap of Al2O3 is between −3.5 and −11 (FIG.2) which encompasses the commonly used II-VI and III-V QD core and shell materials. In addition to having a bandgap energy that encompasses the commonly used QD core-shell materials, Al2O3, at a thickness of 4-5 atomic layers, has the additional property of providing an absolute or near-absolute barrier to the diffusion of oxygen and water. This provides a high barrier of protection from chemical attack by water and oxygen on the sensitive core-shell semiconductor materials. FIG.3shows the improved stability achieved by coating a traditional CdSe/ZnS core-shell quantum dot with an Al2O3 passivation layer. 3. The Dispersive Matrix (i.e. the Polymer) The Al2O3 surface layer offers unique synergistic opportunities to provide a matrix for QD dispersion that is chemically stable and electronically stable at the QD/matrix interface. The surface of Al2O3 is characterized by a repeating pattern of electropositive and electronegative regions as seen inFIGS.4and5. QDs with an Al2O3 surface show very tight binding affinities to organic ligands containing —COOH and —SH groups and also polymers with repeating carbonyl groups, such as polymers described in invention disclosures by Nulwala assigned to Crystalplex (U.S. Patent Application Ser. Nos. 62/338,888 and 62/338,915 both filed on May 19, 2016 and incorporated herein by reference) and Ser. No. 14/725,658, which is hereby incorporated by reference. This tight bonding has multiple desirable effects in the resulting QD/ligand/polymer matrix. 3.1 Stability of the Electronic Configuration Immediately Outside of the QD Volume It is known that the electronic configuration of the volume immediately adjacent to the QD surface and extending out to the Exciton Bohr Radius can affect the overall QE of a QD population. (see, X. Ji, D. Copenhaver, C. Sichmeller, and X. Peng, “Ligand bonding and dynamics on colloidal nanocrystals at room temperature: the case of alkylamines on CdSe nanocrystals,” J. Am. Chem. Soc. 130(17), 5726-5735 (2008). S. F. Wuister, C. de Mello Donegá, and A. Meijerink, “Influence of Thiol Capping on the Exciton Luminescence and Decay Kinetics of CdTe and CdSe Quantum Dots,” J. Phys. Chem. B 108(45), 17393-17397 (2004).) This is commonly seen when exchanging small MW organic ligands on the surface of a QD. Even though the QD nanocrystal is not physically changed by the process, a change in photoluminescent QE is observed. What is desired is a local electronic configuration that results in high QE for the QD and a very stable interface between the QD surface and the external matrix that remains unchanged even under extremes of temperature, high photon flux and destructive chemical environments. This can be achieved by binding the Al2O3 surface of the QD to polymers such as those disclosed by Nulwala. The overall binding energy of the matrix polymer to the Al2O3 surface can exceed the energy of a 280° C. extrusion process and provide a stable QD/matrix interface. 3.2 Chemical Stability of the OD/Matrix Interface In addition to heat, the stability of the QD/matrix interface also can be compromised by the presence of oxygen free radicals. These destructive free radicals can be produced at the QD/matrix interface by a combination of high photon flux and the presence of O2 molecules. The destructive radicals can result in the breaking of covalent bonds in the polymer chains in the matrix (chain scission) and/or disruption of the multiple ionic bonds between the matrix polymer chains and the Al2O3 surface of the QDs. The QD/matrix interface can be made resistant to oxygen free radical attack by a combination of the redundancy of ionic bonds between matrix polymers and the Al2O3 surface and the intrinsic high O2 barrier properties of the matrix polymer. Specific polymers, notably homopolymers of cyclohexyl acrylate and cyclohexyl acrylate copolymers with methyl methacrylate or heptyl acrylate have repeating carbonyl units oriented in 3D space such that the electronegative carbonyl oxygen repeat distance matches with the repeat distance of the electropositive regions on the surface of Al2O3. This leads to very tight bonding of the polymer to the Al2O3 surface due to a multitude of binding sites per polymer chain. In addition, these acrylic polymers have high O2 barrier properties. The combined effect of suspending QDs in these matrices is very stable bonding of the polymers to the QD surface and minimal O2 diffusion to the binding site. 3.3 Stable Dispersion in the 3D Matrix Volume In addition to the chemical stability of the OD/matrix interface, the QDs must be dispersed without clumping to function properly in photoluminescent mode. The polymers described in 3.2, and others disclosed by Nulwala, disperse QDs in this fashion. This is due to the fact that the polymer-QD bonding is more stable than QD-QD self bonding. Once bound in this fashion the QD/matrix is stable throughout downstream processing such as thermoplastic, thermoset and solvent-casting operations. In addition, the physical properties of the polymer matrix can be improved by the interaction with the QD nanoparticles. The physical crosslinking sites provided by the QDs can change and improve the physical properties of the polymer such as glass transition temperature, durometer, impact resistance, tensile strength and chemical resistance. 4. Processing 4.1 Preparation of the Composite The QD/polymer composite can be prepared by multiple methods. Polymers can be polymerized in a continuous reactor and QDs can be introduced into the continuous stream either before or after complete polymerization. The resulting QD/polymer composite stream can then be collected and the solvent removed for use as a thermoplastic material to produce an optical component. Solvent may be retained or added to produce a solvent casting composite to produce an optical film. Polymers can be completely polymerized then mixed with QDs in an appropriate solvent. Mixing, such as high shear mixing, can be applied to increase binding of polymers to the QD surface. The QD/polymer composite can be left as is for use in solvent film casting or the solvent can be removed to produce a dry composite for thermoplastic processing to produce optical components. QDs can be suspended in monomer or a mixture of monomers or a mixture of monomers and oligomers or a mixture of monomers and multifunctional monomers with multiple vinyl groups that produce crosslinking in the final polymer. This thermoset material can later be cured by heat or UV radiation to produce the final optical component. 4.1 Downstream Processing of the Composite The three commonly used processes to produce optical components from plastics are thermoplastic, thermoset, and solvent casting. Included in these general categories are injection molding, extrusion, thermoset potting, thermoset film, solvent cast film, solvent cast ink jet printing, solvent cast 3D printing, thermoset ink jet printing, thermoset 3D printing, thermoplastic 3D printing, and other techniques. Other than in the operating examples or where otherwise indicated, all numbers or expressions referring to quantities of ingredients, reaction conditions, etc. used in the specification and claims are to be understood as modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that can vary depending upon the desired properties, which the present invention desires to obtain. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “1 to 10” is intended to include all sub-ranges between and including the recited minimum value of 1 and the recited maximum value of 10; that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10. Because the disclosed numerical ranges are continuous, they include every value between the minimum and maximum values. Unless expressly indicated otherwise, the various numerical ranges specified in this application are approximations. As used herein, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%. As used herein, the term “copolymer” means a polymer resulting from the polymerization of two or more polymerizable unsaturated molecules and is meant to include terpolymers, tetra polymers, etc. As used herein, the term “core/shell” means particles that have a quantum dot as a core and one or more shells or coatings generally uniformly surrounding the quantum dot core. Non-limiting examples of shell materials include Cd or Zn salts of S or Se and/or metal oxides. The terms “include,” “comprise,” and “have” and their conjugates, as used herein, mean “include but not necessarily limited to.” As used herein, the term “Group II element” is meant to include one or more elements from the IUPAC group 2 of the periodic table selected from Cd, Zn and Hg, except when discussing Cd-free embodiments, in which case Group II element refers one or more elements from the IUPAC group 2 of the periodic table selected from Cu, Zn and Hg. As used herein, the term “Group VI element” is meant to include one or more elements from the IUPAC group 16 of the periodic table selected from S, Se, Te, Po, and O. As used herein, the terms “nanoparticles”, “nanocrystals”, and “passivated nanocrystals” refer to small structures in which the ordinary properties of their constituent materials are altered by their physical dimensions due to quantum-mechanical effects, often referred to as “quantum confinement.” For the sake of clarity, the use of these terms in this disclosure refers to objects possessing quantum-confinement properties, which are separated from one another in all three dimensions; enabling incorporation into liquids, vapors, or solids. “Optional” or “optionally” means that the subsequently described structure, event, or circumstance may or may not be present or occur, and that the description includes instances where the structure is present and where it is not or instances where the event occurs and instances where it does not. As used herein, the term “polymer” is meant to encompass, without limitation, oligomers, homopolymers, copolymers and graft copolymers. As used herein, the term “quantum dot” typically refers to a nanocrystalline particle made from a material that in the bulk is a semiconductor or insulating material, which has a tunable photophysical property in the near ultraviolet (UV) to far infrared (IR) range, and in particular, the visible range. In many embodiments of the present invention the term quantum dot includes semiconductor nanocrystals (SCN) that include transition metals, non-limiting examples being Cd and Zn, and anions from the IUPAC group 16 of the periodic table, non-limiting examples being Se, S, Te, and O. As used herein, the term “composite” refers to materials that contain quantum dots and a polymer combined into a matrix that includes quantum dots dispersed throughout the matrix. In some embodiments, the quantum dots are dispersed substantially evenly throughout the matrix. Aspects of this disclosure relate to semiconductor nanocrystals tuned to a predetermined emission wavelength (i.e. a quantum dot). In some instances, the quantum dots may be a plurality of quantum dots containing a ranges of predetermined emission wavelengths. Particularly, in some embodiments, a plurality of quantum dots contains a homogenous mixture of quantum dots emitting a desired plurality of desired wavelengths. Aspects of the present invention relate to films and 3-D structures comprising core/shell quantum dot particles dispersed in a acrylate resin. The films and 3-D structures provide the ability to cast films and place 3-D structures onto commercially applicable equipment resulting in highly stable quantum dot—polymer composite films and 3-D structures. The inventive films and 3-D structures can be used in display and lighting applications. In particular aspects, a single-coat down-conversion film (SCDF) that includes a single layer of the quantum dot—polymer composite film, sandwiched between at least two transparent films and 3-D structures can be used. The single and multilayer inventive films and 3-D structures enable a simpler and more cost effective product that provides at least the performance of more complicated structures, The Quantum Dot Core Any semiconductor nanocrystals known in the art may be used as the core for the quantum dots for incorporation into the polymers described herein, non-limiting examples being the relevant semiconductor nanocrystals disclosed in U.S. Pat. Nos. 6,207,229; 6,322,901; 6,576,291; 6,821,337; 7,138,098; 7,825,405; 7,981,667; 8,071,359; 8,288,152; 8,288,153; 8,420,155; 8,454,927; 8,481,112; 8,481,113; 8,648,524; 9,063,363; and 9,182,621 and U.S. Published Patent Application Nos. 2006/0036084, 2010/0270504, 2010/0283034; 2012/0039859; 2012/0241683; 2013/0335677; 2014/0131632; and 2014/0339497. The quantum dots employed herein may be any quantum dot, and may be:a) cadmium-containing or cadmium freeb) alloy-gradient or non-gradient (i.e. homogenous)c) size-tunable, stoichiometrically-tunable, or not, ord) any combination of these. Additionally, contemplated herein are new methods of making quantum dots, particularly a method of making same-size stoichiometrically and pH controller-tuned quantum dots and Cd-free quantum dots are disclosed herein, in and of themselves, and also for incorporation into the polymers as disclosed herein. Thus, traditional core/shell quantum dots such as those that are commercially available, other Cd-free quantum dots, as well as the same-size stoichiometrically and pH controller-tuned quantum dots and Cd-free quantum dots described and disclosed herein may be incorporated into the polymers as described further below. Cd-Free Quantum Dots As used herein, the term “Cd-free” means the object so described is substantially free of cadmium or was made without using cadmium, or does not contain cadmium. For example, the terms “Cd-free semiconductor nanocrystals” and Cd-free semiconductor “quantum dots” refer to semiconductor nanocrystals or quantum dots that are substantially free of, made without using or do not contain cadmium. “Substantially free of cadmium” means containing less than 5% cadmium, less than 3% cadmium, less than 1%, less than 0.5%, less than 0.3%, less than 0.1% or any range of values between any two of these values and any value there between. As used herein, with respect to Cd-Free quantum dots, the term “Group II element” is meant to include one or more elements from the IUPAC group 2 of the periodic table selected from Cu, Zn and Hg. As used herein, the term “Group III element” is meant to include one or more elements selected from In, Ga, Al, and Tl. As used herein, the term “Group VI element” is meant to include one or more elements from the IUPAC group 16 of the periodic table selected from S, Se, Te, Po, and O. In some embodiments, suitable Cd-free semiconductor nanocrystals that can provide useful quantum dot cores include, but are not limited to, II-II-III-VI semiconductor nanocrystals (SCN) of the formula ABCD where A is a Group II element, B is another group II element, C is a group III element, and D is a group VI element. In particular embodiments the Group II element can be one or more selected from Cu, Zn and Hg, the group III element can be one or more selected from In, Ga, Al, and the group VI element can be can be one or more selected from S, Se, Te, Po, and O. In particular embodiments, the Cd-free nanoparticles are ZnCuInS and/or ZnCuGaS In other particular embodiments, suitable semiconductor nanocrystals that can provide useful Cd-free quantum dot cores in the invention include II-II-III-III-VI semiconductor nanocrystals (SCN) of the formula ABCDE where A is a first Group II element, B is second group II element, C is a first group ill element, D is a second III group element, and E is a group VI element. In further aspects of this particular embodiment the Group II element can be one or more selected from Cu, Zn and Hg, the group III element can be selected from In, Ga, Al, and the group Vi element can be selected from S, Se, Te, Po, and O. In additional specific aspects of this particular embodiment, the Cd-free nanoparticles are ZnCuInAlS and/or ZnCuInGaS. In further embodiments, suitable Cd-free semiconductor nanocrystals that can provide quantum dot cores useful in the invention include II-II-III-VI-VI semiconductor nanocrystals (SCN) of the formula ABCDE where A is a first Group II element, B is second group II element, C is a group III element, D is a first group VI element, and E is a second group element. In aspects of this further embodiment the Group II element can be one or more selected from Cu, Zn and Hg, the group III element In, Ga, Al, and the group Vi element can be selected from S, Se. Te, Po, and O. In a specific aspect of this further embodiment, the Cd-free nanoparticles are ZnCuInSSe, ZnCuGaSSe, ZnCuAlSSe and combinations thereof. In additional embodiments, suitable Cd-free semiconductor nanocrystals that can provide quantum dot cores useful in the invention II-II-III-III-VI-VI include semiconductor nanocrystals (SCN) of the formula ABCDEF, where A is a first Group II element, B is a second group II element, C is a first group III element, D is a second group III element, and D is a group element, E is a first group VI element, and F is a second group VI element. In aspects of this additional embodiment the Group II elements can be one or more selected from Cu, Zn and Hg, the group III elements can be one or more selected from In, Ga, Al, and the group Vi elements can be one or more selected from S, Se. Te, Po, and O. In specific aspects of this additional embodiment, the Cd-free nanoparticles can be ZnCuInAlSSe, ZnCuInGaSSe, ZnCuAlGaSSe and combinations thereof. Source of Group II and Group III Elements In some embodiments, the source of the group II and group III elements are metal oxides. In particular embodiments, source of the group II and group III elements can be selected from ZnO, CuO, In2O3, Al2O3. In some embodiments, the source of the group II and III elements are fatty acid salts. In particular embodiments, the group II and group III elements can be selected from ZnX, CuX, InX, AlX, X can be a carboxylic acid with chain length from C1 to C22. Any suitable carboxylic acid can be used. In some embodiments, the carboxylic acids used can be one or more selected from acetic acid, propionic acid, butyric acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-Linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, stearic acid, palmitic acid, and arachidic acid. In a particular embodiment, the carboxylic acid is oleic acid. In a specific embodiment, the carboxylic acid is acetic acid. Source of VI Elements In some embodiments, the source of the group VI elements is a pure elemental powder. In particular embodiments, the group VI elements can be selected from elemental S, Se, Te, Po, and O. In some embodiments, the source of the group VI elements are group VI element containing molecules. In particular embodiments, the group VI element is present as the corresponding thiolate of a single functional alkyl thiol containing molecule, such as but not limited to, alkyl thiols with a chain length of from C1 to C22. In specific embodiments, the group VI element is the thiolate of 1-Dodecanthiol. In particular embodiments, the group VI element can be a dithiolate of the corresponding dithiol molecules, such as but not limited to those dithiol molecules having a chain length of from C1 to C22. Ligands In embodiments, the Cd-free nanoparticles are coated with ligands. In particular embodiments, the ligands can be selected from single chain fatty acids with chain lengths from C8 to C22. Any suitable fatty acid can be used. In some embodiments, the fatty acids used can be one or more selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-Linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, stearic acid, palmitic acid, caprylic acid and arachidic acid. In specific embodiments, the fatty acid ligands include caprylic or octanoic acid. In particular embodiments, the ligands can be selected from single chain thiols with chain lengths from C1 to C22. In specific embodiments, the ligands include 1-Dodecanthiol. In particular embodiments, the ligands can be a mixture of fatty acid and long chain thiols with a chain length of from C1 to C22. In specific embodiments, the ligands are a mixture of 1-Dodecanthiol and Octanoic acid. In some embodiments, the solvents used for the synthesis of Cd-free nanoparticles include one or more C12 to C20 hydrocarbons. In many embodiments, the precursor solution solvents can be chosen as required by the physical properties of the materials used in the precursor solution and as required by the apparatus available for synthesis. In particular embodiments, a high boiling organic solvent is employed, typically with a boiling point above about 150, in some cases above about 200, and in other cases above about 225° C., In particular embodiments, the solvent includes one or more selected from octadecane, dodecane, hexadecane and icosane. In some embodiments, tributylphosphine (TBP) is used as a solvent in the precursor solution. In other embodiments, a mixture of TBP and C12 to C20 hydrocarbons are used in the precursor solution. In these embodiments, including TBP can be advantageous because it provides a strong dipole moment, which can aid in dissolving the Group VI elements. In many embodiments, the precursor solution solvents can be chosen as required by the physical properties of the materials used in the precursor solution and as required by the apparatus available for synthesis. Cd-Free Core Syntheses Some embodiments provide a method for synthesizing Cd-free semiconductor nanocrystal cores. The method includes heating a precursor solution that includes the desired mixture of Group II element(s), Group III elements(s) and Group VI element(s) as described above in one or more solvents that include one or more C12 to C20 hydrocarbons and one or more fatty acids to a temperature sufficient to produce the Cd-free semiconductor nanocrystal cores. In some embodiments, the emission wavelength of the synthesized Cd-free nanoparticles is determined by molar ratio of the precursors, and the concentration in and type of C12 to C20 hydrocarbon solvent. Once the proper amounts of chemicals needed for the syntheses are weighed, they are placed in a suitable reaction vessel. Without degassing the temperature is raised sufficiently to initiate the reaction, and keep at that temperature for a period of time sufficient to allow the reaction to equilibrate. In some embodiments, the reaction temperature is at least about 200° C., in some cases at least about 220° C., in other cases at least about 240° C. and in some instances at least about 250° C. and can be up to about 300° C., in some cases up to about 280° C. and in other cases up to about 270° C. The temperature employed will depend on the particular precursors and solvents used. The reaction temperature can be any value or range between any of the values recited above. In some embodiments, the reaction time is at least about 5 minutes, in some cases at least about 8 minutes and in other cases at least about 9 minutes and can be up to about 60 minutes, in some cases up to about 45 minutes, in other cases up to about 30 minutes and in some instances up to about 15 minutes. The reaction time employed will depend on the particular precursors and solvents used. The reaction time can be any value or range between any of the values recited above. In a specific embodiment, the reaction time is about 10 minutes. Core Purification Purification of the Cd-free nanoparticle cores is performed to substantially reduce or eliminate unreacted precursors and byproducts generated during the reaction. In some embodiments, purification of the Cd-free nanoparticle cores can be accomplished by:1) Transferring the Cd-free nanoparticle core synthesis solution to a centrifuge tube and diluting to 7.5 times its volume with a 1:3 mixture of a nopolar and polar solvent (a non-limiting example being hexanes and butanol).2) Centrifuging the solution from (1) until crystal pellets form, and pouring off the supernatant.3) Washing the crystals three times with a 1:3 mixture of a nonpolar and polar solvent (a non-limiting example being hexane and methanol), using 6.5 times the volume of the original Cd-free nanoparticle core synthesis solution for each wash. First adding the nonpolar solvent to suspend the crystals and then adding the polar solvent to precipitate them.4) Suspending the crystals in a nonpolar solvent (a non-limiting example being hexane) at 81% the volume of the synthesis solution. Non-Traditional QDs: Stoichiometrically/pH Controlled Tuning The embodiments below relate to a quantum dot made in accordance with the teachings of U.S. Provisional Patent Application No. 62/338,888, employing a pH controller in methods for stoichiometrically tuning QDs to aid in establishing the desired emission wavelength. In some embodiments, the core is a II-VI-VI semiconductor nanocrystal (SCN) having a predetermined emission wavelength. In some embodiments, these are made by heating a II-VI-VI SCN precursor solution that includes a Group II element, a first Group VI element, a second Group VI element, and a pH controller in one or more solvents that together include one or more C12to C20hydrocarbons and one or more fatty acids to a temperature sufficient to produce the II-VI-VI SCNs. The amount of pH controller is adjusted to provide the predetermined emission wavelength from the SCNs. Without wishing to be bound by theory, Applicants believe that the use of oleic acid creates superior quantum dots because they are well-suited for subsequent capping, particularly with ZnS. Pre-Cursor Solution In some embodiments, suitable semiconductor nanocrystals that can provide quantum dot cores useful in the present invention include II-VI-VI semiconductor nanocrystals (SCN) of the formula WYxZ(1-x) where W is a Group H element, Y and Z are different Group VI elements, and 0<X<1. In particular embodiments the Group II element can be one or more selected from Cd, Zn and Hg and the Group VI element can be one or more selected from S, Se, Te, Po, and O. In some embodiments, the source of the group VI elements is soluble in C12 to C20 hydrocarbons and are organic miscible with the one or more fatty acids used to make the II-VI-VI II-VI-VI SCN. In many embodiments, pure group VI elements in powder form are used. In particular embodiments, a desired predetermined emission wavelength to be emitted from the SCNs is identified and the amount of pH controller is adjusted such that the resultant SCNs have the predetermined emission wavelength. pH Controller In some embodiments, the amount of pH controller is selected to tune the emission maximum wavelength of the SCN to the desired predetermined emission wavelength. When a specific wavelength is desired, a few synthesis reactions using different concentrations of pH controllers and, optionally, different molar ratios of precursors are run to construct a calibration curve. The required concentration of pH adjuster and, if determined, the required ratio of precursors are then identified for the desired wavelength from the calibration curve. In particular aspects of this embodiment, the emission wavelength from the SCNs, without pH controller, can be any wavelength in the visible range, and in particular from about 400 nm to about 700 nm, and any wavelength between those values. That is, SCNs can be made with a known emission wavelength. Then by introduction of a pH controller that emission wavelength can be “tuned” from that known emission wavelength to a desired predetermined wavelength. When the pH controller is included in the precursor solution, the emission wavelength of the SCN shifts to a longer wavelength. In some aspects, the SCN emission wavelength can increase at least 3 nm, in some cases at least 5 nm and in other cases at least 7 nm and can increase up to 25 nm, in some cases up to 20 nm, and in other cases up to 17 nm for each 0.1 weight percent of pH controller included in the precursor solution. The amount of SCN emission wavelength can be any value or range between any of the values recited above. The amount of SCN emission wavelength increase can vary based on the size of the semiconductor nanocrystals, the particular pH controller used and the particular Group II and Group VI elements used. Through manipulation of these factors, the emission wavelength can be precisely tuned to a desired emission wavelength. The pH controller is included in the precursor solution at a level that provides the desired SCN emission wavelength increase, often referred to as “tuning” the SCN. The pH controller can be present in the precursor solution at a level of from about 0.01 weight percent of the precursor solution, in some cases about 0.1 weight percent of the precursor solution, in other cases about 0.15 weight percent of the precursor solution and in some instances about 0.2 weight percent of the precursor solution and can be up to about 1 weight percent of the precursor solution, in some cases up to about 0.9 weight percent of the precursor solution, in other cases up to about 0.8 weight percent of the precursor solution and in some instances up to about 0.7 weight percent of the precursor solution. The amount of pH controller will be an amount sufficient to achieve the desired tuning and will typically not exceed an amount that will increase the SCN emission wavelength beyond the visible spectrum. The amount of pH controller in the precursor solution can be any value or range between any of the values recited above. Any pH controller that can maintain a desired pH and effect the emission wavelength tuning described above can be used in the SCN solution. In some embodiments, the pH controller can be an oxide or carboxylic acid salt of a Group II element. In particular embodiments the pH controller can be selected from zinc salts of acetic acid, citric acid, lactic acid, propionic acid, butyric acid, tartaric acid, and valeric acid. In particular embodiments, the pH controller is an oxide or carboxylic acid salt of a Group II element. In some aspects of the invention, the pH controller is selected from zinc salts of acetic acid, citric acid, lactic acid, propionic acid, butyric acid, tartaric acid, and valeric acid. In some embodiments, the C12 to C20 hydrocarbons used in the SCN solution can be one or more selected from hexadecene, octadecene, eicosene, hexadecane, octadecane and Icosane. In other embodiments, the fatty acids used in the SCN solution can be one or more selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-Linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, stearic acid, palmitic acid, and arachidic acid. Any pH controller that can maintain a desired pH and effect the emission wavelength tuning described above can be used in the precursor solution. In some embodiments, the pH controller can be an oxide or carboxylic acid salt of a Group II element. In particular embodiments the pH controller can be a salt of an acid selected from the group consisting of acetic acid, citric acid, lactic acid, propionic acid, butyric acid, tartaric acid, and valeric acid. In some embodiments, the salt is a zinc salt of an acid selected from the group consisting of acetic acid, citric acid, lactic acid, propionic acid, butyric acid, tartaric acid, and valeric acid. In embodiments, the pH controller is soluble in the one or more fatty acids used in the precursor solution. Hydrocarbon Solvent Any suitable C12 to C20 hydrocarbons can be used in the precursor solution. In some embodiments, the C12 to C20 hydrocarbons in the precursor solution can include one or more hydrocarbons selected from hexadecene, octadecene, eicosene, hexadecane, octadecane and icosane. In some some embodiments, tributylphosphine (TBP) is used as a solvent in the precursor solution. In other embodiments, a mixture of TBP and C12 to C20 hydrocarbons are used in the precursor solution. In these embodiments, including TBP can be advantageous because it provides a strong dipole moment, which can aid in dissolving the Group VI elements. In many embodiments, the precursor solution solvents can be chosen as required by the physical properties of the materials used in the precursor solution and as required by the apparatus available for synthesis. Fatty Acid Any suitable fatty acid can be used in the precursor solution. In some embodiments, the fatty acids used in the precursor solution can be one or more fatty acids selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-Linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, stearic acid, palmitic acid, and arachidic acid. In a particular embodiment of the invention, the fatty acid is oleic acid. In particular embodiments, the II-VI-VI SCN precursor is prepared by dissolving the Group II element, the first Group VI element, and the second Group VI element in a solvent that includes the pH controller, octadecene and a fatty acid to provide the II-VI-VI SCN precursor solution. In other embodiments, the II-VI-VI SCN precursor is prepared by preparing a first solution by dissolving the Group II element and the first Group VI element in a first solvent that includes octadecene and a fatty acid; preparing a second solution by dissolving the second Group VI element in a second solvent that includes octadecene; mixing the first and second solutions to provide a II-VI-VI SCN precursor solution. In this embodiment, both of the first and second solutions include the pH controller. In additional embodiments, the II-VI-VI II-VI-VI SCN precursor is made by preparing a first solution by dissolving a Group II element in a first solvent that includes octadecene and a fatty acid; preparing a second solution by dissolving a first Group VI and a second. Group VI element in a second solvent that includes octadecene; and mixing the first and second solutions to provide a II-VI-VI SCN precursor solution. In this embodiment, both of the first and second solutions include the pH controller. In further embodiments, the II-VI-VI SCN precursor is prepared by preparing a first solution by dissolving a Group II element in a first solvent that includes octadecene and a fatty acid; preparing a second solution by dissolving a first Group VI element in a second solvent that includes octadecene; preparing a third solution by dissolving a second Group VI element in a third solvent that includes tributylphosphine; and mixing the first, second, and third solutions to provide a II-VI-VI SCN precursor solution. In this embodiment, one or more of the first, second and third solutions include the pH controller. In all of the embodiments described above, the II-VI-VI semiconductor nanocrystals are synthesized by heating the II-VI-VI SCN precursor solution to a temperature sufficient to form the desired quantum dot core. In embodiments, the precursor solution temperature is at least 200°, in some cases at least 225°, in many cases at least 250° and in many instances at least 270° C. and can be up to about 400°, in some cases up to about 350° and in other cases up to about and 330° C. The temperature at which the II-VI-VI semiconductor nanocrystals are grown will vary depending on the particular Group II and Group VI elements and ratios used as well as the solvents, fatty acids and pH controller employed. In all of the embodiments described above, the II-VI-VI semiconductor nanocrystals are synthesized by heating the II-VI-VI SCN precursor solution to a temperature described above for a period of time that is at least sufficient to form the desired quantum dot core. In some embodiments, the reaction time is at least 40, in some cases at least 50, in many cases at least 60 and in many instances at least 70 minutes and can be up to about 120, in some cases up to about 110 and in other cases up to about 100 minutes. The reaction time over which the II-VI-VI semiconductor nanocrystals are grown will vary depending on the temperature, the particular Group II and Group VI elements and ratios used as well as the solvents, fatty acids and pH controller employed. In a particular embodiment of the invention, the quantum dot core can be prepared by selecting Group II elements that are soluble in the fatty acid. Non-limiting examples of suitable fatty acids being stearic acid and oleic acid. The pH controller soluble in the fatty acid, an oxide or acetate of a Group II element, is used. The source of the Group VI elements are chosen such that they are soluble in an organic solvent that is miscible with the fatty acid used to dissolve the Group II. In this embodiment, the organic solvent can be tributylphosphine and/or octadecene. In this embodiment, the pH, or electrical environment of the reaction system is determined by introducing the pH controller into the reaction system. The pH controller is selected based on having a negative or positive charge depending on the desired type of nanocrystal and the properties of precursors being used; and are miscible with the reaction system employed. In particular embodiments, the pH controller is Zinc acetate. Further to this embodiment, once the pH controller, solvents, and elements are selected, solutions of the elements are prepared in aliquots that are mixed together for nanocrystal synthesis. After mixing, the reaction is allowed to go to completion. In this embodiment, the emission maximum is determined by 1) the molar ratio of the two group six elements; and 2) the concentration of PH controller. The present invention provides methods of tuning a quantum dot core. The inventive, convenient method for tuning the emission maximum wavelength of the resulting quantum dot cores includes identifying a desired emission maximum. Once a specific wavelength is identified, a few synthesis reactions varying the molar ratios of the precursors and the concentration of the pH controller can be performed to identify the molar ratios of the elements and concentration of pH controller that provide the desired wavelength. In many some embodiments, a calibration curve can be constructed by performing the synthesis reactions outlined above using different ratios of elements and concentrations of pH controller. Once the calibration curve is constructed, the ratios of elements and concentration of pH controller can be identified for any desired emission maximum. Particular advantages to some of the embodiments of the present invention include not having to rely on a particular reaction time. Once the pH controller and stock solutions are prepared, aliquots of each can be mixed together and stirred at a temperature sufficient to support crystal growth, in many embodiments from about 200° C. to about 400° C., for about 40 to about 120 minutes. Advantageously, it is not important to end the reaction at a specific time. Once the method according to the invention is followed and the reaction is complete the solution can continue to be stirred at growth temperatures without altering the final quantum dot core product. In many prior art methods of synthesizing nanocrystals, an additional 1 to 5 seconds of extra reaction time substantially alters the product. In particular embodiments, the semiconductor materials of the quantum dot cores may have a gradient of one or more of the semiconductor materials radiating from the center of the nanocrystal or quantum dot to the outermost surface of the nanocrystal. Such nanocrystals or quantum dots are referred to herein as “concentration-gradient quantum dots.” For example, in some embodiments, a concentration-gradient quantum dot having at least a first semiconductor and a second semiconductor may be prepared such that the concentration of the first semiconductor gradually increases from the center of the concentration-gradient quantum dot to the surface of the quantum dot. In such embodiments, the concentration of the second semiconductor can gradually decrease from the core of the concentration-gradient quantum dot to the surface of the quantum dot. Without wishing to be bound by theory, concentration-gradient quantum dot may have a band gap energy that is non-linearly related to the molar ratio of the at least two semiconductors. Concentration-gradient quantum dots may be prepared from any semiconductor material known in the art including those semiconductor materials listed above, and concentration-gradient quantum dots may be composed of two or more semiconductor materials. In particular embodiments, concentration-gradient quantum dots may be alloys of CdSeTe having a molecular formula CdS1-xTex, CdSSe having a molecular formula CdS1-xSex, CdSTe having a molecular formula CdS1-x Tex, ZnSeTe having a molecular formula ZnSe1-x Tex, ZnCdTe having a molecular formula Zn1-x CdxTe, CdHgS having a molecular formula Cd1-x HgxS, HgCdTe having a molecular formula HgCdTe, InGaAs having a molecular formula InGaS, GaAlAs having a molecular formula GaAlAs, or InGaN having a molecular formula InGaN, where x in each example can be any fraction between 0 and 1. The methods described above provide various uncapped semiconductor nanocrystals, referred to collectively as quantum dot cores herein. Some embodiments provide quantum dot cores and in particular II-VI-VI semiconductor nanocrystals made according to the methods described above. Some embodiments provide quantum dot cores and II-VI-VI semiconductor nanocrystal that include Cd, S and Se, where the nanocrystal has been modified by a zinc alkylcarboxylate (such as zinc acetate). The quantum dot cores and II-VI-VI semiconductor nanocrystals generally correspond to the formula WYxZ(1-x) where W is a Group II element, Y and Z are different Group VI elements, and 0<X<1. In particular embodiments, the quantum dot cores and II-VI-VI semiconductor nanocrystals have a predetermined emission wavelength. The II-VI-VI semiconductor nanocrystals of the invention can have any diameter, and, thus, be of any size, provided that quantum confinement is achieved. In certain embodiments, the II-VI-VI semiconductor nanocrystals described herein have a primary particle size of less than about 10 nm in diameter. According to other embodiments, the II-VI-VI semiconductor nanocrystals have a primary particle size of between about 1 to about 500 nm in diameter. In other embodiments, a primary particle size of between about 1 to about 100 nm in diameter, and in still other embodiments, a primary particle size of between about 5 to about 15 nm in diameter. As used herein, the phrase “primary particle” refers to the smallest identifiable subdivision in a particulate system. Primary particles can also be subunits of aggregates. Standard Core/Shell Quantum Dots (CdSe/ZnS) Standard core/shell quantum dots of the CdSe/ZnS variety were obtained from a commercial source. The quantum dots were processed to assess the stability of the quantum dots with and without an Al2O3 passivation layer, and the stability of the quantum dots with and without the Al2O3 passivation layer additionally with an without incorporation into the polymer matrix described herein.FIG.6depicts the results of those tests. To assess the effect of the Al2O3 passivation layer, QDs with and without the Al2O3 passivation layer were coated naked on glass slides and exposed to 85/85 conditions (85° C., 85% humidity.) There was a marked difference as seen inFIG.6between Al2O3passivated QDs and those that without the Al2O3 passivation. The relative intensity is not necessarily important in this analysis, but the drop in the intensity of the QDs without Al2O3passivation layer indicates a much less stable QD. FIG.6shows that a core/shell. QD with or without the Al2O3 passivation layer benefits from incorporation in the polymer as described below herein. Here, QDs with and without the passivation layer were dispersed and embedded in the polymer described herein and tested under the 85/85 test conditions.FIG.6shows that the dispersion in the polymer lead to stable QDs for both samples. Thus, dispersion within the polymer as disclosed herein leads to stable QDs. Shell Growth (Capping) of Cd-Free Nanoparticle Cores Capping the purified Cd-free nanoparticle cores can be accomplished by the following methods. Method 1: Maintaining an oxygen free environment during the capping process. Take a sample of the purified Cd-free nanoparticle cores and perform the steps below. The quantities indicated are for every 0.1 mmol of Group II element in the Cd-free nanoparticle core solution.)1) Vacuum purging until the nonpolar solvent has evaporated.2) Adding 4.00 g trioctylphosphine oxide, and vacuum purging for 10 minutes. Optionally, 0.2 g stearic acid can be added along with the trioctylphosphine oxide prior to performing the vacuum purge, if a shell comprising stearic acid is desired.3) Heating to about 100° C. for about 30 minutes under vacuum and then to 200° C. without vacuum for 30 minutes.4) Preparing a capping solution by mixing 40 μL Zn(CH3)2, 80 μL Hexamethyldisilathiane (CAS #3385-94-2), and 2.00 mL trioctylphosphine in an oxygen-free environment.5) Dripping the capping solution into solution (3) at about 200-220° C. over about 5 minutes for every 2.0 mL trioctylphosphine used.6) Stirring for about 30 minutes to about 2 hours at 200° C. under nitrogen.7) Allowing the solution to cool to room temperature. A graph of ratios of elements versus emission wavelength can be prepared to provide a calibration curve. The calibration curve can be used to determine the proper fraction of elements needed to obtain crystals that fluoresce at the desired wavelength. Method 2: Load purified Cd-free nanoparticle cores into a three-neck flask with desired amounts of Zinc Acetate, elemental sulfur, 1-dodecanethiol, octadecane and octanoic acid. Degassing for 20 about minutes, then filling the flask with nitrogen, raising the temperature high enough to allow the reaction to proceed for about 60 minutes at that temperature. Capping the Quantum Dot Core Embodiments of the present invention relate to a method of capping a semiconductor nanocrystal. Any of the quantum dot cores disclosed hereinabove can be used in the methods according to these embodiments. One or more of the semiconductor nanocrystals described above are provided and heated in a solution containing one or more C12 to C20 hydrocarbons and one or more fatty acids to form an SCN solution. A solution containing dialkyl zinc, hexaalkyldisilathiane and trialkylphosphine is added to the SCN solution and heated to a temperature sufficient to produce a capped II-VI-VI semiconductor nanocrystal. In particular embodiments, a predetermined emission wavelength from the capped semiconductor nanocrystal is identified and an amount of pH controller may be added to provide the predetermined emission wavelength from the capped semiconductor nanocrystal. In some embodiments, the amount of pH controller is selected to tune the emission maximum wavelength of the capped SCN. When a specific wavelength is desired, a few synthesis reactions using different concentrations of pH controllers and the particular SCN to be capped are run to construct a calibration curve. The required concentration of pH adjuster is then identified for the desired wavelength from the calibration curve. In particular aspects of this embodiment, the emission wavelength from the capped semiconductor nanocrystal when no pH controller is present can be any wavelength in the visible range and in particular from about 400 nm to about 700 nm and any wavelength between those values. When the pH controller is included in the SCN solution, the emission wavelength of the capped semiconductor nanocrystal shifts to a longer wavelength. In some aspects of the invention, the SCN emission wavelength can increase at least 2 nm, in some case at least 3 nm and in other cases at least 4 nm and can increase up to 15, in some cases up to 12 and in other cases up to 10 nm for each 0.1 weight percent of pH controller included in the SCN solution. The amount of capped semiconductor nanocrystal emission wavelength can increase and can be any value or range between any of the values recited above. The amount of capped semiconductor nanocrystal emission wavelength can increase and vary based on the size of the capped semiconductor nanocrystal, the particular pH adjuster used and the particular Group II and Group VI elements used. The pH controller is included in the SCN solution at a level that provides the desired capped semiconductor nanocrystal emission wavelength increase, often referred to as “tuning” the capped semiconductor nanocrystal. The pH controller can be present in the SCN solution at a level of from about 0.01, in some cases about 0.1, in other cases about 0.15 and in some instances about 0.2 weight percent of the SCN solution and can be up to about 1, in some cases up to about 0.9, in other cases up to about 0.8 and in some instances up to about 0.7 weight percent of the SCN solution. The amount of pH controller will be an amount sufficient to achieve the desired tuning and will typically not exceed an amount that will increase the capped semiconductor nanocrystal emission wavelength beyond the visible spectrum. The amount of pH controller in the SCN solution can be any value or range between any of the values recited above. Any pH controller that can maintain a desired pH and effect the emission wavelength tuning described above can be used in the SCN solution. In some embodiments, the pH controller can be an oxide or carboxylic acid salt of a Group II element. In particular embodiments the pH controller can be selected from zinc salts of acetic acid, citric acid, lactic acid, propionic acid, butyric acid, tartaric acid, and valeric acid. In particular embodiments, the pH controller is an oxide or carboxylic acid salt of a Group II element. In some aspects of the invention, the pH controller is selected from zinc salts of acetic acid, citric acid, lactic acid, propionic acid, butyric acid, tartaric acid, and valeric acid. In some embodiments, the C12 to C20 hydrocarbons used in the SCN solution can be one or more selected from hexadecene, octadecene, eicosene, hexadecane, octadecane and Icosane. In other embodiments, the fatty acids used in the SCN solution can be one or more selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-Linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, stearic acid, palmitic acid, and arachidic acid. In embodiments, the dialkyl zinc is dimethyl zinc, the hexaalkyldisilathiane is hexamethyldisilathiane and the trialkylphosphine is trioctylphosphine. In many embodiments, the temperature the SCN solution containing dialkyl zinc, hexaalkyldisilathiane and trialkylphosphine is heated to in order to form the capped quantum dot is between about 150° C. and 350° C. The methods described herein above provide capped semiconductor nanocrystals. The capped semiconductor nanocrystals of the invention can have any diameter, and, thus, be of any size, provided that quantum confinement is achieved. In certain embodiments, the capped semiconductor nanocrystals described herein have a primary particle size of less than about 10 nm in diameter. According to other embodiments, the II-VI-VI semiconductor nanocrystals have a primary particle size of between about 1 to about 500 nm in diameter. In other embodiments, a primary particle size of between about 1 to about 100 nm in diameter, and in still other embodiments, a primary particle size of between about 5 to about 15 nm in diameter. As used herein, the phrase “primary particle” refers to the smallest identifiable subdivision in a particulate system. Primary particles can also be subunits of aggregates. Cd-Free Al2O3 Capping In some embodiments a passivation layer is applied to a capped Cd-free nanoparticle core prepared as described above. In these embodiments, an aluminum capping material is prepared by mixing trimethylaluminum and trioctylphosphine to form a capping solution. The capping solution is added to a solution of core/shell Cd-free nanoparticles at a temperature sufficient to grow monolayers of aluminum on the surface of the core/shell Cd-free nanoparticles to provide aluminum coated core/shell Cd-free nanoparticle cores. In particular embodiments, the monolayers can be from at least 1 atom thick, in some cases at least two atoms thick and in other cases at least 3 atoms thick and can be up to 20 atoms thick, in some cases up to 15 atoms thick, in other cases up to 10 atoms thick and in some instances up to 5 atoms thick. In many instances the capping solution is mixed with the solution of capped Cd-free nanoparticle cores at a temperature of from 100° C., in some cases at least 150° C. and in other cases at least 175° C. and can be mixed at a temperature up to about 300° C., in some cases up to about 250° C. and in other cases up to about 225° C. The aluminum coated capped Cd-free nanoparticle cores are then allowed stand in air at a temperatures of less than 100° C. to oxidize for a time sufficient to convert all or some of the monolayers of aluminum to monolayers of Al2O3, providing aluminum oxide coated capped Cd-free nanoparticle cores (“passivated core/shell Cd-free nanoparticles”). The fabrication methods for the passivated core/shell Cd-free nanoparticles may be further modified in some embodiments to achieve desired features. For example, nanoparticle characteristics such as surface functionality, surface charge, particle size, zeta (ζ) potential, hydrophobicity, and the like, may be optimized depending on the particular application of the passivated nanocrystals. For example, in some embodiments, modified surface chemistry and small particle size may contribute to reduced clearance of the nanoparticles. In other embodiments, the passivated nanoparticles are stable in water or other liquid medium without substantial agglomeration and substantial precipitation for at least 30 days, preferably for at least 90 days, and more preferably for at least 120 days. The term “stable” or “stabilized” means a solution or suspension in a fluid phase wherein solid components (i.e., nanoparticles) possess stability against aggregation and agglomeration sufficient to maintain the integrity of the compound and preferably for a sufficient period of time to be useful for the purposes detailed herein. As used herein, the term “agglomeration” refers to the formation of a cohesive mass consisting of particulate subunits held together by relatively weak forces (for example, van der Waals or capillary forces) that may break apart into particulate subunits upon processing, for example. The resulting structure is called an “agglomerate.” The passivated core/shell Cd-free nanoparticles can have any diameter, and, thus, be of any size, provided that quantum confinement is achieved. In certain embodiments, the passivated core/shell Cd-free nanoparticles described herein have a primary particle size of less than about 10 nm in diameter. According to other embodiments, the passivated core/shell Cd-free nanoparticles have a primary particle size of between about 1 nm to about 500 nm in diameter. In other embodiments, a primary particle size of between about 1 to about 100 am in diameter, and in still other embodiments, a primary particle size of between about 5 nm to about 15 nm in diameter. As used herein, the phrase “primary particle” refers to the smallest identifiable subdivision in a particulate system. Primary particles can also be subunits of aggregates. Passivating a Capped II-VI-VI Semiconductor Nanocrystal (e.g. Al2O3 Passivation) In some embodiments a passivation layer is applied to a capped II-VI-VI semiconductor nanocrystal prepared as described above. In these embodiments, an aluminum capping material is prepared by mixing trimethylaluminum and trioctylphosphine to form a capping solution. The capping solution is added to a solution of core/shell nanocrystals at a temperature sufficient to grow monolayers of aluminum on the surface of the core/shell nanocrystals to provide aluminum coated core/shell nanocrystals. In particular embodiments, the monolayers can be from at least 1, in some cases at least two and in other cases at least 3 atoms thick and can be up to 20, in some cases up to 15, in other cases up to 10 and in some instances up to 5 atoms thick. In many instances the capping solution is mixed with the solution of capped II-VI-VI semiconductor nanocrystal at a temperature of from 100, in some cases at least 150 and in other cases at least 175° C. and can be mixed at a temperature up to about 300, in some cases up to about 250 and in other cases up to about 225° C. The aluminum coated capped II-VI-VI semiconductor nanocrystal are then allowed stand in air at temperatures less than 100° C. and oxidize for a time sufficient to convert the all or some of the monolayers of aluminum to monolayers of Al2O3, to provide aluminum oxide coated capped II-VI-VI semiconductor nanocrystal (“passivated core/shell nanocrystals”). The fabrication methods for the passivated nanocrystals of the invention may be further modified in some embodiments to achieve desired features. For example, nanoparticle characteristics such as surface functionality, surface charge, particle size, zeta (ζ) potential, hydrophobicity, and the like, may be optimized depending on the particular application of the passivated nanocrystals. For example, in some some embodiments, modified surface chemistry and small particle size may contribute to reduced clearance of the nanoparticles. In other embodiments, the passivated nanoparticles are stable in water or other liquid medium without substantial agglomeration and substantial precipitation for at least 30 days, preferably for at least 90 days, and more preferably for at least 120 days. The term “stable” or “stabilized” means a solution or suspension in a fluid phase wherein solid components (i.e., nanoparticles) possess stability against aggregation and agglomeration sufficient to maintain the integrity of the compound and preferably for a sufficient period of time to be useful for the purposes detailed herein. As used herein, the term “agglomeration” refers to the formation of a cohesive mass consisting of particulate subunits held together by relatively weak forces (for example, van der Wants or capillary forces) that may break apart into particulate subunits upon processing, for example. The resulting structure is called an “agglomerate.” The passivated capped nanocrystals of the invention can have any diameter, and, thus, be of any size, provided that quantum confinement is achieved. In certain embodiments, the passivated nanocrystals described herein have a primary particle size of less than about 10 nm in diameter. According to other embodiments, the passivated nanocrystals have a primary particle size of between about 1 to about 500 nm in diameter. In other embodiments, a primary particle size of between about 1 to about 100 nm in diameter, and in still other embodiments, a primary particle size of between about 5 to about 15 nm in diameter. As used herein, the phrase “primary particle” refers to the smallest identifiable subdivision in a particulate system. Primary particles can also be subunits of aggregates. Particular embodiments described above provide a capped II-VI-VI semiconductor nanocrystal that includes a core that includes a II-VI-VI semiconductor nanocrystal containing Cd, S and Se, where the nanocrystal has been modified by a zinc alkylcarboxylate and a cap layer selected from a layer containing ZnS, a layer containing Al2O3 and a layer containing ZnS and a second layer containing Al2O3. As a non-limiting more particular description of the capped semiconductor nanocrystals according to the invention the source of the various elements should be soluble in a fatty acid such as stearic acid or oleic acid. As a non-limiting example, an oxide or acetate compound of the group two elements are often soluble in stearic acid. The source of both group VI elements should be chosen such that they are soluble in an organic solvent that is miscible with the fatty acid used to dissolve the group two element. Pure group six elements in powder form are often suitable. Tributylphosphine (TBP) and octadecene are examples of solvents that are miscible with oleic acid. In many embodiments, IBP provides a strong dipole moment, if needed, to dissolve the group six element. The solvents should be chosen as required by the physical properties of the elements and as required by the apparatus available for synthesis. In many embodiments, the pH, or electrical environment of the reaction system is determined by introducing additional materials to the reaction system. These materials should 1) have a negative or positive charge depending on the type of nanocrystal desired and the properties of precursors used; and 2) are mixable with the chosen reaction system. In particular embodiments, Zinc acetate is the pH controller. Continuing with this embodiment, it is important to remember that the method according to the invention does not require timing of a critical end point. The reaction can be allowed to go to completion. The emission maximum is determined by 1) the molar ratio of the two group six elements; and 2) the concentration of the pH controller, not the reaction time. Further to this embodiment and the description above, tuning the emission maximum wavelength to a specific desired wavelength requires only a few synthesis reactions using different molar ratios of precursors and concentrations of pH controllers. This allows for fine tuning the molar ratios and the concentration of pH controllers to the desired wavelength. In embodiments, a calibration curve is generated by performing a number of syntheses using different concentrations of PH controller. Stock solutions of pH controller are prepared and aliquots of each are mixed together and stirred at a high enough temperature to support crystal growth. Suitable temperatures can be between about 200° C. and about 400° C., for about 40 to about 120 minutes. It is not important to end the reaction at a specific time. In embodiments, once the reaction is complete the solution can be stirred at growth temperatures without altering the product. As a non-limiting example, stirring at growth temperatures for 10, 20, and 30 minutes at temperature does not change the end product semiconductor nanocrystals when a CdSeS system is used. As indicated above, prior art methods of nanocrystal synthesis where 1-5 seconds of extra reaction time is employed substantially alters the product. The method of this embodiment produces uncapped semiconductor nanocrystals, referred to as “cores”. Capping the cores makes them more stable and increases their quantum efficiency. As a non-limiting example, capping with ZnS is known to those skilled in the art. Prior to capping the cores of this embodiment, it is helpful, though not required, to purify the crystals. The cores according to this embodiment can be purified by first diluting the synthesis mixture to 7.5 times its volume with a 1:3 mixture of hexane and butanol. This causes the nanocrystals to precipitate which can then be pelletized via centrifugation. The crystals are then washed three times by first suspending the crystals in hexane and then adding three times the volume of methanol, which causes the crystals to re-precipitate. After the final wash, the crystals are dissolved in hexane for capping. Other particular embodiments provide a method of providing capped CdSeS cores. The method includes three steps; core synthesis, core purification, and core capping. The particular core synthesis of this embodiment includes:1A) Preparing a desired amount of pH controller and precursor by mixing the pH controller and precursor with octadecene and a fatty acid (oleic acid and/or stearic acid), thoroughly sparging with nitrogen gas, and heating to about 250-350° C. until the solution is clear.2A) Preparing solutions of sulfur and selenium in an oxygen free environment and mixing aliquots of each mixed to achieve the desired fluorescent wavelength, so that when added to the cadmium precursor solution the molar ratios of Cd:S:Se are 2:X:(1−X), where 0<X<1.3A) Combining the mixture of sulfur and selenium with octadecene to about 45-50 volume percent of the cadmium precursor solution while maintaining an oxygen free environment.4A) Injecting the solution from step (3A) into solution from step (1A) at 250-350° C. and then maintain a temperature of from about 250-350° C. The resulting solution is stirred about 40-120 minutes, until the reaction is complete, while maintaining an oxygen free environment. The resulting cores are purified according to this particular embodiment using the following method:1B) Transferring the core synthesis solution from step (4A) to a centrifuge tube and diluting to 7.5 times its volume with a 1:3 mixture of hexane and butanol.2B) Centrifuging the mixture from (1B) until crystal pellets are formed and pouring off the supernatant.3B) Washing the crystal pellets from step (2B) three times with 1:3 hexane:methanol, using about 6.5 times the volume of the original core synthesis solution for each wash. Adding hexane to the suspend crystals and then adding methanol to precipitate the crystals.4B) Suspending the precipitated crystals from step (3B) in hexane at about 75-85% of the volume of the synthesis solution. The resulting purified cores are capped according to this particular embodiment by maintaining an oxygen free environment during the capping process and taking a sample of the purified cores from step (4B) and using the following method (The quantities indicated are used with about 0.1 mmol of cadmium in the core solution in step (4B)):1C) Vacuum purging until substantially all of the hexane has evaporated.2C) Adding about 0.2 g of Zinc Acetate (pH controller), 10 ml of octadecene and a fatty acid, and vacuum purging for 10 minutes.3C) Heating to about 75-125° C. for about 30 minutes and then to about 175-225° C. for about 30 minutes.4C) Preparing a capping solution by mixing about 35-45 μL Zn(CH3)2, about 75-85 μL Hexamethyldisilathiane (CAS #3385-94-2), and about 1.85-2.15 mL trioctylphosphine in an anaerobic environment.5C) Slowly adding the capping solution of step (4C) into the solution of step (3C), over a period of about 4-6 minutes for every 2.0 mL trioctylphosphine used.6C) Stirring the solution from step (5C) for about 1.5-2.5 hours at 175-225° C. under nitrogen.7C) Allowing the solution from (6C) to cool to room temperature. Polymer Containing the Capped Quantum Dot Core As used herein, the term “acrylate” is meant to include esters of both acrylic and methacrylic acid, such as the corresponding alkyl esters often referred to as acrylates and methacrylates, and other esters which may contain one or more of N, P, Si and S, which the term “acrylate” is meant to encompass. Acrylates, as used herein, have the formula: wherein R1, is hydrogen or methyl; and R2is selected from the group consisting of methyl; ethyl; propyl; dodecyl; steryl; isopropyl; butyl; isobutyl; pentyl; cyclopentyl; isopentyl; linear C1-18alkyl; linear, branched, and cyclic C6-8alkyl. As used herein, the term “acrylate resin” refers to polymers resulting from the polymerization of one or more acrylates and optionally one or more other polymerizable unsaturated molecules together with any (non-quantum dot) additives that may be blended into the polymer. Unless otherwise specified, all molecular weight values are determined using gel permeation chromatography (GPC) using appropriate polystyrene standards. Unless otherwise indicated, the molecular weight values indicated herein are weight average molecular weights (Mw). Various embodiments are directed to polymers, resins, films or 3-D structures that contain semiconductor nanocrystals as described above dispersed in an acrylate resin. Any suitable acrylate resin can be used in the invention. A non-limiting example of suitable acrylate resins include those that include repeat or monomer units derived from polymerizing one or more monomers according to the formula: wherein R1is hydrogen or methyl and R2is selected from the group consisting of methyl; ethyl; propyl; dodecyl; steryl; isopropyl; butyl; isobutyl; pentyl; cyclopentyl; isopentyl; linear containing from 1-18 Carbon atoms, branched and cyclic hexyl; linear, branched and cyclic heptyl; and linear branched and cyclic octyl. Compounds of formula I are referred to herein as acrylate monomers. The amount and type of the acrylate monomers in the acrylate resin is determined based on the desired properties of the resulting film and/or 3-D structure or other product and the particular semiconductor nanocrystals used in the film. In some embodiments, the acrylate resin is made from methyl methacrylate (i.e. R1=R2=methyl) and, optionally, one or more other monomers according to structure I. In this embodiment, the amount of methyl methacrylate can be at least 1%, in some cases at least 5%, in other cases at least 10%, in some instances at least 20% and in other instances at least 25% and can be 100%, in some cases up to 95%, in other cases up to 90%, in some instances up to 80%, in other instances up to 70%, in some situations up to 60% and in other situations up to 50% based on the weight of the acrylate resin. The amount of methyl methacrylate in the acrylate resin can be any value or range between any of the values recited above. In some embodiments, the acrylate resin is made from methyl acrylate (i.e. R1=R2=methyl) and, optionally, one or more other monomers according to structure I. In this embodiment, the amount of methyl acrylate can be at least 1%, in some cases at least 5%, in other cases at least 10%, in some instances at least 20% and in other instances at least 25% and can be 100%, in some cases up to 95%, in other cases up to 90%, in some instances up to 80%, in other instances up to 70%, in some situations up to 60% and in other situations up to 50% based on the weight of the acrylate resin. The amount of methyl acrylate in the acrylate resin can be any value or range between any of the values recited above. The amount of methyl methacrylate and/or methyl acrylate in the acrylate resin is determined based on the desired properties of the resulting film or structure and the particular capped or capped and passivated semiconductor nanocrystals used in the film. In these embodiments, the other acrylate monomer(s) are used at a level that brings the total percentage of monomers used in the acrylate resin to 100%. In particular some embodiments, the acrylate resin is made from cyclohexyl acrylate (i.e. R1=H, R2=cyclohexyl) and, optionally, one or more other monomers according to structure I. In this embodiment, the amount of cyclohexyl acrylate can be at least 1%, in some cases at least 5%, in other cases at least 10%, in some instances at least 20% and in other instances at least 25% and can be 100%, in some cases up to 95%, in other cases up to 90%, in some instances up to 80%, in other instances up to 70%, in some situations up to 60% and in other situations up to 50% based on the weight of the acrylate resin. The amount of cyclohexyl acrylate in the acrylate resin can be any value or range between any of the values recited above. In these embodiments, the other acrylate monomer(s) are used at a level that brings the total percentage of monomers used in the acrylate resin to 100%. The amount of cyclohexyl acrylate in the acrylate resin is determined based on the desired properties of the resulting film or structure and the particular capped or capped and passivated semiconductor nanocrystals used in the film. Other embodiments are directed to films and 3-D structures that contain semiconductor nanocrystals as described above dispersed in polymers derived from polymerizing one or more acrylate monomers of formula I with one or more monomers according to following formulae: wherein each of R3 and R4 in structures II through V, is independently selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, cyclopentyl, isopentyl. C6 to C12 linear, branched, cyclic and aromatic hydrocarbyl, and polyethylene glycol; and R5 is selected from of hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, cyclopentyl, isopentyl C6 to C12 linear, branched, cyclic and aromatic hydrocarbyl, and polyethylene glycol. Monomers of Formulae II-V are referred to herein as nitrogen containing monomers. In particular embodiments, the acrylate resin is made from one or more acrylate monomers and one or more nitrogen containing monomers. In this embodiment, the amount of acrylate monomer can be at least 1%, in some cases at least 5%, in other cases at least 10%, in some instances at least 20% and in other instances at least 25% and can be up to 99%, in some cases up to 95%, in other cases up to 90%, in some instances up to 80%, in other instances up to 70%, in some situations up to 60% and in other situations up to 50% based on the weight of the acrylate resin. The amount and type of acrylate monomer and the corresponding amount and type of nitrogen containing monomers in the acrylate resin can be any value or range between any of the values recited above, in these embodiments, the nitrogen containing monomers are used at a level that brings the total percentage of monomers used in the acrylate resin to 100%. The amount and type of acrylate monomer and the amount and type of nitrogen containing monomer in the acrylate resin is determined based on the desired properties of the resulting film and the particular capped or capped and passivated semiconductor nanocrystals used in the film. Other embodiments are directed to films and 3-D structures that contain capped or capped and passivated 2-6-6 semiconductor nanocrystals as described above dispersed in polymers derived from polymerizing one or more acrylate monomers according structure I and one or more nitrogen containing monomers according to one or more of structures II, III, IV and V. In some embodiments, the films and 3-D structures described herein can be prepared using any suitable method. A non-limiting example of preparing the films and 3-D structures described herein include dispersing the capped nanocrystals in a suitable solution of polymers derived from polymerizing one or more acrylate monomers according structure I and/or one or more nitrogen containing monomers according to one or more of structures II, III, IV and V. Typically an organic solvent is used in the polymer solution. Any good solvent for the polymers can be used, however, solvents that can be removed to promote film formation are often used. Suitable solvents include, but are not limited to C6-C20 linear, branched and cyclic aliphatic and aromatic solvents. In particular embodiments, hexane, octane, decene, benzene, toluene, and xylene are suitable solvents. The solution of capped nanocrystals, polymer, and solvent is typically homogenized to uniformly disperse the capped nanocrystals in the polymer solution and then drawn into a film, and the solvent allowed to evaporate. In some embodiments, the nanocrystal/polymer composite described herein typically contain nanocrystals at a level of at least 0.0001 wt %, in some cases at least 0.01 wt %, in other cases at least 0.1 wt %, in some instances at least 1 wt %, and in other instances at least 5 weight percent of nanocrystals to composite and can contain up to about 75%, in some cases about 60%, in other cases about 50%, in some instances about 40% and in other instances about 30% weight percent nanocrystals to composite. The amount of nanocrystals will depend on the intended end use, the particular nanocrystals used as well as the particular polymer used. The amount of nanocrystals in the nanocrystal/polymer composite can be any value or range between any of the values recited above, (e.g. 0.0001 to 75% by weight of the composite). The nanocrystal/polymer composite of the current invention, may also contain additives, such as for example, primary antioxidants (such as hindered phenols, including vitamin E); secondary antioxidants (such as phosphites and phosphonites); nucleating agents, plasticizers or process aids (such as fluoroelastomer and/or polyethylene glycol bound process aid), acid scavengers, stabilizers, anticorrosion agents, blowing agents, other ultraviolet light absorbers such as chain-breaking antioxidants, etc., quenchers, antistatic agents, slip agents, anti-blocking agent, pigments, dyes and fillers and cure agents such as peroxide. The particular additives used are chosen so as not to interfere with the desired properties to be obtained from the nanocrystal/polymer composite. These and other common additives in the composite industry may be present in nanocrystal/polymer composite at from about 0.01 to about 50 wt % in some embodiments, and from about 0.1 to about 20 wt % in another embodiment, and from about 1 to about 5 wt % in yet another embodiment, wherein a desirable range may include any combination of any upper wt % limit with any lower wt % limit. Multilayer Films and 3-D Structures Including Films and 3-D Structures Containing the Capped Quantum Dot Core Various embodiments are directed to multilayer films and 3-D structures that include one or more layers that include the films and 3-D structures containing capped or capped and passivated quantum dot cores as described above. The quantum dot cores may be uncapped, capped, passivated, or any combination thereof. As a non-limiting example,FIG.7shows multilayer film10that includes first layer12and last layer16and a middle layer14that includes a film containing capped or capped and passivated quantum dot cores as described above. In some embodiments, first layer12and last layer16can have a refractive index of from at least 1.47, in some cases at least 1.5 and in other cases at least 1.52 and can have a refractive index of up about 1.7, in some cases up to about 1.65 and in other cases up to about 1.6. Generally, multilayer films and 3-D structures according to the invention as depicted inFIG.7can be made by first dispersing quantum dots in a suitable solvent and dissolving a acrylate resin, resin containing nitrogen monomers, and/or a resin made from acrylate monomers and nitrogen containing monomers into the quantum dot dispersion. The resulting dispersion is then coated onto a first film, which is then dried. A second film, and any subsequent film, is then heat laminated over the dispersion coated surface of to the first film. In many prior art systems, the reabsorption behavior of quantum dots and their lack of resistance to environmental degradation has been addressed using expensive multi-laminate structures. These structures are used to efficiently convert blue light from light emitting diodes (“LEDs”) into longer quantum dot emitted wavelengths (“downconversion”) and to protect the quantum dots for extended use in optoelectronic devices. Examples of such structures include cutoff filters, dichroic layers, separation of quantum dots into multiple single-color layers and other complicated multilaminate structures. However, these structures are complex and expensive to manufacture. The invention disclosed herein, as exemplified inFIG.7provides a single-coat downconversion film (SCDF) that includes a single, layer14of a quantum dot containing matrix sandwiched between two transparent films (12,16) and 3-D structures, which can be easily manufactured at low cost. A combination of maximum dispersion and refractive index (RI) matching enables a simple and cost effective product that, at a minimum, provides the performance of more complicated structures. Thus, embodiments of the multilayer films and 3-D structures according to the invention rely on a combination of maximum quantum dot dispersion and refractive index matching to achieve optimal performance. Referring toFIG.8, quantum dots in photoluminescent mode emit light isotropically (in all possible directions). In many applications it is desirable for the light produced by quantum dots to escape the matrix in which they are dispersed and travel in a preferred direction. The simplest structure to achieve some degree of directionality is to coat a layer of quantum dots in a polymer matrix on a film of material with a higher refractive index than the polymer matrix. With quantum dots dispersed in first material (20) with a lower refractive index (n1) than second material (22) with a refractive index (n2), and with an excitation source (24) coming from the side opposite second material (22) (i.e. through the first material20), a percentage of light emitted isotropically from QDs in first material (20) will be refracted toward the normal line and will be preferentially emitted away from the excitation source compared to a situation where n1=n2. If a reflector is placed behind the excitation source then with each pass of reflected quantum dot light the quantum dot light will be directed toward the normal line, amplifying the directionality during each pass. If a sandwich is constructed with first material (20) having refractive index n1layered between two second material (22) layers having refractive index n2, then the light is further directed toward the normal line with each pass. Further embodiments are shown inFIG.9, which shows multilayer film50that includes first layer52and last layer56and a middle layer54that includes a film containing capped or capped and passivated quantum dot cores as described above. First barrier layer58and second barrier layer60are situated between middle layer54and first layer52and middle layer54and last layer56respectively. In particular some embodiments, first layer52and last layer56can have a refractive index of from at least 1.47, in some cases at least 1.5 and in other cases at least 1.52 and can have a refractive index of up about 1.7, in some cases up to about 1.65 and in other cases up to about 1.6. Generally, multilayer films and 3-D structures according to the invention as depicted inFIG.9can be made by first dispersing quantum dots in a suitable solvent and dissolving a acrylate resin, resin containing nitrogen monomers, and/or a resin made from acrylate monomers and nitrogen containing monomers into the quantum dot dispersion. The resulting dispersion is then coated onto a first barrier film, which is then dried. A second barrier film is then heat laminated over the dispersion coated surface of the first barrier film. Suitable first and last films and 3-D structures are then heat laminated over the first and second barrier films and 3-D structures. In some embodiments, and referring to first layer12and last layer16inFIG.7and first layer52and last layer56inFIG.9, the layers can be any suitable material independently selected from polyethylene, polycarbonate, polypropylene, modified cellulosic resins, clear polyvinyl chloride, acrylic resins, polysiloxanes, epoxy resins, Safire, quartz and glass. In many embodiments of the films and 3-D structures and multilayer films and 3-D structures containing capped or capped and passivated 2-6-6 semiconductor nanocrystals as described above are advantageous compared to films and 3-D structures using crosslinked polymers as is often used in the art. The photostability of the resins used in the films and 3-D structures as described hereinabove provide quantum dots and films and 3-D structures containing quantum dots with improved photolytic stability. In many embodiments of the films and 3-D structures the composite material is prepared by combining the nanocrystals with the polymer during or after polymerization in a suitable solvent, then removing the solvent to produce a material that consists of 95-100% solid material that is essentially solvent-free. This composite can then be injection molded, extruded, compression molded, transfer molded and pressed or formed using a process that first melts the composite and converts the composite into the desirable 3-D shape. These 3-D parts are then used in an optoelectronic device. The quantum dots described herein may be included in solutions, inks, films, resin pellets, thermoplastic pellets. Solutions containing the quantum dots described herein may be prepared simply by leaving the QDs in solution without drying or by placing purified QDs in a suitable solution for later use. As described above, the QDs can be embedded in a polymer matrix to form films or 3-D structures. The composite (QD-matrix) can also be pelletized for later use as resin pellets or thermoplastic pellets which may then be used in subsequent molding processes, much as traditional resin or polymer pellets are used. The QDs may be incorporated into an ink such as those suitable for ink jet printing, 3-D printing, or other printing techniques. The inks are generally prepared from the quantum dots as described herein mixed with polymer, such as the acrylate polymer described herein, and a solvent. Any suitable solvent, such as, but not limited to, toluene, may be used. Other additives useful in inks may also be employed, such as, but not limited to flow agents, self-leveling agents, viscosity modifiers, de-bubbling agents, binders, surfactants, etc. In some embodiments, the polymer and solvent components account for about 1 to about 80% of the ink composition. The quantum dots are present from about 0.1 mg to about 100 mg of quantum dots per gram of polymer. The present invention will further be described by reference to the following examples. The following examples are merely illustrative and are not intended to be limiting. Unless otherwise indicated, all percentages are by weight unless otherwise specified. EXAMPLES Example A1-530 nm Cd-Free Quantum Dots 0.25 g of zinc acetate, 0.3 g of Indium Acetate, 0.01 g of copper acetate along with 5 ml of octadecane, 0.5 ml of octanoic acid, and 2 ml of 1-dodecanthiol were loaded into a three-neck flask. Without degassing, the temperature was increased to 270° C. The heat was removed after 10 minutes. The reaction provided. Cd-free quantum dots with an emission wavelength of about 530 nm. Example A2-750 nm Cd-Free Quantum Dots 0.25 g of zinc acetate, 0.3 g of Indium Acetate, 0.05 g of copper acetate along with 5 ml of octadecane, 0.5 ml of Oleic acid, and 2 ml of 1-dodecanthiol were loaded into a three-neck flask. Without degassing, the temperature was increased to 270° C. The heat was removed after 10 minutes. The reaction provided Cd-free ZnInCuS quantum dots with an emission wavelength of about 750 nm. Examples A3-A7: Cd-Free N Quantum Dots with Emission Wavelengths Between 530 and 750 nm By changing the Zn/Cu ratio, the emission wavelength of the Cd-free quantum dots can be tuned to between 530 and 750 nm. In examples 3 to 7, the reactions were carried out as in example 1, except the amount of Copper Acetate used was as indicated in Table 1, which shows the resulting emission spectrum for some of the wavelengths. TABLE 1ExampleCopper Acetate(g)Emission Wavelength (nm)A30.015540A40.018560A50.025600A60.035660A70.045720 FIG.10shows the emission spectrum for some of the wavelengths. Example A8 Capping with Method 1 In a glovebox, a solution was prepared for use in the deposition of one or more layers of ZnS onto the Cd-free nanocrystals of example 1. When no change in emission wavelength was observed of the Cd-free nanocrystals, the solution was injected slowly into the nanocrystal solution. This injection process lasted approximately two minutes. The resultant solution was added to a 50 ml conical centrifuge tube and 5 ml hexanes and 15 ml of butanol were added. After sonication for about 1 minute, 20 ml methanol was added. The nanocrystals were centrifuged and the supernatant was discarded. The nanocrystals were washed two more times with 10 ml of hexanes, precipitated with 20 ml of methanol and re-centrifuged. The purified nanocrystals were transferred to a three-neck round bottom flask and hexanes were removed by vacuum. Trioctylphosphine oxide (8.0 g) and stearic acid (0.2 g) were added. The flask was vacuum purged for 10 minutes and heated to 100° C. for 30 minutes and then to 200° C. for 30 minutes. The capping material was prepared in a glovebox as follows: 40 ul of dimethylzinc, 80 ul of hexamethyldisilathiane and 4 ml of trioctylphosphine were mixed in a glass vial and sealed with a robber stopper. The capping solution was put in a syringe, removed from the glovebox, and slowly injected into the core solution over at least 10 minutes. The resulting solution was stirred for 30 minutes at 200° C., then removed from heat and allowed to cool to room temperature. This example provided capped Cd-free nanocrystals. Example A9 Capping with Method 2 0.25 g of purified Cd-free cores from example 1 were placed in a three-neck flask with 1 g of Zinc Acetate. 0.032 g of S, 2 ml of 1-dodecanethiol, 1.0 ml of ODE and 2 ml of Octanoic acid. Degassing was conducted for 20 minutes, then the flask was filled with nitrogen, and the temperature raised to 240° C., and the reaction was allowed to progress for about 60 minutes. This example provided capped Cd-free nanocrystals. Example A10 Al2O3Capping In a glovebox, a solution was prepared for use in the deposition of one or more layers of ZnS onto the Cd-free nanocrystals of example 1. When no change in emission wavelength was observed of the Cd-free nanocrystals, the solution was injected slowly into the nanocrystal solution. This injection process lasted approximately two minutes. The resultant solution was added to a 50 ml conical centrifuge tube and 5 ml hexanes and 15 ml of butanol were added. After sonication for about 1 minute, 20 ml methanol was added. The nanocrystals were centrifuged and the supernatant was discarded. The nanocrystals were washed two more times with 10 ml of hexanes, precipitated with 20 ml of methanol and re-centrifuged. The purified capped Cd-free nanocrystals were suspended in hexanes for further capping. The purified nanocrystals were transferred to a three-neck round bottom flask and hexanes were removed by vacuum. Trioctylphosphine oxide (8.0 g) and stearic acid (0.2 g) were added. The flask was vacuum purged for 10 minutes and heated to 100° C. for 30 minutes and then to 200° C. for 30 minutes. The capping material was prepared in a glovebox as follows: 40 ul of dimethylzinc, 80 ul of hexamethyldisilathiane and 4 ml of trioctylphosphine were mixed in a glass vial and sealed with a robber stopper. The capping solution was put in a syringe, removed from the glovebox, and slowly injected into the core solution over at least 10 minutes. The resulting solution was stirred for 30 minutes at 200° C., then removed from heat and allowed to cool to room temperature. Several monolayers of aluminum were grown on the capped Cd-free nanocrystals as follows. The aluminum capping materials were prepared in a glovebox by mixing 10 ul of trimethylaluminum and 1 ml of trioctylphosphine to form a capping solution and sealed with robber stopper. The capping solution was put in a syringe, removed from the glovebox, and slowly injected into the core/shell nanocrystal solution over about 5 minutes at 200° C. then removed from the heat and allow to cool to 100° C., at which point the flask was opened to air, which allowed the aluminum outer coating on the core/shell nanocrystals to slowly oxidize over 3 hours at 100° C. Several monolayers of Al2O3were coated on the core/shell nanocrystals providing passivated core/shell Cd-free nanocrystals. Example A11-ZnCuGaS 0.25 g of zinc acetate, 0.3 g of Gallium Acetate, 0.01 g of copper acetate along with 5 ml of octadecane, 0.5 ml of Octanoic acid, and 2 ml of 1-dodecanthiol were loaded into a three flask. Without degassing, the temperature was increases to 270° C. The heat was removed after 10 minutes. This example provided Cd-free quantum dots with emission wavelength around 550 nm. Example A12-ZnCuAlS 0.25 g of zinc acetate. 0.3 g of Aluminum Acetate, 0.01 g of copper acetate along with 5 ml of octadecane, 0.5 ml of Octanoic acid, and 2 ml of 1-dodecanthiol were loaded into a three flask. Without degassing, the temperature was increased to 270° C., The heat was removed after 10 minutes. This example provided Cd-free quantum dots with emission wavelength around 490 nm. Example A13 ZnCuInSSe 0.25 g of zinc acetate. 0.3 g of Indium Acetate, 0.01 g of copper acetate along with 5 ml of octadecane, 0.5 ml of Octanoic acid, 200 ul of TBP/Se solution (concentration was 1 g/10 ml) and 2 ml of 1-dodecanthiol were loaded into a three flask. Without degassing, the temperature was increased to 270° C. The heat was removed after 10 minutes. This example provided Cd-free quantum dots with emission wavelength around 550 nm. Example A14-ZnCuInGaS 0.25 g of zinc acetate, 0.3 g of Indium Acetate, 0.1 g of Gallium Acetate, 0.01 g of copper acetate alone with 5 ml of octadecane, 0.5 ml of Octanoic acid, and 2 ml of 1-dodecanthiol were loaded into a three flask, Without degassing, the temperature was increased to 270° C. The heat was removed after 10 minutes. This example provided Cd-free quantum dots with emission wavelength around 560 nm. Example A15-ZnCuInGaSSe 0.25 g of zinc acetate, 0.3 g of Indium Acetate, 0.1 g of Gallium Acetate, 0.01 g of copper acetate along with 5 ml of octadecane, 0.5 ml of Octanoic acid, 200 ul of TBP/Se solution (concentration was 1 g/10 ml) and 2 ml of 1-dodecanthiol were loaded into a three flask. Without degassing, the temperature was increased to 270° C. The heat was removed after 10 minutes. This example provided Cd-free quantum dots with emission wavelength around 560 nm. Example A16-ZnCuInAlS 0.25 g of zinc acetate, 0.3 g of Indium Acetate, 0.1 g of Aluminum Acetate, 0.01 g of copper acetate along with 5 ml of octadecane, 0.5 ml of Octanoic acid, and 2 ml of 1-dodecanthiol were loaded into a three flask. Without degassing, the temperature was increased to 270° C. The heat was removed after 10 minutes. This example provided quantum dots with emission wavelength around 500 nm. Example A17-ZnCuInAlSSe 0.25 g of zinc acetate, 0.3 g of Indium Acetate, 0.1 g of Aluminum Acetate, 0.01 g of copper acetate along with 5 ml of octadecane, 0.5 ml of Octanoic acid, 200 ul of TBP/Se solution (concentration was 1 g/10 ml) and 2 ml of 1-dodecanthiol were loaded into a three flask. Without degassing, the temperature was increased to 270° C. The heat was removed after 10 minutes. This example provided quantum dots with emission wavelength around 540 nm. Example A18-ZnCuGaAlS 0.25 g of zinc acetate, 0.3 g, of Gallium Acetate, 0.1 g of Aluminum Acetate, 0.01 g of copper acetate along with 5 ml of octadecane, 0.5 ml of Octanoic acid, and 2 ml of 1-dodecanthiol were loaded into a three flask. Without degassing, the temperature was increased to 270° C. The heat was removed after 10 minutes. This example provided quantum dots with emission wavelength around 500 nm. Example A19-ZnCuGaAlSSe 0.25 g of zinc acetate, 0.3 g of Gallium Acetate, 0.1 g of Aluminum Acetate, 0.01 g of copper acetate along with 5 ml, of octadecane, 0.5 ml of Octanoic acid, 200 ul of TBP/Se solution (concentration was 1 g/10 ml) and 2 ml of 1-dodecanthiol were loaded into a three flask. Without degassing, the temperature was increased to 270° C. The heat was removed after 10 minutes. This example provided quantum dots with emission wavelength around 540 nm. Example B1: pH Controller Tuned Qds Core Synthesis Zinc Acetate (0.2 g)(as pH controller), octadecene (80 mL) was mixed with oleic acid (4 ml) and added to CdO (0.512 g) in a three neck round bottom flask. The flask was flushed with 99.999% nitrogen for 20 minutes and then heated to 300° C. until the solution was clear. Stock solutions of selenium and sulfur were prepared in a glove box under 99.999% nitrogen. Selenium powder (1.00 vas mixed with tributylphosphine 0.00 mL) and sulfur powder (0.050 g) was mixed with octadecene (20.00 mL). 200 μL selenium precursors were mixed with 20 mL sulfur precursors in a 20 mL glass vial, diluted to 2.00 mL with octadecene, and then added to the cadmium precursors via a syringe and stirred for 60 minutes, or until no change in emission wavelength is observed. This produces cores that fluoresce at 570 nm. Examples B2-B6 The same procedure for Example B1 was conducted for examples B2-B6, except the amount of Zinc Acetate, as pH controller, in the core synthesis was changed as indicated in the table below. ExampleZinc Acetate(g)Emission maximum (nm)B10.20570B20.25590B30.30600B40.40640B50.50660B60.70680 This data can be graphed to provide a calibration curve to determine the proper amount of Zinc Acetate for the desired wavelength by plotting emission maximum on the Y-axis and Zinc Acetate on the X-axis. A calibration curve based on this data is shown inFIG.12. Examples B7-B11 The same procedure for Example B1 was conducted to produce the cores for examples B7-B11. The cores were then subjected to purification and capping. Purification The entire core solution was added to 80 mL of hexanes and 180 mL of butanol. The resultant solution was centrifuged (2,680 G for 5 minutes) and the supernatant was discarded leaving nanocrystals. The nanocrystals were washed three times by being suspended in hexanes (10 mL), precipitated with methanol (30 mL) and centrifuged (2,680 G for 10 minutes). The crystals were then suspended in 5 mL hexanes. Capping The purified nanocrystals were transferred to a three neck round bottom flask and the solvent (hexanes) removed by vacuum. Zinc Acetate (see table below), octadecene (20 ml) and Oleic acid (10 ml) were added to the flask. The flask was vacuum purged for 10 minutes and then heated to 100° C. for 30 minutes and then to 200° C. for another 30 minutes. While the nanocrystals were heating, the capping solution was prepared in a glove box as follows: Dimethyl zinc (40 μL) was mixed with hexamethyldisilathiane (80 μL, CAS #3385-94-2) and trioctylphosphine (2.00 mL). The capping solution was put in a syringe, removed from the glovebox, and added to the nanocrystals drop-by-drop over five minutes. The resulting solution was stirred for two hours at 200° C. and then allowed to cool to room temperature. The amount of Zinc Acetate (pH controller) was changed in the capping step as indicated in the table below. The red shift after capping indicates the longer the red shift, the thicker the shell. A thicker shell nanocrystal can increase the photo and chemical stability. ExampleZinc Acetate(g)Emission maximum red shift (nm)B70.004B80.106B90.208B100.4010B110.7011 A calibration curve for the shift in emission wavelength based on this data is shown inFIG.13. Example B12 (Comparative) CdZnSSe nanocrystals were fabricated as follows. To a 100 ml three-neck round bottom flask, 0.16 mmol of CdO, 0.4 mmol of Zn(AC)2, 200 μl of oleic acid and 8 ml of octadecene were added. The flask was connected to a vacuum and degassed for about 10 minutes, then filled with high purity nitrogen, heated up to 300° C., and stirred until a colorless solution was formed. Stock solution of sulfur and selenium were prepared in a glovebox filled with 99.999% nitrogen. Selenium powder (1.00 g) was mixed with tributylphosphine (1000 ml) and sulfur powder (0.05 g) was mixed with octadecene (25.00 ml). An amount of the above sulfur and selenium stock solutions were mixed together in a glass vial and diluted with octadecene up to 4 ml resulting in a solution herein called an injection solution. The amount of sulfur and selenium was 1 mmol in total, the S to Se ratio was determined by the final emission wavelength of the derived nanocrystals. The injection solution was removed from the glovebox using a syringe and injected into the Cd and Zn precursor solution quickly while the growth temperature was raised to 270° C. This temperature was maintained for 40 to 60 minutes to allow the nanocrystals to grow to the desired size as determined by the desired emission wavelength. In the glovebox, a solution was prepared for use in the deposition of one or more layers of ZnS onto the prepared nanocrystals. When no change in emission wavelength was observed of the above-prepared nanocrystals, the solution was injected slowly into the nanocrystal solution. This injection process lasted approximately two minutes. The resultant solution was added to a 50 ml conical centrifuge tube and 5 ml hexanes and 15 ml of butanol were added. After sonication for about 1 minute, 20 ml methanol was added. The nanocrystals were centrifuged and the supernatant was discarded. The nanocrystals were washed two more times with 10 ml of hexanes, precipitated with 20 ml of methanol and re-centrifuged. The purified nanocrystals were suspended in hexanes for further capping. The purified nanocrystals were transferred to a three-neck round bottom flask and hexanes were removed by vacuum. Trioctylphosphine oxide (8.0 g) and stearic acid (0.2 g) were added. The flask was vacuum purged for 10 minutes and heated to 100° C. for 30 minutes and then to 200° C. for 30 minutes. Capping material was prepared in a glovebox as follows: 40 ul of dimethylzinc, 80 ul of hexamethyldisilathiane and 4 ml of trioctylphosphine were mixed in a glass vial and sealed with a robber stopper. The capping solution was put in a syringe, removed from the glovebox, and slowly injected into the core solution over at least 10 minutes. The resulting solution was stirred for 30 minutes at 200° C., then removed from heat and allowed to cool to room temperature. Examples B13 and B14 To compare the photo stability of the nanocrystals made in this invention, a nanocrystal—polymethylmethacrylate (PMMA) film was deposited and illuminated by an ultra-intense blue (450 nm) LED to monitor the intensity decay. Films were prepared by dispersing the nanocrystals in a toluene solution of PMMA using a Brinkman Homogenizer and then coating films using an Elcometer 4340 Automatic Film Applicator and allowing the films to dry at room temperature. In this way, the nanocrystals (5 mg), from example B11 and example B12, were added to PMMA (5 g) to make a thin film. Under ultra-intense blue (450 nm) LED for continuous illumination.FIG.11shows the stability testing result (Example B13 contains the nanocrystals from example B11 and Example B14 contains the nanocrystals from Example B12. The data demonstrate the photostability of the nanocrystals made according to the invention. As can be seen, the film using nanocrystals according got Example B11 in a PMMA film maintains emission for at least 120 minutes while the comparative nanocrystals of Example B12 in a PMMA film drop significantly, even after only 20 minutes. Examples B15-B20 Polymers were synthesis via free radical polymerization in toluene. Vinyl-based monomers (as indicated in the table below, where weight ratios of comonomers are indicated) with varied amounts were used in the polymerization. Monomer(s) was (were) dissolved in toluene (1 mL to 1 g of monomers). The initiator, azobisisobutyronitrile (AIBN, 0.5 wt % to monomers), was added. The mixture was purged with N2 for 30 min. The mixture was then heated to 70° C. and stirred overnight. The resulting product was colorless viscous liquid. MMA=methyl methacrylate, BA=butyl acrylate, CHA=cyclohexyl acrylate, NNDMT=Formula V where R3 and R4 are both methyl. Mw and PDI values were determined by GPC using analytical standards. Ex.MwTgTolueneNo.Monomer(s)(Kg/mol)PDI(° C.)SolutionB1580/20 MMA/BA511.765transparentB1690/10 MMA/BA501.793transparentB1795/5 MMA/BA391.7110transparentB1860/40 MMA/BA1082.3PhaseseparatedB19100 CHA100transparentB20100 NNDMT19-30transparent Cast films were prepared by dispersing the nanocrystals of Example B11 in a toluene solution of the polymers in Examples B15-B20 using a Brinkman Homogenizer and then casting films using an Elcometer 4340 Automatic Film Applicator and allowing the films to dry at room temperature as was described in Examples B13 and B14. All made acceptable films with improved stability as demonstrated in Example B13, except for Example B18. Extruded films were prepared by dispersing the nanocrystals of Example B11 in a toluene solution of the polymers in Examples B15-B20 using a Brinkman Homogenizer and then removing the toluene in a vacuum oven at 125° C. and 30 mm Hg vacuum. The resulting material was then melted in a heated tube to 175° C. and extruded onto a glass slide and allowed to cool forming a composite containing a concentration of 0.5 mg of nanocrystals per 1000 mg of polymer. Example B21 Passivated CdZnSSe nanocrystals were fabricated as follows. To a 100 ml three-neck round bottom flask, 0.16 mmol of CdO, 0.4 mmol of Zn(AC)2, 200 μl of oleic acid and 8 ml of octadecene were added. The flask was connected to a vacuum and degassed for about 10 minutes, then filled with high purity nitrogen, heated up to 300° C., and stirred until a colorless solution was formed. Stock solution of sulfur and selenium were prepared in a glovebox filled with 99.999% nitrogen. Selenium powder (1.00 g) was mixed with tributylphosphine (10.00 ml) and sulfur powder (0.05 g) was mixed with octadecene (25.00 ml). An amount of the above sulfur and selenium stock solutions were mixed together in a glass vial and diluted with octadecene up to 4 ml resulting in a solution herein called an injection solution. The amount of sulfur and selenium was 1 mmol in total, the S to Se ratio was determined by the final emission wavelength of the derived nanocrystals. The injection solution was removed from the glovebox using a syringe and injected into the Cd and Zn precursor solution quickly while the growth temperature was raised to 270° C. This temperature was maintained for 40 to 60 minutes to allow the nanocrystals to grow to the desired size as determined by the desired emission wavelength. In the glovebox, a solution was prepared for use in the deposition of one or more layers of ZnS onto the prepared nanocrystals. When no change in emission wavelength was observed of the above-prepared nanocrystals, the solution was injected slowly into the nanocrystal solution. This injection process lasted approximately two minutes. The resultant solution was added to a 50 ml conical centrifuge tube and 5 ml hexanes and 15 ml of butanol were added. After sonication for about 1 minute. 20 ml methanol was added. The nanocrystals were centrifuged and the supernatant was discarded. The nanocrystals were washed two more times with 10 ml of hexanes, precipitated with 20 ml of methanol and re-centrifuged. The purified nanocrystals were suspended in hexanes for further capping. The purified nanocrystals were transferred to a three-neck round bottom flask and hexanes were removed by vacuum. Trioctylphosphine oxide (8.0 g) and stearic acid (0.2 g) were added. The flask was vacuum purged for 10 minutes and heated to 100° C. for 30 minutes and then to 200° C. for 30 minutes. Capping material was prepared in a glovebox as follows: 40 ul of dimethylzinc, 80 ul of hexamethyldisilathiane and 4 ml of trioctylphosphine were mixed in a glass vial and sealed with a robber stopper. The capping solution was put in a syringe, removed from the glovebox, and slowly injected into the core solution over at least 10 minutes. The resulting solution was stirred for 30 minutes at 200° C., then removed from heat and allowed to cool to room temperature. Several monolayers of aluminum were grown on the nanocrystals as follows. The aluminum capping materials were prepared in a glovebox by mixing 10 ul of trimethylaluminum and 1 ml of trioctylphosphine to form a capping solution and sealed with robber stopper. The capping solution was put in a syringe, removed from the glovebox, and slowly injected into the core/shell nanocrystal solution over about 5 minutes at 220° C. then removed from the heat and allow to cool to 100° C., at which point the flask was opened to air, which allowed the aluminum outer coating on the core/shell nanocrystals to slowly oxidize over one hour at 100° C. Several monolayers of Al2O3 were coated on the core/shell nanocrystals providing passivated core/shell nanocrystals. Example B22 A solution cast film containing the passivated nanocrystals of Example B2.1 was prepared as follows: The passivated nanocrystals of Example 16 were added to a 50/50 w/w solution of cyclohexylacrylate homopolymer and toluene. The Mw of the polymer was approximately 125,000. The passivated nanocrystals were added at a concentration of 0.5 mg nanocrystals per gram of polymer. The mixture was then mixed for 2 minutes with a high-shear mixer (Brinkman, Model #PT/35). The mixture was them dried on a glass slide to a thickness of 0.5 mm. FIG.14shows an emission spectra of the solvent cast film made using excitation at 450 nm and the emission in the red wavelengths of the spectra. Example B23 A melt extruded film containing the passivated nanocrystals of Example B21 was prepared as follows: The passivated nanocrystals of Example B21 were added to a 50/50 w/w solution of cyclohexylacrylate homopolymer (Mw about 125,000) in toluene. The mixture was then homogenized for 2 minutes with a high-shear mixer (Brinkman. Model #PT/35). The homogenized mixture was dried to form a nanocrystal/polymer composite material, which was ground into 1-5 mm chips and loaded into a glass syringe and heated to 175° C. The molten mixture was then extruded onto a glass slide at a thickness of 0.5 mm. FIG.15shows an emission spectra of the melt extruded film made using excitation at 450 nm and the emission in the red wavelengths of the spectra. The present invention has been described with reference to certain details of particular embodiments thereof it is not intended that such details be regarded as limitations upon the scope of the invention except insofar as and to the extent that they are included in the accompanying claims. Thermoset Example: An example of a thermoset acrylic formula that cures in the presence of QDs is as follows: heptyl acrylate 60% (weight), cyclohexyl acrylate 30%, trimethylolpropane triacrylate (TMPTA) 10%. To this is added a thermal initiator such as benzoyl peroxide at 0.1% and QDs in the range of 0.001-20% wt/wt. The mixture is polymerized by heating to 85 deg C. for 10 min. It is contemplated herein that any quantum dot can be subjected to the capping and passivation methods disclosed herein and further incorporated into a polymer matrix as described herein. The fact that the disclosure or examples above are directed to specific combinations of particular quantum dot types, particular capping, particular passivation layers, and a particular polymers is not meant to suggest that this disclosure is limited to those particular combinations. The disclosure is exemplary, and not limiting, in nature. Those of skill in the art will recognize variations of the theme without departing from the scope and spirit of this disclosure. | 122,900 |
11859119 | DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be specifically described. The following constituents will be described based on typical embodiments of the present invention in some cases, but the present invention is not limited to the embodiments. In the present specification, the range of numerical values described using “to” means a range including the numerical values listed before and after “to” as the lower limit and the upper limit. Furthermore, in the present invention, “ppm” means “parts-per-million (10−6)”, “ppb” means “parts-per-billion (10−9)”, and “ppt” means “parts-per-trillion (10−12)”. In the present specification, “room temperature” is “25° C.”. In the present specification, the pH of the chemical liquid is a value measured at room temperature (25° C.) by using F-51 (trade name) manufactured by Horiba, Ltd. [Chemical Liquid] The chemical liquid according to an embodiment of the present invention contains water, a hydroxylamine compound selected from the group consisting of hydroxylamine and a hydroxylamine salt, and a specific compound represented by Formula (1) which will be described later. It is unclear what mechanism works for the chemical liquid constituted as above to achieve the above objects. According to the inventors of the present invention, the mechanism is assumed to be as below. The chemical liquid according to the embodiment of the present invention contains both the hydroxylamine compound and the specific compound. Presumably, as a result, in the presence of the hydroxylamine compound capable of acting as a reducing agent, the specific compound containing a double bond and an amide group could interact with the surface of the first metal-containing substance, which may bring about the desired effect. Furthermore, the chemical liquid according to the embodiment of the present invention excellently dissolves the first metal-containing substance and excellently improves the smoothness (roughness) of the surface of a metal-containing substance after being used for dissolving the first metal-containing substance. Hereinafter, the characteristics of the chemical liquid according to the embodiment of the present invention, such as reducing the variation in the dissolving amount of the first metal-containing substance, excellently dissolving the first metal-containing substance, and/or being capable of improving the smoothness (roughness) of the surface of a metal-containing substance after being used for dissolving the first metal-containing substance, will be described as “improving the effects of the present invention” as well. Hereinafter, the components contained in the chemical liquid according to the embodiment of the present invention (hereinafter, also simply called “chemical liquid”) will be specifically described. <Water> The chemical liquid contains water. The water is not particularly limited, and examples thereof include distilled water, deionized water, and pure water. The content of water in the chemical liquid is not particularly limited. The content of water with respect to the total mass of the chemical liquid is preferably 50% by mass or more, more preferably 70% by mass or more, and even more preferably 80% by mass or more. The upper limit of the content of water is less than 100% by mass. <Hydroxylamine Compound> The chemical liquid contains a hydroxylamine compound selected from the group consisting of hydroxylamine and a hydroxylamine salt. “Hydroxylamine” for the hydroxylamine compound refers to hydroxylamine compounds including substituted or unsubstituted alkylhydroxylamine and the like in a broad sense. Any of the hydroxylamine compounds can bring about the effects of the present invention. The hydroxylamine compound is not particularly limited, but unsubstituted hydroxylamine, a hydroxylamine derivative, and a salt thereof are preferable. Examples of hydroxylamine derivatives include O-methylhydroxylamine, O-ethylhydroxylamine, N-methylhydroxylamine, N,N-dimethylhydroxylamine, N,O-dimethylhydroxylamine, N-ethylhydroxyl amine, N,N-diethylhydroxylamine, N,O-diethylhydroxyl amine, O,N,N-trimethylhydroxylamine, N,N-dicarboxyethylhydroxylamine, and N,N-disulfoethylhydroxylamine. As the salt of the unsubstituted hydroxylamine or hydroxylamine derivative, an inorganic or organic salt of the aforementioned unsubstituted hydroxylamine or hydroxylamine derivative is preferable, a salt of an inorganic acid formed by binding of a non-metal atom, such as Cl, S, N, or P, to a hydrogen atom is more preferable, and a salt of any of hydrochloric acid, sulfuric acid, and nitric acid is even more preferable. Among these, hydroxylamine nitrate, hydroxylamine sulfate, hydroxylamine hydrochloride, hydroxylamine phosphate, N,N-diethylhydroxylamine sulfate, N,N-diethylhydroxylamine nitrate, or a mixture of these is preferable. Examples of the aforementioned organic acid salt of the unsubstituted hydroxylamine or hydroxylamine derivative include hydroxylammonium citrate, hydroxylammonium oxalate, hydroxylammonium fluoride, and the like. Among these, in view of further improving the effects of the present invention, hydroxylamine, hydroxylamine sulfate, hydroxylamine hydrochloride, hydroxylamine phosphate, hydroxylamine nitrate, or N,N-diethylhydroxylamine is preferable, and hydroxylamine is more preferable. The content of the hydroxylamine compound is not particularly limited. In view of further improving the effects of the present invention, the content of the hydroxylamine compound with respect to the total mass of the chemical liquid is preferably 0.001% to 20% by mass, and more preferably 0.1% to 18% by mass. One kind of hydroxylamine compound may be used alone, or two or more kinds of hydroxylamine compounds may be used. In a case where two or more kinds of hydroxylamine compounds are used, the total amount thereof is preferably within the above range. <Specific Compound> The chemical liquid contains a specific compound represented by Formula (1). The specific compound is assumed to interact with the surface of the first metal-containing substance. In a case where the chemical liquid contains the specific compound, the desired effect can be obtained. In Formula (1), R1to R3each independently represent a hydrogen atom or a substituent. Here, at least one of R1, R2, or R3represents a specific substituent containing —CO—NH—. In other words, one to three of R1to R3represent the specific substituent, and the others represent a hydrogen atom or a substituent other than the specific substituent (non-specific substituent). The specific substituent is a group containing —CO—NH—. The direction of binding of —CO—NH— is not particularly limited. For example, —CO— of —CO—NH— may be on the double bond side specified in Formula (1), or —NH— of —CO—NH— may be on the double bond side specified in Formula (1). Particularly, it is preferable that —CO— of —CO—NH— be on the double bond side specified in Formula (1). Furthermore, the number of —CO—NH— present in the specific substituent is not limited, and is preferably 1 to 5 and more preferably 1. In view of further improving the effects of the present invention, the specific substituent is preferably a group represented by Formula (T). -LT-CO—NH—RT In Formula (T), LTrepresents a single bond or a divalent linking group. Examples of the divalent linking group include an ether group (—O—), a carbonyl group (—CO—), an ester group (—COO—), a thioether group (—S—), —SO2—, —NRN— (RNrepresents a hydrogen atom or an alkyl group), a divalent hydrocarbon group (an alkylene group, an alkenylene group (such as —CH═CH—), an alkynylene group (such as —C≡C—), or an arylene group), —SiRSX2— (RSNrepresents a hydrogen atom or a substituent), and a group formed by combining one or more groups selected from the group consisting of the above groups. These groups may have a substituent if possible, or may not have a substituent. Particularly, as the aforementioned divalent linking group, a divalent hydrocarbon group having 1 to 10 carbon atoms is preferable, an alkylene group is more preferable, and a methylene group is even more preferable. In Formula (T), RTrepresents a hydrogen atom or a substituent. RTis preferably a hydrogen atom or a hydroxyl group, and more preferably a hydroxyl group. That is, the specific substituent is preferably a group containing —CO—NH—OH. In view of further improving the effects of the present invention, one of R1to R3in Formula (1) is preferably the specific substituent. In view of further improving the effects of the present invention, it is preferable that one or two of R1to R3in Formula (1) be the non-specific substituent, and it is more preferable that two of R1to R3in Formula (1) be the non-specific substituent. The non-specific substituent is a substituent that does not contain —CO—NH—. In view of further improving the effects of the present invention, the non-specific substituent is preferably a group represented by Formula (2). -L2-COOH (2) In Formula (2), L2represents a single bond or a divalent linking group. Examples of the divalent linking group represented by L2in Formula (2) are the same as the examples of the divalent linking group represented by LTin Formula (T), and preferred forms of the divalent linking group are also the same. Here, the divalent linking group represented by L2does not contain —CO—NH—. In Formula (1), R1and R2may be bonded to each other to form an aromatic ring which may have a substituent. The aromatic ring which is formed by the bonding of R1and R2and may have a substituent may be a monocyclic ring or a polycyclic ring, and is preferably a monocyclic ring. The aromatic ring may or may not have a heteroatom (such as an oxygen atom, a sulfur atom, and/or a nitrogen atom). It is preferable that the aromatic ring do not have a heteroatom. The number of atoms as members of the aromatic ring is preferably 5 to 12, more preferably 6. The aromatic ring may have a substituent other than R3if possible, or may not have such a substituent. It is preferable that the aromatic ring do not have such a substituent. In a case where R1and R2are bonded to each other to form an aromatic ring which may have a substituent, R3represents the aforementioned specific substituent. Especially, it is preferable that the chemical liquid contain one or more kinds of compounds among the following compounds A to C as the specific compound. It does not matter whether each of the following compounds A to C is a cis isomer or a trans isomer. In view of further improving the effects of the present invention, the content of the specific compound with respect to the total mass of the chemical liquid is preferably 0.10 ppm by mass to 10% by mass, and more preferably 0.01% to 1% by mass. One kind of specific compound may be used alone, or two or more kinds of specific compounds may be used. In a case where two or more kinds of specific compounds are used, the total amount thereof is within the above range. In the present specification, in a case where the specific compound includes cis and trans isomers (for example, in a case where the specific compound includes cis and trans isomers distinguished based on the C═C double bond specified in Formula (1)), all of the specific compounds including cis and trans isomers that differ from each other only in terms of the structure are regarded as one kind of specific compound. For example, the compound A may include either or both of a compound in which “—CO—NH—OH” and “—COOH” bonded to a C═C double bond are arranged at cis position (cis isomer) and a compound in which “—CO—NH—OH” and “—COOH” are arranged at trans position (trans isomer). In a case where the chemical liquid contains, as the specific compound, the compound A as a cis isomer and the compound A as a trans isomer, the chemical liquid is considered as having only one kind of specific compound. It is preferable that the chemical liquid contain two or more kinds of specific compounds. In a case where the chemical liquid contains two or more kinds of specific compounds, the mass ratio of the content of a specific compound which takes up the highest proportion of the specific compounds to the content of a specific compound which takes up the second highest proportion of the specific compounds (content of specific compound which takes up the highest proportion of specific compounds/content of specific compound which takes up the second highest proportion of specific compounds) is preferably 500 or less, and more preferably 50 or less. In a case where “content of a specific compound which takes up the highest proportion of the specific compounds” and “content of a specific compound which takes up the second highest proportion of the specific compounds” are very close to each other, the mass ratio may be 1. That is, “content of a specific compound which takes up the highest proportion of the specific compounds” and “content of a specific compound which takes up the second highest proportion of the specific compounds” may be substantially the same amount. In a case where the chemical liquid contains two or more kinds of specific compounds, it is also preferable that at least one kind of specific compound be the compound A. In this case, the mass ratio of the content of the compound A to the content of a specific compound which is not the compound A and takes up the highest proportion of the specific compounds is preferably 0.001 to 500, and more preferably 0.01 to 50. In view of further improving the effects of the present invention, the mass ratio of the content of the hydroxylamine compound to the content of the specific compound (content of hydroxylamine compound/content of specific compound) is preferably 1.0×10−6to 1.0×108, and more preferably 2.0×10−4to 1.5×106. The chemical liquid may contain components other than water, the hydroxylamine compound, and the specific compound. <Chelating Agent> The chemical liquid may contain a chelating agent. In a case where the chemical liquid contains a chelating agent, the chemical liquid exhibits higher etching performance to the first metal-containing substance. The chelating agent means an acid capable of functioning as a chelating ligand. As the chelating agent, a compound having one or more acid groups is preferable. The chelating agent does not include the specific compound and a reducing agent different from hydroxylamine which will be described later. The acid group is not particularly limited, and is preferably at least one kind of functional group selected from the group consisting of a carboxylic acid group, a sulfonic acid group, and a phosphonic acid group. Examples of the chelating agent having a carboxylic acid group include polyaminopolycarboxylic acids, aliphatic dicarboxylic acids, aliphatic polycarboxylic acids containing a hydroxyl group, and ascorbic acids. The polyaminopolycarboxylic acids are compounds having a plurality of amino groups and a plurality of carboxylic acid groups. Examples thereof include mono- or polyalkylene polyamine polycarboxylic acid, polyaminoalkane polycarboxylic acid, polyaminoalkanol polycarboxylic acid, and hydroxyalkyl ether polyamine polycarboxylic acid. Examples of the polyaminopolycarboxylic acids include butylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetrapropionic acid, triethylenetetraminehexacetic acid, 1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic acid, propylenediaminetetraacetic acid, ethylenediaminetetraacetic acid (EDTA), trans-1,2-diaminocyclohexanetetraacetic acid, ethylenediaminediaminediacetic acid, ethylenediaminedipropionic acid, 1,6-hexamethylene-diamine-N,N,N′,N′-tetraacetic acid, N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid, diaminopropanetetraacetic acid, 1,4,7,10-tetraazacyclododecane-tetraacetic acid, diaminopropanol tetraacetic acid, and (hydroxyethyl)ethylenediaminetriacetic acid. Among these, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), or trans-1,2-diaminocyclohexanetetraacetic acid is preferable. Examples of the aliphatic dicarboxylic acids include oxalic acid, malonic acid, succinic acid, and maleic acid. Among these, oxalic acid, malonic acid, or succinic acid is preferable. Examples of the aliphatic polycarboxylic acids containing a hydroxyl group include malic acid, tartaric acid, and citric acid. Among these, citric acid is preferable. Examples of ascorbic acids include ascorbic acid, isoascorbic acid, ascorbic acid sulfate, ascorbic acid phosphate, ascorbic acid 2-glucoside, ascorbic acid palmitate, ascorbyl tetraisopalmitate, ascorbic acid isopalminate, and ascorbic acids of these salts and the like. Among these, ascorbic acid is preferable. Examples of the chelating agent having a sulfonic acid group include methanesulfonic acid. Examples of the chelating agent having a phosphonic acid group include methyldiphosphonic acid, aminotri(methylenephosphonic acid), 1-hydroxyethylidene-1,1-diphosphonic acid, nitrilotrismethylenephosphonic acid (NTMP), ethylenediaminetetrakis(methylenephosphonic acid) (EDTPO), hexamethylenediaminetetra(methylenephosphonic acid), propylenediaminetetra(methylenephosphonic acid), diethylenetriaminepenta(methylenephosphonic acid), triethylenetetraminehexa(methylenephosphonic acid), triaminotriethylaminehexa(methylenephosphonic acid), trans-1,2-cyclohexanediaminetetra(methylenephosphonic acid), glycol ether diaminetetra(methylenephosphonic acid), tetraethylenepentaminehepta(methylenephosphonic acid), and glycine-N,N-bis(methylenephosphonic acid) (glyphosine). In view of further improving the effects of the present invention, the chelating agent is preferably a compound selected from the group consisting of citric acid, diethylenetriaminepentaacetic acid, ethylenediaminetetraacetic acid, trans-1,2-diaminocyclohexanetetraacetic acid, oxalic acid, malonic acid, succinic acid, methanesulfonic acid, 1-hydroxyethylidene-1,1-diphosphonic acid, and nitrilotrismethylenephosphonic acid, and more preferably citric acid. In a case where the chemical liquid contains a chelating agent, in view of further improving the effects of the present invention, the content of the chelating agent (particularly in a case where the first metal-containing substance is a cobalt-containing substance such as simple cobalt, a cobalt alloy, a cobalt oxide, or a cobalt nitride) with respect to the total mass of the chemical liquid is preferably 0.01% to 20% by mass, more preferably 0.1% to 15% by mass, even more preferably 0.1% to 5% by mass, and particularly preferably more than 0.2% by mass and 1% by mass or less. In a case where the first metal-containing substance is a ruthenium-containing substance (such as simple ruthenium, a ruthenium alloy, a ruthenium oxide, or a ruthenium nitride), the content of the chelating agent is preferably 10% to 20% by mass with respect to the total mass of the chemical liquid. In a case where the first metal-containing substance is a molybdenum-containing substance (such as simple molybdenum, a molybdenum alloy, a molybdenum oxide, or a molybdenum nitride), the content of the chelating agent is preferably 0.5% to 15% by mass with respect to the total mass of the chemical liquid. In a case where the first metal-containing substance is an aluminum-containing substance (such as simple aluminum, an aluminum alloy, an aluminum oxide, or a aluminum nitride), the content of the chelating agent is preferably more than 0.2% by mass and 20% by mass or less (more preferably 5% to 15% by mass) with respect to the total mass of the chemical liquid. In a case where the first metal-containing substance is a copper-containing substance (such as simple copper, a copper alloy, a copper oxide, or a copper nitride), the content of the chelating agent is preferably more than 0.2% by mass and 1% by mass or less with respect to the total mass of the chemical liquid. One kind of chelating agent may be used alone, or two or more kinds of chelating agents may be used. In a case where two or more kinds of chelating agents are used, the total amount thereof is preferably within the above range. In view of further improving the effects of the present invention, the mass ratio of the content of the chelating agent to the content of the specific compound (content of chelating agent/content of specific compound) is (particularly in a case where the first metal-containing substance is a cobalt-containing substance) preferably 1.0×10−2to 5.0×106, more preferably 1.0×100to 5.0×104, and even more preferably 7.0×100to 5.0×101. In a case where the first metal-containing substance is a molybdenum-containing substance, the above mass ratio is preferably 1.5×101to 5.0×102. In a case where the first metal-containing substance is a ruthenium-containing substance, the above mass ratio is preferably 1.0×102to 1.0×103. In a case where the first metal-containing substance is an aluminum-containing substance, the above mass ratio is preferably 7.0×100to 5.0×103(more preferably 1.0×102to 1.0×103). In a case where the first metal-containing substance is a copper-containing substance, the above mass ratio is preferably 7.0×100to 5.0×101. <Metal Component> The chemical liquid may contain a metal component. Examples of the metal component include metal particles and metal ions. For example, the content of the metal component means the total content of metal particles and metal ions. The chemical liquid may contain either metal particles or metal ions, or may contain both of them. It is preferable that the chemical liquid contain both the metal particles and metal ions. Examples of the metal atom contained in the metal component include metal atoms selected from the group consisting of Ag, Al, As, Au, Ba, Ca, Cd, Co, Cr, Cu, Fe, Ga, Ge, K, Li, Mg, Mn, Mo, Na, Ni, Pb, Sn, Sr, Ti, and Zn. The metal component may contain one kind of metal atom or two or more kinds of metal atoms. The metal particles may be a simple metal or an alloy, and may be in the form of particles in which a metal and an organic substance are aggregated. The metal component may be a metal component which is inevitably incorporated into each component (raw material) of the chemical liquid or a metal component inevitably incorporated into the chemical liquid during the manufacturing, storage, and/or transfer of the chemical liquid. Alternatively, the metal component may be intentionally added. In a case where the chemical liquid contains a metal component, the content of the metal component is usually 0.01 ppt by mass to 10 ppm by mass with respect to the total mass of the chemical liquid. In view of further improving the effects of the present invention, the content of the metal component is preferably 0.1 ppt by mass to 1 ppm by mass, and more preferably 0.01 ppb by mass to 100 ppb by mass. The mass ratio of the content of the metal component to the content of the specific compound is not particularly limited, and is usually 10−9to 108. In view of further improving the effects of the present invention, the mass ratio is preferably 10−8to 107. The type and content of the metal component in the chemical liquid can be measured by single nano particle inductively coupled plasma mass spectrometry (SP-ICP-MS). The device used in SP-ICP-MS is the same as the device used in general inductively coupled plasma mass spectrometry (ICP-MS). The only difference between SP-ICP-MS and ICP-MS is how to analyze data. With SP-ICP-MS, data can be analyzed using commercial software. With ICP-MS, the content of a metal component as a measurement target is measured regardless of the way the metal component is present. Accordingly, the total mass of metal particles and metal ions as a measurement target is quantified as the content of the metal component. With SP-ICP-MS, the content of metal particles can be measured. Accordingly, by subtracting the content of the metal particles from the content of the metal component in a sample, the content of metal ions in the sample can be calculated. Examples of the device for SP-ICP-MS include Agilent 8800 triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS, for semiconductor analysis, option #200) manufactured by Agilent Technologies, Inc. By using this device, the content of the metal-containing particles can be measured by the method described in Examples. In addition to the device described above, it is possible to use NexION350S manufactured by PerkinElmer Inc. and Agilent 8900 manufactured by Agilent Technologies, Inc. <Reducing Agent Different from Hydroxylamine Compound> The chemical liquid may contain a reducing agent different from the hydroxylamine compound. Here, the chelating agent is not included in the reducing agent different from the hydroxylamine compound. The reducing agent different from the hydroxylamine compound is not particularly limited. The reducing agent is preferably a reducing substance such as a compound having a OH group or a CHO group or a compound containing a sulfur atom. The reducing agent is oxidative and has a function of oxidizing OW ions, dissolved oxygen, and the like which cause decomposition of the hydroxylamine compound. Among the reducing substances such as the compound having a OH group or a CHO group and the compound containing a sulfur atom, one kind of compound selected from the group consisting of a compound represented by Formula (4) and a compound having a sulfur atom is preferable. In Formula (4), R4ato R4eeach independently represent a hydrogen atom, a hydroxyl group, or a hydrocarbon group which may have a heteroatom. In a case where R4ato R4ehave a hydrocarbon group which may have a heteroatom, the hydrocarbon group may have a substituent. Examples of the hydrocarbon group represented by R4ato R4ein Formula (4) that may have a heteroatom include a hydrocarbon group and a hydrocarbon group having a heteroatom. Examples of the hydrocarbon group represented by R4ato R4einclude an alkyl group (preferably having 1 to 12 carbon atoms, and more preferably having 1 to 6 carbon atoms), an alkenyl group (preferably having 2 to 12 carbon atoms, and more preferably having 2 to 6 carbon atoms), an alkynyl group (preferably having 2 to 12 carbon atoms, and more preferably having 2 to 6 carbon atoms), an aryl group (preferably having 6 to 22 carbon atoms, more preferably having 6 to 14 carbon atoms, and even more preferably having 6 to 10 carbon atoms), and an aralkyl group (preferably having 7 to 23 carbon atoms, more preferably having 7 to 15 carbon atoms, and even more preferably having 7 to 11 carbon atoms). Examples of the hydrocarbon group represented by R4ato R4ehaving a heteroatom include a group formed in a case where —CH2— in the aforementioned hydrocarbon group is substituted, for example, with one kind of substituent selected from the group consisting of —O—, —S—, —CO—, —SO2—, and —NRa— or with a divalent group formed by combining two or more substituents among the above. Rarepresents a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms (preferably an alkyl group having 1 to 5 carbon atoms). Examples of substituents include a hydroxyl group, a carboxyl group, and a substituted or unsubstituted amino group (the substituent is preferably an alkyl group having 1 to 6 carbon atoms and more preferably an alkyl group having 1 to 3 carbon atoms). Examples of the compound represented by Formula (4) include gallic acid, resorcinol, ascorbic acid, tert-butylcatechol, catechol, isoeugenol, o-methoxyphenol, 4,4′-dihydroxyphenyl-2,2-propane, isoamyl salicylate, benzyl salicylate, methyl salicylate, and 2,6-di-t-butyl-p-cresol. In view of adding reducing properties, the compound represented by Formula (4) preferably has two or more hydroxyl groups, and more preferably has three or more hydroxyl groups. The position of substitution with a hydroxyl group is not particularly limited. In view of adding reducing properties, the position is preferably R4aand/or R4b. Examples of the compound represented by Formula (4) having two or more hydroxyl groups include catechol, resorcinol, tert-butylcatechol, and 4,4′-dihydroxyphenyl-2,2-propane. Examples of the compound represented by Formula (4) having three or more hydroxyl groups include gallic acid. Examples of the compound containing a sulfur atom include mercaptosuccinic acid, dithiodiglycerol [S(CH2CH(OH)CH2(OH))2], bis(2,3-dihydroxypropylthio)ethylene [CH2CH2(SCH2CH(OH)CH2(OH))2], sodium 3-(2,3-dihydroxypropylthio)-2-methyl-propylsulfonate [CH2(OH)CH(OH)CH2SCH2CH(CH3)CH2SO3Na], 1-thioglycerol [HSCH2CH(OH)CH2(OH)], sodium 3-mercapto-1-propanesulfonate [HSCH2CH2CH2SO3Na], 2-mercaptoethanol [HSCH2CH2(OH)], thioglycolic acid [HSCH2CO2H], and 3-mercapto-1-propanol [HSCH2CH2CH2OH]. Among these, a compound having a SH group (mercapto compound) is preferable, 1-thioglycerol, sodium 3-mercapto-1-propanesulfonate, 2-mercaptoethanol, 3-mercapto-1-propanol, or thioglycolic acid is more preferable, and 1-thioglycerol or thioglycolic acid is even more preferable. One kind of reducing agent different from the hydroxylamine compound may be used alone, or two or more kinds of such reducing agents may be used in combination. <pH Adjuster> The chemical liquid may contain a pH adjuster other than the components described above. Examples of the pH adjuster include an acid compound and a base compound. (Acid Compound) Examples of the acid compound include sulfuric acid, hydrochloric acid, acetic acid, nitric acid, hydrofluoric acid, perchloric acid, hypochlorous acid, and periodic acid. (Base Compound) Examples of the base compound include aqueous ammonia, an amine compound different from a hydroxylamine compound, and a quaternary ammonium hydroxide salt. Examples of the amine compound different from a hydroxylamine compound include a cyclic compound (a compound having a cyclic structure). Examples of the cyclic compound include an amine compound having a cyclic structure that will be described later. The quaternary ammonium hydroxide salt is not included in the amine compound different from a hydroxylamine compound. As the amine compound different from a hydroxylamine compound, an amine compound having a cyclic structure is preferable. In the amine compound having a cyclic structure, an amino group may be in either or both of the aforementioned cyclic structure and any position other than the cyclic structure. Examples of the amine compound having a cyclic structure include tetrahydrofurfurylamine, N-(2-aminoethyl)piperazine, 1,8-diazabicyclo[5.4.0]-7-undecene, 1,4-diazabicyclo[2.2.2]octane, hydroxyethyl piperazine, piperazine, 2-methylpiperazine, trans-2,5-dimethylpiperazine, cis-2,6-dimethylpiperazine, 2-piperidinemethanol, cyclohexylamine, and 1,5-diazabicyclo[4,3,0]-5-nonene. As the amine compound, among these, tetrahydrofurfurylamine, N-(2-aminoethyl)piperazine, 1,8-diazabicyclo[5.4.0]-7-undecene, or 1,4-diazabicyclo[2.2.2]octane is preferable. In a case where the chemical liquid contains the amine compound different from a hydroxylamine compound, the content of the amine compound different from a hydroxylamine compound with respect to the total mass of the chemical liquid is preferably 0.1% to 50% by mass, and more preferably 0.5% to 30% by mass. Examples of the quaternary ammonium hydroxide salt include a compound represented by Formula (5). In Formula (5), R5ato R5deach independently represent an alkyl group having 1 to 16 carbon atoms, an aryl group having 6 to 16 carbon atoms, an aralkyl group having 7 to 16 carbon atoms, or a hydroxyalkyl group having 1 to 16 carbon atoms. At least two of R5ato R5dmay be bonded to each other to form a cyclic structure. Particularly, the groups in at least either a combination of R5aand R5bor a combination of R5cand R5dmay be bonded to each other to form a cyclic structure. As the compound represented by Formula (5), tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, methyltripropylammonium hydroxide, methyltributylammonium hydroxide, ethyltrimethylammonium, dimethyldiethylammonium hydroxide, benzyltrimethylammonium hydroxide, hexadecyltrimethylammonium hydroxide, or (2-hydroxyethyl)trimethylammonium hydroxide is preferable. In a case where the chemical liquid contains a quaternary ammonium hydroxide salt, the content of the quaternary ammonium hydroxide salt with respect to the total mass of the chemical liquid is preferably 0.05% to 10% by mass, and more preferably 0.1% to 5% by mass. As the base compound, a water-soluble amine other than the above compounds can also be used. A pka of the water-soluble amine is preferably 7.5 to 13.0 at room temperature. In the present specification, the water-soluble amine means an amine which can dissolve in an amount of 50 g or more in 1 L of water at room temperature. Aqueous ammonia is not included in the water-soluble amine. Examples of the water-soluble amine having a pKa of 7.5 to 13 include diglycolamine (DGA) (pKa=9.80), methylamine (pKa=10.6), ethylamine (pKa=10.6), propylamine (pKa=10.6), butylamine (pKa=10.6), pentylamine (pKa=10.0), ethanolamine @Ka=9.3), propanolamine (pKa=9.3), butanol amine (pKa=9.3), methoxyethylamine (pKa=10.0), methoxypropylamine (pKa=10.0), dimethylamine (pKa=10.8), diethylamine (pKa=10.9), dipropylamine (pKa=10.8), trimethylamine (pKa=9.80), and triethylamine @Ka=10.72). As the water-soluble amine, unsubstituted hydroxylamine and a hydroxylamine derivative may also be used. In the present specification, the pka of the water-soluble amine is an acid dissociation constant in water. The acid dissociation constant in water can be measured using a spectrometer and potentiometry in combination. As the pH adjuster, particularly, one or more kinds of compounds are preferable which are selected from the group consisting of sulfuric acid, hydrochloric acid, acetic acid, nitric acid, hydrofluoric acid, perchloric acid, hypochlorous acid, periodic acid, tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetrabutylammonium hydroxide, aqueous ammonia, and a water-soluble amine. <Anticorrosive> The chemical liquid may contain an anticorrosive. The anticorrosive has a function of preventing overetching of the object to be treated. The anticorrosive mentioned herein does not include the aforementioned reducing agent different from hydroxylamine and the chelating agent. The anticorrosive is not particularly limited, and examples thereof include 1,2,4-triazole (TAZ), 5-aminotetrazole (ATA), 5-amino-1,3,4-thiadiazole-2-thiol, 3-amino-1H-1,2,4-triazole, 3,5-diamino-1,2,4-triazole, 3-amino-5-mercapto-1,2,4-triazole, 1-amino-1,2,4-triazole, 1-amino-1,2,3-triazole, 1-amino-5-methyl-1,2,3-triazole, 3-mereapto-1,2,4-triazole, 3-isopropyl-1,2,4-triazole, 1H-tetrazole-5-acetic acid, 2-mercaptobenzothiazole (2-MBT), 1-phenyl-2-tetrazoline-5-thione, 2-mercaptobenzimidazole (2-MBI), 4-methyl-2-phenylimidazole, 2-mercaptothiazoline, 2,4-diamino-6-methyl-1,3,5-triazine, thiazole, imidazole, benzimidazole, triazine, methyltetrazole, bismuthiol I, 1,3-dimethyl-2-imidazolidinone, 1,5-pentamethylenetetrazole, 1-phenyl-5-mercaptotetrazole, diaminomethyltriazine, imidazolinethione, 4-methyl-4H-1,2,4-triazole-3-thiol, 5-amino-1,3,4-thiadiazole-2-thiol, benzothiazole, 2,3,5-trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, quinoxaline, acetylpyrrole, pyridazine, and pyrazine. Furthermore, as the anticorrosive, benzotriazoles other than those listed above are also preferable. Examples of the benzotriazoles include benzotriazole (BTA), 1-hydroxybenzotriazole, 5-phenylthiol-benzotriazole, 5-chlorobenzotriazole, 4-chlorobenzotriazole, 5-bromobenzotriazole, 4-bromobenzotriazole, 5-fluorobenzotriazole, 4-fluorobenzotriazole, naphthotriazole, tolyltriazole, 5-phenyl-benzotriazole, 5-nitrobenzotriazole, 4-nitrobenzotriazole, 3-amino-5-mercapto-1,2,4-triazole, 2-(5-amino-pentyl)-benzotriazole, 1-amino-benzotriazole, 5-methyl-1H-benzotriazole, benzotriazole-5-carboxylic acid, 4-methylbenzotriazole, 4-ethylbenzotriazole, 5-ethylbenzotriazole, 4-propylbenzotriazole, 5-propylbenzotriazole, 4-isopropylbenzotriazole, 5-isopropylbenzotriazole, 4-n-butylbenzotriazole, 5-n-butylbenzotriazole, 4-isobutylbenzotriazole, 5-isobutylbenzotriazole, 4-pentylbenzotriazole, 5-pentylbenzotriazole, 4-hexylbenzotriazole, 5-hexylbenzotriazole, 5-methoxybenzotriazole, 5-hydroxybenzotriazole, dihydroxypropylbenzotriazole, 1-[N,N-bis(2-ethylhexyl)aminomethyl]-benzotriazole, 5-t-butylbenzotriazole, 5-(1′,1′-dimethylpropyl)-benzotriazole, 5-(1′,1′,3′-trimethylbutyl)benzotriazole, 5-n-octylbenzotriazole, and 5-(1′,1′,3′,3′-tetramethylbutyl)benzotriazole. In view of further improving anticorrosion performance, a compound represented by Formula (6) is particularly preferable as the anticorrosive. In Formula (6), R6a, R6b, and R6ceach independently represent a hydrogen atom or a substituted or unsubstituted hydrocarbon group. Furthermore, R6aand R6bmay be bonded to each other to form a ring. Examples of the hydrocarbon group represented by R6aand R6bin Formula (6) include an alkyl group (preferably having 1 to 12 carbon atoms, more preferably having 1 to 6 carbon atoms, and even more preferably having 1 to 3 carbon atoms), an alkenyl group (preferably having 2 to 12 carbon atoms, and more preferably having 2 to 6 carbon atoms), an alkynyl group (preferably having 2 to 12 carbon atoms, and more preferably having 2 to 6 carbon atoms), an aryl group (preferably having 6 to 22 carbon atoms, more preferably having 6 to 14 carbon atoms, and even more preferably having 6 to 10 carbon atoms), and an aralkyl group (preferably having 7 to 23 carbon atoms, more preferably having 7 to 15 carbon atoms, and even more preferably having 7 to 11 carbon atoms). The substituent is not particularly limited, and examples thereof include a hydroxyl group, a carboxyl group, and a substituted or unsubstituted amino group (the substituent is preferably an alkyl group having 1 to 6 carbon atoms and more preferably an alkyl group having 1 to 3 carbon atoms). R6aand R6bmay be bonded to each other to form a ring. Examples of the ring include a benzene ring and a naphthalene ring. In a case where R6aand R6bare bonded to each other to form a ring, the ring may further have a substituent (such as a hydrocarbon group having 1 to 5 carbon atoms or a carboxyl group). Examples of the compound represented by Formula (6) include 1H-1,2,3-triazole, benzotriazole, and carboxybenzotriazole 5-methyl-1H-benzotriazole. In a case where the chemical liquid contains an anticorrosive, the content of the anticorrosive with respect to the total mass of the chemical liquid is preferably 0.01% to 10% by mass, and more preferably 0.05% to 5% by mass. <Chemical Liquid Manufacturing Method> The method for manufacturing the chemical liquid is not particularly limited, and known manufacturing methods can be used. For example, a method of mixing together water, a hydroxylamine compound, and a predetermined amount of specific compound may be used. In mixing the above components, if necessary, other optional components may be mixed together. Furthermore, in manufacturing the chemical liquid, if necessary, the chemical liquid may be purified by being filtered using a filter. In view of further improving the effects of the present invention, the pH of the chemical liquid is preferably 1 to 13, and more preferably 2 to 12. The chemical liquid according to the embodiment of the present invention is a chemical liquid used for an object to be treated containing a first metal-containing substance (material containing a metal selected from the group consisting of cobalt, ruthenium, molybdenum, aluminum, and copper). The object to be treated for which the chemical liquid is to be used may contain at least one kind of first metal-containing substance. It is preferable that the object to be treated also contain another metal-containing substance (material containing a metal). The aforementioned metal-containing substance is a material containing a metal, and may either or both of a first metal-containing substance other than the aforementioned “at least one kind of first metal-containing substance” and a second metal-containing substance which is a material different from the first metal-containing substance and contains a metal. For example, it is preferable that the chemical liquid be used for an object to be treated which contains at least two kinds of first metal-containing substances or used for an object to be treated which contains the first metal-containing substance and the second metal-containing substance. In addition, the chemical liquid may be used for an object to be treated which contains at least two kinds of first metal-containing substances and the second metal-containing substance. It is preferable that the chemical liquid be used for an object to be treated which is a treatment target containing at least two kinds of metal-containing substances in which the absolute value of a difference in a corrosion potential between the two kinds of metal-containing substances in the chemical liquid is 0.5 V or less (more preferably 0.3 V or less). It is preferable that at least one of the two kinds of metal-containing substances be the first metal-containing substance. For example, in a case where the chemical liquid is used for an object to be treated which contains at least two kinds of first metal-containing substances, it is preferable that the absolute value of a difference in a corrosion potential between the two kinds of first metal-containing substances in the chemical liquid be 0.5 V or less (more preferably 0.3 V or less). In a case where the object to be treated contains more than two kinds of first metal-containing substances, it is preferable that at least a combination (preferably all combinations) consisting of two kinds of first metal-containing substances in the object to be treated satisfy the range of the absolute value described above. Furthermore, for example, In a case where the object to be treated contains the first metal-containing substance and the second metal-containing substance, it is preferable that the absolute value of a difference between a corrosion potential of the first metal-containing substance in the chemical liquid and a corrosion potential of the second metal-containing substance in the chemical liquid be 0.5 V or less (more preferably 0.3 V or less). In a case where the object to be treated contains two or more kinds of first metal-containing substances and/or two or more kinds of second metal-containing substances, it is preferable that at least a combination (preferably all combinations) consisting of one kind of first metal-containing substance and one kind of second metal-containing substance in the object to be treated satisfy the range of the absolute value described above. Details of the first metal-containing substance and the second metal-containing substance will be described later. The corrosion potential is measured by the following method. First, as measurement targets, a silicon wafer with a first metal-containing substance disposed on the wafer surface and a silicon wafer with a first metal-containing substance different from the above first metal-containing substance or a second metal-containing substance disposed on the wafer surface are prepared, and used as electrodes. Then, the prepared electrodes are immersed in a predetermined chemical liquid, the corrosion potential is measured based on the Tafel plot obtained using a potentiostat/galvanostat (Princeton Applied Research VersaSTAT 4), and the absolute value of a difference between corrosion potentials obtained from the two electrodes is determined. The corrosion potential corresponds to the potential of the inflection point of the curve of the Tafel plot. The measurement conditions are as follows.Current range: ±0.2 V (vs open circuit potential)Scan rate: 1.0 mV/s (0.5 mV per session)Counter electrode: PtReference electrode: Ag/AgClMeasurement temperature: 25° C. <Chemical Liquid Storage Body> The chemical liquid may be stored in a container and kept as it is until use. The container and the chemical liquid stored in the container are collectively called chemical liquid storage body. The stored chemical liquid is used after being taken out of the chemical liquid storage body. Furthermore, the chemical liquid may be transported as a chemical liquid storage body. It is preferable to use a container for semiconductors which has a high internal cleanliness and hardly causes elution of impurities. Examples of usable containers include a “CLEAN BOTTLE” series manufactured by AICELLO CORPORATION, and “PURE BOTTLE” manufactured by KODAMA PLASTICS Co., Ltd. It is preferable that the inner wall of the container be formed of one or more kinds of resins selected from the group consisting of a polyethylene resin, a polypropylene resin, and a polyethylene-polypropylene resin, or formed of a resin different from these. It is also preferable that the inner wall of the container be formed of a metal having undergone a rustproofing treatment or a metal elution preventing treatment, such as stainless steel, Hastelloy, Inconel, or Monel. As “resin different from these” described above, a fluororesin (perfluororesin) is preferable. In a case where a container having inner wall made of a fluororesin is used, the occurrence of problems such as elution of an ethylene or propylene oligomer can be further suppressed, than in a case where a container having inner wall formed of a polyethylene resin, a polypropylene resin, or a polyethylene-polypropylene resin is used. Examples of the container having inner wall made of a fluororesin include a FluoroPure PFA composite drum manufactured by Entegris, and the like. In addition, it is also possible to use the containers described on page 4 of JP1991-502677A (JP-H03-502677A), page 3 of WO2004/016526A, pages 9 and 16 of the WO99/46309A, and the like. Furthermore, in addition to the fluororesin described above, quartz and an electropolished metallic material (that is, a metallic material having undergone electropolishing) are also preferably used for the inner wall of the container. For manufacturing the electropolished metallic material, it is preferable to use a metallic material which contains at least one kind of metal selected from the group consisting of chromium and nickel, and in which the total content of chromium and nickel is more than 25% by mass with respect to the total mass of the metallic material. Examples of such a metallic material include stainless steel and a nickel-chromium alloy. The total content of chromium and nickel in the metallic material is preferably 30% by mass or more with respect to the total mass of the metallic material. The upper limit of the total content of chromium and nickel in the metallic material is not particularly limited, but is preferably 90% by mass or less with respect to the total mass of the metallic material. The stainless steel is not particularly limited, and known stainless steel can be used. Particularly, an alloy with a nickel content of 8% by mass or more is preferable, and austenite-based stainless steel with a nickel content of 8% by mass or more is more preferable. Examples of the austenite-based stainless steel include Steel Use Stainless (SUS) 304 (Ni content: 8% by mass, Cr content: 18% by mass), SUS304L (Ni content: 9% by mass, Cr content: 18% by mass), SUS316 (Ni content: 10% by mass, Cr content: 16% by mass), and SUS316L (Ni content: 12% by mass, Cr content: 16% by mass). The nickel-chromium alloy is not particularly limited, and known nickel-chromium alloys can be used. Among these, a nickel-chromium alloy is preferable in which the nickel content is 40% to 75% by mass and the chromium content is 1% to 30% by mass. Examples of the nickel-chromium alloy include HASTELLOY (trade name, the same is true of the following description), MONEL (trade name, the same is true of the following description), and INCONEL (trade name, the same is true of the following description). More specifically, examples thereof include HASTELLOY C-276 (Ni content: 63% by mass, Cr content: 16% by mass), HASTELLOY C (Ni content: 60% by mass, Cr content: 17% by mass), and HASTELLOY C-22 (Ni content: 61% by mass, Cr content: 22% by mass). Furthermore, if necessary, the nickel-chromium alloy may further contain boron, silicon, tungsten, molybdenum, copper, or cobalt, in addition to the aforementioned alloy. The method of electropolishing the metallic material is not particularly limited, and known methods can be used. For example, it is possible to use the methods described in paragraphs “0011” to “0014” in JP2015-227501A, paragraphs “0036” to “0042” in JP2008-264929A, and the like. It is preferable that the metallic material have undergone buffing. As the buffing method, known methods can be used without particular limitation. The size of abrasive grains used for finishing the buffing is not particularly limited, but is preferably #400 or less because such grains make it easy to further reduce the surface asperity of the metallic material. The buffing is preferably performed before the electropolishing. Furthermore, one of the multistage buffing carried out by changing the size of abrasive grains, acid pickling, magnetorheological finishing, and the like or a combination of two or more treatments selected from the above may be performed on the metallic material. It is preferable that the inside of these containers be washed before the containers are filled with the chemical liquid. For washing, it is preferable to use a liquid with a lower metal impurity content. After being manufactured, the chemical liquid may be bottled using a container, such as a gallon bottle or a quart bottle, and transported or stored. In order to prevent changes in the components of the chemical liquid during storage, the inside of the container may be purged with an inert gas (such as nitrogen or argon) having a purity of 99.99995% by volume or higher. Particularly, a gas with a low moisture content is preferable. Although the chemical liquid may be transported and stored at room temperature, in order to prevent deterioration, the temperature may be controlled in a range of −20° C. to 20° C. The chemical liquid may be prepared as a kit composed of a plurality of separated raw materials of the chemical liquid. Furthermore, the chemical liquid may be prepared as a concentrated solution. In a case where the chemical liquid is prepared as a concentrated solution, the concentration factor is appropriately determined depending on the composition, but is preferably 5× to 2,000×. That is, the concentrated solution is used after being diluted 5× to 2,000×. [Method for Treating Object to be Treated] In the method for treating an object to be treated according to an embodiment of the present invention (hereinafter, also simply described as “the present treatment method”), it is preferable to use the chemical liquid by bringing the chemical liquid into contact with the object to be treated containing the first metal-containing substance, so that the first metal-containing substance is dissolved (etched). The chemical liquid effectively functions as a so-called etching treatment liquid. The object to be treated may contain both the first metal-containing substance and second metal-containing substance (material which is different from the first metal-containing substance and contains a metal). In this case, the chemical liquid may be used for etching only the first metal-containing substance, or used for etching both the first metal-containing substance and second metal-containing substance. The form of the object to be treated is not particularly limited. For example, the object may be an object10to be treated shown inFIG.1having a substrate12, an insulating film14with hole portions that is disposed on the substrate12, a second metal-containing substance portion16disposed in the form of a layer along the inner wall of the hole portions of the insulating film14, and a first metal-containing substance portion18with which the hole portions are filled. InFIG.1, the second metal-containing substance portion can function as a barrier metal layer. AlthoughFIG.1shows an aspect in which the object to be treated has one first metal-containing substance portion, the object to be treated is not limited to this aspect. For example, the object may be an object20to be treated shown inFIG.2having a substrate12, an insulating film14with a plurality of hole portions that is disposed on the substrate12, a second metal-containing substance portion16disposed in the form of a layer along the inner wall of each of the hole portions of the insulating film14, and a first metal-containing substance portion18with which each of the hole portions is filled. That is, the object to be treated may have an aspect in which each of the first metal-containing substance and the second metal-containing substance is at a plurality of sites. The type of substrate that may be contained in the object to be treated is not particularly limited. Examples of the substrate include various substrates such as a semiconductor wafer, a glass substrate for a photomask, a glass substrate for liquid crystal display, a glass substrate for plasma display, a substrate for field emission display (FED), a substrate for an optical disk, a substrate for a magnetic disk, and a substrate for a magneto-optical disk. Examples of materials constituting the semiconductor substrate include silicon, silicon germanium, a Group III-V compound such as GaAs, and any combination of these. The size, thickness, shape, layer structure, and the like of the substrate are not particularly limited, and can be appropriately selected as desired. As the insulating film, known insulating films are used. The insulating film inFIGS.1and2has hole portions. However, the insulating film is not limited to this aspect, and may be an insulating film having groove portions. The first metal-containing substance may be a material that can be etched with the chemical liquid and contains metal atoms. Particularly, the metal atoms contained in the first metal-containing substance are preferably one or more kinds of metals selected from the group consisting of cobalt, ruthenium, molybdenum, aluminum, and copper. The first metal-containing substance is preferably one kind of simple metal listed above or an oxide or nitride of one kind of metal listed above. Furthermore, the first metal-containing substance is preferably an alloy, a composite oxide, or a composite nitride of two or more kinds of metals listed above. Among these, a cobalt-containing substance that contains cobalt atoms (for example, simple cobalt, a cobalt alloy, a cobalt oxide, or a cobalt nitride) is preferable as the first metal-containing substance. The metal atoms contained in the second metal-containing substance are preferably one or more kinds of metals selected from the group consisting of cobalt, ruthenium, titanium, and tantalum. The second metal-containing substance is preferably one kind of simple metal listed above or an oxide or nitride of one kind of metal listed above. Furthermore, the second metal-containing substance is preferably an alloy, a composite oxide, or a composite nitride of two kinds of metals listed above. Among these, a titanium-containing substance that contains titanium atoms (for example, simple titanium, a titanium oxide, or a titanium nitride) is preferable as the second metal-containing substance. In a case where the object to be treated contains the second metal-containing substance, the first metal-containing substance and the second metal-containing substance may be in contact with each other in the object to be treated or may be arranged via other members. The forms of the first metal-containing substance and the second metal-containing substance are not particularly limited. For example, the first and second metal-containing substances may be in the form of a film, wiring, or particles. In a case where the first metal-containing substance and the second metal-containing substance are in the form of a film, the thickness thereof is not particularly limited and may be appropriately selected depending on the use. For example, the thickness is preferably 50 nm or less, more preferably 20 nm or less, and even more preferably 10 nm or less. The first metal-containing substance and the second metal-containing substance may be disposed only on one of the main surfaces of the substrate, or may be disposed on both the main surfaces of the substrate. Furthermore, the first metal-containing substance and the second metal-containing substance may be disposed on the entire main surface of the substrate, or may be disposed on a portion of the main surface of the substrate. As described above, the object to be treated may contain two or more kinds of first metal-containing substances, and may contain, in addition to the first metal-containing substance, the second metal-containing substance which is a material different from the first metal-containing substance and contains a metal. In a case where the object to be treated contains two or more kinds of metal-containing substances, in an aspect, examples of combinations thereof include a combination of a cobalt-containing substance as a first metal-containing substance and a titanium-containing substance or tantalum-containing substance (for example, simple tantalum, a tantalum oxide, or a tantalum nitride) as a second metal-containing substance. The object to be treated may include various layers and/or structures as desired, in addition to the first metal-containing substance and the second metal-containing substance. For example, the substrate may have metal wiring, a gate electrode, a source electrode, a drain electrode, an insulating layer, a ferromagnetic layer, and/or a non-magnetic layer, and the like. The substrate may include the structure of an exposed integrated circuit, for example, an interconnection mechanism such as metal wiring and a dielectric material. Examples of metals and alloys used for the interconnection mechanism include aluminum, a copper-aluminum alloy, copper, titanium, tantalum, cobalt, silicon, titanium nitride, tantalum nitride, and tungsten. The substrate may include a layer of silicon oxide, silicon nitride, silicon carbide, and/or carbon-doped silicon oxide. The method for manufacturing the object to be treated is not particularly limited. For example, the object to be treated shown inFIG.1may be manufactured by a method of forming an insulating film on a substrate, forming hole portions or groove portions in the insulating film, arranging a metal-containing substance layer and a cobalt-containing substance layer in this order on the insulating film by a sputtering method, a chemical vapor deposition (CVD) method, a molecular beam epitaxy (MBE) method, or the like, and then performing a smoothing treatment such as CMP. <First Aspect> Examples of the method for treating an object to be treated according to an embodiment of the present invention include a method having a step A of bringing the object to be treated containing at least the first metal-containing substance into contact with the chemical liquid so that the first metal-containing substance is dissolved. This method for treating an object to be treated is also called a first aspect of the method for treating an object to be treated according to the embodiment of the present invention. The method of bringing the object to be treated into contact with the chemical liquid is not particularly limited, and examples thereof include a method of immersing the object to be treated in the chemical liquid stored in a tank, a method of spraying the chemical liquid onto the object to be treated, a method of causing the chemical liquid to flow on the object to be treated, and a combined method consisting of any of the above methods. Among these, the method of immersing the object to be treated in the chemical liquid is preferable. In order to further enhance the washing ability of the chemical liquid, a mechanical stirring method may also be used. Examples of the mechanical stirring method include a method of circulating the chemical liquid on an object to be treated, a method of causing the chemical liquid to flow on the object to be treated or spraying the chemical liquid onto the object to be treated, and a method of stirring the chemical liquid by using ultrasonic or megasonic waves. The contact time between the object to be treated and the chemical liquid can be adjusted as appropriate. The treatment time (the contact time between the chemical liquid and the object to be treated) is not particularly limited, but is preferably 0.25 to 10 minutes, and more preferably 0.5 to 2 minutes. The temperature of the chemical liquid during the treatment is not particularly limited, but is preferably 20° C. to 75° C. and more preferably 20° C. to 60° C. By the treatment performed as above, mainly the first metal-containing substance in the object to be treated is dissolved. In a case where the object to be treated contains the second metal-containing substance in addition to the first metal-containing substance, the second metal-containing substance may or may not be dissolved together with the first metal-containing substance by this treatment. In a case where the second metal-containing substance is dissolved, the dissolution of the second metal-containing substance may be intentional or inevitable. In a case where the dissolution of the second metal-containing substance is unintentional, it is preferable that the amount of the inevitably dissolved second metal-containing substance be small. In a case where the dissolution of the second metal-containing substance is unintentional, and the amount of the inevitably dissolved second metal-containing substance is small, the chemical liquid is also described as being excellent in member resistance of the second metal-containing substance. For example, the chemical liquid according to the embodiment of the present invention is excellent in member resistance of a tantalum nitride. <Second Aspect> Furthermore, for example, in another aspect, the method for treating an object to be treated according to an embodiment of the present invention has a step A of bringing the object to be treated containing the first metal-containing substance and the second metal-containing substance into contact with the chemical liquid so that the first metal-containing substance is dissolved, and a step B of bringing the object to be treated into contact with a solution selected from the group consisting of a mixed aqueous solution of ammonia and aqueous hydrogen peroxide, a mixed aqueous solution of hydrofluoric acid and aqueous hydrogen peroxide, a mixed aqueous solution of sulfuric acid and aqueous hydrogen peroxide, and a mixed aqueous solution of hydrochloric acid and aqueous hydrogen peroxide (hereinafter, the solution will be also simply called “specific solution”) before or after the step A so that the second metal-containing substance is dissolved. This method for treating an object to be treated is also called a second aspect of the method for treating an object to be treated according to the embodiment of the present invention. The second aspect can also be said to be a form of the first aspect. In the step A, the second metal-containing substance may be intentionally or inevitably dissolved. Furthermore, in the step B, the first metal-containing substance may be intentionally or inevitably dissolved. The procedure of the step A is as described above. In some cases, the second metal-containing substance in the object to be treated may have different solubility in the aforementioned chemical liquid, depending on the type of metal atoms that the chemical liquid contains. In these cases, it is preferable to adjust the extent of dissolution of the first metal-containing substance and the second metal-containing substance by using a solution that excellently dissolves the second metal-containing substance. Such an adjustment procedure corresponds to the step B of bringing the object to be treated, which has been or has not yet been subjected to the step A, into contact with the specific solution so that the second metal-containing substance is dissolved. The specific solution is a solution selected from the group consisting of a mixed aqueous solution of ammonia and aqueous hydrogen peroxide (APM), a mixed aqueous solution of hydrofluoric acid and aqueous hydrogen peroxide (FPM), a mixed aqueous solution of sulfuric acid and aqueous hydrogen peroxide (SPM), and a mixed aqueous solution of hydrochloric acid and hydrogen peroxide (HPM). The composition of APM is, for example, preferably in a range of “aqueous ammonia:aqueous hydrogen peroxide:water=1:1:1” to “aqueous ammonia:aqueous hydrogen peroxide:water=1:3:45” (volume ratio). The composition of FPM is, for example, preferably in a range of “hydrofluoric acid:aqueous hydrogen peroxide:water=1:1:1” to “hydrofluoric acid:aqueous hydrogen peroxide:water=1:1:200” (volume ratio). The composition of SPM is, for example, preferably in a range of “sulfuric acid:aqueous hydrogen peroxide:water=3:1:0” to “sulfuric acid:aqueous hydrogen peroxide:water=1:1:10” (volume ratio). The composition of HPM is, for example, preferably in a range of “hydrochloric acid:aqueous hydrogen peroxide:water=1:1:1” to “hydrochloric acid:aqueous hydrogen peroxide:water=1:1:30” (volume ratio). The preferred compositional ratio described above means a compositional ratio determined in a case where the content of aqueous ammonia is 28% by mass, the content of hydrofluoric acid is 49% by mass, the content of sulfuric acid is 98% by mass, the content of hydrochloric acid is 37% by mass, and the content of aqueous hydrogen peroxide is 30% by mass. Furthermore, the volume ratio is based on a volume at room temperature. “A:B:C=x:y:z to A:B:C=X:Y:Z” used above to describe a preferable range means that it is preferable that at least one (preferably two and more preferably all) of “A:B=x:y to A:B=X:Y”, “B:C=y:z to B:C=Y:Z”, or “A:C=x:z to A:C=X:Z” be satisfied. In the step B, the method of bringing the object to be treated, which has been or has not yet been subjected to the step A, into contact with the specific solution by using the specific solution is not particularly limited. Examples of the method include a method of immersing the object to be treated in the specific solution stored in a tank, a method of spraying the specific solution onto the object to be treated, a method of causing the specific solution to flow on the object to be treated, and a combined method consisting of any of the above methods. The contact time between the object to be treated, which has been or has not yet been subjected to the step A, and the specific solution is, for example, preferably 0.25 to 10 minutes, and more preferably 0.5 to 5 minutes. The step A and the step B may be performed alternately. In a case where the steps are performed alternately, it is preferable that each of the step A and the step B be performed 1 to 20 times. <Third Aspect> In another aspect of the method for treating an object to be treated according to an embodiment of the present invention, for example, the chemical liquid is used and applied to a substrate having undergone dry etching so that the first metal-containing substance is dissolved and dry etching residues on the substrate are removed. More specifically, this is a method of bringing the chemical liquid into contact with an object to be treated which contains the first metal-containing substance and dry etching residues on the surface thereof (and the second metal-containing substance as desired) so that the first metal-containing substance is dissolved and the dry etching residues on the surface of the object to be treated are removed. This method for treating an object to be treated is also called a third aspect of the method for treating an object to be treated according to the embodiment of the present invention. The third aspect can be said to be a form of the first aspect described above that is accomplished by specifically restricting the constitution of the object to be treated and the purpose of the treatment in the first aspect. The chemical liquid according to the embodiment of the present invention can also be suitably used for washing the object to be treated (for removing residues) as described above. FIG.3is a schematic view showing an example of the object to be treated in the third aspect. An object30to be treated shown inFIG.3comprises a metal-containing film34, an etch stop layer36, an interlayer insulating film38, a metal hard mask40in this order on a substrate32. Through a dry etching step or the like, a hole42exposing the metal-containing film34is formed at a predetermined position. That is, the object to be treated shown inFIG.3is a laminate which comprises the substrate32, the metal-containing film34, the etch stop layer36, the interlayer insulating film38, and the metal hard mask40in this order and comprises the hole42that extends from the surface of the metal hard mask40to the surface of the metal-containing film34at the position of the opening portion of the mask40. An inner wall44of the hole42is constituted with a cross-sectional wall44awhich includes the etch stop layer36, the interlayer insulating film38, and the metal hard mask40, and a bottom wall44bwhich includes the exposed metal-containing film34. A dry etching residue46is attached to the inner wall44. The metal hard mask in the object to be treated may turn into a barrier metal after the object to be treated is further processed. In other words, the layer that will turn into a barrier metal in the subsequent step may be used as a metal hard mask in the dry etching step. That is, the barrier metal may be used as a metal hard mask. For example, it is preferable that at least one of the metal-containing film34or the metal hard mask40be the first metal-containing substance. Especially, it is preferable that the metal-containing film34and the metal hard mask40be the first metal-containing substance and the second metal-containing substance respectively, or that the metal-containing film34and the metal hard mask40be the second metal-containing substance and the first metal-containing substance respectively. It is particularly preferable that the metal-containing film34and the metal hard mask40be the first metal-containing substance and the second metal-containing substance respectively. As the interlayer insulating film, known materials can be used. The dry etching residues may contain the first metal-containing substance. Examples of the specific method of the third aspect include a method of bringing the aforementioned object to be treated into contact with the chemical liquid. The method of bringing the object to be treated into contact with the chemical liquid is as described above in the first aspect. By the method of bringing the chemical liquid into contact with the object to be treated, usually, the dry etching residues on the object to be treated are removed, and the first metal-containing substance (preferably the metal-containing film34) is dissolved. The dissolving amount of the first metal-containing substance is not limited. The dissolution of the first metal-containing substance may be intentional dissolution for removing a part or all of the metal-containing film34(first metal-containing substance) on the bottom wall44bof the hole42, or inevitable dissolution resulting from the contact between the chemical liquid and the first metal-containing substance. If necessary, the present treatment method may include a rinsing step of performing a rinsing treatment on the object to be treated by using a rinsing solution. For example, the method for treating an object to be treated in the first aspect, the second aspect, or the third aspect described above may further include the rinsing step after the procedure described above in each aspect. As the rinsing solution, for example, water, hydrofluoric acid (preferably 0.001% to 1% by mass hydrofluoric acid), hydrochloric acid (preferably 0.001% to 1% by mass hydrochloric acid), aqueous hydrogen peroxide (preferably 0.5% to 31% by mass aqueous hydrogen peroxide, and more preferably 3% to 15% by mass aqueous hydrogen peroxide), a mixed solution of hydrofluoric acid and aqueous hydrogen peroxide (FPM), a mixed solution of sulfuric acid and aqueous hydrogen peroxide (SPM), a mixed solution of aqueous ammonia and aqueous hydrogen peroxide (APM), a mixed solution of hydrochloric acid and aqueous hydrogen peroxide (HPM), aqueous carbon dioxide (preferably 10 to 60 ppm by mass aqueous carbon dioxide), aqueous ozone (preferably 10 to 60 ppm by mass aqueous ozone), aqueous hydrogen (preferably 10 to 20 ppm by mass aqueous hydrogen), an aqueous citric acid solution (preferably a 0.01% to 10% by mass aqueous citric acid solution), sulfuric acid (preferably a 1% to 10% by mass aqueous sulfuric acid solution), aqueous ammonia (preferably 0.01% to 10% by mass aqueous ammonia), isopropyl alcohol (IPA), an aqueous hypochlorous acid solution (preferably a 1% to 10% by mass aqueous hypochlorous acid solution), aqua regia (preferably aqua regia obtained by mixing together “37% by mass hydrochloric acid:60% by mass nitric acid” at a volume ratio of “2.6:1.4” to “3.4:0.6”), ultrapure water, nitric acid (preferably 0.001% to 1% by mass nitric acid), perchloric acid (preferably 0.001% to 1% by mass perchloric acid), an aqueous oxalic acid solution (preferably a 0.01% to 10% by mass aqueous oxalic acid solution), acetic acid (preferably a 0.01% to 10% by mass aqueous acetic acid solution or an undiluted acetic acid solution), or an aqueous periodic acid solution (preferably a 0.5% to 10% by mass aqueous periodic acid solution, examples of the periodic acid include orthoperiodic acid and metaperiodic acid) is preferable. The preferred conditions required to FPM, SPM, APM, and HPM are the same as the preferred conditions required, for example, to FPM, SPM, APM, and HPM used as the specific solution described above. The hydrofluoric acid, nitric acid, perchloric acid, and hydrochloric acid mean aqueous solutions obtained by dissolving HF, HNO3, HClO4, and HCl in water respectively. The aqueous ozone, aqueous carbon dioxide, and aqueous hydrogen mean aqueous solutions obtained by dissolving O3, CO2, and H2in water respectively. As long as the purpose of the rinsing step is not impaired, these rinsing solutions may be used by being mixed together. The rinsing solution may also contain an organic solvent. Examples of the specific method of the rinsing step include a method of bringing the rinsing solution into contact with the object to be treated. The method of bringing the rinsing solution into contact with the object to be treated is performed by a method of immersing the object to be treated in the rinsing solution stored in a tank, a method of spraying the rinsing solution onto the object to be treated, a method of causing the rinsing solution to flow on the object to be treated, or a combined method consisting of any of the above methods. The treatment time (contact time between the rinsing solution and the object to be treated) is not particularly limited, but is 5 seconds to 5 minutes for example. The temperature of the rinsing solution during the treatment is not particularly limited. Generally, the temperature of the rinsing solution is, for example, preferably 16° C. to 60° C., and more preferably 18° C. to 40° C. In a case where SPM is used as the rinsing solution, the temperature thereof is preferably 90° C. to 250° C. If necessary, the present treatment method may include a drying step of performing a drying treatment after the rinsing step. The method of the drying treatment is not particularly limited, and examples thereof include spin drying, causing a drying gas to flow on the substrate, heating the substrate by a heating unit such as a hot plate or an infrared lamp, isopropyl alcohol (IPA) vapor drying, Marangoni drying, Rotagoni drying, and any combination of these. The drying time varies with the specific method to be used, but is about 30 seconds to a few minutes in general. The present treatment method may be performed in combination with a semiconductor device manufacturing method, before or after the steps performed in the manufacturing method. While being performed, the present treatment method may be incorporated into those other steps. Alternatively, while those other steps are being performed, the present treatment method may be incorporated into the steps and performed. Examples of those other steps include a step of forming each structure such as metal wiring, a gate structure, a source structure, a drain structure, an insulating layer, a ferromagnetic layer and/or a non-magnetic layer (layer formation, etching, chemical mechanical polishing, modification, and the like), a step of forming resist, an exposure step and a removing step, a heat treatment step, a washing step, an inspection step, and the like. The present treatment method may be performed in the back end process (BEOL: Back end of the line) or in the front end process (FEOL: Front end of the line). In addition, the chemical liquid may be applied, for example, to NAND, dynamic random access memory (DRAM), static random access memory (SRAM), resistive random access memory (ReRAM), ferroelectric random access memory (FRAM (registered trademark)), magnetoresistive random access memory (MRAM), phase change random access memory (PRAM), or the like, or applied to a logic circuit, a processor, or the like. EXAMPLES Hereinafter, the present invention will be more specifically described based on examples. The materials, the amounts and ratios of the materials used, the details of treatments, the procedures of treatments, and the like shown in the following examples can be appropriately changed as long as the gist of the present invention is maintained. Therefore, the scope of the present invention is not limited to the following examples. [Preparation of Chemical Liquid] The compounds listed in the following table (such as a hydroxylamine compound, a chelating agent, a specific compound, a pH adjuster, and water) were mixed together according to a predetermined formulation, thereby preparing chemical liquids used in each test. In a case where a pH adjuster was used, the amount of the pH adjuster added was adjusted so that the chemical liquid had a pH shown in the table. The balance of the chemical liquid other than the components shown in the table is water. In other words, the component other than the hydroxylamine compound, the chelating agent, the specific compound, and the pH adjuster is water. As each raw material, a semiconductor grade high-purity raw material was used. If necessary, a purification treatment was additionally performed on the raw material. Here, as the specific compound, a synthetic product was used, which was added to the chemical liquid after being subjected to the purification treatment. In addition, the specific compound that can include both the cis isomer and trans isomer was synthesized as it was as a mixture of the cis isomer and the trans isomer, and added as it was to the chemical liquid. <Specific Compound> The specific compounds used for preparing the chemical liquids are as below. [Test X (Test Using Cobalt-Containing Substance as First Metal-Containing Substance)] <Dissolvability Evaluation> An object A to be treated having the structure shown inFIG.1was prepared. Specifically, the object A to be treated used in this evaluation includes a substrate, an insulating film with hole portions disposed on the substrate, a titanium nitride layer (corresponding to a second metal-containing substance) disposed in the form of layer along the lateral surface of the hole portions, and metallic cobalt (corresponding to a cobalt-containing substance as a first metal-containing substance) with which the hole portions are filled. The obtained object A to be treated was immersed in SC-1 (28% ammonia:30% hydrogen peroxide:water=1:2:30 (mass ratio)) at 30° C. for 1 minute. Then, the object A to be treated went through treatment cycles each consisting of immersion in each chemical liquid listed in the table for 30 seconds at room temperature. The number of cycles repeated until the metallic cobalt dissolved by 20 nm was counted, and dissolvability was evaluated according to the following standard. The smaller the number of cycles, the higher the dissolvability of the chemical liquid. “A”: The number of cycles is 1 to 5. “B”: The number of cycles is 6 to 10. “C”: The number of cycles is 11 to 15. “D”: The number of cycles is 16 to 20. “E”: The number of cycles is 21 or more. (Variation Evaluation) An object B to be treated having the structure shown inFIG.2was prepared. Specifically, the object B to be treated used in this evaluation includes a substrate, an insulating film with a plurality of (100 or more) hole portions disposed on the substrate, a titanium nitride layer (corresponding to a second metal-containing substance) disposed in the form of layer along the lateral surface of the hole portions, and metallic cobalt (corresponding to a cobalt-containing substance as a first metal-containing substance) with which the hole portions are filled. The obtained object B to be treated was immersed in SC-1 (28% ammonia:30% hydrogen peroxide:water=1:2:30 (mass ratio)) at 30° C. for 1 minute. Then, the object B to be treated went through treatment cycles each consisting of immersion in each chemical liquid listed in Table 1 for 30 seconds at room temperature. The number of treatment cycles in which the object B to be treated was immersed in each chemical liquid was the same as the number of cycles repeated until metallic cobalt in the object A to be treated was dissolved by 20 nm in <Dissolvability evaluation> described above. Fifty cross sections of the hole portions, which were filled with the metallic cobalt in the obtained object B to be treated, were observed with a scanning microscope (Hitachi High-Tech Corporation., S-4800). The variation (standard deviation) in the film thickness of the residual metallic cobalt portion in each region was calculated and evaluated according to the following standard. “A”: 1 nm or less “B”: More than 1 nm and 3 nm or less “C”: More than 3 nm and 5 nm or less “D”: More than 5 nm and 10 nm or less “E”: More than 10 nm <Smoothness (Roughness) Evaluation> Substrates were prepared in which a metallic cobalt layer was formed on one surface of a commercial silicon wafer (diameter: 12 inches) by a chemical vapor deposition (CVD) method. The thickness of the metallic cobalt layer was 15 nm. Each of the obtained substrates was put in a container filled with each chemical liquid, and the chemical liquid was stirred. The removal treatment was interrupted after a short time passed, that is, at a point in time when ½ of the time taken for the metallic cobalt layer to disappear passed after the start of stirring or at a point in time when 30 minutes passed after the start of stirring. Then, after the treatment, the surface of the metallic cobalt layer was observed with a scanning electron microscope, and the smoothness of the treated portion was evaluated according to the following standard. A: The surface of the metallic cobalt layer is smooth, and no roughness is observed. B: The surface of the metallic cobalt layer is smooth, and substantially no roughness is observed (roughness higher than A). C: Although the surface of the metallic cobalt layer is slightly rough, the roughness is at an acceptable level (roughness higher than B). D: Although the surface of the metallic cobalt layer is rough, the roughness is at an acceptable level (roughness higher than C). E: The surface of the metallic cobalt layer is rough, and the roughness is at an unacceptable level. <Measurement of Corrosion Potential Difference> A silicon wafer with metallic cobalt disposed on a wafer surface or a silicon wafer with titanium nitride (TiN) disposed on a wafer surface was used as an electrode for measurement. The electrodes were immersed in each chemical liquid listed in the table, the corrosion potentials thereof were measured based on the Tafel plot obtained using potentiostat/galvanostat (Princeton Applied Research VersaSTAT 4), and the absolute value of a difference between the corrosion potentials was determined. The corrosion potential corresponds to the potential of the inflection point of the curve of the Tafel plot. The measurement conditions are as follows.Current range: ±10.2 V (vs open circuit potential)Scan rate: 1.0 mV/s (0.5 mV per session)Counter electrode: PtReference electrode: Ag/AgClMeasurement temperature: 25° C. <Result of Test X> Table 1 shows the formulation of the chemical liquids used in the series of test X and the test results. In Table 1, each of “Content (% by mass)” in the column of “Hydroxylamine compound”, “Content (% by mass)” in the column of “Specific compound”, and “Content (% by mass)” in the column of “Chelating agent” means the content (% by mass) of each compound with respect to the total mass of the chemical liquid. The column of “Ratio 1” shows the mass ratio of the content of the hydroxylamine compound to the content of the specific compound. The column of “Ratio 2” shows the mass ratio of the content of the chelating agent to the content of the specific compound. The column of “Ratio 3” is for a chemical liquid using two or more kinds of specific compounds, which shows the mass ratio of the content of a specific compound which takes up the highest proportion of the specific compounds to the content of a specific compound which takes up the second highest proportion of the specific compounds. In the chemical liquid of Example 7, the content of a specific compound which takes up the highest proportion of the specific compounds is substantially the same as the content of a specific compound which takes up the second highest proportion of the specific compounds. Therefore, the ratio 3 is “1” in Example 7. “E+number” in each cell represents “×10number”. TABLE 1Composition of chemical liquidHydroxylamineRatio 2Ratio 3Evaluation resultcompoundSpecific compoundRatio 1(Chelating(SpecificCorrosionContentContentChelating agent(HA/agent/compound/potential(% byContent(% byContentpHSpecificSpecificSpecificDissolv-differenceTable 1Typemass)Type(% by mass)Typemass)Type(% by mass)adjusterpHcompound)compound)compound)abilityRoughnessVariation(V)Example1Hydroxylamine0.5Benzohydroxamic0.0381.7E+01CBCLess than 0.1acid2Hydroxylamine0.5Maleic acid0.0381.7E+01CCCLess than 0.1monoamide3Hydroxylaminc0.5Compound A0.0381.7E+01CBBLess than 0.14Hydroxylamine0.5Compound B0.0381.7E+01CBBLess than 0.15Hydroxylamine0.5Compound C0.0381.7E+01CBBLess than 0.16Hydroxylamine0.5Compound A0.0299Com-0.000181.7E+013.0E+02BBBLess than 0.1pound C7Hydroxylamine0.5Compound A0.03Com-0.0388.3E+001.0E+00BABLess than 0.1pound C8Hydroxylamine0.5Compound A0.029Com-0.00181.7E+012.9E+01BABLess than 0.1pound C9Hydroxylamine0.5Compound A0.001Com-0.02981.7E+012.9E+01BABLess than 0.1pound C10Hydroxylamine0.5Compound A0.0001Com-0.029981.7E+013.0E+02BBBLess than 0.1pound C11Hydroxylamine0.5Compound A0.00001105.0E+04CCB0.112Hydroxylamine0.5Compound A0.0001105.0E+03CCBLess than 0.113Hydroxylamine0.5Compound A0.00282.5E+02CCBLess than 0.114Hydroxylamine0.5Compound A0.155.0E+00CBBLess than 0.115Hydroxylamine0.5Compound A145.0E−01CBBLess than 0.116Hydroxylamine0.5Compound A9.935.1E−02CBCLess than 0.117Hydroxylamine0.05Compound A9.92.55.1E−03DCCLess than 0.118Hydroxylamine0.002Compound A9.92.52.0E−04DCC0.219Hydroxylamine0.5Compound A0.03Citric0.0591.7E+011.7E+00CBB0.1acid20Hydroxylamine0.5Compound A0.03Citric0.271.7E+016.7E+00ABALess than 0.1acid21Hydroxylamine0.5Compound A0.03Com-0.01Citric0.361.3E+017.5E+003.0E+00AAALess than 0.1pound Bacid22Hydroxylaminc0.5Compound A0.03Citric231.7E+016.7E+01ABALess than 0.1acid23Hydroxylamine0.5Compound A0.03Citric1521.7E+015.0E+02ABBLess than 0.1acid24Hydroxylamine2Compound A0.03Citric0.586.7E+011.7E+01AAALess than 0.1acid25Hydroxylamine5Compound A0.03Citric0.5101.7E+021.7E+01AAALess than 0.1acid TABLE 2Composition of chemical liquidHydroxylamineSpecific compoundChelating agentRatio 1Ratio 2Ratio 3Evaluation resultCompoundCon-Con-(HA/(Chelating(SpecificCorrosionContentContenttenttentSpecificagent/compound/potential(% by(% by(% by(% bycom-SpecificSpecificDissolv-Rough-Varia-differenceTable 1Typemass)Typemass)Typemass)Typemass)pH adjusterpHpound)compound)compound)abilitynesstion(V)Example26Hydroxylamine15Compound A0.03Citric acid0.5115.0E+021.7E+01AAALess than0.127Hydroxylamine15Compound A0.0001Citric acid0.5111.5E+055.0E+03ACB0.228Hydroxylamine0.5Compound A0.03Nitrilotrismethylene-0.271.7E+016.7E+00BBA0.1phosphonic acid29Hydroxylamine0.5Compound A0.031-Hydroxyethylidene-1,1-0.271.7E+016.7E+00BBA0.2diphosphonic acid30Hydroxylamine0.5Compound A0.03Methanesulfonic acid0.271.7E+016.7E+00BBA0.231Hydroxylamine0.5Compound A0.03Succinic acid0.271.7E+016.7E+00BBA0.132Hydroxylamine0.5Compound A0.03Oxalic acid0.271.7E+016.7E+00BBA0.133Hydroxylamine0.5Compound A0.03Trans-1,2-diaminocyclo-0.271.7E+016.7E+00BBA0.1hexanetetraacetic acid34Hydroxylamine0.5Compound A0.03Ethylenediaminetetra-0.271.7E+016.7E+00BBA0.1acetic acid35Hydroxylamine0.5Compound A0.03Diethylenetriamine-0.271.7E+016.7E+00BBA0.1pentaacetic acid36Hydroxylamine0.5Compound A0.03Citric acid0.2Sulfuric acid51.7E+016.7E+00BBB0.237Hydroxylamine0.5Compound A0.03Citric acid0.2Nitric acid51.7E+016.7E+00ABB0.338Hydroxylamine0.5Compound A0.03Citric acid0.2Periodic acid51.7E+016.7E+00ABB0.339Hydroxylamine0.5Compound A0.03Citric acid0.2Tetramethyl-81.7E+016.7E+00BBA0.1ammoniumhydroxide40Hydroxylamine0.5Compound A0.03Citric acid0.2Aqueous101.7E+016.7E+00BBA0.1ammonia41Hydroxylamine0.5Compound A0.03Tetramethyl-71.7E+01BBA0.1sulfateammoniumhydroxide42Hydroxylamine0.5Compound A0.03Tetramethyl-71.7E+01BBA0.1hydrochlorideammoniumhydroxide43Hydroxylamine0.5Compound A0.03Tetramethyl-71.7E+01BBA0.1phosphateammoniumhydroxide44Hydroxylamine0.5Compound A0.03Tetramethyl-71.7E+01BBA0.1nitrateammoniumhydroxide45N′,N′-diethyl-0.5Compound A0.03Sulfuric acid71.7E+01BBA0.3hydroxylamineComparative1Hydroxylamine0.511EEE0.5Example20.5Compound A0.033DCE0.2 From the results shown in the table, it has been confirmed that the chemical liquid according to an embodiment of the present invention can reduce the variation in the dissolving amount in a case where the chemical liquid dissolves the cobalt-containing substance as the first metal-containing substance. It has been confirmed that in a case where the specific compound contains a group containing —CO—NH—OH as a specific substituent, the effects of the present invention are further improved. Furthermore, it has been confirmed that in a case where one of R1to R3in the specific compound is a specific substituent and the other two are groups represented by Formula (2), the effects of the present invention are further improved. (See the Results of Examples 1 to 5 and the Like.) It has been confirmed that in a case where the content of the specific compound is 0.01% to 1% by mass with respect to the total mass of the chemical liquid, the effects of the present invention are further improved. (See the Results of Examples 16 to 18 and 24 to 26, and the Like.) It has been confirmed that in a case where the content of the hydroxylamine compound is 0.1% to 18% by mass with respect to the total mass of the chemical liquid, the effects of the present invention are further improved. (See the Results of Examples 3 and 11 to 16, and the Like.) It has been confirmed that in a case where the content of the chelating agent is 0.1% to 15% by mass (more preferably 0.1% to 5% by mass, and even more preferably more than 0.2% by mass and 1% by mass or less) with respect to the total mass of the chemical liquid, the effects of the present invention are further improved. (See the Results of Examples 19 to 23 and 26, and the Like.) It has been confirmed that in a case where the chemical liquid contains a chelating agent (preferably citric acid), the effects of the present invention are further improved. (See the Results of Examples 3, 20, and 28 to 35, and the Like.) It has been confirmed that in a case where the mass ratio of the content of the chelating agent to the content of the specific compound (content of chelating agent/content of specific compound) is 7.0×100to 5.0×101in the chemical liquid, the effects of the present invention are further improved. (See the Results of Examples 21 and 24 to 26, and the Like.) It has been confirmed that in a case where the chemical liquid contains two or more kinds of specific compounds, and the mass ratio of the content of a specific compound which takes up the highest proportion of the specific compounds to the content of a specific compound which takes up the second highest proportion of the specific compounds is 500 or less (more preferably 50 or less), the effects of the present invention are further improved. (See the Results of Examples 6 to 10 and the Like.) [Test Y (Test Using Substances Other than Cobalt-Containing Substance as First Metal-Containing Substance)] The dissolvability evaluation, variation evaluation, smoothness (roughness) evaluation, and measurement of a corrosion potential difference were performed in the same manner as in the test X described above, except that cobalt (metallic cobalt) was changed to ruthenium (Ru), molybdenum (Mo), aluminum (Al), or copper (Cu). The results of the test Y are shown in Table 2. Each of the example numbers in Table 2 shows that the same chemical liquid as that in the corresponding examples in Table 1 in the test X was used. For example, in Example 3 in the test Y, the same chemical liquid as that used in Example 3 in Test X was used for the test. TABLE 3CorrosionpotentialdifferenceTable 2DissolvabilityRoughnessVariation(V)Ru Evaluation resultExample3CBB0.220CBB0.123BBB0.226CBB0.339CBB0.2Mo Evaluation resultExample3CBB0.220CBB0.323AAB0.226AAB0.139CBB0.1Al Evaluation resultExample3BABLess than 0.120BAALess than 0.123AAA0.126AAB0.239BABLess than 0.1Cu Evaluation resultExample3BBC0.120BBC0.123CCB0.226ABB0.239BBC0.1 From the results shown in Table 2, it has been confirmed that even though a substance other than a cobalt-containing substance is used as a first metal-containing substance, the desired results are obtained. [Test Z] <Evaluation of Residue Removability (Washing Properties)> An object to be treated (untreated laminate) comprising a metallic cobalt layer, a SiN film, a SiO2film, and a barrier metal (TaN) having a predetermined opening portion on a substrate (Si) in this order was formed. By using the barrier metal as a mask, plasma etching (dry etching) was performed on the obtained object to be treated. The SiN film and the SiO2film were etched until the metallic cobalt layer was exposed and via holes were formed, thereby manufacturing a sample 1 (seeFIG.3). The cross section of the laminate was checked using an image of a scanning electron microscope (SEM). As a result, plasma etching residues (dry etching residues) on the wall surface of the holes were observed. Then, by the following procedure, residue removability was evaluated. First, a section (about 2.0 cm×2.0 cm) of the prepared sample 1 was immersed in (treated with) each chemical liquid controlled to have a temperature of 60° C. After the lapse of a predetermined time, the section of the sample 1 was taken out and immediately washed with ultrapure water and dried with N2. <Evaluation of Residue Removability> The surface of the section of the sample 1 having undergone immersion was observed with SEM, and the removability of the plasma etching residues (“residue removability”) was evaluated according to the following standard. “A”: The plasma etching residues were completely removed within 5 minutes. “B”: The plasma etching residues were completely removed within a time that is longer than 5 minutes and 8 minutes or less. “C”: The plasma etching residues were not completely removed even after 8 minutes. <Evaluation of TaN Member Resistance> The surface of the section of sample 1 having undergone immersion was observed with SEM, and based on the reduction in the film thickness of the barrier metal (TaN) before and after the treatment performed until the plasma etching residues were completely removed, TaN member resistance was evaluated according to the following standard. Based on the evaluation, it is possible to make a conclusion that the smaller the reduction in the film thickness, the higher the TaN member resistance exhibited in the chemical liquid. The film thickness of the barrier metal (TaN) before the treatment was 3.0 nm. “A”: The reduction in the film thickness of the barrier metal was 0.5 nm or less before and after the treatment. “B”: The reduction in the film thickness of the barrier metal was more than 0.5 nm before and after the treatment. <Variation Evaluation> Furthermore, 100 via holes in the sample 1 having undergone immersion were observed with SEM. As a result, until the plasma etching residues were completely removed, the variation in the dissolving amount of the metallic cobalt layer exposed on the bottom portion of the via holes tended to be the same as the variation shown in the results of the test X. The results of the test Z are shown in the following Table 3. Each of the example numbers in Table 3 shows that the same chemical liquid as that in the corresponding examples in the test X was used. For example, in Example 3 in the test Z, the same chemical liquid as that used in Example 3 in Test X was used for the test. TABLE 4Evaluation resultResidueTaN memberTable 3removabilityresistanceExample3BA20AA39AA From the results shown in Table 3, it has been confirmed that the chemical liquid according to an embodiment of the present invention is also excellent in the residue removability and TaN member resistance. Furthermore, it has been confirmed that the residue removability is further improved in a case where the chemical liquid contains a chelating agent. EXPLANATION OF REFERENCES 20: object to be treated12: substrate14: insulating film16: second metal-containing substance portion18: first metal-containing substance portion30: object to be treated32: substrate34: metal-containing film36: etch stop layer38: interlayer insulating film40: metal hard mask42: hole46: dry etching residue44: inner wall44a: cross-sectional wall44b: bottom wall | 99,436 |
11859120 | The use of the same reference symbols in different drawings indicates similar or identical items. DETAILED DESCRIPTION The following is also directed to methods of forming shaped abrasive particles and features of such shaped abrasive particles. The shaped abrasive particles may be used in various abrasive articles, including for example bonded abrasive articles, coated abrasive articles, and the like. Alternatively, the shaped abrasive particles of the embodiments herein may be utilized in free abrasive technologies, including for example grinding and/or polishing slurries. Referring initially toFIG.1, an exemplary process is shown and is generally designated100. As shown, a backing102may be paid from a roll104. The backing102may be coated with a binder formulation106dispensed from a coating apparatus108. An exemplary coating apparatus includes a drop die coater, a knife coater, a curtain coater, a vacuum die coater or a die coater. Coating methodologies can include either contact or non-contact methods. Such methods include 2 roll, 3 roll reverse, knife over roll, slot die, gravure, extrusion, or spray coating applications. In a particular embodiment, the binder formulation106may be provided in a slurry that includes the binder formulation and abrasive grains. In an alternative embodiment, the binder formulation106may be dispensed separate from the abrasive grains. Then, the abrasive grains may be provided following the coating of the backing102with the binder formulation106, after partial curing of the binder formulation106, after patterning of the binder formulation106, or after fully curing the binder formulation108. The abrasive grains may, for example, be applied by a technique, such as electrostatic coating, drop coating or mechanical projection. In a particular aspect, the abrasive grains may be any combination of one or more of the shaped abrasive grains described herein. The binder formulation106may be cured after passing under an energy source110. The selection of the energy source110may depend in part upon the chemistry of the binder formulation106. For example, the energy source110may be a source of thermal energy or actinic radiation energy, such as electron beam, ultraviolet light, or visible light. The amount of energy used may depend on the chemical nature of the reactive groups in the precursor polymer constituents, as well as upon the thickness and density of the binder formulation106. For thermal energy, an oven temperature of about 75° C. to about 150° C. and a duration of about 5 minutes to about 60 minutes may be generally sufficient. Electron beam radiation or ionizing radiation may be used at an energy level of about 0.1 MRad to about 100 MRad, particularly at an energy level of about 1 MRad to about 10 MRad. Ultraviolet radiation includes radiation having a wavelength within a range of about 200 nanometers to about 400 nanometers, particularly within a range of about 250 nanometers to 400 nanometers. Visible radiation includes radiation having a wavelength within a range of about 400 nanometers to about 800 nanometers, particularly in a range of about 400 nanometers to about 550 nanometers. Curing parameters, such as exposure, are generally formulation dependent and can be adjusted via lamp power and belt speed. In an exemplary embodiment, the energy source110may provide actinic radiation to the coated backing, partially curing the binder formulation106. In another embodiment, the binder formulation106is thermally curable and the energy source110may provide heat for thermal treatment. In a further embodiment, the binder formulation106may include actinic radiation curable and thermally curable components. As such, the binder formulation may be partially cured through one of thermal and actinic radiation curing and cured to complete curing through a second of thermal and actinic radiation curing. For example, an epoxy constituent of the binder formulation may be partially cured using ultraviolet electromagnetic radiation and an acrylic constituent of the binder formulation may be further cured through thermal curing. Once the binder formulation106is cured a structured abrasive article112is formed. Alternatively, a size coat may be applied over the patterned abrasive structures. In a particular embodiment, the structured abrasive article112may be rolled into a roll114. In other embodiments, fully curing may be performed after rolling a partially cured abrasive article112. In one or more alternative embodiments, a size coat may be applied over the binder formulation106and abrasive grains. For example, the size coat may be applied before partially curing the binder formulation106, after partially curing the binder formulation106or after further curing the binder formulation106. The size coat may be applied, for example, by roll coating or spray coating. Depending on the composition of the size coat and when it is applied, the size coat may be cured in conjunction with the binder formulation106or cured separately. A supersize coat including grinding aids may be applied over the size coat and cured with the binder formulation106, cured with the size coat or cured separately. Referring toFIG.2, a structured abrasive article is shown and is generally designated200. As illustrated, the structured abrasive article200may include a backing202and a plurality of shaped abrasive grains204deposited thereon. In a particular aspect, the structured abrasive article200may be manufactured using the process described in conjunction withFIG.1. In a particular aspect, the shaped abrasive grains204may be one or more of the shaped abrasive grains described herein. Further, the shaped abrasive grains may include one or more, or any combination, of the shaped abrasive grains described herein. Further, one or more of the shaped abrasive grains described herein may include an upright orientation probability. The upright orientation may be considered an orientation that corresponds to a favorable abrasive/cutting position for each shaped abrasive grain and the probability is a simple mathematical probability that the grain lands in the upright orientation. In a particular aspect, the upright orientation is at least fifty percent (50%). In another aspect, the upright orientation is at least fifty-five percent (55%). In another aspect, the upright orientation is at least sixty percent (60%). In another aspect, the upright orientation is at least sixty-five percent (65%). In another aspect, the upright orientation is at least seventy percent (70%). In another aspect, the upright orientation is at least seventy-five percent (75%). In another aspect, the upright orientation is at least eighty percent (80%). In another aspect, the upright orientation is at least eighty-five percent (85%). In another aspect, the upright orientation is at least ninety percent (90%). In another aspect, the upright orientation is at least ninety-five percent (95%). In another aspect, the upright orientation is one hundred percent (100%). The body of each of the shaped abrasive grains described herein may include a polycrystalline material. The polycrystalline material may include abrasive grains. The abrasive grains may include nitrides, oxides, carbides, borides, oxynitrides, diamond, or a combination thereof. Further, the abrasive grains may include an oxide selected from the group of oxides consisting of aluminum oxide, zirconium oxide, titanium oxide, yttrium oxide, chromium oxide, strontium oxide, silicon oxide, and a combination thereof. In another aspect, the abrasive grains may include alumina. In yet another aspect, the abrasive grains consist essentially of alumina. Further, the abrasive grains may have an average grain size of not greater than about 500 microns. Alternatively, the average grain size is not greater than about 250 microns. In another aspect, the average grain size is not greater than about 100 microns. In another aspect, the average grain size is not greater than about 50 microns. In another aspect, the average grain size is not greater than about 30 microns. In another aspect, the average grain size is not greater than about 20 microns. In another aspect, the average grain size is not greater than about 10 microns. In another aspect, the average grain size is not greater than about 1 micron. In another aspect, the average grain size is at least about 0.01 microns. In another aspect, the average grain size is at least about 0.05 microns. In another aspect, the average grain size is at least about 0.08 microns. In another aspect, the average grain size is at least about 0.1 microns. In another aspect, the body of each of the shaped abrasive grains described herein may be a composite that includes at least about 2 different types of abrasive grains. FIG.3andFIG.4illustrate a first embodiment of a shaped abrasive grain300. As shown inFIG.3, the shaped abrasive grain300may include a body301that is generally prismatic with a first end face302and a second end face304. Further, the shaped abrasive grain300may include a first side face310extending between the first end face302and the second end face304. A second side face312may extend between the first end face302and the second end face304adjacent to the first side face310. As shown, the shaped abrasive grain300may also include a third side face314extending between the first end face302and the second end face304adjacent to the second side face312and the first side face310. As depicted inFIG.3andFIG.4, the shaped abrasive grain300may also include a first edge320between the first side face310and the second side face312. The shaped abrasive grain300may also include a second edge322between the second side face312and the third side face314. Further, the shaped abrasive grain300may include a third edge324between the third side face314and the first side face312. As shown, each end face302,304the shaped abrasive grain300may be generally triangular in shape. Each side face310,312,314may be generally rectangular in shape. Further, the cross-section of the shaped abrasive grain300in a plane parallel to the end faces302,304is generally triangular. It can be appreciated that the shaped abrasive grain300may include more than the three side faces310,312,314, and three edges320,322,324. It may be further appreciated that depending on the number of side faces310,312,314, the end faces302,304and cross-section of the shaped abrasive grain300through a plane parallel to the end faces302,304may have that shape of any polygon, e.g., a quadrilateral, a pentagon, a hexagon, a heptagon, an octagon, a nonagon, a decagon, etc. Further, the polygon may be convex, non-convex, concave, or non-concave. FIG.5andFIG.6illustrate a second embodiment of a shaped abrasive grain500. As shown inFIG.5, the shaped abrasive grain500may include a body501that is generally prismatic with a first end face502and a second end face504. Further, the shaped abrasive grain500may include a first side face510extending between the first end face502and the second end face504. A second side face512may extend between the first end face502and the second end face504adjacent to the first side face510. As shown, the shaped abrasive grain500may also include a third side face514extending between the first end face502and the second end face504adjacent to the second side face512and the first side face510. As depicted inFIG.5andFIG.6, the shaped abrasive grain500may also include a first edge face520between the first side face510and the second side face512. The shaped abrasive grain500may also include a second edge face522between the second side face512and the third side face514. Further, the shaped abrasive grain500may include a third edge face524between the third side face514and the first side face512. As shown, each end face502,504the shaped abrasive grain500may be generally triangular in shape. Each side face510,512,514may be generally rectangular in shape. Further, the cross-section of the shaped abrasive grain500in a plane parallel to the end faces502,504is generally triangular. FIG.7andFIG.8illustrate a third embodiment of a shaped abrasive grain700. As shown inFIG.7, the shaped abrasive grain700may include a body701that is generally prismatic with a first end face702and a second end face704. Further, the shaped abrasive grain700may include a first side face710extending between the first end face702and the second end face704. A second side face712may extend between the first end face702and the second end face704adjacent to the first side face710. As shown, the shaped abrasive grain700may also include a third side face714extending between the first end face702and the second end face704adjacent to the second side face712and the first side face710. As depicted inFIG.7andFIG.8, the shaped abrasive grain700may also include a first concave edge channel720between the first side face710and the second side face712. The shaped abrasive grain700may also include a second concave edge channel722between the second side face712and the third side face714. Further, the shaped abrasive grain700may include a third concave edge channel724between the third side face714and the first side face712. As shown, each end face702,704the shaped abrasive grain700may be generally triangular in shape. Each side face710,712,714may be generally rectangular in shape. Further, the cross-section of the shaped abrasive grain700in a plane parallel to the end faces702,704is generally triangular. FIG.9andFIG.10illustrate a fourth embodiment of a shaped abrasive grain900. As shown inFIG.9, the shaped abrasive grain900may include a body901that is generally prismatic with a first end face902and a second end face904. Further, the shaped abrasive grain900may include a first side face910extending between the first end face902and the second end face904. A second side face912may extend between the first end face902and the second end face904adjacent to the first side face910. As shown, the shaped abrasive grain900may also include a third side face914extending between the first end face902and the second end face904adjacent to the second side face912and the first side face910. As depicted inFIG.9andFIG.10, the shaped abrasive grain900may also include a first V-shaped edge channel face920between the first side face910and the second side face912. The shaped abrasive grain900may also include a second V-shaped edge channel face922between the second side face912and the third side face914. Further, the shaped abrasive grain900may include a third V-shaped edge channel face924between the third side face914and the first side face912. As shown, each end face902,904the shaped abrasive grain900may be generally triangular in shape. Each side face910,912,914may be generally rectangular in shape. Further, the cross-section of the shaped abrasive grain900in a plane parallel to the end faces902,904is generally triangular. In the exemplary embodiments shown inFIG.3throughFIG.10, it can be appreciated that the edges320,322,324; the edge faces520,522,524; the concave edge channels720,722,724; and the V-shaped edge channels920,922,924may be considered edge structures. Further, the edge structures ensure that when the shaped abrasive grains300,500,700,900are deposited, or otherwise disposed, on a backing, a side face will land on the backing and an edge structure will face up, or outward, from the backing. Further, the edge structures provide sharp edges that provide substantially increased grinding performance. Additionally, it may be appreciated that in each of the exemplary embodiments shown inFIG.3throughFIG.10, the face of the shaped abrasive grain300,500,700,900, i.e., the base, that is touching a backing has an area that is substantially greater than the area of the portion of the shaped abrasive grain300,500,700,900that is pointed outward, or upward, e.g., the edge structure. In particular, the base may comprise at least about thirty percent (30%) of the total surface area of the particle. In another aspect, the base may comprise at least about forty percent (40%) of the total surface area of the particle. In another aspect, the base may comprise at least about fifty percent (50%) of the total surface area of the particle. In another aspect, the base may comprise at least about sixty percent (60%) of the total surface area of the particle. In another aspect, the base may comprise no greater than ninety-nine percent (99%) of the total surface area of the particle. In another aspect, the base may comprise no greater than ninety-five percent (95%) of the total surface area of the particle. In another aspect, the base may comprise no greater than ninety percent (90%) of the total surface area of the particle. In another aspect, the base may comprise no greater than eighty percent (80%) of the total surface area of the particle. In another aspect, the base may comprise no greater than seventy-five percent (75%) of the total surface area of the particle. Referring toFIG.11andFIG.12, a fifth embodiment of a shaped abrasive grain is shown and is generally designated1100. As shown, the shaped abrasive grain1100may include a body1101that is generally pyramid-shaped with a generally triangle-shaped bottom face1102. Further, the shaped abrasive grain1100may be formed with a hole1104, i.e., an opening, therein. In a particular aspect, the hole1104may define a central axis1106that passes through a center of the hole1104. Further, the shaped abrasive grain1100may also define a central axis1108that passes through a center of the shaped abrasive grain1100. It may be appreciated that the hole1104may be formed in the shaped abrasive grain1100such that the central axis1106of the hole1104is spaced a distance1110above the central axis1108of the shaped abrasive grain1100. As such, a center of mass of the shaped abrasive grain1100may be moved below the geometric midpoint of the shaped abrasive grain1100. Moving the center of mass below the geometric midpoint of the shaped abrasive grain may ensure that the shaped abrasive grain1100lands on the same face, e.g., the bottom face1102, when dropped, or otherwise deposited, onto a backing, such that the shaped abrasive grain has an upright orientation. In a particular embodiment, the center of mass of is displaced from the geometric midpoint by a distance that is equal to 0.05 the height (h) along a vertical axis of the body1102defining a height. In another aspect, the center of mass may be displaced by a distance of at least about 0.1(h). In another aspect, the center of mass may be displaced by a distance of at least about 0.15(h). In another aspect, the center of mass may be displaced by a distance of at least about 0.18(h). In another aspect, the center of mass may be displaced by a distance of at least about 0.2(h). In another aspect, the center of mass may be displaced by a distance of at least about 0.22(h). In another aspect, the center of mass may be displaced by a distance of at least about 0.25(h). In another aspect, the center of mass may be displaced by a distance of at least about 0.27(h). In another aspect, the center of mass may be displaced by a distance of at least about 0.3(h). In another aspect, the center of mass may be displaced by a distance of at least about 0.32(h). In another aspect, the center of mass may be displaced by a distance of at least about 0.35(h). In another aspect, the center of mass may be displaced by a distance of at least about 0.38(h). In another aspect, the center of mass is displaced a distance no greater than 0.5(h). In yet another aspect, the center of mass is displaced a distance no greater than 0.49(h). In still another aspect, the center of mass is displaced a distance no greater than 0.48(h). In another aspect, the center of mass is displaced a distance no greater than 0.45(h). In still another aspect, the center of mass is displaced a distance no greater than 0.43(h). In yet still another aspect, the center of mass is displaced a distance no greater than 0.40(h). In another aspect, the center of mass is displaced a distance no greater than 0.39(h). In another aspect, the center of mass is displaced a distance no greater than 0.38(h). Further, the center of mass may be displaced so that the center of mass is closer to a base, e.g., the bottom face1102, of the body1101, than a top of the body1101when the shaped abrasive grain1100is in an upright orientation as shown inFIG.11. In another embodiment, the center of mass may be displaced from the geometric midpoint by a distance1110that is equal to 0.05 the width (w) along a horizontal axis of the of the body1102defining the width. In another aspect, the center of mass may be displaced by a distance of at least about 0.1(w). In another aspect, the center of mass may be displaced by a distance of at least about 0.15(w). In another aspect, the center of mass may be displaced by a distance of at least about 0.18(w). In another aspect, the center of mass may be displaced by a distance of at least about 0.2(w). In another aspect, the center of mass may be displaced by a distance of at least about 0.22(w). In another aspect, the center of mass may be displaced by a distance of at least about 0.25(w). In another aspect, the center of mass may be displaced by a distance of at least about 0.27(w). In another aspect, the center of mass may be displaced by a distance of at least about 0.3(w). In another aspect, the center of mass may be displaced by a distance of at least about 0.32(w). In another aspect, the center of mass may be displaced by a distance of at least about 0.35(w). In another aspect, the center of mass may be displaced by a distance of at least about 0.38(w). In another aspect, the center of mass is displaced a distance no greater than 0.5(w). In yet another aspect, the center of mass is displaced a distance no greater than 0.49 (w). In still another aspect, the center of mass is displaced a distance no greater than 0.48(w). In another aspect, the center of mass is displaced a distance no greater than 0.45(w). In still another aspect, the center of mass is displaced a distance no greater than 0.43(w). In yet still another aspect, the center of mass is displaced a distance no greater than 0.40(w). In another aspect, the center of mass is displaced a distance no greater than 0.39(w). In another aspect, the center of mass is displaced a distance no greater than 0.38(w). In another embodiment, the center of mass may be displaced from the geometric midpoint by a distance that is equal to 0.05 the length (l) along a longitudinal axis of the body1102defining a length. In another aspect, the center of mass may be displaced by a distance of at least about 0.1(l). In another aspect, the center of mass may be displaced by a distance of at least about 0.15(l). In another aspect, the center of mass may be displaced by a distance of at least about 0.18(l). In another aspect, the center of mass may be displaced by a distance of at least about 0.2(l). In another aspect, the center of mass may be displaced by a distance of at least about 0.22(l). In another aspect, the center of mass may be displaced by a distance of at least about 0.25(l). In another aspect, the center of mass may be displaced by a distance of at least about 0.27(l). In another aspect, the center of mass may be displaced by a distance of at least about 0.3(l). In another aspect, the center of mass may be displaced by a distance of at least about 0.32(l). In another aspect, the center of mass may be displaced by a distance of at least about 0.35(l). In another aspect, the center of mass may be displaced by a distance of at least about 0.38(l). In another aspect, the center of mass is displaced a distance no greater than 0.5(l). In yet another aspect, the center of mass is displaced a distance no greater than 0.49(l). In still another aspect, the center of mass is displaced a distance no greater than 0.48(l). In another aspect, the center of mass is displaced a distance no greater than 0.45(l). In still another aspect, the center of mass is displaced a distance no greater than 0.43(l). In yet still another aspect, the center of mass is displaced a distance no greater than 0.40(l). In another aspect, the center of mass is displaced a distance no greater than 0.39(l). In another aspect, the center of mass is displaced a distance no greater than 0.38(l). FIG.13andFIG.14illustrate a sixth embodiment of a shaped abrasive grain that is generally designated1300. As depicted, the shaped abrasive grain1300may include a body1301that may include a central portion1302that extends along a longitudinal axis1304. A first radial arm1306may extend outwardly from the central portion1302along the length of the central portion1302. A second radial arm1308may extend outwardly from the central portion1302along the length of the central portion1302. A third radial arm1310may extend outwardly from the central portion1302along the length of the central portion1302. Moreover, a fourth radial arm1312may extend outwardly from the central portion1302along the length of the central portion1302. The radial arms1306,1308,1310,1312may be equally spaced around the central portion1302of the shaped abrasive grain1300. As shown inFIG.13, the first radial arm1306may include a generally arrow-shaped distal end1320. The second radial arm1308may include a generally arrow-shaped distal end1322. The third radial arm1310may include a generally arrow-shaped distal end1324. Further, the fourth radial arm1312may include a generally arrow-shaped distal end1326. FIG.13also indicates that the shaped abrasive grain1300may be formed with a first void1330between the first radial arm1306and the second radial arm1308. A second void1332may be formed between the second radial arm1308and the third radial arm1310. A third void1334may also be formed between the third radial arm1310and the fourth radial arm1312. Additionally, a fourth void1336may be formed between the fourth radial arm1312and the first radial arm1306. As shown inFIG.13, the shaped abrasive grain1300may include a length1340, a height1342, and a width1344. In a particular aspect, the length1340is greater than the height1342and the height1342is greater than the width1344. In a particular aspect, the shaped abrasive grain1300may define a primary aspect ratio that is the ratio of the length1340to the height1342(length:height). Further, the shaped abrasive grain1300may define a secondary aspect ratio that is the ratio of the height1342to the width1344(height:width). Finally, the shaped abrasive grain1300may define a tertiary aspect ratio that is the ratio of the length1340to the width1342(length:width). In a particular aspect, the primary aspect ratio is at least 1:1. In another aspect, the primary aspect ratio is at least 2:1. In another aspect, the primary aspect ratio is at least 2.5:1. In another aspect, the primary aspect ratio is at least 3:1. In another aspect, the primary aspect ratio is at least 3.5:1. In another aspect, the primary aspect ratio is at least 4:1. In another aspect, the primary aspect ratio is at least 4.5:1. In another aspect, the primary aspect ratio is at least 5:1. In another aspect, the primary aspect ratio is at least 5.5:1. In another aspect, the primary aspect ratio is at least 6:1. In another aspect, the primary aspect ratio is at least 6.5:1. In another aspect, the primary aspect ratio is at least 7:1. In another aspect, the primary aspect ratio is at least 7.5:1. In another aspect, the primary aspect ratio is at least 8:1. In another aspect, the primary aspect ratio is at least 8.5:1. In another aspect, the primary aspect ratio is at least 9:1. In another aspect, the primary aspect ratio is at least 9.5:1. In another aspect, the primary aspect ratio is at least 10:1. In a particular aspect, the secondary aspect ratio is at least 1:1. In another aspect, the secondary aspect ratio is at least 1.5:1. In another aspect, the secondary aspect ratio is 2:1. In another aspect, the secondary aspect ratio is at least 2.5:1. In another aspect, the secondary aspect ratio is at least 3:1. In another aspect, the secondary aspect ratio is at least 3.5:1. In another aspect, the secondary aspect ratio is at least 4:1. In another aspect, the secondary aspect ratio is at least 4.5:1. In another aspect, the secondary aspect ratio is at least 5:1. In another aspect, the secondary aspect ratio is at least 5.5:1. In another aspect, the secondary aspect ratio is at least 6:1. In another aspect, the secondary aspect ratio is at least 6.5:1. In another aspect, the secondary aspect ratio is at least 7:1. In another aspect, the secondary aspect ratio is at least 7.5:1. In another aspect, the secondary aspect ratio is at least 8:1. In another aspect, the secondary aspect ratio is at least 8.5:1. In another aspect, the secondary aspect ratio is at least 9:1. In another aspect, the secondary aspect ratio is at least 9.5:1. In another aspect, the secondary aspect ratio is at least 10:1. In a particular aspect, the tertiary aspect ratio is at least 1:1. In another aspect, the tertiary aspect ratio is at least 1.5:1. In another aspect, the tertiary aspect ratio is 2:1. In another aspect, the tertiary aspect ratio is at least 2.5:1. In another aspect, the tertiary aspect ratio is at least 3:1. In another aspect, the tertiary aspect ratio is at least 3.5:1. In another aspect, the tertiary aspect ratio is at least 4:1. In another aspect, the tertiary aspect ratio is at least 4.5:1. In another aspect, the tertiary aspect ratio is at least 5:1. In another aspect, the tertiary aspect ratio is at least 5.5:1. In another aspect, the tertiary aspect ratio is at least 6:1. In another aspect, the tertiary aspect ratio is at least 6.5:1. In another aspect, the tertiary aspect ratio is at least 7:1. In another aspect, the tertiary aspect ratio is at least 7.5:1. In another aspect, the tertiary aspect ratio is at least 8:1. In another aspect, the tertiary aspect ratio is at least 8.5:1. In another aspect, the tertiary aspect ratio is at least 9:1. In another aspect, the tertiary aspect ratio is at least 9.5:1. In another aspect, the tertiary aspect ratio is at least 10:1. In a particular aspect, the shape of the shaped abrasive grain1300with respect to the primary aspect ratio is generally rectangular, e.g., flat, or curved. Moreover, the shape of the shaped abrasive grain1300with respect to the secondary aspect ratio may be any polyhedral shape, e.g., a triangle, a square, a rectangle, a pentagon, etc. The shape of the shaped abrasive grain1300with respect to the secondary aspect ratio may also be the shape of any alphanumeric character, e.g., 1, 2, 3, etc., A, B, C, etc. Further, the shape of the shaped abrasive grain1300with respect to the secondary aspect ratio may be a character selected from the Greek alphabet, the modern Latin alphabet, the ancient Latin alphabet, the Russian alphabet, any other alphabet, or any combination thereof. Further, the shape of the shaped abrasive grain1300with respect to the secondary aspect ratio may be a Kanji character. In another aspect of the shaped abrasive grain1300, the width1344is greater than the height1342and the height1342is greater than the length1340. In this aspect, the shaped abrasive grain1300may define a primary aspect ratio that is the ratio of the width1344to the height1342(width:height). Further, the shaped abrasive grain1300may define a secondary aspect ratio that is the ratio of the height1342to the length1340(height:length). Finally, the shaped abrasive grain1300may define a tertiary aspect ratio that is the ratio of the width1342to the length1340(width:length). In a particular aspect, the primary aspect ratio is at least 2:1. In another aspect, the primary aspect ratio is at least 2.5:1. In another aspect, the primary aspect ratio is at least 3:1. In another aspect, the primary aspect ratio is at least 3.5:1. In another aspect, the primary aspect ratio is at least 4:1. In another aspect, the primary aspect ratio is at least 4.5:1. In another aspect, the primary aspect ratio is at least 5:1. In another aspect, the primary aspect ratio is at least 5.5:1. In another aspect, the primary aspect ratio is at least 6:1. In another aspect, the primary aspect ratio is at least 6.5:1. In another aspect, the primary aspect ratio is at least 7:1. In another aspect, the primary aspect ratio is at least 7.5:1. In another aspect, the primary aspect ratio is at least 8:1. In another aspect, the primary aspect ratio is at least 8.5:1. In another aspect, the primary aspect ratio is at least 9:1. In another aspect, the primary aspect ratio is at least 9.5:1. In another aspect, the primary aspect ratio is at least 10:1. In a particular aspect, the secondary aspect ratio is at least 1.5:1. In another aspect, the secondary aspect ratio is 2:1. In another aspect, the secondary aspect ratio is at least 2.5:1. In another aspect, the secondary aspect ratio is at least 3:1. In another aspect, the secondary aspect ratio is at least 3.5:1. In another aspect, the secondary aspect ratio is at least 4:1. In another aspect, the secondary aspect ratio is at least 4.5:1. In another aspect, the secondary aspect ratio is at least 5:1. In another aspect, the secondary aspect ratio is at least 5.5:1. In another aspect, the secondary aspect ratio is at least 6:1. In another aspect, the secondary aspect ratio is at least 6.5:1. In another aspect, the secondary aspect ratio is at least 7:1. In another aspect, the secondary aspect ratio is at least 7.5:1. In another aspect, the secondary aspect ratio is at least 8:1. In another aspect, the secondary aspect ratio is at least 8.5:1. In another aspect, the secondary aspect ratio is at least 9:1. In another aspect, the secondary aspect ratio is at least 9.5:1. In another aspect, the secondary aspect ratio is at least 10:1. In a particular aspect, the tertiary aspect ratio is at least 1.5:1. In another aspect, the tertiary aspect ratio is 2:1. In another aspect, the tertiary aspect ratio is at least 2.5:1. In another aspect, the tertiary aspect ratio is at least 3:1. In another aspect, the tertiary aspect ratio is at least 3.5:1. In another aspect, the tertiary aspect ratio is at least 4:1. In another aspect, the tertiary aspect ratio is at least 4.5:1. In another aspect, the tertiary aspect ratio is at least 5:1. In another aspect, the tertiary aspect ratio is at least 5.5:1. In another aspect, the tertiary aspect ratio is at least 6:1. In another aspect, the tertiary aspect ratio is at least 6.5:1. In another aspect, the tertiary aspect ratio is at least 7:1. In another aspect, the tertiary aspect ratio is at least 7.5:1. In another aspect, the tertiary aspect ratio is at least 8:1. In another aspect, the tertiary aspect ratio is at least 8.5:1. In another aspect, the tertiary aspect ratio is at least 9:1. In another aspect, the tertiary aspect ratio is at least 9.5:1. In another aspect, the tertiary aspect ratio is at least 10:1. In a particular aspect, the shape of the shaped abrasive grain1300with respect to the secondary aspect ratio is generally rectangular, e.g., flat, or curved. Moreover, the shape of the shaped abrasive grain1300with respect to the primary aspect ratio may be any polyhedral shape, e.g., a triangle, a square, a rectangle, a pentagon, etc. The shape of the shaped abrasive grain1300with respect to the primary aspect ratio may also be the shape of any alphanumeric character, e.g., 1, 2, 3, etc., A, B, C, etc. Further, the shape of the shaped abrasive grain1300with respect to the primary aspect ratio may be a character selected from the Greek alphabet, the modern Latin alphabet, the ancient Latin alphabet, the Russian alphabet, any other alphabet, or any combination thereof. Moreover, the shape of the shaped abrasive grain1300with respect to the primary aspect ratio may be a Kanji character. Referring now toFIG.15andFIG.16, a seventh embodiment of a shaped abrasive grain is shown and is generally designated1500. As shown, the shaped abrasive grain1500may include a body1501that includes a flat bottom1502and a generally dome-shaped top1504. The domed-shaped top1504may be formed with a first edge1506, a second edge1508, a third edge1510, a fourth edge1512, and a fifth edge1514. It may be appreciated that the shaped abrasive grain1500may include more or less than five edges1506,1508,1510,1512,1514. Further, the edges1506,1508,1510,1512,1514may be equally spaced radially around a center of the dome-shaped top1504. In a particular aspect, the edges1506,1508,1510,1512,1514in the dome-shaped top1504may be formed by injecting the material comprising the shaped abrasive grain1500through a generally star-shaped nozzle. It may be appreciated that the shape of the shaped abrasive grain1500may facilitate orientation of the shaped abrasive grain1500as it is dropped, or otherwise deposited, on a backing. Specifically, the dome-shaped top1504will allow the shaped abrasive grain1500to roll onto the flat bottom1502ensuring that the edges face out, or up, from the backing. FIG.17andFIG.18illustrate an eighth embodiment of a shaped abrasive grain, designated1700. As depicted, the shaped abrasive grain1700may include a body1701that includes a flat bottom1702and a generally dome-shaped top1704. The domed-shaped top1704may be formed with a peak1706. In a particular aspect, the peak1706in the dome-shaped top1704may be formed by injecting the material comprising the shaped abrasive grain1700through a generally round, generally small nozzle. It may be appreciated that the shape of the shaped abrasive grain1700may facilitate orientation of the shaped abrasive grain1700as it is dropped, or otherwise deposited, on a backing. Specifically, the dome-shaped top1704and the peak1706will allow the shaped abrasive grain1700to roll onto the flat bottom1702ensuring that the peak1706and the dome-shaped top1704face out, or up, from the backing. Referring now toFIG.19andFIG.20, a ninth embodiment of a shaped abrasive grain is shown and is generally designated1900. As shown, the shaped abrasive grain1900may include a body1901that is generally box-shaped with six exterior faces1902and twelve1904edges. Further, the shaped abrasive grain1900may be formed with a generally X-shaped hole1906, i.e., an opening, through the shaped abrasive grain1900parallel to a longitudinal axis1908that passes through a center1910of the shaped abrasive grain. Further, a center1912of the X shaped hole1906may be spaced a distance1914from the longitudinal axis1908. As such, a center of mass1916of the shaped abrasive grain1900may be moved below the geometric midpoint1910of the shaped abrasive grain1900. Moving the center of mass below the geometric midpoint of the shaped abrasive grain may ensure that the shaped abrasive grain1900lands on the same face when dropped, or otherwise deposited, onto a backing. It may be appreciated that the X shaped hole1906may be formed along the longitudinal axis1908through the geometric midpoint1910of the shaped abrasive grain1900. Further, it may be appreciated that the X shaped hole1906may be rotated forty-five degrees (45°) and in such a case the hole1906would appear to be generally + shaped. It may be appreciated that the hole1906formed in the shaped abrasive grain1900may have any shape: polygonal or otherwise. FIG.21throughFIG.23depict a tenth embodiment of a shaped abrasive grain that is generally designated2100. As shown, the shaped abrasive grain2100may include a body2101that may have a first end face2102and a second end face2104. In a particular aspect, depending on the orientation, the first end face2102may be a base surface and the second end face2104may be an upper surface. Further, the shaped abrasive grain2100may include a first lateral face2106extending between the first end face2102and the second end face2104. A second lateral face2108may extend between the first end face2102and the second end face2104. Further, a third lateral face2110may extend between the first end face2102and the second end face2104. A fourth lateral face2112may also extend between the first end face2102and the second end face2104. As shown, the first end face2102and the second end face2104are parallel to each other. However, in a particular aspect, the first end face2102is rotated with respect to the second end face2104to establish a twist angle2114. In a particular aspect, the twist angle2114is at least about one degree. In another aspect, the twist angle2114is at least about two degrees. In another aspect, the twist angle2114is at least about five degrees. In another aspect, the twist angle2114is at least about eight degrees. In another aspect, the twist angle2114is at least about ten degrees. In another aspect, the twist angle2114is at least about twelve degrees. In another aspect, the twist angle2114is at least about fifteen degrees. In another aspect, the twist angle2114is at least about eighteen degrees. In another aspect, the twist angle2114is at least about twenty degrees. In another aspect, the twist angle2114is at least about twenty-five degrees. In another aspect, the twist angle2114is at least about thirty degrees. In another aspect, the twist angle2114is at least about forty degrees. In another aspect, the twist angle2114is at least about fifty degrees. In another aspect, the twist angle2114is at least about sixty degrees. In another aspect, the twist angle2114is at least about seventy degrees. In another aspect, the twist angle2114is at least about eighty degrees. In another aspect, the twist angle2114is at least about ninety degrees. It can be appreciated that the twist angle2100of the shaped abrasive grain may be a horizontal twist angle, i.e., along a longitudinal axis of the body2101defining a length. In another aspect, the twist angle2100of the shaped abrasive grain may be a vertical twist angle, i.e., along a vertical axis defining a height of the body2101. Referring toFIG.24andFIG.25, an eleventh embodiment of a shaped abrasive grain is shown and is generally designated2400. As illustrated, the shaped abrasive grain2400may include a body2401that may include a central portion2402that extends along a longitudinal axis2404. A first radial arm2406may extend outwardly from the central portion2402along the length of the central portion2402. A second radial arm2408may extend outwardly from the central portion2402along the length of the central portion2402. A third radial arm2410may extend outwardly from the central portion2402along the length of the central portion2402. Moreover, a fourth radial arm2412may extend outwardly from the central portion2402along the length of the central portion2402. The radial arms2406,2408,2410,2412may be equally spaced around the central portion2402of the shaped abrasive grain2400. As shown inFIG.24, the first radial arm2406may include a generally box-shaped distal end2420. The second radial arm2408may include a generally box-shaped distal end2422. The third radial arm2410may include a generally box-shaped distal end2424. Further, the fourth radial arm2412may include a generally box-shaped distal end2426. FIG.24andFIG.25further show that the shaped abrasive grain2400may be formed with a hole2428through the shaped abrasive grain2400along the longitudinal axis2404. As shown, the hole2428may be generally triangular in shape. It may be appreciated that in other aspects the hole2428formed in the shaped abrasive grain2400may have any shape: polygonal or otherwise. FIG.26andFIG.27illustrate a twelfth embodiment of a shaped abrasive grain that is generally designated2600. As shown, the shaped abrasive grain2600may include a body2601that may include a central portion2602that extends along a longitudinal axis2604. A first radial arm2606may extend outwardly from the central portion2602along the length of the central portion2602. A second radial arm2608may extend outwardly from the central portion2602along the length of the central portion2602. A third radial arm2610may extend outwardly from the central portion2602along the length of the central portion2602. Moreover, a fourth radial arm2612may extend outwardly from the central portion2602along the length of the central portion2602. The radial arms2606,2608,2610,2612may be equally spaced around the central portion2602of the shaped abrasive grain2600. As shown inFIG.26andFIG.27, the first radial arm2606may include a generally box-shaped distal end2620formed with a V-shaped channel2622. The second radial arm2608may include a generally box-shaped distal end2624formed with a V-shaped channel2626. The third radial arm2610may also include a generally box-shaped distal end2628formed with a V-shaped channel2630. Further, the fourth radial arm2612may include a generally box-shaped distal end2632that is also formed with a V shape channel2634. FIG.28andFIG.29illustrate a thirteenth embodiment of a shaped abrasive grain that is generally designated2800. As shown, the shaped abrasive grain2800may include a body2801that may include a central portion2802that extends along a longitudinal axis2804. A first radial arm2806may extend outwardly from the central portion2802along the length of the central portion2802. A second radial arm2808may extend outwardly from the central portion2802along the length of the central portion2802. A third radial arm2810may extend outwardly from the central portion2802along the length of the central portion2802. Moreover, a fourth radial arm2812may extend outwardly from the central portion2802along the length of the central portion2802. The radial arms2806,2808,2810,2812may be equally spaced around the central portion2802of the shaped abrasive grain2800. As shown inFIG.28andFIG.29, the first radial arm2806may include a generally box-shaped distal end2820formed with a concave channel2822. The second radial arm2808may include a generally box-shaped distal end2824formed with a concave channel2826. The third radial arm2810may also include a generally box-shaped distal end2828formed with a concave channel2830. Further, the fourth radial arm2812may include a generally box-shaped distal end2832that is also formed with a concave channel2834. FIG.30andFIG.31illustrate a fourteenth embodiment of a shaped abrasive grain that is generally designated3000. As depicted, the shaped abrasive grain3000may include a body3001having a central portion3002that extends along a longitudinal axis3004. A first radial arm3006may extend outwardly from the central portion3002along the length of the central portion3002. A second radial arm3008may extend outwardly from the central portion3002along the length of the central portion3002. A third radial arm3010may extend outwardly from the central portion3002along the length of the central portion3002. Moreover, a fourth radial arm3012may extend outwardly from the central portion3002along the length of the central portion3002. The radial arms3006,3008,3010,3012may be equally spaced around the central portion3002of the shaped abrasive grain3000. As shown inFIG.30, the first radial arm3006may include a generally T-shaped distal end3020. The second radial arm3008may include a generally T-shaped distal end3022. The third radial arm3010may include a generally T-shaped distal end3024. Further, the fourth radial arm3012may include a generally T-shaped distal end3026. FIG.30also indicates that the shaped abrasive grain3000may be formed with a first void3030between the first radial arm3006and the second radial arm3008. A second void3032may be formed between the second radial arm3008and the third radial arm3010. A third void3034may also be formed between the third radial arm3010and the fourth radial arm3012. Additionally, a fourth void3036may be formed between the fourth radial arm3012and the first radial arm3006. FIG.32andFIG.33illustrate a fifteenth embodiment of a shaped abrasive grain that is generally designated3200. As depicted, the shaped abrasive grain3200may include a body3201that may include a central portion3202that extends along a longitudinal axis3204. A first radial arm3206may extend outwardly from the central portion3202along the length of the central portion3202. A second radial arm3208may extend outwardly from the central portion3202along the length of the central portion3202. A third radial arm3210may extend outwardly from the central portion3202along the length of the central portion3202. Moreover, a fourth radial arm3212may extend outwardly from the central portion3202along the length of the central portion3202. The radial arms3206,3208,3210,3212may be equally spaced around the central portion3202of the shaped abrasive grain3200. As shown inFIG.32, the first radial arm3206may include a generally rounded T-shaped distal end3220. The second radial arm3208may include a generally rounded T-shaped distal end3222. The third radial arm3210may include a generally rounded T-shaped distal end3224. Further, the fourth radial arm3212may include a generally rounded T-shaped distal end3226. FIG.32also indicates that the shaped abrasive grain3200may be formed with a first void3230between the first radial arm3206and the second radial arm3208. A second void3232may be formed between the second radial arm3208and the third radial arm3210. A third void3234may also be formed between the third radial arm3210and the fourth radial arm3212. Additionally, a fourth void3236may be formed between the fourth radial arm3212and the first radial arm3206. FIG.34andFIG.35illustrate a sixteenth embodiment of a shaped abrasive grain that is generally designated3400. As depicted, the shaped abrasive grain3400may include a body3401having a central portion3402that extends along a longitudinal axis3404. The central portion3402may be formed with a hole3406along the longitudinal axis3404along the entire length of the central portion3402of the shaped abrasive grain3400. A generally triangular first radial arm3410may extend outwardly from the central portion3402of the shaped abrasive grain3400along the length of the central portion3402. A generally triangular second radial arm3412may extend outwardly from the central portion3402of the shaped abrasive grain3400along the length of the central portion3402. A generally triangular third radial arm3414may extend outwardly from the central portion3402of the shaped abrasive grain3400along the length of the central portion3402. A generally triangular fourth radial arm3416may extend outwardly from the central portion3402of the shaped abrasive grain3400along the length of the central portion3402. Further, a generally triangular fifth radial arm3418may extend outwardly from the central portion3402of the shaped abrasive grain3400along the length of the central portion3402. As further depicted inFIG.34andFIG.35, a generally triangular sixth radial arm3420may extend outwardly from the central portion3402of the shaped abrasive grain3400along the length of the central portion3402. A generally triangular seventh radial arm3422may extend outwardly from the central portion3402of the shaped abrasive grain3400along the length of the central portion3402. A generally triangular eighth radial arm3424may extend outwardly from the central portion3402of the shaped abrasive grain3400along the length of the central portion3402. A generally triangular ninth radial arm3426may extend outwardly from the central portion3402of the shaped abrasive grain3400along the length of the central portion3402. Moreover, a generally triangular tenth radial arm3428may extend outwardly from the central portion3402of the shaped abrasive grain3400along the length of the central portion3402. In a particular aspect, the radial arms3410,3412,3414,3416,3418,3420,3422,3424,3426,3428may be equally spaced around the central portion3402of the shaped abrasive grain to form a generally star-shaped first end face3430, a generally star-shaped second end face3432and a generally star-shaped cross-section taken parallel to the end faces3430,3432. Referring now toFIG.36andFIG.37, a seventeenth embodiment of a shaped abrasive grain is shown and is generally designated3600. As shown, the shaped abrasive grain3600may include a body3601having a first end face3602and a second end face3604. In a particular aspect, depending on the orientation, the first end face3602may be a base surface and the second end face3604may be an upper surface. Further, the shaped abrasive grain3600may be formed with a hole3606along a longitudinal axis3608. As shown, the hole3606may be generally box shaped. FIG.36andFIG.37show that the shaped abrasive grain3600may include a generally K-shaped first side face3610extending between the first end face3602and the second end face3604. The shaped abrasive grain3600may also include a generally K-shaped second side face3612extending between the first end face3602and the second end face3604opposite the generally K-shaped first side face3610. As illustrated, the shaped abrasive grain3600may include a generally flat third side face3614extending between the first K shaped side face3610and the second K shaped side face3612and between the first end face3602and the second end face3604. The shaped abrasive grain3600may also include a generally flat fourth side face3616extending between the first K-shaped side face3610and the second K shape side face3612opposite the generally flat third side face3614. FIG.38andFIG.39depict an eighteenth embodiment of a shaped abrasive grain that is generally designated3800. As shown, the shaped abrasive grain3800may include a body3801having a first end face3802and a second end face3804. In a particular aspect, depending on the orientation, the first end face3802may be a base surface and the second end face3804may be an upper surface. The shaped abrasive grain3800may include a generally K-shaped first side face3806extending between the first end face3802and the second end face3804. Further, the shaped abrasive grain3800may include a generally flat second side face3808opposite the generally K-shaped first side face3806and extending between the first end face3802and the second end face3804. As shown, the shaped abrasive grain3800may also include a third side face3810extending between the first end face3802and the second end face3804and between the first side face3806and the second side face3808. Further, the shaped abrasive grain3800may include a fourth side face3812extending between the first end face3802and the second end face3804opposite the third side face3810. FIG.40andFIG.41show a nineteenth embodiment of a shaped abrasive grain4000. As shown inFIG.40andFIG.41, the shaped abrasive grain4000may include a body4001that is generally prismatic with a first end face4002and a second end face4004. In a particular aspect, depending on the orientation, the first end face4002may be a base surface and the second end face4004may be an upper surface. Further, the shaped abrasive grain4000may include a first side face4010extending between the first end face4002and the second end face4004. A second side face4012may extend between the first end face4002and the second end face4004adjacent to the first side face4010. As shown, the shaped abrasive grain4000may also include a third side face4014extending between the first end face4002and the second end face4004adjacent to the second side face4012. Further, the shaped abrasive grain4000may include a fourth side face4016extending between the first end face4002and the second end face4004adjacent to the third side face4014and the first side face4010. As depicted inFIG.40andFIG.41, the shaped abrasive grain4000may also include a first edge4020between the first side face4010and the second side face4012. The shaped abrasive grain4000may also include a second edge4022between the second side face4012and the third side face4014. The shaped abrasive grain4000may include a third edge4024between the third side face4014and the fourth side face4016. Moreover, the shaped abrasive grain4000may include a fourth edge4026between the fourth side face4016and the first side face4010. As shown, each end face4002,4004the shaped abrasive grain4000may be generally diamond-shaped. Each side face4010,4012,4014,4016may be generally rectangular in shape. Further, the cross-section of the shaped abrasive grain4000in a plane parallel to the end faces4002,4004is generally diamond-shaped. As shown, the shaped abrasive grain4000may also include a hole4030formed along a central longitudinal axis4032. The hole4030may pass through the center of the shaped abrasive grain4000. Alternatively, the hole4030may be offset from the center of the shaped abrasive grain4000in any direction. FIG.42andFIG.43illustrate a twentieth embodiment of a shaped abrasive grain that is generally designated4200. As shown, the shaped abrasive grain4200may include a body4201that includes a generally circular first end face4202and a generally circular second end face4204. In a particular aspect, depending on the orientation, the first end face4202may be a base surface and the second end face4204may be an upper surface. In a particular aspect, a diameter of the second end face4204may be larger than a diameter of the first end face4202. As shown, the shaped abrasive grain4200may include continuous side face4206between the first end face4202and the second end face4204. Accordingly, the shaped abrasive grain4200is generally frusto-conically shaped.FIG.42andFIG.43further indicate that the shaped abrasive grain4200may include a generally cylindrical hole4208formed along a central longitudinal axis4210. Referring now toFIG.44throughFIG.46, a twenty-first embodiment of a shaped abrasive grain is shown and is generally designated4400. The shaped abrasive grain4400may include a body4401that may include a generally triangular first end face4402and a generally circular second end face4404. In a particular aspect, depending on the orientation, the first end face4402may be an upper surface and the second end face4404may be a base surface. Further, the shaped abrasive grain4400may include a first side face4410extending between the first end face4402and the second end face4404. A second side face4412may extend between the first end face4402and the second end face4404adjacent to the first side face4410. As shown, the shaped abrasive grain4400may also include a third side face4414extending between the first end face4402and the second end face4404adjacent to the second side face4412and the first side face4410. As depicted inFIG.44andFIG.45, the shaped abrasive grain4400may also include a first edge4420between the first side face4410and the second side face4412. The shaped abrasive grain4400may also include a second edge4422between the second side face4412and the third side face4414. Further, the shaped abrasive grain4400may include a third edge4424between the third side face4414and the first side face4412. Referring now toFIG.47throughFIG.49, a twenty-second embodiment of a shaped abrasive grain is shown and is generally designated4700. The shaped abrasive grain4700may include a body4701having a generally square first end face4702and a generally circular second end face4704. In a particular aspect, depending on the orientation, the first end face4702may be an upper surface and the second end face4704may be a base surface. Further, the shaped abrasive grain4700may include a first side face4710extending between the first end face4702and the second end face4704. A second side face4712may extend between the first end face4702and the second end face4704adjacent to the first side face4710. As shown, the shaped abrasive grain4700may also include a third side face4714extending between the first end face4702and the second end face4704adjacent to the second side face4712. The shaped abrasive grain4700may also include a fourth side face4716adjacent to the third side face4714and the first side face4710. As depicted inFIG.47andFIG.48, the shaped abrasive grain4700may also include a first edge4720between the first side face4710and the second side face4712. The shaped abrasive grain4700may also include a second edge4722between the second side face4712and the third side face4714. Further, the shaped abrasive grain4700may include a third edge4724between the third side face4714and the fourth side face4716. Also, the shaped abrasive grain4700may include a fourth edge4726between the fourth side face4716and the first side face4710. FIG.50throughFIG.52show a twenty-third embodiment of a shaped abrasive grain that is generally designated5000. The shaped abrasive grain5000may include a body5001having a generally plus (+) shaped first end face5002and a generally circular second end face5004. In a particular aspect, depending on the orientation, the first end face5002may be an upper surface and the second end face5004may be a base surface. Further, the shaped abrasive grain5000may include a first side face5010extending between the first end face5002and the second end face5004. A second side face5012may extend between the first end face5002and the second end face5004adjacent to the first side face5010. As shown, the shaped abrasive grain5000may also include a third side face5014extending between the first end face5002and the second end face5004adjacent to the second side face5012. The shaped abrasive grain5000may also include a fourth side face5016adjacent to the third side face5014and the first side face5010. As depicted inFIG.50andFIG.51, the shaped abrasive grain5000may also include a first void5020between the first side face5010and the second side face5012. The shaped abrasive grain5000may also include a second void5022between the second side face5012and the third side face5014. Further, the shaped abrasive grain5000may include a third void5024between the third side face5014and the fourth side face5016. Also, the shaped abrasive grain5000may include a fourth void5026between the fourth side face5016and the first side face5010. FIG.53throughFIG.55show a twenty-fourth embodiment of a shaped abrasive grain that is generally designated5300. The shaped abrasive grain5300may include a body5301having a generally plus (+) shaped first end face5302and a generally rounded plus (+) shaped end face5304. In a particular aspect, depending on the orientation, the first end face5302may be an upper surface and the second end face5304may be a base surface. As shown, the shaped abrasive grain5300may include a first side face5310extending between the first end face5302and the second end face5304. A second side face5312may extend between the first end face5302and the second end face5304adjacent to the first side face5310. As shown, the shaped abrasive grain5300may also include a third side face5314extending between the first end face5302and the second end face5304adjacent to the second side face5312. The shaped abrasive grain5300may also include a fourth side face5316adjacent to the third side face5314and the first side face5310. As depicted inFIG.53throughFIG.55, the shaped abrasive grain5300may also include a first void5320between the first side face5310and the second side face5312. The shaped abrasive grain5300may also include a second void5322between the second side face5312and the third side face5314. Further, the shaped abrasive grain5300may include a third void5324between the third side face5314and the fourth side face5316. Also, the shaped abrasive grain5300may include a fourth void5326between the fourth side face5316and the first side face5310. Referring now toFIG.56throughFIG.58, a twenty-fifth embodiment of a shaped abrasive grain is shown and is generally designated5600. The shaped abrasive grain5600may include a body5601having a generally circular first end face5602and a generally triangular second end face5604. The second end face5604is relatively larger than the first end face5602. In a particular aspect, depending on the orientation, the first end face5602may be an upper surface and the second end face5604may be a base surface. As depicted, the shaped abrasive grain5600may include a first side face5610extending between the first end face5602and the second end face5604. A second side face5612may extend between the first end face5602and the second end face5604adjacent to the first side face5610. As shown, the shaped abrasive grain5600may also include a third side face5614extending between the first end face5602and the second end face5604adjacent to the second side face5612and the first side face5610. As shown inFIG.56throughFIG.58, the shaped abrasive grain5600may also include a first edge5620between the first side face5610and the second side face5612. The shaped abrasive grain5600may also include a second edge5622between the second side face5612and the third side face5614. Further, the shaped abrasive grain5600may include a third edge5624between the third side face5614and the first side face5612. Referring toFIG.59throughFIG.61, a twenty-sixth embodiment of a shaped abrasive grain is shown and is generally designated5900. The shaped abrasive grain5900may include a body5901having a generally circular first end face5902and a generally square second end face5904. In a particular aspect, the second end face5904is relatively larger than the first end face5902. In a particular aspect, depending on the orientation, the first end face5902may be an upper surface and the second end face5904may be a base surface. Further, the shaped abrasive grain5900may include a first side face5910extending between the first end face5902and the second end face5904. A second side face5912may extend between the first end face5902and the second end face5904adjacent to the first side face5910. As shown, the shaped abrasive grain5900may also include a third side face5914extending between the first end face5902and the second end face5904adjacent to the second side face5912. The shaped abrasive grain5900may also include a fourth side face5916adjacent to the third side face5914and the first side face5910. As depicted inFIG.59throughFIG.61, the shaped abrasive grain5900may also include a first edge5920between the first side face5910and the second side face5912. The shaped abrasive grain5900may also include a second edge5922between the second side face5912and the third side face5914. Further, the shaped abrasive grain5900may include a third edge5924between the third side face5914and the fourth side face5916. Also, the shaped abrasive grain5900may include a fourth edge5926between the fourth side face5916and the first side face5910. One or more of the shaped abrasive grains described herein are configured to land in an upright orientation when deposited onto a backing. Further, one or more of the embodiments described herein may provide a relatively high aspect ratio associated with a particular length:height ratio, height:width ratio, length:width ratio, width:height ratio, height:length ratio, width:length ratio, or a combination thereof. A high aspect ratio enables the manufacture of a coated abrasive structure having an open coat, i.e., the distance between adjacent shaped abrasive grains may be increased. Further, the open coat provides greater space for chip clearance and may lower power consumption by making a better cut, or grind. Moreover, in bonded abrasive and thin wheel applications shaped abrasive grains having high aspect ratios with sharp edges allows the manufacture of grinding wheels having greater porosity. Greater porosity provides more space for swarf and chip clearance and may enable more coolant to flow through the grinding wheel to provide greater efficiency. FIGS.62Aand B include illustrations of a system for forming shaped abrasive particles in accordance with an embodiment. The process of forming shaped abrasive particles can be initiated by forming a mixture6201including a ceramic material and a liquid. In particular, the mixture6201can be a gel formed of a ceramic powder material and a liquid, wherein the gel can be characterized as a shape-stable material having the ability to hold a given shape even in the green (i.e., unfired) state. In accordance with an embodiment, the gel can include a powder material that is an integrated network of discrete particles. The mixture6201can be formed to have a particular content of solid material, such as the ceramic powder material. For example, in one embodiment, the mixture6201can have a solids content of at least about 25 wt %, such as at least about 35 wt %, at least about 38 wt %, or even at least about 42 wt % for the total weight of the mixture6201. Still, in at least one non-limiting embodiment, the solid content of the mixture6201can be not greater than about 75 wt %, such as not greater than about 70 wt %, not greater than about 65 wt %, or even not greater than about 55 wt %. It will be appreciated that the content of the solids materials in the mixture6201can be within a range between any of the minimum and maximum percentages noted above. According to one embodiment, the ceramic powder material can include an oxide, a nitride, a carbide, a boride, an oxycarbide, an oxynitride, and a combination thereof. In particular instances, the ceramic material can include alumina. More specifically, the ceramic material may include a boehmite material, which may be a precursor of alpha alumina. The term “boehmite” is generally used herein to denote alumina hydrates including mineral boehmite, typically being Al2O3·H2O and having a water content on the order of 15%, as well as psuedoboehmite, having a water content higher than 15%, such as 20-38% by weight. It is noted that boehmite (including psuedoboehmite) has a particular and identifiable crystal structure, and accordingly unique X-ray diffraction pattern, and as such, is distinguished from other aluminous materials including other hydrated aluminas such as ATH (aluminum trihydroxide) a common precursor material used herein for the fabrication of boehmite particulate materials. Furthermore, the mixture6201can be formed to have a particular content of liquid material. Some suitable liquids may include organic materials, such as water. In accordance with one embodiment, the mixture6201can be formed to have a liquid content less than the solids content of the mixture6201. In more particular instances, the mixture6201can have a liquid content of at least about 25 wt % for the total weight of the mixture6201. In other instances, the amount of liquid within the mixture6201can be greater, such as at least about 35 wt %, at least about 45 wt %, at least about 50 wt %, or even at least about 58 wt %. Still, in at least one non-limiting embodiment, the liquid content of the mixture can be not greater than about 75 wt %, such as not greater than about 70 wt %, not greater than about 65 wt %, not greater than about 60 wt %, or even not greater than about 55 wt %. It will be appreciated that the content of the liquid in the mixture6201can be within a range between any of the minimum and maximum percentages noted above. Furthermore, to facilitate processing and forming shaped abrasive particles according to embodiments herein, the mixture6201can have a particular storage modulus. For example, the mixture6201can have a storage modulus of at least about 1×104Pa, such as at least about 4×104Pa, or even at least about 5×104Pa. However, in at least one non-limiting embodiment, the mixture6201may have a storage modulus of not greater than about 1×107Pa, such as not greater than about 1×106Pa. It will be appreciated that the storage modulus of the mixture6201can be within a range between any of the minimum and maximum values noted above. The storage modulus can be measured via a parallel plate system using ARES or AR-G2 rotational rheometers, with Peltier plate temperature control systems. For testing, the mixture6201can be extruded within a gap between two plates that are set to be approximately 8 mm apart from each other. After extruding the get into the gap, the distance between the two plates defining the gap is reduced to 2 mm until the mixture6201completely fills the gap between the plates. After wiping away excess mixture, the gap is decreased by 0.1 mm and the test is initiated. The test is an oscillation strain sweep test conducted with instrument settings of a strain range between 0.1% to 100%, at 6.28 rad/s (1 Hz), using 25-mm parallel plate and recording 10 points per decade. Within 1 hour after the test completes, lower the gap again by 0.1 mm and repeat the test. The test can be repeated at least 6 times. The first test may differ from the second and third tests. Only the results from the second and third tests for each specimen should be reported. Furthermore, to facilitate processing and forming shaped abrasive particles according to embodiments herein, the mixture6201can have a particular viscosity. For example, the mixture6201can have a viscosity of at least about 4×103Pa s, at least about 5×103Pa s, at least about 6×103Pa s, at least about 8×103Pa s, at least about 10×103Pa s, at least about 20×103Pa s, at least about 30×103Pa s, at least about 40×103Pa s, at least about 50×103Pa s, at least about 60×103Pa s, or even at least about 65×103Pa s. In at least one non-limiting embodiment, the mixture6201may have a viscosity of not greater than about 1×106Pa s, not greater than about 5×105Pa s, not greater than about 3×105Pa s, or even not greater than about 2×105Pa s. It will be appreciated that the viscosity of the mixture6201can be within a range between any of the minimum and maximum values noted above. The viscosity can be calculated by dividing the storage modulus value by 6.28 s−1. Moreover, the mixture6201can be formed to have a particular content of organic materials, including for example, organic additives that can be distinct from the liquid, to facilitate processing and formation of shaped abrasive particles according to the embodiments herein. Some suitable organic additives can include stabilizers, binders, such as fructose, sucrose, lactose, glucose, UV curable resins, and the like. Notably, the embodiments herein may utilize a mixture6201that is distinct from slurries used in conventional tape casting operations. For example, the content of organic materials within the mixture6201, particularly, any of the organic additives noted above may be a minor amount as compared to other components within the mixture6201. In at least one embodiment, the mixture6201can be formed to have not greater than about 30 wt % organic material for the total weight of the mixture6201. In other instances, the amount of organic materials may be less, such as not greater than about 15 wt %, not greater than about 10 wt %, or even not greater than about 5 wt %. Still, in at least one non-limiting embodiment, the amount of organic materials within the mixture6201can be at least about 0.1 wt %, such as at least about 0.5 wt % for the total weight of the mixture6201. It will be appreciated that the amount of organic materials in the mixture6201can be within a range between any of the minimum and maximum values noted above. Moreover, the mixture6201can be formed to have a particular content of acid or base distinct from the liquid, to facilitate processing and formation of shaped abrasive particles according to the embodiments herein. Some suitable acids or bases can include nitric acid, sulfuric acid, citric acid, chloric acid, tartaric acid, phosphoric acid, ammonium nitrate, ammonium citrate. According to one particular embodiment, the mixture6201can have a pH of less than about 5, and more particularly, within a range between about 2 and about 4, using a nitric acid additive. ReferencingFIG.62, the system6200can include a die6203. As illustrated, the mixture6201can be provided within the interior of the die6203and configured to be extruded through a die opening6205positioned at one end of the die6203. As further illustrated, forming can include applying a force6280(that may be translated into a pressure) on the mixture6201to facilitate moving the mixture6201through the die opening6205. In accordance with an embodiment, a particular pressure may be utilized during extrusion. For example, the pressure can be at least about 10 kPa, such as at least about 500 kPa. Still, in at least one non-limiting embodiment, the pressure utilized during extrusion can be not greater than about 4 MPa. It will be appreciated that the pressure used to extrude the mixture6201can be within a range between any of the minimum and maximum values noted above. In certain systems, the die6203can include a die opening6205having a particular shape. It will be appreciated that the die opening6205may be shaped to impart a particular shape to the mixture6201during extrusion. In accordance with an embodiment, the die opening6205can have a rectangular shape. Furthermore, the mixture6201extruded through the die opening6205can have essentially the same cross-sectional shape as the die opening6205. As further illustrated, the mixture6201may be extruded in the form of a sheet6211and onto a belt6209underlying the die6203. In specific instances, the mixture6201can be extruded in the form of a sheet6211directly onto the belt6209, which may facilitate continuous processing. According to one particular embodiment, the belt can be formed to have a film overlying a substrate, wherein the film can be a discrete and separate layer of material configured to facilitate processing and forming of shaped abrasive particles. The process can include providing the mixture6201directly onto the film of the belt to form the sheet6211. In certain instances, the film can include a polymer material, such as polyester. In at least one particular embodiment, the film can consist essentially of polyester. In some embodiments, the belt6209can be translated while moving the mixture6201through the die opening6205. As illustrated in the system6200, the mixture6201may be extruded in a direction6291. The direction of translation6210of the belt6209can be angled relative to the direction of extrusion6291of the mixture. While the angle between the direction of translation6210and the direction of extrusion6291are illustrated as substantially orthogonal in the system6200, other angles are contemplated, including for example, an acute angle or an obtuse angle. The belt6209may be translated at a particular rate to facilitate processing. For example, the belt6209may be translated at a rate of at least about 3 cm/s, such as at least about 4 cm/s, at least about 6 cm/s, at least about 8 cm/s, or even at least about 10 cm/s. Still, in at least one non-limiting embodiment, the belt6209may be translated in a direction6210at a rate of not greater than about 5 m/s, such as not greater than about 1 m/s, or even not greater than about 0.5 m/s. It will be appreciated that the belt6209may be translated at a rate within a range between any of the minimum and maximum values noted above. For certain processes according to embodiments herein, the rate of translation of the belt6209as compared to the rate of extrusion of the mixture6201in the direction6291may be controlled to facilitate proper processing. For example, the rate of translation of the belt6209can be essentially the same as the rate of extrusion to ensure formation of a suitable sheet6211. After the mixture6201is extruded through the die opening6205, the mixture6201may be translated along the belt6209under a knife edge6207attached to a surface of the die6203. The knife edge6207may facilitate forming a sheet6211. More particularly, the opening defined between the surface of the knife edge6207and belt6209may define particular dimensions of the extruded mixture6201. For certain embodiments, the mixture6201may be extruded in the form of a sheet6211having a generally rectangular cross-sectional shape as viewed in a plane defined by a height and width of the sheet6211. While the extrudate is illustrated as a sheet, other shapes can be extruded, including for example cylindrical shapes and the like. The process of forming the sheet6211from the mixture6201can include control of particular features and process parameters to facilitate suitable formation of shaped abrasive particles having one or more features as provided in the embodiments herein. For example, in certain instances, the process of forming a sheet6211from the mixture6201can include forming a sheet6211having a particular height6281controlled in part by a distance between the knife edge6207and a surface of the belt6209. Moreover, it is noted that the height6281of the sheet6211can be controlled by varying a distance between the knife edge6207and the surface of the belt6209. Additionally, forming the mixture6201into the sheet6211can include controlling the dimensions of the sheet6211based in part upon the viscosity of the mixture6201. In particular, forming the sheet6211can include adjusting the height6281of the sheet6211based on the viscosity of the mixture6201. The sheet6211can have particular dimensions, including for example a length (l), a width (w), and a height (h). In accordance with an embodiment, the sheet6211may have a length that extends in the direction of the translating belt6209, which can be greater than the width, wherein the width of the sheet6211is a dimension extending in a direction perpendicular to the length of the belt6209and to the length of the sheet. The sheet6211can have a height6281, wherein the length and width are greater than the height6281of the sheet6211. Notably, the height6281of the sheet6211can be the dimension extending vertically from the surface of the belt6209. In accordance with an embodiment, the sheet6211can be formed to have a particular dimension of height6281, wherein the height may be an average height of the sheet6211derived from multiple measurements. For example, the height6281of the sheet6211can be at least about 0.1 mm, such as at least about 0.5 mm. In other instances, the height6281of the sheet6211can be greater, such as at least about 0.8 mm, at least about 1 mm, at least about 1.2 mm, at least about 1.6 mm, or even at least about 2 mm. Still, in one non-limiting embodiment, the height6281of the sheet6211may be not greater than about 10 mm, not greater than about 5 mm, or even not greater than about 2 mm. It will be appreciated that the sheet6211may have an average height within a range between any of the minimum and maximum values noted above. According to one embodiment, the sheet6211can have a length (l), a width (w), and a height (h), wherein the length≥width≥height. Moreover, the sheet6211can have a secondary aspect ratio of length:height of at least about 10, such as at least about 100, at least about 1000, or even at least about 1000. After extruding the mixture6201from the die6203, the sheet6211may be translated in a direction6212along the surface of the belt6209. Translation of the sheet6211along the belt6209may facilitate further processing to form precursor-shaped abrasive particles. For example, the sheet6211may undergo a shaping process within the shaping zone6213. In particular instances, the process of shaping can include shaping a surface of the sheet6211, including for example, an upper major surface6217of the sheet6211, which may be completed using a shaping article6215. In other embodiments, other major surfaces of the sheet may undergo shaping, including for example, the bottom surface or side surfaces. For certain processes, shaping can include altering a contour of the sheet through one or more processes, such as, embossing, rolling, cutting, engraving, patterning, stretching, twisting, and a combination thereof. In accordance with an embodiment, the process of forming a shaped abrasive particle can further include translation of the sheet along the belt6209through a forming zone6221. In accordance with an embodiment, the process of forming a shaped abrasive particle can include sectioning the sheet6211to form precursor shaped abrasive particles6223. For example, in certain instances, forming can include perforating a portion of the sheet6211. In other instances, the process of forming can include patterning the sheet6211to form a patterned sheet and extracting shapes from the patterned sheet. Particular processes of forming can include cutting, pressing, punching, crushing, rolling, twisting, bending, drying, and a combination thereof. In one embodiment, the process of forming can include sectioning of the sheet6211. Sectioning of the sheet6211can include the use of at least one mechanical object, which may be in the form of a gas, liquid, or solid material. The process of sectioning can include at least one or a combination of cutting, pressing, punching, crushing, rolling, twisting, bending, and drying. Moreover, it will be appreciated that sectioning can include perforating or creating a partial opening through a portion of the sheet6211, which may not extend through the entire height of the sheet6211. In one embodiment, sectioning of the sheet6211can include use of a mechanical object including one or a plurality of a blade, a wire, a disc, and a combination thereof. The process of sectioning can create different types of shaped abrasive particles in a single sectioning process. Different types of shaped abrasive particles can be formed from the same processes of the embodiments herein. Different types of shaped abrasive particles include a first type of shaped abrasive particle having a first two-dimensional shape and a second type of shaped abrasive particle having a different two-dimensional shape as compared to the first two-dimensional shape. Furthermore, different types of shaped abrasive particles may differ from each other in size. For example, different types of shaped abrasive particles may have different volumes as compared to each other. A single process which is capable of forming different types of shaped abrasive particles may be particularly suited for producing certain types of abrasive articles. Sectioning can include moving the mechanical object through a portion of a sheet6211and creating an opening within the sheet6211. In particular, the sheet can be formed to have an opening extending into the volume of the sheet and defined by certain surfaces. The opening can define a cut extending through at least a fraction of the entire height of sheet. It will be appreciated that the opening does not necessarily need to extend through the full height of the sheet. In certain instances, the method of sectioning can include maintaining the opening in the sheet. Maintaining the opening after sectioning the sheet has been sectioned by a mechanical object may facilitate suitable formation of shaped abrasive particles and features of shaped abrasive particles and features of a batch of shaped abrasive particles. Maintaining the opening can include at least partially drying at least one surface of the sheet defining the opening. The process of at least partially drying can include directing a drying material at the opening. A drying material may include a liquid, a solid, or even a gas. According to one particular embodiment, the drying material can include air. Controlled drying may facilitate the formation of shaped abrasive particles according to embodiments herein. In certain instances, the process of sectioning can be conducted prior to sufficient drying of the sheet. For example, sectioning can be conducted prior to volatilization of not greater than about 20% of the liquid from the sheet as compared to the original liquid content of the sheet during initial formation of the sheet. In other embodiments, the amount of volatilization allowed to occur before or during sectioning can be less, such as, not greater than about 15%, not greater than about 12%, not greater than about 10%, not greater than about 8%, or even not greater than about 4% of the original liquid content of the sheet. Referring again toFIGS.62A and62B, after forming precursor-shaped abrasive particles6223, the particles may be translated through a post-forming zone6225. Various processes may be conducted in the post-forming zone6225, including for example, heating, curing, vibration, impregnation, doping, and a combination thereof. In one embodiment, the post-forming zone6225includes a heating process, wherein the precursor-shaped abrasive particles6223may be dried. Drying may include removal of a particular content of material, including volatiles, such as water. In accordance with an embodiment, the drying process can be conducted at a drying temperature of not greater than 300° C. such as not greater than 280° C. or even not greater than about 250° C. Still, in one non-limiting embodiment, the drying process may be conducted at a drying temperature of at least 50° C. It will be appreciated that the drying temperature may be within a range between any of the minimum and maximum temperatures noted above. Furthermore, the precursor-shaped abrasive particles6223may be translated through a post-forming zone at a particular rate, such as at least about 0.2 feet/min and not greater than about 8 feet/min. Furthermore, the drying process may be conducted for a particular duration. For example, the drying process may be not greater than about six hours. After the precursor-shaped abrasive particles6223are translated through the post-forming zone6225, the particles may be removed from the belt6209. The precursor-shaped abrasive particles6223may be collected in a bin6227for further processing. In accordance with an embodiment, the process of forming shaped abrasive particles may further comprise a sintering process. The sintering process can be conducted after collecting the precursor-shaped abrasive particles6223from the belt6209. Sintering of the precursor-shaped abrasive particles6223may be utilized to densify the particles, which are generally in a green state. In a particular instance, the sintering process can facilitate the formation of a high-temperature phase of the ceramic material. For example, in one embodiment, the precursor-shaped abrasive particles6223may be sintered such that a high-temperature phase of alumina, such as alpha alumina is formed. In one instance, a shaped abrasive particle can comprise at least about 90 wt % alpha alumina for the total weight of the particle. In other instances, the content of alpha alumina may be greater, such that the shaped abrasive particle may consist essentially of alpha alumina. FIG.63includes an illustration of a system for forming a shaped abrasive particle in accordance with an embodiment. In particular, the system6300can generally include a screen printing process of forming shaped abrasive particles. However, as noted herein, certain portions of the system may be modified to conduct a molding process. As illustrated, the system6300can include a screen6351configured to be translated between rollers6370and6371. It will be appreciated that the screen6351can be translated over a greater number of rollers or other devices if so desired. As illustrated, the system6300can include a belt6309configured to be translated in a direction6316over rollers6372and6373. It will be appreciated that the belt6309may be translated over a greater number of rollers or other devices if so desired. As illustrated, the system6300can further include a die6303configured to conduct extrusion of a mixture6301contained within a reservoir6302of the die6303. The process of forming shaped abrasive particles can be initiated by forming a mixture6301including a ceramic material and a liquid as described herein. The mixture6301can be provided within the interior of the die6303and configured to be extruded through a die opening6305positioned at one end of the die6303. As further illustrated, extruding can include applying a force (or a pressure) on the mixture6301to facilitate extruding the mixture6301through the die opening6305. In accordance with an embodiment, a particular pressure may be utilized during extrusion. For example, the pressure can be at least about 10 kPa, such as at least about 500 kPa. Still, in at least one non-limiting embodiment, the pressure utilized during extrusion can be not greater than about 4 MPa. It will be appreciated that the pressure used to extrude the mixture6301can be within a range between any of the minimum and maximum values noted above. In particular instances, the mixture6301can be extruded through a die opening6305at the end of the die6303proximate to the screen6351. The screen6351may be translated in a direction6353at a particular rate to facilitate suitable processing. Notably, the screen6351can be translated through the application zone6383including the die opening6305to facilitate the formation of precursor-shaped abrasive particles. The screen6351may be translated through the application zone at a rate of at least about 3 cm/s, such as at least about 4 cm/s, at least about 6 cm/s, at least about 8 cm/s, or even at least about 10 cm/s. Still, in at least one non-limiting embodiment, the screen6351may be translated in a direction6353at a rate of not greater than about 5 m/s, such as not greater than about 1 m/s, or even not greater than about 0.5 m/s. It will be appreciated that the screen6351may be translated at a rate within a range between any of the minimum and maximum values noted above. Additionally, the belt6309can be translated in a direction6316at a particular rate to facilitate suitable processing. For example, the belt6309can be translated at a rate of at least about 3 cm/s, such as at least about 4 cm/s, at least about 6 cm/s, at least about 8 cm/s, or even at least about 10 cm/s. Still, in at least one non-limiting embodiment, the belt6309may be translated in a direction6316at a rate of not greater than about 5 m/s, such as not greater than about 1 m/s, or even not greater than about 0.5 m/s. It will be appreciated that the belt6309may be translated at a rate within a range between any of the minimum and maximum values noted above. In accordance with a particular embodiment, the screen6351may be translated at a particular rate as compared to the rate of translation of the belt6309. For example, within the application zone6383, the screen6351may be translated at substantially the same rate of translation of the belt6309. That is, the difference in rate of translation between the screen and the belt may be not greater than about 5%, such as not greater than about 3%, or even not greater than about 1% based on the rate of the translation of the screen6351. As illustrated, the system6300can include an application zone6383, including the die opening6305. Within the application zone6383, the mixture6301may be extruded from the die6303and directly onto the screen6351. More particularly, a portion of the mixture6301may be extruded from the die opening6305, and further extruded through one or more openings in the screen6351and onto the underlying belt6309. Referring briefly toFIG.64, a portion of a screen6451is illustrated. As shown, the screen6451can include an opening6452, and more particularly, a plurality of openings6452. The openings can extend through the volume of the screen6451, to facilitate passable of the mixture6301through the openings and onto the belt6309. In accordance with an embodiment, the openings6452can have a two-dimensional shape as viewed in a plane defined by the length (l) and width (w) of the screen. While the openings6452are illustrated as having a three-pointed star two-dimensional shape, other shapes are contemplated. For example, the openings6452can have a two-dimensional shape such as polygons, ellipsoids, numerals, Greek alphabet letters, Latin alphabet letters, Russian alphabet characters, complex shapes including a combination of polygonal shapes, and a combination thereof. In particular instances, the openings6452may have two-dimensional polygonal shapes such as, a triangle, a rectangle, a quadrilateral, a pentagon, a hexagon, a heptagon, an octagon, a nonagon, a decagon, and a combination thereof. Moreover, a screen6451can be formed to include a combination of openings6452having a plurality of different two-dimensional shapes. Certain aspects of processing were found to facilitate the formation of shaped abrasive particles according to embodiments herein. Notably, the orientation of the openings relative to the die head was found to have an effect on the shape of the shaped abrasive particles. In particular, it was noted, that when the openings are aligned as illustrated inFIG.64, wherein a point6455of the opening is first filled with the mixture6301the shaped abrasive particles are suitably formed having the features described herein. In other orientations, wherein for example, a side6456of the opening would be first filled, as opposed to a point (e.g.,6455) of the opening6452, it was noted that the shaped abrasive particles had certain less suitable features. Referring again toFIG.63, after forcing the mixture6301through the die opening6305and a portion of the mixture6301through the openings6352in the screen6351, precursor-shaped abrasive particles6353may be printed on a belt6309disposed under the screen6351. According to a particular embodiment, the precursor-shaped abrasive particles6353can have a shape substantially replicating the shape of the openings6352. After extruding the mixture6301into the openings6352of the screen6351, the belt6309and screen6351may be translated to a release zone6385, wherein the belt6309and screen6351can be separated to facilitate the formation of precursor shaped abrasive particles. In accordance with an embodiment, the screen6351and belt6309may be separated from each other within the release zone6385at a particular release angle6355. In accordance with specific embodiment, the release angle6355can be a measure of the angle between a lower surface6354of the screen6351and an upper surface6356of the belt6309. Notably, the mixture6301can be forced through the screen6351in rapid fashion, such that the average residence time of the mixture6301within the openings152can be less than about 2 minutes, less than about 1 minute, less than about 40 second, or even less than about 20 seconds. In particular non-limiting embodiments, the mixture6301may be substantially unaltered during printing as it travels through the screen openings6352, thus experiencing no change in the amount of components, and may experience no appreciable drying in the openings6352of the screen6351. In an alternative embodiment, the process of forming can include a molding process. The molding process may utilize some of the same components of the system6300, however, the screen can be replaced with a molding blank having openings within a substrate material for molding the mixture6301. Notably, unlike a screen, the molding blank can have openings that extend partially through the entire thickness of the blank, such that the openings are not apertures extending from one major surface to the opposite major surface of the blank. Instead, the mold openings can have a bottom surface within the interior volume, which are intended to form a major surface of the precursor-shaped abrasive particle formed therein. Moreover, a molding system may not necessarily utilize a belt underlying the molding blank. The forming process may also utilize a particular drying process to facilitate formation of shaped abrasive particles having features of the embodiments herein. In particular, the drying process may include drying under conditions including humidity, temperature, and atmospheric pressure and composition suitable for limiting distortions to the shaped abrasive particles. It was found that unlike the formation of shaped abrasive particles having typical polygonal shapes, the process of forming complex shapes, particularly using replication processes, required control of one or more process parameters, including drying conditions, amount and type of lubricant, pressure applied to the mixture during extrusion, material of the blank or belt, and the like. In particular instances, it was found that a belt or blank of stainless steel or polycarbonate polymer could be used. Moreover, it was found that the use of a natural oil material (e.g., canola oil) as a lubricant on the openings of the blank or belt may facilitate improved forming of shaped abrasive particles. The body of the shaped abrasive particles may include additives, such as dopants, which may be in the form of elements or compounds (e.g., oxides). Certain suitable additives can include alkali elements, alkaline earth elements, rare-earth elements, hafnium (Hf), zirconium (Zr), niobium (Nb), tantalum (Ta), molybdenum (Mo), and a combination thereof. In particular instances, the additive can include an element such as lithium (Li), sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), scandium (Sc), yttrium (Y), lanthanum (La), cesium (Ce), praseodymium (Pr), niobium (Nb), hafnium (Hf), zirconium (Zr), tantalum (Ta), molybdenum (Mo), vanadium (V), chromium (Cr), cobalt (Co), iron (Fe), germanium (Ge), manganese (Mn), nickel (Ni), titanium (Ti), zinc (Zn), and a combination thereof. The body of a shaped abrasive article may include a specific content of additive (e.g., dopant). For example, the body of a shaped abrasive particle may include not greater than about 12 wt % additive for the total weight of the body. In still other embodiments, the amount of additive may be less, such as not greater than about 11 wt %, not greater than about 10 wt %, not greater than about 9 wt %, not greater than about 8 wt %, not greater than about 7 wt %, not greater than about 6 wt %, or even not greater than about 5 wt %. Still, the amount of additive in at least one non-limiting embodiment can be at least about 0.5 wt %, such as at least about 1 wt %, at least about 1.3 wt %, at least about 1.8 wt %, at least about 2 wt %, at least about 2.3 wt %, at least about 2.8 wt %, or even at least about 3 wt %. It will be appreciated that the amount of additive within a body of a shaped abrasive particle may be within a range between any of the minimum and maximum percentages noted above. FIG.65Aincludes a top view image of a shaped abrasive particle formed according to a particular embodiment. As illustrated, the shaped abrasive particle6500can define a star-shaped body, as viewed in two dimensions. In particular, the shaped abrasive particle6500can include a body6501having a central portion6502and a first arm6503, a second arm6504, and a third arm6505extending from the central portion6502. The body6501can have a length (l) measured as the longest dimension along a side of the particle and a width (w), measured as the longest dimension of the particle between a midpoint6553of a side through the midpoint6590of the body6501to a first tip6506of the first arm6503. The width can extend in a direction perpendicular to the dimension of the length. The body6501can have a height (h), extending in a direction perpendicular to the upper surface6510of the body6501defining the third side surface6556between the upper surface and the base surface6511as illustrated inFIG.65B, which is a side view illustration of the image of the particle ofFIG.65A. The shaped abrasive particle6500can have a body6501in the form of a three-pointed star defined by the first arm6503, second arm6504, and the third arm6505extending from the central portion6502. According to one particular embodiment, at least one of the arms, including for example, the first arm6503, can have a midpoint width6513that is less than a central portion width6512. The central portion6502can be defined as a region between the midpoints6551,6552, and6553of the first side surface6554, second side surface6555, and third side surface6556, respectively. The central portion width6512of the first arm6503can be the width of the dimension between the midpoints6551and6552. The midpoint width6513can be the width of the line at a midpoint between the line of the central portion width6510and the tip6506of the first arm6503along a first axis6560. In certain instances, the midpoint width6513can be not greater than about 90% of the central portion width6512, such as not greater than about 80%, not greater than about 70%, not greater than about 65%, or even not greater than about 60%. Still, the midpoint width6513can be at least about 10%, such as at least about 20%, at least about 30%, or even at least about 40% of the central portion width6510. It will be appreciated that the midpoint width6513can have a width relative to the central portion width6512within a range between any of the above minimum and maximum percentages. Moreover, the body6501can have at least one arm, such as the first arm6503, having a tip width6514at the tip6506of the first arm6503that is less than a midpoint width6513. In such instances wherein the tip6506is sharply formed, the tip width6514may be considered 0. In instances wherein the tip6506has a radius of curvature, the tip width6514may be considered the diameter of the circle defined by the radius of curvature. According to one embodiment, the tip width6514can be not greater than about 90% of the midpoint width6513, such as not greater than about 80%, not greater than about 70%, not greater than about 60%, not greater than about 50%, not greater than about 40%, not greater than about 30%, not greater than about 20%, or even not greater than about 10%. Still, in certain non-limiting embodiments, the tip width6514can be at least about 1%, such as at least about 2%, at least about 3%, at least about 5%, or even at least about 10% of the midpoint width6513. It will be appreciated that the tip width6514can have a width relative to the midpoint width6513within a range between any of the above minimum and maximum percentages. As further illustrated, the body6501can have a first arm6503including a first tip6506defining a first tip angle6521between the first side surface6554and the second side surface6555. According to an embodiment, the first tip angle can be less than about 60 degrees, such as not greater than about 55 degrees, not greater than about 50 degrees, not greater than about 45 degrees, or even not greater than about 40 degrees. Still, the first tip angle can be at least about 5 degrees, such as at least about 8 degrees, at least about 10 degrees, at least about 15 degrees, at least about 20 degrees, at least about 25 degrees, or even at least about 30 degrees. The first tip angle can be within a range between any of the minimum and maximum values noted above. The body6501can include a second arm6504having a second tip6507defining a second tip angle6522between the second side surface6555and third side surface6556. The second tip angle can be substantially the same as the first tip angle, such as within 5% of the angle numerical value. Alternatively, the second tip angle can be substantially different relative to the first tip angle. The body6501can include a third arm6505having a third tip6508defining a third tip angle6523between the first side surface6554and third side surface6556. The third tip angle can be substantially the same as the first tip angle or second tip angle, such as within 5% of the angle numerical value. Alternatively, the third tip angle can be substantially different relative to the first tip angle or the second tip angle. The body6501can have a total angle, which is a sum of the value of the first tip angle, second tip angle, and third tip angle which can be less than about 180 degrees. In other embodiments, the total angle can be not greater than about 175 degrees, such as not greater than about 170 degrees, not greater than about 165 degrees, not greater than about 150 degrees, such as not greater than about 140 degrees, not greater than about 130 degrees, not greater than about 125 degrees, or even not greater than about 120 degrees. Still, in one non-limiting embodiment, the body6501can have a total angle of at least about 60 degrees, such as at least about 70 degrees, at least about 80 degrees, at least about 90 degrees, such as at least about 95 degrees, at least about 100 degrees, or even at least about 105 degrees. It will be appreciated that the total sum angle can be within a range between any of the minimum and maximum values noted above. As noted herein, the body6501can have a first side surface6554extending between the first arm6506and the third arm6508. In certain instances, the first side surface6554can have an arcuate contour. For example, turning briefly toFIG.65C, a top view image of a shaped abrasive particle according to an embodiment is provided. Notably, the shaped abrasive particle ofFIG.65Ccan include a three-pointed star having a body6581and an arcuate side surface6582extending between two points. In particular instances, the side surface6582can have a concave contour defining a curved portion extending inward toward the central portion6583of the body6581. Referring again toFIG.65A, the body6501can have a first side surface6554having a first side section6558and a second side section6559. The first side section6558can extend between the first tip6506and the midpoint6551and the second side section6559can extend between the third tip6508and the midpoint6551. The first side section6558and second side section6559can define an interior angle6562that can be obtuse. For example, the interior angle6562can be greater than about 90 degrees, such as greater than about 95 degrees, greater than about 100 degrees, greater than about 110 degree, or even greater than about 120 degrees. Still, in one non-limiting embodiment, the interior angle6562can be not greater than about 320 degrees, such as not greater than about 300 degrees, or even not greater than about 270 degrees. It will be appreciated that the interior angle can be within a range between any of the minimum and maximum values noted above. The first side section6558can extend for a significant portion of the length of the first side surface6554. For example, the first side section6558can extend for at least about 20%, such as at least about 25%, at least about 30%, at least about 35%, or even at least about 40% of a total length of the first side surface6554. Still, in one non-limiting embodiment, the first side section6558can have a length (ls1) between the midpoint6551and the first tip6506of not greater than about 80%, such as not greater than about 75%, not greater than about 70%, or even not greater than about 65% of the total length of the side surface6554. It will be appreciated that the length of the first side section6558can be within a range between any of the minimum and maximum percentages noted above. The second side section6559can extend for a significant portion of the length of the first side surface6554. For example, the second side section6559can extend for at least about 20%, such as at least about 25%, at least about 30%, at least about 35%, or even at least about 40% of a total length of the first side surface6554. Still, in one non-limiting embodiment, the second side section6559can have a length (ls2) between the midpoint6551and the third tip6508of not greater than about 80%, such as not greater than about 75%, not greater than about 70%, or even not greater than about 65% of the total length of the side surface6554as a straight line between the first tip6506and the third tip6508. It will be appreciated that the length of the second side section6559can be within a range between any of the minimum and maximum percentages noted above. The body6501can further include a fractured region6570on at least a portion of one side surface. For example, the body6501can have a fractured region6570on a portion of the side surface6554between the midpoint6551and the third tip6508. The fracture region6570can be intersecting at least a portion of an edge defining the base surface6511. Alternatively, or additionally, the fracture region6570can be intersecting at least a portion of an edge defining the upper surface6510. The fractured region can be characterized by having a surface roughness greater than a surface roughness of at least the upper surface6510or the base surface6511of the body6501. The fractured region6570can define a portion of the body extending from the base surface6511. In certain instances, the fractured region6570can be characterized by irregularly shaped protrusions and grooves extending from the base surface6511along the first side surface6554. In certain instances, the fractured region6570can appear as and define a serrated edge. A fracture region6583is also illustrated on the side surface6582of the shaped abrasive particle ofFIG.65C. In certain instances, the fracture region6570can be preferentially located at or near the tips of the arms of the body. The fractured region6570can extend from the bottom surface1703and extend vertically for a fraction of the entire height of the side surface or even for the entire height of the side surface. While the foregoing body6501of the three-pointed star has been shown to have an upper surface6510having a two-dimensional shape, as viewed in the plane of the length and width of the body, that is substantially the same as the two-dimensional shape of the base surface6511of the body6501, other shapes are contemplated. For example, in one embodiment, the cross-sectional shape of the body at the base surface can define a base surface shape from the group consisting of a three-pointed star, a four-pointed star, a cross-shape, a polygon, ellipsoids, numerals, Greek alphabet characters, Latin alphabet characters, Russian alphabet characters, complex shapes having a combination of polygonal shapes, and a combination thereof. Moreover, the cross-sectional shape of the body at the upper surface can define an upper surface shape, which can be different than the base surface shape and selected from the group of a three-pointed star, a four-pointed star, a cross-shape, a polygon, ellipsoids, numerals, Greek alphabet characters, Latin alphabet characters, Russian alphabet characters, complex shapes having a combination of polygonal shapes, and a combination thereof. In particular instances, the upper surface shape can have an arcuate form of the base surface shape. For example, the upper surface shape can define an arcuate three-pointed two-dimensional shape, wherein the arcuate three-pointed two-dimensional shape defines arms having rounded ends. In particular, the arms as defined at the base surface can have a smaller radius of curvature at the tip as compared to the radius of curvature of the corresponding tip at the upper surface. As described in other embodiments herein, it will be appreciated that at least one of the arms of the body6501may be formed to have a twist, such that the arm twists around a central axis. For example, the first arm6503may twist around the axis6560. Moreover, the body6501can be formed such that at least one arm extends in an arcuate path from the central region. FIG.66Aincludes a top view image of a shaped abrasive particle formed according to a particular embodiment. As illustrated, the shaped abrasive particle6600can define a star-shaped body, as viewed in a plane defined by the two dimensions of length and width. In particular, the shaped abrasive particle6600can include a body6601having a central portion6602and a first arm6603, a second arm6604, a third arm6605, and a fourth arm6606extending from the central portion6602. The body6601can have a length (l), measured as the longest dimension along a side of the particle and a width (w), and measured as the longest dimension of the particle between two points of opposite arms and through the midpoint6609of the body6601. The width can extend in a direction perpendicular to the dimension of the length. The body6601can have a height (h), extending in a direction perpendicular to the upper surface6610of the body6601defining the third side surface6656between the upper surface and the base surface6611as illustrated inFIG.66B. Notably, the body6601can have more than one height as will be described in more detail herein. The shaped abrasive particle6600can have a body6601in the form of a four-pointed star defined by the first arm6603, a second arm6604, a third arm6605, and the fourth arm6606extending from the central portion6602. The body6601can have any of the features described in the embodiments herein. For example, according to one particular embodiment, at least one of the arms, including for example, the first arm6603, can have a midpoint width that is less than a central portion width, as described in accordance with the embodiment ofFIG.65A. Moreover, the body6601can have at least one arm, such as the first arm6603, having a tip width at the tip of the first arm that is less than a midpoint width as described in accordance with the embodiment ofFIG.65A. In one aspect, the body6601can have a first arm6603including a first tip6607defining a first tip angle6621between the first side surface6654and the second side surface6655. According to an embodiment, the first tip angle can be less than about 60 degrees, such as not greater than about 55 degrees, not greater than about 50 degrees, not greater than about 45 degrees, or even not greater than about 40 degrees. Still, the first tip angle6621can be at least about 5 degrees, such as at least about 8 degrees, at least about 10 degrees, at least about 15 degrees, or even at least about 20 degrees. The first tip angle6621can be within a range between any of the minimum and maximum values noted above. Likewise, any of the other tips, including the second tip6608of the second arm6604, the third tip6609of the third arm6605, or fourth tip6610of the fourth arm6606can have a tip angle having the same features described in accordance with the first tip angle6621above. According to one embodiment the second tip6608can define a second tip angle that is substantially the same as the first tip angle6621, such as within 5% of the angle numerical value. Alternatively, the second tip angle can be substantially different relative to the first tip angle6621. The third tip6609can define a third tip angle that is substantially the same as the first tip angle6621, such as within 5% of the angle numerical value. Alternatively, the third tip angle can be substantially different relative to the first tip angle6621. The fourth tip6610can define a fourth tip angle that is substantially the same as the first tip angle6621, such as within 5% of the angle numerical value. Alternatively, the fourth tip angle can be substantially different relative to the first tip angle6621. According to one embodiment, the body6601can include a first arm6603, second arm6604, third arm6605, and fourth arm6606that are substantially evenly spaced apart with respect to each other. As illustrated, the arms6603-6606can be spaced substantially evenly around the central portion6602. In one particular embodiment, the arms6603-6606can be spaced apart from each other at substantially orthogonal angles relative to each other. In other embodiments, the first arm6603and second arm6604can be spaced apart from each other based on the spacing angle6631defined by the angle between the axis6690extending between opposite tips6609and6607and through the midpoint6609relative to the axis6691extending between tips6608and6610and through the midpoint6609. The first arm6603and second arm6604can be spaced apart from each other as define by the spacing angle6631by at least about 45 degrees, such as at least about 60 degrees, or even at least about 70 degrees. Still, in other embodiments, the spacing angle6631can be not greater than about 120 degrees, such as not greater than about 110 degrees, or even approximately 90 degrees. The spacing angle6631can be within a range between any of the minimum and maximum values noted above. In certain instances, the body6601can be formed such that at least one side surface, such as the first side surface6654can have an arcuate contour. In more particular embodiments, at least one side surface can have a concave curvature for at least a portion of the length of the entire side surface. In still another embodiment, at least one side surface of the body6601, such as the first side surface6654, can have a first section6625and a second section6626, which can be joined together at a first side surface midpoint6627and defining a first interior angle6628. According to one embodiment, the first interior angle can be greater than about 90 degrees, such as greater than about 95 degrees, greater than about 100 degrees, greater than about 130 degrees, greater than about 160 degrees, greater than about 180 degrees, or even greater than about 210 degrees. Still, in one non-limiting embodiment, the first interior angle can be not greater than about 320 degrees, not greater than about 300 degrees, or even not greater than about 270 degrees. The first interior angle can be within a range between any of the minimum and maximum values noted above. Moreover, the body can include a second interior angle6629at the second side surface6655, a third interior angle6632at the third side surface6656, and a fourth interior angle6633at the fourth side surface6657. Each of the interior angles can have the features described with respect to the first interior angle6628. Moreover, each and any of the second side surface6655, the third side surface6656, and the fourth side surface6657can have any of the features of the first side surface6654. The body6601can have a first arm6603and the third arm6605extending in opposite directions from the central portion6602of the body6601relative to each other. Moreover, the second arm6604and the fourth arm6606can extend in opposite directions relative to each other. According to one embodiment, the second arm6604can have a length, as measured between from the boundary of the central portion6602to the tip6608along the axis6691that can be substantially the same as a length of the fourth arm6606. In yet another instance, the second arm6604can have a length that is substantially different than (e.g., less than or greater than) a length of the first arm6603or third arm6605. While the foregoing body6601of the four-pointed star has been shown to have an upper surface6640having a two-dimensional shape, as viewed in the plane of the length and width of the body, that is substantially the same as the two-dimensional shape of the base surface6641of the body6501, other shapes are contemplated. For example, in one embodiment, the cross-sectional shape of the body at the base surface can define a base surface shape from the group consisting of a three-pointed star, a four-pointed star, a cross-shape, a polygon, ellipsoids, numerals, Greek alphabet characters, Latin alphabet characters, Russian alphabet characters, complex shapes having a combination of polygonal shapes, and a combination thereof. Moreover, the cross-sectional shape of the body at the upper surface can define an upper surface shape, which can be different than the base surface shape and selected from the group of a three-pointed star, a four-pointed star, a cross-shape, a polygon, ellipsoids, numerals, Greek alphabet characters, Latin alphabet characters, Russian alphabet characters, complex shapes having a combination of polygonal shapes, and a combination thereof. In particular instances, the upper surface shape can have an arcuate form of the base surface shape. For example, the upper surface shape can define an arcuate four-pointed two-dimensional shape, wherein the arcuate four-pointed two-dimensional shape defines arms having rounded ends. In particular, the arms as defined at the base surface can have a smaller radius of curvature at the tip as compared to the radius of curvature of the corresponding tip at the upper surface. According to one particular aspect, the body can be formed to have limited deformation or warping of the body. For example, the body can have a curling factor (ht/hi) of not greater than about 10, wherein the curling factor is defined as a ratio between the greatest height of the body at one tip of an arm (ht) as compared to a smallest dimension of height of the body at the interior (hi) (e.g., within the central portion6602). For example, turning to a side-view illustration of a shaped abrasive particle ofFIG.66B, the body6601can have an interior height, which represents the smallest height of the particle as viewed from the side. The greatest height (ht) of the body is represented by the distance between the bottom surface (or plane of the bottom surface) and the highest point of the body6601as viewed from the side, which can be tip of a curled up arm. The shaped abrasive particles of the embodiments herein demonstrate limited warping, having a curling factor of not greater than about 5, not greater than about 3, not greater than about 2, not greater than about 1.8, not greater than about 1.7, not greater than about 1.6, not greater than about 1.5, not greater than about 1.3, not greater than about 1.2, not greater than about 1.14, or even not greater than about 1.10. Suitable computer programs, such as ImageJ software, may be used to conduct an accurate analysis from images of the shaped abrasive particles to measure curling factor. FIG.67includes a top view image of a shaped abrasive particle formed according to a particular embodiment. As illustrated, the shaped abrasive particle6700can define a cross-shaped body, as viewed in a plane defined by the two dimensions of length and width. In particular, the shaped abrasive particle6700can include a body6701having a central portion6702and a first arm6703, a second arm6704, a third arm6705, and a fourth arm6706extending from the central portion6702. The body6701can have a length (l), measured as the longest dimension along a side of the particle and a width (w), and measured as the longest dimension of the particle between two points of opposite arms and through the midpoint6709of the body6701. The width can extend in a direction perpendicular to the dimension of the length. The body6701can have a height (h), extending in a direction perpendicular to the upper surface6710of the body6701defining a side surface between the upper surface6710and the base surface6711. The body6701can have any one or a combination of features described in any of the embodiments herein. The body6701can have at least one arm, such as the first arm6703having a midpoint width6714that is substantially the same as a central portion width6712of the first arm6703. Moreover, the length of the arm between points6715and6716on the axis6790defining the width of the body6701can be less than the width of the first arm6703. In particular instances, the length can be not greater than about 90% of the width, such as not greater than about 80%, not greater than about 70%, not greater than about 60%. Still, in one non-limiting embodiment, the length of the first arm6703can be at least about 10%, such as at least about 20% of the width of the first arm6703. The length can have a dimension relative to the width within a range between any of the minimum and maximum percentages noted above. Reference to the width of the first arm6703can be reference to the central portion width6712, or midpoint width6714. Any of the arms of the body6701can have the same features of the first arm6703. FIG.68includes a top view image of a shaped abrasive particle according to an embodiment. As shown, the shaped abrasive particle6800can define a generally cross-shaped body, as viewed in a plane defined by the two dimensions of length and width. In particular, the shaped abrasive particle6800can include a body6801having a central portion6802and a first arm6803, a second arm6804, a third arm6805, and a fourth arm6806extending from the central portion6802. The body6801can have a length (l), measured as the longest dimension along a side of the particle and a width (w), and measured as the longest dimension of the particle between two points of opposite arms and through the midpoint6809of the body6801. The width can extend in a direction perpendicular to the dimension of the length. The body6801can have a height (h), extending in a direction perpendicular to the upper surface6810of the body6801defining a side surface between the upper surface6810and the base surface6811. The body6801can have any one or a combination of features described in any of the embodiments herein. In the particular embodiment ofFIG.68, the body can have a particular combination of two-dimensional shapes of the base surface6811and the upper surface6810. For example, the body can have a two-dimensional shape (i.e., cross-sectional shape) of the body at the base surface defining a base surface shape, and a two-dimensional shape of the body at the upper surface defining an upper surface shape, and in particular, the base surface shape can be a generally cross-shaped the upper surface shape can be a rounded quadrilateral shape. The rounded quadrilateral shape can be defined by an upper surface6810(edges shown by the dotted line) that has four sides joined by rounded corners, wherein the corners generally correspond to the arms of the cross-shape defined by the base surface. Notably, the upper surface may not define arm portions separated by a side surface having at least two side surface sections angled with respect to each other, which are shown by the cross-shaped contour of the base surface shape. FIG.69Aincludes an illustration of a side view of a shaped abrasive particle according to an embodiment. As illustrated, the shaped abrasive particle6900can include a body6901including a first layer6902and a second layer6903overlying the first layer6902. According to an embodiment, the body6901can have layers6902and6903that are arranged in a stepped configuration relative to each other. A stepped configuration can be characterized by at least one plateau region6920on an upper surface6910of the first layer6902between a side surface6904of the first layer6902and a side surface6905of the second layer6903. The size and shape of the plateau region6920may be controlled or predetermined by one or more processing parameters and may facilitate an improved deployment of the abrasive particles into an abrasive article and performance of the abrasive article. In one embodiment, the plateau region6902can have a lateral distance6921, which can be defined as the greatest distance between an edge6907between the upper surface6910of the first layer6902and a side surface6904of the first layer to the side surface6905of the second layer. Analysis of the lateral distance6921may be facilitated by a top-view image of the body6901, such as shown inFIG.69B. As illustrated, the lateral distance6921can be the greatest distance of the plateau region6902. In one embodiment, the lateral distance6921may have a length that is less than the length6910of the first layer6902(i.e., larger layer). In particular, the lateral distance6921can be not greater than about 90%, such as not greater than about 80%, not greater than about 70%, not greater than about 60%, not greater than about 50%, not greater than about 40%, not greater than about 30%, or even not greater than about 20% of the length6910of the first layer6902of the body6901. Still, in one non-limiting embodiment, the lateral distance6921can have a length that is at least about 2%, at least about 5%, at least about 8%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, or even at least about 50% of the length of the first layer6902of the body6901. It will be appreciated that the lateral distance6921can have a length within a range between any of the minimum and maximum percentages noted above. The second layer6903can have a particular length6909, which is the longest dimension of a side, such as shown inFIG.69B, relative to a length6910of the first layer6902that may facilitate improved deployment of the abrasive particles into an abrasive article and/or performance of the abrasive article. For example, the length6909of the second layer6903can be not greater than about 90%, such as not greater than about 80%, not greater than about 70%, not greater than about 60%, not greater than about 50%, not greater than about 40%, not greater than about 30%, or even not greater than about 20% of the length6910of the first layer6902of the body6901. Still, in one non-limiting embodiment, the second layer6903can have a length69909that can be at least about 2%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or even at least about 70% of the length6910of the first layer6902of the body6901. It will be appreciated that the length6909of the second layer6903relative to the length6910of the first layer6902can be within a range between any of the minimum and maximum percentages noted above. The foregoing shaped abrasive particle ofFIGS.69A and69Bcan be formed using multiple sheets of material, multiple screens, and/or multiple molding blanks. For example, one process can include the use of a first screen, which is completely or partially filled with a first mixture, and provision of a second screen, which can be different in size, shape or orientation with respect to the first screen, and provision of a second mixture within the openings of the second screen. The second screen can be placed over the first screen or over precursor-shaped abrasive particles formed from the first screen. The second mixture can be provided on the precursor-shaped abrasive particles of the first mixture to form precursor-shaped abrasive particles having the stepped and layered configuration. Notably, the openings of the second screen can be smaller than the openings of the first screen. It will be appreciated that the first screen and second screen can have, but need not necessarily utilize, different size openings, different two-dimensional shapes of openings, and a combination thereof. Moreover, in certain instances, the first screen and second screen can be used at the same time as a composite screen to shape the mixture. In such instances, the first screen and second screen may be affixed to each other to facilitate proper and continuous alignment between the openings of the first screen and second screen. The second screen can be oriented on the first screen to facilitate alignment between the openings in the first screen and openings in the second screens to facilitate suitable delivery of the mixture into the openings of the first screen and second screen. Still, the first screen and second screen may be used in separate processes. For example, wherein the first mixture is provided in the first screen at a first time and the second mixture is provided in the second screen at a second time. More particularly, the first mixture can be provided in the openings of the first screen, and after the first mixture has been formed in the openings of the first screen, the second mixture can be provided on the first mixture. Such a process may be conducted while the first mixture is contained in the first openings of the first screen. In another instance, the first mixture may be removed from the openings of the first screen to create precursor-shaped abrasive particles of the first mixture. Thereafter, the precursor shaped abrasive particles of the first mixture can be oriented with respect to openings of the second screen, and the second mixture can be placed in the openings of the second screen and onto the precursor shaped abrasive particles of the first mixture to facilitate formation of composite precursor shaped abrasive particles including the first mixture and the second mixture. The same process may be used with one mold and one screen. Moreover, the same process may be completed using first and second molds to form the first and second layers, respectively. It will be appreciated that any of the characteristics of the embodiments herein can be attributed to a batch of shaped abrasive particles. A batch of shaped abrasive particles can include, but need not necessarily include, a group of shaped abrasive particles made through the same forming process. In yet another instance, a batch of shaped abrasive particles can be a group of shaped abrasive particles of an abrasive article, such as a fixed abrasive article, and more particularly, a coated abrasive article, which may be independent of a particular forming method, but having one or more defining features present in a particular population of the particles. For example, a batch of particles may include an amount of shaped abrasive particles suitable for forming a commercial grade abrasive product, such as at least about 20 lbs. of particles. Moreover, any of the features of the embodiments herein (e.g., aspect ratio, multiple portions, number of arms, midpoint width to central portion width, two-dimensional shape, curling factor, etc.) can be a characteristic of a single particle, a median value from a sampling of particles of a batch, or an average value derived from analysis of a sampling of particles from a batch. Unless stated explicitly, reference herein to the characteristics can be considered reference to a median value that is a based on a statistically significant value derived from a random sampling of a suitable number of particles of a batch. Notably, for certain embodiments herein, the sample size can include at least 10, and more typically, at least 40 randomly selected particles from a batch of particles. Any of the features described in the embodiments herein can represent features that are present in at least a portion of a batch of shaped abrasive particles. The portion may be a minority portion (e.g., less than 50% and any whole number integer between 1% and 49%) of the total number of particles in a batch, a majority portion (e.g., 50% or greater and any whole number integer between 50% and 99%) of the total number of particles of the batch, or even essentially all of the particles of a batch (e.g., between 99% and 100%). The provision of one or more features of any shaped abrasive particle of a batch may facilitate alternative or improved deployment of the particles in an abrasive article and may further facilitate improved performance or use of the abrasive article. A batch of particulate material can include a first portion including a first type of shaped abrasive particle and a second portion including a second type of shaped abrasive particle. The content of the first portion and second portion within the batch may be controlled at least in part based upon certain processing parameters. Provision of a batch having a first portion and a second portion may facilitate alternative or improved deployment of the particles in an abrasive article and may further facilitate improved performance or use of the abrasive article. The first portion may include a plurality of shaped abrasive particles, wherein each of the particles of the first portion can have substantially the same features, including for example, but not limited to, the same two-dimensional shape of a major surface. The batch may include various contents of the first portion. For example, the first portion may be present in a minority amount or majority amount. In particular instances, the first portion may be present in an amount of at least about 1%, such as at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or even at least about 70% for the total content of portions within the batch. Still, in another embodiment, the batch may include not greater than about 99%, such as not greater than about 90%, not greater than about 80%, not greater than about 70%, not greater than about 60%, not greater than about 50%, not greater than about 40%, not greater than about 30%, not greater than about 20%, not greater than about 10%, not greater than about 8%, not greater than about 6%, or even not greater than about 4% of the total portions within the batch. The batch can include a content of the first portion within a range between any of the minimum and maximum percentages noted above. The second portion of the batch can include a plurality of shaped abrasive particles, wherein each of the shaped abrasive particles of the second portion can have substantially the same feature, including for example, but not limited to, the same two-dimensional shape of a major surface. The second portion can have one or more features of the embodiments herein, which can be distinct compared to the plurality of shaped abrasive particles of the first portion. In certain instances, the batch may include a lesser content of the second portion relative to the first portion, and more particularly, may include a minority content of the second portion relative to the total content of particles in the batch. For example, the batch may contain a particular content of the second portion, including for example, not greater than about 40%, such as not greater than about 30%, not greater than about 20%, not greater than about 10%, not greater than about 8%, not greater than about 6%, or even not greater than about 4%. Still, in at least on non-limiting embodiment, the batch may contain at least about 0.5%, such as at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 10%, at least about 15%, or even at least about 20% of the second portion for the total content of portions within the batch. It will be appreciated that the batch can contain a content of the second portion within a range between any of the minimum and maximum percentages noted above. Still, in an alternative embodiment, the batch may include a greater content of the second portion relative to the first portion, and more particularly, can include a majority content of the second portion for the total content of particles in the batch. For example, in at least one embodiment, the batch may contain at least about 55%, such as at least about 60% of the second portion for the total portions of the batch. It will be appreciated that the batch can include other portions, including for example a third portion, comprising a plurality of shaped abrasive particles having a third feature that can be distinct from the features of the particles of the first and second portions. The batch may include various contents of the third portion relative to the second portion and first portion. The third portion may be present in a minority amount or majority amount. In particular instances, the third portion may be present in an amount of not greater than about 40%, such as not greater than about 30%, not greater than about 20%, not greater than about 10%, not greater than about 8%, not greater than about 6%, or even not greater than about 4% of the total portions within the batch. Still, in other embodiments the batch may include a minimum content of the third portion, such as at least about 1%, such as at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or even at least about 50%. The batch can include a content of the third portion within a range between any of the minimum and maximum percentages noted above. Moreover, the batch may include a content of diluent, randomly shaped abrasive particles, which may be present in an amount the same as any of the portions of the embodiments herein. EXAMPLES Example 1 A mixture in the form of a gel is obtained having approximately 42% solids loading of boehmite commercially available as Catapal B from Sasol Corp. combined with 58 wt % water containing a minority content of nitric acid and organic additives. The gel has a viscosity of approximately 3×103to 4×104Pa. and a storage modulus of 3×104to 2×105Pa. The gel is extruded from a die using a pressure of up to 80 psi (552 kPa) onto a mold blank of polycarbonate and into a plurality of openings, wherein each of the openings are in the shape of a three-pointed star. The surfaces of the openings within the mold blank have been coated with canola oil. The openings define three-pointed star two-dimensional shapes having a length of approximately 5-7 mm, a width of 3-5 mm, and a depth of approximately 0.8 mm. The openings have tip angles of approximately 35 degrees, and an interior angle between the three arms of approximately 225 degrees. The gel is extruded into the openings and the gel is then dried for approximately 24-48 hours in air under atmospheric conditions and within the mold to form precursor-shaped abrasive particles. The precursor-shaped abrasive particles were calcined in a box furnace at approximately 600° C. for 1 hour and then, the precursors shaped abrasive particles were sintered in a tube furnace up to 1320° C. for 3 to 20 minutes.FIG.65Ais an image of a representative particle formed Example 1. The body has a curling factor of less than 5. Example 2 The process of Example 1 was used with the exception that the mold blank utilized openings defining a four-point star-shaped two-dimensional shape having a length of approximately 7-9 mm, a width of 7-9 mm, and a depth of approximately 0.8 mm. The openings have tip angles of approximately 25 degrees, and an interior angle between the three arms of approximately 250 degrees.FIG.66Ais an image of a representative particle formed from Example 2. The body has a curling factor of less than 5. Example 3 The process of Example 1 was used with the exception that the mold blank utilized openings defining a cross-shaped two-dimensional shape having a length of approximately 5-6 mm, a width of 5-6 mm, and a depth of approximately 0.8 mm. The arms have a width of approximately 2 mm and a length of approximately 1 mm.FIG.67is an image of a representative particle formed from Example 3. The body has a curling factor of less than 5. The present application represents a departure from the state of the art. While the industry has recognized that shaped abrasive particles may be formed through processes such as molding and screen printing, the processes of the embodiments herein are distinct from such processes. Moreover, the resulting shaped abrasive particles have one or a combination of distinct features from particles formed according to conventional approaches. The shaped abrasive particles of the embodiments herein can have a particular combination of features distinct from other conventional particles including, but not limited to, aspect ratio, composition, additives, two-dimensional shape, three-dimensional shape, stepped configuration, curling factor, tip angles, interior angles, and the like. Notably, the embodiments herein include a combination of features facilitating the formation of batches of shaped abrasive particle having particular features. And in fact, one or more such features facilitate alternative deployment of the particles in abrasive articles, and further, may facilitate improved performance in the context of fixed abrasives, such as bonded abrasives or coated abrasives. The above-disclosed subject matter is to be considered illustrative, and not restrictive, and the appended claims are intended to cover all such modifications, enhancements, and other embodiments, which fall within the true scope of the present invention. Thus, to the maximum extent allowed by law, the scope of the present invention is to be determined by the broadest permissible interpretation of the following claims and their equivalents, and shall not be restricted or limited by the foregoing detailed description. The Abstract of the Disclosure is provided to comply with Patent Law and is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, various features may be grouped together or described in a single embodiment for the purpose of streamlining the disclosure. This disclosure is not to be interpreted as reflecting an intention that the claimed embodiments require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter may be directed to less than all features of any of the disclosed embodiments. Thus, the following claims are incorporated into the Detailed Description, with each claim standing on its own as defining separately claimed subject matter. | 148,759 |
11859121 | DETAILED DESCRIPTION OF THE INVENTION The present disclosure relates to anti-icing surfaces, and particularly to anti-icing surfaces that have nano-viscoelastic characteristics and are highly flexible, durable, and universal in application. The present icephobic material shows extremely low ice adhesion while having long-term mechanical, chemical and environmental durability. The icephobic material, stress-localized viscoelastic material, utilizes elastic energy localization at the ice-material interface to shear the interface. With minimal applied force, cracks are formed at the interface generating local stress fields. This shear stress advances cracks at the interface to detach ice form the material. This icephobic material is a smooth coating and would not affect the aerodynamic properties of a surface such as airfoil. Once ice forms on a surface, the interaction between ice and the substrate is governed by van der Waal's force, electrostatic forces or hydrogen-bonding forces. A wide range of surfaces has been studied to reduce ice adhesion strength. Among those, elastomers have shown minimum ice adhesion and have the potential to achieve exceptional icephobic properties. Consider a rigid ice phase attached to an elastomer as shown inFIG.1A. If a shear force is applied in the ice-elastomer plane, the ice would only slide with no detachment from the surface. However, if the force is applied at a plane higher than the interface, the ice would detach at a critical stress. It has been shown that the elastic instability at the interface of a rigid body and an elastomer is responsible for fracture. The fingers developed at the contact line by elastic instability elongate and break down in the form of bubbles that help in propagation of crack at the interface. The threshold for bubble formation depends on the shear modulus of the elastomer. For a uniform elastomer with isotropic properties, one finds that the adhesion stress at the interface (as) is written as: σS≅(al)WaGh where a and l are the geometrical parameters as shown inFIG.1A, Wais the work of adhesion, G is the shear modulus, and h is the thickness of the elastomer. This formulation suggests that low ice adhesion can be achieved through low values of G and Wa. Note that the value of G can be tuned by several orders of magnitude, but the value of Wain the best case can be tuned by an order of magnitude (e.g. introduction of perfluorinated groups on a surface). By tuning the substrate from hard elastomers (G˜1 GPa) to gel (G˜1 Pa), low values of ice adhesion has been achieved. However, low values of G lead to low mechanical durability of the icephobic coatings, which results in poor long-term performance. The values of a, l and h are determined by dimensions of experimental instrument and icephobic material. Inconsistency in these dimensions in measuring of ice adhesion has resulted in scattered data of ice adhesion for the same substrate. For example, the reported values of ice adhesion for PDMS varies in the range of 100-800 kPa. A standard method to measure ice adhesion is described in Example 2 below. In the above formulation, an isotropic elastomer was considered, which resulted in a direct dependence of GS on G. However, once local phases with low shear modulus, such as those in the present icephobic surfaces, are introduced at the ice-material interface, as shown inFIG.1B, with minimal force, ice is detached from local phases and forms a local crack. This local crack induces an elastic stress field around the crack. This induced shear stress field opens the crack front and leads to propagation of crack at the interface. That is, the induced stress field by local phases leads to crack growth and failure. Through mathematical formulation of the discussed physics, the ice adhesion strength on these surfaces is written as σS~g(φI1)(al)Wa_Gmh where g(ϕII) denotes the stress-localization function, (p H is the volumetric fraction of phase II,Wais the work of adhesion of the material, and Gmis the shear modulus of the material. The values ofWaand Gmdepend on properties of individual phase I and II, their volumetric fraction and their geometry. The salient feature of this formulation is the stress-localization function, which plays a critical role in the adhesion of ice to the material and its impact is far more effective than other parameters studied before (i.e. work of adhesion and shear modulus). This localization function reduces the adhesion of a solid on an elastomer by an order of magnitude as demonstrated and discussed below. Based on the developed stress-localization concept, a new form of icephobic surface, stress-localized viscoelastic material was developed. The material includes a matrix as Phase I with high shear modulus and highly dispersed phase II with low shear modulus. An exemplary procedure for development of one embodiment of these materials is given in Example 2 below. Phase I is a silicon elastomer and Phase II is a silicon-based organogel. As the matrix of this material plays a major role in long-term mechanical durability, it is crucial to choose an elastomer with high shear modulus. The preferred silicone elastomers are room temperature vulcanizing (RTV) with certain mechanical properties. To form a homogenous material, compatibility of the matrix and the dispersed phase is critical. Thus, silicon-based organogel particles with dimension of 2-20 μm are preferred. Other combinations of elastomers and the dispersed phases may be used as long as they provide a homogenous material. Once the material is developed, its viscosity can be adjusted through a solvent. Here, hexamethyldisiloxane is used in preferred embodiments to reduce the viscosity of the material. In the dilute form, the material can be brushed or sprayed to form a uniform coating. Once applied, the material is completely cured after 24 hrs. The surface of these materials was examined through Scanning Probe Microscopy (SPM) (Bruker Multimedia 8 SPM) to determine distribution of Phase II on the surface.FIG.1Cshows modulus of elasticity of both phases. As shown Phase II has much smaller modulus than that of the matrix.FIG.1Dshows a representation of a formation of a crack at a coordinate of Phase II with minimal forces. FIG.2shows a schematic of a viscoelastic anti-icing surface prepared in accordance with preferred embodiments of this disclosure. The icephobic surface includes a phase of organogel particles (also referred to as Phase II) dispersed throughout an elastomer matrix (also referred to as Phase I). Preferred concentrations of organogel particles in the elastomer matrix are about 1% to about 99% based on volumetric ratio, and more preferred concentrations are about 5% to about 85%. The Phase II particles should be generally dispersed throughout the elastomer matrix to avoid accumulation of particles in isolated regions. The viscoelastic icephobic surfaces can utilize a variety of different elastomers that serve as a host or matrix. In certain embodiments the elastomer can be a room-temperature-vulcanizing (RTV) silicone rubber prepared using a suitable base and a curing agent. Additional preferred elastomers may include polyurethane, poly isoprene, fluoroelastomers, and the like. The selected elastomer should have a high shear modulus. Different types of gels can also be used as Phase II particle beads to be integrated within the elastomer matrix. The gel beads may be made of organogels (gels made of hydrocarbons), polyacrylamide, polydimethylsiloxane (PDMS), or other suitable materials. The gel beads may be mixed with a variety of different surfactants, including butyl butyrate, propylene glycol, and silicone (Si) oil, and crushed prior to incorporation into the rubber or polymer matrix. In preferred embodiments, the organogel particles include tuned liquid organic phases (non-crosslinked components in the gel matrices) entrapped within a solid phase (three-dimensionally crosslinked gel network). In certain preferred embodiments, the organogel particles are made up of combinations of siloxanes, silicas, and ethyl benzene. In additional preferred embodiments, the organogel particles are made up of a combination of dimethyl siloxane, dimethylivinyl terminated silica, dimethylvinylated silica, trimethylated silica, tetra (trimethoxysiloxy) silane, ethyl benzene), dimethyl, methylhydrogen siloxane, and tetramethyl tetravinyl cyclotetra siloxane. In additional preferred embodiments, the organogel particle beads are polydimethylsiloxane based. The gel beads incorporated into the elastomer matrix are preferably about 10 nm to about 100 microns in diameter, and more preferably about 2 to about 20 microns. Generating the nano-viscoelastic surfaces from different materials allows for alteration of the properties of the product, which also allows for adjusting the desired durability and ice adhesion properties based on the desired function for the surface. Table 1 below illustrates some types of materials that may be used to develop preferred embodiments of the nano-viscoelastic surfaces. TABLE 1Gel beadsOrganogelPDMSbeads + ButylPolyacrylamide +(SYLGARDMatrixbutyratepropylene glycol184) + Si oilPolyurethanePU1PU2PU3Poly isoprenePO1PO2PO3Silicone rubberSI1SI2SI3 The nano-viscoelastic anti-icing surfaces are physically and chemically stable while maintaining extremely low ice adhesion properties. In preferred embodiments, the nano-viscoelastic surfaces are applied to a surface in need of protection from icing by spraying the uncured material to the base surface and allowing the material to cure to form the anti-icing surface. An important factor for the long term durability of anti-icing surfaces is their ability to adhere to the surface and also their ability to withstand severe abrasion. These factors become increasingly relevant for outdoor operation. Most current anti-icing technologies cannot demonstrate this physical stability for prolonged durations. The current nano-viscoelastic surfaces have been tested and verified to have physical stability in these conditions. The present anti-icing surfaces are highly durable icephobic materials. These materials utilize stress-localization to initiate cracks at the ice-material interface and consequently minimize ice adhesion on the surface. Stress-localization leads to a shear force at the interface for detachment of ice from the material. The developed concept is implemented in elastomers and the superior icephobicity of these materials compared to state-of-the-art materials is demonstrated. These forms of icephobic materials demonstrate excellent mechanical, chemical and environmental durability with no change of characteristics under extreme air and water shear flows. Furthermore, these icephobic materials do not change the aerodynamic characteristics of airfoils thereby providing a promising solution for aerospace application. In contrast to surface modified coatings, the icephobicity of these materials is a volumetric property and no degradation in the performance occurs in long-term operation under mechanical loadings. The developed concept of stress-localization reduces adhesion of solids on a material by an order of magnitude with no compromise in mechanical properties. The developed icephobic materials could be used to minimize adhesion of any solid species (i.e. ice, gas hydrate, dust, and even bio-species) on a surface with omnipresent application in transportation systems (aviation, cars and vessels), energy systems, and bio-sciences. Example 1 To verify the properties of the nano-viscoelastic surfaces, testing was carried out on a preferred embodiment identified in Table 1 above as SI3. Sample SI3 was created by preparing polydimethylsiloxane (PDMS) beads (SYLGARD® 184, The Dow Chemical Company), then mixing and crushing the PDMS beads in a silicone (Si) oil surfactant until the beads are nano-micro sized, or about 10 nm to about 200 microns. A polymer base of silicone rubber was separately prepared and the crushed beads were added to the silicone rubber base. Prior to curing, a portion of the polymer bead mixture was applied to a surface made of glass at a thickness of about 400 microns and a width of about 25 mm and a length of about 70 mm, then allowed to cure for 30 minutes to prepare a SI3 sample surface. To test chemical stability, the SI3 sample surfaces were submerged in separate containers containing the solvents alcohol, acetone, or toluene at room temperature overnight.FIG.3shows the sample products before and after submersion overnight. No changes were observed on the surfaces after complete submersion in the chemicals overnight. Thus, sample SI3 was chemically inert to these materials, demonstrating long term durability. An abrasion test was carried out with 2 newton force directly applied onto the surface of the SI3 sample using linear TABER® Abraser equipment (Taber Industries, New York, USA) with CS-10 as the fine abrader and H-18 as the medium abrader. The as-prepared sample was clamped down and tested for 10,000 abrasion cycles.FIG.4shows the SI3 sample surface before and after running the abrasion test. Only 97 microns of the surface was removed, which is considerably less material loss compared to current state-of the art technologies, thereby proving physical stability and durability. To further evaluate the physical durability of these anti-icing surfaces, sample SI3 was tested for UV radiation effects. The sample was placed in a fluorescent chamber for 500 hours to be fully exposed to UV radiation.FIG.5shows the SI3 sample before and after spending 500 hours in a UV radiation chamber with a wavelength of 250-400 nm and a lamp power of 40 W. After removing the sample from the UV chamber, no cracks or material degradation were spotted. The sample was then re-examined using the abrasion test under 2 N force after UV radiation exposure. The amount of material removed from the product was 101 microns, again a very small amount of material. The anti-icing characteristics of the sample surfaces identified in Table 1 were also studied. Sample surfaces were generally prepared according to the details provided above for the preparation of the SI3 sample surface, using different gel beads, surfactants, and polymer, to provide similarly sized sample surfaces. The ice adhesion strength on the sample surfaces was measured through direct applied shear stress. In this approach, a rectangular cuvette was placed on the cold sample. The cuvette was filled with water for ice formation on the sample. The formed ice was left for 1 hour on the surface before the measurement. A shear force was applied tangentially to the ice cube and measured using a digital force gauge such as the IMADA DS2-110 (Imada, Inc., Northbrook, Illinois) to determine the detachment force required to remove the ice from the surface. The detachment force divided by the ice-sample surface area gave the ice adhesion strength.FIG.6shows the results of ice adhesion measurements taken at −15° C. for various sample surfaces shown in Table 1. Sample surface SI3 provided a consistent average ice adhesion of 4.5 kPa±2 at −15° C. which was independent of the number of icing/de-icing cycles. This also demonstrates the durability of these surfaces for anti-icing applications. The anti-icing properties of sample SI3 were also re-examined after the abrasion test and the average ice adhesion was still found to be 4.5 kPa±2 at −15° C. Multiple ice adhesion measurements were taken for sample SI3 to evaluate the performance of the sample at different temperatures using the process described above.FIG.7shows the average ice adhesion measurements for sample SI3 at different surface temperatures. Even at very low temperatures of −30° C., the ice adhesion strength on the sample surface was relatively low. Additional tests were also carried out on sample surfaces identified in Table 1. The mass change of the surfaces was studied several months after the surfaces were prepared, and no sign of any mass change was observed. The sample surfaces were also stored at 100° C. for more than 24 hours, which no change in mass or other characteristics observed following this heat treatment. The sample surfaces were also stored at −30° C. for more than 5 hours to measure shrinkage effects, but the results were found to be negligible. Low ice adhesion properties were demonstrated in sample SI3 by inducing a 17 m/s average air velocity across the surface having ice droplets.FIG.8shows a schematic of the sample surface having an ice droplet before applying the wind, then after applying the wind at 17 m/s. Example 2 Exemplary stress-localized icephobic materials were developed. Phase I, the elastomer, was a RTV-1 silicone rubber. The RTV-1 silicone rubber had the material properties of: Elongation at break—500%, Hardness Shore A—30, Tensile strength—8 N/mm2, Viscosity, dynamic at 20° C.—300000 mPa·s, Density at 23° C. in water—1.1 g/cm3, and tear-strength—13.5 N/mm. Phase II, organogel particles, consisted of tuned liquid organic phases (non-crosslinked components in the gel matrices) entrapped within a solid phase (three-dimensionally crosslinked gel network). The procedure for development of these organogels was: 10 mL of base (SYLGARD 184, Dow Corning—Dimethyl siloxane, dimethylivinyl terminated, Dimethylvinylated and trimethylated silica, Tetra (trimethoxysiloxy) silane, and Ethyl benzene) was mixed with 1 mL of curing agent (SYLGARD 184, Dow Corning—Dimethyl, methylhydrogen siloxane, Dimethyl siloxane, dimethylvinyl terminated, Dimethylvinylated and trimethylated silica, Tetramethyl tetravinyl cyclotetra siloxane, and Ethyl benzene). 100 mL of an organic liquid (i.e. Polydimethylsiloxane (PDMS), or silicone oil) was added to this mixture. The solution was then vigorously mixed to obtain a homogeneous solution. The precursor sample was heated at 100° C. for 4 hrs in a petri dish. The final product was a non-syneresis organogel. Non-syneresis property of organogel comes from miscibility of the components and silicone oil with PDMS before and after gelation. Generally, the organogel particles are made up of a cross-linked polydimethylsiloxane network with entrapped silicone oil. Once phase II was developed, it was crushed in the presence of silicone oil for ten minutes to avoid aggregation of gel particles. The solution was filtered to remove excess oil. The final product was a batch of gel particles with dimension in the range of 2-20 μm. The particles were mixed with the elastomer in a pre-defined concentration, preferably about 1 to 99% based on volumetric ratio. The solution was diluted with a solvent, hexamethyldisilaxane, to reduce viscosity for spraying on a surface. A standard procedure to examine ice adhesion on various materials was developed and utilized. Standard protocol was followed for all the measurements. The schematic of experiments is shown inFIG.9. The test chamber was cooled at a rate of −2° C./min to the target temperature. Temperature of the cooling plate was monitored using a thermocouple on top of the plate. Four exemplary types of icephobic materials were created through tuning the volumetric ratio of phase II in the material. AI-10, AI-11, AI-12, and AI-13 stand for 67%, 50%, 33%, 25% of phase II, respectively. The icephobic sample was placed on the cooling plate. A square acrylic cuvette with dimension of 15 mm by 15 mm was fabricated with laser cutter with an accuracy of 100 μm. The edges of cuvette were coated with Silane in order to achieve low surface energy and minimize adhesion of cuvette to the icephobic surface. This step minimizes the errors in ice adhesion measurements. The cuvette was filled with deionized water and was allowed to freeze for 1 hr. Ice column encased in acrylic columns was adhered to the test samples. The force required to detach each ice column was measured by propelling the 0.8 cm diameter probe of a force transducer (Imada, model DS2-110) to the side of the ice columns at a constant velocity of 0.1 mm/s. The probe velocity was controlled using a syringe pump. The center of probe was located at 1 mm above the material surface. The measured maximum force at break was converted into ice adhesion strength by dividing by the known cross-sectional area (2.25 cm2) of the ice-substrate interface. The entire experiment was conducted in a low-humidity nitrogen atmosphere to minimize frost formation on the samples and the test apparatus. The measured values of ice adhesion at temperature of −25° C. on all these samples are shown inFIG.10A. With the same experimental protocol, ice adhesion was measured on other state-of-the-art icephobic coatings and included inFIG.10A. The reported value of ice adhesion (σS) was the average of ten measurements. In the protocol of ice adhesion. All the samples had the same thickness of 300+20 CD. As shown, ice adhesion on AI-10 is an order of magnitude lower than other state-of-the-art surfaces. This low ice adhesion is believed to be achieved through stress-localization. Another important metric for assessment of ice adhesion on coatings of uniform thickness is ice adhesion reduction factor (ARF) which is defined as ARF=σS(Al)/σS(icephobic material). This criterion is a non-dimensional figure to determine ice adhesion, independent of geometry of measurement setup. The ARF values for various samples are included inFIG.10A, showing that AI-10 reduces ice adhesion by 800 times compared to Aluminum substrate. For some of the state-of-the-art materials, ice adhesion depends on the number of icing/deicing cycles as the properties of these materials (i.e. surface characteristics) changes. For example, for liquid-infused surfaces, the depletion of liquid on the surface adversely affects cyclic ice adhesion. For the developed stress-localized icephobic surfaces, ice adhesion up to 100 icing/deicing cycles was determined. For these experiments, once the ice column was detached from the substrate, a new cuvette was placed on the sample and the procedure for ice formation was repeated. After complete formation of ice column, standard procedure was followed to measure ice adhesion. For the same sample, these experiments were conducted up to 100 times during a week to demonstrate consistency of ice adhesion on these icephobic surfaces and no change was observed. These experiments were conducted for various grades of these materials.FIG.10Bshows results. To assess ice adhesion of these materials in harsh environments, the icephobic coating was exposed to high shear flow of water and air up to Reynolds number of 2×104and 3×104respectively for one month. For these experiments, icephobic material was coated on a glass substrate through spraying. The sample was left to cure for 24 hr. The ice adhesion on the sample was measured through the protocol described above. Next, the coated glass substrate was placed in a tube and initially was exposed to shear flow of water with Reynolds number of 20000. The sample was left under high shear flow for one month. After this time period, the ice adhesion on the sample was re-measured. The same sample was moved to another setup and was exposed to shear flow of air with Reynolds number of 30000 for one month. The ice adhesion on the samples was remeasured after this experiment. No change in the ice adhesion was observed.FIG.10Cshows results. To resemble samples exposed to various chemical environment, the icephobic samples were exposed to solutions with pH ranging 1-13 and re-examined using the standard ice adhesion protocol.FIG.10Dshows results. Furthermore, to demonstrate long-term ice adhesion of samples exposed to UV radiation in the environment, the samples were placed in a UV chamber and kept for 4 weeks. The ice adhesion before and after UV exposure remained unchanged, as shown inFIG.10D. Mechanical, chemical and environmental durability of the developed icephobic materials were also examined. The mechanical durability of the icephobic coatings was examined through Taber abrasion test (Taber Reciprocating Abraser, Model 5900) according to ASTM D4060. In these experiments, material removal for different samples as various loading conditions (i.e. 1, 5, and 10 N) was measured. Samples were placed firmly on a horizontal plate in the Taber instrument and 1000 abrasion cycles applied in each experiment. Superhydrophobic surfaces and SLIPS failed all the tests. AI-10 (67% phase II concentration) failed the 10 N abrasion test. However, other AI samples passed the tests in all loading conditions. The thickness removal in the abrasion tests are shown inFIG.11A. After abrasion test under 5 N loading for 1000 cycles, the icephobic performance of coatings exposed to mechanical loadings was re-examined. The ice adhesion for these samples along with state-of-the-art icephobic surfaces are shown inFIG.11B. As shown, no measurable change in ice adhesion was observed and the AI samples offered minimal ice adhesion. In contrast to surface-modified materials (i.e. superhydrophobic surfaces or hydrated-surfaces), the stress-localization property of these materials is volumetric and does not change as they abrade. This feature ensures low ice adhesion on these stress localized viscoelastic surfaces for long-term performance. As another metric for its mechanical durability, the icephobic coating was abraded through sand paper and iron file. No change in its properties was measured. The coating holds its low ice adhesion as the icephobic characteristics is a volumetric property and not a surface property. Depending on the application, the icephobic coatings may be exposed to various chemical environments. The chemical stability of the AI coatings was examined in a range of solutions with pH between 1-13. The acidic solutions were prepared through various HCl and water concentrations. The basic solutions were Tris 0.15 mM NaCl (pH=8) and Sodium hydroxide (pH=13) solutions. The samples were soaked in these solutions for 48 hrs. There was no change in the integrity of the coatings after being exposed to these chemical environments. No change in the ice adhesion on these coatings after chemical stability test was detected. To assess environmental durability of icephobic coatings, the samples were tested for UV radiation effects. The icephobic sample was placed in a chamber for 500 hours under UV radiation. No cracks or material degradation or changes to the material's durability were spotted. After UV exposure, the icephobic coating was re-examined under abrasive loading of 5 N. The amount of material removed from the coating remained the same as before UV radiation. That is, the integrity of the coating is not affected by UV radiation. Finally, to demonstrate on-field repairability of this coating, the coating was damaged with a sharp blade to remove a part of material. The coating was then repaired by spraying of a new coating. The newly sprayed icephobic material was integrated within the coating and no visible change in the coating was observed. The repaired surface kept its integrity and icephobic properties. In aerospace applications, icephobic coatings should have minimal effect on the aerodynamic characteristic of the airfoil (i.e. drag and lift). To examine these characteristics, a wing with a cross section close to NACA 6415 airfoil profile was chosen. The experimental setup included two wing sections, which were removed from a small, commercially available wind turbine (ALEKO Vertical Wind Power Generator) in which they were used as the turbine blades to generate torque for a small generator. Of the two wing sections, one was coated with an example of the icephobic coating and the other one was left uncoated. Before conducting any experiments, the lift and drag coefficients were estimated for different angles of attack using XFOIL, a program developed to analyze subsonic isolated airfoils. XFOIL analyzes the 2D airfoil profile of a NACA 6415 under viscous flow conditions with a Reynolds number of 90,000 and a Mach number of 0.09 to compute the lift and drag characteristics of the airfoil. The mounting system was designed using Autodesk Inventor and was tailored specifically for use with the NACA 6415 cross-section and the 6-Axis load cell. The mounting system consisted of an airfoil mount and two circular plates as part of the load cell mounts, one of which was fixed to the base of the wind tunnel and the other was fixed to the bottom of the load cell. The two load cell plates were designed in such a way that the top plate could rotate on top of the bottom plate, with increments of 1°, covering the complete 360° range. This design feature was used to change the angle of attack of the wing section attached to the load cell. The plates were also designed to have 360 holes so that the plates could be pinned to hold the testing system at a certain angle of attack. After the CAD drawing was made, the mounting system was 3D-printed using PLA (Polylactic Acid) filament with a 100 infill to provide structural rigidity. Each wing section was attached to a 6-Axis load cell in the wind tunnel, which in turn was attached to the base of the wind tunnel. The 6-Axis load cell measured the forces and torques acting on the surface of the load cell and had a left-handed coordinate system. The wings were placed in a recirculating wind tunnel with a rectangular test section with a cross-section measuring 1.05 m×1.65 m. The wings were tested at a constant wind speed of 17 m/s, so as to match the conditions used in XFOIL, which corresponds to a chord Reynolds number of approximately 50,000. The forces and torques acting on the wing were measured simultaneously by the load cell for a given angle of attack. The force and torque measurements were used to determine the 2D drag and lift curves for the airfoil, with and without the icephobic coating. The experimental setup is shown inFIG.12A. The lift and drag coefficients for the coated and uncoated wing sections were plotted against angle of attack inFIG.12BandFIG.12C. Furthermore, the ratio of lift/drag versus of angle of attack is plotted inFIG.12D. The experimental data sets for both coefficients of lift and drag are accompanied by error bars, which were calculated based off the resolution of the load cell. The XFOIL data sets do not have any corresponding error bars, since this was a computational value. The results indicate that lift and drag for the coated wing and the uncoated wing have a similar trend for different angle of attacks and the difference in magnitudes on both is small. The magnitude of lift and drag coefficients of the airfoils found experimentally differs from the XFOIL computational results because XFOIL is a 2D computational tool that does not account for three dimensional effects, such as the 3D characteristics of the finite wing. In a finite wing, the higher-pressure air from beneath the wing tries to move towards the lower pressure above the wing. Moreover, the new experimental data indicate that the coating does not affect the lift and drag characteristics of a wing, which is important in any passive alternative for deicing aerospace systems. To demonstrate the role of stress localization function on ice adhesion, an experimental procedure was designed to probe crack nucleation at the material-ice interface. A form of the icephobic material was developed and was applied to a glass substrate. The coating included PDMS matrix and black organogel particles to provide contrast for visualization of crack nucleation at the material-ice interface. Organogel particles were included at 5% concentration according to volume in the PDMS matrix. The dimension of the organogel particles was between about 100 nm and 20 microns. A silanized glass prism (15 mm×15 mm×25 mm) was placed on the icephobic material to resemble interaction of ice with the coating. The glass slide was placed on a moving stage, the movement of which is controlled by a motorized motion controller and computer. The motorized stage was a syringe pump with forward velocity variation of 0.5 μm/s to 5 mm/s. A firmly held beam load cell (Imada, model DS2-110) was used to measure the force. The force was applied at a distance of 1 mm above the interface. The interface of the icephobic material-prism was viewed as shown inFIG.13A. Through a coupled optical microscope and a high-speed camera system, the crack nucleation at the interface was probed.FIG.13Bshows micrograph of interfacial cracks observed during these experiments. As shown, all the interfacial cracks were formed at the coordinate of phase II particles having low shear modulus. That is, phase II particles were responsible for cavitation and crack initiation at the interface. The fringes observed at the crack coordinates indicated the ellipsoidal form of these cavities. The generated crack induces a local stress field and the stored elastic energy depends on shear modulus of phase I and the dimension of these cracks. This stored elastic energy leads to a shear force at the front of crack, propagation of crack, and detachment of ice from the material. To determine the value of the stress-localization function for examples of the stress-localized icephobic materials, the values ofWa, the work of adhesion of the material, and Gm, the shear modulus of the material, were determined. Work adhesion is determined as Wa=γw(1+cos θ), where ywdenotes surface tension of liquid (i.e. water) at −20° C. and θ is the contact angle of sessile droplet on the surface. The contact angle of water was determined for the various samples and the work of adhesion was consequently determined. The shear modulus of the example materials was also measured using a Dynamic Mechanical Analyzer (DMA). The measured values are shown in Table 2 below. TABLE 2PhasePhase IIIAI-10AI-11AI-12AI-13Wa487057535149(mN/m)Gm3.5 +/−NA0.6 +/−0.9 +/−1.4 +/−1.8 +/−(MPa)0.50.50.50.50.5 Using the figures in Table 2, the values of the stress localization function were determined and plotted as shown inFIG.13C. The stress localization function depends on the concentration of phase II in the material structure as predicted. This stress-localization function reduces ice adhesion on the icephobic material up to an order of magnitude. The role of the stress localization function on reduction of ice adhesion is several times higher than the role of shear modulus. For example, comparing the sample AI-10 and pure silicon elastomer, the difference of shear modulus is approximately six times which results in ˜2.5 times reduction in ice adhesion. However, for the same samples, stress localization reduces the ice adhesion by more than 12 times. The stress localization function depends on geometrical parameters (a and l) along with volumetric fraction of phase II. For high values of all the role of normal force is dominant in the fracture and the role of stress localization (i.e. shear force) is small. However, for low values of all, the fracture is governed by shear forces and the stress localization is the dominant factor. The developed physic of stress-localization is applicable in detachment of any solid material (ice, dust and even bio-species) from elastomers. | 35,315 |
11859122 | DETAILED DESCRIPTION It should be understood at the outset that although an illustrative implementation of one or more embodiments are provided below, the disclosed systems and/or methods can be implemented using any number of techniques, whether currently known or in existence. The disclosure should in no way be limited to the illustrative implementations, drawings, and techniques below, including the exemplary designs and implementations illustrated and described herein, but can be modified within the scope of the appended claims along with their full scope of equivalents. Disclosed herein are settable compositions (e.g., cement compositions) and methods for carbon sequestration utilizing such settable compositions. In embodiments, the settable compositions comprise cement compositions (also referred to herein as “cement slurry compositions”), resins, or other hardenable materials. Although intended to include cement compositions, resins, and other hardenable materials, for simplicity, such settable compositions will be referred to herein simply as “cement compositions” or “cement slurry compositions”. It is to be understood that, although described hereinbelow with reference to typical Portland style hydraulic cements, other settable materials, such as, without limitation, sorel cements, resins, etc., are intended to be within the scope of this disclosure. Description of a method of according to this disclosure will now be made with reference toFIG.1, which is a schematic flow diagram of a method I. As seen inFIG.1, in embodiments, a method I of this disclosure can comprise, as indicated at step105, entraining carbon dioxide (CO2) in a cement slurry composition and subjecting the cement slurry composition to conditions under which the CO2achieves and maintains a supercritical state; and, as indicated at step110, allowing the cement slurry composition to harden to form a hardened cement having CO2sequestered therein. Method I can further include separating CO2from a gas comprising CO2(e.g., an exhaust gas, produced gas, etc.), as indicated at step101, and/or compressing the CO2, as indicated at step102. As noted above, method I comprises, at step105, entraining carbon dioxide (CO2) in a cement slurry composition and subjecting the cement slurry composition to conditions under which the CO2achieves and maintains a supercritical state. The conditions under which the CO2achieves and maintains a supercritical state can generally comprise a temperature of greater than about 87.8° F. (31° C.) and/or a pressure of greater than or equal to about 1070 psi (7.3 MPa). A CO2phase diagram, such as depicted inFIG.6, can be utilized to determine conditions at which CO2is supercritical. The CO2becomes and remains supercritical when the conditions exceed and remain above the critical point (i.e., 31.1° C. and 73.8 bar) as illustrated by the phase diagram ofFIG.6. In embodiments the CO2can be combined with the other cement slurry components (e.g., injected into a cement slurry composition as described herein) as a liquid, and introduced downhole. As the cement slurry composition is positioned downhole it will get warmer and experience an increase in pressure, such that the CO2becomes supercritical. Alternatively, in embodiments, parameters can be adjusted to introduced the CO2into the cement slurry composition as gas or supercritical at the surface. As the conditions will not reduce below the critical point during placement of the cement slurry composition downhole, the CO2can remain supercritical. The supercritical CO2remains entrapped in the hardened cement after the cement slurry composition hardens. Without being limited by theory, chemical absorption can assist in this process by decreasing the permeability of the system. The increased pressure (i.e., having the CO2achieve and maintain the supercritical state) allows for a greatly increased mass of CO2per unit volume of hardened cement than possible via conventional techniques. By introducing the cement slurry composition comprising the entrained CO2downhole, a density change can be effected, as described further in the Example below. In embodiments, subjecting the cement slurry composition to conditions under which the CO2achieves and maintains a supercritical state at step105comprises positioning the cement slurry composition downhole at a wellsite. In embodiments, step105of entraining CO2in the cement slurry composition under conditions whereby the CO2reaches the supercritical state can comprise: combining liquid CO2with the cement slurry composition and compressing such that the CO2becomes supercritical CO2. Alternatively or additionally, entraining CO2in the cement slurry composition under conditions whereby the CO2reaches the supercritical state can comprise combining supercritical CO2with the cement slurry composition. Compressing such that the CO2becomes supercritical CO2can occur when the cement slurry composition and the liquid CO2are placed downhole, whereby downhole pressure and temperature cause the liquid CO2to become the supercritical CO2. Entraining the CO2in the cement slurry composition under conditions whereby the CO2achieves the supercritical state, in step105, can comprise combining a fluid stream comprising CO2with the cement slurry composition. The fluid stream can comprise liquid or gaseous CO2. The fluid stream can comprise CO2produced at a jobsite at which the method is performed and/or at another jobsite. For example, in embodiments, the fluid stream comprises all or a portion (e.g., one or more components) of an exhaust gas (also referred to as a “flue gas”), a produced gas, methane, or a combination thereof, optionally produced at the jobsite (e.g., a wellsite). The exhaust gas can be a product of combustion of a fuel, such as, natural gas, gasoline (petrol), diesel fuel, fuel oil, biodiesel, coal, or a combination thereof. In addition to CO2, the exhaust gas can comprise nitrogen (N2), carbon monoxide (CO), hydrogen sulfide (H2S), water vapor, hydrocarbons CxHy(or “HC”), nitrogen oxides (NOx), particulate matter (soot), or a combination thereof. Method I can further comprise separating CO2gas from gas comprising CO2(e.g., exhaust gas, produced gas, etc.), as indicated at step101. As depicted at step102, method I can further comprise compressing the (e.g., separated) CO2gas, e.g., to provide liquid or the supercritical CO2. The compressing at step102can be effected with hydrocarbons (e.g., wellhead gas) recovered or produced at the or another jobsite (e.g., wellsite). For example, and without limitation, in embodiments, rather than flaring wellhead gas, the wellhead gas is utilized to run a compressor for the compressing at step102. At step110, allowing the cement slurry composition to harden to form a hardened cement having CO2sequestered therein can comprise positioning or leaving the cement slurry composition downhole, for example as described hereinbelow with reference toFIG.3,FIG.4, andFIG.5. In embodiments, the cement slurry composition further comprises methane. The methane can be incorporated into the cement slurry composition as a component of another gas (e.g., exhaust gas, produced gas) or separately. For example, the method I can further comprise capturing methane in a wellhead gas and incorporating the methane gas into the cement slurry composition. The cement slurry composition, the hardened cement, or both can contain therein from about 5 to about 60, from about 40 to about 50, from about 45 to about 50, and/or greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 vol % CO2at the conditions under which the CO2achieves and/or maintains the supercritical state. In embodiments, the hardened cement comprises from about 1, 5, 10, 50, 100, 150, or 200 to about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 kg CO2per m3of hardened cement, and/or greater than or equal to about 1, 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more kg CO2per m3of hardened cement. In embodiments, the cement slurry composition is a foamed cement slurry comprising a base slurry, one or more foaming agents, one or more foam stabilizers, one or more additional cement components (e.g., cementitious materials or additives, as described further hereinbelow), or a combination thereof. As utilized herein, ‘foamed’ includes the supercritical fluid regime. In some such embodiments, a method of this disclosure, which will now be described with reference toFIG.2, which is a schematic flow diagram of a Method II of this disclosure, can comprise forming a foamed cement composition comprising carbon dioxide (CO2), wherein the CO2is in a supercritical state, as indicated at step205; and allowing the foamed cement composition to cure under conditions at which the CO2remains supercritical, to provide a hardened cement, at step210. Forming the foamed cement composition at step205can comprise adding to a base cement slurry: (i) non-supercritical CO2(e.g., gaseous CO2) and/or (ii) a non-CO2foaming agent (e.g., another gas). Forming the foamed cement composition at step205can comprise adding liquid CO2to the base cement slurry and reducing the pressure to obtain gaseous CO2to provide the foamed cement composition, and increasing the pressure (e.g., to a pressure at which the CO2becomes supercritical (e.g., a pressure of greater than or equal to 1070 psi) to convert remaining CO2in the foamed cement composition to supercritical. Increasing the pressure to the pressure at which the CO2becomes supercritical can comprise compressing with a compressor or introducing the foamed slurry composition downhole, whereby the foamed cement composition is subjected to downhole pressures (e.g., downhole pressures of greater than or equal to about 1070, 2000, or 3000 psi). Allowing the foamed cement composition to cure under the conditions at which the CO2remains supercritical at step210can comprise positioning or leaving the foamed cement composition downhole. The conditions at which the CO2becomes supercritical can comprise a temperature of greater than or equal to about 87.8° F. (31° C.) and/or a pressure of greater than or equal to about 1070 psi (7.39 MPa). As noted hereinabove, the foamed cement composition and/or the hardened cement can contain therein from about 5 to about 60, from about 40 to about 50, from about 45 to about 50, and/or greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 vol % CO2therein at the conditions under which the CO2becomes supercritical. The cement slurry composition (e.g., foamed cement composition) can further comprise water and one or more additional cement components. Exemplary such additional cement components and amounts of the water and one or more additional components will be described hereinbelow. Although a variety of cementitious materials and additives are described hereinbelow, cementitious materials and cement additives other than or in addition to those described herein can be utilized in the cement slurry composition according to this disclosure. A design consideration for a cement slurry composition may be slurry density. Providing a cement slurry composition with a density within a safe operational envelope may be important to ensure that the set cement provides effective zonal isolation. Most subterranean formations may have an upper density limit defined by the fracture gradient of the subterranean formation. If a cement has a high density whereby the pressure of the cement column on the subterranean formation exceeds the fracture gradient, the cement may cause the formation to fracture, leading to loss of cement and potential formation damage. Even if the cement does not fracture the formation, providing a cement with too high density may cause cement to leak off into the formation which may lead to formation damage and additional cost of cement to “make up” the cement lost. However, a lower density limit may be defined by the formation fluid pressure at the wellbore walls, for example. The cement slurry composition generally must have sufficient density to minimize or prevent formation fluids from entering the wellbore before the cement has set. Without sufficient density, the formation fluids may flow into the cement column which may weaken the cement. Slurry density may be controlled by adjusting the amount of water in the cement slurry composition. For example, a cement may be produced with relatively higher amounts of water if a lower density cement is desired or relatively lower amounts of water if a higher density cement is desired. The slurry may also include lightweight cement additives such as hollow beads or other relatively low-density additives that may aid in lowering density or heavy cement additives such as weighting agents or other relatively high-density additives which may increase density. However, adjusting cement density by changing water content or adding cement additives may affect other properties of the cement slurry composition such as compressive strength, thickening time, rheology, fluid loss, free fluid, and fluid stability, among others. Furthermore, some additives may be incompatible with each other or require excessive water to hydrate. A cement slurry composition generally should have a water content that does not result in undesirable free water or separation of water from the bulk cement slurry composition. Free water may be an aqueous phase that separates from a slurry or mixture of fluids. In cementing operations, free water is generally undesirable since channels can form through the set cement, providing potential gas migration paths. When processing reservoir fluids, the water that separates easily under gravity separation is known as free water. In some cases, additional water may be locked in an emulsion, contributing to the aqueous phase but not available as free water. API RP 10B-2, Recommended Practice for Testing Well Cements, First Edition, July 2005 provides methods to determine free water for a cement slurry composition. Free water may cause problems in wellbore cementing, especially in deviated wellbores such as horizontal wellbores. Water that separates from the bulk cement slurry composition may migrate to the top of a conduit to be cemented resulting in pockets where the cement slurry composition is not in contact with the subterranean formation. These pockets of free water may cause problems such as loss of zonal isolation, conduit corrosion, wellbore collapse, and other problems. It is therefore typically preferred that cement slurry compositions have little to no free water present in the set composition. A common and inexpensive method to decrease cement slurry composition density may be to add additional water during preparation of the cement slurry composition as water is typically less dense than other components in the cement slurry composition. The additional water may allow the cement slurry composition to be prepared to lower densities but may also result in free water separating from the cement slurry composition as the cement slurry composition is introduced into a wellbore and allowed to set. The water in the cement slurry composition can be from any source provided that it does not contain an excess of compounds that may undesirably affect other components in the cement slurry composition. For example, a cement slurry composition may include fresh water, salt water such as brine (e.g., saturated saltwater produced from subterranean formations) or seawater, or any combination thereof. Salt water generally may include one or more dissolved salts therein and may be saturated or unsaturated as desired for a particular application. Seawater or brines may be suitable for use in some examples of the cement slurry composition. Further, the water may be present in an amount sufficient to form a pumpable slurry. Generally, the water may be added to the cement slurry composition in any desired concentration, including at a point in a range of from about 30 10% to about 80% by weight of the cement slurry composition. Alternatively, the water may be present in the cement slurry composition at a point in a range of from an amount of about 10% to about 30% by weight of the cement slurry composition, at a point in a range of from about 30% to about 50% by weight of the cement slurry composition, at a point in a range of from about 50% to about 60% by weight of the cement slurry composition, at a point in a range of from about 60% to about 70% by weight of the cement slurry composition, at a point in a range of from about 70% to about 80% by weight of the cement slurry composition or any points therebetween. The cement slurry composition can have a density suitable for a particular application. By way of example, the cement slurry composition may have a density at a point in a range of from about 4 pounds per gallon (“lb/gal”) (479 kg/m3) to about 20 lb/gal (2396 kg/m3). Alternatively, the cement slurry composition may have a density at a point in a range of from about 4 lb/gal (479 kg/m3) to about 7 lb/gal (839 kg/m3), at a point in a range of from about 7 lb/gal (839 kg/m3) to about 10 (1198 kg/m3), at a point in a range of from about 10 lb/gal (1198 kg/m3) to about 13 lb/gal (1558 kg/m3), at a point in a range of from about 13 lb/gal (1558 kg/m3), to about 16 lb/gal (1917 kg/m3), at a point in a range of from about 16 lb/gal (1917 kg/m3) to about 20 lb/gal (2396 kg/m3), or any points therebetween. As discussed above, the density of cement may be an important design factor as the density range of cement may be limited by the formation properties. As mentioned hereinabove, one method to control density may be to increase the fraction of water included in the cement slurry composition. However, increasing water fraction generally leads to a cement with a lower compressive strength and increased free water which may be unsuitable for some applications. The cement slurry composition may include hydraulic cement. In some instances, the hydraulic cement may be included in the cement slurry composition as a source of hydroxide ions. Any of a variety of hydraulic cements may be suitable including those including calcium, aluminum, silicon, oxygen, iron, and/or sulfur, which set and harden by reaction with water. Specific examples of hydraulic cements that may be suitable include, but are not limited to, Portland cements, pozzolana cements, gypsum cements, alumina based cements, silica cements, and any combination thereof. Examples of suitable Portland cements may include those classified as Classes A, B, C, G, or H cements according to American Petroleum Institute, API Specification for Materials and Testing for Well Cements, API Specification 10, Fifth Ed., Jul. 1, 1990. Additional examples of suitable Portland cements may include those classified as ASTM Type I, II, III, IV, or V. The hydraulic cement may be included in the cement slurry composition in an amount (or concentration). The amount of the hydraulic cement may be selected, for example, to provide a particular compressive strength for the cement slurry composition after setting (i.e., the hardened cement). Where used, the hydraulic cement may be included in an amount in a range of from about 1% to about 80% by weight of the cement slurry composition. By way of example, the hydraulic cement may be present in an amount ranging between any of and/or including any of about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% by weight of the cement slurry composition. In embodiments, the hydraulic cement may be present in an amount in a range of from about 25% to about 75% by weight of the cement slurry composition and, alternatively, from about 40% to 60% by weight of the cement slurry composition. In some examples, the cement slurry composition may further include a lightweight additive. The lightweight additive may be included to reduce the density of examples of the cement slurry composition. For example, the lightweight additive may be used to form a lightweight cement slurry composition, for example, having a density of less than about 13 lb/gal (1558 kg/m3). The lightweight additive typically may have a specific gravity of less than about 2.0. Examples of suitable lightweight additives may include sodium silicate, hollow microspheres, gilsonite, perlite, and combinations thereof. Where used, the lightweight additive may be present in an amount in the range of from about 0.1% to about 20% by weight of the cement slurry composition, for example. In alternative examples, the lightweight additive may be present in an amount in the range of from about 1% to about 10% by weight of the cement slurry composition. In some examples, as noted above, the cement slurry composition is foamed. In such embodiments, the cement slurry composition can include a foaming agent, and a gas (e.g., CO2). Optionally, to provide a cement slurry composition with a lower density and more stable foam, the foamed cement composition may further comprise a lightweight additive, for example. With the lightweight additive, a base slurry may be prepared that may then be foamed to provide an even lower density. In some embodiments, the foamed cement composition may have a density in the range of from about 4 lb/gal (479 kg/m3) to about 13 lb/gal (1558 kg/m3) and, alternatively, about 7 lb/gal (839 kg/m3) to about 9 lb/gal (1078 kg/m3). In embodiments, a base slurry may be foamed from a density of in the range of from about 9 lb/gal (1078 kg/m3) to about 13 lb/gal (1558 kg/m3) to a lower density, for example, in a range of from about 7 lb/gal (839 kg/m3) to about 9 lb/gal (1078 kg/m3). The gas used in embodiments of the foamed cement composition may be any suitable gas for foaming the cement composition, including, but not limited to CO2, air, nitrogen, and combinations thereof. Generally, the gas should be present in examples of the foamed cement composition in an amount sufficient to form the desired foam. In certain embodiments, the gas may be present in an amount in the range of from about 5% to about 80% by volume of the cement slurry composition at atmospheric pressure, alternatively, about 5% to about 55% by volume, and, alternatively, about 15% to about 30% by volume. Where foamed, examples of the cement slurry composition may include a foaming agent for providing a suitable foam. As used herein, the term “foaming agent” refers to a material or combination of materials that facilitate the formation of a foam in a liquid. Any suitable foaming agent for forming a foam in an aqueous liquid may be used in embodiments of the cement slurry composition. Examples of suitable foaming agents may include, but are not limited to: anionic, nonionic, amphoteric (including zwitterionic surfactants), cationic surfactant, or mixtures thereof, betaines; anionic surfactants such as hydrolyzed keratin; amine oxides such as alkyl or alkene dimethyl amine oxides; cocoamidopropyl dimethylamine oxide; methyl ester sulfonates; alkyl or alkene amidobetaines such as cocoamidopropyl betaine; alpha-olefin sulfonates; quaternary surfactants such as trimethyltallowammonium chloride and trimethylcocoammonium chloride; C8 to C22 alkylethoxylate sulfates; and combinations thereof. Specific examples of suitable foaming additives include, but are not limited to: mixtures of an ammonium salt of an alkyl ether sulfate, a cocoamidopropyl betaine surfactant, a cocoamidopropyl dimethylamine oxide surfactant, sodium chloride, and water; mixtures of an ammonium salt of an alkyl ether sulfate surfactant, a cocoamidopropyl hydroxysultaine surfactant, a cocoamidopropyl dimethylamine oxide surfactant, sodium chloride, and water; hydrolyzed keratin; mixtures of an ethoxylated alcohol ether sulfate surfactant, an alkyl or alkene amidopropyl betaine surfactant, and an alkyl or alkene dimethylamine oxide surfactant; aqueous solutions of an alpha-olefinic sulfonate surfactant and a betaine surfactant, mixtures of an ammonium salt of an alkyl ether sulfate, and combinations thereof. Generally, the foaming agent may be present in embodiments of the cement slurry composition fluids in an amount sufficient to provide a suitable foam. In some embodiments, the foaming agent may be present in an amount in the range of from about 0.8% to about 5% by volume of the water (“bvow”). The cement slurry compositions may further include a pozzolan composition, such as fly ash, silica fume, metakaolin, volcanic glasses, other natural glasses or combinations thereof, for example, to prevent cement compressive. An example of a suitable pozzolan may include fly ash. An additional example of a suitable pozzolan may include a natural pozzolan. Natural pozzolans are generally present on the Earth's surface and set and harden in the presence of hydrated lime and water. Examples including of natural pozzolans may include natural glasses, diatomaceous earth, volcanic ash, opaline shale, tuff, and combinations thereof. The cement slurry composition can include fly ash. A variety of fly ashes may be suitable, including fly ash classified as Class C or Class F fly ash according to American Petroleum Institute, ASTM C618-15, “Standard Specification for Coal Fly Ash and Raw or Calcined Natural Pozzolan for Use in Concrete” published 2015. Where used, the fly ash generally may be included in the cement slurry composition in an amount desired for a particular application. In some examples, the fly ash may be present in the cement slurry composition in an amount in the range of from about 1% to about 60% by weight of the cement slurry composition (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, etc.). In some examples, the fly ash may be present in the cement slurry composition in an amount in the range of from about 1% to about 35% by weight of the cement slurry composition. In some examples, the fly ash may be present in the cement slurry composition in an amount in the range of from about 1% to about 10% by weight of the cement slurry composition. Alternatively, the amount of fly ash may be expressed by weight of dry solids. For example, the fly ash may be present in an amount in a range of from about 1% to about 99% by weight of dry solids (e.g., about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, etc.). In some examples, the fly ash may be present in an amount in the range of from about 1% to about 20% and, alternatively, from about 1% to about 10% by weight of dry solids. The cement slurry composition may further include slag. Slag is generally a granulated, blast furnace by-product from the production of cast iron including the oxidized impurities found in iron ore. The slag may be included in examples of the cement slurry composition in an amount suitable for a particular application. Where used, the slag may be present in an amount in the range of from about 0.1% to about 40% by weight of the cement slurry composition. For example, the slag may be present in an amount ranging between any of and/or including any of about 0.1%, about 10%, about 20%, about 30%, or about 40% by weight of the cement slurry composition. The cement slurry composition may further include shale in an amount sufficient to provide the desired compressive strength, density, and/or cost. A variety of shales are suitable, including those including silicon, aluminum, calcium, and/or magnesium. Examples of suitable shales include vitrified shale and/or calcined shale. Where used, the shale may be present in an amount in the range of from about 0.1% to about 40% by weight of the cement slurry composition. For example, the shale may be present in an amount ranging between any of and/or including any of about 0.1%, about 10%, about 20%, about 30%, or about 40% by weight of the cement slurry composition. Some examples of the cement slurry compositions may include silica sources; for example, crystalline silica and/or amorphous silica. Crystalline silica is a powder that may be included in examples of the cement slurry composition, for example, to prevent cement compressive strength retrogression. Amorphous silica is a powder that may be included in examples of the cement slurry composition as a lightweight filler and/or to increase cement compressive strength. Amorphous silica is generally a byproduct of a ferrosilicon production process, wherein the amorphous silica may be formed by oxidation and condensation of gaseous silicon suboxide, SiO, which is formed as an intermediate during the process. Examples including a silica source may utilize the silica source as needed to enhance compressive strength or set times. The cement slurry composition may further include kiln dust. “Kiln dust,” as that term is used herein, refers to a solid material generated as a by-product of the heating of certain materials in kilns. The term “kiln dust” as used herein is intended to include kiln dust made as described herein and equivalent forms of kiln dust. In some instances, the kiln dust may be included in the cement slurry composition as a source of hydroxide ions. Depending on its source, kiln dust may exhibit cementitious properties in that it can set and harden in the presence of water. Examples of suitable kiln dusts include cement kiln dust, lime kiln dust, and combinations thereof. Cement kiln dust may be generated as a by-product of cement production that is removed from the gas stream and collected, for example, in a dust collector. Usually, large quantities of cement kiln dust are collected in the production of cement that are commonly disposed of as waste. The chemical analysis of the cement kiln dust from various cement manufactures varies depending on a number of factors, including the particular kiln feed, the efficiencies of the cement production operation, and the associated dust collection systems. Cement kiln dust generally may include a variety of oxides, such as SiO2, Al2O3, Fe2O3, CaO, MgO, SO3, Na2O, and K2O. Problems may also be associated with the disposal of lime kiln dust, which may be generated as a by-product of the calcination of lime. The chemical analysis of lime kiln dust from various lime manufacturers varies depending on several factors, including the particular limestone or dolomitic limestone feed, the type of kiln, the mode of operation of the kiln, the efficiencies of the lime production operation, and the associated dust collection systems. Lime kiln dust generally may include varying amounts of free lime and free magnesium, limestone, and/or dolomitic limestone and a variety of oxides, such as SiO2, Al2O3, Fe2O3, CaO, MgO, SO3, Na2O, and K2O, and other components, such as chlorides. In some examples, the kiln dust may be present in the cement slurry composition in an amount in the range of from about 1% to about 60% by weight of the cement slurry composition (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, etc.). In some examples, the fly ash may be present in the cement slurry composition in an amount in the range of from about 1% to about 35% by weight of the cement slurry composition. In some examples, the fly ash may be present in the cement slurry composition in an amount in the range of from about 1% to about 10% by weight of the cement slurry composition. Alternatively, the amount of fly ash may be expressed by weight of dry solids. For example, the fly ash may be present in an amount in a range of from about 1% to about 99% by weight of dry solids (e.g., about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, etc.). In some examples, the fly ash may be present in an amount in the range of from about 1% to about 20% and, alternatively, from about 1% to about 10% by weight of dry solids. The cement slurry composition may further include a set retarder. A broad variety of set retarders may be suitable for use in the cement slurry composition. For example, the set retarder may include phosphonic acids, such as ethylenediamine tetra(methylene phosphonic acid), diethylenetriamine Penta(methylene phosphonic acid), lignosulfonates, such as sodium lignosulfonate, calcium lignosulfonate, salts such as stannous sulfate, lead acetate, monobasic calcium phosphate, organic acids, such as citric acid, tartaric acid, cellulose derivatives such as hydroxyl ethyl cellulose (HEC) and carboxymethyl hydroxyethyl cellulose (CMHEC), synthetic co- or ter-polymers including sulfonate and carboxylic acid groups such as sulfonate-functionalized acrylamide-acrylic acid co-polymers; borate compounds such as alkali borates, sodium metaborate, sodium tetraborate, potassium pentaborate; derivatives thereof, or mixtures thereof. Examples of suitable set retarders include, among others, phosphonic acid derivatives. Generally, the set retarder may be present in the cement slurry composition in an amount sufficient to delay the setting for a desired time. In some examples, the set retarder may be present in the cement slurry composition in an amount in the range of from about 0.01% to about 10% by weight of the cement slurry composition. In specific examples, the set retarder may be present in an amount ranging between any of and/or including any of about 0.01%, about 0.1%, about 1%, about 2%, about 4%, about 6%, about 8%, or about 10% by weight of the cement slurry composition. The cement slurry composition may further include a set accelerator. Set accelerators may be included in the cement slurry compositions to, for example, increase the rate of setting reactions. Control of setting time may allow for the ability to adjust to wellbore conditions or customize set times for individual jobs. Examples of suitable set accelerators may include, but are not limited to, aluminum sulfate, alums, calcium chloride, calcium sulfate, gypsum-hemihydrate, sodium aluminate, sodium carbonate, sodium chloride, sodium silicate, sodium sulfate, ferric chloride, or a combination thereof. In some examples, the set accelerator may be present in the cement slurry composition in an amount in the range of from about 0.01% to about 10% by weight of the cement slurry composition. In specific examples, the set accelerator may be present in an amount ranging between any of and/or including any of about 0.01%, about 0.1%, about 1%, about 2%, about 4%, about 6%, about 8%, or about 10% by weight of the cement slurry composition. The cement slurry composition may further include a dispersant. Examples of suitable dispersants include, without limitation, sulfonated-formaldehyde-based dispersants (e.g., sulfonated acetone formaldehyde condensate) and polycarboxylated ether dispersants. In some examples, a dispersant may be included in the cement slurry compositions in an amount in the range of from about 0.01% to about 5% by weight of the cement slurry composition. In specific examples, the dispersant may be present in an amount ranging between any of and/or including any of about 0.01%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5% by weight of the cement slurry composition. The cement slurry composition may further include a free water control additive. As used herein, the term “free water control additive” refers to an additive included in a liquid for, among other things, reducing or preventing the presence of free water in the cement slurry composition. Free water control additive may also reduce or prevent the settling of solids. Examples of suitable free water control additives include, but are not limited to, bentonite, amorphous silica, hydroxyethyl cellulose, and combinations thereof. The free water control additive may be provided as a dry solid in some embodiments. Where used, the free water control additive may be present in an amount in the range of from about 0.1% to about 16% by weight of dry solids, for example. In alternative embodiments, the free water control additive may be present in an amount in the range of from about 0.1% to about 2% by weight of dry solids. The cement slurry composition may further include a fluid-loss-control additive. A fluid-loss-control additive may decrease the volume of fluid that is lost to the subterranean formation. Examples of suitable fluid-loss-control additives include, but not limited to, certain polymers, such as hydroxyethyl cellulose, carboxymethylhydroxyethyl cellulose, copolymers of 2-acrylamido-2-methylpropanesulfonic acid and acrylamide or N,N-dimethylacrylamide, and graft copolymers including a backbone of lignin or lignite and pendant groups including at least one member selected from the group consisting of 2-acrylamido-2-methylpropanesulfonic acid, acrylonitrile, and N,N-dimethylacrylamide, for example. The cement slurry composition can include a source of hydroxide ions. The source of hydroxide ions can be any source of hydroxide ions suitable for use in a cement slurry composition. Some examples of sources of hydroxide ions may be compounds which release hydroxide when mixed with water, such as calcium hydroxide, or compounds which react with water or other compounds and release hydroxide ions such as Portland cement. Other sources of hydroxide ions may include, but are not limited to, hydrated lime, cement kiln dust, and lime kiln dust, for example. The source of hydroxide ions may include hydrated lime. As used herein, the term “hydrated lime” will be understood to mean calcium hydroxide. In some examples, the hydrated lime may be provided as quicklime (calcium oxide) which hydrates when mixed with water to form the hydrated lime. The hydrated lime may be included in examples of the cement slurry compositions, for example, to form a hydraulic composition with a pozzolan or silica source. For example, the hydrated lime may be included in a pozzolan or silica source-to hydrated-lime weight ratio of about 10:1 to about 1:1 or a ratio of about 3:1 to about 5:1. Where present, the hydrated lime may be included in the cement slurry composition in an amount at a point in a range of from about 1% to about 40% by weight of the cement slurry composition, for example. In some examples, the hydrated lime may be present in an amount ranging between any of and/or including any of about 1%, about 10%, about 20%, about 30%, or about 40% by weight of the cement slurry composition. Other additives (e.g., suitable for use in subterranean cementing operations) may also be included in examples of the cement slurry composition. Examples of such additives include, but are not limited to weighting agents, gas-generating additives, mechanical-property-enhancing additives, lost-circulation materials, filtration-control additives, defoaming agents, thixotropic additives, and combinations thereof. In examples, one or more of these additives may be added to the cement sherry composition prior to the placement of the cement slurry composition (e.g., into a wellbore). In some examples, the cement slurry composition may set to have a desirable compressive strength. Compressive strength is generally the capacity of a material or structure to withstand axially directed pushing forces. The compressive strength may be measured at a specified time after the cement slurry composition has been maintained under specified temperature and pressure conditions. Compressive strength can be measured by either destructive or non-destructive methods. The destructive method physically tests the strength of treatment fluid samples at various points in time by crushing the samples in a compression-testing machine. The compressive strength can be calculated from the failure load divided by the cross-sectional area resisting the load and is reported in units of pound-force per square inch (psi). Non-destructive methods may employ a USA™ ultrasonic cement analyzer, available from Fann Instrument Company, Houston, Tex. Compressive strength values may be determined in accordance with API RP 10B-2, Recommended Practice for Testing Well Cements, First Edition, July 2005. By way of example, the cement slurry composition may develop a 24-hour compressive strength in the range of from about 50 psi (345 kPa) to about 5000 psi (34474 kPa), alternatively, from about 100 psi (689 kPa) to about 4500 psi (31026 kPa), or alternatively from about 500 psi (3447 kPa) to about 4000 psi (27578 kPa). In some examples, the cement slurry composition may develop a compressive strength in 24 hours of at least about 50 psi (345 kPa), at least about 100 psi (689 kPa), at least about 500 psi (3447 kPa), or more. In some examples, the compressive strength values may be determined using destructive or non-destructive methods at a temperature ranging from 100° F. (38° C.) to 200° F. (93° C.). The exemplary cement slurry compositions disclosed herein may be used in a variety of subterranean operations, including primary and remedial cementing. The cement slurry composition may be introduced into a wellbore and allowed to set therein, as described hereinbelow with reference toFIG.5, which is a schematic illustration of an exemplary placement of a cement slurry composition into a wellbore annulus, according to embodiments of this disclosure. As used herein, introducing the cement slurry composition into a subterranean formation includes introduction into any portion of the subterranean formation, including, without limitation, into a wellbore drilled into the subterranean formation, into a near wellbore region surrounding the wellbore, such as a subterranean formation, or into both. In primary cementing, the cement slurry composition may be introduced into an annular space between a conduit located in a wellbore and the walls of a wellbore (and/or a larger conduit in the wellbore), wherein the wellbore penetrates the subterranean formation. The cement slurry composition may be allowed to set in the annular space to form an annular sheath of hardened cement. The cement slurry composition may form a barrier that prevents the migration of fluids in the wellbore. The cement slurry composition may also, for example, support the conduit in the wellbore. In remedial cementing, a cement slurry composition may be used, for example, in squeeze-cementing operations or in the placement of cement plugs. By way of nonlimiting example, the cement slurry composition may be placed in a wellbore to plug an opening (e.g., a void or crack) in the formation, in a gravel pack, in the conduit, in the cement sheath, and/or between the cement sheath and the conduit (e.g., a microannulus). As described further hereinbelow with reference toFIG.3, the components of the cement slurry composition may be combined in any order desired to form a cement slurry composition that can be placed into a subterranean formation. In addition, the components of the cement slurry compositions may be combined using any mixing device compatible with the cement slurry composition, including a bulk mixer, for example. In some embodiments, a cement slurry composition may be prepared by dry blending the solid components of the cement slurry composition at a bulk plant, for example, and thereafter combining the dry blend with water when desired for use. For example, a dry blend may be prepared that includes the dry cement components. Liquid additives (if any) can be combined with the water before the water is combined with the dry components or added directly to a mixer tub. The method I/II can provide for an increased mass and/or volume capacity of CO2capture relative to a mass and/or volume capacity of CO2capture obtained via foam cementing employing a gaseous CO2at atmospheric pressure and/or chemical absorption methods of sequestering CO2. In embodiments, the method I/II can provide for an increased amount (e.g., mass and/or volume) of C1 (e.g., components having a single carbon atom, such as methane) for carbon capture and storage (CCS) relative to an amount (e.g., mass and/or volume, respectively) of C1 for CCS obtained via foam cementing employing a gaseous CO2at atmospheric pressure and/or chemical absorption methods of sequestering CO2. The mass and/or volume capacity of CO2capture relative to the volume capacity of CO2capture provided by the Method I can be from about 50 to about 1000, from about 100 to about 1000, from about 200 to about 500, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the mass and/or volume capacity of CO2capture, respectively, obtained via the foam cementing employing the gaseous CO2at atmospheric pressure and/or the chemical absorption methods of sequestering CO2. The amount (e.g., mass and/or volume) of C1 for CCS relative to the amount of C1 for CCS provided by the Method I can be from about 50 to about 1000, from about 100 to about 1000, from about 200 to about 500, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the amount (e.g., mass and/or volume, respectively) of C1 for CCS obtained via the foam cementing employing the gaseous CO2at atmospheric pressure and/or the chemical absorption methods of sequestering CO2. The Method I/II of this disclosure provides for simultaneous curing of the cement slurry composition (e.g., downhole) and bulk storage of the entrained CO2in the hardened cement. Also provided herein is a hardened foamed cement comprising: from about 5 to about 60, from about 40 to about 50, from about 45 to about 50, and/or greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 vol % CO2entrained per m3of the hardened foamed cement at conditions under which the CO2is supercritical. In embodiments, the hardened foamed cement is downhole. Referring now toFIG.3, the preparation of a cement slurry composition in accordance with example embodiments will now be described.FIG.3is a schematic illustration of a system III for the preparation of a cement slurry composition (e.g., a foamed cement composition) and subsequent delivery of the cement slurry composition to a final destination at which the cement slurry composition will be allowed to harden, for example, a wellbore. The cement slurry composition may be prepared according to any method disclosed herein such that the cement slurry composition remains in a pumpable fluid state for an extended period of time. As shown, the cement slurry composition may be mixed in mixing equipment305, such as, without limitation, a jet mixer, re-circulating mixer, or a batch mixer, for example, and then pumped via pumping equipment307to a final destination, for example, a wellbore. In some embodiments, the mixing equipment305and the pumping equipment307may be disposed on one or more cement trucks. One or more lines301/302can be utilized to introduce water, cementitious materials, and additives to mixer305. One or more lines303can be utilized to introduce the CO2into the cement slurry composition. In embodiments, a compressor304can be utilized to increase the pressure of the CO2in line303, e.g., to provide liquid or supercritical CO2. The CO2in CO2line303(optionally after compression in compressor304) can be introduced into System III at a variety of locations. For example, in embodiments, CO2, optionally after passage through compressor304, is combined, via line303A, with one or more components of the cement slurry composition in the one or more lines301/302prior to introduction into mixer305. Alternatively or additionally, CO2, optionally after passage through compressor304, is introduced separately, via line303B, into mixer305. Alternatively or additionally, CO2, optionally after passage through compressor304, is introduced, via line303C, into line306downstream of mixer305and upstream of pump307. Alternatively or additionally, CO2, optionally after passage through compressor304, is introduced, via line303D, into line308downstream of pump307. In embodiments, the CO2in line303,303A,303B,303C,303D,306, or308is gaseous, liquid, or supercritical phase. An example technique for placing the cement slurry composition into a subterranean formation will now be described with reference toFIGS.4and5.FIG.4is a schematic illustration of surface equipment400that may be used in placement of a cement slurry composition in accordance with certain embodiments. It should be noted that whileFIG.4generally depicts a land-based operation, those skilled in the art will readily recognize that the principles described herein are equally applicable to subsea operations that employ floating or sea-based platforms and rigs, without departing from the scope of the disclosure. As illustrated byFIG.4, the surface equipment400may include a cementing unit412, which may include one or more cement trucks. The cementing unit412may include mixing equipment305and pumping equipment307(e.g., as depicted inFIG.3). The cementing unit412may pump a cement slurry composition414through a feed pipe416and to a cementing head418which conveys the cement slurry composition414downhole. Turning now toFIG.5, the cement slurry composition414may be placed into a subterranean formation420in accordance with example embodiments. As illustrated, a wellbore422may be drilled into the subterranean formation420. While wellbore422is shown extending generally vertically into the subterranean formation420, the principles described herein are also applicable to well bores that extend at an angle through the subterranean formation420, such as horizontal and slanted wellbores. As illustrated, the wellbore422comprises walls424. In the illustrated embodiment, a surface casing426has been inserted into the wellbore422. The surface casing426may be cemented to the walls424of the wellbore422by cement sheath428. In the illustrated embodiment, one or more additional conduits (e.g., intermediate casing, production casing, liners, etc.), shown here as casing430may also be disposed in the wellbore422. As illustrated, there is a wellbore annulus432formed between the casing430and the walls424of the wellbore422and/or the surface casing426. One or more centralizers434may be attached to the casing430, for example, to centralize the casing430in the wellbore422prior to and during the cementing operation. With continued reference toFIG.5, the cement slurry composition414may be pumped down the interior of the casing430. The cement slurry composition414may be allowed to flow down the interior of the casing430through a casing shoe442at the bottom of the casing430and up around the casing430into the wellbore annulus432. The cement slurry composition414may be allowed to set in the wellbore annulus432, for example, to form a cement sheath that supports and positions the casing430in the well bore422. While not illustrated, other techniques may also be utilized for introduction of the cement slurry composition414. By way of example, reverse circulation techniques may be used that include introducing the cement slurry composition414into the subterranean formation420by way of the wellbore annulus432instead of through the casing430. As it is introduced, the cement slurry composition414may displace other fluids436, such as drilling fluids and/or spacer fluids that may be present in the interior of the casing430and/or the wellbore annulus432. At least a portion of the displaced fluids436may exit the wellbore annulus432via a flow line438and be deposited, for example, in one or more retention pits440(e.g., a mud pit), as shown onFIG.4. Referring again toFIG.5, a bottom plug444may be introduced into the wellbore422ahead of the cement slurry composition414, for example, to separate the cement slurry composition414from the fluids436that may be inside the casing430prior to cementing. After the bottom plug444reaches the landing collar446, a diaphragm or other suitable device should rupture to allow the cement slurry composition414through the bottom plug444. InFIG.5, the bottom plug444is shown on the landing collar446. In the illustrated embodiment, a top plug448may be introduced into the wellbore422behind the cement slurry composition414. The top plug448may separate the cement slurry composition414from a displacement fluid450and also push the cement slurry composition414through the bottom plug444. This method described herein provides a novel approach to carbon capture by intentionally foaming cement with CO2and allowing the CO2to be trapped in the cement matrix while potentially also providing strengthening, energized fluid properties, and density reduction. Via embodiments of this disclosure, CO2can be utilized rather than N2for foam cementing (e.g., as foaming gas). Rather than simply treating cement/concrete with CO2to promote hardening, sequestration, etc., the herein disclosed methods can utilize the high pressure found in a wellbore to compress CO2whereby substantially more CO2(and/or C1 for CCS, for example, when the cement slurry composition includes methane) can be captured than can be done at atmospheric pressure or via conventional chemical absorption methods Under wellbore conditions, CO2will be supercritical (except possibly for at the surface or during transport downhole) and thus the capacity (e.g., mass and/or volume) of CO2capture can be increased by about 100 to 1000 times via embodiments of this disclosure relative to atmospheric treatment. Above about 88° F. (31° C.), CO2will be gas or supercritical but can be injected/entrained into the cement slurry composition, in embodiments, as a liquid to enhance mixing efficiency below 1071 psi. Previous research indicates that pure CO2has limited effect on the bulk cement, and calcite formation appears to be limited to the bubble structure likely due to the presence of water and the permeability reduction that occurs. Some prior art cementing methods sequester small amounts of CO2by reaction to carbonate. Such prior art methods involve CO2curing rather than entrainment, which entrainment, as described herein, accomplishes the curing and bulk storage simultaneously, and provides much greater CO2sequestration. Other advantages will be apparent to those of skill in the art and with the help of this disclosure. EXAMPLES The embodiments having been generally described, the following examples are given as particular embodiments of the disclosure and to demonstrate the practice and advantages thereof. It is understood that the examples are given by way of illustration and not intended to limit the specification or the claims in any manner. Example 1 In this Example 1, a study was performed of the density of carbon dioxide under varying conditions. Table 1 provides density values for carbon dioxide under various conditions of temperature and pressure. In Table 1, “change from atmospheric” indicates the density change from normal conditions of normal temperature and pressure (i.e., a temperature of 20° C. and atmospheric pressure (i.e., 101.3 kPa)). As can be seen in Table 1, increasing the pressure substantially increases the density relative to the density at normal temperature and pressure (as seen in the “change from atmospheric”). TABLE 1Data from Example 1DensityDensityTemperaturePressure[g/L] =Change fromState[K][° C.][° F.][MPa][bara][psia][kg/m3]atmosphericGas30026.980.30.1114.51.77330026.980.311014518.5810.48Supercritical450177350101001450131.674.22phase6503777101010014508246.25850577107010100145061.3134.581050777143010100145049.3127.81Supercritical250−23.2−9.71001000145041236697.12phase450177350100100014504847477.72650377710100100014504601.9339.488505771070100100014504468.1264.0210507771430100100014504387218.27 The methods of this disclosure enable a greater mass percent of CO2in the hardened cement (e.g., kg CO2per m3of hardened cement) relative to conventional methods, due to the increased density of the CO2under supercritical conditions. Example 2 The herein described compositions and methods provide that the pressures (e.g., downhole) enable compression of CO2and sequestration of an increased mass of CO2per unit volume of composition. Compared to ambient conditions, albeit the volumes may not be much different, the mass is very different.FIG.7is a plot of density (kg/m3) of CO2as a function of pressure (psia) at a temperature of 180° F. (82° C.). For example, in a cement composition of this disclosure comprising 50% by volume CO2at 13,000 psi and 180° F., the CO2will have a specific density of about 1000 kg/m3and, if 50% of the volume is the CO2, then 500 kg of CO2are captured per m3of the cement. By comparison, for a same example at atmospheric (14.5 psi) pressure, the CO2will be in the vapor phase and have a density of just 1.5 kg/m3. Accordingly, the comparative cement could capture only 750 g of CO2per m3of the comparative cement. ADDITIONAL DISCLOSURE The following are non-limiting, specific embodiments in accordance with the present disclosure: In a first embodiment, a method comprises: entraining carbon dioxide (CO2) in a cement slurry composition and subjecting the cement slurry composition to conditions under which the CO2achieves and maintains a supercritical state; and allowing the cement slurry composition to harden to form a hardened cement having CO2sequestered therein. A second embodiment can include the method according to the first embodiment, wherein subjecting the cement slurry composition to conditions under which the CO2achieves and maintains a supercritical state comprises positioning the cement slurry composition downhole at a wellsite. A third embodiment can include the method according to any one of the first or second embodiments, wherein the cement slurry composition comprises from about 5 to 60, from about 40 to about 50, from about 45 to about 50, and/or greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 vol % CO2entrained therein at the conditions under which the CO2achieves and maintains the supercritical state. A fourth embodiment can include the method according to any one the first to third embodiments, wherein the hardened cement contains therein from about 5 to 60, from about 40 to about 50, from about 45 to about 50, and/or greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 vol % CO2per m3of the hardened cement. A fifth embodiment can include the method according to any one of the first to fourth embodiments, wherein the cement slurry composition further comprises water and one or more additional cement components. A sixth embodiment can include the method according to any one of the first to fifth embodiments, wherein entraining carbon dioxide (CO2) in the cement slurry composition under conditions whereby the CO2reaches the supercritical state comprises: combining liquid and/or gaseous CO2with the cement slurry composition and compressing such that the CO2becomes supercritical CO2; and/or combining supercritical CO2with the cement slurry composition. A seventh embodiment can include the method according to the sixth embodiment, wherein compressing such that the CO2becomes supercritical CO2occurs when the cement slurry composition and the liquid and/or gaseous CO2are placed downhole, whereby downhole pressure and temperature cause the liquid and/or gaseous CO2to become the supercritical CO2. An eighth embodiment can include the method according to any one of the first to seventh embodiments, wherein entraining the CO2in the cement slurry composition under conditions whereby the CO2achieves the supercritical state comprises combining a fluid stream comprising CO2with the cement slurry composition. A ninth embodiment can include the method according to the eighth embodiment, wherein the fluid stream comprises a liquid or gaseous CO2. A tenth embodiment can include the method according to the eighth or ninth embodiments, wherein the fluid stream comprises CO2produced at a jobsite at which the method is performed. An eleventh embodiment can include the method according to any one of the eighth to tenth embodiments, wherein the fluid stream comprises all or a portion of a gas comprising CO2(e.g., an exhaust gas, a produced gas, etc.). A twelfth embodiment can include the method according to the eleventh embodiment further comprising: separating CO2gas from a gas comprising CO2(e.g., an exhaust gas, produced gas, etc.) produced at a wellsite. A thirteenth embodiment can include the method according to the twelfth embodiment further comprising compressing the CO2gas to provide the liquid CO2. A fourteenth embodiment can include the method according to the thirteenth embodiment, wherein the compressing is effected with hydrocarbons (e.g., wellhead gas) recovered or produced at the wellsite. A fifteenth embodiment can include the method according to any one of the eleventh to fourteenth embodiments, wherein the gas comprising CO2(e.g., exhaust gas, produced gas, etc.) further comprises nitrogen (N2), carbon monoxide (CO), hydrogen sulfide (H2S), water vapor, hydrocarbons (CxHy), nitrogen oxides (NOx), particulate matter, or a combination thereof. A sixteenth embodiment can include the method according to any one of the first to fifteenth embodiments, wherein the cement slurry composition is a foamed cement slurry comprising a base slurry, one or more foaming agents, one or more foam stabilizers, accelerators, set retarders, fluid loss control agents, or a combination thereof. A seventeenth embodiment can include the method according to any one of the first to sixteenth embodiments, wherein the method provides for an increased mass and/or volume capacity of CO2capture (and/or an increased amount of C1 for carbon capture and storage (CCS)) relative to a mass and/or volume capacity of CO2capture (and/or an amount of C1 for carbon capture and storage (CCS)) obtained via foam cementing employing a gaseous CO2at atmospheric pressure and/or chemical absorption methods of sequestering CO2. An eighteenth embodiment can include the method according to any one of the first to seventeenth embodiments, wherein the cement slurry composition further comprises methane. A nineteenth embodiment can include the method according to the eighteenth embodiment further comprising capturing methane in a wellhead gas and incorporating the methane gas into the cement slurry composition. A twentieth embodiment can include the method according to any one of the seventeenth to nineteenth embodiments, wherein the mass and/or volume capacity of CO2capture (and/or the amount of C1 for carbon capture and storage (CCS)) relative to the mass and/or volume capacity of CO2capture (and/or the amount of C1 for carbon capture and storage (CCS)), respectively, provided by the method is from about 50 to about 1000, from about 100 to about 1000, from about 200 to about 500, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the mass and/or volume capacity of CO2capture (and/or the amount of C1 for carbon capture and storage (CCS)) obtained via the foam cementing employing the gaseous CO2at atmospheric pressure and/or the chemical absorption methods of sequestering CO2. A twenty first embodiment can include the method according to any one of the first to twentieth embodiments, wherein the method provides for simultaneous curing of the cement slurry composition downhole and bulk storage of the entrained CO2in the hardened cement. A twenty second embodiment can include the method according to any one of the first to twenty first embodiments, wherein the conditions comprise a temperature of greater than or equal to about 87.8° F. (31° C.) and a pressure of greater than or equal to about 1070 psi (7.3 MPa). A twenty third embodiment can include the method according to the twenty second embodiment, wherein the conditions comprise a temperature of greater than about 100° F. (37.7° C.) and a pressure of greater than or equal to about 1070 psi (7.3 MPa). In a twenty fourth embodiment, a method comprises: forming a foamed cement composition comprising carbon dioxide (CO2), wherein the CO2is in a supercritical state; and allowing the foamed cement composition to cure under conditions at which the CO2remains supercritical, to provide a hardened cement. A twenty fifth embodiment can include the method according to the twenty second embodiment, wherein forming the foamed cement composition comprises adding to a base cement slurry: (i) non-supercritical CO2(e.g., gaseous CO2) and/or (ii) a non-CO2foaming agent (e.g., another gas). A twenty sixth embodiment can include the method according to the twenty fourth embodiment, wherein forming the foamed cement composition comprises adding liquid CO2to the base cement slurry and reducing the pressure to obtain gaseous CO2to provide the foamed cement composition, and increasing the pressure to convert remaining CO2in the foamed cement composition to supercritical. A twenty seventh embodiment can include the method according to any one of the twenty fourth to twenty sixth embodiments, wherein allowing the foamed cement composition to cure under the conditions at which the CO2remains supercritical comprises positioning the foamed cement composition downhole. A twenty eighth embodiment can include the method according to any one of the twenty fourth to twenty seventh embodiments, wherein the conditions comprise a temperature of greater than or equal to about 87.8° F. (31° C.) and/or a pressure of greater than or equal to about 1070 psi (7.39 MPa). A twenty ninth embodiment can include the method according to any one of the twenty fourth to twenty eighth embodiments, wherein the hardened cement contains therein from about 5 to 60, from about 40 to about 50, from about 45 to about 50, and/or greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 vol % CO2per m3of the hardened cement at the conditions. A thirtieth embodiment can include the method according to any one of the twenty fourth to twenty ninth embodiments, wherein the foamed cement composition comprises from about 5 to 60, from about 40 to about 50, from about 45 to about 50, and/or greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 vol % CO2entrained therein at the conditions at which the CO2remains supercritical. In a thirty first embodiment, a hardened foamed cement comprises therein from about 5 to 60, from about 40 to about 50, from about 45 to about 50, and/or greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 vol % CO2per m3of the hardened foamed cement. A thirty second embodiment can include the hardened foamed cement of the thirty first embodiment, wherein the hardened foamed cement is downhole. While embodiments have been shown and described, modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of this disclosure. The embodiments described herein are exemplary only, and are not intended to be limiting. Many variations and modifications of the embodiments disclosed herein are possible and are within the scope of this disclosure. Where numerical ranges or limitations are expressly stated, such express ranges or limitations should be understood to include iterative ranges or limitations of like magnitude falling within the expressly stated ranges or limitations (e.g., from about 1 to about 10 includes, 2, 3, 4, etc.; greater than 0.10 includes 0.11, 0.12, 0.13, etc.). For example, whenever a numerical range with a lower limit, Rl, and an upper limit, Ru, is disclosed, any number falling within the range is specifically disclosed. In particular, the following numbers within the range are specifically disclosed: R=Rl+k*(Ru−Rl), wherein k is a variable ranging from 1 percent to 100 percent with a 1 percent increment, i.e., k is 1 percent, 2 percent, 3 percent, 4 percent, 5 percent, . . . 50 percent, 51 percent, 52 percent, . . . , 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or 100 percent. Moreover, any numerical range defined by two R numbers as defined in the above is also specifically disclosed. Use of broader terms such as comprises, includes, having, etc. should be understood to provide support for narrower terms such as consisting of, consisting essentially of, comprised substantially of, etc. When a feature is described as “optional,” both embodiments with this feature and embodiments without this feature are disclosed. Similarly, the present disclosure contemplates embodiments where this “optional” feature is required and embodiments where this feature is specifically excluded. Accordingly, the scope of protection is not limited by the description set out above but is only limited by the claims which follow, that scope including all equivalents of the subject matter of the claims. Each and every claim is incorporated into the specification as embodiments of the present disclosure. Thus, the claims are a further description and are an addition to the embodiments of the present disclosure. The discussion of a reference herein is not an admission that it is prior art, especially any reference that can have a publication date after the priority date of this application. The disclosures of all patents, and patent applications, cited herein are hereby incorporated by reference, to the extent that they provide exemplary, procedural, or other details supplementary to those set forth herein. | 69,792 |
11859123 | DETAILED DESCRIPTION It should be understood at the outset that although an illustrative implementation of one or more embodiments are provided below, the disclosed systems and/or methods may be implemented using any number of techniques, whether currently known or in existence. The disclosure should in no way be limited to the illustrative implementations, drawings, and techniques illustrated below, including the exemplary designs and implementations illustrated and described herein, but may be modified within the scope of the appended claims along with their full scope of equivalents. It is to be understood that “subterranean formation” encompasses both areas below exposed earth and areas below earth covered by water such as ocean or fresh water. Disclosed herein are methods and compositions for making and using a wellbore servicing fluid, more specifically, a wellbore servicing fluid comprising a suspension composition. In some embodiments, a wellbore servicing fluid of the type disclosed herein can be a cementitious fluid or cement slurry, and can be used for cementing a wellbore penetrating a subterranean formation. In other embodiments, a wellbore servicing fluid of the type disclosed herein can be a spacer fluid, and can be used for separating a drilling fluid from a cementitious fluid. Disclosed herein are methods of preparing a suspension composition, methods of preparing a wellbore servicing fluid comprising the suspension composition, and methods of servicing a wellbore (e.g., cementing; using a spacer fluid) by placing the wellbore servicing fluid comprising the suspension composition into the wellbore. In some embodiments, the wellbore servicing fluid can be a cementitious fluid, wherein the cementitious fluid comprises the suspension composition, water, and a cement blend. In other embodiments, the wellbore servicing fluid can be a spacer fluid, wherein the spacer fluid comprises the suspension composition and water. The spacer fluid may be used to separate the cementitious fluid from another fluid, such as a drilling fluid, as will be described on more detail later herein. In embodiments, the suspension composition can comprise a particulate material, an organic carrier fluid, and a suspension viscosifier; alternatively a particulate material, an organic carrier fluid, a suspension viscosifier and a biocide; or alternatively a particulate material, an organic carrier fluid, a suspension viscosifier, water and a biocide. In embodiments, the suspension composition is a substantially homogenous mixture (e.g., a suspension) in which the particulate material does not dissolve, but gets uniformly suspended throughout the bulk of an organic carrier fluid. For example, the particulate material is uniformly dispersed (e.g., floating around freely) in the suspension composition. A discontinuous internal phase (e.g., particulate material) of the suspension composition can be uniformly dispersed throughout a continuous external phase (e.g., organic carrier fluid) of the homogenous suspension composition through preparation (e.g., mixing or blending), with the use of a viscosifying suspending agent (e.g., the suspension viscosifier). The suspension composition may be prepared by mixing or blending the components of the suspension composition to form the homogenous suspension. The suspension composition can function as a suspending agent in the wellbore servicing fluid. Generally, a suspending agent is a substance that prevents particulates (e.g., a cementitious material, a weighting agent, etc.) from settling in the wellbore servicing fluid during storage and/or before reaching a downhole target (e.g., a portion of the wellbore and/or subterranean formation). The particulate material is substantially insoluble in the organic carrier fluid. In embodiments, the particulate material can be characterized by a solubility in the organic carrier fluid of less than about 100 mmol/L, alternatively less than about 50 mmol/L, alternatively less than about 25 mmol/L, alternatively less than about 10 mmol/L, alternatively less than about 1 mmol/L, alternatively less than about 0.1 mmol/L, alternatively less than about 0.01 mmol/L, or alternatively less than about 0.001 mmol/L. The particulate material may comprise a water-interactive material and/or a water-insoluble material. While the current disclosure is discussed in detail in the context of the suspension composition comprising a water-interactive material and/or a water-insoluble material, it should be understood that any material that is substantially insoluble in the organic carrier fluid may be used as the particulate material in the suspension composition. In embodiments, the particulate material may comprise a water-interactive material. While the water-interactive material is substantially insoluble in the organic carrier fluid, the water-interactive material may react with water and/or be water soluble. For example, the water-interactive material can be substantially insoluble in the organic carrier fluid (e.g., characterized by a solubility in the organic carrier fluid of less than about 10 mmol/L) and can be soluble in water (e.g., characterized by a solubility in water of equal to or greater than about 10 mmol/L, alternatively equal to or greater than about 100 mmol/L, or alternatively equal to or greater than about 1 mol/L). As another example, the water-interactive material can be substantially insoluble in the organic carrier fluid (e.g., characterized by a solubility in the organic carrier fluid of less than about 10 mmol/L) and can interact with water, whereby the water-interactive material is consumed once in contact with water (e.g., by dissolution in water; by reacting with water and/or aqueous solution components). Suspension compositions as disclosed herein comprising a particulate material comprising a water-interactive material enable suspending the water-interactive material in aqueous-compatible suspensions, when the formation of water suspensions is not feasible owing to the intrinsic material properties of the particulate material with respect to water. For example, attempting to form aqueous suspensions of water-interactive materials can significantly and undesirably increase the viscosity of the aqueous suspension, thereby undesirably limiting the aqueous suspensions to relatively low concentrations of water-interactive material. In embodiments, the water-interactive material may comprise an expansion agent, a viscosifying clay, a delayed viscosifier, a fluid loss agent, and the like, or combinations thereof. The expansion agents may comprise alkali metal oxides, alkaline earth metal oxides, metal powders, and the like, or combinations thereof. For example, the expansion agents may comprise magnesium oxide, lightly burned magnesium oxide, hard burned magnesium oxide, deadburned magnesium oxide, aluminum powder, a gypsum blend (e.g., a calcium aluminate/calcium sulfate blend), and the like, or combinations thereof. Expansion agents can provide for a bulk volumetric increase of a composition, for example a cementitious composition comprising an expanding agent may exhibit a bulk volumetric increase upon setting. For example, an expansion agent may be any material that enables a gas to become incorporated into the cement composition. The viscosifying clay and the delayed viscosifier are viscosifiers for aqueous solutions, but do not substantially increase the viscosity of the suspension composition. The suspension composition acts as a carrier for the particulate material comprising the viscosifying clay and/or the delayed viscosifier, such that the viscosifying clay and/or the delayed viscosifier may increase the viscosity of a wellbore servicing fluid, such as a cementitious fluid or a spacer fluid. The viscosifying clay may comprise bentonite, sepiolite, hectorite, and the like, or combinations thereof. The delayed viscosifier may comprise crosslinked materials, such as crosslinked guar, crosslinked vinyl alcohols, crosslinked acrylamide polymers, and the like, or combinations thereof. In embodiments, the particulate material comprises a crosslinked guar. A crosslinked guar, also referred to as crosslinked guar gum, can be formed by crosslinking guar gum molecules by a crosslinker. Guar gum (GG) is a galactomannan polysaccharide extracted from guar beans that has thickening and stabilizing properties. Guar gum can be prepared by mechanically and/or chemically treating guar beans to liberate the guar seed endosperm, or “guar splits.” from the guar beans. Guar splits primarily comprise a polymannose backbone with galactose side chains and mannose, and contain a fair concentration of contaminates, such as cellulose, protein, and glycolipids. The guar splits are generally treated under relatively high pressures and temperatures with chemicals, after which treated guar splits are subjected to multiple washings to remove impurities and salts (which are byproducts of some of the treatments) from the guar splits. The treated and washed guar splits are then ground and dried to yield guar gums. Guar gum molecules can be crosslinked by crosslinkers to form crosslinked guar gums. Nonlimiting examples of crosslinkers suitable for crosslinking guar gum include chromium, aluminum, antimony, zirconium, boron, and the like, or combinations thereof. For example, without being limited by theory, boron, in a form of B(OH)4, reacts with hydroxyl groups of guar gum molecules in a two-step process to link two guar molecule strands together. The fluid loss agent may comprise an acrylic-based polymer, a polyacrylate, an acrylamide-based polymer, a polyacrylamide, an acrylamide copolymer, an acrylic acid copolymer, a polymer of acrylamide-tertiary-butyl sulfonate (ATBS), an ATBS/acrylamide copolymer, 2-acrylamido-2-methylpropane sulfonic acid/acrylamide copolymers, 2-acrylamido-2-methylpropane sulfonic acid/N,N-dimethyl-acrylamide copolymers, vinylpyrrolidone/2-acrylamido-2-methylpropane sulfonic acid/acrylamide terpolymers, acrylamide/t-butyl acrylate/N-vinylpyrrolidone terpolymers, acrylamide/t-butyl acrylate/2-acrylamido-2-methylpropane sulfonic acid terpolymers, 2-acrylamido-2-methylpropane sulfonic acid/N-N-dimethylacrylamide/acrylamide terpolymers, acrylamide/t-butyl acrylate/N-vinylpyrrolidone/2-acrylamido-2-methylpropane sulfonic acid tetrapolymers, acrylamide/t-butyl acrylate copolymers, poly(2-hydroxyethyl methacrylate), poly(2-hydroxypropyl methacrylate), derivatives thereof, and the like, or combinations thereof. Generally, a fluid loss agent may control the loss of fluid to a wellbore and/or subterranean formation. In embodiments, the particulate material may comprise a water-insoluble material; such as pozzolana cement, sand, a weighting agent (e.g., an iron oxide, such as hematite; a manganese oxide, such as hausmannite; a titanium-iron oxide, such as ilmenite, etc.), a fiber (e.g., carbon fiber, acrylonitrile fiber, polypropylene fiber, rubber fiber, glass fiber, etc.), a rubber particle; a hollow glass sphere; a hollow pozzolanic sphere; a glass bubble; a glass ball; a ceramic ball; graphite; pozzolan; pumice; trass; clay; calcined clay; silica, fume silica, amorphous silica, micro-sized silica, nano-sized silica; and the like; or combinations thereof. A weighting agent can increase a density of a fluid. Fibers suitable for use as particulate material in the present disclosure can be further characterized by any suitable aspect ratio. The aspect ratio of a fiber may be calculated by dividing the length of the fiber by the diameter of the fiber. For example, fibers suitable for use as a particulate material as disclosed herein may be characterized by an aspect ratio of equal to or greater than about 2:1, alternatively equal to or greater than about 5:1, or alternatively equal to or greater than about 10:1. In embodiments where the particulate material comprises a water-insoluble material, the suspension composition as disclosed herein does not require the use of a biocide (although a biocide may be used), while an aqueous suspension comprising the water-insoluble material would necessitate the use of a biocide to mitigate shelf life issues due to degradation of the aqueous suspension over time. Organic carrier fluids as disclosed herein may be biocidic when largely water-free. In embodiments, the particulate material can be characterized by a particle size of from about 1 nm to about 10,000 μm, alternatively about 10 nm to about 9,000 μm, alternatively from about 0.1 μm to about 7.500 μm, alternatively about 0.5 μm to about 5,000 μm, or alternatively about 1 μm to about 1,000 μm. For purposes of the disclosure herein, the particle size refers to the largest dimension of any two-dimensional cross section through the particle. Nonlimiting examples of particulate material shapes suitable for use in the present disclosure include cylindrical, discoidal, spherical, tabular, ellipsoidal, equant, irregular, cubic, acicular, and the like, or combinations thereof. In embodiments, the particulate material can be present in the suspension composition in an amount of from about 1 wt. % to about 80 wt. %, alternatively from about 1 wt. % to about 70 wt. %, alternatively from about 1 wt. % to about 60 wt. %, alternatively from about 1 wt. % to about 50 wt. %, alternatively from about 5 wt. % to about 40 wt. %, alternatively from about 10 wt. % to about 30 wt. %, alternatively from about 10 wt. % to about 80 wt. %, alternatively from about 20 wt. % to about 70 wt. %, alternatively from about 30 wt. % to about 60 wt. %, or alternatively from about 40 wt. % to about 55 wt. %, based on a total weight of the suspension composition. In some embodiments, the crosslinked guar can be present in the suspension composition in an amount of from about 1 wt. % to about 50 wt. %, alternatively from about 5 wt. % to about 50 w-t. %, alternatively from about 5 wt. % to about 40 w-t. %, or alternatively from about 10 wt. % to about 30 wt %, based on a total weight of the suspension composition. The organic carrier fluid may comprise a glycol and/or a glycol ether. Glycols suitable for use in the present disclosure may comprise monoethylene glycol (MEG, also known as ethylene glycol), propylene glycol, butylene glycol, and the like, or combinations thereof. Generally, when substantially water-free, glycols may be biocidic, and consequently the use of a biocide in the suspension composition may be unnecessary. In embodiments, the organic carrier fluid as disclosed herein excludes a polyol, which is an organic compound containing multiple hydroxyl groups, such as 3 or more hydroxyl groups. For example, the organic carrier fluid as disclosed herein excludes a polyethylene glycol (PEG). Glycol ethers suitable for use in the present disclosure may comprise methyl ethers and/or ethyl ethers of the glycols that are suitable for use as organic carrier fluid as disclosed herein. For example, glycol ethers suitable for use in the present disclosure may comprise ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, butylene glycol monomethyl ether, butylene glycol monoethyl ether, and the like, or combinations thereof. The glycol and/or a glycol ether that are suitable for use as organic carrier fluid in the suspension compositions as disclosed herein may degrade within a few weeks upon contacting seawater. The organic carrier fluid is water miscible. As opposed to an oil (e.g., oleaginous fluid), the organic carrier fluids as disclosed herein are water miscible, such that a compatibilizer-type material (e.g., a surfactant) is not necessary in order to be able to homogenously mix the suspension composition with an aqueous fluid (e.g., an aqueous wellbore servicing fluid). Further, in certain applications, oleaginous fluid may be undesirable. In embodiments, the organic carrier fluid comprises MEG. MEG, also referred to as ethylene glycol, is an organic compound characterized by the formula (CH2OH)2. Pure MEG is an odorless and colorless liquid at room temperature and is miscible in water. MEG biodegrades relatively quickly in soil (e.g., half-life of about 2-12 days), surface water (e.g., half-life of about 2-12 days), and ground water (e.g., half-life of about 4-24 days). MEG can be used as an organic carrier fluid in the suspension composition and can degrade within a few weeks upon contacting seawater. In embodiments, the organic carrier fluid can be present in the suspension composition in an amount of from about 20 wt. % to about 98.99 wt. %, alternatively from about 30 wt. % to about 98.99 wt. %, alternatively from about 40 wt. % to about 98.99 wt. %, alternatively from about 50 wt. % to about 98.99 wt. %, alternatively from about 60 wt. % to about 95 wt. %, alternatively from about 70 wt. % to about 90 wt. %, alternatively from about 20 wt. % to about 90 wt. %, alternatively from about 30 wt. % to about 80 wt. %, alternatively from about 40 wt. % to about 70 wt. %, or alternatively from about 45 wt. % to about 60 wt. %, based on a total weight of the suspension composition. In some embodiments, MEG can be present in the suspension composition in an amount of from about 49 wt. % to about 98.99 wt. %, alternatively from about 49 wt. % to about 95 wt. %, alternatively from about 59 wt. % to about 94 wt. %, or alternatively from about 69 wt. % to about 89 wt. %, based on a total weight of the suspension composition. The suspension composition may comprise a suspension viscosifier. In embodiments, the suspension viscosifier functions as a suspending agent in the suspension composition. The suspension viscosifier herein can also be referred to as a viscosifying suspending agent. Without being limited by theory, the suspension viscosifier can prevent the particulate material (e.g., crosslinked guar) from settling in the suspension composition after preparation. In embodiments, the suspension viscosifier comprises Guar gum, Xanthan gum. Welan gum, Diutan, hydroxyethyl cellulose (HEC), modified cellulose, diatomaceous earth, starch, modified/crosslinked starch, viscoelastic surfactants (VES), precipitated silica, derivatives thereof, and the like, or combinations thereof. The suspension viscosifier can be present in the suspension composition in an amount of from about 0.01 wt. % to about 20 wt. %, alternatively from about 0.05 wt. % to about 20 wt. %, alternatively from about 0.05 wt. % to about 15 wt. %, or alternatively from about 0.1 wt. % to about 10 wt. %, based on a total weight of the suspension composition. In embodiments, the suspension composition can comprise water. The water can be selected from a group including freshwater, seawater, saltwater, brine (e.g., underground natural brine, formulated brine, etc.), and combinations thereof. Generally, the water may be from any source, provided that it does not contain an amount of components that may undesirably affect the other components in the suspension composition. The water can be present in the suspension composition in an amount effective to provide a suspension composition having desired rheological properties. In embodiments, the water can be present in the suspension composition in an amount of from about 0 wt. % to about 30 wt. %, alternatively from about 5 wt. % to about 30 wt. %, or alternatively from about 10 wt. % to about 30 wt. %, based on a total weight of the suspension composition. In embodiments of the suspension composition comprising water, the suspension composition may further comprise a biocide. A biocide refers to a diverse group of substances including preservatives, insecticides, disinfectants, pesticides, and the like, or combinations thereof used for the control of organisms that are harmful to human or animal health or that cause damage to natural or manufactured products, according to the US Environmental Protection Agency (EPA). In the European legislation, a biocide is defined as a chemical substance or microorganism intended to destroy, deter, render harmless, or exert a controlling effect on any harmful organism. In embodiments, the biocide comprises 3,3′-methylenebis[5-methyloxazolidine]. A nonlimiting example of a biocide suitable for use in the present disclosure is GROTAN® OX broad spectrum bactericide and fungicide, which is a type of organic, non-oxidizing, formaldehyde-releasing biocide, and a preservative for control of microbial growth commercially available from VINK Chemicals. In embodiments, a biocide can be present in the suspension composition in an amount of from about 0 wt. % to about 1 wt. %, alternatively from about 0.1 wt. % to about 0.9 wt. %, or alternatively from about 0.2 wt. % to about 0.8 wt. %, based on the total weight of the suspension composition. In embodiments, the suspension composition can comprise a particulate material (e.g., a crosslinked guar), an organic carrier fluid (e.g., monoethylene glycol (MEG)), and a suspension viscosifier. In some embodiments, the suspension composition can comprise a particulate material (e.g., a crosslinked guar), an organic carrier fluid (e.g., monoethylene glycol (MEG)), a suspension viscosifier, and water; alternatively a particulate material (e.g., a crosslinked guar), an organic carrier fluid (e.g., monoethylene glycol (MEG)), a suspension viscosifier, and a biocide; or alternatively a particulate material (e.g., a crosslinked guar), an organic carrier fluid (e.g., monoethylene glycol (MEG)), a suspension viscosifier, water, and a biocide. In embodiments, the suspension composition is a substantially homogenous mixture (e.g., a suspension) in which the particulate material (e.g., a crosslinked guar) does not dissolve, but gets uniformly suspended throughout the bulk of the organic carrier fluid (e.g., MEG). For example, the particulate material (e.g., a crosslinked guar) can be uniformly dispersed (e.g., floating around freely) in the suspension composition. A discontinuous internal phase (e.g., particulate material such as crosslinked guar) of the suspension composition can be uniformly dispersed throughout a continuous organic carrier fluid (e.g., MEG) external phase of the homogenous suspension composition through preparation (e.g., mixing or blending), with the use of a viscosifying suspending agent (e.g., the suspension viscosifier). In embodiments, the suspension composition has a density of from about 9 pounds per gallon (ppg) to about 12 ppg, alternatively from about 9.2 ppg to about 11.5 ppg, or alternatively from about 9.5 ppg to about 11 ppg. In embodiments, the suspension composition has a specific gravity of from about 0.5 to about 3, alternatively from about 0.8 to about 2.5, alternatively from about 1 to about 2, alternatively from about 1.1 to about 1.4, alternatively from about 1.1 to about 1.3, alternatively from about 1.12 to about 1.28, or alternatively from about 1.14 to about 1.26. In embodiments, a 1 vol. % dilution of the suspension composition in water has a pH1 in a range of from about 4 to about 12, alternatively from about 5 to about 11, or alternatively from about 6 to about 9. In embodiments, the suspension composition has a Brookfield viscosity of from about 50 cP to about 600 cP, alternatively from about 75 cP to about 500 cP, or alternatively from about 100 cP to about 400 cP, wherein the viscosity is measured at 75° F. and 100 rpm. In embodiments, the suspension composition has a flash point of equal to or greater than about 230° F., alternatively equal to or greater than about 240° F. or alternatively equal to or greater than about 250° F. In embodiments, the suspension composition has a freezing point of from about 8° F. to about 24° F., alternatively from about 9° F. to about 19° F., or alternatively from about 10° F. to about 14° F. In embodiments, the suspension composition has a boiling point of from about 210° F. to about 410° F., alternatively from about 380° F. to about 405° F. alternatively from about 385° F. to about 405° F., or alternatively from about 390° F. to about 400° F. In embodiments, the suspension composition stays substantially homogeneous and in a pourable fluid form during a storage period after being prepared. During the storage period, the suspension composition can be kept static and the densities of samples from different portions of the suspension composition can have a difference between each other of equal to or less than about 10%, alternatively equal to or less than about 8%, or alternatively equal to or less than about 5%. The storage period can be equal to or greater than about 1 day, alternatively equal to or greater than about 7 days, alternatively equal to or greater than about 14 days, alternatively equal to or greater than about 21 days, or alternatively equal to or greater than about 28 days. In embodiments, a suspension composition of the type disclosed herein can be prepared using any suitable method. For example, a method of the present disclosure can comprise contacting components of the suspension composition (e.g., a particulate material, such as a crosslinked guar; an organic carrier fluid, such as MEG; suspension viscosifier; and optionally water; biocide; etc.) to form the suspension composition. The contacting can comprise placing the components into a suitable suspension container (e.g., a mixer, a blender, a sonicator, a bid mill, a homogenizer) to form a suspension mixture, and blending the suspension mixture until the suspension mixture becomes a pumpable fluid (e.g., a suspension composition). The suspension container can be any container that is compatible with the suspension mixture and has sufficient space for the suspension mixture. A blender can be used for blending. In embodiments, a suspension composition of the type disclosed herein can be prepared by contacting a suspension viscosifier and an organic carrier fluid. The contacting can comprise placing the components into a suitable suspension container (e.g., a mixer, a blender, a sonicator, a bid mill, a homogenizer) to form a base mixture which can be characterized as a clear solution. The base mixture can then be contacted with a material to be dispersed which is then mixed, as described herein, to form a uniform suspension comprising the particulate material. In some embodiments, the uniform suspension may be contacted with water, alternatively a biocide, or alternatively water and a biocide to form a suspension composition. In embodiments, a suspension composition of the type disclosed herein can be prepared by contacting a suspension viscosifier and organic carrier fluid (e.g., MEG). The contacting can comprise placing the components into a suitable suspension container (e.g., a mixer, a blender, a sonicator, a bid mill, a homogenizer) to form a base mixture which can be characterized as a clear solution. The base mixture can then be contacted with a crosslinked guar which is then mixed, as described herein, to form a uniform suspension. In some embodiments, the uniform suspension may be contacted with water, alternatively a biocide, or alternatively water and a biocide to form a suspension composition. The suspension composition as disclosed herein can be used in any suitable fluid, such as a wellbore servicing fluid. In embodiments, the wellbore servicing fluid may comprise a cementitious fluid. In embodiments, the wellbore servicing fluid may comprise a spacer fluid. In embodiments, the suspension composition can be prepared at the wellsite. Components of the suspension composition can be transported to the wellsite and combined (e.g., mixed/blended) proximate the wellsite to form the suspension composition. The components of the suspension composition can be pre-combined such that the suspension composition is prepared at a location remote from the wellsite and transported to the wellsite, and, if necessary, stored at an on-site location for use in making a wellbore servicing fluid. When it is desirable to prepare the suspension composition at the wellsite, the components of the suspension composition can be added into a suspension container (e.g., a blender tub, for example mounted on a trailer), and the suspension mixture is then blended until the suspension mixture becomes a pumpable fluid (e.g., a suspension composition). In some other embodiments, the suspension composition is prepared at a location remote from the wellsite, transported to the wellsite, optionally stored at the wellsite and combined with water, and other necessary components (e.g., a cement blend), and optionally one or more additives, such as weighting agents, or weight-reducing agents to form a wellbore servicing fluid. Transporting of the suspension composition and/or the components of the suspension composition can be done by a ship, a pipeline, tanker truck, or any suitable transportation method. In embodiments, the suspension composition can be present in a wellbore servicing fluid in an amount ranging from about 0.1 wt. % to about 60 wt. %, alternatively from about 0.1 wt. % to about 40 wt. %, alternatively from about 0.1 wt. % to about 20 wt. %, alternatively from about 0.5 wt. % to about 10 wt. %, or alternatively from about 1 wt. % to about 5 wt., based on the total weight of the wellbore servicing fluid. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid; spacer fluid) further comprises one or more additives. The one or more additives may comprise a defoamer, a cement retarder, a cement dispersant, a fluid loss control additive, a fume silica, a free fluid control additive, a viscosifying agent, an acid, a base, an emulsifier, a salt, a corrosion inhibitor, a mutual solvent, a conventional breaking agent, a relative permeability modifier, lime, a gelling agent, a crosslinker, a flocculant, a water softener, a proppant, an oxidation inhibitor, a thinner, a scavenger, a gas scavenger, a lubricant, a friction reducer, a bridging agent, a vitrified shale, a thixotropic agent, a surfactant, a scale inhibitor, a clay, a clay control agent, a clay stabilizer, a silicate-control agent, a biostatic agent, a storage stabilizer, a filtration control additive, a foaming agent, a foam stabilizer, latex emulsions, a formation conditioning agent, elastomers, gas/fluid absorbing materials, resins, superabsorbers, mechanical property modifying additives, inert particulates, and the like, or combinations thereof. A wellbore servicing fluid (e.g., cementitious fluid; spacer fluid) of the type disclosed herein can exclude a biocide. In embodiments, a wellbore servicing fluid of the type disclosed comprises equal to or less than about 1%, 0.1%, 0.001%, 0.0001%, 0.00001%, 0.000001%, or 0.0000001% biocide, based on the total weight of the wellbore servicing fluid. In embodiments, components of the wellbore servicing fluid (e.g., the particulate material, such as the crosslinked guar; the organic carrier fluid, such as the MEG; the cementitious material; the one or more additives; etc.) are materials described to Pose Little or No Risk to the Environment (PLONOR). The PLONOR list is an OSPAR (Oslo and Paris Conventions) list of substances and/or preparations used and discharged offshore that are deemed to cause no or little harm to the environment. In other words, the components of the wellbore servicing fluid are PLONOR materials. In embodiments, the wellbore servicing fluid comprises, consists essentially of, or consists of PLONOR materials. In embodiments, a wellbore servicing fluid of the type disclosed herein excludes any material that does not have a PLONOR designation (i.e., materials that are not on the PLONOR list). In embodiments, a wellbore servicing fluid of the type disclosed comprises equal to or less than about 25%, 20%, 15%, 10%, 5%, 1%, 0.1%, 0.001%, 0.0001%, 0.00001%, 0.000001%, or 0.0000001% non-PLONOR materials based on the total weight of the wellbore servicing fluid. The wellbore servicing fluid (e.g., cementitious fluid; spacer fluid) can be used in a wellbore having a Bottomhole Circulating Temperature (BHCT) from about 70° F. to about 400° F. alternatively from about 120° F. to about 400° F., or alternatively from about 160° F. to about 370° F. In embodiments, the wellbore servicing fluid is used in a wellbore having a Bottomhole Static Temperature (BHST) from about 100° F. to about 400° F. alternatively from about 150° F. to about 400° F. or alternatively from about 190° F. to about 400° F. In some embodiments, the wellbore servicing fluid comprising the suspension composition may be a cementitious fluid. A cementitious fluid refers to the material used to permanently seal an annular space between a casing and a wellbore wall. A cementitious fluid can also be used to seal formations to prevent loss of drilling fluid (e.g., in squeeze cementing operations) and for operations ranging from setting kick-off plugs to plug and abandonment of a wellbore. Generally, a cementitious fluid used in oil field is less viscous and has less strength than cement or concrete used for construction, since the cementitious fluid is required to be pumpable in a relatively narrow annulus over long distances. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) comprises a cement blend. The cement blend can comprise, consist essentially of, or consist of a cementitious material. The cementitious material can comprise Portland cement, pozzolana cement, gypsum cement, shale cement, acid cement, base cement, phosphate cement, high alumina content cement, slag cement, silica cement, high alkalinity cement, magnesia cement, lime, amorphous silica, siliceous material, fly ash, any cementitious material, and the like, or combinations thereof. As used herein, the term “high alumina content cement” refers to a cement having an alumina concentration in the range of from about 40 wt. % to about 80 wt. %, by a weight of the high alumina content cement. The term “high alkalinity cement” refers to a cement having a sodium oxide concentration in the range of from about 1.0 wt. % to about 2.0 wt. %, by a weight of the high alkalinity cement. In embodiments, the cementitious material is present in the cement blend in an amount of from about 1% BWOB (by weight of blend) to about 100% BWOB, alternatively from about 5% BWOB to about 100% BWOB, alternatively from about 10% BWOB to about 80% BWOB, or alternatively from about 20% BWOB to about 60% BWOB, based on a total weight of the cement blend. In embodiments, the cement blend further comprises an expansion agent. Without limitation, examples of expansion agents suitable for use in the cement blend of the present disclosure include metal powders, aluminum powder, a gypsum blend, alkali metal oxides, alkaline earth metal oxides, magnesium oxide, lightly burned magnesium oxide, hard burned magnesium oxide, deadburned magnesium oxide, and the like, or combinations thereof. In embodiments where both the particulate material and the cement blend comprise an expansion agent, the expansion agent of the particulate material and the expansion agent of the cement blend can be the same or different. For example, in some embodiments, both the particulate material and the cement blend can independently comprise lightly burned magnesium oxide. As another example, in some embodiments, the particulate material can comprise aluminum powder, while the cement blend can comprise lightly burned magnesium oxide. In embodiments, the expansion agent can be present in the cement blend in an amount of from about 1% BWOB to about 10% BWOB, alternatively from about 1.5% BWOB to about 7.5% BWOB, or alternatively from about 2% BWOB to about 5% BWOB, based on a total weight of the cement blend. In embodiments, the cement blend further comprises one or more cement blend additives. The one or more cement blend additives can comprise quartz flour, bulk flow enhancer, aggregate, particles, filler, amorphous silica, siliceous material, fly ash, and the like, or combinations thereof. In embodiments, the one or more cement blend additives can be present in the cement blend in an amount of from about 5% BWOB to about 95% BWOB, alternatively from about 5% BWOB to about 80% BWOB, alternatively from about 10% BWOB to about 60% BWOB, or alternatively from about 15% BWOB to about 40% BWOB, based on a total weight of the cement blend. A cement blend of the type disclosed herein can be prepared using any suitable method. Components of the cement blend can be predetermined. In embodiments, the cement blend comprises more than one component (e.g., a cementitious material, an expansion agent, a bulk flow enhancer, and one or more cement blend additives), which can be dry mixed to form the cement blend. The dry mixing can be at a location away from the wellsite and the cement blend can be transported to the wellsite. In embodiments, the components of the cement blend can be prepared at a location remote from the wellsite and transported to the wellsite, and, if necessary, stored at an on-site location. When desired, the components of the cement blend can be dry mixed at the wellsite. In embodiments, the cement blend contains (e.g., consists essentially of or consists of) one component (i.e., a cementitious material) and can be transported and stored at the wellsite. Transporting of the cement blend and/or the components of the cement blend can be by a ship or any suitable transportation. In embodiments, the components of the cement blend can be added to a dry-mixing container (e.g., a mixing head of a solid feeding system) and be dry mixed therein. The dry-mixing container can be any container that is compatible with the components of the cement blend and has sufficient space for the components of the cement blend. A blender can be used for dry mixing. In embodiments, the cement blend can be present in the wellbore servicing fluid in an amount ranging from about 20 wt. % to about 90 wt. %, alternatively from about 40 wt. % to about 80 wt. %, or alternatively from about 60 wt. % to about 70 wt. %, based on the total weight of the wellbore servicing fluid. The wellbore servicing fluid (e.g., cementitious fluid) can comprise water. The water can be selected from a group including freshwater, seawater, saltwater, brine (e.g., underground natural brine, formulated brine, etc.), and combinations thereof. Generally, the water may be from any source, provided that it does not contain an amount of components that may undesirably affect the other components in the wellbore servicing fluid. The water can be present in the wellbore servicing fluid in an amount effective to provide a slurry having desired (e.g., job or service specific) rheological properties. The water can be present in the wellbore servicing fluid in an amount of from about 10 L/100 kg to about 400 L/100 kg, alternatively from about 20 L/100 kg to about 150 L/100 kg, or alternatively from about 30 L/100 kg to about 65 L/100 kg, based on a total weight of the cement blend. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) further comprises a weighting agent and/or a weight-reducing agent. In embodiments, a weighting agent and/or a weight-reducing agent may be included within the cement blend (e.g., as part of a dry cement blend or dry cementitious composition) prior to formation of a wellbore servicing fluid by mixing the cement blend with water to form a pumpable cement slurry. A weighting agent can increase a density of the wellbore servicing fluid. Nonlimiting examples of suitable weighting agents for the present disclosure include barium sulfate, (i.e., barite), iron oxide (i.e., hematite), manganese oxide (i.e., hausmannite), and combinations thereof. An example of weighting agent suitable for use in this disclosure includes without limitation a synthetic hausmannite known as MICROMAX® FF weight additive, which is commercially available from Elkem Materials Inc. A weight-reducing agent can reduce a density of the wellbore servicing fluid. Nonlimiting examples of suitable weight-reducing agents suitable for use in the present disclosure include hollow glass and ceramic beads. In embodiments where both the particulate material and the cement blend comprise a weighting agent, the weighting agent of the particulate material and the weighting agent of the cement blend can be the same or different. For example, in some embodiments, both the particulate material and the cement blend can independently comprise hausmannite. As another example, in some embodiments, the particulate material can comprise ilmenite, while the cement blend can comprise hausmannite. The amount of the weighting agent or weight-reducing agent in the wellbore servicing fluid (e.g., cementitious fluid) may be an amount effective to produce a desired density of the wellbore servicing fluid. In embodiments, the weighting agent or the weight-reducing agent can be present in the wellbore servicing fluid in an amount of from about 1% BWOB to about 200% BWOB, alternatively from about 5% BWOB to about 150% BWOB, or alternatively from about 10% BWOB to about 100% BWOB, based on a total weight of the cement blend. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) comprising a cement blend further comprises a foaming agent. In such embodiments, the wellbore servicing fluid comprising a cement blend forms a foamed cement having a density that is reduced in comparison to an otherwise similar composition prepared in the absence of the foaming agent. The foaming agent may be introduced (e.g., added into the wellbore servicing fluid) prior to placing the wellbore servicing fluid in the wellbore. The addition of a foaming agent to the cement composition may be accomplished by any suitable method. In embodiments, the foaming agent comprises a gas such as air, an inert gas such as nitrogen, and combinations thereof. The gas (e.g., nitrogen) may be introduced by direct injection into the wellbore servicing fluid. In such embodiments, the gas is present in the wellbore servicing fluid in an amount of from about 10 vol. % to about 30 vol. %, based on a total volume of the wellbore servicing fluid placed in the wellbore. In embodiments, the foamed cement can have a density (e.g., a target density of the wellbore servicing fluid) of from about 5 ppg to about 16 ppg, alternatively from about 8 ppg to about 15 ppg, or alternatively from about 10 ppg to about 14 ppg. In embodiments, the one or more additives can be present in the wellbore servicing fluid (e.g., cementitious fluid) in a total amount of from about 0.1 L/100 kg to about 50 L/100 kg, based on a total weight of the cement blend, alternatively from about 1 L/100 kg to about 35 L/100 kg, or alternatively from about 5 L/100 kg to about 20 L/100 kg, based on a total weight of the wellbore servicing fluid. Additives suitable for use in the present disclosure may be in solid form and in such embodiments the additive may be included in the wellbore servicing fluid in amounts of from about 0.05% BWOB to about 100% BWOB, alternatively from about 0.5% BWOB to about 50% BWOB, or alternatively from about 5% BWOB to about 20% BWOB, based on a total weight of the cement blend. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) has a density of from about 9 pounds per gallon (ppg) to about 26 ppg, alternatively from about 11 ppg to about 22 ppg, or alternatively from about 13 ppg to about 18 ppg. In embodiments, a wellbore servicing fluid (e.g., cementitious fluid) suitable for use in the present disclosure comprises about 20 wt. % of a cement blend based on the total weight of the wellbore servicing fluid and about 400 L/100 kg of water BWOB and has a density of about 9.6 ppg. In some other embodiments, a wellbore servicing fluid suitable for use in the present disclosure comprises about 40 wt. % of a cement blend based on the total weight of the wellbore servicing fluid and about 150 L/100 kg of water BWOB and has a density of about 11.5 ppg. In some other embodiments, a wellbore servicing fluid suitable for use in the present disclosure comprises about 60 wt. % of a cement blend based on the total weight of the wellbore servicing fluid and about 65 L/100 kg of water BWOB and has a density of about 14.1 ppg. In some other embodiments, a wellbore servicing fluid suitable for use in the present disclosure comprises about 70 wt. % of a cement blend based on the total weight of the wellbore servicing fluid and about 40 L/100 kg of water BWOB and has a density of about 16.0 ppg. In some other embodiments, a wellbore servicing fluid suitable for use in the present disclosure comprises about 75 wt. % of a cement blend based on the total weight of the wellbore servicing fluid and about 35 L/100 kg of water BWOB and has a density of about 17.1 ppg. In yet some other embodiments, a wellbore servicing fluid suitable for use in the present disclosure comprises about 90 wt. % of a cement blend based on the total weight of the wellbore servicing fluid and about 15 L/100 kg of water BWOB and has a density of about 21 ppg. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) has a specific gravity of from about 0.5 to about 3, alternatively from about 1.1 to about 2.5, alternatively from about 1.3 to about 2.3, or alternatively from about 1.5 to about 2.0. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) has a mixability rating of from about 3 to about 5, alternatively from about 4 to about 5. The mixability rating is on a 0 to 5 scale, where 0 is not mixable and 5 is fully mixable. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) has a fluid loss of from about 10 ml per 30 minutes to about 250 ml per 30 minutes, alternatively from about 20 ml per 30 minutes to about 100 ml per 30 minutes, or alternatively from about 30 ml per 30 minutes to about 50 ml per 30 minutes, when measured on a 325 mesh screen at about 129° F. and 1.000 psig differential pressure in accordance with a test standard API-RP-10B-2. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) has a 10-second static gel strength of from about 1 to about 50, alternatively from about 5 to about 40, or alternatively from about 10 to about 30, when measured at about 129° F. in accordance with the test standard API-RP-10B-2. In embodiments, the wellbore servicing fluid has a 10-minute static gel strength of from about 1 to about 300, alternatively from about 5 to about 150, or alternatively from about 10 to about 75, when measured at about 129° F. in accordance with the test standard API-RP-10B-2. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) has a thickening time of from about 3 hour to about 24 hours, alternatively from about 4 hours to about 16 hours, or alternatively from about 5 hours to about 8 hours, when measured in accordance with the test standard API-RP-10B-2 to achieve about 70 Bearden units (Bc) at about 129° F. and 5.000 psig. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) has a 50 psi Ultrasonic Cement Analyzer (UCA) compressive strength of from about 1 hour to about 48 hours, alternatively from about 4 hours to about 24 hours, or alternatively from about 6 hours to about 18 hours, when measured at about 168° F. and 5.000 psig. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) has a 500 psi UCA compressive strength of from about 2 hours to about 72 hours at, alternatively from about 6 hours to about 36 hours, or alternatively from about 8 hours to about 24 hours, when measured at about 168° F. and 5.000 psig in accordance with the test standard API-RP-10B-2. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) has a 24 hr UCA compressive strength of from about 50 psig to about 10,000 psig, alternatively from about 250 psig to about 6.000 psig, or alternatively from about 500 psig to about 4,000 psig, when measured at about 168° F. and 5,000 psig in accordance with the test standard API-RP-10B-2. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid) has rheology readings in a range of from about 1 to about 350 at about 68° F., alternatively from about 2 to about 350 at about 129° F., or alternatively from about 2 to about 350 at about 190° F. when measured by a Fann® Model 35 viscometer at a speed of 3 rpm to 300 rpm in accordance with the test standard API-RP-10B-2. In embodiments, after being cured at about 212° F. and 5.000 psig for about 7 days, the wellbore servicing fluid (e.g., cementitious fluid) forms a set cement having a crush compressive strength of from about 500 psig to about 12,000 psig, alternatively from about 1,500 psig to about 9,000 psig, or alternatively from about 3,000 psig to about 7,000 psig. In embodiments, after being cured at about 212° F. and 5.000 psig for about 7 days, the wellbore servicing fluid (e.g., cementitious fluid) forms a set cement having a Young's Modulus of from about 0.3 Mpsig to about 3 Mpsig, alternatively from about 0.8 Mpsig to about 2 Mpsig, or alternatively from about 1.2 Mpsig to about 1.6 Mpsig. In embodiments, after being cured at about 212° F. and 5,000 psig for about 7 days, the wellbore servicing fluid (e.g., cementitious fluid) forms a set cement having a Brazilian Tensile Strength of from about 50 psig to about 1.600 psig, alternatively from about 100 psig to about 900 psig, or alternatively from about 200 psig to about 700 psig. In some embodiments, the wellbore servicing fluid comprising the suspension composition may be a spacer fluid. In embodiments, the spacer fluid as disclosed herein may comprise the suspension composition and a base fluid, and optionally any suitable additives. The base fluid may comprise water or an aqueous fluid. Alternatively, the base fluid may comprise a hydrocarbon fluid such as mud base oil, diesel, etc. A spacer fluid is generally used to physically separate one special purpose liquid from another, and a spacer fluid should be compatible with each of the special purpose fluids. For example, a spacer fluid can separate a drilling fluid from a cementitious fluid. The spacer fluid may have a density that is different from the density of the fluids it separates. In embodiments where the spacer fluid separates a drilling fluid from a cementitious fluid, the spacer fluid may have a density that is greater than the density of the drilling fluid, and the spacer fluid may have a density that is lower than the density of the cementitious fluid. In embodiments, the spacer fluid can have a density in a range of from about 4 ppg to about 25 ppg, alternatively from about 7 ppg to about 21 ppg, or alternatively from about 9 ppg to about 17 ppg. The spacer fluid can comprise water. The water can be selected from a group including freshwater, seawater, saltwater, brine (e.g., underground natural brine, formulated brine, etc.), and combinations thereof. Generally, the water may be from any source, provided that it does not contain an amount of components that may undesirably affect the other components in the wellbore servicing fluid. The water can be present in the spacer fluid in an amount effective to provide a slurry having desired (e.g., job or service specific) rheological properties. The water can be present in the spacer fluid in an amount of from about 10 wt. % to about 99.9 wt. %, alternatively from about 20 wt. % to about 80 wt. %, or alternatively from about 30 wt. % to about 60 wt. %, based on a total weight of the spacer fluid. In embodiments where the density of the spacer fluid is relatively low (e.g., about 4 ppg), the spacer fluid may be a foamed fluid, for example comprising a gas such as air, nitrogen, or any other suitable gas; may comprise hollow beads or bubbles, for example glass bubbles; or combinations thereof. In embodiments where the spacer fluid is a foamed fluid, the spacer fluid may comprise a gas (e.g., air, nitrogen, or any other suitable gas) in an amount of from about 1 vol. % to about 90 vol. %, alternatively from about 5 vol. % to about 85 vol. %, or alternatively from about 10 vol. % to about 80 vol. %, based on the total volume of the spacer fluid. A wellbore servicing fluid of the type disclosed herein can be prepared using any suitable method. In embodiments, a method of making the wellbore servicing fluid comprises contacting water with the suspension composition, and optionally cement blend and/or additives to form the wellbore servicing fluid. In embodiments, the wellbore servicing fluid (e.g., cementitious fluid; spacer fluid) can be prepared at the wellsite. Components of the wellbore servicing fluid can be transported to the wellsite and combined (e.g., mixed/blended) proximate the wellsite to form the wellbore servicing fluid. The components of the wellbore servicing fluid can be added into a container (e.g., a blender tub, for example mounted on a trailer), and the wellbore servicing fluid is then blended until the wellbore servicing fluid becomes a pumpable fluid. The methods disclosed herein for preparing the wellbore servicing fluid (e.g., cementitious fluid; spacer fluid) can comprise a continuous process (also referred to as an “on-the-fly” process). A continuous process or an “on-the-fly” process means one or more steps in the process are running on a continuous basis. For example, a contacting step can be continuous in which wellbore servicing fluid components are contacted in a container (e.g., a blender or mixer) in a manner that yields an about constant output of the wellbore servicing fluid from the container. The pumps, the blender, and other process equipment can operate at about steady state conditions during a continuous process, with the understanding that one or more operational parameters (e.g., rate, pressure, etc.) in the continuous process can be adjusted during the process. The continuous process can be performed by using proper equipment (e.g., a mixer, a blender, feeders, pumps, etc.) and process management/control. For example, forming the suspension composition can be continuous using pumps and a blender; forming the cement blend can be continuous using a blender and solid feeders; conveying water, the suspension composition, and/or the one or more additives can be continuous using pumps; combining the cement blend with a mixture in the container can be continuous using a feeder; blending the cementitious fluid in the container can be continuous by generating a whirlpool continuously; and any combination thereof may be employed in a continuous process of the type described herein. As another example, the suspension composition can be contacted with water and optional additives in a container on a continuous basis to yield a continuous output of the spacer fluid from the container. In embodiments, a spacer fluid of the type disclosed herein can be prepared using any suitable method. Generally, spacer fluids may be prepared in a pit or any suitable tank by recirculating and/or agitating. Usually, spacer fluids may be prepared at the well site; although spacer fluids may be prepared remotely and then transported to the well site. For example, a method of the present disclosure can comprise contacting components of the spacer fluid (e.g., a suspension composition; water; optional additives) to form the spacer fluid at a location proximate a wellsite. The wellsite can comprise an offshore platform (e.g., an offshore oil and gas platform) and/or a floating vessel and the wellbore can be offshore. The contacting of the components of the spacer fluid can comprise placing the components into a suitable spacer fluid container (e.g., a mixer, a blender, a sonicator, a bid mill, a homogenizer) to form a spacer fluid mixture, and blending the spacer fluid mixture until the spacer fluid mixture becomes a pumpable fluid (e.g., a spacer fluid). The spacer fluid container can be any container that is compatible with the spacer fluid and has sufficient space for the spacer fluid. A blender can be used for blending. The spacer fluid container may provide a continuous spacer fluid output. In embodiments, a cementitious fluid of the type disclosed herein can be prepared using any suitable method. In embodiments, a method of making the cementitious fluid comprises contacting water with the suspension composition and a cement blend prepared using the methods disclosed hereinabove at a location proximate a wellsite. The wellsite can comprise an offshore platform (e.g., an offshore oil and gas platform) and/or a floating vessel and the wellbore can be offshore.FIG.1depicts a process flow diagram of a method200of making a wellbore servicing fluid (e.g., cementitious fluid) of the type disclosed herein. Referring toFIG.1, the water can be conveyed via a water flow line201from any resource, for example, seawater around the wellsite, produced water, and water conveyed from onshore. The method can comprise contacting a suspension composition of the type disclosed herein with water to form a mixture. Contacting the suspension composition with water can comprise conveying (e.g., via a suspension flow line202) the suspension composition into the water in the water flow line201. In some embodiments, a liquid suspension aid (e.g., suspension viscosifier) may be added directly into the mixing water (e.g., water flow line201). The combination of water and the suspension composition can be referred to as a diluted suspension composition or a first mixture. In embodiments, one or more additives of the type disclosed herein optionally can be added into the first mixture in a mixture flow line205, for example by conveying the one or more additives (e.g., via one or more additive flow lines204) into the first mixture in the mixture flow line205to form a second mixture. In some embodiments, the fluid flowing via the mixture flow line205may be formulated and used as a spacer fluid. After contacting the suspension composition with water to form the first mixture, and optionally adding the one or more additives into the first mixture to form the second mixture, the first or second mixture can be further contacted with a cement blend prepared using the methods disclosed hereinabove. In embodiments, the first or second mixture is conveyed via the mixture flow line205to a container. The container can be any container that is compatible with the first or second mixture and the cement blend and has sufficient space. The cement blend can be added (e.g., metered by a solids feeding system such as a conveyor or auger) into the container and blended with the first or second mixture. The blending can be conducted using any suitable method/tool (e.g., a blender) until a pumpable fluid (e.g., the wellbore servicing fluid; cementitious fluid) is formed. In embodiments, the blending comprises generating whirlpools (e.g., vortexes) in the cementitious fluid. Whirlpools can be generated by any suitable method, for example by a nozzle that releases a jet of the contents of the container therein (e.g., a pump-around loop). In embodiments, prior to and/or concurrent with contacting the cement blend with the first or second mixture, the method further comprises adding a weighting agent or a weight-reducing agent to the cement blend, to the first mixture, to the second mixture, directly to the container, or any combination thereof. The weighting agent or the weight-reducing agent can be placed into the container having the other components of the wellbore servicing fluid therein. The methods disclosed herein for preparing the cementitious fluid can comprise a continuous process (also referred to as an “on-the-fly” process). For example, a contacting step can be continuous in which the cement blend and the first or second mixture are contacted in a container (e.g., a blender or mixer) in a manner that yields an about constant output of the wellbore servicing fluid from the container. The pumps, the blender, and other process equipment can operate at about steady state conditions during a continuous process, with the understanding that one or more operational parameters (e.g., rate, pressure, etc.) in the continuous process can be adjusted during the process of making the cementitious fluid. The continuous process of making the cementitious fluid can be performed by using proper equipment (e.g., a mixer, a blender, feeders, pumps, etc.) and process management/control. For example, forming the suspension composition can be continuous using pumps and a blender; forming the cement blend can be continuous using a blender and solid feeders; conveying water, the suspension composition, and/or the one or more additives can be continuous using pumps; combining the cement blend with the first or second mixture in the container can be continuous using a feeder; blending the wellbore servicing fluid in the container can be continuous by generating a whirlpool continuously; and any combination thereof may be employed in a continuous process of the type described herein. In embodiments, referring toFIG.2, a method300disclosed herein comprises contacting a particulate material (e.g., a crosslinked guar), an organic carrier fluid (e.g., MEG), a suspension viscosifier of the types disclosed herein, and optionally water, a biocide, or both, to form a suspension composition of the type disclosed herein. The contacting can be in a suspension container, and can occur proximate a wellsite or remote from a wellsite (e.g., prepared remotely and transported to a wellsite such as an offshore platform or a floating vessel). The method can take place at a location proximate an offshore platform and/or a floating vessel, where a water flow line301conveys water from a water resource (e.g., seawater around the offshore platform or the floating vessel). The method can further comprise conveying the suspension composition via a suspension flow line302into the water in the water flow line301to form a diluted suspension. In some embodiments, a liquid suspension aid (e.g., suspension viscosifier) may be added directly into the mixing water (e.g., water flow line301). In embodiments, the method further comprises conveying one or more additives via one or more additive flow lines304into the diluted suspension in a diluted suspension flow line303to form a mixture in a mixture flow line305. One or more pumps can be used on each of the water flow line301, the suspension flow line302, the diluted suspension flow line303, the one or more additive flow lines304, and the mixture flow line305. In some embodiments, the fluid flowing via the mixture flow line305may be formulated and used as a spacer fluid. In other embodiments, the method further comprises placing the mixture in a container (e.g., a mixing container). A cement blend of the type disclosed herein from a cement blend resource (e.g., a holding tank) can be added into the mixture to form a slurry within the container. A solid feeding system (e.g., a solid feeder such as an auger feeder or a screw feeder) can be used for adding the cement blend into the container. The method can further comprise blending the slurry to form a pumpable fluid (e.g., the cementitious fluid). The blending can be by a blender. In embodiments, the blending is by generating whirlpools in the slurry within the container (e.g., by a nozzle in the container that releases a jet of the slurry). The wellbore servicing fluid can then be placed downhole. In embodiments, a wellbore servicing fluid of the type disclosed herein is used as a cementitious fluid, for example comprising a cement blend of the type disclosed herein. The method of the present disclosure can further comprise placing the wellbore servicing fluid in an offshore wellbore penetrating a subterranean formation and allowing at least a portion of the wellbore servicing fluid to set. The wellbore servicing fluid can be used to permanently seal the annular space between the conduit (e.g., casing) and the wellbore wall or the annular space between two casings. The wellbore servicing fluid can also be used to seal formations to prevent loss of drilling fluid (e.g., in squeeze cementing operations) and for operations ranging from setting kick-off plugs to plug and abandonment of a wellbore. In embodiments, a wellbore servicing fluid (e.g., cementitious fluid) of the type disclosed herein can be employed in well completion operations such as primary and secondary cementing operations. The cementitious fluid may be placed into an annulus of the wellbore (e.g., an annulus formed between casing and a wellbore wall) and allowed to set such that it isolates the subterranean formation from a different portion of the wellbore. The cementitious fluid thus forms a barrier that prevents fluids in that subterranean formation from migrating into other subterranean formations. Within the annulus, the cementitious fluid also serves to support a conduit, e.g., casing, in the wellbore. In embodiments, the wellbore in which the cementitious fluid is positioned belongs to a multilateral wellbore configuration. It is to be understood that a multilateral wellbore configuration includes at least two principal wellbores connected by one or more ancillary wellbores. In secondary cementing, often referred to as squeeze cementing, the wellbore servicing fluid (e.g., cementitious fluid) can be strategically positioned in the wellbore to plug a void or crack in the conduit, to plug a void or crack in the hardened sealant (e.g., cement sheath) residing in the annulus, to plug a relatively small opening known as a microannulus between the hardened sealant and the conduit, to plug a permeable zone, and so forth. In embodiments, a method of servicing a wellbore penetrating a subterranean formation (e.g., offshore wellbore penetrating a subterranean formation) comprises placing a wellbore servicing fluid (e.g., spacer fluid) of the type described herein into the wellbore, wherein the wellbore servicing fluid comprises the suspension composition as disclosed herein. In some embodiments, the method of servicing a wellbore comprises placing (e.g., pumping) a first fluid (e.g., a drilling fluid) into the wellbore, thereafter placing (e.g., pumping) the spacer fluid into the wellbore, and thereafter placing (e.g., pumping) a second fluid (e.g., cementitious fluid) into the wellbore, wherein the spacer fluid physically spaces the first fluid apart from the second fluid such that the first fluid and the second fluid do not comingle while being placed (e.g., pumped) into the wellbore. For example, the spacer fluid can be used to space apart two fluids (e.g., a drilling fluid and a cementitious fluid) that are being flowed from the surface down through a conduit (e.g., casing) present in the wellbore, exiting the conduit and flowing back upward in the annular space between the outside conduit wall and interior face of the wellbore. In embodiments, a wellbore may have casing disposed therein to form an annular space between the wellbore wall and the outer surface of the casing, wherein a drilling fluid (or other fluid) is present in at least a portion of the annular space. The drilling fluid herein refers to any liquid and gaseous fluid and mixtures of fluids and solids used in operations of drilling a borehole into the earth. The drilling fluid can be a water-based fluid. In embodiments, the method of servicing a wellbore penetrating a subterranean formation comprises placing a spacer fluid into at least a portion of the annular space and displacing at least a portion of the drilling fluid from the annular space, wherein the spacer fluid comprises the suspension composition as disclosed herein, and wherein the density of the spacer fluid is greater than the density of the drilling fluid. In some embodiments, the method of servicing a wellbore penetrating a subterranean formation further comprises placing a cementitious fluid into at least a portion of the annular space and displacing at least a portion of the spacer fluid from the annular space, wherein the density of the cementitious fluid is greater than the density of the spacer fluid. In embodiments, the spacer fluid (e.g., comprising the suspension composition, water, and optional additives) may be used to separate the drilling fluid from the cementitious fluid. In embodiments, a method of servicing a wellbore penetrating a subterranean formation (e.g., offshore wellbore penetrating a subterranean formation) can further comprise placing a spacer fluid into at least a portion of the tubular space inside the casing; and displacing at least a portion of the cementitious fluid from the tubular space. For example, a spacer fluid of the type described herein can be pumped into the wellbore following release of a cement plug, and the spacer fluid can be used to push the cement plug through the casing, which in turn pushes the cementitious fluid out of the casing and into the annular space between the casing and the wellbore wall. FIG.3illustrates a method100in accordance with the present disclosure. Block101includes forming a suspension composition of the type disclosed herein, either at (e.g., proximate) a wellsite or remote from the wellsite and transported to the wellsite. The forming can comprise contacting a particulate material (e.g., a crosslinked guar), an organic carrier (e.g., MEG), a suspension viscosifier, and optionally water, a biocide, or both. Block102includes contacting the suspension composition, water, and a cement blend of the type disclosed herein to form a wellbore servicing fluid (e.g., cementitious fluid) at a location proximate a wellsite. The wellsite can be an offshore oil and gas platform and/or a floating vessel. Block103includes placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation, for example as a primary or secondary cementing operation. The wellbore can be an offshore wellbore. Block104includes allowing at least a portion of the wellbore servicing fluid (e.g., cementitious fluid) to set. At least a portion of block101, block102, and block103can comprise a continuous process as disclosed herein. Referring toFIG.4, in embodiments, block102further comprises block401, block402, block403, and block404. Block401includes contacting the suspension composition and water to form a mixture. Block402is optional and includes adding one or more additives of the type disclosed herein into the mixture. Block403is optional and includes contacting a weighting agent or a weight-reducing agent with the mixture. Block404includes contacting the mixture with the cement blend to form a wellbore servicing fluid (e.g., cementitious fluid). FIG.5illustrates a method500in accordance with the present disclosure. Block501includes forming a suspension composition of the type disclosed herein, either at (e.g., proximate) a wellsite or remote from the wellsite and transported to the wellsite. The forming can comprise contacting a particulate material (e.g., a crosslinked guar), an organic carrier (e.g., MEG), a suspension viscosifier, and optionally water, a biocide, or both. Block502includes contacting the suspension composition, water, a cement blend of the type disclosed herein (e.g., a cement blend comprising a cementitious material), and optionally one or more additives and/or weighting agents or weight-reducing agents, to form a wellbore servicing fluid (e.g., cementitious fluid) at a location proximate a wellsite. The wellsite can be an offshore oil and gas platform and/or a floating vessel. Block503includes placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation, for example as a primary or secondary cementing operation. The wellbore can be an offshore wellbore. Block504includes allowing at least a portion of the wellbore servicing fluid to set. At least a portion of block501, block502, and block503can comprise a continuous process as disclosed herein. FIG.6illustrates a method600in accordance with the present disclosure. Block601includes forming a suspension composition of the type disclosed herein, either at (e.g., proximate) a wellsite or remote from the wellsite and transported to the wellsite. The forming can comprise contacting a particulate material (e.g., a crosslinked guar), an organic carrier (e.g., MEG), a suspension viscosifier, and optionally water, a biocide, or both. Block602includes contacting the suspension composition, water, and optional additives of the type disclosed herein to form a wellbore servicing fluid (e.g., spacer fluid) at a location proximate a wellsite. The wellsite can be an offshore oil and gas platform and/or a floating vessel. Block603includes placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation, for example to separate a drilling fluid from a cementitious fluid. The wellbore can be an offshore wellbore. At least a portion of block601, block602, and block603can comprise a continuous process as disclosed herein. Referring toFIG.7, in embodiments, block602further comprises block701and block702. Block701includes contacting the suspension composition and water to form a mixture. Block702is optional and includes adding one or more additives of the type disclosed herein into the mixture to form a wellbore servicing fluid (e.g., spacer fluid). FIG.8illustrates a method800in accordance with the present disclosure. Block801includes forming a suspension composition of the type disclosed herein, either at (e.g., proximate) a wellsite or remote from the wellsite and transported to the wellsite. The forming can comprise contacting a particulate material (e.g., a crosslinked guar), an organic carrier (e.g., MEG), a suspension viscosifier, and optionally water, a biocide, or both. Block802includes contacting the suspension composition, water, and optionally one or more additives to form a wellbore servicing fluid (e.g., spacer fluid) at a location proximate a wellsite. The wellsite can be an offshore oil and gas platform and/or a floating vessel. Block803includes placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation, for example to separate a drilling fluid from a cementitious fluid. The wellbore can be an offshore wellbore. At least a portion of block801, block802, and block803can comprise a continuous process as disclosed herein. Various benefits may be realized by utilization of the presently disclosed methods and compositions. For example, the wellbore servicing compositions (e.g., cementitious fluid; spacer fluid) of the present disclosure may exclude a biocide resulting in a reduced risk to organisms in surrounding environment. In addition, components of the wellbore servicing fluid disclosed herein have been deemed environment-friendly and on the PLONOR list, thus the wellbore servicing fluid can be used in offshore areas with relatively strict environmental protection regulations. Another advantage of the present disclosure is that the suspension composition used in the disclosed compositions and methods is easier to handle and allows improved accuracy with regard to metering an amount to add to a system, compared with a dry powder suspending agent. For example, some fluid preparation systems that cannot process a dry powder suspending agent due to limited equipment/tools (e.g., equipment/tools at an offshore platform) can process the suspension composition. Also, the suspension composition can be stable for more than 28 days which allows a sufficient time for transportation and storage. EXAMPLES The embodiments having been generally described, the following examples are given as particular embodiments of the disclosure and to demonstrate the practice and advantages thereof. It is understood that the examples are given by way of illustration and are not intended to limit the specification or the claims in any manner. Example 1 A formulation of a suspension composition of the type in the present disclosure for 1000 gram fluid is listed in Table 1 below. The suspension composition was prepared and used in Examples 1 and 2. TABLE 1Suspension CompositionComponentAmount (g)Crosslinked guar gum100.00Monoethylene glycol (MEG)898.80Suspension Viscosifier1.20 The suspension composition was firstly used in stability tests. The suspension composition was kept static in a standing 25 ml measuring cylinder to observe mixture stability. After 21 days from preparation, density of the suspension composition was checked from top, middle and bottom portion of the suspension composition and shown in Table 2. TABLE 2Density across different portionsDensitySpecificSection(lbm/gal)gravity (SG)Top portion9.561.146Middle portion9.551.144Bottom portion9.561.146 As shown inFIG.9, after 28 days from preparation, there was no visible separation of the suspension composition and the suspension composition was pourable. The results show that suspension composition was stable and uniformly dispersed throughout the suspension composition. Physical properties were measured for the suspension composition and shown in Table 3 below. TABLE 3Physical propertiesFormLiquidAppearanceYellow fluidwt. % Solids10pH (1% suspension7-8composition in water)Brookfield viscosity (cP)*100-400SG1.145Density (lbm/gal)9.555*B1, 75° F., 100 rpm Example 2 Wellbore servicing fluids were prepared using a dry powder suspending agent or the suspension composition in Example 1. Test conditions and formulas of the wellbore servicing fluids are listed in Tables 4 and 5. The amounts of the cement blend composition are based on the total weight of the cement blend. The amount of the dry powder suspending agent is based on the total weight of the cement blend, while the dry powder suspending agent is not a part of the cement blend. Both of the wellbore servicing fluids had a density of 14.60 lbm/gal and a specific gravity of 1.75. The amount of the dry powder suspending agent in wellbore servicing fluid 1 (WSF1) was 1.3 g per 600 ml WSF1, which was equivalent to the amount of the crosslinked guar gum in wellbore servicing fluid 2 (WSF2). TABLE 4Test conditionsBottomhole circulating129° F.temperature (BHCT)Bottomhole static168° F.temperature (BHST)Heating Time60 minPressure5000 psi TABLE 5Wellbore servicing fluids, 14.6 lbm/galMixingDescriptionUnitWSF1WSF2procedureCement Blend CompositionCementitious materialwt. %98.0498.04PBExpansion agentwt. %1.961.96PBOther MaterialsDry powder suspending%0.20—PHagentBWOBSuspension compositionL/100 kg—1.76PHDefoamerL/100 kg0.090.09PHRetarderL/100 kg1.001.00PHFluid loss control agentL/100 kg7.707.70PHFree fluid control additiveL/100 kg3.603.60PHWaterL/100 kg48.6747.30BWOB: By Weight of Cement BlendPB: Pre-blend (added to the cementitious material as a part of the cement blend)PH: Pre-hydrate (added to water before adding the cement blend) Table 6 below shows 24 hr sonic compressive strength is lower in WSF2 compared to WSF1, however other properties are comparable. TABLE 6Performance comparisonPerformance TestsWSF1WSF2Mixability rating (0-5), 0 is not mixable44Free Fluid, 45 degree inclination00angle (%)API Fluid loss (ml/30 min)4438API Static Gel Strength (10 sec/10 min)1/92/16Thickening Time, 70 Bc (hh:mm)07:5007:0050 psi UCA Compressive Strength10:1510:26(hh:mm)500 psi UCA Compressive Strength13:4015:05(hh:mm)24 hr UCA Compressive Strength (psig)1253956 Table 7 shows that the rheology data measured by a Fann® Model 35 viscometer for WSF 1 and WSF 2 are comparable. TABLE 7FANN ® 35 Rheology Data68° F.129º F.190º F.RPMWSF1WSF2WSF1WSF2WSF1WSF231123456223557308711141622601313192326351002121283336482003839485560763005355667580102 Further, WSF1 and WSF2 were cured at 168° F./5.000 psig for 7 days and then tested for mechanical properties. The results are in Table 8 below. TABLE 8Mechanical propertiesTestsWSF1WSF2Crush Compressive Strength (psig)35823926Std. Dev. (psig)7643Young's Modulus (Mpsig)0.8900.954Std. Dev. (Mpsig)0.0140.015Brazilian Tensile Strength (psig)464472Std. Dev. (psig)1354 The experiments demonstrate the following. 7 days curing data shows there was no adverse effect of the use of the suspension composition on mechanical properties of set cement. UCA Compressive Strength shows a slight delay in strength development for WSF2. Regarding to other slurry properties such as mixability, free fluid, rheology, gel strength, and fluid loss, there was no adverse effect of the use of the suspension composition by comparing WSF1 and WSF2. Example 3 A formulation of a suspension composition for 1235 gram fluid is listed in Table 9 below, and the suspension composition was prepared and used in Examples 3 and 4. The preparation procedures included: taking a 2-liter clean acrylic wearing blender, adding 1 liter of monoethylene glycol into the blender, starting mixing at 1000 rpm and adding 1.5 g of suspension viscosifier, continuing stirring for 10 minutes to get a clear solution, adding 123.5 g of crosslinked guar gum under stirring and continuing mixing at 1500 rpm for 15 min. and storing the produced suspension composition in a closed container. TABLE 9Suspension CompositionComponentgmmlCrosslinked guar gum123.5080.72Monoethylene glycol (MEG)1110.001000.00Suspension Viscosifier1.501.02 Physical properties were measured for the suspension composition and shown in Table 10 below. TABLE 10Physical propertiesFormLiquidAppearanceYellow fluidOdorSmells like the crosslinkedguar gum, no specific solvent odorwt. % Solids10-50Solubility in waterMisciblepH (1% suspension7-8composition in water)Brookfield viscosity100-600(B1, 75° F.,100 rpm) (cP)SG1.1-1.3Density (lbm/gal)9-11Flash Point>230° F. (110° C.)Freezing pointAbout 10° F. (−12° C.)Boiling pointAbout 387° F. (197° C.) Example 4 Wellbore servicing fluids were prepared using a dry powder suspending agent or the suspension composition in Example 3. Formulas of the wellbore servicing fluids are listed in Table 11. The amounts of the cement blend composition are based on the total weight of the cement blend. The amounts of the weighting agent and the dry powder suspending agent are based on the total weight of the cement blend while the weighting agent and the dry powder suspending agent are not a part of the cement blend. Both of the wellbore servicing fluids have a density of 17.53 lbm/gal and a specific gravity of 2.1. The amount of the dry powder suspending agent in wellbore servicing fluid 3 (WSF3) is equivalent to the amount of the crosslinked guar gum in wellbore servicing fluid 4 (WSF4). TABLE 11Wellbore servicing fluids, 17.53 lbm/galMixingDescriptionUnitWSF3WSF4procedureCement Blend CompositionCementitious materialwt. %72.4272.42PBQuartz flourwt. %25.3425.34PBBulk flow enhancerwt. %0.070.07PBExpansion agentwt. %2.172.17PBOther MaterialsWeighting agent% BWOB20.0020.00PHDry powder% BWOB0.20—PHsuspending agentSuspensionL/100 kg—1.76PHcompositionDefoamerL/100 kg0.100.10PHRetarderL/100 kg4.004.00PHFluid loss control agentL/100 kg4.504.50PHCement dispersantL/100 kg4.004.00PHMicrosilica liquidL/100 kg8.008.00PHwaterL/100 kg21.2619.80BWOB: By Weight of Cement BlendPB: Pre-blend (added to the cement material as a part of the cement blend)PH: Pre-hydrate (added to water before adding the cement blend) WSF3 and WSF4 were cured at 212° F./5000 psi for 7 days and then tested for mechanical properties. The results in Table 12 below show there was no adverse effect of the use of the suspension composition on mechanical properties of set cement. TABLE 12Mechanical propertiesTestsWSF3WSF4Crush Compressive Strength (psig)51835446Young's Modulus (Mpsig)1.4541.383Brazilian Tensile Strength (psig)650633 Example 5 A formulation of a suspension composition of comprising clay as the particulate material was prepared as follows. Clay suspension #1 contained 36 w-t. % bentonite, 63.98 wt. % MEG, and 0.02 wt. % diutan, based on the total weight of the clay suspension. Clay suspension #2 contained 36 wt. % bentonite, 63.96 wt. % MEG, and 0.04 wt. % diutan, based on the total weight of the clay suspension. Clay suspension #1 was prepared by taking a 2-liter clean acrylic wearing blender, adding 1 liter of monoethylene glycol into the blender, starting mixing at 1,000 rpm and adding 0.37 g of diutan, continuing stirring for 10 minutes to get a clear solution, adding 627 g of bentonite under stirring, and continuing mixing at 1,000 rpm for 10 min, followed by storing the produced suspension composition in a closed container. Clay suspension #2 was prepared by taking a 2-liter clean acrylic wearing blender, adding 1 liter of monoethylene glycol into the blender, starting mixing at 1,000 rpm and adding 0.74 g of diutan, continuing stirring for 10 minutes to get a clear solution, adding 627 g of bentonite under stirring, and continuing mixing at 1000 rpm for 10 min, followed by storing the produced suspension composition in a closed container. Rheology data were measured by a Fann® Model 35 viscometer for the clay suspensions #1 and #2, 10 weeks after preparing the suspensions, and the data are displayed in Tables 13 and 14, respectively. TABLE 133 rpm6 rpm30 rpm60 rpm100 rpm200 rpm300 rpm152159101153269>300 TABLE 143 rpm6 rpm30 rpm60 rpm100 rpm200 rpm300 rpm283998154224>300>300 After 10 weeks from preparation, there was no visible separation of the suspension compositions and the suspension compositions remained pourable. The data in Tables 13 and 14 show that the suspension compositions were stable and the bentonite was uniformly dispersed throughout the suspension composition. These clay-based suspension composition can be used for suspending particles in a cement slurry (e.g., cementitious fluid). Example 6 A formulation of a suspension composition as disclosed herein was prepared as follows. The suspension composition contained 20 wt. % clay (e.g., a specially formulated clay which imparts thixotropic properties to a cement slurry, such as a cementitious fluid) and 80 wt. % MEG, based on the total weight of the suspension composition. The suspension composition was prepared by taking a 2-liter clean acrylic wearing blender, adding 1 liter of monoethylene glycol into the blender, starting mixing at 1,000 rpm and adding 280 g of clay, and continuing stirring for 5 minutes, followed by storing the produced suspension composition in a closed container. Rheology data were measured by a Brookfield viscometer for the suspension composition, and the viscosity was 370 cP when measured after 2 days at 22° C., 100 rpm/170 l/s. After preparation, there was no visible separation of the suspension composition and the suspension composition was pourable. The results show that suspension composition was stable and the clay was uniformly dispersed throughout the suspension composition. This clay-based suspension composition can be used for suspending particles in a cement slurry (e.g., cementitious fluid). Example 7 A formulation of a suspension composition of comprising magnesium oxide as the particulate material was prepared as follows. The composition of the suspension compositions, along with the rheology data are displayed in Table 15. Compositions S #1 and S #2 in Table 15 do not contain magnesium oxide and are used as controls (e.g., baseline points). The suspension compositions were prepared by taking a 0.5-liter clean glass blender, adding 4 times the mass fraction (in grams) of monoethylene glycol into the blender, starting mixing at 1,000 rpm and adding the corresponding amount of diutan (per Table 15), continuing stirring for 10 minutes to get a clear solution, adding magnesium oxide under stirring, and continuing mixing at 1.000 rpm for 10 min. followed by storing the produced suspension composition in a closed container. TABLE 15EthyleneFreeFreeBob Deflection (Degrees) at rotor speedGlycolDiutanMagnesiumFluidFluid300200100603063(mf)(mf)Oxide (mf)(24 h)(21 d)rpmrpmrpmrpmrpmrpmrpmS#1100.00%0.00%0.00%19.212.46.53.71.2S#299.96%0.04%0.00%30.823.113.08.45.11.00.6S#343.46%0.05%56.49%1.00%1.00%219.782.159.4S#449.98%0.05%49.98%10.00%20.00%277.6180.199.729.020.8S#554.98%0.05%44.97%15.00%25.00%270.6190.999.862.634.310.97.7S#654.96%0.09%44.95%10.00%20.00%259.1170.4100.733.322.3S#759.94%0.02%40.04%162.5111.251.931.916.74.72.7mf = mass fraction The data in Table 15 indicate that the free fluid increases as the yield point decreases but more importantly, there are no solids settling out. The very high rheologies (such as for composition S #3) also develop very strong static gels. Without being limited by theory, it appears that the majority of the free fluid is due to varying amounts of syneresis. The data in Table 15 indicate that one can modify the rheological properties of the fluid via adjustment of the suspending aid to prevent sedimentation. Further, and without being limited by theory, the suspension composition is viscoelastic. ADDITIONAL DISCLOSURE Embodiment A: A method comprising contacting a suspension composition, water, and a cement blend to form a wellbore servicing fluid at a location proximate a wellsite, wherein the suspension composition comprises a crosslinked guar, monoethylene glycol (MEG), and a suspension viscosifier; placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation; and allowing the wellbore servicing fluid to set. Embodiment B: The method of Embodiment A, wherein the wellsite comprises an offshore platform, a floating vessel, or combinations thereof; and wherein the wellbore is offshore. Embodiment C: The method of any one of Embodiments A and B, wherein the contacting comprises contacting the suspension composition with water to form a mixture; and contacting the mixture with the cement blend to form the wellbore servicing fluid. Embodiment D: The method of Embodiment C, wherein contacting the suspension composition with water comprises conveying the suspension composition via a suspension flow line into the water in a water flow line to form the mixture. Embodiment E: The method of any one of Embodiments A through D, further comprising adding one or more additives to the wellbore servicing fluid prior to placing the wellbore servicing fluid in the wellbore. Embodiment F: The method of Embodiment E, wherein the one or more additives are added to the mixture prior to contacting the mixture with the cement blend. Embodiment G: The method of any one of Embodiments E and F, wherein adding one or more additives to the mixture comprises conveying the one or more additives via one or more additive flow lines into the mixture in a mixture flow line. Embodiment H: The method of any one of Embodiments A through G, further comprising adding a weighting agent or a weight-reducing agent to the wellbore servicing fluid prior to placing the wellbore servicing fluid in the wellbore. Embodiment I: The method of Embodiment H, wherein the weighting agent or the weight-reducing agent is added to the mixture prior to or concurrent with contacting the mixture with the cement blend. Embodiment J: The method of any one of Embodiments A through I, wherein at least a portion of contacting the suspension composition, water, and the cement blend comprises a continuous process. Embodiment K: The method of any one of Embodiments A through J, wherein the crosslinked guar is present in the suspension composition in an amount of from about 1 wt. % to about 50 wt. %, based on a total weight of the suspension composition. Embodiment L: The method of any one of Embodiments A through K, wherein the MEG is present in the suspension composition in an amount of from about 49 wt. % to about 98.99 wt. %, based on a total weight of the suspension composition. Embodiment M: The method of any one of Embodiments A through L, wherein the suspension viscosifier comprises Guar gum, Xanthan gum, Welan gum, Diutan, hydroxyethyl cellulose (HEC), modified cellulose and derivatives thereof, diatomaceous earth, starch, modified and/or crosslinked starch, viscoelastic surfactants (VES), precipitated silica, or combinations thereof. Embodiment N: The method of any one of Embodiments A through M, wherein the suspension viscosifier is present in the suspension composition in an amount of from about 0.01 wt. % to about 20 wt. %, based on a total weight of the suspension composition. Embodiment O: The method of any one of Embodiments A through N, wherein the suspension composition further comprises water, a biocide, or combinations thereof. Embodiment P: The method of Embodiment O, wherein the water is selected from a group consisting of freshwater, saltwater, brine, seawater, and combinations thereof. Embodiment Q: The method of any one of Embodiments O and P, wherein the water is present in the suspension composition in an amount of from about 0 wt. % to about 30 wt. %, based on a total weight of the suspension composition. Embodiment R: The method of any one of Embodiments O through Q, wherein the biocide comprises 3,3′-methylenebis[5-methyloxazolidine]. Embodiment S: The method of any one of Embodiments O through R, wherein the biocide is present in the suspension composition in an amount of from about 0 wt. % to about 1 wt. %, based on a total weight of the suspension composition. Embodiment T: The method of any one of Embodiments A through S, wherein the suspension composition is present in the wellbore servicing fluid in an amount of from about 0.1 wt. % to about 20 wt. %, based on a total weight of the wellbore servicing fluid. Embodiment U: The method of any one of Embodiments A through T, wherein the suspension composition has a density of from about 9 pounds per gallon (ppg) to about 12 ppg. Embodiment V: The method of any one of Embodiments A through U, wherein the suspension composition has a specific gravity of from about 1.1 to about 1.4. Embodiment W: The method of any one of Embodiments A through V, wherein the suspension composition has a pH in a range of from about 4 to about 12, when measured for 1 vol. % dilution of the suspension composition in water. Embodiment X: The method of any one of Embodiments A through W, wherein the suspension composition has a Brookfield viscosity of from about 50 cP to about 600 cP at 75° F. and 100 rpm. Embodiment Y: The method of any one of Embodiments A through X, wherein the suspension composition has a flash point of equal to or greater than about 230° F. Embodiment Z: The method of any one of Embodiments A through Y, wherein the suspension composition has a freezing point of from about 8° F. to about 24° F. Embodiment AA: The method of any one of Embodiments A through Z, wherein the suspension composition has a boiling point of from about 210° F. to about 410° F. Embodiment BB: The method of any one of Embodiments A through AA, wherein the cement blend comprises a cementitious material. Embodiment CC: The method of Embodiment BB, wherein the cementitious material comprises Portland cement, pozzolana cement, gypsum cement, shale cement, acid cement, base cement, phosphate cement, high alumina content cement, slag cement, silica cement, high alkalinity cement, magnesia cement, lime, or combinations thereof. Embodiment DD: The method of any one of Embodiments BB and CC, wherein the cementitious material is present in the cement blend in an amount of from about 1% BWOB (by weight of blend) to about 100% BWOB, based on a total weight of the cement blend. Embodiment EE: The method of any one of Embodiments BB through DD, wherein the cement blend further comprises an expansion agent. Embodiment FF: The method of Embodiment EE, wherein the expansion agent comprises metal powders, aluminum powder, a gypsum blend, alkali metal oxides, alkaline earth metal oxides, magnesium oxide, deadburned magnesium oxide, lightly burned magnesium oxide, hard burned magnesium oxide, or combinations thereof. Embodiment GG: The method of any one of Embodiments EE and FF, wherein the expansion agent is present in the cement blend in an amount of from about 1% BWOB to about 10% BWOB, based on a total weight of the cement blend. Embodiment HH: The method of any one of Embodiments EE through GG, wherein the method further comprises dry mixing the cementitious material and the expansion agent to form the cement blend prior to contacting the cement blend with the water and the suspension composition. Embodiment II: The method of any one of Embodiments BB through HH, wherein the cement blend further comprises one or more cement blend additives. Embodiment JJ: The method of Embodiment II, wherein the one or more cement blend additives comprise quartz flour, bulk flow enhancer, amorphous silica, siliceous material, fly ash, or combinations thereof. Embodiment KK: The method of any one of Embodiments II and JJ, wherein the one or more cement blend additives are present in the cement blend in an amount of from about 5% BWOB to about 95% BWOB, based on a total weight of the cement blend. Embodiment LL: The method of any one of Embodiments II through KK, wherein the method further comprises dry mixing the cementitious material and the one or more cement blend additives to form the cement blend prior to contacting the cement blend with the water and the suspension composition. Embodiment MM: The method of any one of Embodiments A through LL, wherein the cement blend is present in the wellbore servicing fluid in an amount ranging from about 20 wt. % to about 90 wt. %, based on a total weight of the wellbore servicing fluid. Embodiment NN: The method of any one of Embodiments H1 through MM, wherein the weighting agent or the weight-reducing agent is present in the wellbore servicing fluid in an amount of from about 1% BWOB to about 200% BWOB, based on a total weight of the cement blend. Embodiment OO: The method of any one of Embodiments E through NN, wherein the one or more additives comprise a defoamer, a cement retarder, a cement dispersant, a fluid loss control additive, a fume silica, a free fluid control additive, a viscosifying agent, an acid, a base, an emulsifier, a salt, a corrosion inhibitor, a mutual solvent, a conventional breaking agent, a relative permeability modifier, lime, a gelling agent, a crosslinker, a flocculant, a water softener, a proppant, an oxidation inhibitor, a thinner, a scavenger, a gas scavenger, a lubricant, a friction reducer, a bridging agent, a vitrified shale, a thixotropic agent, a surfactant, a scale inhibitor, a clay, a clay control agent, a clay stabilizer, a silicate-control agent, a biostatic agent, a storage stabilizer, a filtration control additive, a foaming agent, a foam stabilizer, latex emulsions, a formation conditioning agent, elastomers, gas/fluid absorbing materials, resins, superabsorbers, mechanical property modifying additives, inert particulates, and the like, or combinations thereof. Embodiment PP: The method of any one of Embodiments E through 00, wherein the one or more additives are present in the wellbore servicing fluid in a total amount of from about 0.1 L/100 kg to about 50 L/100 kg, based on a total weight of the cement blend. Embodiment QQ: The method of any one of Embodiments E through PP, wherein the one or more additives are present in the wellbore servicing fluid in a total amount of from about 0.05% BWOB to about 100% BWOB, based on a total weight of the cement blend. Embodiment RR: The method of any one of Embodiments A through QQ, wherein the water is selected from a group consisting of freshwater, saltwater, brine, seawater, and combinations thereof. Embodiment SS: The method of any one of Embodiments A through RR, wherein the water is present in the wellbore servicing fluid in an amount of from about 10 L/100 kg to about 400 L/100 kg, based on a total weight of the cement blend. Embodiment TT: The method of any one of Embodiments A through SS, wherein components of the wellbore servicing fluid are PLONOR (Pose Little or No Risk to the Environment) materials. Embodiment UU: The method of any one of Embodiments A through TT, wherein the wellbore servicing fluid has a density of from about 9 pounds per gallon (ppg) to about 26 ppg. Embodiment VV: The method of any one of Embodiments A through UU, wherein the wellbore servicing fluid has a specific gravity of from about 1.1 to about 2.5. Embodiment WW: The method of any one of Embodiments A through VV, wherein the wellbore servicing fluid has a mixability rating of from about 3 to about 5. Embodiment XX: The method of any one of Embodiments A through WW, wherein the wellbore servicing fluid has a fluid loss of from about 10 ml per 30 minutes to about 250 ml per 30 minutes on 325 mesh screen at about 129° F. and about 1,000 psig differential pressure, when measured in accordance with a test standard API-RP-10B-2. Embodiment YY: The method of any one of Embodiments A through XX, wherein the wellbore servicing fluid has a 10-second static gel strength of from about 1 to about 50, and a 10-minute static gel strength of from about 1 to about 300, at about 129° F., when measured in accordance with a test standard API-RP-10B-2. Embodiment ZZ: The method of any one of Embodiments A through YY, wherein the wellbore servicing fluid has a thickening time of from about 3 hours to about 24 hours at about 129° F. and about 5,000 psig, when measured in accordance with a test standard API-RP-10B-2. Embodiment AAA: The method of any one of Embodiments A through ZZ, wherein the wellbore servicing fluid has a 50 psi UCA compressive strength of from about 1 hour to about 48 hours, a 500 psi UCA compressive strength of from about 2 hours to about 72 hours, and a 24 hr UCA compressive strength of from about 50 psig to about 10.000 psig, when measured at about 168° F. and about 5.000 psi in accordance with a test standard API-RP-10B-2. Embodiment BBB: The method of any one of Embodiments A through AAA, wherein the wellbore has a Bottomhole Circulation Temperature (BHCT) of from about 70° F. to about 400° F. Embodiment CCC: The method of any one of Embodiments A through BBB, wherein the wellbore has a Bottomhole Static Temperature (BHST) of from about 100° F. to about 400° F. Embodiment DDD: The method of any one of Embodiments A through CCC, wherein a cement cured from the wellbore servicing fluid has a crush compressive strength of from about 500 psig to about 12,000 psig. Embodiment EEE: The method of any one of Embodiments A through DDD, wherein a cement cured from the wellbore servicing fluid has a Young's Modulus of from about 0.3 Mpsig to about 3 Mpsig. Embodiment FFF: The method of any one of Embodiments A through EEE, wherein a cement cured from the wellbore servicing fluid has a Brazilian Tensile Strength of from about 50 psig to about 1.600 psig. Embodiment GGG: A method comprising forming a suspension composition comprising a crosslinked guar, monoethylene glycol (MEG), and a suspension viscosifier; contacting the suspension composition, water, a cement blend comprising a cementitious material, and optionally one or more additives, weighting agents or weight-reducing agents to form a wellbore servicing fluid at a location proximate a wellsite; placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation; and allowing the wellbore servicing fluid to set. Embodiment HHH: A method comprising forming a suspension composition comprising a crosslinked guar, monoethylene glycol (MEG), a suspension viscosifier, and optionally water, a biocide, or both; contacting the suspension composition with water to form a mixture at a location proximate a wellsite; contacting the mixture with a cement blend and optionally one or more additives, weighting agents or weight-reducing agents to form a wellbore servicing fluid; placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation; and allowing the wellbore servicing fluid to set. Embodiment III: A method comprising forming a suspension composition comprising a crosslinked guar, monoethylene glycol (MEG), a suspension viscosifier, and brine; contacting the suspension composition with water to form a mixture at a location proximate a wellsite; contacting the mixture with a cement blend and optionally one or more additives, weighting agents or weight-reducing agents to form a wellbore servicing fluid; placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation; and allowing the wellbore servicing fluid to set. Embodiment JJJ: The method of any one of Embodiments A through Ill, wherein the method further comprises adding a gas to the wellbore servicing fluid, prior to placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation. Embodiment KKK: The method of Embodiment JJJ, wherein a target density of the wellbore servicing fluid is from about 5 pounds per gallon (ppg) to about 16 ppg. Embodiment LLL: The method of any one of Embodiments JJJ and KKK, wherein the gas is present in the wellbore servicing fluid placed in the wellbore in an amount of from about 10 vol. % to about 30 vol. %, based on a total volume of the wellbore servicing fluid placed in the wellbore. Embodiment MMM: The method of any one of Embodiments JJJ through LLL, wherein the gas comprises nitrogen. Embodiment NNN: A method comprising (a) contacting a crosslinked guar, monoethylene glycol (MEG), and a suspension viscosifier to form a suspension composition; (b) conveying the suspension composition via a suspension flow line into water in a water flow line at a location proximate an offshore platform to form a diluted suspension; (c) conveying one or more additives via one or more additive flow lines into the diluted suspension in a diluted suspension line to form a mixture; (d) placing the mixture in a container; (e) adding a cement blend and optionally a weighting agent or a weight-reducing agent into the container to form a slurry; (f) blending the slurry to form a wellbore servicing fluid; (g) placing the wellbore servicing fluid in an offshore wellbore penetrating a subterranean formation; and (h) allowing the wellbore servicing fluid to set. Embodiment OOO: A suspension composition comprising a crosslinked guar, monoethylene glycol (MEG), and a suspension viscosifier. Embodiment PPP: The suspension composition of Embodiment OOO further comprising water, a biocide, or combinations thereof. Embodiment QQQ: A wellbore servicing composition comprising the suspension composition of any one of Embodiments 000 and PPP, water, a cement blend, and optionally one or more additives, weighting agents or weight-reducing agents. Embodiment A1: A method comprising (a) contacting a suspension composition, water, and optionally one or more additives to form a wellbore servicing fluid at a location proximate a wellsite; wherein the suspension composition comprises a particulate material, an organic carrier fluid, and a suspension viscosifier; and (b) placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation. Embodiment B1: The method of Embodiment A1, wherein the wellsite comprises an offshore platform, a floating vessel, or combinations thereof; and wherein the wellbore is offshore. Embodiment C1: The method of any one of Embodiments A1 and B1, wherein the particulate material comprises a water-interactive material and/or a water-insoluble material; wherein the water-interactive material comprises an expansion agent, alkali metal oxides, alkaline earth metal oxides, magnesium oxide, lightly burned magnesium oxide, hard burned magnesium oxide, deadburned magnesium oxide, metal powders, aluminum powder, a gypsum blend; a viscosifying clay, bentonite, sepiolite, hectorite; a delayed viscosifier, crosslinked guar, crosslinked vinyl alcohols, crosslinked acrylamide polymers; a fluid loss agent, an acrylic-based polymer, a polyacrylate, an acrylamide-based polymer, a polyacrylamide, an acrylamide copolymer, an acrylic acid copolymer, a polymer of acrylamide-tertiary-butyl sulfonate (ATBS), an ATBS/acrylamide copolymer, 2-acrylamido-2-methylpropane sulfonic acid/acrylamide copolymers, 2-acrylamido-2-methylpropane sulfonic acid/N,N-dimethyl-acrylamide copolymers, vinylpyrrolidone/2-acrylamido-2-methylpropane sulfonic acid/acrylamide terpolymers, acrylamide/t-butyl acrylate/N-vinylpyrrolidone terpolymers, acrylamide/t-butyl acrylate/2-acrylamido-2-methylpropane sulfonic acid terpolymers, 2-acrylamido-2-methylpropane sulfonic acid/N-N-dimethylacrylamide/acrylamide terpolymers, acrylamide/t-butyl acrylate/N-vinylpyrrolidone/2-acrylamido-2-methylpropane sulfonic acid tetrapolymers, acrylamide/t-butyl acrylate copolymers, poly(2-hydroxyethyl methacrylate), poly(2-hydroxypropyl methacrylate), derivatives thereof; or combinations thereof; and wherein the water-insoluble material comprises pozzolana cement; sand; a weighting agent, an iron oxide, hematite, a manganese oxide, hausmannite, a titanium-iron oxide, ilmenite; a fiber, a carbon fiber, an acrylonitrile fiber, a polypropylene fiber, a glass fiber, a rubber fiber; a rubber particle; a hollow glass sphere; a hollow pozzolanic sphere; a glass bubble; a glass ball; a ceramic ball; graphite; pozzolan; pumice; trass; clay; calcined clay; silica, fume silica, amorphous silica, micro-sized silica, nano-sized silica; or combinations thereof. Embodiment D1: The method of any one of Embodiments A1 through C1, wherein the particulate material is present in the suspension composition in an amount of from about 1 wt. % to about 80 wt. %, based on a total weight of the suspension composition. Embodiment E1: The method of any one of Embodiments A1 through D1, wherein the organic carrier fluid comprises a glycol and/or a glycol ether; wherein the glycol comprises monoethylene glycol, propylene glycol, butylene glycol, or combinations thereof; and wherein the glycol ether comprises ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, butylene glycol monomethyl ether, butylene glycol monoethyl ether, or combinations thereof. Embodiment F1: The method of any one of Embodiments A1 through E1, wherein the organic carrier fluid is present in the suspension composition in an amount of from about 20 wt. % to about 98.99 wt. %, based on a total weight of the suspension composition. Embodiment G1: The method of any one of Embodiments A1 through F1, wherein the suspension viscosifier comprises Guar gum, Xanthan gum, Welan gum, Diutan, hydroxyethyl cellulose (HEC), diatomaceous earth, starch, modified and/or crosslinked starch, modified cellulose, viscoelastic surfactants (VES), precipitated silica, derivatives thereof, or combinations thereof. Embodiment H1: The method of any one of Embodiments A1 through G1, wherein the suspension viscosifier is present in the suspension composition in an amount of from about 0.01 wt. % to about 20 wt. %, based on a total weight of the suspension composition. Embodiment I1: The method of any one of Embodiments A1 through H1, wherein the suspension composition is present in the wellbore servicing fluid in an amount of from about 0.1 wt. % to about 60 wt. %, based on a total weight of the wellbore servicing fluid. Embodiment J1: The method of any one of Embodiments A1 through I1, wherein the suspension composition has (A1) a density of from about 4 pounds per gallon (ppg) to about 25 ppg; (A2) a specific gravity of from about 0.5 to about 3; (A3) a pH in a range of from about 4 to about 12, when measured for 1 vol. % dilution of the suspension composition in water; (A4) a Brookfield viscosity of from about 50 cP to about 600 cP at 75° F. and 100 rpm; (A5) a flash point of equal to or greater than about 230° F.; (A6) a freezing point of from about 8° F. to about 24° F.; (A7) a boiling point of from about 210° F. to about 410° F.; or (A8) any combination of (A1)-(A7). Embodiment K1: The method of any one of Embodiments A1 through J1, wherein the wellbore servicing fluid is a cementitious fluid; wherein the contacting comprises (i) contacting the suspension composition with water to form a mixture, and (ii) contacting the mixture with a cement blend to form the wellbore servicing fluid; wherein the one or more additives are optionally added to the mixture prior to contacting the mixture with the cement blend; and wherein the wellbore servicing fluid is allowed to set. Embodiment L1: The method of Embodiment K1 further comprising adding a weighting agent or a weight-reducing agent to the wellbore servicing fluid prior to placing the wellbore servicing fluid in the wellbore; wherein the weighting agent or the weight-reducing agent is added to the mixture prior to or concurrent with contacting the mixture with the cement blend. Embodiment M1: The method of any one of Embodiments K1 and L1 further comprising adding a weighting agent or a weight-reducing agent to the wellbore servicing fluid prior to placing the wellbore servicing fluid in the wellbore; wherein the weighting agent or the weight-reducing agent is added to the mixture prior to or concurrent with contacting the mixture with the cement blend. Embodiment N1: The method of any one of Embodiments K1 through M1, wherein the cement blend is present in the wellbore servicing fluid in an amount ranging from about 20 wt. % to about 90 wt. %, based on a total weight of the wellbore servicing fluid; and wherein the weighting agent or the weight-reducing agent is present in the wellbore servicing fluid in an amount of from about 1% by weight of blend (BWOB) to about 200% BWOB, based on a total weight of the cement blend. Embodiment O1: The method of any one of Embodiments K1 through N1, wherein the wellbore servicing fluid has (B1) a density of from about 9 pounds per gallon (ppg) to about 26 ppg; (B2) a specific gravity of from about 1.1 to about 2.5; (B3) a mixability rating of from about 3 to about 5; (B4) a fluid loss of from about 10 ml per 30 minutes to about 250 nil per 30 minutes on 325 mesh screen at about 129° F. and about 1,000 psig differential pressure, when measured in accordance with a test standard API-RP-10B-2; (B5) a 10-second static gel strength of from about 1 to about 50, and a 10-minute static gel strength of from about 1 to about 300, at about 129° F. when measured in accordance with a test standard API-RP-10B-2; (B6) a thickening time of from about 3 hours to about 24 hours at about 129° F. and about 5000 psi when measured in accordance with a test standard API-RP-10B-2; (B7) a 50 psi UCA compressive strength of from about 1 hour to about 48 hours, a 500 psi UCA compressive strength of from about 2 hours to about 72 hours, and a 24 hr UCA compressive strength of from about 50 psig to about 10,000 psig, when measured at about 168° F. and about 5,000 psig in accordance with a test standard API-RP-10B-2; or (B8) any combination of (B1)-(B7). Embodiment P1: The method of any one of Embodiments K1 through O1, wherein a cement cured from the wellbore servicing fluid has (C1) a crush compressive strength of from about 500 psig to about 12,000 psig; (C2) a Young's Modulus of from about 0.3 Mpsig to about 3 Mpsig; (C3) a Brazilian Tensile Strength of from about 50 psig to about 1,600 psig; or (C4) any combination of (C1)-(C3). Embodiment Q1: The method of any one of Embodiments A1 through J1, wherein the wellbore servicing fluid is a spacer fluid; and wherein the spacer fluid has a density in a range of from about 4 pounds per gallon (ppg) to about 25 ppg. Embodiment R1: A method comprising (a) forming a suspension composition comprising a crosslinked guar, monoethylene glycol (MEG), and a suspension viscosifier; (b) contacting the suspension composition with water to form a mixture at a location proximate a wellsite; (c) contacting the mixture with a cement blend and optionally one or more additives, weighting agents or weight-reducing agents to form a wellbore servicing fluid; (d) placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation; and (e) allowing the wellbore servicing fluid to set. Embodiment S1: The method of Embodiment R1, wherein the method further comprises adding a gas to the wellbore servicing fluid, prior to placing the wellbore servicing fluid in a wellbore penetrating a subterranean formation; wherein a target density of the wellbore servicing fluid is from about 5 pounds per gallon (ppg) to about 16 ppg. Embodiment T1: The method of Embodiment SL, wherein the gas is present in the wellbore servicing fluid placed in the wellbore in an amount of from about 10 vol. % to about 30 vol. %, based on a total volume of the wellbore servicing fluid placed in the wellbore. Embodiment U1: A method comprising (a) contacting a crosslinked guar, monoethylene glycol (MEG), and a suspension viscosifier to form a suspension composition; (b) conveying the suspension composition via a suspension flow line into water in a water flow line at a location proximate an offshore platform to form a diluted suspension; (c) conveying one or more additives via one or more additive flow lines into the diluted suspension in a diluted suspension line to form a mixture; (d) placing the mixture in a container; (e) adding a cement blend and optionally a weighting agent or a weight-reducing agent into the container to form a slurry; (f) blending the slurry to form a wellbore servicing fluid; (g) placing the wellbore servicing fluid in an offshore wellbore penetrating a subterranean formation; and (h) optionally allowing the wellbore servicing fluid to set. Embodiment V1: A suspension composition comprising a particulate material, an organic carrier fluid, and a suspension viscosifier; wherein the particulate material is substantially insoluble in the organic carrier fluid: wherein the particulate material comprises a water-interactive material and/or a water-insoluble material; and wherein the organic carrier fluid comprises a glycol and/or a glycol ether. Embodiment W1: The suspension composition of Embodiment V1, wherein the particulate material comprises a crosslinked guar; and wherein the organic carrier fluid comprises monoethylene glycol (MEG). Embodiment X1: The suspension composition of any one of Embodiments V1 and W1 further comprising water, a biocide, or both water and a biocide. Embodiment Y1: The suspension composition of Embodiment X1, wherein the biocide comprises 3,3′-methylenebis[5-methyloxazolidine]. Embodiment Z1: A wellbore servicing composition comprising the suspension composition of Embodiment V1, water, a cement blend, and optionally one or more additives, weighting agents or weight-reducing agents. Embodiment Z2: A wellbore servicing composition comprising the suspension composition of Embodiment V1, water, and optionally one or more additives. While embodiments of the disclosure have been shown and described, modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the disclosure. The embodiments described herein are exemplary only, and are not intended to be limiting. Many variations and modifications of the disclosure disclosed herein are possible and are within the scope of the disclosure. Where numerical ranges or limitations are expressly stated, such express ranges or limitations should be understood to include iterative ranges or limitations of like magnitude falling within the expressly stated ranges or limitations (e.g., from about 1 to about 10 includes, 2, 3, 4, etc.; greater than 0.10 includes 0.11, 0.12, 0.13, etc.). For example, whenever a numerical range with a lower limit. RL, and an upper limit. RU, is disclosed, any number falling within the range is specifically disclosed. In particular, the following numbers within the range are specifically disclosed: R=RL+k*(RU−RL), wherein k is a variable ranging from 1 percent to 100 percent with a 1 percent increment, i.e., k is 1 percent, 2 percent, 3 percent, 4 percent, 5 percent, . . . , 50 percent, 51 percent, 52 percent, . . . , 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or 100 percent. Moreover, any numerical range defined by two R numbers as defined in the above is also specifically disclosed. When a feature is described as “optional,” both embodiments with this feature and embodiments without this feature are disclosed. Similarly, the present disclosure contemplates embodiments where this feature is required and embodiments where this feature is specifically excluded. Both alternatives are intended to be within the scope of the claim. Use of broader terms such as comprises, includes, having, etc. should be understood to provide support for narrower terms such as consisting of, consisting essentially of, comprised substantially of, etc. Accordingly, the scope of protection is not limited by the description set out above but is only limited by the claims which follow, that scope including all equivalents of the subject matter of the claims. Each and every claim is incorporated into the specification as an embodiment of the present disclosure. Thus, the claims are a further description and are an addition to the embodiments of the present disclosure. | 121,507 |
11859124 | DETAILED DESCRIPTION The present disclosure may generally relate to polymers and treatment fluids that include polymers. Provided herein are methods that may include identifying and developing an analytical relationship between polymer properties and performance. The techniques disclosed herein may include measuring physicochemical properties of a polymer and correlating the properties to desirable properties of the polymer. The techniques may be used to design and prepare a treatment fluid, for example. At least one embodiment may include measuring physicochemical properties of a polymer. The physicochemical properties may include, but are not limited to, number average number average, weight average, polydispersity index, radius of gyration Rg, wet time, rheology, degree of branching, number of carbon atoms, ratio of carbon atoms to sulfur, nitrogen, and/or oxygen, and/or fluid loss property. An example embodiment of correlating desirable properties with at least one physicochemical property will now be described for an example polymer. For a polymeric fluid loss control additive, the fluid loss performance may be correlated to the molecular weight of the polymer. However, as will be illustrated below, in addition to the number or mass average molecular weight, there is a contribution from the dispersity to the performance of the polymeric fluid loss control additive. Polymerization reactions typically generate a distribution of polymer sizes around an average value. As used herein, “polydispersity index” (PDI) refers to a measure of the distribution of molecular mass in a given polymer sample. The polydispersity index is calculated by dividing the weight average molecular weight (Mw) by the number average molecular weight (Mn). As used herein, the term “weight average molecular weight” generally refers to a molecular weight measurement that depends on the contributions of polymer molecules according to their sizes. As used herein, the term “number average molecular weight” generally refers to a molecular weight measurement that is calculated by dividing the total weight of all the polymer molecules in a sample with the total number of polymer molecules in the sample. These terms are well-known by those of ordinary skill in the art. There may exist an optimum PDI at which fluid loss performance and mixability of a polymer are balanced such that fluid loss is minimized while mixability is maximized. Another factor which may contribute to the performance of a polymer may be the degree of branching of the polymer. The degree of branching may be measured in terms of the radius of gyration of a polymer molecule, wherein the larger radius of gyration for a given molecular weight equates to a lower degree of branching. As will be illustrated below, polymeric fluid loss control additive specifications that do not include PDI and radius of gyration may pass as a given product but may have vastly different performance characteristic. To illustrate the effects of PDI and radius of gyration on polymeric fluid loss control additives, a test was prepared to compare a polymeric fluid loss control additive from different vendors, each polymeric fluid loss control additive having the same specification. Five samples were obtained from different vendors and formulated with Portland class H cement. The density of the prepared slurries was fixed at 16.2 pounds per gallon (1941 kg/m3). The concentration of the polymeric fluid loss control additive was varied from 0.2% to 0.6% BWOC (by weight of cement). The slurries were evaluated for fluid loss, rheology and for the wet (or mix) time, at room temperature. The results are shown in Table 1. The number average (Mn) and weight average (Mw) are expressed in kilo Daltons (kDa), fluid loss (FL) is expressed in mL, wet time is expressed in seconds, polydispersity index (PDI) is dimensionless, radius of gyration Rg is expressed in nano-meters, and concentration is expressed by weight of cement. Each test was performed according to API 10B-2 and manufacturer's instructions for a gel permeation chromatography and multi angle light scattering instrument. One of ordinary skill in the art should recognize the laboratory tests used. TABLE 1Polymer PropertiesSlurry Dial Reading,Fluid Loss10% BWOWWet Time (sec)(100 rpm)(mL, doubled)MnMwRg(Dial reading,0.3%0.4%0.6%0.3%0.4%0.6%0.3%0.4%0.6%Polymer(kDa)(kDa)PDI(nm)100 rpm)BWOCBWOCBWOCBWOCBWOCBWOCBWOCBWOCBWOC182322022.68150.288.518182644.062.597.572402229342428.32.60161.515515151957.072.5116.86234263962.72627.82.73181.5154.514172357.073.5119.0117542441237.63655.42.95214.4250304410052.575.5130.05430265136437982.78209.423620256957.874.8135.0703626 FIG.1is a graph of the fluid loss at 80° F. (26.67° C.) of the 0.3% BWOC samples from Table 1. It can be observed that increasing molecular weight does not necessarily lead to a decrease in fluid loss. As such, molecular weight alone is not able to explain the differences between observed fluid loss performances between the samples. A model may be used that incorporates the effects of PDI, Rg, concentration, and molecular weight. One form of the model may be expressed in Equation 1. ln(FL)=A+B*PDI+C*Rg+D[CONC]+E*Mw (1) Another form of the model may be expressed in Equation 2. FL=K*P(PDI)α*R(Rg)β*C([Conc])γ*D(Mw)δ(2) In either model from Equation 1 or Equation 2, the constants A, B, C, D, and E and α, β, γ, and δ may be determined by multivariate linear regression or any other regression technique. K, P, R, C and D represent functions. P(PDI) indicates a function of PDI. The functions may be exponential, logarithmic, trigonometric, polynomial, power law or combinations thereof. A parity plot of model 1, using the data of Table 1, is illustrated inFIG.2. The constants for the parity plot model are derived from the data of Table 1. It is observed that the higher PDI indicates that as PDI increases, the fluid loss decreases. A higher PDI indicates presence of a larger distribution of molecular weights present in the sample which may lead to better control over fluid loss. However, increasing PDI may also have a negative impact on mixing.FIG.3illustrates a plot of PDI versus wetting times from the data of Table 1. It is observed that the wet time generally increases with increasing PDI. Wet time is the time it takes for an amount of polymer to hydrate. For the particular molecular weight polymers of Table 1, the optimum PDI is observed to be about 2.7 at which wet times are minimized and PDI is maximized. The optimum PDI may be a function of mean molecular weight of the polymers, for example. For polymers with lower average molecular weight than those in Table 1, the optimum PDI may be higher, and for polymers with higher average molecular weight than those in Table 1, the optimum PDI may be lower. The models presented above may be useful to characterize a polymer and may provide insight into polymer specifications that may affect polymer performance such as PDI and radius of gyration. The models may also be used to tune a polymer for a desired performance characteristic or to design a polymer to have a desired physicochemical property. For example, the models may be used to design a polymer that has a desired wet time and/or fluid loss property. The models may be used to determine the number average number average, weight average, polydispersity index, and radius of gyration Rg that is required to achieve the desired wet time, rheology, degree of branching, number of carbon atoms, ratio of carbon atoms to sulfur, nitrogen, and/or oxygen, and/or fluid loss property. The models may also be used to design a treatment fluid with a desired property with a given polymer additive. The models may be used to determine the required concentration of the polymer to include to achieve the desired fluid loss, for example. FIG.4illustrates multiple plots of PDI, radius of gyration, concentration of polymer, and molecular weight for a set of polymeric fluid loss control additives. For the given set of materials, it is observed that the fluid loss may decrease as a function of increasing PDI, increasing molecular weight, and increasing concentration. For a given radius of gyration and PDI, and increase in molecular weight may be inversely correlated to fluid loss. As one of ordinary skill in the art will appreciate, radius of gyration and molecular weight are covariates. Increasing molecular weight without affecting radius of gyration may be accomplished by introducing branching into the polymer backbone, for example, with may lead to a decrease in fluid loss performance. The previously discussed properties and models of polymers may be beneficial when designing a cement that has a fluid loss requirement and/or a mixability requirement. As discussed above, physicochemical properties such as PDI may affect the fluid loss performance and wet time for a slurry comprising a polymer as a polymeric fluid loss control additive. A cement operator may have a plurality of polymers available to include in a cement slurry. The plurality of polymers may have varying chemistries as the plurality of polymers may be sourced from various vendors. Generally, a test slurry may be made comprising a selected polymer and the test slurry may then be tested for fluid loss and mixability. Given that there may be wide variability between polymers of the same chemistry from different manufacturers, several tests may need to be prepared before a suitably mixable and fluid loss controlled slurry is found. The trial-and-error nature of the testing may require time to complete and may be inefficient. Additionally, the resultant slurry may be complex as the trial-and-error methodology may not find the optimum PDI to wet time at a concentration. A method to design a cement slurry, or other wellbore treatment fluids comprising a polymer, may include providing a plurality of polymers, providing at least one of a required wet time, fluid rheology, and required fluid loss performance of a cement slurry comprising a polymer, correlating the fluid loss performance and/or wet time of each of the available polymers based on one or more physicochemical properties, and selecting a polymer based at least in part on the correlation. The selecting may further include selecting a concentration of the polymer. The correlation may include a model as previously described. The model may be specific to the chemistry of the polymer. In some examples, the correlation, and by extension the model, may use physicochemical parameters of the polymer inputs or as constants, for example. Cement slurries may generally include water and a cement along with a polymer. The polymer may be included as a polymeric fluid loss control additive. A cement blend may include one or more bulk dry materials of various cementitious components, which may be dry blended to form the cement dry blend prior to combination with the water. In some cases, “chemical additives” may be also dry blended with the cement blend. Alternatively, some of the chemical additives may not be combined until the blend (sometimes referred to as bulk blend) has been mixed with the water. The cement components may generally be described as alkali soluble. In one embodiment, a cement slurry may include water and a cement blend, wherein the cement blend includes hydraulic cement and two or more silica sources, such as cement kiln dust and a natural pozzolan. The cement slurries may have a density suitable for a particular application. The cement slurries may have a density in the range of about 7 pounds per gallon (“ppg”) (840 kg/m3) to about 23 ppg (2760 kg/m3). In the foamed examples, the foamed cement slurries may have a density in the range of about 7 ppg to about 15 ppg (or even lower). The water used in the cement slurries may include, for example, freshwater, saltwater (e.g., water containing one or more salts dissolved therein), brine (e.g., saturated saltwater produced from subterranean formations), seawater, or combinations thereof Generally, the water may be from any source, provided that it does not contain an excess of compounds that may undesirably affect other components in the cement slurry. The water may be included in an amount sufficient to form a pumpable slurry. The water may be included in the cement slurries in the range of about 40% to about 200% by weight of the cement slurry (“bwoc”). In some examples, the water may be included in an amount in the range of about 40% to about 150% bwoc. The cement slurry may include two or more cement components. A variety of hydraulic cements may be utilized in accordance with the present disclosure, including, but not limited to, those comprising calcium, aluminum, silicon, oxygen, iron, and/or sulfur, which set and harden by reaction with water. Suitable hydraulic cements may include Portland cements, gypsum, and high alumina content cements, among others. Portland cements that are suited for use in the present disclosure may be classified as Classes A, C, G, and H cements according to American Petroleum Institute, API Specification for Materials and Testing for Well Cements, API Specification 10, Fifth Ed., Jul. 1, 1990. In addition, in some examples, Portland cements suitable may be classified as ASTM Type I, II, III, I/II, III/V and V. Cement slurries that may be considered “low Portland” may be designed by use of the techniques disclosed herein. Where present, the hydraulic cement generally may be included in the cement slurries in an amount sufficient to provide the desired compressive strength and/or density. The hydraulic cement may be present in the cement slurries in any suitable concentration, including in an amount in the range of about 0% to about 99% bwoc. The hydraulic cement may be present in an amount ranging between any of and/or including any of about 1%, about 5%, about 10%, about 20%, about 40%, about 60%, about 80%, or about 90% bwoc. The cement component may be considered “low Portland” in that the Portland cement (where used) may be present in the cement slurry in an amount of about 40% or less bwoc and, alternatively, about 10% or less. Cement slurries may also be designed that are free (or essentially free) of Portland cement. In addition to Portland cement, additional cement components may be used that can be considered alkali soluble. A cement component is considered alkali soluble where it is at least partially soluble in an aqueous solution of pH 7.0 or greater. Certain of the alkali soluble cement components may include a geopolymer cement, which may include an aluminosilicate source, a metal silicate source, and an activator. The geopolymer cement may react to form a geopolymer. A geopolymer is an inorganic polymer that forms long-range, covalently bonded, non-crystalline networks. Geopolymers may be formed by chemical dissolution and subsequent re-condensation of various aluminosilicates and silicates to form a 3D-network or three-dimensional mineral polymer. The activator may include, but is not limited to, metal hydroxides chloride salts such as KCl, CaCl2, NaCl, carbonates such as Na2CO3, silicates such as sodium silicate, aluminates such as sodium aluminate, and ammonium hydroxide. The aluminosilicate source may include any suitable aluminosilicate. Aluminosilicate is a mineral comprising aluminum, silicon, and oxygen, plus counter-cations. A wide variety of suitable minerals may be an aluminosilicate source in that they may include aluminosilicate minerals. Each aluminosilicate source may potentially be used in a particular case if the specific properties, such as slurry, may be known. Some minerals such as andalusite, kyanite, and sillimanite are naturally occurring aluminosilicate sources that have the same slurry, Al2SiO5, but differ in crystal structure. Each mineral andalusite, kyanite, or sillimanite may react more or less quickly and to different extents at the same temperature and pressure due to the differing crystal structures. Other suitable aluminosilicate sources may include, but are not limited to, calcined clays, partially calcined clays, kaolinite clays, lateritic clays, illite clays, natural glass, mine tailings, blast furnace slag, and coal fly ash. The metal silicate source may include any suitable metal silicate. A silicate is a compound containing an anionic silicon compound. Some examples of a silicate include the orthosilicate anion also known as silicon tetroxide anion, SiO44−as well as hexafluorosilicate [SiF6]2−. Other common silicates include cyclic and single chain silicates which may have the general formula [SiO2+n]2n−and sheet-forming silicates ([SiO2.5]−)n. Each silicate example may have one or more metal cations associated with each silicate molecule. Some suitable metal silicate sources and may include, without limitation, sodium silicate, magnesium silicate, and potassium silicate. Where present, the geopolymer cement generally may be included in the cement slurries in an amount sufficient to provide the desired compressive strength and/or. The geopolymer cement may be present in the cement slurries in any suitable concentration, including an amount in the range of about 0% to about 99% bwoc. The geopolymer cement may be present in an amount ranging between any of and/or including any of about 1%, about 5%, about 10%, about 20%, about 40%, about 60%, about 80%, or about 90% bwoc. Those of ordinary skill in the art, with the benefit of this disclosure, would be able to select an appropriate amount of geopolymer cement for a particular application. Additional cement components that are alkali soluble may include a silica source. The silica source may be any suitable material that provides silica to the cement slurry. By inclusion of the silica source, a different path may be used to arrive at a similar product as from Portland cement. A pozzolanic reaction may be induced wherein silicic acid (H4SiO4) and portlandite (Ca(OH)2react to form a cement product (calcium silicate hydrate). If other compounds, such as, aluminate, are present in the silica source, additional reactions may occur to form additional cement products, such as calcium aluminate hydrates. Calcium hydroxide necessary for the reaction may be provide from other cement components, such as Portland cement, or may be separately added to the cement slurry. Examples of suitable silica sources may include fly ash, slag, silica fume, crystalline silica, silica flour, cement kiln dust (“CKD”), natural glasses, metakaolin, diatomaceous earth, zeolite, shale, and agricultural waste ash (e.g., rice husk ash, sugar cane ash, and bagasse ash), among other. Some specific examples of the silica source will be discussed in more detail below. Where present, the silica source generally may be included in the cement slurries in an amount sufficient to provide the desired compressive strength and/or density. The silica source may be present in the cement slurries in any suitable concentration, including in an amount in the range of about 0% to about 99% bwoc. The silica source may be present in an amount ranging between any of and/or including any of about 1%, about 5%, about 10%, about 20%, about 40%, about 60%, about 80%, or about 90% bwoc. Those of ordinary skill in the art, with the benefit of this disclosure, would be able to select an appropriate amount of silica source for a particular application. An example of a suitable silica source may include fly ash. A variety of fly ash may be suitable, including fly ash classified as Class C and Class F fly ash according to American Petroleum Institute, API Specification for Materials and Testing for Well Cements, API Specification 10, Fifth Ed., Jul. 1, 1990. Class C fly ash includes both silica and lime, so it may set to form a hardened mass upon mixing with water. Class F fly ash generally does not contain a sufficient amount of lime to induce a cementitious reaction, therefore, an additional source of calcium ions is necessary for a set-delayed cement slurry comprising Class F fly ash. Where used, lime may be mixed with Class F fly ash in any suitable amount, including in an amount in the range of about 0.1% to about 100% by weight of the fly ash. In some instances, the lime may be hydrated lime. Suitable examples of fly ash include, but are not limited to, POZMIX® A cement additive, commercially available from Halliburton Energy Services, Inc., Houston, Texas. Another example of a suitable silica source may include slag. Slag is generally a by-product in the production of various metals from their corresponding ores. By way of example, the production of cast iron can produce slag as a granulated, blast furnace by-product with the slag generally comprising the oxidized impurities found in iron ore. Slag generally does not contain sufficient basic material, so slag cement may be used that further may include a base to produce a settable slurry that may react with water to set to form a hardened mass. Examples of suitable sources of bases include, but are not limited to, sodium hydroxide, sodium bicarbonate, sodium carbonate, lime, and combinations thereof. Another example of a suitable silica source may include CKD. Cement kin dust or “CKD”, as that term is used herein, refers to a partially calcined kiln feed which is removed from the gas stream and collected, for example, in a dust collector during the manufacture of cement. Usually, large quantities of CKD are collected in the production of cement that are commonly disposed of as waste. CKD is another component that may be included in examples of the cement slurries. Another example of a suitable silica source may include natural glass. Certain natural glasses can exhibit cementitious properties, in that they may set and harden in the presence of hydrated lime and water. The natural glass may also be ground, for example. Generally, the natural glass may have any particle size distribution as desired for a particular application. In certain embodiments, the natural glass may have a mean particle size in a range of from about 1 micron to about 200 microns. The mean particle size corresponds to d50 values as measured by particle size analyzers such as those manufactured by Malvern Instruments, Worcestershire, United Kingdom. One of ordinary skill in the art, with the benefit of this disclosure, would be able to select a particle size for the natural glass suitable for use for a chosen application. Another example of a suitable silica source may include metakaolin. Generally, metakaolin is a white pozzolan that may be prepared by heating kaolin clay, for example, to temperatures in the range of about 600° to about 800° C. Another example of a suitable silica source may include shale. Among other things, shale included in the cement slurries may react with excess lime to form a suitable cementing material, for example, calcium silicate hydrate. A variety of shales are suitable, including those comprising silicon, aluminum, calcium, and/or magnesium. An example of a suitable shale includes vitrified shale. Generally, the shale may have any particle size distribution as desired for a particular application. In certain embodiments, the shale may have a particle size distribution in the range of about 37 micrometers to about 4,750 micrometers. Another example of a suitable silica source may include zeolite. Zeolites generally are porous alumino-silicate minerals that may be either a natural or synthetic material. Synthetic zeolites are based on the same type of structural cell as natural zeolites, and may include aluminosilicate hydrates. As used herein, the term “zeolite” refers to all natural and synthetic forms of zeolite. Examples of zeolites may include, without limitation, mordenite, zsm-5, zeolite x, zeolite y, zeolite a, etc. Furthermore, examples comprising zeolite may include zeolite in combination with a cation such as Na+, K+, Ca2+, Mg2+, etc. Zeolites comprising cations such as sodium may also provide additional cation sources to the cement slurry as the zeolites dissolve. The cement slurries may further include hydrated lime. As used herein, the term “hydrated lime” will be understood to mean calcium hydroxide. In some examples, the hydrated lime may be provided as quicklime (calcium oxide) which hydrates when mixed with water to form the hydrated lime. The hydrated lime may be included in examples of the cement slurries, for example, to form a hydraulic slurry with the silica source. The hydrated lime may be included in any suitable concentration, including, but not limited to, in a silica source-to-hydrated-lime weight ratio of about 10:1 to about 1:1 or a ratio of about 3:1 to about 5:1. Where present, the hydrated lime may be included in the cement slurries in an amount in the range of from about 10% to about 100% by weight of the silica source, for example. The hydrated lime may be present in an amount ranging between any of and/or including any of about 10%, about 20%, about 40%, about 60%, about 80%, or about 100% by weight of the silica source. One of ordinary skill in the art, with the benefit of this disclosure, would recognize the appropriate amount of hydrated lime to include for a chosen application. The cement slurries may also include a calcium source other than hydrated lime, which may be used in addition, or in place of the hydrated lime. In general, calcium and a high pH, for example a pH of 7.0 or greater, may be needed for certain cementitious reactions to occur. A potential advantage of hydrated lime may be that calcium ions and hydroxide ions are supplied in the same molecule. In another example, the calcium source may be Ca(NO3)2or CaCl2with the hydroxide being supplied form NaOH or KOH, for example. One of ordinary skill would understand the alternate calcium source and hydroxide source may be included in a cement slurry in the same way as hydrated lime. For example, the calcium source and hydroxide source may be included in any suitable amount, including, but not limited to, a silica source-to-hydrated-lime weight ratio of about 10:1 to about 1:1 or a ratio of about 3:1 to about 5:1. Where present, the alternate calcium source and hydroxide source may be included in the cement slurries in an amount in the range of from about 10% to about 100% by weight of the silica source, for example. The alternate calcium source and hydroxide source may be present in an amount ranging between any of and/or including any of about 10%, about 20%, about 40%, about 60%, about 80%, or about 100% by weight of the silica source. One of ordinary skill in the art, with the benefit of this disclosure, would recognize the appropriate amount of alternate calcium source and hydroxide source to include for a chosen application. Other additives suitable for use in cementing operations also may be included in the cement slurries as needed for a particular application. Examples of such additives include, but are not limited to: weighting agents, activators, lightweight additives, gas-generating additives, mechanical-property-enhancing additives, lost-circulation materials, filtration-control additives, fluid-loss-control additives, defoaming agents, foaming agents, dispersants, thixotropic additives, and combinations thereof. One of ordinary skill in the art, with the benefit of this disclosure, would be able to select an appropriate additive for a particular application. The following statements may describe certain aspects of the disclosure but should not be read to be limiting to a particular embodiment. Statement 1. A method of well treatment comprising: providing a polymer; correlating performance of the polymer to a least one physical property of the polymer; and preparing a treatment fluid comprising the polymer. Statement 2. The method of statement 1 wherein the at least one physical property includes number average molecular weight, weight averaged molecular weight, polydispersity index, radius of gyration, or combinations thereof. Statement 3. The method of any of statements 1-2 wherein the step of correlating includes using a model of fluid loss, the model being a function of a least one physical property of the polymer. Statement 4. The method of any of statements 1-3 wherein the model is specific to a chemistry of the polymer. Statement 5. The method of any of statements 1-4 wherein the model is of the form of: ln(FL)=A+B*PDI+C*R9+D [CONC]+E*Mw where A, B, C, D, and E are constants, PDI is polydispersity index, Rgis radius of gyration, conc is concentration, Mw is number average molecular weight, and FL is fluid loss. Statement 6. The method of any of statements 1-5 wherein the model is in the form of: FL=K*P(PDI)α*R(Rg)β*C([Conc])γ*D(Mw)δwhere α, β, γ, δ, K, P, R, C, and D are constants, PDI is polydispersity index, Rgis radius of gyration, conc is concentration, Mw is number average molecular weight, and FL is fluid loss. Statement 7. The method of any of statements 1-6 wherein the step of correlating includes correlating polydispersity index to wet time of the polymer. Statement 8. The method of any of statements 1-7 wherein the step of designing the treatment fluid includes selecting a concentration of the polymer based at least in part of the correlation of polydispersity index to wet time. Statement 9. The method of any of statements 1-8 further comprising providing a required wet time, a required fluid loss performance, or both and wherein selecting a concentration of the polymer includes selecting a concentration of the polymer with a such that the polymer provides the required wet time and/or the required fluid loss performance. Statement 10. The method of any of statements 1-9 wherein the fluid includes at least one fluid selected from the group consisting of a cement slurry, a spacer fluid, a displacement fluid, a flushing fluid, and combinations thereof. Statement 11. A method of cementing comprising: providing a plurality of polymers; correlating fluid loss performance and wet time of each of the plurality of polymers to a least one physical property and/or concentration of each of the plurality of polymers; selecting at least one polymer from the plurality of poly polymers wherein the selecting is based at least in part on the correlation; preparing a cement slurry comprising the at least one polymer. Statement 12. The method of statement 11 wherein the step of correlating includes using a model of fluid loss, the model being a function of a least one physical property of a polymer. Statement 13. The method of any of statements 11-12 wherein the model is of the form of: ln(FL)=A+B*PDI+C*Rg+D[CONC]+E*Mw where A, B, C, D, and E are constants, PDI is polydispersity index, Rgis radius of gyration, conc is concentration, Mw is number average molecular weight, and FL is fluid loss. Statement 14. The method of any of statements 11-13 wherein the model is in the form of: FL=K*P(PDI)α*R(Rg)β*C([Conc])γ*D(Mw)δwhere α, β, γ, δ, K, P, R, C, and D are constants, PDI is polydispersity index, Rgis radius of gyration, conc is concentration, Mw is number average molecular weight, and FL is fluid loss. Statement 15. The method of any of statements 11-14 wherein the step of selecting includes selecting a polymer that meets or exceeds the required wet time, the required fluid loss performance, or both. Statement 16. The method of any of statements 11-15 wherein selecting further includes selecting a concentration of the polymer based at least in part on the correlation and the required fluid loss performance. Statement 17. The method of any of statements 11-16 wherein the cement slurry includes the at least one polymer, a cementitious material, and water. Statement 18. The method of any of statements 11-17 further comprising placing the cement slurry in a wellbore. Statement 19. A method comprising: providing a plurality of polymers; correlating fluid loss performance and wet time, using a model, of each of the plurality of polymers to a least one physical property and/or concentration of each of the plurality of polymers; selecting at least one polymer and concentration thereof from the plurality of polymers, wherein the selected at least one polymer and concentration thereof meets or exceeds a required wet time and a required fluid loss performance, wherein the selecting is at least partially based on the correlation; and preparing a cement slurry comprising the at least one polymer and the concentration thereof. Statement 20. The method of statement 19 wherein the cement slurry includes the at least one polymer, a cementitious material, and water. Example methods of using the cement slurries will now be described in more detail with reference toFIGS.5-10. Any of the previous examples of the cement slurries and or slurries may apply in the context ofFIGS.5-10. Referring now toFIG.5, the preparation of a cement slurry in accordance with examples will now be described.FIG.5illustrates a system300for the preparation of a cement slurry and subsequent delivery of the cement slurry to a wellbore in accordance with certain examples. As shown, the cement slurry may be mixed in mixing equipment305, such as a jet mixer, re-circulating mixer, or a batch mixer, for example, and then pumped via pumping equipment310to the wellbore. In some examples, the mixing equipment305and the pumping equipment310may be disposed on one or more cement trucks as will be apparent to those of ordinary skill in the art. If a cement slurry is to be used, a bulk dry cement may be preformulated and prepared at a bulk cement plant, for example. A cement slurry may be mixed by combing the bulk dry cement in mixing equipment305or in other mixing equipment. Liquid additives may be blended with the cement slurry in mixing equipment305. Pumping equipment310may pump the cement slurry to the wellbore. An example primary cementing technique using a cement slurry will now be described with reference toFIGS.5and6.FIG.6illustrates surface equipment400that may be used in the placement of a cement slurry in accordance with certain examples. It should be noted that whileFIG.6generally depicts a land-based operation, those skilled in the art will readily recognize that the principles described herein are equally applicable to subsea operations that employ floating or sea-based platforms and rigs, without departing from the scope of the disclosure. As illustrated byFIG.6, the surface equipment400may include a cementing unit405, which may include one or more cement trucks. The cementing unit405may include mixing equipment410and pumping equipment415(e.g.,FIG.5) as will be apparent to those of ordinary skill in the art. Cementing unit405, or multiple cementing units405, may pump a cement slurry430through a feed pipe420and to a cementing head425which conveys the cement slurry430downhole. Cement slurry420may displace other fluids present in the wellbore, such as drilling fluids and spacer fluids, which may exit the wellbore through an annulus and flow through pipe435to mud pit440. FIG.7generally depicts the placement of cement slurry420into a subterranean formation500in accordance with example examples. As illustrated, a wellbore505may be drilled into the subterranean formation500. While wellbore505is shown extending generally vertically into the subterranean formation500, the principles described herein are also applicable to wellbores that extend at an angle through the subterranean formation500, such as horizontal and slanted wellbores. As illustrated, the wellbore505includes walls506. In the illustrated example, a surface casing508has been inserted into the wellbore505. The surface casing508may be cemented in the wellbore505by a cement sheath510. In alternative examples, surface casing508may be secured in the wellbore505by a hardened resin or hardened resin-cement composite sheath in place of cement sheath510. In the illustrated example, one or more additional conduits (e.g., intermediate casing, production casing, liners, etc.), shown here as casing512may also be disposed in the wellbore505. As illustrated, there is a wellbore annulus514formed between the casing512and the walls506of the wellbore505and/or the surface casing508. One or more centralizers516may be attached to the casing512, for example, to centralize the casing512in the wellbore505prior to and during the cementing operation. With continued reference toFIG.7, a first spacer fluid518may be pumped down the interior of the casing512. The first spacer fluid518may be allowed to flow down the interior of the casing512through the casing shoe520at the bottom of the casing512and up around the casing512into the wellbore annulus514. After the first spacer fluid518has been pumped into the casing512, cement slurry240may be pumped into the casing512. In a manner similar to pumping the first spacer fluid518, the cement slurry420may be allowed to flow down the interior of the casing512through the casing shoe520at the bottom of the casing512and up around the casing512into the wellbore annulus514. After the cement slurry420has been pumped into the casing512, a second spacer fluid522may be pumped into casing512and allowed to flow down the interior of the casing512. The first spacer fluid518and the second spacer fluid522may be used to separate the cement slurry420from fluids introduced into the wellbore505either in front of or behind the cement slurry420. Once the cement slurry420has been placed into the desired position in the wellbore annulus514, the cement slurry420may be allowed to set in the wellbore annulus514, for example, to form a hardened resin sheath that supports and positions the casing512in the wellbore505. Alternatively, one or no spacer fluids may be used, and cement slurry420may not need to be separated from other fluids introduced previously or subsequently into wellbore505. While not illustrated, other techniques may also be utilized for introduction of the cement slurry420. By way of example, reverse circulation techniques may be used that include introducing the cement slurry420into the subterranean formation500by way of the wellbore annulus514instead of through the casing512. These techniques may also utilize a first spacer fluid518and a second spacer fluid522, or they may utilize one or none spacer fluids. As it is introduced, the cement slurry420may displace the first spacer fluid518. At least a portion of the first spacer fluid518may exit the wellbore annulus514via a flow line38and be deposited, for example, in one or more mud pits440, as shown onFIG.6. FIGS.8-10illustrate methods of remedial or secondary cementing. Turning now toFIG.8, there is shown a partial cross-section of a conventional producing wellbore505that has a primary cemented casing512. The cement sheath510around the casing512may have defects potentially caused by a variety of issues, such as improper curing of the cement sheath510while it was being formed. Alternatively, the primary cementing may have been successful, but due to adverse temperatures and pressures within the subterranean formation500, the casing512and/or the cement sheath510surrounding the casing512may form cracks or other types of small perforations600. The small perforations600may be problematic since they may facilitate the introduction of undesirable fluids into the casing512. As shown inFIG.8, a small perforation600has formed in the cement sheath510and the casing512, potentially allowing the introduction of undesirable fluids into the interior of the casing512. Referring now toFIG.7, a small perforation600may be filled or plugged by a cement slurry420or a resin-cement composite. A plug700(the plug700may be any type of plug, e.g., bridge plug, etc.) may be initially placed adjacent and below the small perforation600, to form a barrier to prevent cement slurry420from flowing down the wellbore505and therefore allow cement slurry420of the present disclosure to fill the small perforations600in the casing512and cement sheath510. As shown inFIG.9, tubing705(e.g., coiled tubing, drill pipe, etc.) may be lowered into wellbore505. A first spacer fluid518may be pumped into the wellbore505via the tubing705and allowed to flow down the interior of the tubing705and into the blocked section of the wellbore505created by the plug700. A portion of the first spacer fluid518may then flow through the small perforation600while another portion may reside in the annulus514. After pumping the first spacer fluid518through the tubing705, the cement slurry420may be pumped through the tubing705. The cement slurry420may be pumped down the interior of the tubing705and into the blocked section of the wellbore505created by the plug700. A portion of the cement slurry420may then flow through the small perforation600while another portion may reside in the annulus514. The cement slurry420may be allowed to set in the small perforation600and in a portion of the wellbore annulus514, for example, to form a hardened mass that seals small perforation600to prevent the migration of undesirable fluids into the interior of the casing512. After the cement slurry420has been pumped into the tubing705, a second spacer fluid522may be pumped into the tubing705and allowed to flow down the interior of the tubing705into the blocked section of the wellbore505created by the plug700and up around the tubing705into the wellbore annulus514. Alternatively, one or no spacer fluids may be used, and cement slurry420may not need to be separated from other fluids introduced previously or subsequently into wellbore505. The tubing705may then be removed. The plug700may also be removed. In alternative examples, plug700may remain in the wellbore505and be drilled through. After tubing705is removed, the portion of the hardened cement slurry420remaining in the wellbore505(i.e., the portion not in the small perforation600) may then be drilled through. FIG.10describes another example of filling a small perforation600with a cement slurry420. A plug700(the plug700may be any type of plug, e.g., bridge plug, etc.) may be initially placed adjacent and below the small perforation600, to form a barrier that may allow pressurized pumping of a cement slurry420of the present disclosure to fill any small perforations600in the casing512and cement sheath510. As shown inFIG.10, tubing705(e.g., coiled tubing, drill pipe, etc.) may be lowered into wellbore505. Tubing705may be attached to a retainer800or may be inserted into a retainer800already placed into the wellbore505. Retainer800may allow for the pressurized pumping of the cement slurry420into any small perforations600. Retainer800must be placed adjacent to and above the small perforations600to be filled by cement slurry420. Retainer800may be any type of retainer, for example, a cement retainer. After plug700, tubing705, and retainer800are placed, a first spacer fluid518may be pumped into the wellbore505via the tubing705and allowed to flow down the interior of the tubing705and into the blocked section of the wellbore505created by the plug700. A portion of the first spacer fluid518may then flow through the small perf oration600. After pumping the first spacer fluid518through the tubing705, the cement slurry420may be pumped through the tubing705. The cement slurry420may be pumped down the interior of the tubing705and into the blocked section of the wellbore505created by the plug700. A portion of the cement slurry420may then flow through the small perforation600while another portion may reside in the space formed between the plug700and retainer800. The cement slurry420may be allowed to set in the small perforation600and in the space formed between the plug700and retainer800. The cement slurry420may then harden to form a hardened mass that seals small perforation600to prevent the migration of undesirable fluids into the interior of the casing512. After the cement slurry420has been pumped into the tubing705, a second spacer fluid522may be pumped into the tubing705and allowed to flow down the interior of the tubing705into the blocked section of the wellbore505created by the plug700and into the space formed between the plug700and retainer800. Alternatively, one or no spacer fluids may be used, and cement slurry420may not need to be separated from other fluids introduced previously or subsequently into wellbore505. The tubing705may then be removed. The plug700may also be removed. In alternative examples, plug700may remain in the wellbore505and be drilled through. Retainer800may also be removed. Conversely, in alternative examples, retainer800may be drilled through. After tubing705is removed, the portion of the hardened cement slurry420remaining in the wellbore505(i.e., the portion not in the small perforation600) may then be drilled through. The disclosed cement slurries and associated methods may directly or indirectly affect any pumping systems, which representatively includes any conduits, pipelines, trucks, tubulars, and/or pipes which may be coupled to the pump and/or any pumping systems and may be used to fluidically convey the cement slurries downhole, any pumps, compressors, or motors (e.g., topside or downhole) used to drive the cement slurries into motion, any valves or related joints used to regulate the pressure or flow rate of the cement slurries, and any sensors (i.e., pressure, temperature, flow rate, etc.), gauges, and/or combinations thereof, and the like. The cement slurries may also directly or indirectly affect any mixing hoppers and retention pits and their assorted variations. It should be understood that the slurries and methods are described in terms of “comprising,” “containing,” or “including” various components or steps, the slurries and methods can also “consist essentially of” or “consist of” the various components and steps. Moreover, the indefinite articles “a” or “an,” as used in the claims, are defined herein to mean one or more than one of the element that it introduces. For the sake of brevity, only certain ranges are explicitly disclosed herein. However, ranges from any lower limit may be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit may be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit may be combined with any other upper limit to recite a range not explicitly recited. Additionally, whenever a numerical range with a lower limit and an upper limit is disclosed, any number and any included range falling within the range are specifically disclosed. In particular, every range of values (of the form, “from about a to about b,” or, equivalently, “from approximately a to b,” or, equivalently, “from approximately a-b”) disclosed herein is to be understood to set forth every number and range encompassed within the broader range of values even if not explicitly recited. Thus, every point or individual value may serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited. Therefore, the present disclosure is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. The particular examples disclosed above are illustrative only, as the present disclosure may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Although individual examples are discussed, the disclosure covers all combinations of all those examples. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. Also, the terms in the claims have their plain, ordinary meaning unless otherwise explicitly and clearly defined by the patentee. It is therefore evident that the particular illustrative examples disclosed above may be altered or modified and all such variations are considered within the scope and spirit of the present disclosure. If there is any conflict in the usages of a word or term in this specification and one or more patent(s) or other documents that may be incorporated herein by reference, the definitions that are consistent with this specification should be adopted. | 48,393 |
11859125 | EXAMPLE 1—PREPARATIONS OF INVERSE WATER-IN-OIL EMULSIONS CONTAINING CATIONIC MICROGELS Water-in-oil emulsions containing cationic microgels were prepared by inverse suspension polymerization according to the following procedure. An oily continuous phase was prepared by mixing under mechanical stirring LAMIX 30 as an oily fluid and a mixture of the commercial non-ionic surfactants SPAN80 and TWEEN80. The weight ratio between the two surfactants was selected so as to cause the polymerization reaction at the desired HLB value. In the specific example of an HLB value equal to 6, the SPAN80 surfactant was used with a massive concentration, referring to the sum of the two surfactants, equal to 84%. An aqueous discontinuous phase was prepared by dissolving in water, with the aid of an ultrasonic sonicator and keeping the temperature below 50° C., a cationic monomer ([2-(methacryloxy))ethyl] trimethylammonium chloride—MADQUAT), a cross-linking agent (N,N′-methylenbis(acrylamide)—MBA) and optionally a (poly(ethylene glycol)methyl ether methacrylate comonomer of molecular weight 500 Da (PEGMEMA 500) or 2000 Da (PEGMEMA 2000)). An aqueous solution of 2,2′-azobis(2-methylpropianimidine) dihydrochloride (AAPH) (thermally activatable radical polymerization initiator) was prepared separately, using a minimum amount of water sufficient to dissolve the compound. The oily continuous phase and the aqueous discontinuous phase were mixed in a reactor with a volume equal to 2 liters, heated by means of a thermostatic oil bath. The mixing took place by means of a mechanical stirrer. The reactor was equipped with a water cooling jacket to remove the heat generated during the polymerization reaction. During polymerization, the reactor was kept under constant N2flow so as to remove the air inside it. The weight ratio between the aqueous discontinuous phase and the total weight of the water-in-oil emulsion was selected equal to 16% for the PEG2 sample and 18% for the PEG4 and PEG11 samples. The polymerization reaction was initiated by pouring the solution of the AAPH initiator drop by drop into the reaction mixture, previously heated to the polymerization temperature, equal to 70° C. or 80° C. The reaction duration was selected equal to 2 hours or 2.5 hours. The following Table 1 shows the compositions of the prepared inverse emulsions of microgels. TABLE 1Composition of the emulsions of microgelsPEGMEMAPEGMEMAMADQUAT5002000MBAAAPHSurfactantsSample(%)a(%)a(%)a(%)a(%)c(%)bHLB1100——0.350.57.06(A4)d295—50.350.55.04.3(PEG2)e390—100.350.55.04.3(PEG4)e492.57.5—0.350.55.04.3(PEG11)eapercentage by weight with respect to the weight of MADQUAT + PEGMEMA comonomers;bpercentage by weight referred to the total weight of the emulsion;cpercentage by weight with respect to the total weight of MADQUAT + PEGMEMA comonomers + MBA;dpolymerization temperature = 80° C.; polymerizationduration 2.5 hours;epolymerization temperature = 70° C.; polymerization duration 2.0 hours. EXAMPLE 2—PREPARATION OF INVERSE WATER-IN-OIL EMULSIONS CONTAINING CATIONIC NANOGELS Water-in-oil emulsions containing cationic nanogels were prepared by inverse miniemulsion polymerization according to the following procedure. An oily continuous phase was prepared by mixing under mechanical stirring Eni LAMIX 30 as an oily fluid and a mixture of the commercial non-ionic surfactants SPAN80 and TWEEN80. The weight ratio between the two surfactants was selected so as to cause the polymerization reaction at the desired HLB value. In the specific example of an HLB value equal to 10, the SPAN80 surfactant was used with a massive concentration, referring to the sum of the two surfactants, equal to 47%. An aqueous discontinuous phase was prepared by dissolving in water, with the aid of an ultrasonic sonicator and keeping the temperature below 50° C., a cationic monomer ([2-(methacryloxy))ethyl]trimethylammonium chloride MADQUAT), a cross-linking agent (N,N′-methylenbis(acrylamide)—MBA) and ammonium persulfate as a first initiator of the pair of redox initiators, ammonium persulfate (APS)/sodium metabisulfite (SMBS). After conditioning the oily continuous phase in an ice bath (T equal to about 0-5° C.), the aqueous discontinuous phase was added to the oily continuous phase by keeping the mixture of the two phases under sonication. A SMBS aqueous solution was then added drop by drop to the mixture to initiate the polymerization reaction (polymerization duration 50 minutes). The following Table 2 shows the compositions of the prepared inverse emulsions of nanogels. TABLE 2Composition of the emulsions of nanogelsMADQUATMBAAPSSMBSSurfactantsSample(%)a(%)b(%)b(%)b(%)bHLB5 (MZ17)350.352.52.52110apercentage by weight with respect to the weight of LAMIX 30 ®;bpercentage by weight with respect to the tota weight of MADQUAT. 3. Characterization of Emulsions of Microgels and Nanogels 3.1 Swelling Test The capacity of water absorption of the prepared microgels were determined by measuring the average particle diameter by means of a compound light microscope, before and after the swelling test. The swelling test was carried out by depositing a few drops of a water-in-oil emulsion in a vial previously filled with water with two different degrees of salinity or with Lamix 30®. The samples were allowed to rest for 24 hours to allow the thermodynamic equilibrium to be reached. The samples were then observed under a microscope to determine the final size of the microgel particles. The average particle diameter and the polydispersion index (PDI) of the polymer of the nanogels of the MZ17 sample were determined by means of dynamic light scattering (DLS) measurements. The particle size distribution of the nanogels was monomodal. The results of the DLS measurements are shown in Table 5. Table 3 shows the chemical compositions of the saline waters used in the test. Table 4 shows the diameter values of the microgels determined in Lamix 30® and in the different waters tested. TABLE 3Composition of saline watersNa+Ca2+Mg2+(g/L)(g/L)(g/L)Field A855.80.6Field B857.91.5 TABLE 4Microgel diameterStd.Std.Std.LamixDev.Field BDev.Field ADev.(μm)(μm)(μm)(μm)(μm)(μm)A410.172.4435.9511.5939.7310.52PEG29.375.6332.7711.7430.9313.77PEG410.422.0745.7226.1861.8634.69PEG119.9042.37267.2233.38163.91629.087 It has been observed that in the samples placed in contact with Lamix 30® the sizes of the microgels before and after the swelling test are substantially identical; this shows that the microgels do not swell in contact with oily fluids. On the other hand, in the samples in contact with water, the sizes of the microgels after the swelling test are greater than the sizes of the same microgels before the test. TABLE 5Nanogel diameterDiameterPolydispersity(nm)(PDI)5 (MZ17)299.20.226 3.2 Compatibility Assessment of the Emulsions of Microgels with Production Fluids The following test was conducted to assess the behaviour of cationic microgels in contact with production fluids (formation water and hydrocarbon fluids). A 10 ml aliquot of saline water was placed in a glass container. A 2 g aliquot of oil was added to it. The container was closed and conditioned in a stove at a temperature of 85° C. (to simulate the temperature of the well bottom). The sample was then taken from the stove and added with the emulsion containing the microgels. The tested emulsions, having different concentrations of microgel particles, were dosed in the respective containers containing water and oil in amounts such as to obtain a concentration by weight of microgels equal to 26-28% referring to the weight of the mixture. The following waters with different salinity originating from extraction fields of hydrocarbon oils were used in the test:Field C (total salinity: 2.3 g/l)Field D (total salinity: 84 g/l)Standard Sea Water (total salinity: 35 g/l) In tests a hydrocarbon oil having a density between 1.012 and 1.017 g/cm3 also originating from an extraction field of hydrocarbon oils was used as a heavy oil. The glass containers containing the water-oil mixtures were placed in a stove and conditioned at 85° C. At the end of the conditioning, the emulsion containing the microgels in the amounts indicated above was added to each container. The containers were then overturned repeatedly to mix all the components thoroughly and put back in the stove at 85° C. for 24 hours. At the end of the thermal conditioning, the degree of separation of the water and oil phases, the settlement of the microgels on the bottom of the container and the volume of the container occupied by the microgels following the swelling as a result of water absorption were assessed visually. Samples 2, 3 and 4 all showed a good separation of the water and oil phases and the settlement of the particles of microgels with water absorption in all the tests, i.e. with all three of the aforementioned waters with different salinity. Sample 4, in particular, showed the best separation results of the water and oil phases (clearer aqueous phase and greater volume occupied by the swollen microgels). The test therefore proved that the inverse emulsions containing microgels are compatible with the production fluids, in particular their contact with these fluids does not lead to the emulsion of oil with water that may be present, which in a real situation could worsen the oil extraction effectiveness, increasing the amount of co-produced water. The tests also show the effectiveness of the emulsions prepared according to the present invention in a very wide range of water salinity. 3.3 Assessment of the Emulsions of Microgels with Production Fluids in the Presence of Calcium Carbonate The following test was performed to assess the effectiveness of the interaction of the inverse emulsions according to the present invention with a carbonatic rock. 10 g of solid calcium carbonate was weighed in one vial. 3 g of saline water or 4.5 g (Standard Sea Water having total salinity equal to 35 g/l) were then added to the vial. The amount equal to 3 g was sufficient to completely cover the present calcium carbonate (Series 1). The amount equal to 4.5 g produced an excess water condition (Series 2). 2 ml of an oil phase (Field D) were slowly deposited on the water phase. The vials were then placed in a stove and conditioned at 85° C. At the end of the conditioning, the emulsion containing the microgels was slowly added to each vial, in an amount equal to 2 ml, taking care not to create turbulence. The vials were then put back in the stove at 85° C. for 24 hours. At the end, it was assessed visually whether the particles of microgels were able to cross the oil phase without causing any emulsion thereof and where these particles were positioned. The vials, after a period of 24 hours, were overturned so as to assess the degree of adhesion to the calcium carbonate. For comparison, the test was repeated with an inverse emulsion of microgels containing copolymers of methacrylic acid (partially neutralized with NaOH) and poly(ethylene glycol)methyl ether methacrylate (HEMA-PEG, MW=2000 Da, 42 polyoxyethylene units) prepared as described in Example 2 of WO 2016/166672. In the Series 1 samples added with the emulsions containing the cationic microgels No. 2, 3 and 4, the total penetration of the particles of microgels between the calcium carbonate grains was observed. This penetration was not observed substantially in the comparative sample. Probably, the observed penetration is attributable to the electrostatic attraction between the positive charges of the cationic microgels and the negative charges of the calcium carbonate, as well as the smaller sizes of the particles of the cationic microgels (about 10 micrometers) with respect to the comparative microgel particles (about 20 micrometers). Furthermore, after overturning the vials, it was observed that samples No. 2, 3 and 4 help to compact the carbonate grains together, such that the solid phase remains firm on the bottom of the vial even when it is overturned. On the contrary, in the comparative sample, when the vial was overturned, the grain break-up of the calcium carbonate grains was observed and their sliding downwards. The same behaviour of the cationic microgels according to the invention and of the comparative one was observed in the Series 2 samples containing excess water. The test has proved a greater capability of the particles of the microgels of the emulsions according to the present invention of interacting with the carbonatic rocks with respect to the emulsions of microgels of the prior art. 4. Characterization of the Emulsions of Nanogels 4.1 Test 1—Flushing of Cores Saturated with Oil and Saline Water The injectability of sample 5 containing particles of nanogels inside a sandy medium (Berea Sandstone) and its ability to modify water permeability in a formation was assessed by means of flux measurements in porous medium. For this purpose, a cylindrical core with a length equal to 5.09 cm and a diameter equal to 2.47 cm, having a porosity of 16.5% was used. The core was placed in a core holder under confining pressure (40 bar) to avoid fluid leaks. The core was initially filled with synthetic sea water (salinity: 33 g/L) and brought to the temperature of 40° C. in the stove. The core was then flushed with Lamix 30® until it reached the conditions in which water is no longer produced (Core under residual water saturation). At this point sample 5 was injected for about 24 times the pore volume. The core was then allowed to rest at 40° C. for 24 hours (shut-in) to allow for the action of the nanogels. At the end of the shut-in period, the core was again flushed with synthetic sea water, to verify the possible effect of reduction of permeability to water generated by the cationic nanogels. Sample 5 was easily injectable and no pressure increases were observed during its injection. The final flushing with saline water showed a reduction in the permeability of the core to water with respect to flushing with water before the treatment with the inverse emulsion according to the invention. The value of the initial water permeability was in fact 36 mD and drops to 1.4 mD at the end of the test, due to the desired behaviour of the cationic nanogels. | 14,307 |
11859126 | DETAILED DESCRIPTION The description, being of exemplary embodiments, is not intended to limit the claims of this disclosure. On the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the claims. Many changes may be made to the particular embodiments and details disclosed herein without departing from such spirit and scope. Certain terms are used herein and in the appended claims to refer to particular components. As one skilled in the art will appreciate, different persons may refer to a component by different names. This document does not intend to distinguish between components that differ in name but not function. Also, the terms “including” and “comprising” are used herein and in the appended claims in an open-ended fashion, and thus should be interpreted to mean “including, but not limited to . . . .” Further, reference herein and in the appended claims to components and aspects in a singular tense does not necessarily limit the present disclosure or appended claims to only one such component or aspect, but should be interpreted generally to mean one or more, as may be suitable and desirable in each particular instance. All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. All references cited herein are incorporated herein by reference. Fluid loss from the fluid loss control pills described herein into the well or the subterranean formation penetrated by the well is minimal. Further, the highly viscous pill reduces friction pressure and thus is highly useful in pumping operations. Further, the fluid has been demonstrated to minimize formation impairment. The fluid loss pill exhibits high viscosity and is stable at bottom hole temperatures in excess of 275° F., typically greater than 300° F. The fluid loss pill may be stable for extended periods, in some cases greater than 20 hours and in some cases greater than 24 hours, for instance, at 275° F. and in some cases greater than 20 hours at 300° F. Thus, unlike conventional polymer based fluid loss control additives, the fluid loss pill disclosed herein is stable even in high-temperature wells. The enhanced stability of the fluid loss pill allows it to be stored for later use. Because of the constantly changing conditions in the wellbore, it is often desirable to have the flexibility to store a fluid loss pill for a period of time, prior to use. The fluid loss pill contains a brine and a crosslinked polymer of a galactomannan and a puffed boron or ulexite crosslinking agent. The reference herein to “crosslinked polymer” shall include a fully crosslinked polymer as well as a partial crosslinked polymer. Typically, the crosslinked polymer in the fluid loss pill exhibits sufficient viscoelastic properties, in particular relatively high viscosities (e.g., at least about 300-500 cP at a shear rate of 100 sec−1). The partial crosslinked polymer typically is a gel which exhibits “lipping”. A lipping gel is distinguishable from a liquid which freely pours out of a container. Characteristics of a lipping gel are discussed in U.S. Pat. No. 7,814,980. As used herein, “lipping” shall refer to a gel having sufficient homogeneous, three-dimensional elasticity on a macroscopic level with very few, if any, distinct micro-domains. Thus, a lipping gel thus may consist of the galactomannan not being fully crosslinked with the puffed boron or ulexite crosslinking agent. In an embodiment, the fluid loss pill is introduced into a targeted location within the well. At the targeted location, the viscosity of the fluid loss pill may increase under in-situ operating conditions. At this stage, the partial crosslinked polymer becomes fully crosslinked and prevents fluid leak-off into the well or into the formation penetrated by the well. Once emplaced and gelled, the fluid loss pill may be optionally removed by injecting a conventional breaker fluid into the wellbore. An embodiment of the disclosure is a two-component fluid loss pill comprising the crosslinked polymer and the brine. While other components may be added to alter or stabilize fluid properties if necessary, typically, the fluid loss pill is free of such solid components. For instance, the fluid loss pill does not require solid additives or particulates such as starch, sized salts, carbonate chips, mica or other particulates (though the fluid is compatible and can be used with these materials). Such additives and particulates are known to cause formation damage. The fluid loss pill provided disclosed provides viscosity controlled fluid loss rather than particulate (filter cake) controlled fluid loss, making the fluid loss easier to remove and non-damaging. The base fluid of the brine may be fresh water or sea water. Preferred galactomannans are underivatized guar and guar derivatives such as hydroxypropyl guar, carboxymethyl guar and carboxymethyl hydroxypropyl guar as well as mixtures thereof. There is no need to further derivatize the galactomannan. In a preferred embodiment, the hydratable polymer is hydroxypropyl guar. The crosslinker is typically a puffed boron or ulexite. Typically, the crosslinked polymer contains from about 0.1 to about 2.5 volume percent of the crosslinking agent; the remainder being the galactomannan gum. In a preferred embodiment, the crosslinking agent is a puffed boron consisting of low density particles or beads having a high surface area and large quantities of voids. Generally, each particle is comprised of a plurality of expanded cells adjacent to and attached to each other. The density of the particles typically ranges from about 0.05 to about 0.40 g/cm3, the bulk density is typically from about 150 to about 350 g/1, and the particle diameter is typically from about 420 to about 1,000 μm. The particle size of the puffed boron may be non-uniform as demonstrated by the puffing (the ratio between the diameter of the puffed particle and the diameter of the feed particle yielding the puffed particle). For instance, the smaller or lighter feed particles may be puffed in excess over the larger denser feed particles to provide a higher puff ratio. In other embodiments, the puffed boron consists of a more uniform ratio of expanded particles wherein a particle size distribution (PDS) curve of the puffed boron is substantially similar in shape to the PSD curve for the feed borax, thereby providing a lower puff ratio. The bead strength of the puffed boron is greater when the puff ratio is lower. See, for instance, U.S. Pat. No. 4,412,978. Typically, the void volume of the puffed boron is from about 2.1 to about 3.5 cm3/g. Puffed boron may be prepared by rapidly heating particulate borax pentahydrate particles to above the melting temperature of the pentahydrate, wherein the borate dissolves in its own water of hydration causing the solution to erupt through the partially dehydrated crystal surface. Other methods of making puffed boron are disclosed in U.S. Pat. Nos. 3,454,357; 3,944,651; 4,412,978; and 4,547,352. Suitable methods for making puffed boron are disclosed in R. C. Rhees and H. H. Hammar, “Puffed Borax”,Soap and Chemical Specialties, Vol. XLII, January, 1966 (pp. 58-61 and 118-120). Further suitable as puffed boron is the reticulated particle set forth in U.S. Pat. No. 4,547,352 which exhibits an enhanced absorptive capacity. Though not preferred ulexite, having the formula (Na2Ca2B10O18·H16H2O), may be used in some instances as crosslinking agent. The fluid loss pill may be composed of any brine including ammonium chloride and monovalent salt brines such as sodium bromide, potassium chloride, sodium acetate, sodium formate, sodium chloride, potassium acetate, potassium formate and mixtures thereof, such as mixtures of sodium chloride and sodium bromide, mixtures of potassium chloride and calcium chloride, etc. The disclosure, however, is of particular value when used with heavy brines, i.e., in particular calcium and/or zinc containing brines and brines having a density in excess of 11.0, typically greater than 12.5. Such brines include divalent salts such as calcium chloride, calcium bromide, zinc bromide, as well as binary, mixtures of heavy salts such as calcium chloride and calcium bromide, zinc bromide and calcium bromide, etc. as well as brines composed of three or more salts. Such brines include, for example, a mixture of zinc bromide, calcium bromide and calcium chloride. The disclosure is particularly applicable to the use of calcium and/or zinc containing brines. Further, a brine within the same density range as a zinc and/or calcium brine may be attained using the process disclosed herein. Where single salt brines and mixtures of salts are used, the density of such brines may be increased to be within the same range as zinc containing brines. Where a monovalent salt is used as the brine, the fluid loss pill may be formed by introducing the crosslinked polymer directly to the brine. Typically, the volume ratio of the crosslinked polymer to brine is from about 1:50 to about 1:500. The fluid may further contain glycerol. In an embodiment, the fluid loss pill contains brine and a slurry of the crosslinked polymer in glycerol. The glycerol in the slurry enhances hydration of the galactomannan in the brine. The fluid loss pill is composed of between about 2 to about 10 volume percent of the slurry and about 90 to about 98 volume percent of the brine. The volumetric ratio of crosslinked polymer to glycerol in the slurry is typically from about 1:1.5 to about 1:7. The fluid loss pill may further contain a linear (non-crosslinkable) viscosifying polymer. In an embodiment, especially when the density of the brine having substantial divalent metal ions (such as calcium and/or zinc), the linear viscosifying polymer, with the glycerol, maintains the crosslinked polymer in a suspended state in the slurry. The linear viscosifying polymer may be a salt of a sulfonated acrylamide. In an embodiment, the linear viscosifying polymer is a copolymer of the sulfonated acrylamide salt and a vinyl lactam. In an embodiment, the sulfonated acrylamide salt is of the structural formula (I): wherein R is selected from the group consisting of alkenyl groups having from about 1 to about 4 carbon atoms; R1and R2are selected from the group consisting of hydrogen and methyl groups; and, X is a cation, preferably an alkali metal, such as sodium. A preferred sulfonate monomer is 2-acrylamido-2-methylpropane sulfonic acid, sodium salt (commonly referred to as AMPS). The N-vinyl lactam may be of the structural formula (II): wherein R9, R10, R11and R12independently are selected from the group consisting of hydrogen, methyl, and ethyl. In an embodiment, the N-vinyl lactam may be N-vinyl-2-pyrrolidone (NVP). Typically, the mole ratio of the sulfonated acrylamide salt, and the N-vinyl lactam in the copolymer is from about 50 to about 60 mole % of sulfonated monomer and between from about 25 to about 50 mole % of N-vinyl lactam. In a less preferred embodiment, the linear non-crosslinkable viscosifying polymer is a water-soluble copolymer of polyacrylamide (PAM) and the sulfonated acrylamide salt. The polyacrylamide may originate from the olefinic amide monomer of the formula R(CO)N(R1)—CH2—R2wherein R is a 1-alkenyl group and R1and R2independently are selected from the group consisting of hydrogen and alkyl groups having from 1 to 4 carbon atoms. In an embodiment, the linear viscosifying polymer has an average molecular weight (Mw) from about 0.5 to about 30 million Daltons, in some cases from about 1 to about 25 million Daltons, in other cases from about 4 to about 25 million Daltons; and in some cases, at least 2 million Daltons. Suitable copolymers include those set forth in U.S. Pat. No. 9,902,894. The linear viscosifying polymer is typically a solid, most notably a dry powder, and is water-soluble. The average particle size of the powder in some instances is at least 3000 μm, in other embodiments from about 35 to about 2000 μm; in other embodiments between from about 150 to about 450 μm. The amount of the viscosifying polymer in the slurry is an amount sufficient to keep the crosslinked galactomannan suspended in the glycerol. When present, the volumetric ratio of the crosslinked galactomannan to linear viscosifying polymer is from about 5:1 to about 1500:1, the ratio of the crosslinked galactomannan to glycerol being from about 1:1.5 to about 1:7. The linear viscosifying polymer has not been observed to crosslink with the crosslinking agent. The fluid loss pill may further may contain a dispersant. When present, the volumetric ratio of the crosslinked galactomannan to dispersant in the slurry is from about 1:25 to about 15:1. Suitable dispersants include sorbitan derivatives, phenyl-polymer with oxirane, monooctyl ether, polymer of phenylundoxiran, alkyl-modified organomodified siloxane, a siloxane polyolefin or an amphiphilic copolymer. Suitable sorbitan derivatives include sorbitan trioleate and polyoxyethylene sorbitan, polyoxyethylene-sorbitan-fatty acid esters, for example polyoxyethylene(20)sorbitan monolaurate, polyoxyethylene(4)sorbitan monolaurate, polyoxyethylene(20)sorbitan monopalmitate, polyoxyethylene(20)sorbitan monostearate, polyoxyethylene(20)sorbitan tristearate, polyoxyethylene(20)sorbitan monooleate, polyoxyethylene(5)sorbitan monooleate and polyoxyethylene(20)sorbitan trioleate. The dispersant may be a polyorganosiloxane derived from siloxane units having the general formula —(R2SiO)— in which the two monovalent groups R, which may be identical or different, are linear or branched alkyl groups having 1 to 18 carbon atoms, cycloaliphatic groups having 4 to 8 C atoms, linear or branched alkyl groups having 2 to 4 carbon atoms, phenyl- or alkyl phenyl groups having 1 to 12 carbon atoms in the aliphatic group (halogens or hydroxyl-, carboxyl-, carbonic acid anhydride, amino-, epoxy-, alkoxy- or alkenyloxy groups may be substituted for the hydrocarbon groups), polyether- or polyolefin groups and hydrogen, the groups being bonded together directly or via an oxygen or nitrogen atom with a silicon atom of the polysiloxane chain. Examples of such groups R are methyl-, ethyl-, isopropyl-, isobutyl, dodecyl- and octadecyl groups, cyclopentyl-, cyclohexyl- and cyclooctyl groups, vinyl-, allyl-, isopropenyl and 3-butenyl groups, ethylphenyl-, dodecyl groups, and groups having hydrocarbon groups that are partially substituted, e.g., by halogens such as fluorine or chlorine, as is the case, e.g., with chloropropyl or the 1,1,1-trifluoropropyl group. At least a portion of the groups R may also consist of polymeric groups, in particular polyethers, such as polyethylene-, polypropylene-, polybutylene or polyhexamethylene glycol or polytetrahydrofuran and mixed polymers of these ethers, as well as polyolefins, e.g., polybutadiene, polyisoprene, polybutene, polyisobutene, or the like. Finally, a portion of the groups R may be hydrogen. It is also possible to use mixtures of the aforementioned polyorganosiloxanes. Further, the dispersant may be at least one organo-modified siloxane compound, wherein the at least one organo-modified siloxane compound contains organoalkoxysiloxane units according to general formula (III): wherein:R1is an alkyl radical and/or aryl radical,R2is H and/or alkyl radical with 1 to 4 carbon atoms,a is ≥0 and ≤2; andb is >0 and ≤3 with the proviso that a+b≥1 and ≤4. Formula (III) is an average formula of the organoalkoxy-siloxane units of the organo-modified siloxane compound. The proportion of H for —R2can be ≥0% and ≤10%, preferably ≥0% and ≤5%, particularly preferably ≥0% and ≤1%, and especially preferably 0%. Preferably, the organo-modified siloxane disclosed herein is liquid at 25° C. Preferably, the substituents R 1 and/or R2of the organoalkoxysiloxanes of the liquid, organo-modified siloxane compound/s are those wherein R1is phenyl and/or C1-C16alkyl radical, preferably R1is C1-C12alkyl radical, more preferably R1is a C1-C8alkyl radical, particularly preferably R1is a C1-C4alkyl radical, wherein most preferably R1is methyl and/or ethyl; and/or R2is —H, methyl, ethyl, propyl, isopropyl, butyl or tert-butyl, where methyl and/or ethyl are most preferred. Furthermore, it is preferable that a is 0.5 to 1.8, preferably 0.7 to 1.7, and more preferably 1.0 to 1.5, with the proviso that a+b≤4 and preferably a+b≤3. In addition, it is also specified according to the invention that b is 0.1 to 2.5, preferably b is 0.2 to 2.3, still more preferably b is >0.3 to 2.0 and particularly preferably b is >0.3 to 1.2, with the proviso that a+b≤4 and preferably a+b is ≤3. The liquid organo-modified siloxane may have a molecular weight from 120 to 100,000, preferably 250 to preferably 500 to 60,000, more preferably 750 to 50,000 and particularly preferably 1,000 to 30,000. Further, suitable dispersants include polyethers containing units of 2-pentyloxirane, 2-methyl-3-phenyloxirane, 2,3-epoxypropylbenzene, 2-(4-fluorophenyl)oxirane. Suitable amphiphilic dispersants include polyethersiloxanes such as those disclosed in U.S. Pat. No. 8,034,848, as well as phospholipids, lecithins, betaines, sulfobetaines, as well as those based on nonionic fatty alcohol ethoxylates or alkylphenol ethoxylates and/or their anionically modified derivatives and tri- and partial glycerides and also fatty acids). The slurry may further contain a hydrophilic oxygenated liquid, a hydrophobic oxygenated liquid or a combination thereof. Suitable hydrophilic and hydrophobic oxygenated liquids include propylene glycol n-propyl ether, propylene glycol methyl ether, tripropylene glycol methyl ether, dipropylene glycol methyl ether acetate, propylene glycol butyl ether, dipropylene glycol butyl ether, tripropylene glycol butyl ether and dipropylene glycol methyl ether and mixtures. Preferred hydrophobic and hydrophilic components are dipropylene glycol n-butyl ether and dipropylene glycol methyl ether, respectively. When present, the volumetric ratio of crosslinked galactomannan to hydrophilic oxygenated liquid, hydrophobic oxygenated liquid or the combination thereof in the slurry may be from about 1:0.8 to about 1:4. Typically, the volumetric ratio of the crosslinked polymer to glycerol is from about 1:1.4 to about 1:7 and the hydrophilic oxygenated liquid to the hydrophobic oxygenated liquid in the slurry is from about 1:3 to 3:1, preferably about 1:1. In an embodiment, the fluid loss pill may contain the brine and a slurry comprising the crosslinked polymer, linear viscosifying polymer, glycerol, hydrophilic oxygenated liquid and/or hydrophobic oxygenated solvent as well as dispersant. In an embodiment, the fluid loss pill may contain from about 90 to about 98 volume percent of a brine having a density of 11.0 ppg or higher (such as a brine containing divalent metals like calcium and/or zinc) and from about 2 to about 10 volume percent of the slurry, the slurry comprising between from about 10 to about 20 volume percent of the crosslinked polymer, between from about 0.05 to about 3 volume percent of the linear viscosifying polymer, between from about 30 to about 75 volume percent of glycerol, between from 16 to about 40 volume percent of the hydrophilic oxygenated solvent/hydrophobic oxygenated solvent and between from about 2 to about 8 volume percent of the dispersant. The pH of the fluid loss pill may be less than 6.0 and in some cases is between 3.5 and 6.5. Depending on the source of the brine, the pH may fluctuate. This is unlike conventional aqueous borate crosslinked well treatment fluids where the pH of the fluid must be increased to 8 or higher in order to crosslink the galactomannan with the borate salt. However, at such an elevated pH, divalent salts, such as calcium and/or zinc, precipitate. The viscosity of the fluid loss pill thereby decreases. In the past, galactomannan-based fluid loss pills containing a brine having substantial divalent metal ion (e.g., calcium and zinc) content have exhibited instability and have not been used at elevated downhole temperatures, such as in excess of 250° F. With the fluid loss pill described herein, precipitation of salts is less of a concern and an alkaline oxide may be included in the fluid loss pill. Typically, the pH of the disclosed fluid loss pill may be increased by the addition of an alkaline oxide. In light of the low pH of the disclosed fluid loss pill, the alkaline oxide may be included. The addition of the alkaline oxide enhances thermal stability of the fluid loss pill as well as enhances crosslinking (gelation) of the guar in the brine. The alkaline oxide may be added either prior to, with or after the addition of the crosslinking agent. The metal oxide is typically a Group II metal of the Periodic Table such as magnesium. Further, the alkaline oxide may be added to the fluid loss pill as a buffer. When used, the amount of amount of alkaline oxide in the fluid loss pill is from about 0.5 to about 7.5 volume percent. In an embodiment, a fluid loss pill may be prepared by forming a slurry of the linear viscosifying polymer and glycerol. The brine portion contains slurry (glycerol, linear viscosifying polymer, optional dispersant, galactomannan, optional hydrophobic oxygenated ether and optional hydrophilic oxygenated ether), optional metal oxide and crosslinking agent. The two polymer systems may then be co-mingled into one solution to form the fluid loss control pill. The volume ratio of slurry to brine is from about 1:3 to about 1:20. The volumetric ratio of linear viscosifying polymer in the brine may be from about 1:30 to about 1:80. The volume percent of crosslinkable galactomannan is from about 0.5 to about 2 percent. After viscosifying the brine to about 30 to about 60% of saturation, additional brine may be added. The salt of the additional brine may be dry salt. The dry salt may constitute all or a portion of the additional brine. Typically, the salt of the additional brine is the same salt as the salt of the brine initially added to the slurry. The amount additional brine may be an amount sufficient to render about 75 to about 95% of saturated brine. (As used herein, “saturated brine” refers to a brine having the maximum level of salt at which no further salt can be added to the brine without the salt falling out of solution. For instance, the amount of salt in sodium chloride, sodium bromide, potassium chloride, calcium chloride and calcium bromide brines is 26, 40, 24, 40 and 57%, respectively.) In an embodiment, the amount of additional brine is about 2 to 4 times the amount of brine initially added to the slurry. For example, where 60 mls of brine is added initially to 20 mls of the slurry, an additional 60 mls of brine may be added over a period of time until the amount of brine in the resulting viscous fluid is about 350 mls (1 bbl equivalent). The final density of the liquid is the same or substantially the same (within 10%, or within 5%, or within 0.5 to 1%) as the density of the brine initially added to the slurry. Once the brine is about 75 to 95% saturated, the crosslinking agent may then be added (optionally with an alkaline oxide) to render the fluid loss pill. In an embodiment, thermal stability of the fluid loss pill may be enhanced by hydrating the galactomannan with a fluid containing the brine and a slurry of linear viscosifying polymer in glycerol. The fluid containing brine and slurry is then contacted with the initial slurry containing the linear viscosifying polymer in glycol, galactomannan, optional dispersant and/or hydrophobic organic/hydrophilic organic liquid. Hydration of the galactomannan in the brine then occurs. Typically, the linear viscosifying polymer in the brine-containing fluid is the same as the linear viscosifying polymer in the initial slurry. After being pumped into the well, the fluid loss pill may slowly further gel as crosslinking of the galactomannan with the crosslinking agent occurs. Such further crosslinking occurs upon the fluid loss pill being situated at its desired location within the well The fluid loss pill brines disclosed herein may be used in any field of oil/gas technology that requires use of a fluid loss pill. As such, the fluid loss pill may be a component of an acid diverting system, fracturing fluid, completion fluid, workover fluid, drill-in fluid, insulating packer fluid, displacement spacer, cementing cleaning spacer, gravel pack carrier fluid, drilling mud, a coil tubing fluid for clean out, etc. Further, the viscous brines may be used to clean sand and silt deposits hampering the productivity of a producing well. In an embodiment, the fluid loss pill may be selectively emplaced in the wellbore. A downhole anemometer or similar tool may be used to detect fluid flow downhole and thus where fluid may be lost to the formation. The relative location of the fluid loss may be determined by conventional methods. In an embodiment, the fluid loss pill may be injected into a work string, flow to the bottom of the wellbore, and then out of the work string and into the annulus between the work string and the casing or wellbore. The pill may also be pushed by injection of other wellbore fluids, such as completion fluids, behind the pill to the site where fluid loss is suspected. Injection of fluids into the wellbore may then be stopped, as the pill then moves toward or into the fluid loss location. The fluid loss pill may then form a plug near or at the surface where fluid loss occurs. Fluid flow into the formation is thereby reduced. While the above embodiments describe the brine fluid for use as a fluid loss pill, the brine fluid may be used for other applications such as killing a well. Another application is to introduce the fluid loss pill during pre-conditioning of the well, especially in high-permeable reservoirs prior to injecting a polymer-based fluid. The injection of the fluid loss pill minimizes the volume of polymer invasion into the formation before the polymer-based fluid forms a filter cake. Consequently, formation damage associated with the stimulation of highly permeable formations is reduced. EXAMPLES The following examples are illustrative of some of the embodiments of the present invention. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the description set forth herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow. All percentages set forth in the Examples are given in terms of volume percentages except as may otherwise be indicated. Example 1. A slurry was prepared containing about 55 ml glycerol and 0.1 g of a sodium salt of sulfonated acrylamide and vinyl lactam, commercially available as Diacel® HE® Polymer 400 from Chevron Phillips Chemical Company LP. About 3 ml of sorbitan trioleate was added and then about 20 g of hydroxpropyl guar followed by about 14 mls dipropylene glycol n-butyl ether and about 14 mls of dipropylene glycol methyl ether. Upon thickening, 20 ml of the resulting slurry was contacted with about 350 ml of calcium bromide brine (density of 14.2 ppg). Viscosity was increased by the addition of approximately 5 pounds per barrel (ppb) MgO followed by about 5 ppb of puffed boron. Fluid loss was then tested in an OFITE filter pressure with a permeable 40μ aloxite disk at a temperature of 300° F. and using a top pressure of 600 psi and a bottom pressure of 100 psi. Volume of fluid loss over time was recorded. Testing was complete when the pressure equalized after 1260 minutes (21 hours). The results are shown in Table I. TABLE ITime, minutesVolume, millilitersSpurt44148554105715613062607212010318011524012012001761260206 Example 2. The procedure of Example 1 was repeated using a 11.0 ppg calcium bromide brine. Testing was conducted using a permeable 35μ aloxite disk at a temperature of 275° F. Testing was complete when the pressure equalized after 1298 minutes. The results are shown in Table II. TABLE IITime, minutesVolume, millilitersSpurt5916156410651569307460831209518010624011712602341298276 The spurt in Examples 1 and 2 forms a filter cake. Tables I and II demonstrate the filter cake starts to disintegrate in about 21 hours or greater. The data demonstrates the fluid loss pill controls fluid loss for at least 21 hours (pressure equalization in Tables I and II). Example 3. A slurry was prepared from about 55 mls glycerol and about 0.1 grams of sodium salt of sulfonated acrylamide and vinyl lactam. About 3 ml of sorbitan trioleate was added to the slurry followed by about 20 grams of hydroxypropyl guar. As thickening occurred, about 14 mls dipropylene glycol n-butyl ether (hydrophobic glycol ether) and 14 mls dipropylene glycol methyl ether (hydrophilic glycol ether) was added. The hydroxypropyl guar was then hydrated by the addition of about 20 mls of the resulting slurry to about 220 mls of calcium bromide brine (density of 14.2 ppg). The calcium bromide brine further contained a slurry of about 9 ppb HE 400 in 105 mls glycerol. The amount of the slurry in the brine was 105 mls. Upon thickening, magnesium oxide was added (approximately 5 pounds per barrel (ppb) followed by about 5 ppb puffed boron. Then about 95 g of dry calcium bromide was added to provide a final density of 14.2 ppg. Fluid loss was then tested in an OFITE high temperature high pressure fluid loss cell with a 35 micron aloxite disk at a temperature of 325° F. and using a top pressure of 600 psi and a bottom pressure of 100 psi. Volume of fluid loss over time was recorded. Testing was complete when the pressure equalized after 1260 minutes (21 hours). The results are shown in Table III. TABLE IIITime, minutesVolume, millilitersSpurt3313854410482052305660791201201801492401683751941395211As illustrated in Table III, the fluid loss pill was stable up to 1395 minutes at 325° F.As illustrated in Table III, the fluid loss pill was stable up to 1395 minutes at 325° F. While the invention has been described with respect to a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the invention as disclosed herein. Accordingly, the scope of the invention should be limited only by the following claims. | 30,638 |
11859127 | DETAILED DESCRIPTION OF THE INVENTION Various preferred features and embodiments will be described below by way of non-limiting illustration. Composition The compositions herein will include carbon dioxide. Carbon dioxide consists of two oxygen atoms covalently bonded to a single carbon atom. Carbon dioxide can exist as a solid, liquid, gas, or, at temperatures above its critical point, as a supercritical fluid. Supercritical fluids are those that exhibit properties of both liquids, such as the ability to dissolve other substances, and of gases, such as the ability to effuse through solids. As a supercritical fluid, carbon dioxide has the ability to mix homogeneously, or in other words is miscible with, hydrocarbons, such as crude oil, and can therefore improve the recovery of such hydrocarbons. The carbon dioxide composition will also include at least one branched polyolefin polymer. Polyolefins are well known in the art. In one embodiment, the polyolefin employed in the carbon dioxide composition may be derivable (or derived) from olefins with 2 to 24 carbon atoms. As used herein, the term “olefin” refers to an unsaturated hydrocarbon compound having a hydrocarbon chain containing at least one carbon-to-carbon double bond in the structure thereof, wherein the carbon-to-carbon double bond does not constitute a part of an aromatic ring. The olefin may be straight-chain, branched-chain or cyclic. “Olefin” is intended to embrace all structural isomeric forms of olefins, unless it is specified to mean a single isomer or the context clearly indicates otherwise. By derivable or derived it is meant the polyolefin is polymerized from the starting polymerizable olefin monomers having the noted number of carbon atoms or mixtures thereof. In embodiments, the polyolefin employed in the carbon dioxide composition may be derivable (or derived) from olefins with 3 to 24 carbon atoms. In some embodiments, the polyolefin employed in the carbon dioxide composition may be derivable (or derived) from olefins with 4 to 24 carbon atoms. In further embodiments, the polyolefin employed in the carbon dioxide composition may be derivable (or derived) from olefins with 5 to 20 carbon atoms. In still further embodiments, the polyolefin employed in the carbon dioxide composition may be derivable (or derived) from olefins with 6 to 18 carbon atoms. In still further embodiments, the polyolefin employed in the carbon dioxide composition may be derivable (or derived) from olefins with 8 to 14 carbon atoms. In alternate embodiments, the polyolefin employed in the carbon dioxide composition may be derivable (or derived) from olefins with 8 to 12 carbon atoms. As used herein, the term “carbon backbone” of a polyolefin is defined as the straight carbon chain therein having the largest number of carbon atoms. As used herein, the term “branching group” with respect to a polyolefin refers to any group other than hydrogen attached to the carbon backbone of the polyolefin, other than those attached to the carbon atoms at the very ends of the carbon backbone. Often the polymerizable olefin monomers comprise one or more of propylene, isobutene, 1-butene, isoprene, 1,3-butadiene, or mixtures thereof. An example of a useful polyolefin is polyisobutylene. Polyolefins also include poly-α-olefins derivable (or derived) from α-olefins. As used herein, the term “alpha-olefin” refer to an olefin having a terminal carbon-to-carbon double bond ((R1R2)—C═CH2) in the structure thereof. As used herein, “polyalpha-olefin(s)” (“PAO(s)”) includes any oligomer(s) and polymer(s) of one or more alpha-olefin monomer(s). PAOs are oligomeric or polymeric molecules produced from the polymerization reactions of alpha-olefin monomer molecules in the presence of a catalyst system, optionally further hydrogenated to remove residual carbon-carbon double bonds therein. Thus, the PAO can be a dimer, a trimer, a tetramer, or any other oligomer or polymer comprising two or more structure units derived from one or more alpha-olefin monomer(s). The PAO molecule can be highly regio-regular, such that the bulk material exhibits an isotacticity, or a syndiotacticity when measured by13C NMR. The PAO molecule can be highly regio-irregular, such that the bulk material is substantially atactic when measured by13C NMR. A PAO material made by using a metallocene-based catalyst system is typically called a metallocene-PAO (“mPAO”), and a PAO material made by using traditional non-metallocene-based catalysts (e.g., Lewis acids, supported chromium oxide, and the like) is typically called a conventional PAO (“cPAO”). The poly-α-olefins used herein may be mPAOs. The poly-α-olefins used herein may also be cPAOs. The α-olefins may be linear or branched or mixtures thereof. Examples include mono-olefins such as propylene, 1-butene, isobutene, 1-pentene, 1-hexene, 1-heptene, 1-octene, 1-nonene, 1-decene, etc. Other examples of α-olefins include 1-decene, 1-undecene, 1-dodecene, 1-tridecene, 1-tetradecene, 1-pentadecene, 1-hexadecene, 1-heptadecene 1-octadecene, and mixtures thereof. Other examples of α-olefins include 1-nonadecene, 1-eicosene, 1-heneicosene, 1-docosene, 1-tricosene, 1-tetracosene in yet another embodiment. Preferred LAO feeds are 1-hexene, 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene and 1-octadecene. Examples of preferred α-olefin mixtures as monomers for making the poly-α-olefins include, but are not limited to: C6/C8; C6/C10; C6/C12; C6/C14; C6/C16; C6/C8/C10; C6/C8/C12; C6/C8/C14; C6/C8/C16; C8/C10; C8/C12; C8/C14; C8/C16; C8/C10/C12; C8/C10/C14; C8/C10/C16; C10/C12; C10/C14; C10/C16; C10/C12/C14; C10/C12/C16; and the like. An example of a useful α-olefin is 1-decene. An example of a useful poly-α-olefin is poly-decene. The polyolefin may also be a copolymer of at least two different olefins, also known as an olefin copolymer (OCP). These copolymers are preferably copolymers of α-olefins having from 2 to about 28 carbon atoms, preferably copolymers of ethylene and at least one α-olefin having from 3 to about 28 carbon atoms, typically of the formula CH2═CHR1wherein R1is a straight chain or branched chain alkyl radical comprising 1 to 22 carbon atoms. Preferably R1in the above formula can be an alkyl of from 1 to 8 carbon atoms, and more preferably can be an alkyl of from 1 to 2 carbon atoms. The composition may be substantially free of ethylene and polymers thereof. The composition may be completely free of ethylene and polymers thereof. By substantially free, it is meant that the composition contains less than of the given material. In some embodiments, substantially free means less than 0.005 wt. % of the given material. Substantially free can also mean less than 1000 ppm of the given material. In some embodiments, substantially free means less than 500 ppm of the given material. Substantially free can also mean less than 250 ppm of the given material. In some embodiments, substantially free means less than 100 ppm of the given material. Substantially free can also mean less than 50 ppm of the given material. In some embodiments, substantially free means less than 30 ppm of the given material. Substantially free can also mean less than 10 ppm, or less than 5 ppm, or even less than 1 ppm of the given material. The composition may be substantially free of propylene and polymers thereof. The composition may be completely free of propylene and polymers thereof. The polyolefin polymers prepared from the aforementioned olefin monomers can have a number average molecular weight of from 140 to 5000. The polyolefin polymers prepared from the aforementioned olefin monomers can also have a number average molecular weight of from 200 to 4750. The polyolefin polymers prepared from the aforementioned olefin monomers can also have a number average molecular weight of from 250 to 4500. The polyolefin polymers prepared from the aforementioned olefin monomers can also have a number average molecular weight of from 500 to 4500. The polyolefin polymers prepared from the aforementioned olefin monomers can also have a number average molecular weight of from 750 to 4000 as measured by gel permeation chromatography with a polystyrene standard. Some of the polyolefins will include branching by virtue of the structure of the polymer. For example, the polymerization of α-olefins along the α bond results in the tails of these monomers branching along the polymerized α olefin. The longer the α-olefin, the longer the branches off of the resultant polyolefin. Polydecene for example can result in a polymer having branches of 8 carbon atoms. A typical, hydrogenated PAO molecule can be represented by the following formula I: where R1, R2, R3, each of R4and R5, R6, and R7,the same or different at each occurrence, independently represents a hydrogen or a substituted or unsubstituted hydrocarbyl (preferably an alkyl) group, and n is an non-negative integer corresponding to the degree of polymerization. Thus, where n=0, formula I represents a dimer produced from the reaction of two monomer molecules after a single addition reaction between two carbon-carbon double bonds. Where n=m, m being a positive integer, formula I represents a molecule produced from the reactions of m+2 monomer molecules after m+1 steps of addition reactions between two carbon-carbon double bonds. Thus, where n=1, formula I represents a trimer produced from the reactions of three monomer molecules after two steps of addition reactions between two carbon-carbon double bonds. Assuming a straight carbon chain starting from R1and ending with R7has the largest number of carbon atoms among all straight carbon chains existing in formula I, then the straight carbon chain starting from R1and ending with R7having the largest number of carbon atoms constitutes the carbon backbone of the poly-α-olefin molecule formula I. R2, R3, each of R4and R5, and R6, which can be substituted or unsubstituted hydrocarbyls (preferably alkyl) groups, are branching groups (if not hydrogen). If only alpha-olefin monomers are used in the polymerization process, and no isomerization of the monomers and oligomers ever occurs in the reaction system during polymerization, about half of R1, R2, R3, all R4and R5, R6, and R7would be hydrogen, and one of R1, R2, R6, and R7would be a methyl, and about half of groups R1, R2, R3, all R4and R5, R6, and R7would be hydrocarbyl groups introduced from the alpha-olefin monomer molecules. In a specific example of such case, assuming R2is methyl, R3, all R5, and R6are hydrogen, and R1, all R4, and R7have 8 carbon atoms in the longest carbon chains contained therein, and n=8, then the carbon backbone of the formula I PAO molecule would comprise 35 carbon atoms, and the average branching group length of the branching groups (R2, all of R4) would be 7.22 (i.e., (1+8*8)/9). This PAO molecule, which can be produced by polymerizing 1-decene using certain metallocene catalyst systems described in greater detail below, can be represented by formula II below: Depending on the polymerization catalyst system used, however, different degrees of isomerization of the monomers and/or oligomers can occur in the reaction system during the polymerization process, resulting in different degrees of substitution on the carbon backbone. In a specific example of such case, assuming R2, R3, all R5are methyls, and R6is hydrogen, le has 8 carbon atoms in the longest straight carbon chain contained therein, and all R4and R7have 7 carbon atoms in the longest straight carbon chain contained therein, and n=8, then the carbon backbone of the formula I PAO molecule would comprise 34 carbon atoms, and the average branching group length of the branching groups (R2, all R4, and R5) would be 3.67 (i.e., (1+1+7*8+1*8)/18). This PAO molecule, which may be produced by polymerizing 1-decene using certain non-metallocene catalyst systems described in greater detail below, can be represented by the following formula III: PAO base stocks useful for the present invention may be a homopolymer made from a single alpha-olefin monomer or a copolymer made from a combination of two or more alpha-olefin monomers. The branching groups on the PAO molecules can be straight chain alkyls having at least 6 carbon atoms. The branching groups on the PAO molecules can be straight chain alkyls having at least 8 carbon atoms. In one embodiment, there is provided a 1000 to 5000 molecular weight branched PAO polymer, such as a polydecene polymer. The branched PAO polymer, such as polydecene, can also have a number average molecular weight of from 1250 to 4750. The branched PAO polymer, such as polydecene, can also have a number average molecular weight of from 1500 to 4500. The branched PAO polymer, such as polydecene, can have a number average molecular weight of from 2000 to 4250. The branched PAO polymer, such as polydecene, can also have a number average molecular weight of from 2500 to 4000 as measured by gel permeation chromatography with a polystyrene standard. The polyolefins may also be functionalized with substituents to add branching along the polyolefin backbone. For example, the polyolefin may be functionalized with at least one of an aromatic hydrocarbyl group, aliphatic hydrocarbyl group, cyclic hydrocarbyl group, and mixtures thereof, so that the branching of the branched polyolefin polymer includes at least one of an aromatic hydrocarbyl group, aliphatic hydrocarbyl group, cyclic hydrocarbyl group, and mixtures thereof. In some embodiments, the polyolefin may be functionalized with at least one aromatic hydrocarbyl group so that the branching of the branched polyolefin polymer includes at least one aromatic hydrocarbyl group. The aromatic hydrocarbyl group may be, for example, a hydroxyl containing aromatic group, such as, for example, a phenol group, an amine containing aromatic group, such as, for example, aniline, and mixtures thereof. Other aromatic groups can include, for example, phenylmethylene; o-heptyl-phenylmethylene; and p-heptylphenylmethylene; aniline and alkyl anilines; indole and alkyl indoles; quinoline and alkyl quinoline; isoquinoline and alkyl isoquinoline; pyrazine and alkyl pyrazine; quinoxaline and alkyl quinoxaline; acridine and alkyl acridine; pyrimidine and alkyl pyrimidine; quinazoline and alkyl quinazoline. The aromatic group can also be a polyaromatic group, such as, for example, naphthalene, naphthol or other homologues of phenol with fused aromatic rings, naphthylamine or other homologues of aniline. In an embodiment, the aromatic group is a hydroxyl containing aromatic group. In an embodiment, the aromatic group is a phenol group. In an embodiment, the aromatic group is an amine containing aromatic group. In an embodiment, the aromatic group is a hydroxyl and amine containing aromatic group. In an embodiment, the aromatic group is a 2-((dimethylamino)methyl)phenol group. In some embodiments, the polyolefin may be functionalized with at least one aliphatic hydrocarbyl group, so that the branching of the branched polyolefin polymer includes at least one aliphatic hydrocarbyl group. In some embodiments, the polyolefin may be functionalized with at least one cyclic hydrocarbyl group, so that the branching of the branched polyolefin polymer includes at least one cyclic hydrocarbyl group. An example cyclic group includes, for example, cyclohexylmethylene. Other cyclic groups can include heterocyclic groups, such as, for example, pyridines and alkyl pyridines, pyrrole and alkyl pyrroles, piperidine and alkyl piperidines, pyrrolidine and alkyl pyrrolidines, imidazole and alkyl imidazole. Other cyclic groups can include, in particular, vinyl-pyridine and/or vinylimidazole, as well as styrene. In some embodiments, the polyolefin can be a polyisobutylene polymer with a number average molecular weight from 140 to 5000. The polyisobutylene polymer can also have a number average molecular weight of from 200 to 4500. The polyisobutylene polymer can also have a number average molecular weight of from 250 to 4000. The polyisobutylene polymer can have a number average molecular weight of from 300 to 3500. The polyisobutylene polymer can have a number average molecular weight of from 350 to 3000. The polyisobutylene polymer can also have a number average molecular weight of from 400 to 2500 as measured by gel permeation chromatography with a polystyrene standard. The carbon dioxide composition can include the branched polyolefin polymer at from 0.01 to 5 wt. % based on the weight of the composition. The carbon dioxide composition may include the branched polyolefin polymer at from 0.05 to 4.5 wt. % based on the weight of the composition. The carbon dioxide composition may also include the branched polyolefin polymer at from 0.1 to 4 wt. % based on the weight of the composition. The carbon dioxide composition could also include the branched polyolefin polymer at from 0.5 to 3.5 wt. % based on the weight of the composition. One purpose of the polyolefin polymer is to increase the viscosity of supercritical carbon dioxide. The absolute viscosity of supercritical carbon dioxide will vary depending on the temperature and pressure at which the viscosity is measured, but has been seen to be about 0.07 cP at 2000 psi and 0.09 cP at 2900 psi as measured by viscometer. The polyolefin polymer can be dosed into the supercritical carbon dioxide to increase the viscosity of the composition relative to the starting supercritical carbon dioxide viscosity, up to the point at which the supercritical carbon dioxide becomes un-flowable. In some embodiments, the polyolefin polymer can be dosed into the carbon dioxide composition to increase the relative viscosity of the composition by at least 100% which can also be referenced as 2 times or “2×;” meaning the absolute viscosity of the carbon dioxide composition is 100% greater than the absolute viscosity of the supercritical carbon dioxide on its own. For example, if the absolute viscosity of the supercritical carbon dioxide is 0.05, a relative viscosity 100% greater would be 0.05+(0.05)*100%=0.01, or 2 times. In some embodiments, the polyolefin polymer can be dosed into the carbon dioxide composition to increase the relative viscosity of the composition from about 100% to about the point at which the composition does not flow freely, such as about 1 or 2 or 3 or 3.5 or 4 or 4.5 or 5 orders of magnitude and more. As used herein “order of magnitude” means approximately a factor of 10. In some embodiments, the polyolefin polymer can be dosed into the carbon dioxide composition to increase the relative viscosity of the composition by at least 150% which can also be referenced as 2.5 times or “2.5×.” In some embodiments, the polyolefin polymer can be dosed into the carbon dioxide composition to increase the relative viscosity of the composition from about 150% to about the point at which the composition does not flow freely, such as about 1 or 2 or 3 or 3.5 or 4 or 4.5 or 5 orders of magnitude and more. In some embodiments, the polyolefin polymer can be dosed into the carbon dioxide composition to increase the relative viscosity of the composition by at least 200%, which can also be referenced as 3 times or “3×.” In some embodiments, the polyolefin polymer can be dosed into the carbon dioxide composition to increase the relative viscosity of the composition from about 200% to about the point at which the composition does not flow freely, such as about 1 or 2 or 3 or 3.5 or 4 or 4.5 or 5 orders of magnitude and more. In some embodiments, the polyolefin polymer can be dosed into the carbon dioxide composition to increase the relative viscosity of the composition by at least 250% or 3.5×. In some embodiments, the polyolefin polymer can be dosed into the carbon dioxide composition to increase the relative viscosity of the composition from about 250% to about the point at which the composition does not flow freely, such as about 1 or 2 or 3 or 3.5 or 4 or 4.5 or 5 orders of magnitude and more. In some embodiments, the polyolefin polymer can be dosed into the carbon dioxide composition to increase the relative viscosity of the composition by at least 300% or 4×. In some embodiments, the polyolefin polymer can be dosed into the carbon dioxide composition to increase the relative viscosity of the composition from about 300% to about the point at which the composition does not flow freely, such as about 1 or 2 or 3 or 3.5 or 4 or 4.5 or 5 orders of magnitude and more. Given the temperatures and pressures involved with obtaining supercritical carbon dioxide, measurements of absolute viscosity are difficult and may provide slightly differing results from well to well. However, when comparing viscosity between two samples out of the same well by the same measurement method (i.e., viscosity of supercritical carbon dioxide to viscosity of carbon dioxide composition containing the polyolefin polymer) the relative viscosity trends should be the same or similar between methods. Thus, the relative viscosity numbers herein may be arrived at by measure of the absolute viscosity of the comparable samples by any reasonable test method. One method may be to employ a viscometer. One useful measure to screen polyolefin polymers can be to check the solubility of the polymer in supercritical carbon dioxide. In general, the more soluble a second substance is in a first substance, the more available the second substance is to act on the first substance. While complete solubility is desired, a partially soluble polymer can also provide viscosity improvements. The solubility of the polymer can be measured by methods known in the art, such as, for example, by visual inspection or cloud point. In an embodiment, the solubility of the polyolefin polymer in the carbon dioxide composition can be measured by a sapphire rocking cell test. The sapphire rocking cell test employs an apparatus having two rocking cells generally of about 20 mL volume, each equipped with a stainless steel ball to aid agitation. Each cell is charged with a designated volume of the chosen branched polyolefin polymer and injected with carbon dioxide to a desired supercritical carbon dioxide pressure. The cells are then submerged in a constant temperature water bath. The cells are rocked in the water bath from a 45° angle to a −45° angle at a pre-determined rocking frequency, for example, 15 times/min. The water bath is brought to the desired temperature and the sapphire cells are observed for solubility of the polyolefin polymer in the supercritical carbon dioxide. If the polyolefin is completely soluble in the supercritical carbon dioxide at the given pressure and temperature, the supercritical mixture will appear homogeneous. Otherwise, separate phases will be observed in the cells. In some cases, the pressure actually necessitated for solubility of the hydrocarbons in the supercritical carbon dioxide could depend upon the minimum miscibility pressure (MMP) of the hydrocarbons present. The MMP may be found by simple experiment, using samples of the hydrocarbons from the reservoir and the carbon dioxide composition, which anyone skilled in the art would be readily able to perform. Method An aspect of the disclosed technology is the use of branched polyolefin polymers to thicken carbon dioxide. Thus, the technology provides a method of increasing the viscosity of supercritical carbon dioxide. The method can include adding to carbon dioxide a thickener. The thickener is at least one branched polyolefin polymer that increases the viscosity of supercritical carbon dioxide. The method can further include pressurizing the carbon dioxide composition at a temperature to cause the formation of supercritical carbon dioxide. The carbon dioxide composition described herein can be employed to sequester carbon dioxide in an underground formation, as well as to recover hydrocarbons from an underground hydrocarbon containing formation. Hydrocarbons can be recovered from an underground hydrocarbon containing formation or reservoir by injecting a solvent (carbon dioxide in this case) into the reservoir through an injection well and recovering hydrocarbon containing fluids from a production well which is at a horizontal distance or offset from the injection well. In practice, more than one injection well and more than one production well may be used and these may be arranged in a number of different patterns suitable for solvent drive operations of this kind. For simplicity, however, the present invention is described below with reference only to a single injection well and a single production well. The carbon dioxide composition as described herein should be injected under sufficient pressure so that under the conditions which prevail in the reservoir, the carbon dioxide in the composition is present as a dense phase, that is, it is under supercritical conditions and present neither as a liquid or a dense vapor. Generally, this will be achieved by maintaining pressure in the reservoir sufficiently high to maintain the carbon dioxide in the required dense phase state, i.e. at a density greater than approximately 0.468 g cm−3. This pressure, in itself, increases with increasing reservoir temperature and the pressure should therefore be chosen in accordance with reservoir temperature. The method of viscosity increase discussed herein can be employed at pressures of 500 psia or greater, such as up to 10,000 psia, or for example 750 to 6000 psia. Typical minimum pressures for maintaining the dense phase state are 900 psia at 85° F., 1200 psia at 100° F., 1800 psia at 150° F., 2500 psia at 200° F. and 3100 psia at 250° F. (6205 kPa at 30° C., 8275 kPa at 38° C., 12410 kPa at 65° C., 17235 kPa at 93° C. and 21375 kPa at 120° C.). Thus, the method of recovering hydrocarbons from an underground hydrocarbon containing formation can involve at least some, if not all, of the following steps, not necessarily in the following order:determining the temperature and pressure of the hydrocarbon formation;optionally, screening for a suitable branched polyolefin polymer by, for example, either determining the solubility of the at least one branched polyolefin polymer at the temperature and pressure encountered in the formation, which may be done, for example, by performing the sapphire rocking cell test, or determining the MMP of the hydrocarbons present;selecting the at least one branched polyolefin polymer;injecting into the hydrocarbon formation a carbon dioxide composition containing carbon dioxide and the at least one branched polyolefin polymer; andrecovering released hydrocarbons from the hydrocarbon containing formation. The amount of each chemical component described is presented exclusive of any solvent or diluent oil, which may be customarily present in the commercial material, that is, on an active chemical basis, unless otherwise indicated. However, unless otherwise indicated, each chemical or composition referred to herein should be interpreted as being a commercial grade material which may contain the isomers, by-products, derivatives, and other such materials which are normally understood to be present in the commercial grade. As used herein, the term “hydrocarbyl substituent” or “hydrocarbyl group” is used in its ordinary sense, which is well-known to those skilled in the art. Specifically, it refers to a group having a carbon atom directly attached to the remainder of the molecule and having predominantly hydrocarbon character. Examples of hydrocarbyl groups include: hydrocarbon substituents, that is, aliphatic (e.g., alkyl or alkenyl), alicyclic (e.g., cycloalkyl, cycloalkenyl) substituents, and aromatic-, aliphatic-, and alicyclic-substituted aromatic substituents, as well as cyclic substituents wherein the ring is completed through another portion of the molecule (e.g., two substituents together form a ring); substituted hydrocarbon substituents, that is, substituents containing nonhydrocarbon groups which, in the context of this invention, do not alter the predominantly hydrocarbon nature of the substituent (e.g., halo (especially chloro and fluoro), hydroxy, alkoxy, mercapto, alkylmercapto, nitro, nitroso, amino, and sulfoxy); hetero substituents, that is, substituents which, while having a predominantly hydrocarbon character, in the context of this invention, contain other than carbon in a ring or chain otherwise composed of carbon atoms and encompass substituents as pyridyl, furyl, thienyl and imidazolyl. Heteroatoms include sulfur, oxygen, and nitrogen. In general, no more than two, or no more than one, nonhydrocarbon substituent will be present for every ten carbon atoms in the hydrocarbyl group; alternatively, there may be no non-hydrocarbon substituents in the hydrocarbyl group. It is known that some of the materials described above may interact in the final formulation, so that the components of the final formulation may be different from those that are initially added. For instance, metal ions (of, e.g., a detergent) can migrate to other acidic or anionic sites of other molecules. The products formed thereby, including the products formed upon employing the composition of the present invention in its intended use, may not be susceptible of easy description. Nevertheless, all such modifications and reaction products are included within the scope of the present invention; the present invention encompasses the composition prepared by admixing the components described above. EXAMPLES Sample 1—un-hydrogenated decene dimer of 280 number average molecular weight Sample 2—un-hydrogenated metallocene poly-decene of 2300 number average molecular weight Sample 3—un-hydrogenated metallocene poly-decene of 2900 number average molecular weight Sample 4—polyolefin phenol derived from polyisobutylene of 590 number average molecular weight Sample 5—polyolefin phenol derived from polyisobutylene of 950 number average molecular weight Sample 6—Mannich reaction product of sample 5 and dimethylamine Sample 7—reaction product of polyisobutylene succinic anhydride and aromatic amine Sample 8—reaction product of polyisobutylene succinic anhydride, maleinated ethylene/propylene copolymer and aromatic amine Sample 9—reaction product of maleinated ethylene/propylene copolymer and aromatic amine Sample 10—maleinated product of sample 2 Sample 11—reaction product of Sample 10 and polyethylene polyamine Sample 12—polyisobutylene of 2700 number average molecular weight Sample 13—polyisobutylene of 2060 number average molecular weight Sample 14—polyisobutylene of 1000 number average molecular weight Sample 15—polyisobutylene of 1000000 number average molecular weight Each of Samples 1 to 15 were tested for solubility in carbon dioxide in the sapphire rocking cell test. The designated weight of Sample polymer was charged into two separate rocking cells containing 20 mL carbon dioxide, each cell containing a stainless steel ball to aid agitation. The cells were charged to 2500 psi and then submerged in a constant temperature water bath at 35° C. The cells were rocked in the water bath from a 45° angle to a −45° angle at a rocking frequency of 15 times/min and observed for solubility of the Sample polymer in the supercritical carbon dioxide. If the Sample was soluble, the supercritical mixture appeared homogeneous. Otherwise, separate phases were observed in the cells. Solubility of polymer at specific concentration withrespect to CO2 at 2500 psi, 35° C.Polymer/wt. % in CO20.50.7511.51.752Sample 1——NoNo——Sample 2YesYesYesYes——Sample 3YesYesYesNo——Sample 4——YesYesYesYesSample 5——YesYesYesNoSample 6Yes—YesNo—NoSample 7No————NoSample 8No————NoSample 9No————NoSample 10No————NoSample 11No—YesNo—NoSample 12No—No———Sample 13No—No———Sample 14No—No———Sample 15No—No———“—” means not tested A ViscoPro2100 moving piston viscometer unit was used to measure the viscosity of blends of each of the samples with carbon dioxide. The viscometer unit was connected in line with the rocking cell setup. There were 2 cells with valves, and as both valves were opened, the mixture of CO2and Sample polymer was passed from the rocking cell setup to the viscometer. The ViscoPro 2100 viscometer consists of a sensor in which a piston is moved from one end of the sensor to the other with the help of electromagnetic coils. The sensor also consists of a temperature probe. Once solubility of a Sample polymer in CO2was achieved in the rocking cell, the valve connecting the rocking cell to the viscometer was opened, allowing the fluid to pass into the viscometer sensor. Temperature control was achieved in the lines connecting the viscometer and rocking cell setup with the help of heating bands. Real time readings of viscosity and temperature of the fluid in the sensor were provided. The results of the measurements and the pressures at which each measurement was taken are provided in the table below. wt. %ViscosityRelativePolymerin CO2(cP)viscosity2000 psiBlank—0.071Sample 310.324.5Sample 410.162.2Sample 510.22.82500 psiBlank—0.081Sample 10.750.172.1Sample 210.212.6Sample 310.394.9Sample 410.22.5Sample 510.243Sample 610.243Sample 71*1Sample 101*1.6Sample 141*1.52900 psiBlank—0.091Sample 310.455.1*complete single phase was not achieved Each of the documents referred to above is incorporated herein by reference, including any prior applications, whether or not specifically listed above, from which priority is claimed. The mention of any document is not an admission that such document qualifies as prior art or constitutes the general knowledge of the skilled person in any jurisdiction. Except in the Examples, or where otherwise explicitly indicated, all numerical quantities in this description specifying amounts of materials, reaction conditions, molecular weights, number of carbon atoms, and the like, are to be understood as modified by the word “about.” It is to be understood that the upper and lower amount, range, and ratio limits set forth herein may be independently combined. Similarly, the ranges and amounts for each element of the invention can be used together with ranges or amounts for any of the other elements. As used herein, the transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, un-recited elements or method steps. However, in each recitation of “comprising” herein, it is intended that the term also encompass, as alternative embodiments, the phrases “consisting essentially of” and “consisting of,” where “consisting of” excludes any element or step not specified and “consisting essentially of” permits the inclusion of additional un-recited elements or steps that do not materially affect the essential or basic and novel characteristics of the composition or method under consideration. There is provided a carbon dioxide composition comprising a major amount of carbon dioxide and at least one branched polyolefin polymer. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity at least 100% greater than supercritical carbon dioxide. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity at least 150% greater than supercritical carbon dioxide. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity at least 200% greater than supercritical carbon dioxide. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity at least 300% greater than supercritical carbon dioxide. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 100% to about 1 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 100% to about 2 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 100% to about 3 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 100% to about 3.5 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 100% to about 4 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 100% to about 5 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 150% to about 5 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 150% to about 4 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 150% to about 3 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 200% to about 5 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 200% to about 4 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 200% to about 3 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 250% to about 5 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 250% to about 4 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 250% to about 3 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 300% to about 5 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 300% to about 4 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the composition has a relative viscosity from about 300% to about 3 orders of magnitude. The composition of any sentence in any previous paragraph, wherein the polyolefin polymer has a number average molecular weight of from 140 to 5000 as measured by gel permeation chromatography with polystyrene standard. The composition of any sentence in any previous paragraph, wherein the polyolefin polymer has a number average molecular weight of from 200 to 4750 as measured by gel permeation chromatography with polystyrene standard. The composition of any sentence in any previous paragraph, wherein the polyolefin polymer has a number average molecular weight of from 250 to 4500 as measured by gel permeation chromatography with polystyrene standard. The composition of any sentence in any previous paragraph, wherein the polyolefin polymer has a number average molecular weight of from 500 to 4500 as measured by gel permeation chromatography with polystyrene standard. The composition of any sentence in any previous paragraph, wherein the polyolefin polymer has a number average molecular weight of from 750 to 4000 as measured by gel permeation chromatography with polystyrene standard. The composition of any sentence in any previous paragraph, wherein the polyolefin polymer has a number average molecular weight of from 1000 to 5000 as measured by gel permeation chromatography with polystyrene standard. The composition of any sentence in any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from a C2-C24 olefin or mixture thereof. The composition of any sentence in any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from a C3-C24 olefin or mixture thereof. The composition of any sentence in any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from a C4-C24 olefin or mixture thereof. The composition of any sentence in any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from a C5-C20 olefin or mixture thereof. The composition of any sentence in any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from a C6-C18 olefin or mixture thereof. The composition of any sentence in any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from a C8-C14 olefin or mixture thereof. The composition of any sentence in any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from a C8-C12 olefin or mixture thereof. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a propylene polymer. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises an isobutene polymer. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a 1-butene polymer. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises an isoprene polymer. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a 1,3-butadiene polymer. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a polyisobutylene polymer. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a polyisobutylene polymer having a number average molecular weight from 140 to 5000. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a polyisobutylene polymer having a number average molecular weight of from 200 to 4500. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a polyisobutylene polymer having a number average molecular weight of from 250 to 4000. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a polyisobutylene polymer having a number average molecular weight of from 300 to 3500. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a polyisobutylene polymer having a number average molecular weight of from 350 to 3000. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a polyisobutylene polymer having a number average molecular weight of from 400 to 2500 as measured by gel permeation chromatography with a polystyrene standard The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from C4-C24 α-olefin or mixture thereof. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-pentene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-hexene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-heptene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-octene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-nonene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-decene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-decene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-undecene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-dodecene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-tridecene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-tetradecene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-pentadecene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-hexadecene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-heptadecene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-octadecene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-nonadecene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-eicosene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-heneicosene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-docosene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-tricosene. The composition of any sentence of any previous paragraph, wherein the at least one branched polyolefin polymer is polymerized from 1-tetracosene. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a 1000 to 5000 Mn polydecene polymer as measured by gel permeation chromatography with a polystyrene standard. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a 1250 to 4750 Mn polydecene polymer as measured by gel permeation chromatography with a polystyrene standard. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a 1500 to 4500 Mn polydecene polymer as measured by gel permeation chromatography with a polystyrene standard. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a 2000 to 4250 Mn polydecene polymer as measured by gel permeation chromatography with a polystyrene standard. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises a 2500 to 4000 Mn polydecene polymer as measured by gel permeation chromatography with a polystyrene standard. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of any of the polymers in the preceding sentences. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C6 and C8 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C6 and C10 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C6 and C12 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C6 and C14 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C6 and C16 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C6, C8 and C10 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C6, C8 and C12 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C6, C8 and C14 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C6, C8 and C16 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C8 and C10 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C8 and C12 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C8 and C14 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C8 and C16 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C8, C10 and C12 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C8, C10 and C14 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C8, C10 and C16 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C10 and C12 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C10 and C14 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C10 and C16 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C10, C12 and C14 α-olefin. The composition of any sentence of any previous paragraph, wherein the branched polyolefin polymer comprises mixtures of C10, C12 and C16 α-olefin. The composition of any sentence of any previous paragraph, wherein the branching of the branched polyolefin polymer comprises an aromatic hydrocarbyl group. The composition of any sentence of any previous paragraph, wherein the branching of the branched polyolefin polymer comprises a hydroxyl containing aromatic group. The composition of any sentence of any previous paragraph, wherein branching of the branched polyolefin polymer comprises an amine containing aromatic group. The composition of any sentence of any previous paragraph, wherein the branching of the branched polyolefin polymer comprises an aliphatic hydrocarbyl group. The composition of any sentence of any previous paragraph, wherein the branching of the branched polyolefin polymer comprises a cyclic hydrocarbyl group. The composition of any sentence of any previous paragraph, wherein the branching of the branched polyolefin polymer comprises mixture of any of the foregoing hydrocarbyl groups. The composition of any sentence of any previous paragraph, wherein the branching of the branched polyolefin polymer is substantially free of, or free of, succinimide or succinic anhydride functionality. The composition of any sentence of any previous paragraph, comprising from 0.01 to 5 wt. % of the branched polyolefin polymer based on the weight of the composition. The composition of any sentence of any previous paragraph, comprising from 0.05 to 4.5 wt. % of the branched polyolefin polymer based on the weight of the composition. The composition of any sentence of any previous paragraph, comprising from 0.1 to 4 wt. % of the branched polyolefin polymer based on the weight of the composition. The composition of any sentence of any previous paragraph, comprising from 0.5 to 3.5 wt. % of the branched polyolefin polymer based on the weight of the composition. A method to increase the production of hydrocarbons from an underground hydrocarbon containing formation comprising injecting into the formation a composition as claimed in any sentence of any previous paragraph, and recovering released hydrocarbons from said hydrocarbon containing formation. A method of increasing the viscosity of supercritical carbon dioxide comprising adding to the carbon dioxide a thickener comprising at least one branched polyolefin polymer that increases the relative viscosity of the combination of the carbon dioxide and thickener at least 100% compared to supercritical carbon dioxide. The method of the previous paragraph wherein the viscosity is increased at a pressure between 500 and 10,000 psi and temperatures between 30 C and 120 C. The method of the previous paragraph wherein the viscosity is increased at a pressure between 750 and 6,000 psi and temperatures between 30 C and 120 C. While certain representative embodiments and details have been shown for the purpose of illustrating the subject invention, it will be apparent to those skilled in this art that various changes and modifications can be made therein without departing from the scope of the subject invention. In this regard, the scope of the invention is to be limited only by the following claims. | 53,797 |
11859128 | DETAILED DESCRIPTION The present disclosure provides aqueous treatment fluids and methods for their use in aqueous brines. The aqueous treatment fluids utilize an advantageous combination of a water soluble polymer, which can be anionic, cationic, amphoteric or non-ionic, and an inverting surfactant composition which comprises one or more surfactants selected from the group consisting of ethoxylated amine compounds, ethoxylated fatty acid compounds, and alkyl polyethyleneglycol ether carboxylic acid compounds, alkyl polyglycol ether carboxylic acid compounds, and salts or esters thereof. The exemplary emulsions, treatment fluids and methods may be used to provide rapid and enhanced friction reduction in aqueous brines, for example in harsh brine conditions. The exemplary emulsions, treatment fluids and methods may be used at a range of temperatures, even at low temperatures, without loss of polymer performance. In exemplary embodiments, the emulsions, treatment fluids and methods can be used to carry proppants into fractures, for example in fracturing applications. High molecular weight polyacrylamides are commonly used in fracturing applications as a friction reducer. Generally, crosslinked fluids are used to carry proppants into the fractures, which typically requires additional chemicals, such as crosslinkers, buffers and breakers, to be incorporated into the fracturing fluid. In exemplary embodiments, the emulsions and treatment fluid can be used to carry proppant while minimizing the use of other chemicals or additives typically required by crosslinked fluids. In slickwater fracturing, the water is made slick by adding friction reducer. Slickwater frac fluids typically have low viscosities; a higher injection flow rate can be used to carry proppant. The exemplary emulsions, treatment fluids and methods can be used in slickwater fracturing applications. Advantageously, the exemplary emulsions and treatment fluids can be used in high brines with very fast inversion of the emulsion, very good friction reduction and with good proppant carrying capabilities at higher loadings. Polymers As used herein, the terms “polymer,” “polymers,” “polymeric,” and similar terms are used in their ordinary sense as understood by one skilled in the art, and thus may be used herein to refer to or describe a large molecule (or group of such molecules) that contains recurring units. Polymers may be formed in various ways, including by polymerizing monomers and/or by chemically modifying one or more recurring units of a precursor polymer. A polymer may be a “homopolymer” comprising substantially identical recurring units formed by, e.g., polymerizing a particular monomer. A polymer may also be a “copolymer” comprising two or more different recurring units formed by, e.g., copolymerizing two or more different monomers, and/or by chemically modifying one or more recurring units of a precursor polymer. A polymer may also be a “terpolymer” which comprises three or more different recurring units. The term “polymer” as used herein is intended to include both the acid form of the polymer as well as its various salts. In exemplary embodiments, the polymer is an emulsion polyacrylamide, for example an emulsion polyacrylamide that can be used as a friction-reducing polymer. The term “friction reducing polymer” refers to a polymer that reduces energy losses due to friction between an aqueous fluid in turbulent flow and tubular goods, e.g. pipes, coiled tubing, and the like, and/or formation. The friction reducing polymer is not intended to be limited to any particular type and may be synthetic polymers, natural polymers, or viscoelastic surfactants. Suitable friction reducing polymers are typically latex polymers or copolymers of acrylamides, acrylates, guar gum, polyethylene oxide, and combinations thereof. The friction reducing polymers may be anionic, cationic, amphoteric or non-ionic depending on desired application. In addition, various combinations can be used including but not limited to hydrophilic/hydrophobic combinations, functionalized natural and/or synthetic blends of the above, or the like. In certain exemplary embodiments, the friction reducing polymer is anionic. In certain exemplary embodiments, the friction reducing polymer is cationic. In certain exemplary embodiments, the friction reducing polymer is non-ionic. In certain exemplary embodiments, the friction reducing polymer is amphoteric. In exemplary embodiments, the polymer is a polymer useful in emulsion compositions or an emulsion polymer. In exemplary embodiments, the polymer is an emulsion polyacrylamide (EPAM). EPAMs are generally inverse emulsions (water-in-oil) in which water droplets containing the polymer are suspended in an oil phase. In exemplary embodiments, the polymer is a polymer useful for enhanced oil recovery applications. The term “enhanced oil recovery” or “EOR” (also known as tertiary mineral oil production) refers to a process for mineral oil production in which an aqueous injection fluid comprising at least a water soluble polymer is injected into a mineral oil deposit. The techniques of tertiary mineral oil production include what is known as “polymer flooding”. Polymer flooding involves injecting an aqueous solution of a water-soluble thickening polymer through the injection boreholes into the mineral oil deposit. As a result of the injection of the polymer solution, the mineral oil is forced through the cavities in the formation, proceeding from the injection borehole, in the direction of the production borehole, and the mineral oil is produced through the production borehole. By virtue of the fact that the polymer formulation has an increased viscosity as compared to the viscosity of water, the risk is reduced that the polymer formulation breaks through to the production borehole. It is thus possible to mobilize additional mineral oil in the formation. Details of polymer flooding and of polymers suitable for this purpose are disclosed, for example, in “Petroleum, Enhanced Oil Recovery, Kirk-Othmer, Encyclopedia of Chemical Technology, online edition, John Wiley & Sons, 2010”. For polymer flooding, a multitude of different water-soluble thickening polymers have been proposed, especially high molecular weight polyacrylamide, copolymers of acrylamide and further comonomers, for example vinylsulfonic acid or acrylic acid. Polyacrylamide may be partly hydrolyzed polyacrylamide, in which some of the acrylamide units have been hydrolyzed to acrylic acid. It is known in the art to use inverse emulsions of polyacrylamide (co)polymers for enhanced oil recovery (EOR) in particular for use on off-shore platforms. Such inverse emulsions typically comprise about 30 wt. % of polymers. For use inverse emulsions are simply diluted with water to the final concentration of the polymer. In exemplary embodiments, the one or more polymers is water soluble. In exemplary embodiments, the one or more polymers comprises an acrylamide-containing polymer. In exemplary embodiments, the one or more polymers consists essentially of acrylamide-containing polymers. In exemplary embodiments, the one or more polymers comprises polyacrylamide, copolymers of acrylamide, sulfonated polyacrylamide, cationic polyacrylamide, anionic polyacrylamide, and partially hydrolyzed acrylamide. In exemplary embodiments, the one or more polymers comprises acrylamide or partially hydrolyzed acrylamide and one or more nonionic and/or anionic monomers. Suitable non-ionic monomers include but are not limited to acrylamide, N-alkylacrylamides, N,N-dialkylacrylamides, methacrylamide, N-vinylmethylacetamide or formamide, vinyl acetate, vinyl pyrrolidone, alkyl methacrylates, acrylonitrile, N-vinylpyrrolidone other acrylic (or other ethylenically unsaturated) ester or other water insoluble vinyl monomers such as styrene or acrylonitrile. The term “anionic monomer” refers to a monomer which possesses a negative charge. Representative anionic monomers include acrylic acid, sodium acrylate, ammonium acrylate, methacrylic acid, 2-acrylamido-2-methylpropanesulfonic acid (AMPS), vinyl sulfonic acid, styrene sulfonic acid, maleic acid, sulfopropyl acrylate or methacrylate or other water-soluble forms of these or other polymerizable carboxylic or sulphonic acids, sulfomethylated acrylamide, allyl sulfonate, itaconic acid, acrylamidomethylbutanoic acid, fumaric acid, vinylphosphonic acid, allylphosphonic acid, phosphonomethylated acrylamide, methacrylate, itaconate, 2-acrylamido 2-methyl propane sulphonate, sulfoalkyl(meth)acrylic acids, sulfonated styrenes, unsaturated dicarboxylic acids, sulfoalkyl(meth)acrylamides, vinyl acetate, n-vinylformamide, n-vinylacetamide, n-vinylcaprolactam, n-vinylimidazole, n-vinylpyridine, n-vinylpyrolidone, acrylamidopropyltrimonium chloride, salts of said acids and the like, or another anionic ethylenically unsaturated compound. In a particular embodiment, the one or more polymers comprises acrylamide or partially hydrolyzed acrylamide and one or more anionic monomers. In exemplary embodiments, the one or more polymers has an overall anionic charge and comprises acrylamide or partially hydrolyzed acrylamide and one or more nonionic and/or anionic monomers. In exemplary embodiments, the one or more polymers comprises about 5% to about 60% anionic monomers by weight. In exemplary embodiments, the one or more polymers comprises an anionic polyacrylamide. In exemplary embodiments, the anionic polyacrylamide is a copolymer comprising one or more anionic monomers and acrylamide monomers. Exemplary salts of these anionic monomers include but are not limited to sodium and ammonium salts. In one embodiment, the polymer is an anionic polymer. In a particular embodiment, the anionic polymer has about 5% to about 60% charge, about 10% to about 50% charge, about 15% to about 45% charge, about 20% to about 40% charge, about 10% to about 15% charge, or about 25% to about 35% charge. In exemplary embodiments, the one or more polymers comprises acrylamide or partially hydrolyzed acrylamide and one or more cationic monomers. The term “cationic monomer” refers to a monomer which possesses a positive charge. Representative cationic monomers include dialkylaminoalkyl acrylates and methacrylates and their quaternary or acid salts, including, but not limited to, dimethylaminoethyl acrylate methyl chloride quaternary salt, dimethylaminoethyl acrylate methyl sulfate quaternary salt, dimethyaminoethyl acrylate benzyl chloride quaternary salt, dimethylaminoethyl acrylate sulfuric acid salt, dimethylaminoethyl acrylate hydrochloric acid salt, diethylaminoethyl acrylate, methyl chloride quaternary salt, dimethylaminoethyl methacrylate methyl chloride quaternary salt, dimethylaminoethyl methacrylate methyl sulfate quaternary salt, dimethylaminoethyl methacrylate benzyl chloride quaternary salt, dimethylaminoethyl methacrylate sulfuric acid salt, dimethylaminoethyl methacrylate hydrochloric acid salt, dimethylaminoethyl methacryloyl hydrochloric acid salt, dialkylaminoalkylacrylamides or methacrylamides and their quaternary or acid salts such as acrylamidopropyltrimethylammonium chloride, dimethylaminopropyl acrylamide methyl sulfate quaternary salt, dimethylaminopropyl acrylamide sulfuric acid salt, dimethylaminopropyl acrylamide hydrochloric acid salt, methacrylamidopropyltrimethylammonium chloride, dimethylaminopropyl methacrylamide methyl sulfate quaternary salt, dimethylaminopropyl methacrylamide sulfuric acid salt, dimethylaminopropyl methacrylamide hydrochloric acid salt, acryloyloxyethyltrimethylammonium chloride, diethylaminoethylacrylate, diethylaminoethylmethacrylate and diallyldialkylammonium halides such as diallyldiethylammonium chloride and diallyldimethyl ammonium chloride. Alkyl groups are generally C1-8alkyl. In a particular embodiment, the one or more polymers comprises acrylamide or partially hydrolyzed acrylamide and one or more cationic monomers. In a particular embodiment, the one or more polymers comprises acrylamide or partially hydrolyzed acrylamide and acryloyloxyethyltrimethylammonium chloride. In exemplary embodiments, the one or more polymers has an overall cationic charge and comprises acrylamide or partially hydrolyzed acrylamide and one or more cationic monomers. In exemplary embodiments, the one or more polymers comprises about 5% to about 60% cationic monomers by weight. In exemplary embodiments, the one or more polymers comprises a cationic polyacrylamide. In exemplary embodiments, the cationic polyacrylamide is a copolymer comprising one or more cationic monomers and acrylamide monomers. In one embodiment, the polymer is a cationic polymer. In exemplary embodiments, the partially hydrolyzed acrylamide is acrylamide wherein about 3% to about 70% of the amide groups have been hydrolyzed to carboxyl groups. In one embodiment, the one or more polymers comprises an amphoteric polymer. In one embodiment, the one or more polymers comprises a non-ionic polymer. In exemplary embodiments, the one or more polymers comprises acrylamide or partially hydrolyzed acrylamide and one or more monomers selected from the group consisting of acrylic acid, acrylate salt, 2-acrylamido-2-methylpropane sulfonic acid, N,N-dimethylacrylamide, vinyl sulfonic acid, N-vinyl sulfonic acetamide, N-vinyl formamide, itaconic acid, methacrylic acid, acryloyloxyethyltrimethylammonium chloride, salts thereof, and combinations thereof. In a particular embodiment, the one or more polymers comprises acrylamide or partially hydrolyzed acrylamide and one or more monomers selected from the group consisting of acrylic acid, 2-acrylamido-2-methylpropane sulfonic acid, and methacrylic acid, and salts thereof. In a particular embodiment, the one or more polymers comprises acrylamide or partially hydrolyzed acrylamide and one or more monomers selected from the group consisting of acrylic acid and salts thereof. In certain embodiments, the polymer comprises acrylamide and one or more monomers selected from the group consisting of: acrylic acid and its salts, methacrylamide, methacrylic acid and its salts, maleic acid and its salts, methyl acrylate, ethyl acrylate, propyl acrylate, methyl methacrylate, ethyl methacrylate, dimethylaminoethyl acrylate and its methylchloride and methosulfate quaternaries, dimethylaminoethyl methacrylate and its methylchloride and methosulfate quaternaries, diethylaminoethyl acrylate and its methylchloride and methosulfate quaternaries, diethylaminoethyl methacrylate and its methylchloride and methosulfate quaternaries, hydroxyethyl acrylate, hydroxyethyl methacrylate, styrene, acrylonitrile, 2-acrylamido-2-methylpropane sulfonic acid and its salts, 3-(methylacrylamido)-propyltrimethylammonium chloride, dimethylaminopropylmethacrylamide, isopropylaminopropylmethacrylamide, methacrylamidopropylhydroxyethyldimethylammonium acetate, vinyl methyl ether, vinyl ethyl ether, alkali metal and ammonium salts of vinyl sulfonic acid, vinyl pyridine, vinyl pyrrolidone, vinyl imidazole, diallyldimethylammonium chloride, styrene sulfonic acid and its salts, and the like. In exemplary embodiments, one or more polymers is a copolymer of acrylamide or partially hydrolyzed acrylamide and one or more anionic monomers. In exemplary embodiments, the one or more polymers comprises at least about 40 mole %, about 50 mole %, about mole 60%, about mole 70%, about mole 80%, or about mole 90% acrylamide or partially hydrolyzed acrylamide. In exemplary embodiments, the one or more polymers comprises at least about 5 mole %, about 10 mole %, about 20 mole %, about 30 mole %, about 40 mole %, about 50 mole %, or about 55 mole % one or more anionic monomers. In exemplary embodiments, the one or more polymers comprises about 40 mole % to about 95 mole %, or about 60 mole % to about 90 mole %, acrylamide or partially hydrolyzed acrylamide. In exemplary embodiments, the one or more polymers comprises about 5 mole % to about 60 mole %, or about 10 mole % to about 40 mole %, one or more anionic monomers. In exemplary embodiments, one or more polymers is a copolymer of acrylamide or partially hydrolyzed acrylamide and one or more cationic monomers. In exemplary embodiments, the one or more polymers comprises at least about 40 mole %, about 50 mole %, about mole 60%, about mole 70%, about mole 80%, or about mole 90% acrylamide or partially hydrolyzed acrylamide. In exemplary embodiments, the one or more polymers comprises at least about 5 mole %, about 10 mole %, about 20 mole %, about 30 mole %, about 40 mole %, about 50 mole %, or about 55 mole % one or more cationic monomers. In exemplary embodiments, the one or more polymers comprises about 40 mole % to about 95 mole %, or about 60 mole % to about 90 mole %, acrylamide or partially hydrolyzed acrylamide. In exemplary embodiments, the one or more polymers comprises about 5 mole % to about 60 mole %, or about 10 mole % to about 40 mole %, one or more cationic monomers. In exemplary embodiments, one or more polymers is a copolymer of acrylamide or partially hydrolyzed acrylamide and acrylic acid or an acrylate salt. In exemplary embodiments, the one or more polymers comprises at least about 40 mole %, about 50 mole %, about mole 60%, about mole 70%, about mole 80%, or about mole 90% acrylamide or partially hydrolyzed acrylamide. In exemplary embodiments, the one or more polymers comprises at least about 5 mole %, about 10 mole %, about 20 mole %, about 30 mole %, about 40 mole %, about 50 mole %, or about 55 mole % acrylic acid or acrylate salts. In exemplary embodiments, the acrylate salt comprises ammonium acrylate. In exemplary embodiments, the one or more polymers comprises about 40 mole % to about 95 mole %, or about 60 mole % to about 90 mole %, acrylamide or partially hydrolyzed acrylamide. In exemplary embodiments, the one or more polymers comprises about 5 mole % to about 60 mole %, or about 10 mole % to about 40 mole %, acrylic acid or an acrylate salt. The exemplary polymers may be included in the treatment fluids in an amount sufficient to provide the desired properties. In some embodiments, a polymer may be present in an amount in the range of about 0.1 to about 10, about 0.1 to about 6, about 0.1 to about 5, or about 0.25 to about 1, Gallons Per Thousand Gallons of the aqueous treatment fluid (GPTG). The polymers may be added to slick water treatments at concentrations of about 0.1 to about 20 GPTG, of treatment fluid. In other embodiments, the polymer is added at a concentration of about 0.25 to about 6 GPTG of treatment fluid. The polymers of the present embodiments should have a molecular weight sufficient to provide desired properties. For example, those polymers used for friction reduction should have higher molecular weights to provide a desirable level of friction reduction. The polymers used for EOR applications should have sufficient molecular weight to provide the desired viscosity to mobilize oil in a desirable manner. In some exemplary embodiments, the weight average molecular weight of a polymer may be in the range of from about 7,500,000 to about 30,000,000 Dalton. Those of ordinary skill in the art will recognize that polymers having molecular weights outside the listed range may still provide desirable properties in the aqueous treatment fluid. In exemplary embodiments, the polymer is used for EOR applications. Suitable polymers of the present embodiments may be in an acid form or in a salt form. A variety of salts may be made by neutralizing the acid form of a monomer, for example acrylic acid or 2-acrylamido-2-methylpropane sulfonic acid, with a base, such as sodium hydroxide, ammonium hydroxide or the like. As used herein, the term “polymer” is intended to include both the acid form of the copolymer and its various salts. Inverting Surfactant Composition In exemplary embodiments, in addition to the one or more polymers, the emulsion or aqueous treatment fluid comprises an inverting surfactant composition. Among other things, an inverting surfactant or inverting surfactant composition may facilitate the inverting of the emulsion upon addition to the treatment fluids of the present embodiments. As those of ordinary skill in the art will appreciate, with the benefit of this disclosure, upon addition to the treatment fluid, the emulsion should invert, releasing the polymer into the treatment fluid. In exemplary embodiments, the inverting surfactant composition comprises one or more surfactants selected from the group consisting of Surfactant A compounds, Surfactant B compounds, and Surfactant C compounds, as described herein. In certain exemplary embodiments, the inverting surfactant composition comprises one or more surfactants selected from the group consisting of Surfactant A compounds. In certain exemplary embodiments, the inverting surfactant composition comprises one or more surfactants selected from the group consisting of Surfactant B compounds. In exemplary embodiments, the inverting surfactant composition comprises two or more surfactants selected from the group consisting of Surfactant A compounds, Surfactant B compounds, and Surfactant C compounds, as described herein. In exemplary embodiments, the inverting surfactant composition comprises one or more Surfactant A compounds. In exemplary embodiments, the inverting surfactant composition comprises one or more Surfactant B compounds. In exemplary embodiments, the inverting surfactant composition comprises one or more Surfactant C compounds. In exemplary embodiments, the inverting surfactant composition comprises one or more Surfactant A compounds and one or more Surfactant B compounds. In exemplary embodiments, the inverting surfactant composition comprises one or more Surfactant A compounds and one or more Surfactant C compounds. In exemplary embodiments, the inverting surfactant composition comprises one or more Surfactant B compounds and one or more Surfactant C compounds. In exemplary embodiments, the inverting surfactant composition comprises two or more types of Surfactant B compounds. In exemplary embodiments, the inverting surfactant composition comprises one or more Surfactant A compounds, one or more Surfactant B compounds, and one or more Surfactant C compounds. In exemplary embodiments, the inverting surfactant composition comprises one or more Surfactant A compounds, two or more types of Surfactant B compounds. In exemplary embodiments, the inverting surfactant composition comprises one or more Surfactant C compounds, two or more types of Surfactant B compounds. In exemplary embodiments, the inverting surfactant composition comprises one or more Surfactant A compounds, two or more types of Surfactant B compounds, one or more Surfactant C compounds. In exemplary embodiments, the inverting surfactant composition may comprise other inverting surfactants in addition to those chosen from Surfactant A, Surfactant B, and Surfactant C compounds. Representative inverting surfactants that may also be added to the exemplary emulsions include those having a hydrophilic-lipophilic balance (HLB) of greater than 10; polyoxyethylene sorbitol tetraoleate; polyethylene glycol monoleate; ethoxylated alcohols, such as C12-14branched ethoxylated alcohol, ethoxylated octyl and nonyl phenols; ethoxylated nonyl phenol formaldehyde resin; polyethylene oxide esters of fatty acids; dioctyl esters of sodium sulfosuccinate; and other inverting surfactants disclosed in U.S. Pat. No. 3,624,019 incorporated herein by reference. The inverting surfactant should be present in an amount sufficient to provide the desired inversion of the emulsion upon contact with the water in the aqueous treatment fluid. In exemplary embodiments, the inverting surfactant composition comprises 0 to about 100%, 0 to about 75%, or about 5 to about 75 wt %, of Surfactant A compounds. In exemplary embodiments, the inverting surfactant composition comprises at least about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, or about 95 wt % of Surfactant A compounds, when Surfactant A is included in the composition. In certain exemplary embodiments, the inverting surfactant composition does not comprise Surfactant A compounds. In exemplary embodiments, the inverting surfactant composition comprises 0 to about 100%, 0 to about 75%, or about 5 to about 75 wt %, of Surfactant B compounds. In exemplary embodiments, the inverting surfactant composition comprises at least about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, or about 95 wt % of Surfactant B compounds, when Surfactant B is included in the composition. In certain exemplary embodiments, the inverting surfactant composition does not comprise Surfactant B compounds. In exemplary embodiments, the inverting surfactant composition comprises 0 to about 75%, or about 5 to about 50 wt %, of Surfactant C compounds. In exemplary embodiments, the inverting surfactant composition comprises at least about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, or about 50 wt % of Surfactant C compounds, when Surfactant C is included in the composition. In certain exemplary embodiments, the inverting surfactant composition does not comprise Surfactant C compounds. In certain exemplary embodiments, the inverting surfactant composition comprises or consists essentially of about 5 to about 95 wt % one or more Surfactant A compounds and about 5 to about 95 wt % one or more Surfactant B compounds. In certain exemplary embodiments, the inverting surfactant composition comprises or consists essentially of about 25 to about 35 wt % one or more Surfactant A compounds and about 65 to about 75 wt % one or more Surfactant B compounds. In certain exemplary embodiments, the inverting surfactant composition comprises or consists essentially of about 5 to about 95 wt % one or more Surfactant A compounds and about 5 to about 75 wt % one or more Surfactant C compounds. In certain exemplary embodiments, the inverting surfactant composition comprises or consists essentially of about 5 to about 95 wt % one or more Surfactant B compounds and about 5 to about 75 wt % one or more Surfactant C compounds. In certain exemplary embodiments, the inverting surfactant composition comprises or consists essentially of about 25 to about 65, about 25 to about 55, about 30 to about 50, or about 30 to about 60, wt % one or more Surfactant B compounds and about 35 to about 75, about 45 to about 75, about 40 to about 70, about 50 to about 70 wt % one or more Surfactant C compounds. In certain exemplary embodiments, the inverting surfactant composition comprises or consists essentially of about 5 to about 95 wt % one or more Surfactant B compounds and about 5 to about 95 wt % a different Surfactant B compound. In certain exemplary embodiments, the inverting surfactant composition comprises or consists essentially of about 5 to about 90 wt % one or more Surfactant A compounds, about 5 to about 90 wt % one or more Surfactant B compounds and about 5 to about 75 wt % one or more Surfactant C compounds. In certain exemplary embodiments, the inverting surfactant composition comprises or consists essentially of about 5 to about 35, or about 10 to about 30, wt % one or more Surfactant A compounds; about 25 to about 70, or about 30 to about 60, wt % one or more Surfactant B compounds; and about 15 to about 70, about 15 to about 55, about 20 to about 50, about 35 to about 65, or about 40 to about 60, wt % one or more Surfactant C compounds. In certain exemplary embodiments, the inverting surfactant composition comprises or consists essentially of about 15 to about 35, about 25 to to about 35, about 20 to about 30, about 15 to about 25, or about 5 to about 15, wt % one or more Surfactant A compounds; about 15 to about 45, about 35 to about 45; about 45 to about 55, about 55 to about 65, about 65 to about 75, or about 20 to about 40, wt % one or more Surfactant B compounds; and about 15 to about 65, about 15 to about 25, about 25 to about 35, about 35 to about 45, about 45 to about 55, about 55 to about 65, or about 30 to about 50, wt % one or more Surfactant C compounds. In certain exemplary embodiments, the inverting surfactant composition comprises or consists essentially of about 5 to about 75 wt % one or more Surfactant A compounds, about 5 to about 75 wt % two or more types of Surfactant B compounds, and about 5 to about 75 wt % one or more Surfactant C compounds. In a particular embodiment, the inverting surfactant composition comprises or consists essentially of about 25 to about 35, or about 28 to about 32, wt % one or more Surfactant A compounds, about 25 to about 35, or about 28 to about 32, wt % one or more Surfactant B compounds, about 25 to about 35, or about 28 to about 32, wt % one or more Surfactant C compounds and about 5 to about 15, or about 8 to about 12, wt % of a different Surfactant B compound. Surfactant A In exemplary embodiments, the emulsion or aqueous treatment fluid comprises one or more Surfactant A compounds. In exemplary embodiments, Surfactant A compounds are selected from ethoxylated amine compounds, such as ethoxylated tallow amine compounds. As referred to herein, “ethoxylated amine compounds” includes, for example, amine or amide compounds comprising two ethoxy or polyethoxy groups and one group selected from hydrogen, alkyl, aryl, C(═O)-alkyl or C(═O)-aryl group. In certain exemplary embodiments, the ethoxylated amine compounds are nonionic amine compounds. In certain embodiments, the ethoxylated amine compounds do not comprise cationic polyoxyethylene tallow amine compounds. In exemplary embodiments the ethoxylated amine compounds are compounds of Formula I: wherein R1is H, alkyl, aryl, C(═O)-alkyl, or C(═O)-aryl; and X and Y are each independently 1-20. In exemplary embodiments, an alkyl group is a saturated or unsaturated alkyl group having 8 to 26 carbon atoms. In exemplary embodiments, an aryl group is an aryl group having 6 to 18 carbon atoms. In exemplary embodiments, the alkyl group can be either saturated or unsaturated, and can be derived from, but not limited to, tallow, soybean oil, coconut oil, or cottonseed oil. In exemplary embodiments, the poly(oxyethylene) content (X+Y) of the ethoxylated amine is in the range of 3 to 20. In certain embodiments, R1is H. In certain embodiments, R1is not H. In certain embodiments, R1is alkyl, for example is a saturated or unsaturated alkyl group having 8 to 26 carbon atoms. In certain embodiments, le is C(═O)-alkyl, for example a carbonyl group bonded to the amine nitrogen and to a saturated or unsaturated alkyl group having 8 to 26 carbon atoms, such as N,N-bis(2-hydroxyethyl)-9-octadecenamide. It should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “1-10” is intended to include all sub-ranges between and including the recited minimum value of 1 and the recited maximum value of 10. Because the disclosed numerical ranges are continuous, they include every value between the minimum and maximum values. In exemplary embodiments, the ethoxylated amine compounds is polyethylene fatty acid amine or a mixture of polyethylene fatty acid amine compounds. In exemplary embodiments, R1is a residue of a saturated or unsaturated fatty acid, for example a residue of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, or docosahaxaenoic acid. In exemplary embodiments, the one or more ethoxylated amine compounds is polyethylene tallow amine. Tallow contains a variety of fatty acids including oleic (37-43%), palmitic (24-32%), stearic (20-25%), myristic (3-6%) and linoleic (2-3%). In exemplary embodiments, the one or more ethoxylated amine compounds includes polyethylene oleic amine, polyethylene palmitic amine, polyethylene stearic amine, polyethylene myristic amine, and polyethylene linoleic amine. Surfactant B In exemplary embodiments, the emulsion or aqueous treatment fluid comprises one or more Surfactant B compounds. In exemplary embodiments, Surfactant B compounds are selected from alkyl polyethyleneglycol ether carboxylic acid compounds, alkyl polyglycol ether carboxylic acid compounds, and salts or esters thereof. As referred to herein, Surfactant B compounds include, for example, compounds comprising a C8to C26unsaturated or saturated alkyl chain substituted with an (OCH2CH2)yOCH2CO2H wherein the average value of y is about 2 to about 20, or about 2 to about 10. In certain exemplary embodiments, the alkyl polyglycol ether carboxylic acid compounds are anionic compounds. In exemplary embodiments, Surfactant B compounds comprise a C14to C22unsaturated alkyl chain, for example an unsaturated alkyl chain derived from a fatty acid residue, such as oleic acid, myristoleic acid, palmitoleic acid, sapienic acid, elaidic acid, vaccenic acid, linoleic acid, α-linoleic acid, linoelaidic, arachidonic acid, eicospentanoic acid, erucic acid, docosahexaenoic acid, and the like. In exemplary embodiments, the Surfactant B is selected from the group consisting of glycolic acid ethoxylate oleyl ether, glycolic acid ethoxylate myristoleyl ether, glycolic acid ethoxylate palmitoleyl ether, glycolic acid ethoxylate sapienyl ether, glycolic acid ethoxylate elaidyl ether, glycolic acid ethoxylate vaccenyl ether, glycolic acid ethoxylated linoleyl ether, glycolic acid ethoxylated α-linoleyl ether, glycolic acid ethoxylate linoelaidyl ether, glycolic acid ethoxylate arachidonyl ether, glycolic acid ethoxylate eicospentanoyl ether, glycolic acid ethoxylate erucyl ether, and glycolic acid ethoxylated docosahexaenoyl ether. In certain exemplary embodiments, Surfactant B is a compound or a mixture of compounds represented by the formula: CH3(CH2)xCH═CH(CH2)8(OCH2CH2)yOCH2CO2H, wherein x is 1-12 and y is 2-20. In certain exemplary embodiments, the Surfactant B is glycolic acid ethoxylate oleyl ether. In certain exemplary embodiments, Surfactant B is the mixture of compounds represented by the formula: CH3(CH2)xCH═CH(CH2)8(OCH2CH2)yOCH2CO2H, wherein the average value for x is 5-7 and the average value for y is about 2. In exemplary embodiments, Surfactant B compounds comprise a C14to C22saturated alkyl chain, for example an saturated alkyl chain derived from a fatty acid residue, such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, and the like. In exemplary embodiments, the Surfactant B is selected from the group consisting of glycolic acid ethoxylate lauryl ether, glycolic acid ethoxylate caprylyl ether, glycolic acid ethoxylate capryl ether, glycolic acid ethoxylate myristyl ether, glycolic acid ethoxylate palmityl ether, glycolic acid ethoxylate stearyl ether, glycolic acid ethoxylated arachidyl ether, glycolic acid ethoxylated behenyl ether, glycolic acid ethoxylate lignoceryl ether, and glycolic acid ethoxylate cerotyl ether. In certain exemplary embodiments, Surfactant B is a compound or a mixture of compounds represented by the formula: CH3(CH2)w(OCH2CH2)yOCH2CO2H, wherein w is 6-24 and y is 2-20. In certain exemplary embodiments, the Surfactant B is a glycolic acid ethoxylate lauryl ether or a polyoxyethylene lauryl ether carboxylic acid or a salt thereof, such as polyoxyethylene(10) lauryl ether carboxylic acid, polyoxyethylene(3) lauryl ether carboxylic acid, polyoxyethylene(5) lauryl ether carboxylic acid, polyoxyethylene(7) lauryl ether carboxylic acid, or polyoxyethylene(4) lauryl ether carboxylic acid. In certain exemplary embodiments, the Surfactant B is B1 or B2 from the Examples, or a combination thereof. Surfactant C In exemplary embodiments, the emulsion or aqueous treatment fluid comprises one or more Surfactant C compounds. In exemplary embodiments, Surfactant C compounds are selected from ethoxylated fatty acid compounds. As referred to herein, “ethoxylated fatty acid compounds” includes, for example, fatty acid compounds which have been reacted with ethylene oxide to form compounds containing at least 20 moles of ethoxy groups per 1 mole of the fatty acid. In certain exemplary embodiments, the ethoxylated fatty acid compounds are unsaturated, for example monounsaturated. In certain exemplary embodiments, the ethoxylated fatty acid compounds are hydroxylated or substituted with one or more hydroxyl groups. In certain exemplary embodiments, the ethoxylated fatty acid compounds are nonionic compounds. In exemplary embodiments, Surfactant C compound contains at least about 20, about 25, about 30, or about 35 units of ethoxylation. In certain exemplary embodiments, the fatty acid is, for example, a monounsaturated hydroxyl fatty acid, such as Ricinoleic acid. Ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid) is an unsaturated omega-9 fatty acid and a hydroxy acid. It is a major component of the seed oil obtained from mature Castor plant (Ricinus communisL., Euphorbiaceae) seeds or in sclerotium of ergot (Claviceps purpureaTul., Clavicipitaceae). About 90% of the fatty acid content in castor oil is the triglyceride formed from ricinoleic acid. In certain exemplary embodiments, Surfactant C is ethoxylated castor oil. In exemplary embodiments, the Surfactant C compound is produced by the ethoxylation of fatty acid materials derived from saturated or unsaturated animal or vegetable fats, such as coconut oil, tall oil, stearic fatty acid, oleic fatty acid or adipic fatty acid. In certain exemplary embodiments, the ethoxylated fatty acid compound is, for example, coconut fatty acid ethoxylate, lauric acid ethoxylate, oleic acid ethoxylate, or myristic acid ethoxylate. Emulsions Exemplary emulsions, for example water-in-oil emulsions or oil-external emulsions, may comprise water, a water-immiscible liquid, one or more polymers, and an inverting surfactant composition comprising one or more surfactants selected from the group consisting of Surfactant A compounds, Surfactant B compounds, and Surfactant C compounds, as described herein. In certain exemplary embodiments, the emulsions comprise water, a water-immiscible liquid, one or more polymers, and an inverting surfactant composition comprising one or more Surfactant A compounds. In certain exemplary embodiments, the emulsions comprise water, a water-immiscible liquid, one or more polymers, and an inverting surfactant composition comprising one or more Surfactant B compounds. In certain exemplary embodiments, the emulsions comprise water, a water-immiscible liquid, one or more polymers, and an inverting surfactant composition comprising one or more Surfactant C compounds. In certain exemplary embodiments, the emulsions comprise water, a water-immiscible liquid, one or more polymers, and an inverting surfactant composition comprising two or more surfactants selected from the group consisting of Surfactant A compounds, Surfactant B compounds, and Surfactant C compounds. In certain exemplary embodiments, the emulsions comprise water, a water-immiscible liquid, one or more polymers, and an inverting surfactant composition comprising three or more surfactants selected from the group consisting of Surfactant A compounds, Surfactant B compounds, and Surfactant C compounds. In certain exemplary embodiments, the emulsions comprise water, a water-immiscible liquid, one or more polymers, and an inverting surfactant composition comprising four or more surfactants selected from the group consisting of Surfactant A compounds, Surfactant B compounds, and Surfactant C compounds. The emulsion may optionally comprise inhibitors, emulsifiers, salts and/or other surfactants. In exemplary embodiments, the emulsion comprises: water; a water-immiscible liquid; greater than about 10% by weight one or more polymers; about 0.1% to about 5% by weight an inverting surfactant composition described herein. In exemplary embodiments, the emulsion comprises: water; a water-immiscible liquid; greater than about 10% by weight one or more polymers; about 0.1% to about 5%, about 1% to about 4%, or about 1.5% to about 3.5%, by weight an inverting surfactant composition comprising one or more surfactants selected from the group consisting of Surfactant A compounds, Surfactant B compounds, and Surfactant C compounds. In exemplary embodiments, the amounts of each individual inverting surfactant included in the emulsion, when two or more exemplary inverting surfactants are used can vary as necessary, for example, each exemplary inverting surfactant can be present in an amount of about 0.01 to about 5%, 0.01 to about 3%, or about 0.02 to about 2%, by weight, based on the total emulsion. The water present in the emulsions generally includes freshwater, but saltwater or combinations with saltwater also may be used. Generally, the water used may be from any source, provided that it does not contain an excess of compounds that may adversely affect other components in the emulsion. In some embodiments, the water may be present in the emulsion in an amount in the range of from about 1% to about 50%, about 1% to about 12%, about 3% to about 50%, about 3% to about 12%, about 1% to about 5%, about 12% to about 50%, or about 30% to about 50% by weight of the emulsion. In some embodiments, the emulsion composition may have less than about 30%, about 20%, about 12%, about 10%, about 7%, about 5%, or about 3% by weight water. In some embodiments, the emulsion composition may have greater than about 1%, about 2%, about 3%, about 5%, about 7%, about 10%, about 12%, or about 20%, by weight water. In certain exemplary embodiments, the emulsion can be water-free or at least substantially water-free. In embodiments wherein the amount of water in the emulsion is kept to a very small amount, the emulsion may be in the form of a liquid dispersion polymer composition or a liquid polymer composition. Suitable water-immiscible liquids may include, but are not limited to, water-immiscible solvents, such as paraffin hydrocarbons, naphthene hydrocarbons, aromatic hydrocarbons, olefins, oils, stabilizing surfactants and mixtures thereof. The paraffin hydrocarbons may be saturated, linear, or branched paraffin hydrocarbons. Examples of suitable aromatic hydrocarbons include, but are not limited to, toluene and xylene. In one embodiment, the water-immiscible liquid is an olefin and paraffin blend. In one embodiment, the water-immiscible liquid comprises oil and one or more emulsifiers. The water-immiscible liquid may be present in the emulsion in an amount sufficient to form a stable emulsion. In some embodiments, the water-immiscible liquid may be present in the emulsions in an amount in the range of from about 20% to about 60%, about 25% to about 55%, about 35% to about 55%, or about 20% to about 30% by weight. In exemplary embodiments, the emulsion comprises one or more emulsifiers. Emulsifiers, among other things, in the emulsion, lower the interfacial tension between the water and the water-immiscible liquid so as to facilitate the formation of a water-in-oil polymer emulsion. In exemplary embodiments, the emulsifier is not a compound of Surfactants A, B, or C. The emulsifier should be present in an amount sufficient to provide the desired stable water-in-oil polymer emulsion. In some embodiments, the emulsifier may be present in an amount in the range of from about 0.5% to about 5% by weight of the emulsion. The polymer should be present in the emulsion in an amount that does not undesirably impact the emulsion's stability. In exemplary embodiments, the one or more polymers may be present in an amount in the range of from about 10% to about 80%, about 10% to about 35%, about 15% to about 30%, or about 20% to about 30%, about 39% to about 80%, or about 40% to about 60%, or about 45% to about 55%, by weight of the emulsion. In exemplary embodiments, the emulsion may comprise greater than about 35%, about 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or about 60% or higher, by weight polymer. In exemplary embodiments, the emulsion may comprise less than about 35%, or about 30%, or less, by weight polymer. In certain exemplary embodiments, the emulsions may further comprise one or more organic or inorganic salts. In certain exemplary embodiments, the emulsions comprise at least about 0.5% of one or more organic or inorganic salts. In exemplary embodiments, the emulsions comprise one or more organic or inorganic salts in an amount about 0.5% to about the point of saturation in the emulsion. Representative organic or inorganic salts include but are not limited to sodium chloride, sodium sulfate, sodium bromide, ammonium sulfate, ammonium chloride, lithium chloride, lithium bromide, potassium chloride, potassium bromide, magnesium sulfate, aluminum sulfate, ammonium hydrogen phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium salts, lithium salts, potassium salts, magnesium salts, aluminum salts, ammonium salts, phosphate salts, sulfate salts, chloride salts, fluoride salts, citrate salts, acetate salts, tartrate salts, hydrogenphosphate salts, water soluble inorganic salts, other inorganic salts, other organic salts and mixtures thereof. In exemplary embodiments, the one or more organic or inorganic salts includes ammonium chloride. In some embodiments, emulsion polymerization may be used to prepare exemplary emulsions. Suitable emulsion polymerization techniques may have a variety of different initiation temperatures depending on, among other things, the amount and type of initiator used, the amount and type of monomers used, and a number of other factors known to those of ordinary skill in the art. In one embodiment, a suitable emulsion polymerization technique may have an initiation temperature of about 25° C. Due to the exothermic nature of the polymerization reaction, the mixture may be maintained at a higher temperature than the initiation temperature during procession of the polymerization reaction, for example, in the range of from about 30° C. to about 70° C., or from about 40° C. to about 60° C. In exemplary embodiments, the one or more polymers are in the form of a emulsion, such as a polyacrylamide emulsion. In exemplary embodiments, the emulsion comprises a hydrophilic polymer contained within water droplets that are dispersed in a continuous oil phase. In exemplary embodiments, the one or more polymers are in the form of an aqueous dispersion, for example an aqueous polymer dispersion prepared by solution polymerization. Methods for the preparation of exemplary aqueous polymer dispersions are well known in the art, for example as described in U.S. Pat. No. 5,200,448. In exemplary embodiments, any suitable emulsion polymerization method may be employed in the preparation of the one or more polymers described here. Descriptions of the steps of an exemplary emulsion preparation provided herein, but are not intended to be limiting with respect to the methods for preparing the exemplary one or more polymers. A preliminary emulsion is made by homogenizing oil and aqueous phases. The oil phase of the emulsion, which generally comprises from about 5 to about 35 percent by weight of the total emulsion, is comprised of one or more inert hydrophobic liquids. Preferably, the oil phase comprises about 20 to 30 percent of the emulsion. The oil used may be selected from a large class of organic liquids which are immiscible with water, including liquid hydrocarbons and substituted liquid hydrocarbons. Representative examples of such oils include benzene, xylene, toluene, mineral oils, kerosenes, naphthas, chlorinated hydrocarbons, such as perchloroethylene, and the like. The oil phase may contain one or more primary or emulsifying surfactants, i.e. conventional emulsion polymerization stabilizers. Such stabilizers are well known to the art to promote the formation and stabilization of water-in-oil emulsions. Normally such emulsifiers have HLB values in the range of about 2 to about 10, preferably less than about 7. Suitable such emulsifiers include the sorbitan esters, phthalic esters, fatty acid glycerides, glycerine esters, as well as the ethoxylated versions of the above and any other well-known relatively low HLB emulsifier. Examples of such compounds include sorbitan monooleate, the reaction product of oleic acid with isopropanolamide, hexadecyl sodium phthalate, decyl sodium phthalate, sorbitan stearate, ricinoleic acid, hydrogenated ricinoleic acid, glyceride monoester of lauric acid, glyceride monoester of stearic acid, glycerol diester of oleic acid, glycerol triester of 12-hydroxystearic acid, glycerol triester of ricinoleic acid, and the ethoxylated versions thereof containing 1 to 10 moles of ethylene oxide per mole of the basic emulsifier. Thus, any emulsifier may be utilized which will permit the formation of the initial emulsion and stabilize the emulsion during the polymerization reaction. These primary surfactants are used alone or in mixtures and are utilized in amounts of not greater than about 5%, about 4%, about 3%, about 2% or about 1% by weight of the total emulsion. The aqueous phase generally comprises about 95 to 65% by weight of the initial emulsion. Preferably, it comprises about 80 to 70% thereof. In addition to water, the aqueous phase contains the monomers being polymerized, generally in an amount of less than about 50%, about 15 to about 40%, or about 22 to about 35%, by weight of the total emulsion, and generally chain transfer agents, initiators and sequesterants. Alternatively, the chain transfer agents, initiators and sequesterants may be added to the system after the preliminary emulsion has been prepared. The initiator may also be added continuously during the polymerization to control the rate of polymerization depending upon the particular monomers used and their reactivities. Alternatively, the initiator may be present in either the oil or the aqueous phase with the monomers being added either continuously or incrementally thereafter. All of these variations are well known in the art. The monomers suitable for use in the preparation of the exemplary polymers are described herein. Any conventional chain transfer agent may be employed, such as propylene glycol, isopropanol, 2-mercaptoethanol, sodium hypophosphite, dodecyl mercaptan and thioglycolic acid. The chain transfer agent is generally present in an amount of about 0.1 to 10 percent by weight of the total emulsion, though more may be used. The initiator may be any free radical producing material well known in the art. The preferred free radical initiators are the redox-type and the azo-type polymerization initiators and they are generally used in an amount of about 0.0005 to 0.5 percent by weight of the total emulsion. Radiation may also be used to initiate the reaction. Any conventional sequesterant may also be present in the aqueous phase, such as ethylenediaminetetraacetic acid or pentasodium diethylenetriamine pentaacetate. The sequesterant is generally present in an amount of about 0.01 to 2 percent by weight of the total emulsion, though more may be utilized. Following preparation of the preliminary emulsion, polymerization of the monomers is commenced at a temperature sufficiently high to break down the initiator to produce the desired free radicals. Generally a suitable temperature is about −20° C. to about 200° C., or about 20° C. to 100° C. Preferably the polymerization is run at a pH of about 2 to 12 and a suitable amount of base or acid may be added to the preliminary emulsion to achieve the desired pH. The polymerization is usually completed in about an hour or two to several days, depending upon the monomers employed and other reaction variables. It is generally carried out at atmospheric pressure, but higher pressures are advantageously used when volatile ingredients are involved. In certain exemplary embodiments, once polymerization is complete, the amount of water in the emulsion may be reduced or removed as desired. For example, the water can be removed to a level of less than about 12%, or less than about 10%, or less than about 7%, or less than about 5%, or less than about 3% by weight. In exemplary embodiments, the removal of water is carried out by any suitable means, for example, at reduced pressure, e.g. at a pressure of about 0.00 to about 0.5 bars, or about 0.05 to about 0.25 bars. The temperature for water removal steps may typically be from about 50° C. to about 150° C., although techniques which remove water at higher temperatures may be used. Following completion of the polymerization, the pH of the emulsion may be adjusted as desired. For an anionic polymer emulsion, this is generally about 4 to 10; for cationic emulsions about 2.0 to 5.5; and for non-ionic emulsions about 2.0 to 7.0. A breaker or inverting surfactant, or blend of inverting surfactants, is generally added to yield a single package of final product. In exemplary embodiments, an inverting surfactant composition, as described herein, is added to the polymer emulsion. Other suitable breaker or inverting surfactants may be used in combination with the exemplary polymer and exemplary inverting surfactant composition in the emulsion. As described herein, the total amount of inverting surfactants present in the emulsion is about 0.1 to about 5% by weight, based on the total emulsion. Once prepared, the emulsions of the present embodiments may be chemically modified in any known manner. “Chemically modified” is intended to cover further treatment of the dispersed water-soluble polymer and/or the addition of components to the dispersed water-soluble polymer which, without the stabilization provided by the emulsion stabilizers, would cause the normally water-soluble polymeric particles to coagulate or agglomerate. Examples of such further treatments are disclosed in U.S. Pat. Nos. 4,052,353 and 4,171,296, incorporated herein by reference. The emulsion of the present embodiments may also be concentrated in any suitable manner, such as is disclosed in U.S. Pat. No. 4,021,399, incorporated herein by reference. A variety of different mixtures may be used to prepare an emulsion for use in the present embodiments. Suitable mixtures may include acrylamide, other monomers, water, a water-immiscible liquid, an initiator, and an emulsifier. Generally the one or more ethoxylated amine compounds can be combined with one or more inverting surfactants to form the inverting surfactant composition. The inverting surfactant composition can be added to the polymer emulsion to form a mixture. Optionally, the mixture further may comprise, a base (e.g., sodium hydroxide) to neutralize the monomers in acid form such that the salt of the monomer is not formed, a complexing agent to allow the gradual release of monomers in the polymerization reaction, an activator to initiate polymerization at a lower temperature, and an inverter. Those of ordinary skill in the art, with the benefit of this disclosure, will, know the amount and type of components to include in the mixture based on a variety of factors, including the desired molecular weight and composition of the polymer and the desired initiation temperature. Generally, the exemplary emulsions are particularly suitable for use in brine. The exemplary emulsions may be used in a range of temperatures, for example between about 5 and about 99° C., or about 50 and about 95° C. In certain exemplary embodiments, the emulsion may be used in combination with a proppant. Treatment Fluids The treatment fluid, for example an aqueous treatment fluid, containing the emulsions described herein, can be used in any well treatment fluid, including but not limited to stimulation, production and completion operations. For example, the well treatment fluid can be used for hydraulic fracturing applications or in an application where friction reduction is desired. Conventional fracturing fluids typically contain natural or synthetic water soluble polymers, which are well known in the art. Water soluble polymers viscosify the aqueous liquids at relatively low concentrations due to their high molecular weight. In an exemplary embodiment, the treatment fluid comprises water and an exemplary emulsion described herein. The treatment fluids may be prepared by mixing an exemplary emulsion with water. The additional water that is mixed with the emulsion to form the treatment fluid may be freshwater, saltwater (e.g. water containing one or more salts dissolved therein), brine (e.g. produced from subterranean formations), seawater, or combinations thereof. Generally, the water used may be from any source, provided that it does not contain an excess of compounds that may adversely affect other components in the aqueous treatment fluid or the formation itself. In certain exemplary embodiments, the water is brine with a total dissolved solids content (TDS) of about 5,000 to about 300,000 ppm, or about 100,000 to about 260,000 ppm. In certain exemplary embodiments, the total divalent cationic species content of the brine is in the range of about 5,000 to about 100,000 ppm, or about 10,000 to about 50,000 ppm. In exemplary embodiments, the polymer may be present in the treatment fluid in an amount of about 0.01% to about 1% by weight of the treatment fluid. In these applications, the treatment fluid, can be configured as a gelled fluid, such as a linear gel, a crosslinked gel, or a foamed gel fluid; acidic fluids, water and potassium chloride treatments, and the like. The fluid is injected at a pressure effective to create one or more fractures in the subterranean formation. Depending on the type of well treatment fluid utilized, various additives may also be added to the fracturing fluid to change the physical properties of the fluid or to serve a certain beneficial function. In one embodiment, the fluid does not contain a sufficient amount of water soluble polymer to form a gel. In exemplary embodiments, the treatment fluid comprises a proppant. In various exemplary embodiments, the proppants may be finely sized sand. Generally sand is referred to by the size of mesh which the sand will pass through, and the size of mesh which the sand will not pass through. Typically, a 20-40 mesh sand is used but other sizes, such as 40-50 or 40-60, may be utilized. Sand is also characterized by the “roundness” of the sand particles. Generally rounder sand is utilized in order to create more uniform void spaces between the particles and therefore better permeability within the propped fracture. Fracturing fluids also contain, for example, viscosifiers to slow the rate at which sand will separate from the fluids and permit the sand to be carried farther into the fractures. In other exemplary embodiments, other types of proppants may be used. For example, the proppant may be a ceramic proppant. The proppant may be a coated proppant, such as proppants with coatings with low coefficients of friction in order to reduce erosion caused by the fracturing fluid. Coatings also may be used to make the sand particles more round. Examples of such coatings include antimony trioxide, bismuth, boric acid, calcium barium fluoride, copper, graphite, indium, fluoropolymers (FTFE), lead oxide, lead sulfide, molybdenum disulfide, niobium dielenide, polytetrafluoroethylene, silver, tin, or tungsten disulfide or zinc oxide. Ceramic proppants are suggested, for example, in U.S. Pat. No. 4,555,493 to Watson et al., and low density ceramic proppants are suggested in U.S. Pat. No. 8,420,578 to Usova et al. Fracturing fluids may also contain other components as necessary or desired. For example, the fracturing fluids may contain acids for breaking the thickening polymers, salts such as calcium chlorides to increase the density of the fluids, corrosion inhibitors or other additives in the fracturing fluids. Also, fluid loss agents may be added to partially seal off the more porous sections of the formation so that the fracturing occurs in the less porous strata. Other oilfield additives that may also be added to the fracturing fluid include emulsion breakers, antifoams, scale inhibitors, H2S and or O2scavengers, biocides, crosslinking agents, surface tension reducers, buffers, fluorocarbon surfactants, clay stabilizers, fluid loss additives, foamers, friction reducers, temperature stabilizers, diverting agents, shale and clay stabilizers, paraffin/asphaltene inhibitors, corrosion inhibitors, and acids. For example, an acid may be included in the aqueous treatment fluids, among other things, for a matrix or fracture acidizing treatment. In fracturing embodiments, propping agent may be included in the aqueous treatment fluids to prevent the fracture from closing when the hydraulic pressure is released. In a particular embodiment, the treatment fluid further comprises a biocide. Methods of Use The emulsions and treatment fluids of the present embodiments may be used in any subterranean treatment. Such subterranean treatments include, but are not limited to, drilling operations, stimulation treatments, and completion operations. Those of ordinary skill in the art, with the benefit of this disclosure, will be able to recognize a suitable subterranean treatment. In exemplary embodiments, the emulsion comprises: water; a water-immiscible liquid; about 10% to about 80% by weight one or more polymers; and about 0.1% to about 5% by weight an exemplary inverting surfactant composition described herein. In exemplary embodiments, the methods may further comprise preparing the treatment fluid, or aqueous treatment fluid. Preparing the treatment fluid may comprise providing an emulsion as described herein, and combining the emulsion with water to form the treatment fluid. In exemplary embodiments, a method of treating a portion of a subterranean formation comprises: providing a treatment fluid of the present embodiments comprising an emulsion as described herein, and introducing the treatment fluid into the portion of the subterranean formation. In some embodiments, the treatment fluid may be introduced into the portion of the subterranean formation at a rate and pressure sufficient to create or enhance one or more fractures in the portion of the subterranean formation. The portion of the subterranean formation that the treatment fluid is introduced will vary dependent upon the particular subterranean treatment. For example, the portion of the subterranean formation may be a section of a well bore, for example, in a well bore cleanup operation. In the stimulation embodiments, the portion may be the portion of the subterranean formation to be stimulated. In exemplary embodiments, the treatment fluid may be introduced into the portion of the subterranean formation at a rate of about 30 bpm to about 250 bpm, or about 50 bpm to about 175 bpm. In exemplary embodiments, a method of treating a subterranean formation comprises: providing a treatment fluid comprising an exemplary emulsion described herein; and introducing the treatment fluid into a subterranean formation. In exemplary embodiments, a method of fracturing a subterranean formation comprises: (a) providing an exemplary emulsion as described herein; (b) mixing the emulsion with additional water to form a treatment fluid, wherein the one or more polymers are present in the treatment fluid in an amount of about 0.01% to about 1% by weight of the treatment fluid; and (c) introducing the treatment fluid into a subterranean formation at or above a pressure sufficient to create one or more fractures in the subterranean formation. In exemplary embodiments, the treatment fluid comprises brine. In exemplary embodiments, the exemplary emulsion or treatment fluid comprises proppant. In certain exemplary embodiments, a propping agent (or proppant) such as sand or other hard material is added to the exemplary emulsions or treatment fluids which serves to keep the fractures open after the fracturing operation. The fractures produced may be propped using proppants, or the fracturing fluid may include reactants to react with the surface of the rock faces to result in permeability along the fracture. The fractures may be utilized in vertical or horizontal wells, to produce natural gas, light tight oil, or for injection of fluids into the formation. Fracturing, or fracking, of formations is generally accomplished by injection of a slurry of fracturing fluid and proppant into the formation at pressures sufficiently great to exceed the tensile strength of the formation and cause the formation to separate at the point of the perforations. Formations will generally have a direction where the formation is under the least amount of stress, and the fracture will initially propagate in a plane perpendicular to the direction of such least stress. In deep formations, the weight of the overburden will generally assure that the direction of minimal stress is a horizontal direction. It is generally the goal to provide horizontal wellbores in such formation in the direction of the minimal formation stress so that fractures from the wellbore will tend to be perpendicular to the wellbore. This allows access to the maximum possible volume of formation from a horizontal wellbore of a limited length. Any method for hydraulic fracturing of formations known in the art may utilize the exemplary emulsions and treatment fluids. Propagation of fractures is typically halted or at least inhibited by interfaces between formations because the force exerted at the tip of the fracture can be dispersed at the interface of the formations. Larger fractures may therefore tend to have more rectangular shapes rather than disk shapes as the dimensions of the fracture exceed the height of the formation, and the fracture therefore grows laterally rather than continuing to grow vertically. In exemplary embodiments, methods for improving friction reduction properties of a treatment fluid, comprising: (i) providing an exemplary emulsion as described herein; and (ii) inverting the emulsion in the treatment fluid comprising brine. In certain embodiments, the resultant treatment fluid has an improvement in friction reduction, when compared to a similar treatment fluid in which the inverted emulsion is other than an exemplary inverting surfactant composition as described herein. In certain embodiments, the emulsion further comprises an emulsifier. In one embodiment, the improved friction reduction property is the percent friction reduction of the treatment fluid. In one embodiment, the improved friction reduction property is the time to achieve maximum friction reduction, or a desired percentage of the maximum friction reduction, for example 90%. In certain exemplary embodiments, the methods described herein provide an energy savings over methods which utilize a similar treatment fluid in which the inverted emulsion is other than an exemplary inverting surfactant composition as described herein. In exemplary embodiments, a method for improving friction reduction properties of a treatment fluid comprises: (i) providing an emulsion comprising: water; a water-immiscible liquid; one or more polymers; and an inverting surfactant composition comprising two or more surfactants selected from the group consisting of Surfactant A compounds, Surfactant B compounds, and Surfactant C compounds; and (ii) inverting the emulsion in the treatment fluid comprising brine; wherein the resultant treatment fluid has an improvement in friction reduction, when compared to a similar treatment fluid in which the emulsion that does not contain an inverting surfactant composition comprising two or more surfactants selected from the group consisting of Surfactant A compounds, Surfactant B compounds, and Surfactant C compounds. In exemplary embodiments, a method for improving friction reduction properties of a treatment fluid comprises: (i) providing an emulsion comprising: water; a water-immiscible liquid; one or more polymers; and an inverting surfactant composition comprising two or more surfactants comprising Surfactant B1 and Surfactant B2 compounds; and (ii) inverting the emulsion in the treatment fluid comprising brine; wherein the resultant treatment fluid has an improvement in friction reduction, when compared to a similar treatment fluid in which the emulsion that does not contain an inverting surfactant composition comprising two or more surfactants comprising Surfactant B1 and Surfactant B2 compounds. The inverting surfactant compositions, emulsions and treatment fluids of the present embodiments may have various uses, for example in crude oil development and production from oil bearing formations that can include primary, secondary or tertiary (enhanced) recovery. Chemical techniques, including for example injecting surfactants (surfactant flooding) to reduce interfacial tension that prevents or inhibits oil droplets from moving through a reservoir or injecting polymers that allow the oil present to more easily mobilize through a formation, can be used before, during or after implementing primary and/or secondary recovery techniques. Such techniques can also be used for enhanced oil recovery, or to complement other enhanced oil recovery techniques. The inverting surfactant compositions, emulsions and treatment fluids of the present embodiments may be used in any oil recovery technique, for example an oil recovery technique where the reduction of friction or interfacial tension is desired, or where mobilization of oil is desired. In exemplary embodiments, a method comprising using an inverting surfactant composition, emulsion or treatment fluid as described herein may be utilized for oil recovery, including but not limited to enhanced oil recovery. In exemplary embodiments, the method comprises providing a treatment fluid comprising an emulsion comprising one or more polymers and an exemplary inverting surfactant composition described herein; and introducing the treatment fluid into a subterranean formation; and recovering hydrocarbons from the subterranean formation. In exemplary embodiments, the method comprises providing an emulsion comprising one or more polymers and an exemplary inverting surfactant composition described herein; and introducing the emulsion into a subterranean formation; and recovering hydrocarbons from the subterranean formation. In certain exemplary embodiments, the methods further comprise adding a proppant. The term “brine” or “aqueous brine” as used herein refers to sea water; naturally-occurring brine; a chloride-based, bromide-based, formate-based, or acetate-based brine containing monovalent and/or polyvalent cations or combinations thereof. Examples of suitable chloride-based brines include without limitation sodium chloride and calcium chloride. Further without limitation, examples of suitable bromide-based brines include sodium bromide, calcium bromide, and zinc bromide. In addition, examples of formate-based brines include without limitation, sodium formate, potassium formate, and cesium formate. The following examples are presented for illustrative purposes only, and are not intended to be limiting. EXAMPLES In these examples, the impact of exemplary inverting surfactant compositions on inversion properties of certain polymer emulsion compositions is evaluated by measuring the friction reduction performance of polymer emulsions. Materials and Methods for Examples 1 and 2: I. Brine Three types of brine were used in the examples (Brine 1, Brine 2, and Brine 3). The composition of each brine is provided in Table 1. TABLE 1Brine compositionsTDSdivalentTDSNaKCaSrBaFeClSO4cationictotalSample(ppm)(ppm)Mg (ppm)(ppm)(ppm)(ppm)(ppm)(ppm)(ppm)(ppm)(ppm)Brine 141,67517,820—10,467——11034,71319010,577104,975Brine 238,8295661,73416,8932,866836143101,269522,472163,141Brine 341,240—11,71235,3601291,740301163,200—49,242253,682 II. Polymer Emulsions A polyacrylamide emulsion was prepared by addition of a monomer phase to a surfactant containing oil phase with homogenization. The resulting monomer emulsion was polymerized using free radical polymerization chemistry in the presence of adequate agitation and cooling, which resulted in a high molecular weight anionic polymer emulsion. The polymerization of acrylamide and co-monomers in an inverse emulsion resulted in a polymer emulsion containing sterically stabilized inverse lattices. The average particle size of the inverse emulsions was typically 0.7-1.5 micron. After polymerization, an inverting surfactant system was added to allow for rapid dilution and dissolution in water. III. Friction Loop Testing The friction loop is a laboratory instrument designed to simulate well fracturing flow conditions. Fracturing in the field often requires pumping over 50 barrels per minute through a ˜4.5″ bore which results in a highly turbulent flow (Reynolds number: 500,000 to 5,000,000). Although it is not possible to achieve this kind of flow in the lab, the friction loop designed simulates the field conditions to the maximum known extent (Reynolds number: 120,000). The data generated by this laboratory scale friction loop is accurate and widely accepted by the industry. The main components of the friction loop are: pump, magnetic flow meter and a differential pressure transmitter to create and monitor necessary conditions. All pipes and other components are constructed using stainless steel 316L/304L material. To test the friction reduction property of the polymer, the friction loop reservoir was filled with 20L of the required brine (see above table for recipes of various brines). This brine was then re-circulated through the friction loop at a flow rate of 24 gallons per minute across a five-foot section of half-inch diameter pipe (required to generate the above mentioned Reynolds number). The baseline pressure drop was measured. The exemplary emulsion containing polymer was now added (at a measured concentration of 0.5 gallons of polymer per thousand gallons of brine or 0.5 GPTG) to the recirculating brine solution, where it inverted and dissolved. The degree of friction reduction (% FRt) at a given time ‘t’ was calculated from the initial pressure drop ΔPi and the pressure drop at time t, ΔPt using the equation: %FRt=ΔPi-ΔPtΔPi×100 Example 1 A cationic polyacrylamide emulsion with a 10 mole % charge was prepared according to standard emulsion procedure without any inverting surfactants. This base emulsion was used to prepare Sample 1 and Sample 2, which included the inverting surfactants as shown in Table 2. Similarly, an anionic base polyacrylamide emulsion with a 15 mole % charge was prepared without any inverting surfactants and this base was used to prepare Sample 3 with the inverting surfactants shown in Table 2. The performance parameters of friction reduction, which include the Max FR (maximum friction reduction), t90(time to 90% friction reduction, a simple measure of inversion rate) and tmax(time to maximum friction reduction) were measured in Brine 1, Brine 2 and Brine 3 with different TDS at a dosage of 0.5 gptg and at 77° F.±3° F. Sample 1 contained 3 wt % of the Surfactants A, B1, B2 and C. Sample 2 contained 2 wt % of Surfactants A, B1 and C. Sample 3 contained 3 wt % of Surfactants A and B1. The results of the friction reduction experiments are provided in Tables 2 and 3, as well as inFIGS.1-5. TABLE 2Friction Reduction Performance of Polymer Emulsions withExemplary Inverting Surfactant CompositionsSurfactantSurfactantSurfactant AB1B2Surfactant CMax FRSample(wt %)(wt %)(wt %)(wt %)Water(%)Tmax(s)T90(s)130301030Brine 1613822130301030Brine 2523016130301030Brine 357542823040030Brine 158513123040030Brine 254904223040030Brine 355103503307000Brine 16053323307000Brine 25954343307000Brine 34911558 TABLE 3Friction Reduction Performance of Polymer Emulsions withComparative Anionic or Cationic Inverting SurfactantMax FRTmaxT90SampleWater(%)(s)(s)Anionic ComparativeBrine 1539248Anionic ComparativeBrine 2474827Anionic ComparativeBrine 2456135Cationic ComparativeBrine 141366155Cationic ComparativeBrine 25021074Cationic ComparativeBrine 3587137 Example 2 A cationic polyacrylamide emulsion with a 10 mole % charge was prepared according to standard emulsion procedure without any inverting surfactants. This base emulsion was used to prepare CPAM1-CPAM8 samples with the 3 wt % of inverting surfactant proportions as shown in Table 4. The surfactant compositions were premixed prior to addition to the emulsion. The friction reduction parameters for each sample were measured in Brine 1 at 77° F.±3° F. at a dosage of 0.5 gptg. The results are shown inFIG.6. TABLE 4Friction Reduction Performance of Polymer Emulsions withExemplary Inverting Surfactant CompositionsProportion ofInverting SurfactantsSampleA:B:C (wt %)Max FR (%)Tmax(s)T90(s)CPAM120:40:40614125CPAM230:40:30585028CPAM30:50:50584527CPAM40:40:60545028CPAM50:100:0547044CPAM60:30:70528336CPAM730:30:405016936CPAM820:30:505017938 Example 3 An anionic polyacrylamide emulsion with a 15 mole % charge was prepared according to standard emulsion procedure without any inverting surfactants. This base emulsion was used to prepare APAM1-APAM5 samples with the 3 wt % of inverting surfactant proportions as shown in Table 5. The surfactant compositions were premixed prior to addition to the emulsion. The friction reduction parameters for each sample were measured in Brine 1 at 77° F.±3° F. at a dosage of 0.5 gptg. The results are shown inFIG.7. TABLE 5Friction Reduction Performance of Polymer Emulsions withExemplary Inverting Surfactant CompositionsProportion ofInvertingSurfactants A:B:CSample(wt %)Max FR (%)Tmax(s)T90(s)APAM130:70:0605332APAM230:50:20559145APAM330:40:30538750APAM410:60:305211771APAM50:60:405011773 In the preceding specification, various embodiments have been described with reference to the examples. It will, however, be evident that various modifications and changes may be made thereto, and additional embodiments may be implemented, without departing from the broader scope of the exemplary embodiments as set forth in the claims that follow. The specification and drawings are accordingly to be regarded in an illustrative rather than restrictive sense. | 79,039 |
11859129 | These drawings are provided to illustrate various aspects of the invention and are not intended to be limiting of the scope in terms of dimensions, materials, configurations, arrangements, or proportions unless otherwise limited by the claims. DETAILED DESCRIPTION While these exemplary embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, other embodiments may be realized and that various changes to the invention may be made without departing from the spirit and scope of the present invention. Thus, the following more detailed description of the embodiments of the present invention is not intended to limit the scope of the invention, as claimed, but is presented for purposes of illustration only and not limitation to describe the features and characteristics of the present invention, to set forth the best mode of operation of the invention, and to sufficiently enable one skilled in the art to practice the invention. Accordingly, the scope of the present invention is to be defined solely by the appended claims. In describing and claiming the present invention, the following terminology will be used. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a particle” includes reference to one or more of such materials and reference to “injecting” refers to one or more such steps. As used herein with respect to an identified property or circumstance, “substantially” refers to a degree of deviation that is sufficiently small enough to not measurably detract from the identified property or circumstance. The exact degree of deviation allowable may in some cases depend on the specific context. As used herein, “adjacent” refers to the proximity of two structures or elements. Particularly, elements that are identified as being “adjacent” may be either abutting or connected. Such elements may also be near or close to each other without necessarily contacting each other. The exact degree of proximity may in some cases depend on the specific context. As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5, but also to include individual numerals such as 2, 3, 4, and sub-ranges such as 1 to 3, 2 to 4, etc. The same principle applies to ranges reciting only one numerical value, such as “less than about 4.5,” which should be interpreted to include all the above-recited values and ranges. Further, such an interpretation should apply regardless of the breadth of the range, or the characteristic being described. Means-plus-function or step-plus-function limitations will only be employed where for a specific claim limitation all the following conditions are present in that limitation: a) “means for” or “step for” is expressly recited; and b) a corresponding function is expressly recited. The structure, material or acts that support the means-plus function are expressly recited in the description herein. Accordingly, the scope of the invention should be determined solely by the appended claims and their legal equivalents, rather than by the descriptions and examples given herein. The present disclosure describes methods of forming a permeable proppant pack in a geothermal formation and particular proppants that can be used in geothermal formations. As mentioned above, proppants have often been used in the oil and gas industry to maintain permeability of fractures. However, such proppants have not been widely used in geothermal wells for a few reasons. First, many previous proppants have been made from materials that dissolve or degrade in water at high temperatures found in geothermal formations. Superhot rock (SHR) in geothermal formations can be at very high temperatures such as greater than 350° C. or greater than 400° C. Sand is a common proppant material, but sand begins dissolving at temperatures greater than about 225° C. Sintered bauxite begins degrading at temperatures above about 275° C. Therefore, these materials can be less effective as proppants in even moderate-temperature geothermal wells down to about 275° C. Dissolved minerals in geothermal fluids can also sometimes form deposits in fractures in the geothermal rock. The dissolved minerals may originate from dissolving proppants or from the geothermal rock itself. In some cases, mineral deposits can build up in fractures over time, which decreases the permeability of the fractures. Thus, the mineral deposition process can reduce the effectiveness of proppants used in the fractures. Additionally, many conventional proppants are made from materials that are more than twice the density of water. As a result, it can be difficult to make dense proppants flow into fractures that propagate horizontally or upward since gravity pulls the proppants downward. In the oil and gas industry, viscosifiers are sometimes used to increase the viscosity of fracturing fluid. The higher-viscosity fracturing fluid can help carry proppants into fractures. However, the viscosifiers that are currently available cannot be used at the high temperatures of geothermal formations and particularly in SHR. Thus, it is difficult to effectively introduce dense proppants into fractures in geothermal formations. The present disclosure describes proppants and methods of using the proppants in geothermal formations. These methods can mitigate the difficulties that have previously been encountered when using proppants in geothermal wells. Proppants can be prepared with a core of a solid material that is thermally stable at the temperature of the geothermal formation. In certain examples, the core can be made of petroleum coke, which is a carbon-rich solid produced in large quantities as a byproduct of oil-refining processes. Petroleum coke can be thermally stable and can withstand temperatures of 500° C. or higher. Petroleum coke can also be chemically resistant, non-soluble, and can have sufficient mechanical strength to prop open fractures without crushing. Petroleum coke also has a low density in the range of about 1.5 g/cm3to about 2.5 g/cm3. If petroleum coke is calcined, some hydrocarbon material and any remaining moisture can be removed to leave behind pores in the calcined petroleum coke. These pores can further reduce the density. For example, a reduced density of 2.04 to 2.07 g/cm3can be achieved. The proppant core can be coated with a resin coating. The resin coating can include a polymer having a decomposition temperature below the temperature of the geothermal formation and a melting temperature below the decomposition temperature. The resin coating can be useful in multiple ways. First, the resin coating can form a seal over open pores in the core. Air can be trapped in the pores in the core beneath the resin coating, which can make the proppants less dense. As mentioned above, it can be useful to use proppants with low density so that the proppants can flow into fractures together with a fracturing fluid without settling out too quickly under the force of gravity. The resin coating itself can also have a low density, such as from about 1 g/cm3to about 1.5 g/cm3. In this manner the density and buoyancy of the proppant can be varied. Additionally, the resin coating can undergo a melting and degradation process after the proppants are injected into a geothermal well. The proppants can be injected with a fracturing fluid at a temperature below the melting temperature of the resin coating. The proppants can flow into fractures in the geothermal rock. Over time the temperature of the rock can rise back toward the original temperature of the rock before the fracturing fluid was injected. As the temperature rises, the resin coating can begin to melt and become sticky. Proppant particles can stick together to form agglomerations of many proppant particles in close contact one with another. The temperature can then rise to the decomposition temperature of the resin coating, at which temperature the resin begins to degrade. Melting of the resin coating can occur over a time sufficient to allow proppants to be oriented in the initial fracturing stages. For example, the resin can chemically react to form other compounds and/or break down into smaller molecules. Eventually, the resin coating can completely degrade into products that dissolve or are washed away and the proppant cores can be left behind in the fracture. Since the proppants agglomerated while the resin coating was sticky, the remaining proppant cores can be arranged in densely packed groups with large open spaces in the fractures between the dense groups of proppant cores. The open spaces can provide high permeability for fluids to flow through the fractures. Further, these proppant cores can generally be free of polymer coating after degradation of the resin. The methods described herein can also include additional ways to modify and enhance the proppants, such as by adding low-density materials to the resin coating, embedding a mineral solubility modifying agent in the proppant cores, injecting the proppants together with a fully thermally degradable material to form islands of proppants separated by open spaces left behind by the thermally-degradable material, and the like. These and other features are explained in more detail below. One example method100of forming a permeable proppant pack in a geothermal formation is shown inFIG.1. This method includes injecting a fracturing fluid into a fracture of a geothermal formation having a formation temperature greater than about 230° C. and in some cases greater than 275° C. In some cases, the formation can be an SHR formation. Similarly, in some cases the formation temperature can be greater than 400° C. The fracturing fluid comprises proppants having a core of a solid material that is thermally stable at the formation temperature. The core is at least partially coated with a resin coating which comprises a polymer having a decomposition temperature below the formation temperature and a melting temperature below the decomposition temperature110. The resin coating can be allowed to be decomposed by heat from the geothermal formation, wherein the core of the proppants remains in the fracture after the resin coating has decomposed120. In one alternative, the proppant can have an exterior surface which is roughened so as to allow control over friction with formation walls. For example, increasing surface roughness can increase probabilities that proppants will remain in place at higher flow rates. As a general rule, surface roughness can be varied from N8 to N12 depending on formation conditions, rock type, and other factors. FIG.2shows a close-up cross-sectional view of an example proppant200. The proppant is a particle including a core210made of a solid material that is thermally stable at geothermal temperatures, such as greater than about 275° C. The core is coated with a resin coating220. The resin coating includes a polymer having a decomposition temperature below the temperature of the geothermal formation in which the proppant is to be used. This allows the resin coating to degrade and fall away over time when the proppants are heated up to the temperature of the formation. The polymer also has a melting temperature that is below the decomposition temperature. This allows the proppants to become tacky when the resin coating begins to melt. As a result, multiple neighboring proppant particles can adhere together to form agglomerations of proppants. The proppant shown inFIG.2has a spherical shape. In other examples, other shapes can be used such as irregular shapes, fiber shapes, rod shapes, block shapes, and others. Combinations of multiple different shapes can also be used. However, in certain examples the proppants can be spherical or nearly spherical. Nearly spherical proppants can have a low aspect ratio, defined as the longest dimension divided by the shortest dimension, from about 1 to about 1.5, or from about 1 to about 1.2, or from about 1 to about 1.1. The size of the proppants can vary, but in some examples the proppants can have an average total diameter from about 1 μm to about 4 mm, and in some cases 0.5 mm to 5 mm. The average total diameter can be a number average of the diameter of proppant particles, including the core and the resin coating together. In further examples, the proppants can have a relatively uniform size. For example, the proppants can have a narrow size distribution in which at least 90% of the proppant particles are within a range from about 75% of the average diameter to about 125% of the average diameter, or from about 80% of the average diameter to about 120% of the average diameter, or from about 90% of the average diameter to about 110% of the average diameter. In one alternative, proppants can have a relatively broad size distribution to accommodate variations in fracture apertures and type. In such cases, proppants can have less than 90% which are within 25% of average diameters. The core of the proppant can be made from a solid material that is thermally stable at the temperature of the geothermal formation in which the proppant is to be used. As used herein, “thermally stable” means that the material can be heated at the temperature of the geothermal formation without chemically decomposing, reacting, dissolving, melting, or otherwise degrading in a way that would cause the proppant core to be removed from a fracture in the geothermal formation. The thermally stable material can be stable for at least a time period of 14 days, or one month, or three months, or six months, or one year, or longer, and in one example at least 30 days. Thus, the proppant core can remain in a fracture in the geothermal formation for an extended period of time while being exposed to the temperature of the surrounding geothermal formation, and the proppant core does not degrade over the extended period of time such that the proppant core can keep the fracture open. In certain examples, the proppant core can be thermally stable up to a temperature greater than 275° C., or greater than 300° C., or greater than 350° C., or greater than 400° C., or greater than 500° C. In certain examples, the maximum stable temperature of the proppant core can be from about 275° C. to about 500° C., or from about 300° C. to about 475° C., or from about 275° C. to about 350° C. The solid material of the proppant core can also have a sufficient mechanical strength to prop open fractures in geothermal formations. Some non-limiting examples of proppant core materials include petroleum coke, calcined petroleum coke, coal coke, ceramic, metal oxides, metal hydroxides, graphite, high temperature silica, high temperature glass (e.g. VYCOR), other high temperature carbonaceous materials, or combinations thereof. In this context, “high temperature” would indicate thermal stability at geothermal formation temperatures for at least a time of operation of the geothermal recovery process. If the proppant core is made from petroleum coke, in some examples the petroleum coke can be raw petroleum coke or calcined petroleum coke. Other specific examples of suitable core materials can include, not are not limited to, kaolin, buckyballs (C60), carbon nanotubes, and the like. The proppant core can also be used as a carrier to carry a mineral solubility modifying agent into a geothermal fracture. The mineral solubility modifying agent can be a compound that either promotes precipitation of minerals in the fracture or promotes dissolution of minerals in the fracture. In some examples, the mineral solubility modifying agent can promote precipitation of minerals in the fracture, or in other words, the agent can cause an increased amount of minerals to precipitate and deposit in the fracture. The mineral solubility modifying agent can be on the proppant cores or held within pores in the proppant cores. Therefore, the increased mineral deposition can occur around the proppant cores in the fracture. In certain examples, minerals can be deposited on and around the proppant cores, and these minerals can act as a cement to hold the proppant cores in place in the fracture. The minerals can also add strength to the proppant pack. As mentioned above, the proppant cores can be agglomerated together due to the stickiness of the resin coating when the resin is near its melting temperature. Thus, in some examples, many proppant cores can be agglomerated near each other, and minerals can be deposited in spaces between the proppant cores and between the cores and the surround geothermal rock. Such a combination of agglomerated proppant cores and deposited minerals can form a very strong proppant pack that can remain in place in the fracture. In other examples, the mineral solubility modifying agent can be a compound that increases solubility of minerals in the fracture. Such compounds can cause increased dissolution of minerals in the fracture. The increased dissolution may counteract mineral deposition that would otherwise occur. The increased dissolution may also cause some minerals in the surrounding geothermal rock to be dissolved and removed, thereby increasing the permeability of the rock. Examples of mineral solubility modifying agents can include anhydrous calcium sulfate, NaOH, HCl, H2SO4(and others that alter pH), EDDHA, EDTA (chelating agents), and the like. In certain examples, when anhydrous calcium sulfate is used as the mineral solubility modifying agent, the proppants can be injected together with a fracturing fluid that includes sodium carbonate. The anhydrous calcium sulfate can react with the sodium carbonate to form calcium carbonate, which precipitates and deposits around the proppant cores in the fracture. The deposited calcium carbonate can act as a cement to hold the proppant cores in place and to strengthen the proppant pack. In the chemical reaction of calcium sulfate with sodium carbonate, sodium sulfate can also be formed. The sodium sulfate is water soluble and will simply be carried away by water flowing in the fracture. Since the proppant cores are initially coated by a resin coating, the anhydrous calcium sulfate in the cores does not react with the sodium carbonate in the fracturing fluid until after the resin coating is removed by degradation at high temperature. In another example, the coating can include a mineral solubility modifying agent which acts as a precursor to formation of a carbonate mineral phase. Such mineral solubility modifying agents can include urea, dimethyl carbonate, or the like. Upon delivery and placement of the proppants within the fractures, a second fluid containing a solution of a divalent cation salt can be injected and flush through the fractures to contact the placed proppants. Non-limiting examples of suitable divalent cation salts can include calcium chloride, magnesium chloride, calcium bromide, calcium iodide, magnesium bromide, magnesium iodide, and the like. Reaction between the mineral solubility modifying agent and the divalent cation salt forms a carbonate mineral phase which deposits on the proppants and surrounding formation. As one example, the mineral solubility modifying agent can be deposited within proppant core porous structure by solution deposition or mixed directly with the resin. Although concentration can vary, the mineral solubility modifying agent can be a minor component of the coating and is typically less than about 10% by weight of the coating, and often less than 5% by weight. Some proppant core materials can be porous. In such examples, a mineral solubility modifying agent can be held within pores of the proppant core. Calcined petroleum coke is one example of a porous proppant core material. Therefore, in certain examples the proppant core can include porous calcined petroleum coke with a mineral solubility modifying agent held within pores of the calcined petroleum coke. In other examples, the proppant core can be made of porous calcined petroleum coke without any mineral solubility modifying agent in the pores. In some examples, the proppant core can consist essentially of porous calcined petroleum coke and an optional mineral solubility modifying agent, i.e., the proppant core can be substantially devoid of any other materials besides the porous calcined petroleum coke and optional mineral solubility modifying agent. In a particular example, the proppant core can consist essentially of porous calcined petroleum coke with anhydrous calcium sulfate held within pores of the porous calcined petroleum coke. The amount of mineral solubility modifying agent included in the proppant can be less than the amount of the thermally stable solid core material such as petroleum coke. In various examples, the proppant core can include from about 75 wt % to 100 wt % of a solid, thermally stable material and up to about 25 wt % of a mineral solubility modifying agent if such agent is included. In certain examples, the proppant core can include the thermally stable solid material in an amount from about 80 wt % to about 99 wt % and the mineral solubility modifying agent in an amount from about 1 wt % to about 20 wt %. In other examples, the proppant core can include the thermally stable solid material in an amount from about 90 wt % to about 99 wt % and the mineral solubility modifying agent in an amount from about 1 wt % to about 10 wt %. The thermally stable solid material of the proppant core can be porous in some examples. As explained above, the pores can be used to carry a mineral solubility modifying agent in some examples. The porosity can also be useful to reduce the overall density of the proppants. Open pores can be filled with air. The resin coating that is added to the surface of the proppant core can seal off the pores and trap air inside the proppant. Thus, the overall density of the proppant can be lower when the porosity of the proppant core is higher. Adding a mineral solubility modifying agent to the proppant core can be useful for the reasons explained above. However, the mineral solubility modifying agent can also occupy some of the pore space and thus increase the density of the proppant. Therefore, the amount of mineral solubility modifying agent that is added can be balanced with the desired amount of open pore space for maintaining a low proppant density. In some examples, the mineral solubility modifying agent can be included in an amount that fills from about 1% to about 99% of the pore volume in the proppant core. In other examples, the mineral solubility modifying agent can be included in an amount that fills from about 5% to about 75%, or from about 5% to about 50%, or from about 5% to about 25% of the pore volume in the proppant core. The resin coating on the proppant core can include a polymer that has a decomposition temperature below the temperature of the geothermal formation in which the proppant is to be used. As used herein, the “decomposition temperature” refers to a temperature at which the polymer undergoes thermal degradation, which includes chemical changes to the polymer. In some examples, the polymer can break down into smaller molecules. The polymer can then physically break down into smaller pieces that can be washed away or dissolved in water or other fluid flowing through the geothermal fracture. Thermal decomposition may occur immediately at the decomposition temperature or over a period of time, such as over a period of 1 minute to 60 minutes, or 60 minutes to 6 hours, or 6 hours to one day, or one day to one week, for example. In various examples, the polymer in the resin coating can have a decomposition temperature from about 150° C. to about 500° C., or from about 200° C. to about 400° C., or from about 300° C. to about 375° C. The polymer can also have a melting temperature that is below the decomposition temperature in some examples. This can allow the polymer to begin to melt as the proppants heat up so that the proppants become sticky and stick together. In some examples, the melting temperature can be from about 120° C. to about 400° C. or from about 200° C. to about 350° C. or from about 250° C. to about 300° C. In other examples, the polymer can have a glass transition temperature (Tg) that is below the decomposition temperature. Heating the proppants above the glass transition temperature can also cause the resin coating to become sticky. In some examples, the glass transition temperature can be from about 120° C. to about 400° C., or from about 200° C. to about 350° C. or from about 250° C. to about 300° C. It is also noted that degradation time for the coating will be a function of reservoir temperature, coating thickness, polymer type, and the like. However, such variables can be considered during choice and design of the proppant coating to achieve a desired degradation time, e.g. generally 3 hours to about 14 days. Some non-limiting examples of polymers that can be included in the resin coating include polyethylene terephthalate, polybutylene terephthalate, polycarbonate, epoxy, acrylonitrile butadiene styrene, and combinations thereof. In some examples, the resin coating can include a single polymer, while in other examples the resin coating can include a mixture of multiple polymers. In addition to the polymer, in some examples the resin coating can include a particulate material distributed within the coating. The particulate material can be a material such as perlite, expanded graphite, silica (e.g. unmodified silica, fluorine-modified silica nanoparticles, etc.), mineral solubility modifying agent, tracer material, or combinations thereof. The particulate material can have an average particle size from about 1 micron to about 1,000 microns, or from about 1 micron to about 500 microns, or from about 1 micron to about 200 microns, or from about 1 micron to about 100 microns, or can be nanoparticles (e.g. less than about 1 micron), in some examples. In various examples, the resin coating can include the particulate material in an amount from about 1 wt % to about 75 wt % with respect to the total weight of the resin coating, or from about 1 wt % to about 50 wt %, or from about 1 wt % to about 25 wt %. The particulate material can have a lower density than the polymer in the resin coating. Therefore, adding the particulate material can decrease the density of the proppants in some cases. In some examples, the proppants can have an overall density, including the core and resin coating, from about 1 g/cm3to about 2.5 g/cm3, or from about 1 g/cm3to about 2 g/cm3, or from about 1 g/cm3to about 1.5 g/cm3, or from about 1.5 g/cm3to about 2 g/cm3. The resin coating can fully enclose the proppant core in some examples. However, in other examples the resin coating may partially coat the proppant core. However, it can be useful to have a resin coating that fully coats the proppant core so that the resin coating can trap air in pores of the proppant and/or prevent a mineral solubility modifying agent from being released from the proppant core until after degradation of the coating within the formation at a desired fracture location. The thickness of the resin coating can vary, but in some examples the thickness can be from about 0.5 mm to about 2 mm. In one aspect, the thickness of the resin coating can be 1% to 20% of a total particle diameter, and in some cases 5% to 15%. These proppants can be injected into a fracture in a geothermal formation. As explained above, the geothermal formation can have a formation temperature that is above the decomposition temperature of the polymer in the resin coating. In various examples, the formation temperature can be from about 275° C. to about 600° C., or from about 300° C. to about 500° C., or from about 350° C. to about 500° C.FIG.3shows an example system300that can be used to inject the proppants200with fracture fluid into fractures in a geothermal formation310. A wellbore320is drilled into the geothermal formation and a frack string330is lowered into the wellbore. In this example, the frack string includes packers332,334to isolate a portion of the wellbore. However, in other examples, the proppants can be used with other methods such as, but not limited to, sliding sleeves, plug and perforation methods, and the like. A fracturing fluid is injected into the space between the packers at high pressure, and the fracturing fluid flows into the geothermal formation through a perforation340in the wall of the wellbore, although more than one perforation can also be used. A dashed outline shows the fracture region350where the fracturing fluid, including the proppants, penetrates into the formation. Note that the size of the proppants200is exaggerated for this description and that the fracture region350is an idealized schematic. In practice, the fracture region may be less defined and irregular, including gradients in fracture pressure and fracture propagation throughout with irregular fractures extending and branching along rock planes and varied formation features. In addition to the proppants, the fracturing fluid352can include water and other ingredients. Additional ingredients can include additives such as biocides, acids, corrosion inhibitors, scale inhibitors, pH adjusting agents, surfactants, and others. In certain examples, the fracturing fluid can be devoid of viscosifiers. The low density of the proppants described herein can allow the proppants to be carried into fractures by the fracturing fluid even without viscosifiers. In some examples, the fracturing fluid can include water in an amount from about 90 wt % to about 99.9 wt %. The proppant particles can be included in the fracturing fluid in an amount from about 0.1 wt % to about 5 wt % in some examples, with respect to the total weight of the fracturing fluid. The fracturing fluid can be injected into an isolated portion of a geothermal well. As such, in some examples the method can include isolating a portion of the geothermal well before injecting the fracturing fluid into the geothermal formation. In certain examples, the portion of the geothermal well can be isolated mechanically using a frack string such as the one illustrated inFIG.3. The frack string can include packers to seal off a portion of the well from the remainder of the well while the fracturing fluid is being injected. Other methods can also be used to isolate a portion of the geothermal well, such as placing casings in the well in other portions where the fracturing fluid is not to be injected. FIG.4Ashows a close-up view of a fracture360in a geothermal formation310with multiple proppants200in the fracture. The proppants include cores210and resin coatings220as explained above. In this figure, the resin coatings have begun to melt together so that the proppant particles have agglomerated into groups. The resin coatings can stick together in this way when heat from the geothermal formation cause the temperature of the proppants to rise sufficiently after the proppants have been injected into the fracture. FIG.4Bshows the fracture360after the temperature has risen further and/or sufficient time has elapsed. The temperature has risen above the decomposition temperature of the resin coating of the proppants, leaving behind the proppant cores210in the fracture. As explained above, the proppant cores are made from a thermally stable material that is also strong enough the prop open the fracture. Therefore, the proppants cores can remain in the fracture and keep the fracture open so that the geothermal formation has increased permeability. It can also be useful to use the proppants described herein together with a thermally degradable material. In particular, a thermally degradable material can be injected into fractures to occupy some of the volume inside the fractures, and then the thermally degradable material can be completely removed by thermal decomposition to leave behind open spaces where the thermally degradable material previously was present. In some examples, a thermally degradable material can be injected into the fractures together with the proppants in such a way that the proppants form discrete islands separated by thermally degradable material. After the formation reheats to its normal temperature, the thermally degradable material can decompose and leave void spaces between the islands of proppants. The proppant cores (which are left behind after the resin coating of the proppants degrades) can hold the fracture open while the larger void spaces left by the thermally degradable material can provide high permeability to allow fluid to flow through the fracture. In a particular example, an amount of fracturing fluid containing proppants can be injected into a fracture, and then an amount of thermally degradable material can be injected into the fracture, and the injection of these materials can be alternated multiple times. This can result in discrete pockets or islands of proppants separated by the thermally degradable material. The resin coatings of the proppants can also stick together, which further encourages the proppants to aggregate and form islands.FIG.5shows an example fracture360that has been injected with proppants200alternated with a thermally degradable material370. The proppants have agglomerated to form islands of proppant particles separated by the thermally degradable material. After the geothermal formation reaches its normal temperature, the thermally degradable material will decompose and leave behind void spaces between the islands of proppants. When thermally degradable material is injected with the proppants, either in a mixture or in an alternated fashion, the relative amounts of thermally degradable material and proppants can vary. In some examples, the ratio of the volume of proppants injected into the fracture compared to the volume of thermally degradable material injected into the fracture can be from about 30:70 to about 70:30. Thus, the volume in the fracture that is occupied by proppants compared to the volume that is occupied by thermally degradable material can have the same ratio of 30:70 to 70:30. In further examples, the ratio can be from 40:60 to 60:40, or from 40:60 to 50:50, or from 35:65 to 45:55. The thermally degradable material can include a variety of materials that decompose at a temperature below the geothermal formation temperature. In some examples, the thermally degradable material can be a low-density viscous polymer gel such as polyvinyl alcohol and polyacrylamide, an anionic polymer of polyacrylamide, or a cross linked copolymer of either of these materials, or another viscous non-cellulosic polymer. In other examples, the thermally degradable material can be a low-density cement including a thermally degradable cement. In further examples, the thermally degradable material can have an increased gel strength or is made to have low density by foaming. Foaming agents may be used with nitrogen added as bubbles to cement or to a polymer, or a thermally degradable foamed polymer pellet such as foamed polylactic acid beads may be used. In yet another example, the thermally degradable material can be a particulate material such as glasses, polyglycolic acid, polylactic acid, polyhydroxybutyrate, co-hydroxyvalerate, polybutylene succinate, polypropylene fumarate, polycaprolactone, polyethylene terephthalate, polyhydroxyalkanoate, polycarbonate, polyoxybenzylmethylenglycolanhydride, polyethylene, polypropylene, polyester, polyaramid, polybutylene succinate, polyether ether ketone, or combinations thereof. In certain examples, the thermally degradable material can include a polyaramid such as poly(isophthaloyl chloride/m-phenylenediamine) or poly-paraphenylene terephthalamide. Non-limiting examples of glasses can include borosilicate glass, soda lime glass, flint glass, fiberglass, and combinations thereof. In other examples, the thermally degradable material can be a thermally degrading foamed cement such as a calcium aluminum cement, ammonium magnesium phosphate sorel cement, magnesium phosphate sorel cement, or magnesium potassium phosphate sorel cement. In further examples, the thermally degradable material can be an inorganic material such as boehmite, alumina, an acid-base cement, sorel cement, magnesium sulfate sorel cement, magnesium chloride sorel cement, calcium aluminum cement, calcium carbonate, ammonium magnesium phosphate sorel cement, magnesium phosphate sorel cement or magnesium potassium phosphate sorel cement, aluminum hydroxide, magnesium oxide, amorphouse silicon dioxide, crystalline silicon dioxide, and other water soluble inorganic material. Non-limiting examples of suitable acid-base cements can include magnesium oxy-acid cement, magnesium ammonium phosphate cement, magnesium potassium phosphate cement, magnesium oxyphosphate cement, calcium aluminate cement, and combinations thereof. Acid-base cements are materials that result from the reaction of a base in powder form with a liquid acid to produce a cementitious matrix and water. These cements are allowed to hydrate or set up to a hard material and then are ground into a target particle size distribution for effective use. Typical bases used for cement formation are oxides or carbonates of divalent and trivalent metals (e.g. calcium, cobalt, copper, and zinc), aluminosilicate glasses, and gelatinizing minerals. The latter minerals are those that contain small silicate groups such as orthosilicates, pyrosilicates, and silicates containing isolated six-membered silicate rings. Also included are minerals with large continuous silicon-oxygen networks that disintegrate into smaller silicate units including disilicates containing appreciable ferric iron in the silicon-oxygen sheets or three-dimensional network silica minerals that contain aluminum in the ratio of at least two aluminum atoms to three silicon atoms. The acid portion of the cement is typically an aqueous solution of inorganic or organic acids including phosphoric acid, multifunctional carboxylic acids, phenolic compounds, polymers bearing carboxylate or phosphate side-groups, and aqueous metal salts (typically chlorides, phosphates, and sulfates). A wide range of acid-base cement particles can be produced due to the large variety of acid and base sources that may be utilized. Thus it is possible to adjust the properties of the particles for different rates and temperatures at which dissolution takes place. As an example, magnesium oxide may be used as a base source, and aqueous magnesium chloride may be used as an acid source to produce an acid-base cement having the chemical formula of 5[Mg(OH)2](MgCl2)·8H2O (different cements can be produced by varying the ratio of MgO and MgCl2). As still another example, magnesium oxide may be used as the base component and aqueous magnesium sulfate may be chosen as an acid source, to produce magnesium oxysulfate acid-base cements such as the 3-form with the composition 3[Mg(OH)2]MgSO4)·8H2O. Similarly, magnesium oxide may be reacted with aqueous dihydrogen phosphate salts to produce an acid-base cement having the chemical formula MMg(PO4)·6H2O (where M=alkali metal cation or ammonium). A different magnesium oxyphosphate cement, MgHPO4·3H2O can be produced from magnesium oxide and aqueous phosphoric acid as the acid source. Although not required, degradation of the thermally degradable material can be accelerated by adding reactive compounds to the geothermal well after the thermally degradable material is in place. For example, when the thermally degradable material is certain types of cement, an acid can be introduced that converts the cement to water-soluble salts. In other examples, a chelating agent can be introduced to cause the dissolution of the cement. However, in other examples, the thermally degradable material can degrade due to high temperature alone without any reactive compounds being added to the geothermal well. The particular choice of thermally degradable material can determine the length of time for degradation as a function of temperature. For example, thermally degradable materials can be chosen to thermally degrade over a period of hours to several days at a particular formation temperature. More specifically, degradation kinetics can be tailored to match fracturing times and conditions. For example, choice of specific materials and particle sizes can affect degradation kinetics. Accordingly, in some examples the thermally degradable material can remain stable during the process of injecting the thermally degradable material and the proppants into the geothermal formation, and then the thermally degradable material can degrade at a desired rate after the injection is finished. The thermally degradable materials used in the methods described herein can be selected to be benign and to have benign breakdown products. Polymeric particles tend to degrade via hydrolysis, typically into non-persistent compounds, which circulate out of fractures. Inorganic particles, on the other hand, most often degrade via dissolution with time and as their temperature increases. Particulate size distributions can be chosen depending on the type of formation, expected fracture width, and desired distribution within a fracture to achieve desirable degree of hydraulic isolation. Although desired particle sizes can vary depending on the formation and desired degree of isolation, typical sizes can range from about 0.005 mm to about 2 cm. Particles shapes can also be varied to achieve desirable packing and degradation characteristics. For example, particles can be spherical, irregular, fibers, rods, blocks, or other shapes, including combinations of these shapes. Corresponding materials can be ground from larger material or grown and formed having a desired morphology. For example, the particles can be formed as a distribution of particles and/or in other shapes to enhance the sealing ability of the thermally degradable material. Some specific examples of thermally degradable materials can include calcium carbonate, soda lime glass, borosilicate glass, 50-100 mesh size crushed glass, 30-50 mesh size crushed glass, fiberglass, TZIM diverters (AltaRock Energy, USA), ENMAT™ PHBV resin (TianAn Biologic Materials Co., China), poly(propylene fumarate), polybutylene succinate, CAPA® 6500 (Ingevity, USA), boehmite, aluminum hydroxide, alumina, calcium aluminate cement, Bakelite, thermoset Bakelite, polycarbonate, polybisphenol carbonate, KEVLAR® (DuPont, USA), NOMEX® (DuPont, USA), polyethylene terephthalate, and polycaprolactone. FIGS.6and7show additional specific types of high-temperature proppants that can be used in the methods described above.FIG.6shows a proppant200that includes a proppant core210including porous calcined petroleum coke with a mineral solubility modifying agent212held within pores of the porous calcined petroleum coke. The mineral solubility modifying agent either promotes precipitation of minerals in a fracture or promotes dissolution of minerals in a fracture. The proppant also includes a resin coating220coated on the core. The resin coating includes a polymer that has a decomposition temperature from about 250° C. to about 400° C. and a melting temperature below the decomposition temperature. This proppant can be particularly useful in geothermal formations that have a formation temperature above the decomposition temperature of the polymer in the resin coating. The mineral solubility modifying agent can include any of the example mineral solubility modifying agents described above. FIG.7is a cross-sectional view of another example proppant200. This proppant includes a resin coating220with particulates of perlite222embedded in the resin. As mentioned above, in some examples the proppants can include particulates such as perlite or expanded graphite in the resin coating. These particulates can reduce the overall density of the proppants. The example shown inFIG.7also includes a proppant core210made of porous calcined petroleum coke with a mineral solubility modifying agent212held within the pores of the porous calcine petroleum coke. It is noted that the figures are not necessarily drawn to scale and any of the components shown therein may vary in size, including the proppants, cores, pores, resin coatings, perlite, or other particulates, etc. as discussed in more detail previously. The foregoing detailed description describes the invention with reference to specific exemplary embodiments. However, it will be appreciated that various modifications and changes can be made without departing from the scope of the present invention as set forth in the appended claims. The detailed description and accompanying drawings are to be regarded as merely illustrative, rather than as restrictive, and all such modifications or changes, if any, are intended to fall within the scope of the present invention as described and set forth herein. | 46,174 |
11859130 | DETAILED DESCRIPTION In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, one skilled in the art will understand that the invention may be practiced without these details. Moreover, while various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of” shall mean excluding more than trace amount of other ingredients and substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of this invention. Numeric ranges are also inclusive of the numbers defining the range. Additionally, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Reference throughout this specification to “one embodiment,” “an embodiment” or “some embodiments” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment,” “in an embodiment” or “in some embodiments” in various places throughout this specification are not necessarily all referring to the same embodiment or embodiments, but may be in some instances. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. The term “about” when used before a numerical value indicates that the value may vary within reasonable range, such as ±10%, ±5%, and ±1%. “Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon (C) and hydrogen (H) atoms, which is saturated or unsaturated (i.e., contains one or more double and/or triple bonds), having from 1 to 30 carbon atoms (C1-C30alkyl), and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted. In some embodiments, alkyl is a straight or branched hydrocarbon chain radical having 1 to 20 carbon atoms (C1-C20alkyl), 1 to 10 carbon atoms (C1-C10alkyl), 1 to 6 carbon atoms (C1-C6alkyl), or 1 to 4 carbon atoms (C1-C4alkyl). “Hydrate” refers to a complex formed by combining water molecules with molecules of a compound, such as a metal oxide, metal bronze or polyoxometalate. “Optional” or “optionally” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution. In some embodiments, provided herein are metal oxides, metal bronzes or polyoxometalates prepared under low temperature that are useful as charge storage materials in electrochromic devices. In some embodiments, the low temperature metal oxides, metal bronzes or polyoxometalates are prepared at a temperature below 150° C. In a flexible electrochromic device, processing temperature of all three layers (electrochromic layer, electrolyte layer and charge storage layer) cannot exceed the glass transition temperature of the plastic substrate materials. In some embodiment, the processing temperature is below 150° C. Providing low temperature solution processable materials is crucially important in order to achieve low cost flexible electrochromic films. Low temperature processable metal oxide, polyoxometalate, or metal bronze as the efficient charge storage materials could significant reduce the processing difficulty of a flexible electrochromic device. Further, the materials are transparent in the visible range and do not exhibit major color changes when changing from clear state to opaque state and vice versa, and therefore they do not interfere significantly with the electrochromic layer. In some embodiments, the charge storage materials described herein are at least about 70% transparent in visible light range. The transparency measurement may be taken by a minolta photospectrometer such as CM-5. In some embodiments, the charge storage materials described herein are about 70% to 90% transparent. Preparation of such materials avoids use of high temperature or vacuum and therefore could also reduce the overall cost of the flexible electrochromic product. In some embodiments, the metal oxide is one or more of TiO2, NiO, Nb2O5, WO3, NiO, V2O5, MoO3, CoO, MoO2, Ni2O3, Co2O3, VOx, and MoOy, etc., wherein x is from about 2 to about 2.5, and y is from about 2 to about 3, and any mixture from such metal oxide complexes or procurers, or polyoxometalates. In some embodiments, the metal oxides is of the formula M1y1M2y2M3y3Ox, wherein each M1, M2and M3is independently a metal, such as Ti, Ni, Nb, W, V, Mo, Si, Zr, Al or Co, each y1, y2 and y3 is independently from 0 to 100, provided that at least one of y1, y2 and y3 is not 0, and x is from 1 to 100. In some embodiments, each y1, y2 and y3 is independently from 0 to 50, or 0 to 20, or 0 to 10, provided that at least one of y1, y2 and y3 is not 0. In some embodiments, x is from 1.5 to 3, each y1, y2, y3 is independently 0 to 1, provided that y1+y2+y3 is 1. In some embodiments, the metal oxide is a hydrate. In some embodiments, the metal oxides is of the formula M1y1M2y2M3y3Ox·nH2O, wherein each M1, M2and M3is independently a metal, such as Ti, Ni, Nb, W, V, Mo, Si, Zr, Al or Co, x is from 1.5 to 3, each y1, y2 and y3 is independently from 0 to 1, provided that y1+y2+y3=1, and n is from 0.001 to 3. In some embodiments, the metal oxide is one or more ofVy1Tiy2Ox·nH2O wherein the ratio of y1 to y2 is from about 1:10 to about 50:1,Vy1Siy2Ox·nH2O wherein the ratio of y1 to y2 is from about 1:10 to about 50:1,Vy1Aly2Ox·nH2O wherein the ratio of y1 to y2 is from about 1:10 to about 50:1,Vy1Moy2Ox·nH2O wherein the ratio of y1 to y2 is from about 1:10 to about 50:1,Vy1Nby2Ox·nH2O wherein the ratio of y1 to y2 is from about 1:10 to about 50:1,Vy1Zry2Ox·nH2O wherein the ratio of y1 to y2 is from about 1:10 to about 50:1,and Vy1Tiy2Aly3Ox·nH2O, or other tri, tetra metal oxide mixture, or its pure form of oxide, wherein the ratio of y1 to y2 is from about 1:1 to about 50:1, the ratio of y2 to y3 is from about 50:1 to about 1:50, the ratio of y1 to y3 is from about 1:1 to about 50:1, wherein x is from about 2 to about 2.75, y1+y2 is about 1 or y1+y2+y3 is about 1, and n is from about 0 to about 3. In some embodiments, the ratio of y1 to y2 is from about 1:1 to about 40:1, or from about 1:1 to about 30:1, or from about 1:1 to about 20:1, or from about 1:1 to about 10:1, or from about 1:1 to about 5:1, or from about 1:10 to about 50:1, or from about 10:1 to about 50:1, or from about 10:1 to about 40:1, or from about 10:1 to about 30:1, or from about 10:1 to about 20:1, or from about 20:1 to about 50:1, or from about 20:1 to about 40:1, or from about 20:1 to about 30:1, or from about 30:1 to about 50:1, or from about 30:1 to about 40:1. In some other embodiments, the ratio of y1 to y2 is about 1:2. In some embodiments, the ratio of y1 to y3 is from about 1:1 to about 40:1, or from about 1:1 to about 30:1, or from about 1:1 to about 20:1, or from about 1:1 to about 10:1, or from about 1:1 to about 5:1, or from about 1:10 to about 50:1, or from about 10:1 to about 50:1, or from about 10:1 to about 40:1, or from about 10:1 to about 30:1, or from about 10:1 to about 20:1, or from about 20:1 to about 50:1, or from about 20:1 to about 40:1, or from about 20:1 to about 30:1, or from about 30:1 to about 50:1, or from about 30:1 to about 40:1. In some other embodiments, the ratio of y1 to y3 is about 1:2. In some embodiments, the ratio of y2 to y3 is from about 40:1 to about 1:50, or from about 40:1 to about 1:40, or from about 40:1 to about 1:30, or from about 40:1 to about 1:20, or from about 40:1 to about 1:10, or from about 40:1 to about 1:1, or from about 40:1 to about 10:1, or from about 40:1 to about 20:1, or from about 40:1 to about 30:1, or from about 30:1 to about 1:50, or from about 30:1 to about 1:40, or from about 30:1 to about 1:30, or from about 30:1 to about 1:20, or from about 30:1 to about 1:10, or from about 30:1 to about 1:1, or from about 30:1 to about 10:1, or from about 30:1 to about 20:1, or from about 20:1 to about 1:50, or from about 20:1 to about 1:40, or from about 20:1 to about 1:30, or from about 20:1 to about 1:20, or from about 20:1 to about 1:10, or from about 20:1 to about 1:1, or from about 20:1 to about 10:1, or from about 10:1 to about 1:50, or from about 10:1 to about 1:40, or from about 10:1 to about 1:30, or from about 10:1 to about 1:20, or from about 10:1 to about 1:10, or from about 10:1 to about 1:1, or from about 1:1 to about 1:50, or from about 1:1 to about 1:40, or from about 1:1 to about 1:30, or from about 1:1 to about 1:20, or from about 1:1 to about 1:10, or from about 1:10 to about 1:50, or from about 1:10 to about 1:40, or from about 1:10 to about 1:30, or from about 1:10 to about 1:20, or from about 1:20 to about 1:50, or from about 1:20 to about 1:40, or from about 1:20 to about 1:30, or from about 1:30 to about 1:50, or from about 1:30 to about 1:40. In some embodiments, the metal bronze (or hydrogen metal oxide bronze) is of the formula HzM1y1M2y2M3y3Ox, wherein each M1, M2and M3is independently a metal, such as Ti, Ni, Nb, W, V, Mo, Si, Zr, Al or Co, each y1, y2 and y3 is independently from 0 to 100, provided that at least one of y1, y2 and y3 is not 0, and each x and z is independently from 1 to 100. In some embodiments, each y1, y2 and y3 is independently from 0 to 50, or 0 to 20, or 0 to 10, provided that at least one of y1, y2 and y3 is not 0. In some embodiments, x is from 1 to 10, each y1, y2, y3 is independently 0 to 5, provided that y1+y2+y3 is 1 to 5, and z is 1 to 5. In some embodiments, x is from 1.5 to 6, each y1, y2, y3 is independently 0 to 2, provided that y1+y2+y3 is 1 to 3, and z is 1. In some embodiments, the metal bronze is HMoO2.4, HMoO2.75, HMoO2.93, HMoO3, HV2O5, or HVO2.46. Polyoxometalates are polyatomic ions, usually anions. In some embodiments, the polyoxometalate comprises two or more transition metal oxyanions linked together by shared oxygen atoms to form a closed 3-dimensional framework. In some embodiments, the polyoxometalate comprises three or more transition metal oxyanions linked together by shared oxygen atoms to form closed 3-dimensional frameworks. In some embodiments, each metal atom is independently a group 6 (e.g., Mo or W) or group 5 (e.g., V, Nb, or Ta) transition metal. In some embodiments, each metal atom is in its high oxidation state. In some embodiments, the polyoxometalate is an isopolymetalate, comprising only one kind of metal and oxide. In some embodiments, the polyoxometalate is a heteropolymetalate, comprising one metal, oxide, and a main group oxyanion (phosphate, silicate, etc.). In some embodiments, the polyoxometalate is a water-soluble fully inorganic early-transition metal-oxygen-anion clusters. In some embodiments, the polyoxometalate comprises an anion of the formula [AsM1y1M2y2M3y3Ox]m-, wherein A is P or Si, each M1, M2and M3is independently a metal, such as Ti, Ni, Nb, W, V, Mo, Si, Zr, Al or Co, each y1, y2 and y3 is independently from 0 to 100, provided that at least one of y1, y2 and y3 is not 0, each s and x is independently from 1 to 100, and m is an integer of from 1 to 10. In some embodiments, each y1, y2 and y3 is independently from 0 to 50, or 0 to 20, or 0 to 10, provided that at least one of y1, y2 and y3 is not 0. In some embodiments, one, two or three of y1, y2 and y3 are integers. In some embodiments, A is P. In some embodiments, A is Si. In some embodiments, s is an integer. In some embodiments, s is 1, 2, 3, 4 or 5. In some embodiments, x is from 1 to 70. In some embodiments, x is an integer. In some embodiments, x is 40 or 62. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, the polyoxometalate comprises [PW12O40]3−, [PMo12O40]3−, [P2W18O62]6−, [P2Mo18O62]6−, [Si2Nb6W18O77]8−, or [SiNb3W9O40]7−. In some embodiments, the polyoxometalate further comprises a cation, such as H+, Li+, Na+, K+, or NH4+. Examples of polyoxometalates include, but are not limited to, H3PW12O40, H3PMo12O40, K6P2W18O62, (NH4)6P2W18O62, (NH4)6P2Mo18O62, and H3PMoO40. In some embodiments, the metal oxide, metal bronze or polyoxometalate is in an amorphous form. In some embodiments, provided is a charge storage material of an electrochromic device comprising a metal oxide, metal bronze or polyoxometalate. The charge storage material described herein provide high performance. In some embodiments, the charge storage material is in the form of a layer of a thin film (charge storage layer) comprising a metal oxide, metal bronze or polyoxometalate and their nanoparticle form described herein. In some embodiments, the thickness of the film is from about 10 nm to about 1000 nm, such as from about 20 nm to about 500 nm, from about 20 nm to about 200 nm, or about 20 nm, about 50 nm, about 100 nm, about 150 nm, or about 200 nm, or any range between any two of the numbers, end points inclusive. In some embodiments, the charge storage material is in the form of nanoparticles. In some embodiments, the sizes of the nanoparticles are from about 1 nm to about 1000 nm, such as from about 10 nm to about 500 nm, from about 20 nm to about 200 nm, or about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 150 nm, or about 200 nm, about 250 nm, about 500 nm, or any range between any two of the numbers, end points inclusive. The present disclosure is also directed to an electrochromic device comprising a charge storage material described herein. Other components of electrochromic devices are generally known in the art. In an electrochromic device, there are two set of materials, anode which is oxidized while apply positive voltage while driving the device, and cathode which is reducing. The metal oxide, metal bronze or polyoxometalate described herein can serve as either the anode or the cathode charge storage materials. In some embodiments, the solution processed low temperature metal oxide, metal bronze or polyoxometalate described herein can serve as either the anode or the cathode charge storage materials. In some embodiments, the electrochromic device further comprises an electrochromic polymer as described in U.S. Pat. No. 9,975,989, which is hereby incorporated by reference in its entirety. In some embodiments, the electrochromic device comprising an electrochromic layer, electrolyte layer and charge storage layer as illustrated byFIG.1, wherein the charge storage layer comprises the metal oxide, metal bronze or polyoxometalate described herein. In some embodiments, provided is a method of preparing the metal oxide, metal bronze or polyoxometalate useful as a charge storage material in an electrochromic device. In some embodiments, solution processed low temperature metal oxide, metal bronze or polyoxometalate can be used as a charge storage material in an electrochromic device. Metal oxides could be synthesized prior coating or during the coating process. In some embodiments, the precursor of the metal oxide, metal bronze or polyoxometalate is one or more metal alkoxide. In some embodiments, the metal alkoxide is of the structure M(OR)p, wherein M is a metal, such as Ti, V, Nb, Zr, Mo, Ni, Cu, or Cr, or a mixture thereof, R is an alkyl group, and p is 1, 2, 3, 4, 5, or 6. In some embodiments, the R is C1-C4alkyl, such as methyl, ethyl or isopropyl. In some embodiments, the method comprises mixing a metal alkoxide in a solvent such as methanol, ethanol, iso-propanol, butanol, methoxyethanol to form a solution or suspension. In some embodiments, the method further comprises coating the solution or suspension on a substrate to form a wet layer. The metal alkoxide hydrolyzes to related metal oxide after being left under ambient conditions. In some embodiments, the method further comprises letting the wet layer dry under ambient conditions to form a dried metal oxide layer. In some embodiments, the method further comprises drying the dried metal oxide layer at a temperature of below about 150° C., to form a charge storage material. In some embodiments, the substrate is a flexible substrate and does not tolerate high temperatures. In some embodiments, the method comprises forming a solution or suspension of nanoparticles of a metal oxide in a solvent, such as methanol, ethanol or isopropanol or water, forming a thin layer of the solution or suspension and then forming a solid film of the metal oxide by drying the thin layer of the solution or suspension. In some embodiments, the method comprises oxidizing a metal in a solvent, such as methanol, ethanol or isopropanol, with an oxidation reagent, such as H2O2, to form a solution or suspension comprising the metal oxide, metal bronze or polyoxometalate. In some embodiments, the method further comprises forming a thin layer of the solution or suspension and then forming a solid film of the metal oxide, metal bronze or polyoxometalate by drying a thin layer of the solution or suspension. In some embodiments, the methods described herein do not comprise a temperature that is higher than 150° C. In some embodiments, the methods described herein are conducted at an ambient temperature. In some embodiments, provided are metal oxides, metal bronzes or polyoxometalates prepared by methods described herein which are useful as charge storage materials of electrochromic devices. EXAMPLES The present technology is further defined by reference to the following examples. It will be apparent to those skilled in the art that many modifications, both to compositions and methods, may be practiced without departing from the scope of the current disclosure. Example 1 Titanium Oxide Such oxide material could be pre-synthesized, like TiO2nano-crystals, and dissolved or dispersed in a proper solvent. For example, 5 nm to 15 nm anatase TiO2nano-crystal dispersion in water was diluted into 10 mg/mL concentration. The solution was dispersed with a slot die coater to form a thin uniform film with 20 micrometer thick liquid layer. The resulted dried TiO2solid layer is about 200 nm thick. Such thin layer could be served as charge storage layer in cathode with an electrochromic polymer material as the anode. The electrolyte was described in US2017/0299932, which is hereby incorporated by reference in its entirety. The electrochromic polymer could be fully bleached at 2.1 V, and reversibly colorized at −1.0V. The absorption spectra of the two states are shown inFIG.2. Example 2 Molybdenum Oxide Metal oxide complex could be also formed during the film formation via reaction such as hydrolysis process. Mo(OC2H5)5with 1% weight ratio in ethanol was applied by a slot die coater, and form a solid film. The film was baked and assembled with an electrochromic polymer and electrolyte (e.g., those described in US2017/0299932) to form an active electrochromic device. Such device could be switched between opaque and clear with 1.5 V and −1 V. The absorption spectra are shown inFIG.3. Example 3 Polyoxometalate Metal oxide or polyoxometalate could be synthesized as the metal bronze or solution. 1 g of Mo was added to 100 mL ethanol and then 3 mL of 30% H2O2was added. The mixture was stirred overnight in room temperature to result a dark blue metal bronze solution. The solution was applied via a slot die coater to form a 100 nm solid film, the composition of such metal bronze material is suggested to be HyMoOx. Such film was assembled with electrochromic polymer and electrolyte to form an active device. Such device could be switched reversibly with 1.2V to −1V. The absorption spectra were shown inFIG.4. Applications/Uses Embodiments of the charge storage materials comprising metal oxide, metal bronze or polyoxometalate disclosed herein may be used in various applications, devices, industries etc. For example, the charge storage materials may be configured for use in smart window and display technology, e.g., anti-glare car mirrors, smart windows configured to modulate the transmission or reflected solar radiation for use in cars, aircrafts, buildings, and the like; protective eyewear; camouflage and/or chameleonic materials; and other electrochromic devices. The invention described and claimed herein is not to be limited in scope by the specific preferred embodiments disclosed herein, as these embodiments are intended as illustrations of several aspects of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. | 22,356 |
11859131 | DESCRIPTION OF EMBODIMENTS The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. In describing embodiments illustrated in the drawings, specific terminology is employed for the sake of clarity. However, the disclosure of this specification is not intended to be limited to the specific terminology so selected and it is to be understood that each specific element includes all technical equivalents that have a similar function, operate in a similar manner, and achieve a similar result. Hereinafter, embodiments of the present invention are described. The embodiments are not limited by the following description and can be appropriately modified without departing from the scope of the present invention. Electrochromic Compound The electrochromic compound according to an embodiment has a triarylamine backbone. Specifically, the electrochromic compound according to an embodiment has a triphenylamine backbone in which the nitrogen atom constituting the triarylamine backbone is substituted with three phenyl groups. Preferably, the electrochromic compound according to an embodiment is a radical-polymerizable compound having a triarylamine backbone, represented by the following general formula (1). In the general formula (1), each of X1to X3independently represents a carbon atom or a silicon atom, and each of R1to R15independently represents a member selected from the group consisting of a hydrogen atom, a halogen atom, a monovalent organic group, and a polymerizable functional group. Specific examples of the halogen atom include, but are not limited to, fluorine, chlorine, bromine, and iodine. Specific examples of the monovalent organic group include, but are not limited to, hydroxyl group, nitro group, cyano group, carboxyl group, carbonyl group, amide group, aminocarbonyl group, sulfonate group, sulfonyl group, sulfonamide group, aminosulfonyl group, amino group, alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, aryloxy group, alkylthio group, arylthio group, heteroaryl group, and silyl group. Each of these groups may have a substituent. Specific examples of the monovalent organic group having a substituent include, but are not limited to: substituted carbonyl groups such as alkoxycarbonyl group, aryloxycarbonyl group, alkylcarbonyl group, arylcarbonyl group, monoalkylaminocarbonyl group, dialkylaminocarbonyl group, monoarylaminocarbonyl group, and diarylaminocarbonyl group; substituted sulfonyl groups such as alkoxysulfonyl group, aryloxysulfonyl group, alkylsulfonyl group, arylsulfonyl group, sulfoneamide group, monoalkylaminosulfonyl group, dialkylaminosulfonyl group, monoarylaminosulfonyl group, and diarylaminosulfonyl group; and substituted alkylamino groups such as monoalkylamino group and dialkylamino group. Specific examples of the substituents include, but are not limited to: alkyl groups or alkenyl groups; alkynyl groups; aryl groups; alkoxy groups; aryloxy groups; alkylthio groups; arylthio groups; and heteroaryl groups. Among these substituents, alkyl groups having 1 or more carbon atoms, alkenyl groups having 2 or more carbon atoms, alkynyl groups having 2 or more carbon atoms, aryl groups having 6 or more carbon atoms, heteroaryl groups having 2 or more carbon atoms, alkoxy groups, aryloxy groups, and heteroaryloxy groups are preferable. Preferred examples of the alkyl groups having 1 or more carbon atoms include, but are not limited to, straight-chain, branched-chain, or cyclic alkyl groups having 1 to 30 carbon atoms, for material availability. Among the cyclic alkyl groups having 1 to 30 carbon atoms, cyclic alkyl groups having 1 to 18 carbon atoms are particularly preferable. Specific examples of the alkyl groups having 1 or more carbon atoms include, but are not limited to, methyl group, ethyl group, propyl group, butyl group, tert-butyl group, isopropyl group, isobutyl group, pentyl group, hexyl group, heptyl group, ethylhexyl group, octyl group, decyl group, dodecyl group, 2-butyloctyl group, octadecyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, and adamantyl group. Preferred examples of the alkenyl groups having 2 or more carbon atoms include, but are not limited to, straight-chain, branched-chain, or cyclic alkenyl groups having 2 to 30 carbon atoms. Among the cyclic alkenyl groups having 2 to 30 carbon atoms, cyclic alkenyl groups having 1 to 18 carbon atoms are particularly preferable. An alkenyl group having 2 or more carbon atoms is a substituent obtained by removing arbitrary two hydrogen atoms from an alkyl group having 1 or more carbon atoms. Specific examples of the alkenyl groups having 2 or more carbon atoms include, but are not limited to, vinyl group (ethenyl group), propenyl group, butenyl group, pentenyl group, hexenyl group, heptanyl group, octenyl group, decenyl group, dodecenyl group, octadecenyl group, cyclobutenyl group, cyclopentenyl group, and cyclohexenyl group. Preferred examples of the alkynyl groups having 2 or more carbon atoms include, but are not limited to, straight-chain, branched-chain, or cyclic alkynyl groups having 2 to 30 carbon atoms. Among the cyclic alkynyl groups having 2 to 30 carbon atoms, cyclic alkynyl groups having 2 to 18 carbon atoms are particularly preferable. An alkynyl group having 2 or more carbon atoms is a substituent obtained by removing arbitrary four hydrogen atoms from an alkyl group having 1 or more carbon atoms. Specific examples of the alkynyl groups having 2 or more carbon atoms include, but are not limited to, ethynyl group, propynyl group, butynyl group, pentynyl group, hexynyl group, heptynyl group, octynyl group, decynyl group, dodecynyl group, and octadecynyl group. Specific examples of the aryl groups having 6 or more carbon atoms include, but are not limited to, phenyl group, o-tolyl group, m-tolyl group, p-tolyl group, p-chlorophenyl group, p-fluorophenyl group, p-trifluorophenyl group, naphthyl group, biphenyl group, anthryl group, phenanthryl group, pyrenyl group, fluorenyl group, benzopyrenyl group, and chrysenyl group. Preferred examples of the heteroaryl groups having 2 or more carbon atoms include, but are not limited to, heteroaryl groups having 2 to 12 carbon atoms. The heteroaryl group having 2 or more carbon atoms may comprise nitrogen atom, sulfur atom, oxygen atom, silicon atom, and/or selenium atom. Preferably, the heteroaryl group having 2 or more carbon atoms comprises at least one of nitrogen atom, sulfur atom, and oxygen atom. Specific examples of the heteroaryl groups having 2 or more carbon atoms include, but are not limited to, monocyclic heteroaryl groups and polycyclic heteroaryl groups. Specific examples of the monocyclic heteroaryl groups include, but are not limited to, pyridine ring, pyrimidine ring, pyridazine ring, pyrazine ring, tetrazine, thiophene ring, furan ring, pyrrole, imidazole, pyrazole, thiazole ring, oxazole ring, isoxazole, oxadiazole ring, triazine ring, tetrazole ring, and triazole ring. Specific examples of the polycyclic heteroaryl groups include, but are not limited to, quinoline group, isoquinoline group, quinazoline group, phthalazine group, indole group, benzothiophene group, benzofuran group, benzimidazole group, benzothiodiazole group, acridine group, phenoxazine group, phenothiazine group, carbazole group, benzodithiophene group, benzodifuran group, dibenzofuran group, and dibenzothiophene group. The polycyclic heteroaryl group may be a group in which an aryl group and a heteroaryl group are bound via a covalent bond, or a group in which an aryl group and a heteroaryl group are condensed into a ring. Specific examples of the group in which an aryl group and a heteroaryl group are bound via a covalent bond and the group in which an aryl group and a heteroaryl group are condensed into a ring include, but are not limited to, biphenyl group, terphenyl group, 1-phneylnaphthalene group, and 2-phenylnaphthalene group. The polymerizable functional group in the general formula (1) is a polymerizable group having a carbon-carbon double bond. Specific examples of the polymerizable functional group include, but are not limited to, 1-substitued ethylene functional groups and 1,1-substituted ethylene functional groups described below. Specific examples of the 1-substituted ethylene functional groups include, but are not limited to, a functional group represented by the following general formula (i). [Chem. 3] CH2═CH—X1— General Formula (i) In the general formula (i), X1represents an arylene group, an alkenylene group, —CO— group, —COO— group, —CON(R100)— group (where R100represents a hydrogen atom, an alkyl group, an aralkyl group, or an aryl group), or —S— group. The arylene group and the alkenylene group each may have a substituent. Specific examples of the arylene group include, but are not limited to, phenylene group and naphthylene group. The phenylene group may have a substituent. Specific examples of the alkenylene group include, but are not limited to, ethenylene group, propenylene group, and butenylene group. Specific examples of the alkyl group include, but are not limited to, methyl group and ethyl group. Specific examples of the aralkyl group include, but are not limited to, benzyl group, naphthylmethyl group, and phenethyl group. Specific examples of the aryl group include, but are not limited to, phenyl group and naphthyl group. Specific examples of the functional group represented by the general formula (i) include, but are not limited to, vinyl group, styryl group, 2-methyl-1,3-butadienyl group, vinyl carbonyl group, acryloyloxy group, acryloylamide group, and vinyl thioether group. Specific examples of the 1,1-substituted ethylene functional groups include, but are not limited to, a functional group represented by the following general formula (ii). [Chem. 4] CH2═C(Y)—X2— General Formula (ii) In the general formula (ii), Y represents an alkyl group, an aralkyl group, an aryl group, a halogen atom, cyano group, nitro group, an alkoxy group, or —COOR101group (where R101represents a hydrogen atom, an alkyl group, an aralkyl group, an aryl group, or CONR102R103(where each of R102and R103independently represents a hydrogen atom, an alkyl group, an aralkyl group, or an aryl group)). Each of these groups may have a substituent. X2represents a substituent such as those of X1in the general formula (i) or an alkylene group. At least one of Y and X2represents oxycarbonyl group, cyano group, an alkenylene group, or an aromatic ring. Specific examples of the alkyl group include, but are not limited to, methyl group and ethyl group. Specific examples of the aralkyl group include, but are not limited to, benzyl group, naphthylmethyl group, and phenethyl group. Specific examples of the aryl group include, but are not limited to, phenyl group and naphthyl group. Specific examples of the alkoxy group include, but are not limited to, in addition to methoxy group and ethoxy group, diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, tetrapropylene glycol, polyethylene glycol, and propylene glycol, in each of which the unit of ethylene glycol or polypropylene glycol is condensed. Specific examples of the polymerizable functional group represented by the general formula (ii) include, but are not limited to, α-acryloyloxy chloride group, methacryloyloxy group, α-cyanoethylene group, α-cyanoacryloyloxy group, α-cyanophenylene group, and methacryloyl amino group. X1, X2, and Y may be further substituted with a substituent, such as a halogen atom, nitro group, cyano group, an alkyl group (e.g., methyl group, ethyl group), an alkoxy group (e.g., methoxy group, ethoxy group), an aryloxy group (e.g., phenoxy group), an aryl group (e.g., phenyl group, naphthyl group), and an aralkyl group (e.g., benzyl group, phenethyl group). Among the functional groups represented by the general formula (i) or (ii), acryloyloxy group and methacryloyloxy group are particularly preferable. In the general formula (1), the polymerizable functional group is preferably substituted at the terminal of an alkyl group having 1 or more carbon atoms, an aryl group having 6 or more carbon atoms, or an alkyl-substituted aryl group having 7 or more carbon atoms, more preferably at the terminal of an alkyl group, for improving resistance to oxidation and reduction. Preferably, the polymerizable functional group is bound to the main backbone of the electrochromic compound via at least an alkyl group having 2 or more carbon atoms. Preferably, each of R1to R3independently represents a member selected from the group consisting of a halogen atom, a monovalent organic group, and a polymerizable functional group. Since the para position of the nitrogen atom of the triphenylamine backbone has a high electron density and is reactive, it is preferable that this site be substituted with any substituent other than hydrogen, i.e., of a halogen atom, a monovalent organic group, or a polymerizable functional group. Preferably, one or more of R1to R9each represent a polymerizable functional group. This is because, when the electrochromic compound according to the present embodiment is used as a polymer film, it makes it possible to impart polymerizable property to the electrochromic compound and easy to introduce a substituent thereto. More preferably, one or more of R1to R3each represent a polymerizable functional group. For preventing the occurrence of color change and a side reaction between molecules at the time when color is developed, preferably, each of R1to R9independently represents an alkyl group, an alkoxy group, an aryl group, an aryloxy group, a halogen atom, or a polymerizable functional group. Among the alkyl groups, a tert-butyl group is particularly preferable. When each of R1to R9is a monovalent organic group, preferred examples of the monovalent organic group include alkyl groups having 1 or more carbon atoms, alkenyl groups having 2 or more carbon atoms, alkynyl groups having 2 or more carbon atoms, aryl groups having 6 or more carbon atoms, heteroaryl groups having 2 or more carbon atoms, alkoxy groups having 1 or more carbon atoms, aryloxy groups, and heteroaryloxy groups. Each of R1to R9may be a group in which one or more aryl groups and/or heteroaryl groups are bound via a covalent bond, or a group in which one or more aryl groups and/or heteroaryl groups are condensed into a ring. The group in which one or more aryl groups and/or heteroaryl groups are bound via a covalent bond, or the group in which one or more aryl groups and/or heteroaryl groups are condensed into a ring, contains 1 to 100 carbon atoms and may further contain a hetero atom. The upper limit of the total number of carbon atoms is preferably 50, and more preferably 36. Specific examples of the hetero atom include, but are not limited to, oxygen atom, sulfur atom, and nitrogen atom. For transparency of the electrochromic compound in the decolored state, preferably, the group in which one or more aryl groups and/or heteroaryl groups are bound via a covalent bond, or the group in which one or more aryl groups and/or heteroaryl groups are condensed into a ring, has an absorption end at 400 nm or less, more preferably at 380 nm or less. The number of the group in which aryl groups and/or heteroaryl groups are bound via a covalent bond or the group in which aryl groups and/or heteroaryl groups are condensed into a ring may be in the range of from 1 to 6, but is preferably from 1 to 3, and more preferably from 1 to 2. This is because, in contrast to triphenylamine that is a chromophore, the group in which aryl groups and/or heteroaryl groups are bound via a covalent bond and the group in which aryl and/or heteroaryl groups are condensed into a ring do not contribute to color development. Therefore, a significant increase in number of these groups is not preferable for color developing efficiency and material cost. Preferably, each of R10to R15independently represents a member selected from the group consisting of a halogen atom, a monovalent organic group, and a polymerizable functional group. This is because hydrogen on the benzyl position has a high acidity and is reactive. Therefore, the benzyl position is preferably substituted with a halogen atom, a monovalent organic group, or a polymerizable functional group, other than a hydrogen atom. Preferably, each of R10to R15independently represents an alkyl group, an alkoxy group, an aryl group, a heteroaryl group, a halogen atom, or a polymerizable functional group, and most preferably any of an alkyl group, an alkoxy group, an aryl group, and a polymerizable functional group. Preferably, the polymerizable functional group is present at a part of an alkyl group or an aryl group, particularly at the terminal thereof. Specific examples of the compound represented by the general formula (1) include, but are not limited to, the following Examples Compounds. In the following Example Compounds, MeO— represents methoxy group. The electrochromic compound according to the present embodiment is not limited to the examples listed below. Example Compound 1 Example Compound 2 Example Compound 3 Example Compound 4 Example Compound 5 Example Compound 6 Example Compound 7 Example Compound 8 Example Compound 9 Example Compound 10 Example Compound 11 Example Compound 12 Example Compound 13 Example Compound 14 Example Compound 15 Example Compound 16 Example Compound 17 Example Compound 18 Example Compound 19 Example Compound 20 Example Compound 21 Example Compound 22 Example Compound 23 Example Compound M1 Example Compound M2 Example Compound M3 Example Compound M4 Example Compound M5 Example Compound M6 Example Compound M7 Example Compound M8 Example Compound M9 Example Compound M10 Example Compound M11 Example Compound M12 The electrochromic compound according to the present embodiment is a radical-polymerizable compound having a triarylamine backbone, represented by the general formula (1) in which each of X1to X3and R1to R15represents a specific element or group. Accordingly, the electrochromic compound according to the present embodiment can improve light durability and durability against repetitive electrostatic charging/charge-removing processes similar to redox processes. To be applied to electrochromic elements, the electrochromic compound is required to provide properties required by electrochromic elements. Electrochromic elements may require, for example, an electrochromic composition that is transparent in the neutral state, an electrochromic composition that has solubility, or electrochromic layers that are stackable. The electrochromic compound according to the present embodiment can provide such properties required by electrochromic elements. Synthesis of Electrochromic Compound The electrochromic compound according to the present embodiment can be efficiently synthesized by the following Synthesis Scheme Example 1 or Synthesis Scheme Example 2. The electrochromic compound according to the present embodiment can be synthesized by applying the method described in J. Mater. Chem., 22, 2017, 15397-15404 or Org. Lett., Vol. 11, No. 7, 2009. Synthesis Scheme Example 1 A halogen or triflate compound (I-I) having an ester group on the ortho position is reacted with an aromatic amine compound (I) having an ester group on the ortho position. In the case of producing a symmetrical triphenylamine, the equivalent may be around 2. For this reaction, a carbon-nitrogen coupling reaction using an organometallic catalyst and a base can be employed, and name reactions such as the Ullman condensation and the Buchwald-Hartwig coupling can be employed. In the case of producing an asymmetric triphenylamine, a compound (I-II) is subsequently reacted under the same conditions as the compound (I-I), thus producing a triphenylamine derivative (III). In the above scheme, each of R1to R15independently represents a hydrogen atom, a halogen atom, or a monovalent organic group. Each of X1and X2independently represents a halogen or a triflate group. Synthesis Scheme Example 2 Subsequently, the triphenylamine derivative (III) is reacted with an organometallic compound (III-I). In this reaction, an organic lithium compound, a Grignard reagent, or an organic zine compound can be suitably used. By this reaction, the ester is converted to a tertiary alcohol and a derivative (IV) is produced. Subsequently, a reagent (IV-I) is added to the triphenylamine derivative (IV) to cause the intramolecular Friedel-Crafts reaction, thereby producing an electrochromic compound (V) according to the present embodiment by cyclodehydration. As the reagent (IV-I), an acid, such as 85% phosphoric acid, hydrochloric acid, acetic acid, trifluoroacetic acid, sulfuric acid, trifluoromethanesulfonic acid, hydrofluoric acid, polyphosphoric acid, and diphosphorus pentoxide, or a dehydrating agent can be suitably used. In addition, Lewis acids such as aluminum (III) chloride and fluoroboric acid can also be used. When the para-position (R1to R3in the formula (V) in the above scheme) of the triphenylamine backbone is a hydrogen atom, halogenation can be performed with high efficiency by a conventionally known method. For example, a bromine atom can be introduced by equivalently reacting N-bromosuccinimide in chloroform. It is also possible to perform iodination by replacing with N-iodosuccinimide or the like. These halogen derivatives can be further derivatized by conventionally known coupling reactions (e.g., the Suzuki-Miyaura coupling with a boron derivative, the Stille-Migita-Kosugi coupling with an organotin compound, the Heck reaction with an alkene or an alkyne compound, and the Sonogashira coupling). Electrochromic Composition An electrochromic composition according to the present embodiment comprises the electrochromic compound according to the present embodiment. The electrochromic compound according to the present embodiment is a radical-polymerizable compound having a triarylamine backbone. Therefore, the electrochromic compound has a role of imparting an electrochromic function for causing redox reactions at the surface of a first electrode of an electrochromic element according to the present embodiment to be described in detail later. Preferably, the electrochromic composition according to the present embodiment further comprises a radical-polymerizable compound (hereinafter “the other radical-polymerizable compound”) other than the electrochromic compound according to the present embodiment. Benzidine Compound The electrochromic composition according to the present embodiment may contain a benzidine compound as the other radical-polymerizable compound. Examples of the benzidine compound include a compound (“tetraphenyl benzidine compound”) having a tetraphenyl benzidine backbone. By containing the tetraphenyl benzidine compound as the benzidine compound, the electrochromic composition according to the present embodiment is transparent in the neutral state and exhibits stable optical properties in the one-electron oxidation state, as described later. In the tetraphenyl benzidine compound, preferably, the para-position of each of four phenyl groups substituted to the amino group of the benzidine backbone is substituted with a substituent other than hydrogen, such as an alkyl group, an alkoxy group, or a radical-polymerizable substituent. A benzidine compound having a terminal hydrogen may react and multimerize in the one-electron oxidation state. In the case of multimerization, there is a possibility that the color developed by the benzidine compound having a terminal hydrogen changes due to a change in oxidation potential or the like. In the tetraphenyl benzidine compound, when the para-position of each of four phenyl groups substituted to the amino group of the benzidine backbone is substituted with a substituent other than hydrogen, electrochemical stability is improved. Thus, multimerization of the tetraphenyl benzidine compound in the one-electron oxidation state can be prevented, so that stable optical properties are exhibited by, for example, preventing a hue change in the developed color. The tetraphenyl benzidine compound can be used as it is or after being copolymerized. Therefore, the tetraphenyl benzidine compound may have a radical-polymerizable substituent. The radical-polymerizable substituent can be appropriately modified within the same scope as the compound of the present application, and may be present at a part of the tetraphenyl benzidine compound such as at a terminal of the alkyl group or alkoxy group at the para position. Particularly preferred are acryloyloxy group and methacryloyloxy group. Preferably, the tetraphenyl benzidine compound in the neutral state is transparent in the visible range, that is, the absorption edge of the UV-visible absorption spectrum is 430 nm or less, more preferably 420 nm or less, and most preferably 410 nm or less. In addition, preferably, the tetraphenyl benzidine compound is colored in yellow or orange upon one-electron oxidation, the peak wavelength in the visible range (from 380 to 780 nm) is around 450 to 550 nm, and the absorption edge is in the range of from 550 to 650 nm. By combining such a tetraphenyl benzidine compound with the electrochromic compound of the present embodiment colored in blue, black color can be developed because their absorption characteristic in the visible region are complementary. Specific examples of the tetraphenyl benzidine compound satisfying the above-described optical properties include the following Example Compounds, but are not limited thereto. Example Compound B1 Example Compound B2 Example Compound B3 Example Compound B4 Example Compound B5 Example Compound B6 Example Compound B7 Example Compound B8 Example Compound B9 Example Compound B10 Example Compound B11 Example Compound B12 Example Compound B13 Example Compound B14 Example Compound B15 Other Radical-Polymerizable Compound The other radical-polymerizable compound is a compound having at least one radical-polymerizable functional group, and is different from the electrochromic compound according to the present embodiment. A plurality of compounds having a triphenylamine backbone or a benzidine backbone can be used. Examples of the other radical-polymerizable compound include, but are not limited to, monofunctional radical-polymerizable compounds, difunctional radical-polymerizable compounds, trifunctional or higher radical-polymerizable compounds, functional monomers, and radical-polymerizable oligomers. Among these compounds, difunctional or higher radical-polymerizable compounds are preferable. The radical-polymerizable functional group in the other radical-polymerizable compound is the same as the radical-polymerizable functional group in the electrochromic compound according to the present embodiment. Among them, acryloyloxy group and methacryloyloxy group are particularly preferable. Specific examples of the monofunctional radical-polymerizable compounds include, but are not limited to, 2-(2-ethoxyethoxy)ethyl acrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monomethacrylate, phenoxypolyethylene glycol acrylate, 2-acryloyloxyethyl succinate, 2-ethylhexyl acrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, tetrahydrofurfuryl acrylate, 2-ethylhexylcarbitol acrylate, 3-methoxybutyl acrylate, benzyl acrylate, cyclohexyl acrylate, isoamyl acrylate, isobutyl acrylate, methoxytriethylene glycol acrylate, phenoxytetraethylene glycol acrylate, cetyl acrylate, isostearyl acrylate, stearyl acrylate, and styrene monomer. Each of these members can be used alone or in combination with others. Specific examples of the difunctional radical-polymerizable compounds include, but are not limited to, 1,3-butanediol diacrylate, 1,4-butanediol diacrylate, 1,4-butanediol dimethacrylate, 1,6-hexanediol diacrylate, 1,6-hexanediol dimethacrylate, diethylene glycol diacrylate, polyethylene glycol diacrylate, neopentyl glycol diacrylate, EO-modified bisphenol A diacrylate, EO-modified bisphenol F diacrylate, and neopentyl glycol diacrylate. Each of these members can be used alone or in combination with others. Specific examples of the trifunctional radical-polymerizable compounds include, but are not limited to, trimethylolpropane triacrylate (TMPTA), trimethylolpropane trimethacrylate, EO-modified trimethylolpropane triacrylate, PO-modified trimethylolpropane triacrylate, caprolactone-modified trimethylolpropane triacrylate, HPA-modified trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate (PETTA), glycerol triacrylate, ECH-modified glycerol triacrylate, EO-modified glycerol triacrylate, PO-modified glycerol triacrylate, tris(acryloxyethyl) isocyanurate, dipentaerythritol hexaacrylate (DPHA), caprolactone-modified dipentaerythritol hexaacrylate, dipentaerythritol hydroxypentaacrylate, alkyl-modified dipentaerythritol pentaacrylate, alkyl-modified dipentaerythritol tetraacrylate, alkyl-modified dipentaerythritol triacrylate, dimethylolpropane tetraacrylate (DTMPTA), pentaerythritol ethoxytetraacrylate, EO-modified phosphoric triacrylate, and 2,2,5,5-tetrahydroxymethylcyclopentanone tetraacrylate. Each of these members can be used alone or in combination with others. In the above descriptions, “EO-modified” and “PO-modified” represent “ethyleneoxy-modified” and “propyleneoxy-modified”, respectively. Specific examples of the functional monomers include, but are not limited to: fluorine-substituted monomers, such as octafluoropentyl acrylate, 2-perfluorooctylethyl acrylate, 2-perfluorooctylethyl methacrylate, and 2-perfluoroisononylethyl acrylate; polysiloxane-group-containing vinyl monomers having 20 to 70 siloxane repeating units disclosed in JP-05-60503-B and JP-06-45770-B, such as acryloyl polydimethylsiloxane ethyl, methacryloyl polydimethylsiloxane ethyl, acryloyl polydimethylsiloxane propyl, acryloyl polydimethylsiloxane butyl, and diacryloyl polydimethylsiloxane diethyl; and acrylates and methacrylates. Each of these members can be used alone or in combination with others. Specific examples of the radical-polymerizable oligomers include, but are not limited to, epoxy acrylate oligomers, urethane acrylate oligomers, and polyester acrylate oligomers. The electrochromic compound according to the present embodiment and the other radical-polymerizable compound can be copolymerized by a polymerization reaction. Preferably, at least one of the electrochromic compound and the other radical-polymerizable compound has two or more radical-polymerizable functional groups, for forming a polymer or cross-linked product. The polymer or cross-linked product is preferable for its mechanical strength, poor solubility in various organic solvents and electrolytes, and little migration between layers in forming a multilayer structure. Preferably, the proportion of the electrochromic compound in the electrochromic composition is from 10% to 100% by mass, more preferably from 30% to 90% by mass. When the proportion is 10% by mass or more, the first electrochromic layer in the electrochromic element, to be described later, sufficiently exhibits an electrochromic function, durability against repeated use under application of voltage is good, and color developing sensitivity is good. When the proportion is 100% by mass or less, the first electrochromic layer exhibits an electrochromic function, and color developing sensitivity is sufficiently high for the thickness. When the proportion is 100% by mass, there may be a case in which the electrochromic composition becomes less compatible with an ionic liquid that is needed for giving and receiving charge, thereby causing deterioration of electric properties by, for example, deterioration of durability against repeated use under application of voltage. Although it depends on the process with which the electrochromic composition is to be used, a preferred proportion is in the range of from 30% to 90% by mass for achieving a good balance between color developing sensitivity and durability against repeated use. Preferably, the electrochromic composition further contains a filler and/or a polymerization initiator. Filler The filler is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include, but are not limited to, inorganic fillers and organic fillers. Specific examples of the inorganic fillers include, but are not limited to, powders of metals (e.g., copper, tin, aluminum, indium), metal oxides (e.g., silicon oxide (silica), tin oxide, zinc oxide, titanium oxide, aluminum oxide (alumina), zirconium oxide, indium oxide, antimony oxide, bismuth oxide, calcium oxide, antimony-doped tin oxide (ATO), tin-doped indium oxide), and metal fluorides (e.g., tin fluoride, calcium fluoride, and aluminum fluoride). Each of these materials can be used alone or in combination with others. Among these, metal oxides are preferable, and silica, alumina, and antimony-doped tin oxide (ATO) are more preferable, for transparency, stability, and the ease in surface modification. Specific examples of the organic fillers include, but are not limited to, resins (e.g., polyester, polyether, polysulfide, polyolefin, silicone, polytetrafluoroethylene), low-molecular-weight compounds (e.g., fatty acids), and pigments (e.g., phthalocyanine). Each of these materials can be used alone or in combination with others. Among these materials, resins are preferable for transparency and insolubility. Preferably, the filler has an average primary particle diameter of 1 μm or less, more preferably from 10 nm to 1 μm. When the average primary particle diameter of the filler is 1 μm or less, the resulting layer has excellent surface smoothness since no coarse particle is present. Preferably, the amount of the filler is from 0.3 to 1.5 parts by mass, more preferably from 0.6 to 0.9 parts by mass, with respect to 100 parts by mass of the total radical-polymerizable compounds on solid basis. When the amount is 0.3 parts by mass or more, the effect of addition of filler is sufficiently exerted and film formation property is excellent. When the amount is 1.5 parts by mass of less, the proportion of triarylamine compounds is appropriate and electrochemical properties of the resulting electrochromic element are excellent. Polymerization Initiator Preferably, the electrochromic composition according to the present embodiment contains a polymerization initiator, as necessary, for improving a cross-linking reaction efficiency between the electrochromic compound and the other radical-polymerizable compound. Examples of the polymerization initiator include, but are not limited to, thermal polymerization initiators and photopolymerization initiators. Photopolymerization initiators are more preferable for polymerization efficiency. The thermal polymerization initiator is not particularly limited and may be appropriately selected depending on the purpose. Specific examples of the thermal polymerization initiators include, but are not limited to: peroxide initiators such as 2,5-dimethylhexane-2,5-dihydroperoxide, dicumyl peroxide, benzoyl peroxide, t-butylcumyl peroxide, 2,5-dimethyl-2,5-di(peroxybenzoyl)hexine-3, di-t-butyl peroxide, t-butyl hydroperoxide, cumene hydroperoxide, and lauroyl peroxide; and azo initiators such as azobisisobutyronitrile, azobiscyclohexanecarbonitrile, azobis(methyl isobutyrate), azobisisobutyl amidine hydrochloride, and 4,4′-azobis-4-cyanovaleric acid. Each of these materials can be used alone or in combination with others. The photopolymerization initiator is not particularly limited and may be appropriately selected depending on the purpose. Specific examples of the photopolymerization initiators include, but are not limited to: acetophenone or ketal photopolymerization initiators such as diethoxyacetophenone, 2,2-dimethoxy-1,2-diphenylethane-1-one, 1-hydroxy-cyclohexyl-phenyl-ketone, 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl) ketone, 2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)butanone-1,2-hydroxy-2-methyl-1-phenylpropane-1-one, 2-methyl-2-morpholino(4-methylthiophenyl)propane-1-one, and 1-phenyl-1,2-propanedione-2-(o-ethoxycarbonyl)oxime; benzoin ether photopolymerization initiators such as benzoin, benzoin methyl ether, benzoin ethyl ether, benzoin isobutyl ether, and benzoin isopropyl ether; benzophenone photopolymerization initiators such as benzophenone, 4-hydroxybenzophenone, methyl o-benzoylbenzoate, 2-benzoyl naphthalene, 4-benzoyl biphenyl, 4-benzoyl phenyl ether, acrylated benzophenone, and 1,4-benzoyl benzene; and thioxanthone photopolymerization initiators such as 2-isopropyl thioxanthone, 2-chlorothioxanthone, 2,4-dimethylthioxanthone, 2,4-diethylthioxanthone, and 2,4-dichlorothioxanthone. Specific examples of the photopolymerization initiators further include, but are not limited to, ethylanthraquinone, 2,4,6-trimethylbenzoyldiphenylphosphine oxide, 2,4,6-trimethylbenzoylphenylethoxyphosphine oxide, bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide, bis(2,4-dimethoxybenzoyl)-2,4,4-trimethylpentylphosphine oxide, methylphenylglyoxy ester, 9,10-phenanthrene, acridine compounds, triazine compounds, and imidazole compounds. Each of these materials can be used alone or in combination with others. In addition, a photopolymerization accelerator may be used alone or in combination with the photopolymerization initiator. Specific examples of the photopolymerization accelerator include, but are not limited to, triethanolamine, methyldimethanolamine, ethyl 4-dimethylaminobenzoate, isoamyl 4-dimethylaminobenzoate, (2-dimethylamino)ethyl benzoate, and 4,4′-dimethylaminobenzophenone. Preferably, the amount of the polymerization initiator is from 0.5 to 40 parts by mass, more preferably from 1 to 20 parts by mass, with respect to 100 parts by mass of the total radical-polymerizable compounds. Other Components The electrochromic composition according to the present embodiment may further contain other components, as necessary. Examples of the other components include, but are not limited to, a solvent, a plasticizer, a leveling agent, a sensitizer, a dispersant, a surfactant, and an antioxidant. In addition, the electrochromic composition according to the present embodiment may contain a cross-linking agent, and may be a copolymer (e.g., a linear copolymer having a linear structure) in which the electrochromic compound according to the present embodiment is polymerized. In addition, the electrochromic composition according to the present embodiment may be a cross-linked product having a branched structure or a three-dimensional network structure, in which the electrochromic compound according to the present embodiment is cross-linked. The cross-linking agent is not particularly limited and can be appropriately selected according to the purpose. Specific examples thereof include, but are not limited to, isocyanates, amino resins, phenol resins, amines, epoxy compounds, monofunctional acrylates and methacrylates, polyfunctional acrylates and methacrylates having at least two ethylenic unsaturated bonds per molecule, and acrylic acid esters and methacrylic acid esters. Among these compounds, isocyanates are preferable, and polyisocyanates having multiple isocyanate groups are particularly preferable. The electrochromic composition according to the present embodiment is able to provide properties required by electrochromic elements because the electrochromic compound according to the present embodiment is contained therein. Electrochromic elements may require, for example, an electrochromic composition that is transparent in the neutral state and has solubility and electrochromic layers that are stackable, as described above. Electrochromic Element An electrochromic element according to the present embodiment includes a first electrode, a second electrode, and an electrolyte layer disposed between the first electrode and the second electrode. The electrochromic element may further include other members, as necessary. The electrochromic element according to the present embodiment further includes, on the first electrode, an electrochromic layer containing the electrochromic composition according to the present embodiment. Alternatively, the electrolyte layer contains the electrochromic composition according to the present embodiment. The electrochromic composition according to the present embodiment has excellent light durability and repetition durability, and can meet the properties required by electrochromic elements. Therefore, the electrochromic element according to the present embodiment uses the electrochromic composition according to the present embodiment under optimum configuration and position. As a result, the electrochromic element according to the present embodiment can provide superior effects than conventional electrochromic elements, in particular, excellent repetition durability and light durability. In the following description, an electrochromic element which contains the electrochromic composition according to the present embodiment in an electrochromic layer disposed on the first electrode is referred to as the electrochromic element according to the first embodiment. In addition, an electrochromic element which contains the electrochromic composition according to the present embodiment in an electrolyte layer is referred to as the electrochromic element according to the second embodiment. Hereinafter, the electrochromic element according to each embodiment is described in detail. Electrochromic Element according to First Embodiment The electrochromic element according to the first embodiment is described in detail below. In the drawings, the scale of each member may be different from the actual scale, for ease of understanding. For the sake of convenience, explanation of a layer structure will be given with the drawings in which a first substrate is illustrated on the lower side, but the arrangement of the layers is not limited thereto in the actual manufacture or use. In the following descriptions, one side of the first substrate in the thickness direction may be referred to as “upper side”, and the other side may be referred to as “lower side”. FIG.1is a schematic cross-sectional view of the electrochromic element according to the first embodiment. Referring toFIG.1, an electrochromic element10A comprises a first substrate11, a display electrode (“first electrode”)12, a first electrochromic layer13, an electrolyte layer14A, a second electrochromic layer15, a counter electrode (“second electrode”)16, and a second substrate17. These members are stacked in this order from the first substrate11side. The display electrode12is disposed on the upper side of the first substrate11, and the first electrochromic layer13is disposed on the upper side of the display electrode12. The counter electrode16is disposed on the lower side of the second substrate17, and the second electrochromic layer15is disposed on the lower side of the counter electrode16. The display electrode12and the counter electrode16are facing each other with a gap therebetween. The electrolyte layer14A is disposed between the display electrode12and the counter electrode16. In the electrochromic element10A, the first electrochromic layer13is colored or decolored in response to a redox reaction occurring at the surface of the display electrode12, and the second electrochromic layer15is colored or decolored in response to a redox reaction occurring at the surface of the counter electrode16. The members constituting the electrochromic element10A are each described in detail below. First Electrochromic Layer The first electrochromic layer contains the above-described electrochromic composition according to the present embodiment. The electrochromic composition used in the first embodiment is hereinafter referred to as the “first electrochromic composition” to be distinguished from the “second electrochromic composition” used in the second embodiment. The first electrochromic composition preferably contains the electrochromic compound according to the present embodiment and the other radical-polymerizable compound, as described above, for solubility and durability of the polymerized product of the first electrochromic composition. The first electrochromic layer may be stacked on the first electrode either in a single layer or in multiple layers. The first electrochromic layer may be stacked on either the whole surface of the first electrode or a partial surface of the first electrode. The first electrochromic layer can be formed by a method for producing an electrochromic element to be described later. Preferably, the first electrochromic layer has an average thickness of from 0.1 to 30 μm, more preferably from 0.4 to 10 μm. First Electrode and Second Electrode The material of the first electrode and the second electrode is not particularly limited and can be appropriately selected depending on the purpose as long as it is a transparent material having conductivity. Specific examples of the material of the first electrode and the second electrode include, but are not limited to, inorganic materials such as tin-doped indium oxide (ITO), fluorine-doped tin oxide (FTO), antimony-doped tin oxide (ATO), and zinc oxide. Among these, InSnO, GaZnO, SnO, In2O3, and ZnO are preferable. Alternatively, a carbon nanotube having transparency, or an electrode made of a highly-conductive non-transmissive material such as Au, Ag, Pt, and Cu formed into a fine network structure to improve conductivity while maintaining transparency, may be used. The thicknesses of the first electrode and the second electrode are so adjusted that these electrodes have proper electric resistance values required for causing redox reactions in the first electrochromic layer and the second electrochromic layer. In a case in which the material of the first electrode and the second electrode is ITO, the thickness of each of the first electrode and the second electrode is preferably from 50 to 500 nm. The first electrode and the second electrode may be formed by, for example, vacuum vapor deposition, sputtering, or ion plating. In addition, the first electrode and the second electrode can also be formed by any coating method, such as spin coating, casting, micro gravure coating, gravure coating, bar coating, roll coating, wire bar coating, dip coating, slit coating, capillary coating, spray coating, nozzle coating, or various printing methods, such as gravure printing, screen printing, flexo printing, offset printing, reverse printing, and inkjet printing. Electrolyte Layer The electrolyte layer is formed of an electrolyte filling the gap between the first electrode and the second electrode. The electrolyte may be injected into the gap between the first electrode and the second electrode through multiple injection holes formed on a sealing material disposed between the first electrode and the second electrode, thereby filling the gap between the first electrode and the second electrode. Examples of the electrolyte include, but are not limited to, inorganic ion salts (e.g., alkali metal salts and alkali-earth metal salts), quatemary ammonium salts, and supporting salts of acids and bases. Specific examples thereof include, but are not limited to, LiClO4, LiBF4, LiAsF6, LiPF6, LiCF3SO3, LiCF3COO, KCl, NaClO3, NaCl, NaBF4, NaSCN, KBF4, Mg(ClO4)2, and Mg(BF4)2. In addition, ionic liquids can also be used as the electrolyte. In particular, organic ionic liquids are preferable because they have a molecular structure that exhibits liquidity in a wide temperature range including room temperature. Specific examples of cationic components in such organic ionic liquids include, but are not limited to, imidazole derivatives (e.g., N,N-dimethylimidazole salt, N,N-methylethylimidazole salt, and N,N-methylpropylimidazole salt), pyridinium derivatives (e.g., N,N-dimethylpyridinium salt and N,N-methylpropylpyridinium salt), and aliphatic quatemary ammonium salts (e.g., trimethylpropylammonium salt, trimethylhexylammonium salt, and triethylhexylammonium salt). Specific preferred examples of anionic components therein include, but are not limited to, fluorine-containing compounds such as BF4−, CF3SO3−, PF4−, (CF3SO2)2N−, and tetracyanobome anion (B(CN)4−), for stability in the atmosphere. Ionic liquids in which the cationic and anionic components are combined are preferably used as the electrolyte. The ionic liquid may be directly dissolved in a photopolymerizable monomer, an oligomer, or a liquid crystal material. When solubility is poor, the ionic liquid may be first dissolved in a small amount of a solvent, and thereafter mixed with a photopolymerizable monomer, an oligomer, or a liquid crystal material. Specific examples of the solvent include, but are not limited to, propylene carbonate, acetonitrile, γ-butyrolactone, ethylene carbonate, sulfolane, dioxolan, tetrahydrofuran, 2-methyltetrahydrofuran, dimethylsulfoxide, 1,2-dimethoxyethane, 1,2-ethoxymethoxyethane, polyethylene glycol, alcohols, and mixed solvents thereof. The electrolyte needs not necessarily be a low-viscosity liquid and may be in the form of a gel, cross-linked polymer, or liquid crystal dispersion. The electrolyte in the form of a gel or solid is advantageous for improving strength and reliability of the element. Preferably, the electrolyte and the solvent are held in a polymer resin for reliable fixation. By this configuration, high ion conductivity and solid strength can be achieved. Preferably, the polymer resin is a photocurable resin. This is because electrochromic element can be prepared at a lower temperature and within a shorter time period compared to a case in which a thin layer is formed by thermal polymerization and/or solvent evaporation. The average thickness of the electrolyte layer containing the electrolyte is not particularly limited and can be appropriately selected according to the purpose, but is preferably from 100 nm to 10 μm. Second Electrochromic Layer The second electrochromic layer may be stacked on the lower surface of the second electrode in a single layer or in multiple layers. The second electrochromic layer may be stacked on either the whole surface of the lower surface of the second electrode or a partial surface of the lower surface of the second electrode. The second electrochromic layer may contain a second electrochromic compound that is a viologen compound represented by the following general formula (I). More specifically, the second electrochromic layer contains an electrochromic complex comprising the viologen compound represented by the general formula (I) in a conductive nanostructural body or a semiconductive nanostructural body (hereinafter “conductive or semiconductive nanostructural body”). The viologen compound represented by the general formula (I) is bindable or adsorbable to the conductive or semiconductive nanostructural body. The electrochromic complex contained in the electrochromic element develops blue color and provides excellent image memory property, i.e., color image maintainability. The second electrochromic layer may further contain, other than the viologen compound represented by the general formula (I), a phosphonic acid compound represented by the following general formula (II) described in JP-2017-111434-A or a straight-chain alkyl phosphonic acid alone or coadsorbed with the viologen compound. Viologen Compound The viologen compound represented by the general formula (I) is described in detail below. In the formula (I), each of R1and R2independently represents a hydrogen atom, an aryl group having 14 carbon atoms at most, a heteroaryl group, a branched alkyl group having 10 carbon atoms at most, an alkenyl group, a cycloalkyl group, or a functional group bindable to hydroxyl group. E of n and m independently represents 0 or an integer of from 1 to 10. X−represents an ion that neutralizes the charge. Preferably, at least one of R1and R2represents a functional group bindable to hydroxyl group. In this case, the viologen compound is adsorbable or fixable to a transparent electrode (e.g., ITO). Such a viologen compound is advantageously adsorbable or fixable to the transparent electrode even when carrier particles comprising metal oxides are disposed on the transparent electrode. More preferably, both of R1and R2each represent a functional group bindable to hydroxyl group. Specific examples of the functional group bindable to hydroxyl group include, but are not limited to, phosphonate group, phosphate group, carboxyl group, sulfonyl group, silyl group, and silanol group. Among these groups, phosphonate group, phosphate group, and carboxyl group are preferable, and phosphonate group is most preferable, for the ease of synthesis, adsorptivity to carrier particles comprising metal oxides disposed on the transparent electrode, and stability of the compound. Specific examples of the phosphonate group include, but are not limited to, methylphosphonate group, ethylphosphonate group, propylphosphonate group, hexylphosphonate group, octylphosphonate group, decylphosphonate group, dodecylphosphonate group, octadecylphosphonate group, benzylphosphonate group, phenylethylphosphonate group, phenylpropylphosphonate group, and biphenylphosphonate group. Specific examples of the phosphate group include, but are not limited to, methylphosphate group, ethylphosphate group, propylphosphate group, hexylphosphate group, octylphosphate group, decylphosphate group, dodecylphosphate group, octadecylphosphate group, benzylphosphate group, phenylethylphosphate group, phenylpropylphosphate group, and biphenylphosphate group. Specific examples of the carboxyl group include, but are not limited to, methylcarboxyl group, ethylcarboxyl group, propylcarboxyl group, hexylcarboxyl group, octylcarboxyl group, decylcarboxyl group, dodecylcarboxyl group, octadecylcarboxyl group, benzylcarboxyl group, phenylethylcarboxyl group, phenylpropylcarboxyl group, biphenylcarboxyl group, 4-propylphenylcarboxyl group, and 4-propylbiphenylcarboxyl group. Specific examples of the sulfonyl group include, but are not limited to, methylsulfonyl group, ethylsulfonyl group, propylsulfonyl group, hexylsulfonyl group, octylsulfonyl group, decylsulfonyl group, dodecylsulfonyl group, octadecylsulfonyl group, benzylsulfonyl group, phenylethylsulfonyl group, phenylpropylsulfonyl group, and biphenylsulfonyl group. Specific examples of the silyl group include, but are not limited to, methylsilyl group, ethylsilyl group, propylsilyl group, hexylsilyl group, octylsilyl group, decylsilyl group, dodecylsilyl group, octadecylsilyl group, benzylsilyl group, phenylethylsilyl group, phenylpropylsilyl group, and biphenylsilyl group. Specific examples of the silanol group include, but are not limited to, methylsilanol group, ethylsilanol group, propylsilanol group, hexylsilanol group, octylsilanol group, decylsilanol group, dodecylsilanol group, octadecylsilanol group, benzylsilanol group, phenylethylsilanol group, phenylpropylsilanol group, and biphenylsilanol group. In the general formula (I), the ion X−for neutralizing the charge is not particularly limited as long as it represents a monovalent anion capable of forming a stable pair with the cation moiety. Specific preferred examples of the ion X−for neutralizing the charge include, but are not limited to, Br ion (Br−), Cl ion (Cl−), I ion (I−), Otf (triflate) ion (Otf−), ClO4ion (ClO4), PF6ion (PF6−), and BF4ion (BF4−). Preferably, the viologen compound is a symmetric system having an alkyl chain with a specific length. In this case, in the formula (I), preferably, each of m and n independently represents an integer of from 4 to 10. More preferably m and n represent the same integer. Specific examples of the viologen compound include the following Example Compounds, but are not limited thereto. Example Compound A Example Compound B Example Compound C Example Compound D Example Compound E Example Compound F Example Compound G Example Compound H Example Compound I Example Compound J Example Compound K Conductive or Semiconductive Nanostructural Body The conductive or semiconductive nanostructural body is described in detail below. Preferably, the conductive or semiconductive nanostructural body is transparent. In the general formula (I), at least one of R1and R2represents a functional group bindable to hydroxyl group. The bonding or adsorption structure of the viologen compound to the conductive or semiconductive nanostructural body includes phosphonate group, sulfonate group, phosphate group, or carboxyl group. In this case, the second electrochromic compound can be easily complexed with the nanostructural body, thus providing an electrochromic complex having excellent color image maintainability. The viologen compound may have multiple phosphonate groups, sulfonate groups, phosphate groups, and/or carboxyl groups. In a case in which the viologen compound has silyl group or silanol group, the viologen compound can be strongly bound to the nanostructural body via siloxane bond, thus providing an electrochromic complex having good stability. Here, the siloxane bond refers to a chemical bond between a silicon atom and an oxygen atom. The electrochromic complex is not limited in bonding structure or configuration as long as it has a configuration in which the viologen compound and the nanostructural body are bound to each other via siloxane bond. The conductive or semiconductive nanostructural body refers to a structural body having nanometer-scale irregularities, such as nanoparticles and nanoporous structural bodies. The conductive or semiconductive nanostructural body is preferably made of a metal oxide for transparency and conductivity. Specific examples of the metal oxide include, but are not limited to, titanium oxide, zinc oxide, tin oxide, zirconium oxide, cerium oxide, yttrium oxide, boron oxide, magnesium oxide, strontium titanate, potassium titanate, barium titanate, calcium titanate, calcium oxide, ferrite, hafnium oxide, tungsten oxide, iron oxide, copper oxide, nickel oxide, cobalt oxide, barium oxide, strontium oxide, vanadium oxide, indium oxide, aluminosilicate, calcium phosphate, and those containing aluminosilicate as the main ingredient. Each of these materials can be used alone or in combination with others. For electric properties (e.g., electric conductivity) and physical properties (e.g., optical property), titanium oxide, zinc oxide, tin oxide, zirconium oxide, iron oxide, magnesium oxide, indium oxide, and tungsten oxide are preferable, and titanium oxide is most preferable. When such a metal oxide or a mixture of these metal oxides is used, a response speed in coloring and decoloring is excellent. Preferably, the metal oxide is in the form of fine particles having an average primary particle diameter of 30 nm or less. As the average primary particle diameter of the metal oxide becomes smaller, light transmittance of the metal oxide is improved and the surface area per unit volume (hereinafter “specific surface area”) of the electrochromic complex is increased. As the specific surface area becomes larger, the second electrochromic compound can be carried by the conductive or semiconductive nanostructural body in a more efficient manner, thus providing a multi-color display with an excellent display contrast ratio between coloring and decoloring. The specific surface area of the electrochromic complex is not particularly limited and may be appropriately selected depending on the purpose, but is preferably 100 m2/g or more. The average primary particle diameter of fine particles of the metal oxide is determined by observing 100 randomly-selected fine particles of the metal oxide with a transmission electron microscope (TEM) to measure the projected area of each fine particle, calculating an equivalent circle diameter of each projected area, and averaging the calculated equivalent circle diameter values. The second electrochromic layer can be formed by vacuum vapor deposition, sputtering, or ion plating. The second electrochromic layer can also be formed by any coating method such as spin coating, casting, micro gravure coating, gravure coating, bar coating, roll coating, wire bar coating, dip coating, slit coating, capillary coating, spray coating, nozzle coating, and various printing methods such as gravure printing, screen printing, flexo printing, offset printing, reverse printing, and inkjet printing. The average thickness of the second electrochromic layer is not particularly limited and may be appropriately selected depending on the purpose, but is preferably from 0.2 to 5.0 μm. When the average thickness is 0.2 μm or greater, proper coloring density can be achieved. When the average thickness is 5.0 μm or less, either an increase of manufacturing cost or a decrease of visibility caused due to coloring can be prevented. The second electrochromic layer can be formed by means of vacuum film formation, but is preferably formed by coating of a particle-dispersed paste for productivity. First Substrate and Second Substrate The first substrate and the second substrate have a function of supporting the first electrode, the first electrochromic layer, the second electrode, the second electrochromic layer, etc. As the substrate, known organic materials and inorganic materials can be used as they are as long as they are transparent materials capable of supporting each layer. Specific examples of the substrate include, but are not limited to, glass substrates made of non-alkali glass, borosilicate glass, float glass, or soda-lime glass. Specific examples of the substrate further include, but are not limited to, resin substrates made of polycarbonate resin, acrylic resin, polyethylene resin, polyvinyl chloride resin, polyester resin, epoxy resin, melamine resin, phenol resin, polyurethane resin, or polyimide resin. The substrate may have a surface coating such as a transparent insulating layer, a UV cut layer, and/or an antireflection layer, for improving vapor barrier property, gas barrier property, ultraviolet resistance, and visibility. The planer shape of the substrate is not particularly limited and can be appropriately selected according to the purpose. For example, the planar shape may be rectangular or circular. The substrate may be a stack of multiple layers. As an example, an electrochromic element sandwiched by two glass substrates provides improved vapor barrier property and gas barrier property. Other Members The other members are not particularly limited and can be appropriately selected according to the purpose. Examples thereof include, but are not limited to, an insulating porous layer, an anti-deterioration layer, and a protective layer. Insulating Porous Layer The insulating porous layer has a function of electrically insulating the first electrode and the second electrode from each other and another function of holding the electrolyte. The material of the insulating porous layer is not particularly limited as long as it is porous. Preferred examples of such materials include, but are not limited to, organic and inorganic materials having high insulating property, durability, and film-formation property and composite materials thereof. The insulating porous layer can be formed by, for example, a sintering method (in which fine polymer particles or inorganic particles are partially fused with each other via a binder to form pores between the particles), an extraction method (in which solvent-soluble organic or inorganic substances and solvent-insoluble binders are formed into a layered structure, and the organic or inorganic substances are dissolved with a solvent to form pores), a foaming method, a phase inversion method in which a mixture of polymers is subjected to phase separation by handling a good solvent and a poor solvent, and a radiation irradiation method in which pores are formed by means of radiation. Anti-Deterioration Layer The function of the anti-deterioration layer is to undergo the reverse reaction of a reaction occurring in the first electrochromic layer and the second electrochromic layer to balance the charges therebetween, so that the first electrode and the second electrode are prevented from being corroded or degraded by an irreversible redox reaction. The reverse reaction includes both a redox reaction of the anti-deterioration layer and an action thereof as a capacitor. The material of the anti-deterioration layer is not particularly limited and can be appropriately selected according to the purpose as long as it has a function of preventing the first electrode and the second electrode from being corroded through an irreversible redox reaction. Specific examples of the material of the anti-deterioration layer include, but are not limited to, antimony tin oxide, nickel oxide, titanium oxide, zinc oxide, tin oxide, and conductive or semiconductive metal oxides containing two or more of these materials. The anti-deterioration layer may be comprised of a porous thin film which does not inhibit injection of an electrolyte. Such a porous thin film providing excellent electrolyte permeability and anti-deterioration property can be obtained by, for example, fixing fine particles of a conductive or semiconductive metal oxide (e.g., antimony tin oxide, nickel oxide, titanium oxide, and zinc oxide, tin oxide) on the second electrode with a binder (e.g., acrylic binder, alkyd binder, isocyanate binder, urethane binder, epoxy binder, and phenol binder). Protective Layer The protective layer has functions of protecting the electrochromic element from external stress and chemicals used in the washing process, preventing leakage of the electrolyte, and preventing intrusion of substances unnecessary for stable operation of the electrochromic element, such as moisture and oxygen in the air. Specific examples of the material of the protective layer include, but are not limited to, ultraviolet-curable or heat-curable resins such as acrylic resin, urethane resin, and epoxy resin. The average thickness of the protective layer is not particularly limited and may be appropriately selected according to the purpose, but is preferably from 1 to 200 μm. Method for Manufacturing Electrochromic Element according to First Embodiment A method for manufacturing the electrochromic element according to the first embodiment is described below. First, the display electrode12is formed on the first substrate11. Next, the display electrode12is coated with a coating liquid (electrolyte liquid) containing the first electrochromic composition containing the electrochromic compound according to the present embodiment and the other radical-polymerizable compound. Thus, a first laminated body is prepared in which the display electrode12and the first electrochromic layer13, in this order, are formed on the first substrate11. Specific examples of the electrochromic compound and the other radical-polymerizable compound used here include the above-described materials exemplified for the electrochromic element according to the first embodiment. The coating liquid may be diluted with a solvent, if necessary, before being applied. Specific examples of the solvent include, but are not limited to, alcohol solvents (e.g., methanol, ethanol, propanol, butanol), ketone solvents (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone), ester solvents (e.g., ethyl acetate, butyl acetate), ether solvents (e.g., tetrahydrofuran, dioxane, propyl ether), halogen solvents (e.g., dichloromethane, dichloroethane, trichloroethane, chlorobenzene), aromatic solvents (e.g., benzene, toluene, xylene), and cellosolve solvents (e.g., methyl cellosolve, ethyl cellosolve, cellosolve acetate). Each of these materials can be used alone or in combination with others. The rate of dilution can be appropriately selected depending on solubility of the first electrochromic composition, the type of coating method, and a target thickness of the first electrochromic layer. The coating method may be, for example, dip coating, spray coating, bead coating, and ring coating. The method for manufacturing the electrochromic element according to the present embodiment may further include a polymerization/cross-linking process for polymerizing/cross-linking the first electrochromic composition by externally applying energy thereto. In the polymerization/cross-linking process, the first electrochromic composition applied onto the first electrode is cured by externally applying energy thereto, thus forming a first electrochromic layer. The external energy may be, for example, heat energy, light energy, or radial rays. A method of applying heat energy may be, for example, performing heating from the coated-surface side or the substrate-side with a gaseous substance (e.g., air, nitrogen gas), vapor, a heat medium, infrared rays, or electromagnetic waves. The heating temperature is not particularly limited and may be appropriately selected depending on the purpose, but is preferably in the range of from 60 to 170 degrees C. Light energy may be given from a UV light source having a main light-emitting wavelength within the ultraviolet range, such as a high-pressure mercury lamp and a metal halide lamp, or a visible light source which emits light corresponding to the absorption wavelength of the radical polymerizable compounds and/or the photopolymerization initiator. The irradiation amount of UV light is not particularly limited and may be appropriately selected according to the purpose, but is preferably in the range of from 5 to 15,000 mW/cm2. Next, the counter electrode16is formed on the second substrate17. The counter electrode16is thereafter coated with a coating liquid containing an electrochromic complex comprising the second electrochromic composition and the conductive or semiconductive nanostructural body. Thus, a second laminated body is prepared in which the counter electrode16and the second electrochromic layer15, in this order, are formed on the second substrate17. Specific examples of the second electrochromic composition and the conductive or semiconductive nanostructural body contained in the electrochromic complex include the above-described materials exemplified for the electrochromic element according to the first embodiment. Next, the gap between the first laminated body and the second laminated body is filled with an electrolyte liquid, so that the first laminated body and the second laminated body are disposed via the electrolyte layer14A. Thus, the electrochromic element10A according to the present embodiment is prepared. In a case in which the electrolyte constituting the electrolyte layer14A is curable by light or heat, the electrolyte is cured after the first laminated body and the second laminated body are bonded to each other via the electrolyte. The method for manufacturing the electrochromic element according to the present embodiment may further include other processes, as necessary. For example, in a case in which the electrochromic element10A further comprises an insulating porous layer, the method may further include a process of forming the insulating porous layer on the first electrochromic layer13. Alternatively, the insulating porous layer may be formed on the lower side of the second electrochromic layer15, or mixed with the electrolyte constituting the electrolyte layer14A. In a case in which the electrochromic element10A further comprises an anti-deterioration layer and/or a protective layer, the method may include a process of forming these layers in the electrochromic element10A. Electrochromic Element according to Second Embodiment The electrochromic element according to the second embodiment is described in detail below. An electrochromic element10B according to the second embodiment is free of the first electrochromic layer13and the second electrochromic layer15that are contained the electrochromic element10A according to the first embodiment illustrated inFIG.1. In addition, the electrochromic element10B according to the second embodiment has an electrolyte layer containing the electrochromic composition according to the present embodiment in place of the electrolyte layer14A contained in the electrochromic element10A according to the first embodiment. FIG.2is a schematic cross-sectional view of the electrochromic element according to the second embodiment. Referring toFIG.2, the electrochromic element10B comprises a first substrate11, a display electrode12, an electrolyte layer14B, a counter electrode16, and a second substrate17. These members are stacked in this order from the first substrate11side. The electrolyte layer14B contains the electrochromic composition according to the present embodiment and an electrolyte. Since the other members constituting the electrochromic element10B are the same as those constituting the electrochromic element10A, detailed explanations thereof are omitted. Method for Manufacturing Electrochromic Element according to Second Embodiment A method for manufacturing the electrochromic element according to the second embodiment is described below. The method for manufacturing the electrochromic element10B according to the second embodiment is free of a process of forming the first electrochromic layer13and the second electrochromic layer15that are formed in the electrochromic element10A according to the first embodiment. The method for manufacturing the electrochromic element10B according to the second embodiment includes a process of forming the electrolyte layer14B containing the electrochromic composition according to the present embodiment in place of the electrolyte layer14A. Thus, the display electrode12is formed on the first substrate11. The counter electrode16is formed on the second substrate17. Next, an electrolyte liquid containing the electrochromic composition according to the present embodiment and an electrolyte is prepared. The gap between the display electrode12and the counter electrode16is filled with the electrolyte liquid, so that the display electrode12and the counter electrode16are disposed via the electrolyte layer14B. Thus, the electrochromic element10B according to the present embodiment is prepared. The electrochromic elements according to the above embodiments have excellent light durability and repetition durability. Therefore, the electrochromic elements according to the above embodiments can be used for, for example, electrochromic display, large-size displays such as stock price display, and light control elements such as anti-glare mirror and light control glass. In addition, the electrochromic elements according to the above embodiments can be preferably used for low-voltage driving elements such as touch-panel-type key switch, optical switch, optical memory, electronic paper, and electronic album. The above-described embodiments are described only as examples for illustration and the present invention is not limited by the above-described embodiments. The above-described embodiments can be implemented in other various forms, and various combinations, omissions, replacements, changes, and the like can be made without departing from the scope of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention. EXAMPLES Further understanding can be obtained by reference to certain specific examples which are provided herein for the purpose of illustration only and are not intended to be limiting. Example 1 Synthesis Example 1 Synthesis of Electrochromic Compound 1 An electrochromic compound 1 was synthesized according to the following synthesis scheme (1). Synthesis of Compound 1-1 The following compound 1-1 was synthesized according to the method described in Zhen Fang, et. Al., J. Mater. Chem., 22, 2017, 15397-15404. Synthesis of Compound 1-2 A four-necked flask was charged with the compound 1-1 (2.78 g, 5.0 mmol), 2-propyne-1-ol (0.34 g, 6.0 mmol), diisopropylethylamine (10 mL), and tetrahydrofuran (hereinafter “THF”, 30 mL). After conducting a degassing with argon gas for 15 minutes, copper (I) iodide (5% by mol, 47 mg) and dichlorobis(triphenylphosphine)palladium (II) (5% by mol, 175 mg) were added to the flask and heat-stirred for 12 hours at an inner temperature of 70 degrees C. under argon atmosphere. The liquid in the flask was cooled to room temperature and filtered through silica gel. The silica gel was washed with ethyl acetate, and the collected filtrate was condensed under reduced pressure. The residue was purified by silica gel column chromatography (eluent: hexane/ethyl acetate=7/3 by volume). The purified residue was thereafter condensed under reduced pressures. Thus, a compound 1-2 was prepared (yield: 1.86 g, 70%) as a pale yellow amorphous. Synthesis of Compound 1-3 A flask was charged with the compound 1-2 (1.85 g, 3.48 mmol), ethanol (20 mL), and THF (30 mL). After the flask was purged with nitrogen gas, palladium carbon (containing 10% of Pd, available from FUJIFILM Wako Pure Chemical Corporatione, 185 mg) was gradually added to the flask. After the inside of the flask was replaced with hydrogen gas, the contents of the flask were stirred at hydrogen pressure (1 atm) for 16 hours. The resulting liquid was filtered through CELITE and the CELITE was washed with THF. The collected filtrate was condensed under reduced pressure. The residue was purified by silica gel column chromatography (eluent: hexane/ethyl acetate=7/3 by volume). The purified residue was thereafter condensed under reduced pressures. Thus, a compound 1-3 was prepared (yield: 1.77 g, 95%) as a pale yellow amorphous. Synthesis of Electrochromic Compound 1 In a four-neck flask purged with argon gas, the compound 1-3 (1.75 g, 3.3 mmol), dimethylaminopyridine (10 mg), THF (30 mL), and pyridine (10 mL) were placed and cooled to 0 degrees C. Acryloyl chloride (358 mg, 4.0 mmol) was dropped therein and stirred at the temperature as it was for 30 minutes. The temperature was then returned to room temperature, and stirring was further performed for 3 hours. Next, ethyl acetate and water were added to separate the organic phase. The aqueous phase was extracted with ethyl acetate three times. The collected organic phase was washed with water twice and with a saturated salt solution once. The organic phase was dried with sodium sulfate and, after the drying agent was separated, condensed. The residue was purified by column chromatography (elute: hexane/ethyl acetate=9/1 by volume). The resulting liquid was added with 2,6-di-tert-butyl-p-cresol (hereinafter “BHT”, 1.6 mg) and condensed. Thus, an electrochromic compound 1 was prepared (yield: 1.65 g, 85%) as a colorless solid. The electrochromic compound 1 was identified by a nuclear magnetic resonance spectrometer1H-NMR (product of JEOL Ltd., 500 MHz) and a mass spectrometer (LCT-Premier with ASAP Probe, product of Waters Corporation). As a result, it was confirmed from the identified structure and molecular weight that the above-prepared compound was the objective electrochromic compound 1. Synthesis Example 2 Synthesis of Electrochromic Compound 2 The electrochromic compound 2 was synthesized according to the following synthesis scheme (2). Synthesis of Compound 2-1 The following compound 2-1 was synthesized according to the method described in Zhen Fang, et. Al., J. Mater. Chem., 22, 2017, 15397-15404. Compounds 2-2 and 2-3 were synthesized in by the same method as the method of synthesizing the electrochromic compound 1 (i.e., the above-described scheme (1)). Thereafter, the solution of the compound 2-3 having been purified by column chromatography was added with 2,6-di-tert-butyl-p-cresol (BHT, 1.5 mg) and condensed into a colorless viscous liquid. Thus, an electrochromic compound 2 was prepared (yield. 1.50 g, 87%). The electrochromic compound 2 was identified by a nuclear magnetic resonance spectrometer1H-NMR (product of JEOL Ltd., 500 MHz) and a mass spectrometer (LCT-Premier with ASAP Probe, product of Waters Corporation). As a result, it was confirmed from the identified structure and molecular weight that the above-prepared compound was the objective electrochromic compound 2. Synthesis Example 3 Synthesis of Electrochromic Compound 3 The electrochromic compound 3 was synthesized according to the following synthesis scheme (3) Synthesis of Compound 3-1 The following compound 3-1 was synthesized according to the method described in Zhen Fang, et. Al., J. Mater. Chem., 22, 2017, 15397-15404. Compounds 3-2 and 3-3 were synthesized by the same method as the method of synthesizing the electrochromic compound 1 (i.e., the above-described scheme (1)). Thereafter, the solution of the compound 3-3 having been purified by column chromatography was added with 2,6-di-tert-butyl-p-cresol (BHT, 1.3 mg) and condensed into a colorless viscous liquid. Thus, an electrochromic compound 3 was prepared (yield: 1.30 g, 75%). The electrochromic compound 3 was identified by a nuclear magnetic resonance spectrometer1H-NMR (product of JEOL Ltd., 500 MHz) and a mass spectrometer (LCT-Premier with ASAP Probe, product of Waters Corporation). As a result, it was confirmed from the identified structure and molecular weight that the above-prepared compound was the objective electrochromic compound 3. Synthesis Example 4 Synthesis of Electrochromic Compound 4 The electrochromic compound 4 was synthesized according to the following synthesis scheme (4). Synthesis of Compound 4-1 The following compound 4-1 was synthesized according to the method described in Zhen Fang, et. Al., J. Mater. Chem., 22, 2017, 15397-15404. Synthesis of Electrochromic Compound 4 In a three-necked flask, the compound 3-1 (1.46 g, 4.0 mmol) and tert-butyl chloride (100 mL) were placed and heated to 60 degrees C. While the contents in the flask were stirred, anhydrous aluminum chloride (533 mg, 4.0 mmol) was gradually added thereto, and heat-stirring was performed at the temperature at it was for 6 hours. The resulting liquid was cooled to room temperature, and the solvent was evaporated under reduced pressure. Chloroform and water were added to the residue to separate the organic phase. The aqueous phase was extracted with chloroform three times. The collected organic phase was washed with water twice and with a saturated salt solution once. The organic phase was dried with sodium sulfate and, after the drying agent was separated, condensed. The residue was purified by column chromatography (elute: hexane/toluene=9/1 by volume). The solid obtained by condensation was recrystallized from toluene/ethanol. Thus, an electrochromic compound 4 was prepared (yield: 1.71 g, 80%) as a colorless plate crystal. The electrochromic compound 4 was identified by a nuclear magnetic resonance spectrometer1H-NMR (product of JEOL Ltd., 500 MHz) and a mass spectrometer (LCT-Premier with ASAP Probe, product of Waters Corporation). As a result, it was confirmed from the identified structure and molecular weight that the above-prepared compound was the objective electrochromic compound 4. Synthesis Example 5 Synthesis of Electrochromic Compound 5 The electrochromic compound 5 was synthesized according to the following synthesis scheme (5). Synthesis of Electrochromic Compound 5 Compounds 5-1 and 5-2 were synthesized according to the method described in Chemical Communications, 2014, 50 (99), 15760-15763. Synthesis of Compound 5-1 Under argon atmosphere, a three-necked flask was charged with tris(2-bromophenyl)amine (4.82 g, 10 mmol) and dehydrated THF (80 mL) and cooled to −78 degrees C. n-Butyllithium (1.6 M, hexane solution, 31.5 mmol, 19.7 mL) was dropped therein and stirred at the temperature as it was for 1.5 hours. Subsequently, chlorodimethylsilane (3.9 mL, 36 mmol) was added thereto, the temperature was returned to room temperature, and stirring was performed for 16 hours. The resulting liquid was quenched by addition of water, and chloroform was further added thereto to separate the organic phase. The aqueous phase was extracted with chloroform twice. The collected organic phase was dried with anhydrous sodium sulfate. The filtrate was condensed, and the residue was purified by column chromatography (elute: hexane/toluene=9/1 by volume). The solid obtained by condensation was recrystallized from toluene/ethanol. Thus, a compound 5-1 was prepared (yield: 2.95 g, 70%) as a colorless solid. Synthesis of Compound 5-2 Under an argon atmosphere, the compound 5-1 (2.95 g, 7 mmol), 3,3-dimethyl-1-butene (4.5 mL, 35 mmol), RhCl(PPh3)3(32 mg, 0.035 mmol), and 1,4-dioxane (70 mL) were placed in a three-necked flask and stirred at 135 degrees C. for 48 hours. The contents in the flask were cooled to room temperature, and the residue was purified by column chromatography (elute: hexane/toluene=9/1 by volume). The solid obtained by condensation was recrystallized from toluene/ethanol. Thus, a compound 5-2 was prepared (yield: 1.73 g, 60%) as a colorless solid. Synthesis of Electrochromic Compound 5 The procedure for preparing the electrochromic compound 4 was repeated except for replacing the compound 4-1 with the compound 5-1. Thus, an electrochromic compound 5 was prepared (yield: 1.95 g, 80%) as a colorless plate crystal. Preparation of First Electrochromic Element Example 1-1 An electrochromic element of Example 1-1 was prepared as follows. Formation of First Electrochromic Layer on First Electrode To form a first electrochromic layer on a first electrode, a first electrochromic composition containing the following materials was prepared. Materials Electrochromic compound 1-1 having acryloxygroup (Example Compound 1): 50 parts bymassIRGACURE 184 (available from BASFJapan Ltd.): 5 parts by massPolyethylene glycol having diacryloxy group(PEG400DA available from Nippon KayakuCo., Ltd.): 50 parts by massMethyl ethyl ketone: 900 parts by mass An ITO glass substrate (having an area of 40 mm×40 mm, a thickness of 0.7 mm, and an ITO film thickness of about 100 nm) serving as a first electrode was coated with the first electrochromic composition by spin coating. The coating layer was exposed to ultraviolet ray emitted from an UV emitter (SPOT CURE available from Ushio Inc.) at 10 mW for 60 seconds, then subjected to an annealing treatment at 60 degrees C. for 10 minutes. Thus, a first electrochromic layer having an average thickness of 400 μm and a cross-linked structure was formed. Formation of Anti-deterioration Layer on Second Electrode Another ITO glass substrate (having an area of 40 mm×40 mm, a thickness of 0.7 mm, and an ITO film thickness of about 100 nm) serving as a second electrode was coated with a titanium oxide nanoparticle dispersion liquid (SP210 available from Showa Titanium Co., Ltd., having an average particle diameter of about 20 nm) by spin coating, to form an anti-deterioration layer. The coating layer was subjected to an annealing treatment at 120 degrees C. for 15 minutes. Thus, a nanostructural semiconductive material comprised of a titanium oxide particle film having a thickness of 1.0 μm was formed. Formation of Second Electrochromic Layer on Second Electrode To form a second electrochromic layer on a second electrode, a second electrochromic composition containing the following materials was prepared. Materials Electrochromic compound 1-2 having afunctional group bindable to hydroxyl group(Example Compound A): 20 parts by massTetrafluoropropanol: 980 parts by mass The nanostructural semiconductive material comprised of a titanium oxide particle film, formed on the second electrode, was then coated and adsorbed with the second electrochromic composition by spin coating. Non-adsorbed compounds were then washed with methanol. Thus, a second electrochromic layer was formed. Filling of Electrolyte Liquid An electrolyte liquid containing the following materials was prepared. Materials IRGACURE 184 (available from BASFJapan Ltd.): 5 parts by massPEG400DA (available from Nippon KayakuCo., Ltd.): 100 parts by mass1-Ethyl-3-methylimidazolium tetracyanoborate(available from Merk KGaA): 50 parts by mass The above-prepared electrolyte liquid in an amount of 30 mg was weighed with a micropipette and dropped onto the ITO glass substrate serving as the second electrode having the anti-deterioration layer and the second electrochromic layer thereon. The ITO glass substrate serving as the first electrode having the cross-linked first electrochromic layer thereon was bonded to the above ITO glass substrate serving as the second electrode while leaving lead portions, thus preparing a bonded element. The bonded element was exposed to ultraviolet ray (having a wavelength of 250 nm) emitted from a UV emitter (SPOT CURE available from Ushio Inc.) at 10 mW for 60 seconds. Thus, an electrochromic element of Example 1-1 was prepared. Coloring/Decoloring Drive Operation Coloring/decoloring of the electrochromic element of Example 1-1 was confirmed as follows. A voltage of −2 V was applied for 5 seconds to between the lead portions of the first electrode and the second electrode. As a result, it was confirmed that the color derived from the electrochromic compound 1 in the first electrochromic layer was developed at the portion where the first electrode and the second electrode were overlapped. In addition, it was confirmed that the color derived from the electrochromic compound 2 in the second electrochromic layer was developed. Next, a voltage of +2 V was applied for 5 seconds to between the lead portions of the first electrode and the second electrode. As a result, the portion where the first electrode and the second electrode were overlapped was decolored and became transparent. FIG.3is a graph showing a ultraviolet-visible absorption spectrum of the electrochromic element of the present Example in the colored state.FIG.3shows an ultraviolet-visible absorption spectrum of the electrochromic element of the present Example which is acquired by subtracting the ultraviolet-visible absorption spectrum at the time when the electrochromic compound 2 is colored, and that at the time when the electrochromic compound 1 and the electrochromic compound 2 are decolored, from the ultraviolet-visible absorption spectrum at the time when the electrochromic compound 1 and the electrochromic compound 2 are colored. Thus,FIG.3shows the ultraviolet-visible absorption spectrum at the time when the electrochromic compound 1 is colored. InFIG.3, the absorption spectrum is illustrated within the wavelength range of from 380 to 780 nm. It was visually confirmed that both of the electrochromic compound 1 and the electrochromic compound 2 were colored in blue, as indicated inFIG.3. Examples 1-2 to 1-20 The procedure in Example 1-1 was repeated except for replacing the electrochromic compound 1-1 (Example Compound 1) with each of Example Compounds 2 to 20. Thus, electrochromic elements of Examples 1-2 to 1-20 were prepared. It was confirmed that the ultraviolet-visible absorption spectrum of each of the electrochromic elements of Examples 1-2 to 1-20 was similar to that of the electrochromic element of Example 1-1. Comparative Examples 1-1 to 1-5 The procedure in Example 1-1 was repeated except for replacing the electrochromic compound 1-1 (Example Compound 1) with each of the following Comparative Compounds 1 to 5. Thus, electrochromic elements of Comparative Examples 1-1 to 1-5 were prepared. It was also confirmed that the ultraviolet-visible absorption spectrum of each of the electrochromic elements of Comparative Examples 1-1 to 1-5 was similar to that of the electrochromic element of Example 1-1. Comparative Compound 1 Comparative Compound 2 Comparative Compound 3 Comparative Compound 4 Comparative Compound 5 Comparative Compound 6 The type and application position of electrochromic compound in each Example or Comparative Example are shown in Table 1. Evaluations Each electrochromic element was subjected to a repetition durability test, a continuous coloring test, a light durability test, and a color test as follows. Test 1-1: Repetition Durability Test Each of the electrochromic elements prepared in Examples and Comparative Examples was subjected to a coloring/decoloring drive operation in which a voltage of −2 V was applied for 5 seconds and thereafter a voltage of +2 V for 5 seconds to between the lead portions of the first electrode and the second electrode. This coloring/decoloring drive operation was repeated 10,000 times. A wavelength λmax at which the absorbance became a local maximum was determined within a visible range (from 400 to 800 nm). (λmax was 700 nm in Example 1.) Repetition durability was evaluated by the change in absorbance at λmax, measured with a spectrometer USB4000, based on the following criteria. The evaluation results are presented in Table 1. Evaluation CriteriaA+: Absorbance at λmax is 90% or more of the initial absorbance.A: Absorbance at λmax is 80% or more of the initial absorbance.B: Absorbance at λmax is 50% or more of the initial absorbance.C: Absorbance at λmax is less than 50% of the initial absorbance. Test 1-2: Continuous Coloring Test In each of the electrochromic elements prepared in Examples and Comparative Examples, a voltage of 1.6 V was applied to between the first electrode and the second electrode, and the electrochromic element was maintained in the colored state for continuous 48 hours. The absorbance within a visible range (from 380 to 800 nm) was measured with a spectrometer USB4000 and a yellow index (YI) was calculated before and after the application of voltage. Continuous coloring property was evaluated by the difference (ΔYI) in yellow index before and after the application of voltage based on the following criteria. The evaluation results are presented in Table 1. Evaluation CriteriaA+: ΔYI is less than 1.A: ΔYI is not less than 1 and less than 5.B: ΔYI is not less than 5 and less than 10.C: ΔYI is 10 or greater. Test 1-3: Light Durability Test In each of the electrochromic elements prepared in Examples and Comparative Examples, a voltage of 1.6 V was applied to between the first electrode and the second electrode. While maintaining the electrochromic element in the colored state, the electrochromic element was irradiated with light emitted from an artificial solar lighting (SOLAX XC-100W available from SERIC Ltd., having an illuminance of 150,000 lux) through an ultraviolet cut filter (LUMICOOL 1501UH available from LINTEC Corporation) for continuous 48 hours. The electrochromic element was further irradiated with light emitted from a deuterium tungsten halogen light source (DH-2000 available from Ocean Optics, Inc.), and the transmitted light was detected by a spectrometer USB4000 to obtain a transmission spectrum. A wavelength λmax at which the transmittance became the minimum was determined within a visible range (from 400 to 800 nm). Light durability was evaluated by the transmittance at λmax based on the following criteria. The evaluation results are presented in Table 1. Evaluation CriteriaA+: Transmittance at λmax is less than 10%.A: Transmittance at λmax is not less than 10% and less than 30%.B: Transmittance at λmax is 30% or greater.C: Transmittance at λmax is 50% or greater. TABLE 1FirstSecondElectrochromic CompositionElectrochromic CompositionType of FirstType of SecondElectrochromicAppliedElectrochromicAppliedTestTestTestCompoundPositionCompoundPosition1-11-23-3Example 1-2ExampleFirstExampleSecondA+A+A+CompoundElectrochromicCompoundElectrochromic1LayerALayerExample 1-2ExampleFirstExampleSecondA+A+A+CompoundElectrochromicCompoundElectrochromic2LayerALayerExample 1-3ExampleFirstExampleSecondA+A+ACompoundElectrochromicCompoundElectrochromic3LayerALayerExample 1-4ExampleFirstExampleSecondA+A+A+CompoundElectrochromicCompoundElectrochromic4LayerALayerExample 1-5ExampleFirstExampleSecondA+A+A+CompoundElectrochromicCompoundElectrochromic5LayerALayerExample 1-6ExampleFirstExampleSecondA+A+A+CompoundElectrochromicCompoundElectrochromic6LayerALayerExample 1-7ExampleFirstExampleSecondAAACompoundElectrochromicCompoundElectrochromic7LayerALayerExample 1-8ExampleFirstExampleSecondA+A+A+CompoundElectrochromicCompoundElectrochromic8LayerALayerExample 1-9ExampleFirstExampleSecondAAACompoundElectrochromicCompoundElectrochromic9LayerALayerExample 1-10ExampleFirstExampleSecondAAACompoundElectrochromicCompoundElectrochromic10LayerALayerExample 1-11ExampleFirstExampleSecondA+AA+CompoundElectrochromicCompoundElectrochromic11LayerALayerExample 1-12ExampleFirstExampleSecondA+AA+CompoundElectrochromicCompoundElectrochromic12LayerALayerExample 1-13ExampleFirstExampleSecondA+A+A+CompoundElectrochromicCompoundElectrochromic13LayerALayerExample 1-14ExampleFirstExampleSecondA+A+A+CompoundElectrochromicCompoundElectrochromic14LayerALayerExample 1-15ExampleFirstExampleSecondA+AA+CompoundElectrochromicCompoundElectrochromic15LayerALayerExample 1-16ExampleFirstExampleSecondA+A+A+CompoundElectrochromicCompoundElectrochromic16LayerALayerExample 1-17ExampleFirstExampleSecondA+A+A+CompoundElectrochromicCompoundElectrochromic17LayerALayerExample 1-18ExampleFirstExampleSecondA+AACompoundElectrochromicCompoundElectrochromic18LayerALayerExample 1-19ExampleFirstExampleSecondABBCompoundElectrochromicCompoundElectrochromic19LayerALayerExample 1-20ExampleFirstExampleSecondABBCompoundElectrochromicCompoundElectrochromic20LayerALayerExample 1-21ExampleFirstExampleSecondA+AA+CompoundElectrochromicCompoundElectrochromic21LayerALayerExample 1-22ExampleFirstExampleSecondA+AA+CompoundElectrochromicCompoundElectrochromic22LayerALayerExample 1-23ExampleFirstExampleSecondAAACompoundElectrochromicCompoundElectrochromic23LayerALayerComparativeComparativeFirstExampleSecondBBCExample 1-1Compound 1ElectrochromicCompoundElectrochromicLayerALayerComparativeComparativeFirstExampleSecondCCBExample 1-2Compound 2ElectrochromicCompoundElectrochromicLayerALayerComparativeComparativeFirstExampleSecondABCExample 1-3Compound 3ElectrochromicCompoundElectrochromicLayerALayerComparativeComparativeFirstExampleSecondCCBExample 1-4Compound 4ElectrochromicCompoundElectrochromicLayerALayerComparativeComparativeFirstExampleSecondCCCExample 1-5Compound 5ElectrochromicCompoundElectrochromicLayerALayer It is clear from Table 1 that the electrochromic elements of Examples 1-1 to 1-23 deliver satisfactory repetition durability, continuous coloring property, and light durability. In particular, continuous driving stability and light durability are excellent. By contrast, the electrochromic elements of Comparative Examples 1-1 to 1-5 are insufficient in at least one of repetition durability, continuous coloring property, and light durability. Thus, the electrochromic composition according to the first embodiment contributes to improvement of continuous driving stability and light durability of the electrochromic element. Example 2 Preparation of Second Electrochromic Element Example 2-1 An electrochromic element of Example 2-1 was prepared as follows. Formation of Spacer on First Electrode An ITO glass substrate (having an area of 40 mm×40 mm, a thickness of 0.7 mm, and an ITO film thickness of about 100 nm) serving as a first electrode was coated with an isopropanol solution of gap control particles (MICROPEARL GS available from Sekisui Chemical Co., Ltd., having a particle diameter of 80 μm) and dried at 80 degrees C. for 3 minutes. Formation of Anti-deterioration Layer on Second Electrode Another ITO glass substrate (having an area of 40 mm×40 mm, a thickness of 0.7 mm, and an ITO film thickness of about 100 nm) serving as a second electrode was coated with a titanium oxide nanoparticle dispersion liquid (SP210 available from Showa Titanium Co., Ltd., having an average particle diameter of about 20 nm) by spin coating, to form an anti-deterioration layer. The coating layer was subjected to an annealing treatment at 120 degrees C. for 15 minutes. Thus, a nanostructural semiconductive material comprised of a titanium oxide particle film having a thickness of 1.0 μm was formed. Bonding of Substrates The ITO substrate as the first electrode and the ITO substrate as the second electrode were bonded to each other with the electrode surfaces facing each other and shifted 5 mm to form lead portions. The end faces of the bonded substrates were coated with a sealing material (TB 3050B available from ThreeBond Group) while leaving two injection holes. The bonded element was irradiated with ultraviolet ray (having a wavelength of 250 nm) emitted from a UV emitter (SPOT CURE available from Ushio Inc.) at 10 mW for 60 seconds. Filling of Electrolyte Liquid An electrolyte liquid containing the following materials was prepared. Materials Electrochromic compound 2 (Example Compound M1): 50 parts by mass1-Ethyl-3-methylimidazoliumbisfluorosulfonylimide (EMIM-FSI) (available from Merk KGaA): 100 parts by massN-methylpyrrolidone (NMP): 600 parts by mass The above-prepared electrolyte liquid in an amount of 30 mg was weighed with a micropipette and injected into the element from the injection holes. The injection holes were sealed with the sealing material and exposed to ultraviolet ray (having a wavelength of 250 nm) emitted from a UV emitter (SPOT CURE available from Ushio Inc.) at 10 mW for 60 seconds. Thus, an electrochromic element of Example 2-1, illustrated inFIG.2, was prepared. Coloring/Decoloring Drive Operation Coloring/decoloring of the electrochromic element of Example 2-1 was confirmed in the same manner as the electrochromic element of Example 1-1. As a result, it was confirmed that, when a voltage of −2 V was applied for 5 seconds to between the lead portions of the first electrode and the second electrode, the color derived from the electrochromic compound 2 in the electrochromic layer was developed at the portion where the first electrode and the second electrode were overlapped. In was also confirmed that, when a voltage of +2 V was applied for 5 seconds to between the lead portions of the first electrode and the second electrode, the portion where the first electrode and the second electrode were overlapped was decolored and became transparent. Examples 2-2 to 2-6 The procedure in Example 2-1 was repeated except for replacing the electrochromic compound 2 (Example Compound M1) with each of Example Compounds M2 to M6. Thus, electrochromic elements of Examples 2-2 to 2-6 were prepared. Comparative Examples 2-1 to 2-5 The procedure in Example 2-1 was repeated except for replacing the electrochromic compound 1 (Example Compound M1) with each of the following Comparative Compounds m1 to m5. Thus, electrochromic elements of Comparative Examples 2-1 to 2-5 were prepared. It was confirmed that the color derived from the electrochromic compound 2 was developed in the electrochromic elements of Comparative Examples 2-1 to 2-6 as in the electrochromic element of Example 2-1. Comparative Compound m1 Comparative Compound m2 Comparative Compound m3 Comparative Compound m4 Comparative Compound m5 Comparative Compound m6 Evaluations Each electrochromic element was subjected to a continuous coloring test, a light durability test, a color test, and a deteriorated matter analysis as follows. Test 2-1: Continuous Coloring Test In each of the electrochromic elements prepared in Examples and Comparative Examples, a voltage of 1.6 V was applied to between the first electrode and the second electrode, and the electrochromic element was maintained in the colored state for continuous 48 hours. The absorbance within a visible range (from 380 to 800 nm) was measured with a spectrometer USB4000 and a yellow index (YI) was calculated before and after the application of voltage. Continuous coloring property was evaluated by the difference (ΔYI) in yellow index before and after the application of voltage based on the following criteria. The evaluation results are presented in Table 2. Evaluation CriteriaA+: ΔYI is less than 1.A: ΔYI is not less than 1 and less than 5.B: ΔYI is not less than 5 and less than 10.C: ΔYI is 10 or greater. Test 2-2: Light Durability Test Each of the electrochromic elements prepared in Examples and Comparative Examples was subjected to a light durability test conducted in the same manner as Test 1-2 described above. The evaluation results are presented in Table 2. Test 2-3: Deteriorated Matter Analysis After the Test 2-2, the sealing material of each electrochromic element was cut to take out the electrolyte liquid from the inside. The electrolyte liquid was dissolved in acetonitrile and analyzed by a liquid chromatography mass spectrometry (LC/MS) instrument (HPLC Alliance/TOF-MS LCT-Premier available from Waters Corporation). Cyclized products, from which one hydrogen molecule had been removed by a photochemical reaction, were detected by a photodiode detector (from 200 to 800 nm) and a mass spectrometer (APCI mode) and quantified by calculating the area ratio to the main component under the absorbance spectrum at 280 nm. The analysis results were evaluated based on the following criteria. The evaluation results are presented in Table 2. Analysis ConditionsColumn: Super ODS (having an inner diameter of 4.6 mm×100 mm, available from Tosoh Corporation)Solvent: Mixed solvent of acetonitrile and water (the ratio of acetonitrile:water was changed from 50:50 to 100:0 with a linear gradient) within a time period of from 0 to 10 minutes, and 100% acetonitrile within a time period of from 10 to 15 minutes. Evaluation CriteriaA: The generation rate of cyclized products from which one hydrogen molecule has been removed is less than 1%.C: The generation rate of cyclized products from which one hydrogen molecule has been removed is 1% or greater. TABLE 2Electrochromic Compound 2AppliedTestTestTestTypePosition2-12-22-3Example 2-1ExampleElectrolyte LayerA+A+ACompound M1Example 2-2ExampleElectrolyte LayerA+A+ACompound M2Example 2-3ExampleElectrolyte LayerA+A +ACompound M3Example 2-4ExampleElectrolyte LayerA+A+ACompound M4Example 2-5ExampleElectrolyte LayerA+A+ACompound M5Example 2-6ExampleElectrolyte LayerA+A+ACompound M6Example 2-7ExampleElectrolyte LayerA+A+ACompound M7Example 2-8ExampleElectrolyte LayerA+AACompound M8Example 2-9ExampleElectrolyte LayerA+A+ACompound M9Example 2-10ExampleElectrolyte LayerA+A+ACompound M10Example 2-11ExampleElectrolyte LayerA+A+ACompound M11Example 2-12ExampleElectrolyte LayerA+A+ACompound M12ComparativeComparativeElectrolyte LayerCBCExample 2-1Compound m1ComparativeComparativeElectrolyte LayerAACExample 2-2Compound m2ComparativeComparativeElectrolyte LayerABCExample 2-3Compound m3ComparativeComparativeElectrolyte LayerCCAExample 2-4Compound m4ComparativeComparativeElectrolyte LayerBCCExample 2-5Compound m5 It is clear from Table 2 that the electrochromic elements of Examples 2-1 to 2-12 deliver satisfactory continuous coloring property and light durability. In particular, continuous driving stability and light durability are excellent. By contrast, the electrochromic elements of Comparative Examples 2-1 to 2-6 are insufficient in continuous coloring property or light durability. In addition, it was confirmed that the electrochromic compound used in the electrochromic elements of Examples 2-1 to 2-6 generates very few cyclized products (carbazole derivatives), which are byproducts produced by light irradiation which degrade the properties of the electrochromic elements. By contrast, when the comparative compound m4 is used as an electrochromic compound as in Comparative Example 2-4, an electrochromic element having continuous driving stability and light durability cannot be provided although generation of cyclized products can be prevented by blocking the hydrogen atom at the position where the cyclized product is formed. Thus, the electrochromic compound according to the second embodiment contributes to improvement of continuous driving stability and light durability of the electrochromic element. Example 3 Preparation of Third Electrochromic Element Example 3-1 The procedure in Example 1-1 was repeated except that a mixture of Example Compound 1 with Example Compound B8 in the same amount (50 parts by mass) was used as the first electrochromic compound in the first electrochromic composition. Thus, an electrochromic element of Example 3-1 was prepared. Coloring/Decoloring Drive Operation Coloring/decoloring of the electrochromic element of Example 3 was confirmed in the same manner as the electrochromic elements of Examples 1 and 2. As a result, it was confirmed that, when a voltage of −2 V was applied for 5 seconds to between the lead portions of the first electrode and the second electrode, black color was observed at the portion where the first electrode and the second electrode were overlapped as the mixture of blue color and orange color derived from the electrochromic compounds in the electrochromic layers. In was also confirmed that, when a voltage of +2 V was applied for 5 seconds to between the lead portions of the first electrode and the second electrode, the colored portion was decolored and became transparent. Comparative Examples 3-1 to 3-3 The procedure in Example 3-1 was repeated except for replacing the Example Compound 1 as the first electrochromic compound in the first electrochromic composition with each of the Comparative Compounds 1, 3, and 6. Thus, electrochromic elements of Comparative Examples 3-1 to 3-3 were prepared. It was also confirmed that black color was developed as the mixture of blue color and orange color in the electrochromic elements of Comparative Examples 3-1 to 3-3 as in the electrochromic element of Example 3-1. Evaluations Each electrochromic element was subjected to a repetition durability test and a continuous coloring test as follows. Test 3-1: Repetition Durability Test Each of the electrochromic elements prepared in Examples and Comparative Examples was subjected to a coloring/decoloring drive operation in which a voltage of −2 V was applied for 5 seconds and thereafter a voltage of +2 V for 5 seconds to between the lead portions of the first electrode and the second electrode. This coloring/decoloring drive operation was repeated 10,000 times. A visible transmittance tmaxwithin a visible range (from 400 to 800 nm) at the time of coloring was measured. A change in transmittance at that time was measured with a fiber multichannel spectrometer USB4000 (available from Ocean Optics, Inc.) and evaluated based on the following criteria. The evaluation results are presented in Table 3. Evaluation CriteriaA+: The change in transmittance tmaxis 90% or more of the initial transmittance.A: The change in transmittance tmaxis 80% or more of the initial transmittance.B: The change in transmittance tax is 50% or more of the initial transmittance.C: The change in transmittance tmaxis less than 50% of the initial transmittance. Test 3-2: Continuous Coloring Test In each of the electrochromic elements prepared in Examples and Comparative Examples, a voltage of 1.6 V was applied to between the first electrode and the second electrode, and the electrochromic element was maintained in the colored state for continuous 48 hours. The absorbance within a visible range (from 380 to 800 nm) was measured with a fiber multichannel spectrometer (USB4000 available from Ocean Optics, Inc.) and a yellow index (YI) was calculated before and after the application of voltage. Continuous coloring property was evaluated by the difference (ΔYI) in yellow index before and after the application of voltage based on the following criteria. The evaluation results are presented in Table 3. Evaluation CriteriaA+: ΔYI is less than 1.A: ΔYI is not less than 1 and less than 5.B: ΔYI is not less than 5 and less than 10.C: ΔYI is 10 or greater. TABLE 3SecondFirst Electrochromic CompositionElectrochromic CompositionFirstSecondType ofType of FirstType of FirstSecondElectrochromicElectrochromicAppliedElectrochromicAppliedTestTestCompoundCompoundPositionCompoundPosition3-13-2Example 3-1ExampleExampleFirstExampleSecondA+A+CompoundCompoundElectrochromicCompoundElectrochromic1B8LayerALayerComparativeComparativeExampleFirstExampleSecondBCexample 3-1CompoundCompoundElectrochromicCompoundElectrochromic1B8LayerALayerComparativeComparativeExampleFirstExampleSecondABexample 3-2CompoundCompoundElectrochromicCompoundElectrochromic3B8LayerALayerComparativeComparativeExampleFirstExampleSecondAAexample 3-3CompoundCompoundElectrochromicCompoundElectrochromic6B8LayerALayer It was confirmed that, in the electrochromic elements of Example 3-1 and Comparative Examples 3-1 to 3-3, black color was developed as the mixture of blue color and orange color. The black color was developed because a benzidine compound was contained in the first electrochromic composition. It is clear from Table 3 that the electrochromic element of Example 3-1 delivers satisfactory repetition durability and continuous coloring property. In particular, continuous driving stability and light durability are excellent. By contrast, the electrochromic elements of Comparative Examples 3-1 to 3-3 are insufficient in repetition durability or continuous coloring property, which is slightly inferior. Thus, the first electrochromic composition according to the third embodiment contributes to improvement of continuous driving stability and light durability of the electrochromic element. Example 4 Preparation of Fourth Electrochromic Element Example 4-1 The procedure in Example 2-1 was repeated except that a mixture of Example Compound M1 with Example Compound B2 in the same amount (50 parts by mass) was used as the electrochromic compound 2. Thus, an electrochromic element of Example 4-1 was prepared. Coloring/Decoloring Drive Operation Coloring/decoloring of the electrochromic element of Example 4 was confirmed in the same manner as the electrochromic elements of Examples 1 and 2. As a result, it was confirmed that, when a voltage of −2 V was applied for 5 seconds to between the lead portions of the first electrode and the second electrode, black color was observed at the portion where the first electrode and the second electrode were overlapped as the mixture of blue color and orange color derived from the electrochromic compounds in the electrochromic layers. In was also confirmed that, when a voltage of +2 V was applied for 5 seconds to between the lead portions of the first electrode and the second electrode, the colored portion was decolored and became transparent. Comparative Examples 4-1 to 4-3 The procedure in Example 4-1 was repeated except for replacing the Example Compound M1 as the first electrochromic compound in the first electrochromic composition with each of the Comparative Compounds m2, m3, and m6. Thus, electrochromic elements of Comparative Examples 4-1 to 4-3 were prepared. It was also confirmed that black color was developed as the mixture of blue color and orange color in the electrochromic elements of Comparative Examples 4-1 to 4-3 as in the electrochromic element of Example 4-1. Evaluations Each electrochromic element was subjected to a repetition durability test and a continuous coloring test as follows. Test 4-1: Repetition Durability Test Each of the electrochromic elements prepared in Examples and Comparative Examples was subjected to a coloring/decoloring drive operation in which a voltage of −2 V was applied for 5 seconds and thereafter a voltage of +2 V for 5 seconds to between the lead portions of the first electrode and the second electrode. This coloring/decoloring drive operation was repeated 10,000 times. A visible transmittance tmaxwithin a visible range (from 400 to 800 nm) at the time of coloring was measured. Repetition durability was evaluated by the change in transmittance, measured with a spectrometer USB4000, based on the following criteria. The evaluation results are presented in Table 4. Evaluation CriteriaA+: The change in transmittance tmaxis 90% or more of the initial transmittance.A: The change in transmittance tmaxis 80% or more of the initial transmittance.B: The change in transmittance tmaxis 50% or more of the initial transmittance.C: The change in transmittance tmaxis less than 50% of the initial transmittance. Test 4-2: Continuous Coloring Test In each of the electrochromic elements prepared in Examples and Comparative Examples, a voltage of 1.6 V was applied to between the first electrode and the second electrode, and the electrochromic element was maintained in a colored state for continuous 48 hours. The absorbance within a visible range (from 380 to 800 nm) was measured with a spectrometer USB4000 and a yellow index (YI) was calculated before and after the application of voltage. Continuous coloring property was evaluated by the difference (ΔYI) in yellow index before and after the application of voltage based on the following criteria. The evaluation results are presented in Table 4. Evaluation CriteriaA+: ΔYI is less than 1.A: ΔYI is not less than 1 and less than 5.B: ΔYI is not less than 5 and less than 10.C: ΔYI is 10 or greater. TABLE 4Electrochromic Compound 2AppliedTestTestFirst TypeSecond TypePosition4-14-2Example 4-1ExampleExampleElectrolyteA+A+CompoundCompound B2LayerM1Comparative Example 4-1Comparative CompoumdExample Compound B2Electrolyle LayerABm2Comparative Example 4-2Comparative CompoundExample Compound B2Electrolyte LayerBCm3Comparative Example 4-3Comparative CompoundExample Compound B2Electrolyte LayerAAm6 It was confirmed that, in the electrochromic elements of Example 4-1 and Comparative Examples 4-1 to 4-3, black color was developed as the mixture of blue color and orange color. The black color was developed because a benzidine compound was contained in the first electrochromic composition. It is clear from Table 4 that the electrochromic element of Example 4-1 delivers satisfactory repetition durability and continuous coloring property. In particular, continuous driving stability and light durability are excellent. By contrast, the electrochromic elements of Comparative Examples 4-1 to 4-3 are insufficient in repetition durability or continuous coloring property, which is slightly inferior. Thus, the electrochromic compound according to the fourth embodiment contributes to improvement of continuous driving stability and light durability of the electrochromic element. The above-described embodiments are illustrative and do not limit the present invention. Thus, numerous additional modifications and variations are possible in light of the above teachings. For example, elements and/or features of different illustrative embodiments may be combined with each other and/or substituted for each other within the scope of the present invention. REFERENCE SIGNS LIST 10A,10B Electrochromic element11First substrate12Display electrode (First electrode)13First electrochromic layer14A,14B Electrolyte layer15Second electrochromic layer16Counter electrode (Second electrode)17Second substrate | 120,144 |
11859132 | DETAILED DESCRIPTION OF THE INVENTION For promoting an understanding of the principles of the present disclosure, reference will now be made to the specific embodiments of the present invention further illustrated in the drawings and specific language will be used to describe the same. The foregoing general description and the following detailed description are explanatory of the present disclosure and are not intended to be restrictive thereof. It will nevertheless be understood that no limitation of the scope of the present disclosure is thereby intended, such alterations and further modifications in the illustrated composition, and such further applications of the principles of the present disclosure as illustrated herein being contemplated as would normally occur to one skilled in the art to which the present disclosure relates. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinarily skilled in the art to which this present disclosure belongs. The methods, and examples provided herein are illustrative only and not intended to be limiting. The process and apparatus as disclosed herein converts the waste plastic pyrolysis oil into valuable products by heat integration of DCU process with pyrolysis oil production, wherein, HCGO stream from delayed coker unit is used as a preheating stream. The process and apparatus further include co-processing of pyrolysis oil with residue feed such as vacuum residue in various configurations for producing more liquid & gaseous products with less coke yields. The process and apparatus also eliminate the use of off-gases of pyrolysis process to get consumed in providing required heat for maintaining reaction temperature, which otherwise can be recovered in the form of valuable products such as ethylene, and propylene. Further, the process as disclosed herein generates valuable liquid products out of waste materials without really disturbing the hardware of the existing delayed coking (DC) technology and in fact improves the overall delayed coking process. The present process and apparatus as disclosed herein also overcomes high energy requirement for producing pyrolysis oil from waste plastics by utilizing HCGO as preheating stream which in turn reduces HCGO temperature to desired level such that it can be utilized in downstream processes such as HCGO hydrotreating. The present invention provides an alternative to the conventional quench oils which not only stops cracking reactions but also helps in increasing liquid yields. The process as disclosed herein includes melting a waste plastic feedstock (1) inside a preheating vessel (3), wherein, the said waste plastic feedstock (1) is melted by a heavy coker gas oil (HCGO) (30) as received from a delayed coker fractionator (19). A pyrolysis step inside a pyrolysis reactor (6) to pyrolyze a molten plastic stream (4) along with nitrogen (5), wherein, the pyrolysis reactor (6) generates a char component (7) from a bottom portion thereof, and a vapour component (8) from a top portion thereof. A condensation step to condense the said vapour component (8) using a heat exchanger (9) into a condensed stream (10), wherein, the said condensed stream (10) is routed to the separator tower (11) to separate the gases (12) and the condensed liquid (13). Then separating the said condensed liquid (13) in two phase separator (14), wherein, the said condensed liquid (13) is separated into pyrolysis oil stream (15) and water (16). The pyrolysis oil stream (15) is further fractionated using fractionator (45) into two cuts, a heavier cut (47) and a lighter cut (46). The lighter cut of pyrolysis oil (46) is fed directly to the LCGO section of delayed coker fractionator (19). The process further includes mixing and separation of a mixed stream of hot heavy residual feedstock (17), and the heavier cut of pyrolysis oil stream (47), coke drum effluent (27,28), wherein, the said mixing is carried out inside a delayed coker fractionator (19) to provide a resulting mixture (20) which is pumped through a coker furnace (21) to achieve a desired coking temperature of 470° C. to 500° C. Then thermal cracking of the said resulting mixture (20) inside the said coker furnace (21), wherein, the thermal cracking step results partial vaporization and mild cracking of the said resulting mixture (20) to give a two-phase mixture (22). After that thermal cracking of the said two-phase mixture (22) inside a plurality of coke drums (25,26), wherein, a heavier part of the pyrolysis oil stream (15) along with a heavy residue go through the thermal cracking (delayed coking) resulting into the coke drum effluent (27,28), coke, and a wash liquid (24). The apparatus as disclosed herein includes a preheating vessel (3) adapted to produce a molten plastic stream (4) by melting a waste plastic feedstock (1) with the help of a heavy coker gas oil (HCGO) (30) having temperature 290-390° C. The apparatus also includes a pyrolysis reactor (6) for pyrolyzing the said molten plastic stream (4) along with nitrogen (5) to produce a char component (7) and a vapour component (8), wherein, the said vapour component (8) is condensed into a condensed stream (10) through a heat exchanger (9). The apparatus also includes a separator tower (11) to separate an off gas (12) and a condensed liquid (13) from the said condensed stream (10), wherein, the said condensed liquid (13) is separated into pyrolysis oil stream (15) and water (16) through a two-phase separator (14). The said off gas (12) from the separator tower (11) is mixed with a coker off gas (36) and fed to light olefin recovery section (39) to get fuel gas (40), ethylene, propylene (41), LPG (42) and naphtha (43), wherein, the fuel gas (40) is used for burning in presence of air (44) to generate heat energy required for pyrolysis reactor (6) for maintaining reaction temperature. The apparatus also includes a delayed coker fractionator (19) for mixing and separation of a mixed stream of hot heavy residual feedstock (17), and the heavier cut of pyrolysis oil stream (47), a coke drum effluent (27,28), wherein, the said mixing provides a resulting mixture (20) which is pumped through a coker furnace (21) to achieve a desired coking temperature of 470° C. to 500° C., wherein, the resulting mixture (20) undergoes thermal cracking resulting in partial vaporization and mild cracking of the said resulting mixture (20) to give a two-phase mixture (22). The lighter cut of pyrolysis oil (46) is fed directly to the LCGO section of delayed coker fractionator (19). Wherein, the said coke drum effluent (27,28) is the outcome of the said plurality of coke drums (25,26). The delayed coker fractionator (19) produces the said heavy coker gas oil (HCGO) (30), a Light coker gas oil (LCGO) (31), and a wet gas (37), wherein the wet gas (37) undergoes through a three-phase separator (35) resulting into gaseous hydrocarbons (36), water as aqueous phase (34) and unstabilized naphtha as liquid phase (32). The apparatus also includes a plurality of coke drums (25,26) to carry out thermal cracking of the said two-phase mixture (22) resulting into coke drum effluent (27,28), coke, and a wash liquid (24). Plastic Feedstock Pyrolysis oil is produced from thermal degradation of different types of waste plastics which includes High density Polyethene (HDPE), Low density Polyethene (LDPE), Polypropylene (PP), Polystyrene (PS) and mixed plastics i.e. mixture of Polyethylene (PE), Polypropylene (PP), and Polystyrene (PS) or mixture of LDPE, HDPE & PP. PET and PVC are not recommended for pyrolysis because both of them produces lower liquid yield compared to gas yields. Moreover, the pyrolysis of PET and PVC resulted in the formation of corrosive benzoic acid and toxic HCl respectively, which can pose major challenge to life of reactor, safety and environment. HDPE finds application in milk bottles, detergent bottles, toys manufacturing and produces a high liquid yield on pyrolysis. LDPE finds application in plastic bags, foils, trash bags and is the second largest amount plastics found in MSW. It produces high liquid yield with low gas and negligible char yield on pyrolysis. PP has diverse applications in office folders, car bumpers, carpets, furniture and storage boxes. It is the largest amount of plastic found in Municipal Solid Wastes (MSW) and it produces decent liquid yields along with coke. PS is widely used in electronics, constructions, medical appliances and toys and on pyrolysis a very high liquid yield with low gas yield is obtained. Mixed plastics produce both liquid yields close to 48 wt % on pyrolysis. Pyrolysis oil obtained from mixed plastics has been used for this study. Pyrolysis Oil Properties The properties of pyrolysis oil include density in the range of 0.7-0.95 g/cc, CCR ranges from 0.05 to 2 wt. %, and sulfur in the range of 10-700 wppm. Heavy residual feedstocks such as vacuum residue, Reduced Crude Oil, VB Tar etc. with CCR in the range of 3 to 30 wt. % is employed along with pyrolysis oil. Process Conditions Delayed Coker reactor & fractionator/section: The operating temperature for delayed coking process varies from 460-520° C. with pressure from 1-5 Kg/cm2and cycle time in the range of 10 to 32 hrs. Temperature of HCGO stream coming out of fractionator is 290-390° C. Pyrolysis reactor/section: The process conditions for production of pyrolysis oil from waste plastic includes temperature which ranges from 250-550° C. at atmospheric pressure with holding time of 1-6 hrs. Operating Temperature range of preheating vessel is 30-290° C. at atmospheric pressure. Process Description The process of the present invention is exemplified by the figures as provided in the present disclosure, but not limited to the figures. FIG.1shows the process flow diagram for the embodiment-1 of the present claimed process. Waste plastics feedstock (1) is fed into the hopper (2) and conveyed into the preheating vessel (3), wherein, HCGO (30) from the delayed coker fractionator (19) serves as a preheating stream which causes melting of waste plastics leaving relatively cooler HCGO stream (38). The molten plastic stream (4) along with Nitrogen (N2) (5) is fed into the pyrolysis reactor (6) leaving char (7) from bottoms while the vapours (8) from top leaves the reactor and is condensed using heat exchanger (9) and condensed stream (10) is routed to the separator tower (11). Wherein, gases (12) leave from the top of the separator tower (11) and condensed liquid (13) leaves from bottom of the separator tower (11) while pyrolysis oil stream (15) and water (16) both are separated in two phase separator (14). The pyrolysis oil stream (15) is further fractionated using fractionator (45) into two cuts, heavier cut (47) & lighter cut (46). The heavier cut of pyrolysis oil stream (47) is mixed with hot heavy residual feedstocks (17) and mixed stream (18) is fed to the delayed coker fractionator (19), wherein, the mixed stream (18) further mixes with condensed recycle. The lighter cut of pyrolysis oil (46) is fed directly to the LCGO section of delayed coker fractionator (19). The resulting mixture (20) is pumped through a coker furnace (21) using fractionator bottom pump(s), for achieving the desired coking temperature (usually between 470° C. and 500° C.) which also results in the partial vaporization and mild cracking of mixture (20). A two-phase mixture (22) usually vapor-liquid comes out of the coker furnace (21) and control valve (23) diverts it to a coke drum(s) (25,26). The said coke drum(s) (25,26) provides sufficient residence time for the thermal cracking to take place till the completion of coking reaction. The vapours of coke drum effluent (27,28) produced during thermal cracking, exits the coke drum(s) (25,26). The temperature of coke drum effluent (27,28) determines the average outlet temperature of the coke drum(s) (25,26). The gas oils from the coke drum effluent (27,28) are used as quenching stream which terminates further thermal cracking to avoid coke formation in vapor transfer line (29). Here, thermal cracking of heavier part of pyrolysis oil along with heavy residue will occur as some lighter part will be fractionated out in fractionator (19). It enhances liquid yields and reduces coke yield. In the present disclosure, a plurality of coke drums is used, specifically, two number of coke drums (25,26) are operated in parallel to ensure smooth functioning of coking cycle. When coke drum (25) is full of coke, the coking cycle ends and the coker furnace (21) outlet flow is then transferred from coke drum (25) to a parallel coke drum (26) to initiate its coking cycle, while coke removal process is initiated in the filled drum (25) which comprises of steaming, water cooling, coke cutting, and vapor heating and draining. The wash liquid (24) from the coke drums (25,26) is discharged into the blow down section. The coke drum effluent (27,28) after quenching are again sent to the bottom of the delayed coker fractionator (19) using vapor transfer line (29), where it is separated and recovered. Further, Heavy coker gas oil (HCGO) (30) and Light coker gas oil (LCGO) (31) are drawn off the delayed coker fractionator (19) at desired boiling temperature ranges using side strippers. The wet gas (37) coming out of the top of the delayed coker fractionator (19) goes to three phase separator (35), wherein, gaseous hydrocarbons (36), water as aqueous phase (34) and unstabilized naphtha as liquid phase (32) are separated and reflux stream (33) is sent back to the delayed coker fractionator (19). Further, the off gas (12) coming from pyrolysis process of the separator tower (11) is mixed with coker off gases (36) and fed to a light olefin recovery section (39) to get fuel gas (40) from the top which is fed back to pyrolysis reactor (6) in presence of air (44) for maintaining reaction temperature. Further, the said light olefin recovery section (39) also generates ethylene, propylene (41), LPG (42) and naphtha (43) from the bottom thereof. FIG.2shows the process flow diagram for the embodiment-2 of the present claimed process. Waste plastics feedstock (1) is fed into the hopper (2) and conveyed into the preheating vessel (3), wherein, HCGO (30) from the delayed coker fractionator (19) serves as a preheating stream which causes melting of waste plastics leaving relatively cooler HCGO stream (38). The molten plastic stream (4) along with Nitrogen (N2) (5) is fed into the pyrolysis reactor (6) leaving char (7) from bottoms while the vapours (8) from top leaves the reactor and is condensed using heat exchanger (9) and condensed stream (10) is routed to the separator tower (11). Wherein, gases (12) leave from the top of the separator tower (11) and condensed liquid (13) leaves from bottom of the separator tower (11) while pyrolysis oil stream (15) and water (16) both are separated in two phase separator (14). The pyrolysis oil stream (15) is mixed with hot heavy residual feedstocks (17) and mixed stream (18) is fed to the delayed coker fractionator (19), wherein, the mixed stream (18) further mixes with condensed recycle to produce a resulting mixture (20). The resulting mixture (20) is pumped through a coker furnace (21) using fractionator bottom pump(s), for achieving the desired coking temperature (usually between 470° C. and 500° C.) which also results in the partial vaporization and mild cracking of mixture (20). A two-phase mixture (22) usually vapor-liquid comes out of the coker furnace (21), and a control valve (23) diverts it to a plurality of coke drums (25,26). The said plurality of coke drums (25,26) provides sufficient residence time for the thermal cracking to take place till the completion of coking reaction. The vapours of coke drum effluent (27,28) produced during thermal cracking, exits the coke drum(s) (25,26). The temperature of coke drum effluent (27,28) determines the average outlet temperature of the coke drum(s) (25,26). The gas oils from the coke drum effluent (27,28) are used as quenching stream which terminates further thermal cracking to avoid coke formation in vapor transfer line (29). Here, thermal cracking of heavier part of pyrolysis oil along with heavy residue will occur as some lighter part will be fractionated out in fractionator (19). It enhances liquid yields and reduces coke yield. In the present disclosure, a plurality of coke drums is used, specifically, two number of coke drums (25,26) are operated in parallel to ensure smooth functioning of coking cycle. When coke drum (25) is full of coke, the coking cycle ends and the coker furnace (21) outlet flow is then transferred from coke drum (25) to a parallel coke drum (26) to initiate its coking cycle, while coke removal process is initiated in the filled drum (25) which comprises of steaming, water cooling, coke cutting, and vapor heating and draining. The wash liquid (24) from the coke drums (25,26) is discharged into the blow down section. The coke drum effluent (27,28) after quenching are again sent to the bottom of the delayed coker fractionator (19) using vapor transfer line (29), where it is separated and recovered. Further, Heavy coker gas oil (HCGO) (30) and Light coker gas oil (LCGO) (31) are drawn off the delayed coker fractionator (19) at desired boiling temperature ranges using side strippers. The wet gas (37) coming out of the top of the delayed coker fractionator (19) goes to three phase separator (35), wherein, gaseous hydrocarbons (36), water as aqueous phase (34) and unstabilized naphtha as liquid phase (32) are separated and reflux stream (33) is sent back to the delayed coker fractionator (19). Further, the off gas (12) coming from pyrolysis process of the separator tower (11) is mixed with coker off gases (36) and fed to a light olefin recovery section (39) to get fuel gas (40) from the top which is fed back to pyrolysis reactor (6) in presence of air (44) for maintaining reaction temperature. Further, the said light olefin recovery section (39) also generates ethylene, propylene (41), LPG (42) and naphtha (43) from the bottom thereof. Further,FIG.3shows the process flow diagram for the embodiment-3 of the invented process. The waste plastics feedstock (1) is fed into the hopper (2) and conveyed into the preheating vessel (3), wherein, HCGO (30) from a delayed coker fractionator (19) serves as a preheating stream which causes melting of waste plastics leaving relatively cooler HCGO stream (38). The molten plastic stream (4) along with Nitrogen (N2) (5) is fed into the pyrolysis reactor (6) leaving char (7) from bottoms while the vapours (8) from top leaves the reactor and is condensed using heat exchanger (9) and condensed stream (10) is routed to the separator tower (11). Wherein, gases (12) leaves from the top of the separator tower (11) and condensed liquid (13) from bottom of the separator tower (11) while pyrolysis oil stream (15) and water (16) both are separated in two phase separator (14). The pyrolysis oil stream (15) is mixed with hot heavy residual feedstock (17) is fed to delayed coker fractionator (19) where it mixes with condensed recycle. The resulting mixture (20) is pumped through a coker furnace (21) using fractionator bottom pump(s), for achieving the desired coking temperature (usually between 470° C. and 500° C.) which also results in the partial vaporization and mild cracking of mixture (20). A two-phase mixture (22) usually vapor-liquid comes out of the coker furnace (21) and control valve (23) diverts it to a coke drum(s) (25,26). The said coking drum(s) (25,26) provides sufficient residence time for the thermal cracking to take place till the completion of coking reaction. The vapours of coke drum effluent (27,28) produced during thermal cracking, exits the coke drum(s) stream (25,26). The temperature of coke drum effluent (27,28) determines the average outlet temperature of the coke drum(s) (25,26). Specifically, the obtained pyrolysis oil stream (15) is then mixed with coke drum effluent (27,28) acting as a quench oil which not only terminates further thermal cracking to avoid coke formation in vapor transfer line (29) but also condenses the heavy part of coke drum vapours. The lighter part of pyrolysis oil will not be cracked as it is fractionated out as naphtha and gases which in turn increases overall the liquid yield. Further, a plurality of coke drums (25,26) are used, specifically, two number of coke drums (25,26) are operated in parallel in order to ensure smooth functioning of coking cycle. When coke drum (25) is full of coke, the coking cycle ends and the coker furnace (21) outlet flow is then transferred from coke drum (25) to a parallel coke drum (26) to initiate its coking cycle, while coke removal process is initiated in the filled drum (25) which comprises of steaming, water cooling, coke cutting, and vapor heating and draining. The wash liquid (24) from the coke drums (25,26) is discharged into the blow down section. The coke drum effluent (27,28) after quenching is again sent to fractionator bottom of the delayed coker fractionator (19) using vapor transfer line (29), where it is separated and recovered. Further, Heavy coker gas oil (HCGO) (30) and Light coker gas oil (LCGO) (31) are drawn off the fractionator at desired boiling temperature ranges using side strippers. The wet gas (37) coming out of fractionator top goes to three phase separator (35), gaseous hydrocarbons (36), water as aqueous phase (34) and unstabilized naphtha as liquid phase (32) are separated with reflux stream (33) going back to the delayed coker fractionator (18). The off gas (12) coming from pyrolysis process separator is mixed with coker off gases (36) and fed to light olefin recovery section (39) to get fuel gas (40) from the top which is fed back to pyrolysis reactor (6) in presence of air (44) for maintaining reaction temperature, ethylene, propylene (41), LPG (42) and naphtha (43) from the bottom. Further,FIG.4shows the process flow diagram for the embodiment-4 of the invented process. The waste plastics feedstock (1) is fed into the hopper (2) and conveyed into the preheating vessel (3) where HCGO (30) from delayed coker fractionator (19) serves as a preheating stream which causes melting of waste plastics leaving relatively cooler HCGO stream (38). The molten plastic stream (4) along with Nitrogen (N2) (5) is fed into the pyrolysis reactor (6) leaving char (7) from bottoms while the vapors (8) from top leaves the reactor and is condensed using heat exchanger (9) and condensed stream (10) is routed to the separator tower (11). Wherein, gases (12) leaves from the top of the separator tower (11) and condensed liquid (13) from bottom of the separator tower (11) while pyrolysis oil stream (15) and water (16) both are separated in two phase separator (14). Specifically, the obtained pyrolysis oil stream (15) is mixed directly with fractionator bottom outlet stream (20) which contains hot heavy residual feedstocks (18) and condensed recycle. The resulting mixture (20) is pumped through a coker furnace (21) using fractionator bottom pump(s), for achieving the desired coking temperature (usually between 470° C. and 500° C.) which also results in the partial vaporization and mild cracking of mixture (20). A two-phase mixture (22) usually vapor-liquid comes out of the coker furnace (21) and control valve (23) diverts it to coke drum(s) (25,26). The coke drum(s) (25,26) provide sufficient residence time for the thermal cracking to take place till the completion of coking reaction. The vapours of coke drum effluent (27,28) produced during thermal cracking, exits the coke drum(s) (25,26). The temperature of coke drum effluent (27,28) determines the average outlet temperature of the drum. Here, thermal cracking of lighter as well as heavier part of pyrolysis oil occurs along with residue which in turn increases overall the liquid yield. A plurality of coke drum(s) (25,26) are used, specifically, two number of coke drum(s) (25,26) are operated in parallel to ensure smooth functioning of coking cycle. When coke drum (25) is full of coke, the coking cycle ends and the furnace outlet flow is then transferred from coke drum (25) to a parallel coke drum (26) to initiate its coking cycle, while coke removal process is initiated in the filled drum (25) which comprises of steaming, water cooling, coke cutting, and vapor heating and draining. The wash liquid (24) from the drums is discharged into the blow down section. The coke drum effluent (27,28) after quenching is again sent to the bottom (19) of the delayed coker fractionator (18) using vapor transfer line (29), where it is separated and recovered. Heavy coker gas oil (HCGO) (30) and Light coker gas oil (LCGO) (31) are drawn off the fractionator at desired boiling temperature ranges using side strippers. The wet gas (37) coming out of the top of delayed coker fractionator (18) goes to three phase separator (35), gaseous hydrocarbons (36), water as aqueous phase (34) and unstabilized naphtha as liquid phase (32) are separated with reflux (33) to the fractionator (19). The off gas (12) coming from pyrolysis process separator is mixed with coker off gases (36) and fed to light olefin recovery section (39) to get Fuel gas (40) from the top which is fed back to pyrolysis reactor (6) in presence of air (44) for maintaining reaction temperature, ethylene, propylene (41), LPG (42) and naphtha (43) from the bottom. EXAMPLE 1 Integration of delayed coking unit (DCU) with waste plastic pyrolysis oil production is explained herein. The pyrolysis oil generated from waste plastics require temperature up to 450° C. which means large amount of energy requirement. The integration of DCU involves initial preheating of waste plastics up to its melting temperature using one of the product streams of DCU fractionator i.e., HCGO which leaves the fractionator at around 360° C. Here, the HCGO coming out-off the fractionator is utilized as a stream which will preheat the waste plastics to its melting temperature as shown inFIG.1-3, thus the amount of energy supplied by the heater will be reduced as compared to earlier know processes. A simulation has been carried out in this regard taking HCGO with inlet temperature as 360° C. and plastic with inlet temperature as 30° C. Accordingly, the heat as required to raise the temperature of waste plastic feedstock becomes half as compared to without preheating. Here, heat required without preheat implies heat required to raise temperature of plastic from 30° C. to reaction temperature i.e. 450° C. Now as depicted in table 1, the heater needs to supply only 2632 KJ/h i.e. 42% of initial heat. This way, HCGO can be utilized as a heat integrating fluid. TABLE 1Indicating heat supplied with preheat and heat required w/o preheatInletOutletHeat suppliedHeat requiredTempMass inTempwith preheatw/o preheat(° C.)(Kg/hr)(° C.)(KJ/h)(KJ/h)Plastic30526035736205HCGO36015249 EXAMPLE 2 Experiments in Micro-Coker Unit was carried out using the waste plastic pyrolysis oil with properties provided in Table-2, by dosing it into two categories of DCU feed samples namely, Low CCR (15 wt. %) & High CCR (24 wt. %). This was carried out to cover both DCUs operating with low and high CCR feedstocks. For each feed case, one base case without pyrolysis oil and two runs with 5 & 10 wt. % dosing was carried out. Experiments were conducted at a reaction temperature of 486° C. & 1 Kg/cm2g pressure. TABLE 2Properties of Waste plastic pyrolysis oilPropertyUnitValueDensity @ 15° C.g/cc0.8732CCRwt %0.18KV@40° C.cSt1.255Sulfurppm525Nitrogen%0.8Asphalteneppm<100ASTM D2887 distillation, wt % vs ° C.5/10/30/50/70/90/95/100107/129/138/147/228/332/384/450 Micro-Coker experimental results for low CCR feed case are provided in Table 3 and are graphically represented inFIGS.4&5. From the table 3, it can be seen that as we increase the pyrolysis oil content in residue feed, the liquid yield increases and coke yield decreases. From these figures, it can be concluded that the liquid yield increases, and coke yield decreases with addition of waste plastic pyrolysis oil in comparison with the base case by 6 wt. % and 2.6 wt. % respectively. TABLE 3Product yields of Micro-Coker runs (low CCR feed)Run #123Pyrolysis Oil added, wt. %0510Gas, wt. %23.523.420.1Light Naphtha, wt. %3.271.442.59Heavy naphtha, wt. %4.386.467.31Light Coker Gas Oil, wt. %24.7323.4825.18Heavy Coker Gas Oil, wt. %12.6814.4115.52Coker Fuel Oil, wt. %1.942.612.40Coke, wt. %29.528.226.9Total100100100 Further, Micro-Coker experimental results for high CCR feed case are provided in Table 4 and are graphically represented inFIGS.6&7. From the table 4, it can be seen that as we increase the pyrolysis oil content in residue feed, the liquid yield increases and coke yield decreases. From these figures, it can be concluded that liquid yield increases, and coke yield decreases with addition of waste plastic pyrolysis oil in comparison with the base case by 3.92 wt. % and 5.2 wt. % respectively. TABLE 4Product yields of Micro-Coker runs (High CCR feed)Run #567Pyrolysis Oil added, wt. %0510Gas, wt. %19.118.420.4Light Naphtha, wt. %3.656.235.23Heavy naphtha, wt. %1.883.464.34Light Coker Gas Oil, wt. %35.4537.2636.95Heavy Coker Gas Oil, wt. %6.824.455.18Coke, wt. %33.130.227.9Total100100100 Further, a pure pyrolysis micro coker run is also carried out and results are provided in Table 5. It can be concluded that liquid yield is close to 88 wt. % while coke yield is very less as the pyrolysis oil has negligible CCR (0.15-0.3 wt. %). TABLE 5Product yields of micro coker run with 100% Pyrolysis OilRun100% Pyrolysis OilGas, wt. %9.3Light Naphtha, wt. %1.31Heavy naphtha, wt. %28.65LCGO, wt. %49.02HCGO, wt. %7.16CFO, wt. %1.86Coke, wt. %2.7 Accordingly, the process and apparatus as disclosed herein generates valuable liquid products out of waste materials without really disturbing the hardware of the existing delayed coking (DC) technology and in fact improves the overall delayed coking process. Further, the process and apparatus as disclosed herein overcomes high energy requirement for producing pyrolysis oil from waste plastics by utilizing HCGO as preheating stream which in turn reduces HCGO temperature to desired level such that it can be utilized in downstream processes such as HCGO hydrotreating. The present disclosure provides an alternative to the conventional quench oils which not only stops cracking reactions but also helps in increasing liquid yields. The present disclosure solves the problem of burning of off gases of pyrolysis section to get consumed in providing required heat for maintaining reaction temperature which otherwise can be recovered in the form of valuable products such as ethylene, propylene etc. Moreover, the process as disclosed herein describes about various processes, wherein, pyrolysis oil from waste plastics can be utilized in delayed coker unit to produce more value distillates with high liquid yields and less coke yields from low value residual hydrocarbons using thermal cracking. Wherein, the thermal cracking is carried out without the requirement of treatment processes such as hydrodesulfurization, dechlorination, hydrogenation etc. and the process is free from hydrogen consumption. Further, the present process does not require any kind the treatment processes for production of high value distillate from thermal cracking of residual hydrocarbon. Moreover, process as disclosed herein does not require catalyst as in case of polymerization which is an added advantage because catalyst effective utilization is a challenge, and it makes process more costly compared to non-catalyst processes. Specifically, the process as disclosed herein involves thermal cracking of pyrolysis oil to produce cracked oil product with liquid yield higher than catalytically cracked oil by 30%. | 32,055 |
11859133 | DETAILED DESCRIPTION OF THE EMBODIMENTS All numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and take into account experimental error and variations that would be expected by a person having ordinary skill in the art. Overview In various aspects, systems and methods are provided for using size-reversing materials in vessels where direct heating is used to at least partially provide heat for reforming reactions under cyclic reforming conditions. An example of a size-reversing material is the combination of NiO and Al2O3. It has been discovered that the phase transition between NiO and NiAl2O4can assist with re-dispersion of Ni on a surface. This can assist with maintaining catalytic activity for reforming over longer time periods in the presence of cyclic reforming conditions. Reforming of hydrocarbons to form synthesis gas is a potentially desirable pathway for reducing or minimizing CO2emissions associated with hydrocarbon fuels. Reforming can convert hydrocarbon fuels into H2, a fuel with no CO2emissions, and CO or CO2. By separating the H2from the carbon oxides in a single location, some of the difficulties associated with preventing CO2emissions can be reduced or minimized. However, due to the elevated temperatures required for hydrocarbon reforming, balancing efficient reforming of hydrocarbons with energy consumption and operating lifetime for equipment remains a challenge. Reverse flow reactors are an example of a type of reactor that can provide heat to a reforming reaction environment by direct heating of the surfaces in the reaction environment. This is achieved using cyclic reforming conditions, where at least a first portion of a cycle involves performing reforming in the reaction environment (an endothermic process), and at least a second portion of the cycle involves direct heating of one or more surfaces within the reaction environment (to provide the heat for the endothermic process). This can reduce or minimize heat loss while attempting to add heat to the reaction zone. Optionally, use of flows in opposing directions can provide further benefits with regard to developing a desirable temperature profile within the reaction zone. Reverse flow reactors will be used herein as an example of this type of system, but it is understood that reverse flow reactors are an example of a reactor that can provide such benefits. Due to the nature of direct heating under cyclic reforming conditions, portions of the reaction environment can be exposed to peak temperatures that are well above the minimum temperatures necessary for performing a reforming reaction. For example, portions of the reaction environment can encounter temperatures of 1000° C. or higher, or 1100° C. or higher, or 1200° C. or higher (such as up to 1600° C. or possibly still higher). During the reforming portion of a cyclic reaction scheme for reforming, H2is formed by the reforming reaction. Due to the reducing nature of an environment containing a substantial amount of H2under reforming conditions, this can cause a substantial portion of the catalytic metals present on exposed surfaces in the reaction environment to be converted from an oxide form to a metallic form. After reforming, the direct heating step can correspond to an oxidizing environment, and a portion of the metals that are in metallic form can be converted back into metal oxides. Unfortunately, the elevated temperatures present in cyclic reforming environment can result in “sintering” of the catalytic metals present on surfaces in the reaction environment. When metals are present in a metallic state at elevated temperatures, the metals can have an increased tendency to coalesce or “sinter” on a surface, resulting in formation of larger metal particles. As a result, this sintering can reduce the available surface area of catalytic metal, as the larger particles of metal have lower ratio of surface area to volume. Over time, this can reduce or minimize the catalytic activity within the reaction environment. It has been discovered that the loss of catalytic activity due to sintering can be reduced or minimized by using a “size-reversing” material in at least a portion of the reaction environment, in combination with modifying at least a portion of the conditions used during the cyclic reforming process. In various aspects, Ni can be used as the catalytic metal (NiO in oxidized form) in at least a portion of the reaction environment, such as a portion of the reaction environment that is exposed to temperatures of 1000° C. or more, or 1100° C. or more, or 1200° C. or more, or 1300° C. or more, such as up to 1600° C. or possibly still higher. The Ni can be provided as part of a catalyst system, such as a system including both NiO and Al2O3. It has been discovered that NiO and Al2O3can undergo a transition to a spinel phase corresponding to NiAl2O4. When NiO and Al2O3are converted to NiAl2O4, it has also been discovered that this phase transition facilitates re-dispersion of the Ni at a surface. It has further been discovered that the size-reversing properties of the NiO+Al2O3/NiAl2O4system can be used to at least partially reverse the effects of sintering, so that the net formation of larger Ni (or NiO) particles is reduced or minimized. In order to achieve cyclic reforming can be performed under modified cyclic reforming conditions. In some aspects, one type of modification can correspond to a modification to increase the amount of metal that is converted from a metallic state to an oxide state during one or more portions of a cyclic reforming process. It has been discovered that the combustion (heating) step used for conventional cyclic reforming conditions can provide a reaction environment that does not contain sufficient O2to allow for substantial conversion of Ni to NiO. By providing additional O2under oxidizing conditions during at least a portion of the cyclic reforming reaction cycle, a higher percentage of metallic Ni can be converted back to NiO. This increase in the amount of available NiO can thus allow for an increased amount of the transition from NiO+Al2O3to the spinel phase composition NiAl2O4. Additionally or alternately, the temperature profile and/or timing during one or more portions of a cyclic reforming process can be modified to provide a sufficient combination of time and temperature to allow for the phase transition to occur from NiO+Al2O3to NiAl2O4. It has been discovered that the phase transition from NiO+Al2O3to NiAl2O4is slow relative to conversion of a nickel oxide (either NiO or NiAl2O4) to metallic Ni. It has further been discovered that elevated temperatures and/or longer times at elevated temperatures can assist with increasing the amount of NiAl2O4that is formed. It is noted that other combinations of oxides are known to undergo a phase transition to form a spinel phase (i.e., the reversible reaction corresponding to <oxide A>+<oxide B>=<spinel oxide AB>). However, such known systems correspond to phase transitions that readily occur at temperatures substantially below 1000° C. It is unexpected that a) the phase transition to form the NiAl2O4phase requires temperatures above 1000° C. to occur at a reasonable rate, and that b) by enabling such a phase transition, sintering of the Ni metal can be at least partially reversed. It is further noted that by increasing the amount of oxygen available during the combustion (heating) step of a reforming reaction cycle, other catalytic metals can also take advantage of formation of a combined phase with alumina (such as a spinel phase) to achieve side-reducing behavior. For example, other metals from Groups 3 to 12 that preferentially form oxides with the stoichiometry “MO” (i.e., metal in a +2 oxidation state) at temperatures of 800° C. or higher, or 1000° C. or higher, such as up to 1600° C. or possibly still higher, can potentially correspond to size-reversing materials. To correspond to size-reversing materials, such metals can also be reduced from an oxide phase corresponding to MO or MAl2O4to metallic M at temperatures of 800° C. or higher, or 1000° C. or higher, such as up to 1600° C. or possibly still higher. As an example, iron (M=Fe) can form FeO as a thermodynamically stable phase at temperatures of 1000° C. or higher, and reduction of FeO to Fe can also occur in the presence of H2at temperature of 1000° C. or higher. Thus, other metals “M” from Groups 3 to 12 of the Periodic Table that both form MO (under oxidation conditions at 800° C. or higher, or 1000° C. or higher) and metallic M (under reducing conditions at 800° C. or higher, or 1000° C. or higher) can also be used while taking advantage of size-reducing behavior to reduce or minimize sintering. Metal Reduction and Oxidation in Cyclic Reforming Environment In a cyclic reforming environment, catalytic metals can be exposed to both reducing conditions and oxidation conditions. In a conventional cyclic reaction cycle for reforming, however, the amount of metal that can be reduced during the reducing portion(s) of the cycle can be substantially greater than the amount of metal that can be oxidized during the oxidizing portion(s) of the cycle. During a reaction cycle for performing reforming, at least a portion of the cycle can correspond to a reforming step. The reforming step can be any convenient type of hydrocarbon reforming, such as reforming based on steam reforming, dry reforming, or a combination thereof. During reforming, a hydrocarbon such as methane is converted into H2, CO, and/or CO2. Due to the generally reducing environment and amount of H2generated, the H2present in the reaction environment during the reforming step typically represents a substantial excess relative to the amount of H2that would be needed for complete conversion of exposed catalytic metal from the oxide state (such as NiO or NiAl2O4) to the metallic state (such as Ni). As a result, the conditions during a reforming step can be suitable for conversion of a substantial amount of the catalytic metal from an oxide state to a metallic state. A reaction cycle for performing reforming that includes direct heating can also include at least one combustion step, where a fuel is combusted to provide heat for the endothermic reforming reaction. Conventionally, the oxygen content for this step can be selected to provide a stoichiometric excess relative to the amount needed for complete combustion of the fuel. Conventionally, addition of a greater amount of gas (such as additional oxygen) has been viewed as undesirable. Cyclic reforming conditions typically involve large flow volumes and high superficial gas velocities. Thus, any additional gas added during a step further increases represents a diluent that can increase the amount of heat that is lost from the process without being transferred to the surfaces of the reaction environment. As a result, under conventional conditions, the excess oxygen present during combustion can correspond to less than 15 mol % of the oxygen that would be needed for complete oxidation of catalyst in a metallic state to catalyst in an oxide state. Based on the above, after only a few cycles of reforming, the catalyst in the reaction zone can primarily be in a reduced (metallic) state, with only a small portion of the catalyst being converted between a metallic state to an oxidized state and then back to metallic during each cycle. In this type of situation, sintering can occur more quickly, as the metal is in a metallic state for a substantial portion of the time during each cycle, including during the combustion portions of the cycle where temperatures are the highest. The above difficulties with a conventional cyclic reforming reaction scheme can be overcome in various manners. One modification can be to increase the amount of available oxygen when the catalytic metal is exposed to oxidizing conditions. For example, instead of selecting the amount of oxygen (O2) during the combustion step based on the stoichiometric need for combusting all of the fuel, the amount of oxygen introduced during the combustion step can be selected based on the combined stoichiometric need for combusting all fuel and converting all catalytic metal in a selected portion of a reactor from a metallic state to an oxide state. As another example, the amount of excess oxygen relative to the stoichiometric need for combustion of all fuel can be increased. In this discussion, 100 mol % of the molar stoichiometric oxygen amount is defined as the stoichiometric amount of oxygen that is needed to combust all fuel introduced during the regeneration step. Amounts of oxygen greater than 100 mol % of the molar stoichiometric oxygen amount correspond to excess oxygen. For example, 120 mol % of the molar stoichiometric oxygen amount corresponds using a number of moles of oxygen that is 20 mol % greater than the number of moles needed for stoichiometric combustion. This can be referred to an excess molar oxygen amount of 20 mol %. In various aspects, the amount of oxygen used during regeneration can correspond to 120 mol % or more of the molar stoichiometric amounts, or 125 mol % or more, or 130 mol % or more, or 140 mol % or more, or 170 mol % or more, or 200 mol % or more, such as up to 400 mol % or possibly still higher. Additionally or alternately, in some aspects the amount of oxygen during combustion can correspond to 100 mol % or more of the combined stoichiometric need for both combustion of fuel and conversion of catalytic metal to metal oxide in a selected region, or 110 mol % or more, or 120 mol % or more, such as up to 200 mol % or possibly still higher. High Temperature Reforming Catalyst System—NiAl2O4and NiO/NiAl2O4 In some aspects, a catalyst system can correspond to a mixture of NiO and Al2O3. Under the cyclic high temperature reforming conditions, the NiO and the Al2O3in the will react to form a mixed phase of NiO, NiAl2O4, and/or Al2O3. Additionally, based on cyclic exposure to oxidizing and reducing conditions, the catalyst can be converted from a substantially fully oxidized state, such as a combination of oxides including NiO, NiAl2O4and Al2O3, to various states including at least some Ni metal supported on a surface. In this discussion, a catalyst system that includes both NiO and Al2O3can be referred to as an NiAl2O4catalyst system. Based on the stoichiometry for combining NiO and Al2O3to form NiAl2O4, a catalyst including a molar ratio of Al to Ni of roughly 2.0 (i.e., a ratio of 2:1) could result in formation of NiAl2O4with no remaining excess of NiO or Al2O3. Thus, one option for forming an NiAl2O4catalyst is to combine NiO and Al2O3to provide a stoichiometric molar ratio of Al to Ni of roughly 2.0. In some other aspects, an excess of NiO can be included in the catalyst relative to the amount of alumina in the support, so that at least some NiO is present in a fully oxidized state. In such aspects, the molar ratio of Al to Ni in the catalyst can be less than 2.0. For example, the molar ratio of Al to Ni in a NiO/NiAl2O4catalyst can be 0.1 to 2.0, or 0.1 to 1.9, or 0.1 to 1.5, or 0.5 to 2.0, or 0.5 to 1.9, or 0.5 to 1.5, or 1.0 to 2.0, or 1.0 to 1.9, or 1.2 to 1.5, or 1.5 to 2.0, or 1.5 to 1.9. In still other aspects, an excess of Al2O3can be included in the catalyst relative to the amount of Ni, so that at least some Al2O3is present in a fully oxidized state. In such aspects, the molar ratio of Al to Ni in the catalyst can be greater than 2.0. For example, the molar ratio of Al to Ni in a NiAl2O4/Al2O3catalyst can be 2.0 to 10, or 2.1 to 10, or 2.0 to 5.0, or 2.1 to 5.0, or 2.0 to 4.0, or 2.1 to 4.0. In various aspects, an NiAl2O4catalyst can be incorporated, for example, into a washcoat that is then applied to a surface or structure within a reactor, such as a monolith. By providing NiO and Al2O3as a catalyst system that is then deposited on a separate monolith (which can then form NiAl2O4under the cyclic conditions), the activity of the catalyst can be maintained for unexpectedly longer times relative to using a monolith that directly incorporates NiO and Al2O3into the monolith structure. When a composition is formed that includes both nickel oxide and alumina, the NiO and Al2O3can react to form a compound corresponding to NiAl2O4. However, when NiO (optionally in the form of NiAl2O4) is exposed to reducing conditions, the divalent Ni can be reduced to form metallic Ni. Thus, under cyclic reforming conditions that include both high temperature oxidizing and reforming environments, at least a portion of NiAl2O4catalyst can undergo cyclic transitions between states corresponding to Ni metal and Al2O3and NiAl2O4. It is believed that this cyclic transition between states can allow an NiAl2O4catalyst to provide unexpectedly improved activity over extended periods of time. Without being bound by any particular theory, it is believed that at least part of this improved activity for extended time periods is due to the ability of Ni to “re-disperse” during the successive oxidation cycles. It is believed this re-dispersion occurs in part due to the formation of NiAl2O4from NiO and Al2O3. Catalyst sintering is a phenomenon known for many types of catalysts where exposure to reducing conditions at elevated temperature can cause catalyst to agglomerate on a surface. Thus, even if the surface area of the underlying surface remains high, the agglomeration of the catalyst may reduce the amount of available catalyst active sites, as the catalyst sinters and forms lower surface area deposits on the underlying surface. By contrast, it is believed that the cyclic transition between states can allow the Ni in an NiAl2O4catalyst system to retain good dispersion, so that catalyst activity can be maintained. It is believed that further advantage can be obtained by using a sufficient amount of excess oxygen during the regeneration step so that all available Ni is oxidized back to NiO and/or NiAl2O4. It is noted that by supplying both NiO as a catalyst and Al2O3as a metal oxide support layer as part of the catalyst system, the alumina for forming NiAl2O4is already provided as part of the catalyst system. It is believed that this reduces or minimizes interaction of Ni with any alumina that may be present in the monolith composition, and therefore reduces or minimizes degradation of the underlying monolith when exposed to successive cycles of high temperature oxidation and reduction. Although NiAl2O4could potentially be used as a structural material for forming a monolith, it has been unexpectedly found that using NiAl2O4as a washcoat for a separate structure (such as a monolith) can allow catalytic activity to be maintained for substantially longer time periods. It is noted that U.S. Patent Application Publication 2020/0030778 describes using a monolith composed of a combination of NiO and Al2O3as a structure to provide reforming catalytic activity under cyclic high temperature reforming conditions. However, it is believed that the cyclic transition of states for a monolith composed at least partially of NiAl2O4can contribute to structural breakdown of the monolith. Because a monolith structure typically includes a large number of cells or passages per unit area, the structural breakdown of the monolith can result in filling or even collapse of the cells, so that the available surface area that the reactant gas flows are exposed to in the reforming environment is greatly reduced. In some aspects, NiAl2O4can be used as a catalyst system when a single catalyst zone is used in a reforming reactor. In some aspects where multiple catalyst zones are present, NiAl2O4can be used as a catalyst system in the highest temperature zone, in an intermediate temperature zone, or a combination thereof. Reforming Catalyst and Metal Oxide Support Layer—General In various aspects, one option for adding a reforming catalyst to a monolith can be to coat the monolith with a mixture of a catalyst (optionally in oxide form) and metal oxide support layer. For example, powders of the catalyst oxide and the metal oxide support layer can be used to form a washcoat that is then applied to the monolith (or other structure). This can result in a catalyst system where the catalyst is mixed within/distributed throughout the metal oxide support layer, as opposed to the catalyst being deposited on top of the metal oxide support layer. In other words, at least a portion of the catalyst system can correspond to a mixture of the catalyst and the support layer. In other aspects, any convenient method for depositing or otherwise coating the catalyst system on the monolith or other structure can be used. The weight of the catalyst system on the monolith (or other structure) can correspond to 0.1 wt % to 10 wt % of the total weight of the catalyst system plus monolith, or 0.5 wt % to 10 wt %, or 2.0 wt % to 10 wt %, or 0.1 wt % to 6.0 wt %, or 0.5 wt % to 6.0 wt %, or 2.0 wt % to 6.0 wt %. In some aspects, the catalyst system can include a thermally stable metal oxide support layer. A thermally stable metal oxide support layer corresponds to a metal oxide that is thermally phase stable with regard to structural phase changes at temperatures between 800° C. to 1600° C. In some aspects, such a thermally stable metal oxide support layer can be formed by coating a surface (such using a washcoat) with a metal oxide powder that has a surface area of 20 m2/g or less. For example, the metal oxide powder used for forming a thermally stable metal oxide coating can have a surface area of 0.5 m2/g to 20 m2/g, or 1.0 m2/g to 20 m2/g, or 5.0 m2/g to 20 m2/g. High temperature reforming refers to reforming that takes place at a reforming temperature of 1000° C. or more, or 1100° C. or more, or 1200° C. or more, such as up to 1500° C. or possibly still higher. In various aspects, a catalyst can be annealed at a temperature of 1000° C. or more, or 1100° C. or more, or 1200° C. or more, or 1300° C. or more, such as up to 1600° C. or possibly still higher. This temperature can be substantially similar to or greater than the peak temperature the catalyst is exposed to during a reforming process cycle. An annealing temperature that is substantially similar to a peak temperature can correspond to an annealing temperature that differs from the peak temperature by 0° C. to 50° C. As an example of a thermally stable metal oxide support layer, alumina has a variety of phases, including α-Al2O3, γ-Al2O3, and θ-Al2O3. A metal powder of α-Al2O3can typically have a surface area of 20 m2/g or less. By contrast, the γ-Al2O3and θ-Al2O3phases have higher surface areas, and a metal powder for use in a washcoat solution of γ-Al2O3and/or θ-Al2O3will have a surface area of greater than 20 m2/g. It is conventionally believed that phases such as θ-alumina or γ-alumina are superior as a supporting structure for a deposited catalyst, as the greater surface per gram of θ-alumina or γ-alumina will allow for availability of more catalyst active sites than α-alumina. However, phases such as γ-Al2O3and θ-Al2O3are not thermally phase stable at temperatures of 800° C. to 1600° C. At such high temperatures, phases such as γ-Al2O3and θ-Al2O3will undergo phase transitions to higher stability phases. For example, at elevated temperatures, γ-Al2O3will first convert to Δ-Al2O3at roughly 750° C.; then Δ-Al2O3will convert to θ-Al2O3at roughly 950° C.; then θ-Al2O3will then convert to α-Al2O3with further exposure to elevated temperatures between 1000° C. and 1100° C. Thus, α-Al2O3is the thermally phase stable version of Al2O3at temperatures of 800° C. to 1600° C. Without being bound by any particular theory, it is believed that such phase changes during exposure to elevated temperature can contribute to degradation of the catalyst and/or the structure supporting the catalyst. By contrast, by using a support that is phase stable at an elevated annealing temperature and then annealing the catalyst (including support) at the elevated annealing temperature, the resulting catalyst can substantially maintain an initial catalytic activity level for an extended period of time. It is noted that the initial catalytic activity achieved by depositing catalyst on a monolith formed from a phase stable, low surface area per gram material may be lower than depositing catalyst on a similar monolith formed from a material having a higher surface area per gram. However, it has been discovered this initial activity advantage for the higher surface area material is quickly lost during exposure to cyclic high temperature reforming conditions. As a further illustration, without being bound by any particular theory, γ-Al2O3is a transitional alumina that may be viewed as a defect oxyhydroxide, with a spinel related crystalline structure. In prior academic work, γ-Al2O3has been formulated an alumina spinel, with defect sites having a formula of Al8/3□1/3O4, where □ symbolizes open cation sites. Including the hydroxyls, it may be viewed as Al2.5□0.5O3.5(OH)0.5. Such γ-Al2O3is thermally unstable with respect to α-Al2O3. Although θ-Al2O3is more crystalline and has less surface area and hydroxide content compared to γ-Al2O3, θ-Al2O3also includes defect sites (i.e., open cation sites, and is also thermally unstable relative to α-Al2O3. Thus, γ- and θ-Al2O3both have defect sites (□) capable of reacting with multivalent cations (or metal oxides). Both Rh(O) and Ni(O) may react to produce denser phases, where Rh and Ni may not be as chemically accessible for catalytic reaction as compared to their oxide or metallic states. Even in the absence of reactions with Rh or Ni, γ- and θ-Al2O3are thermodynamically unstable with respect to α-Al2O3under high temperature conditions. It has been discovered that using a thermally stable metal oxide in a catalyst system, in combination with annealing of the catalyst system on the monolith at high temperature, can provide unexpected activity benefits and structural stability benefits over extended periods of time. Without being bound by any particular theory, when a catalyst system including a non-thermally stable metal oxide is used in a coating for a monolith, exposing such a catalyst system to a cyclic high temperature reforming environment can result in structural degradation of the catalyst system. It is believed that this structural degradation of a catalyst system can contribute to a reduction in available catalyst sites, possibly due to the catalyst becoming buried within a degraded structure and/or additional sintering or agglomeration of the catalyst as the non-thermally stable metal oxide in the catalyst system converts to a lower surface area phase. This structural degradation can be observed, for example, by examining the catalyst system on a monolith after exposure to a cyclic high temperature reaction environment. For a conventional catalyst system, after exposure to a cyclic high temperature reaction environment, the catalyst system can be readily scraped off of the underlying structure. Additionally, a substantial reduction in activity can be observed. By contrast, when a catalyst system is used that includes a thermally stable metal oxide, the activity of the catalyst in the catalyst system can be unexpectedly maintained for extended run lengths with little or no loss of activity. Additionally, after exposure to a cyclic high temperature reforming environment, the catalyst system can unexpectedly remain strongly adhered or coated on the underlying monolith or other structure. It is noted that the initial catalyst activity may be lower than for a conventional system, since thermally stable metal oxides typically have a relatively low surface area. This is believed to initially reduce the number of available catalytic sites. However, because the thermally stable metal oxide does not undergo phase transitions when exposed to heat, the catalytic activity of a catalyst system including a thermally stable metal oxide can be maintained. Due to the rapid deactivation for a conventional catalyst or catalyst system, the activity of a conventional catalyst system can rapidly fall below the activity of a catalyst system using a thermally stable metal oxide. Additionally, further improvements can be achieved by annealing the catalyst system and the underlying monolith (or other supporting structure) at temperatures that are substantially the same as or greater than the peak temperatures the supporting structure is exposed to during the reforming process. One of the distinctions between using a catalyst system including a thermally stable metal oxide and a catalyst system that does not use a thermally stable oxide is that the catalyst system including the thermal stable metal oxide can have improved adhesion to the underlying support structure after exposure to the cyclic high temperature reforming environment. Adhesion of the washcoat after operation can be quantified by the amount of force needed to de-adhere the washcoat. In prior operation, washcoats comprised of theta and gamma alumina were de-adhered with minimal force, such as an amount of force similar to a paint brush stroke (weak). In operation with the phase stable supports, the force needed to de-adhere the washcoat was high, similar to the scraping of dried epoxy off of a glass surface (strong). Due to these differences, only small amounts of washcoat could be de-adhered from the phase stable materials, whereas large amounts of washcoat could be de-adhered from the gamma and theta supports. Other methods for evaluating adhesion of the washcoat include, but are not limited to, (i) a thermal cycling method, (ii) a mechanical attrition method, and (iii) an air-knife method. As a non-limiting example, the thermal cycling method can be performed by heating the washcoated materials to high temperatures in the range of 800 to 1300° C., cooling the heated substrates to ambient temperature, and repeating such a cycle at least five times. As another non-limiting example, the mechanical attrition method can be performed by placing the washcoated materials inside a plastic container and shaking the container on a vibration table for at least 30 minutes. Adhesion of the washcoated materials can be determined based on exposing a washcoated structure to thermal cycling conditions and then measuring the de-adhered material before and after mechanical attrition testing by mass change. Prior to thermal cycling, the weight of the washcoat on the support structure can be determined. The washcoated structure can then be exposed to thermal cycling conditions. The thermal cycling conditions can correspond to the thermal cycling method above, or the washcoated structure can be exposed to cyclic high temperature reforming conditions for at least five reaction cycles. A catalyst system including a thermally phase stable support can provide good adhesion after mechanical attrition testing, corresponding to retaining 80 wt % or more of the initial washcoat, or retaining 90 wt % or more of the initial washcoat, or 95 wt % or more of the initial washcoat. By contrast, a catalyst system not including a thermally phase stable support that is exposed to cyclic high temperature conditions and then exposed to mechanical attrition testing can retain 75 wt % or less of the initial washcoat. A catalyst system can be applied to a monolith or other structure, for example, by applying the catalyst system as a washcoat suspension. To form a washcoat suspension, the catalyst system can be added to water to form an aqueous suspension having 10 wt % to 50 wt % solids. For example, the aqueous suspension can include 10 wt % to 50 wt % solids, or 15 wt % to 40 wt %, or 10 wt % to 30 wt %. Optionally, an acid or a base can be added to the aqueous suspension to reduce or raise, respectively, the pH so as to change the particle size distribution of the alumina catalyst and/or binder particles. For example, acetic acid or another organic acid can be added to achieve a pH of 3 to 4. The suspension can then be ball milled (or processed in another manner) to achieve a desired particle size for the catalyst particles, such as a particle size of 0.5 μm to 5 μm. After milling, the suspension can be stirred until time for use so that the particles are distributed substantially uniformly in the solution. The washcoat suspension can then be applied to a monolith structure to achieve a desired amount of catalyst (such as nickel or rhodium) on the monolith surface. As an example, in one aspect a washcoat thickness of 10 microns was achieved by forming a washcoat corresponding to 10 wt % of the monolith structure. Any convenient type of monolith structure can be used to provide a substantial surface area for support of the catalyst particles. The washcoat can be applied to the monolith to form cells having inner surfaces coated with the catalyst. One option for applying the washcoat can be to dip or otherwise submerge the monolith in the washcoat suspension. After clearing the cell channels of excess washcoat, the catalyst system coated on the monolith can be optionally dried. Drying can correspond to heating at 100° C. to 200° C. for 0.5 hours to 24 hours. After any optional drying, calcination can be performed. In some aspects, calcining can correspond to heating at 200° C. to 800° C. for 0.5 hours to 24 hours. In other aspects, a high temperature calcination step can be used, so that the calcining temperature for the catalyst system coated on the monolith is substantially similar to or greater than the peak temperature the monolith will be exposed to during the cyclic high temperature reforming reaction. For a monolith in a high temperature zone, this can correspond to calcining the catalyst system coated on the monolith at a temperature of 800° C. or more, or 1000° C. or more, or 1200° C. or more, or 1300° C. or more, such as up to 1600° C. or possibly still higher. It is noted that if multiple catalyst zones are present, the calcination for monoliths in different catalyst zones can be different. It has been unexpectedly discovered that performing calcination at a temperature similar to or greater than the peak temperature during the cyclic high temperature reforming process can unexpectedly allow for improved activity for the catalyst system and/or adhesion of the catalyst system to the underlying monolith. Without being bound by any particular theory, it is believed that exposing the monolith and deposited catalyst system to elevated temperatures prior to exposure of the catalyst to a cyclic reaction environment can facilitate forming a stable interface between the catalyst system and the monolith. This stable interface can then have improved resistance to the high temperature oxidizing and/or reducing environment during the reforming process, resulting in improved stability for maintaining the catalyst system on the surface of the monolith. In various aspects, suitable catalytic metals can include, but are not limited to, Ni, Co, Fe, Pd, Rh, Ru, Pt, Ir, Cu, Ag, Au, Zr, Cr, Ti, V, Mo, Nb, and combinations thereof. The catalytic metal can be selected based on the desired type of catalytic activity. Such catalytic metals may be used in a catalyst in the form of a metal oxide. In some aspects, for reforming of hydrocarbons in the presence of H2O and/or CO2to make hydrogen, Ni, Rh, Ru, Pd, Pt, Ir, Cu, Co, or a combination of thereof can be suitable catalytic metals. The weight of catalytic metal oxide in the catalyst system can range from 0.1 wt % to 70 wt %, or 1.0 wt % to 60 wt %, or 2.0 wt % to 50 wt %, relative to the total weight of the catalyst system. In some aspects where the catalytic metal corresponds to a precious metal or noble metal, the weight of catalytic metal oxide in the catalyst system can range from 0.1 wt % to 10 wt %, or 0.2 wt % to 7.0 wt %, or 0.5 wt % to 4 wt %. In some aspects, Ni or other metals capable of forming a metal oxide where the metal is in the +2 oxidation state at temperatures of 800° C. or higher (or 1000° C. or higher), and where the metal oxide is able to be reduced by hydrogen to form the metallic state at temperatures of 800° C. or higher (or 1000° C. or higher), can be used as a catalytic metal. The catalytic metals can be selected to provide long term stable performance at specific temperature zones of the catalytic bed. This can allow for steady methane conversion, phase stability with the metal oxide support, and reduced or minimized sintering of catalytic metals. As an example involving three catalyst zones, the catalyst system in a highest temperature catalytic zone (e.g. 800˜1250° C.), which is exposed to some of highest temperatures and most severe temperature swings, can be composed of Ni as a catalytic metal (NiO as a catalytic metal oxide) and Al2O3as a metal oxide support. It is noted that this catalyst system can at least partially convert to NiAl2O4during portions of the cyclic reforming process. This catalyst system can be formed, for example, by using a mixture of NiO and Al2O3, as a washcoat on α-Al2O3monoliths. In such an example, a catalyst system in a medium temperature catalytic zone (e.g. 600˜1150° C.) can be composed of Ni and Rh as catalytic metals (NiO and Rh2O3as catalytic metal oxide), and Al2O3as a metal oxide support. To form this catalyst system, a mixture of NiO and Rh2O3, as the catalytic material and Al2O3(optionally but preferably α-Al2O3) as a metal oxide support material can be washcoated on a monolith comprising of 95 wt % α-Al2O3, 4 wt % SiO2and 1 wt % TiO2. In such an example, a catalyst system in a low temperature catalytic zone (e.g. 400˜1050° C.) can be composed of Rh as catalytic metal (Rh2O3as catalytic metal oxide) and α-Al2O3as a metal oxide support. To form this catalyst system, a mixture of Rh2O3and α-Al2O3as the catalytic material can be washcoated on a monolith comprising 93 wt % α-Al2O3, 5 wt % SiO2and 2 wt % MgO. In various aspects, suitable metals for the metal oxide support layer in the catalyst system can include, but are not limited to, Al, Si, Mg, Ca, Sr, Ba, K, Na, Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Fe, Ni, Co, Y, La, Ce, and combinations thereof. The metal (or metals) for the metal oxide support can be selected so that the metal oxide support substantially does not convert to metallic form under the reducing conditions present in the cyclic reaction environment. As an example, when the catalytic metal oxide is NiO, one option for a metal oxide support is Al2O3, preferably α-Al2O3. Another example of a suitable metal oxide support, optionally, in combination with NiO as the catalytic metal oxide, is a mixture of Al2O3with SiO2, MgO and/or TiO2. In such an example, SiO2can combine with Al2O3to form a mullite phase that could increase resistance to thermal shock and/or mechanical failure. Additionally or alternately, in such an example, MgO and/or TiO2can be added. The weight of metal oxide support in the catalyst bed can range from 1.0 wt % to 40 wt %, or 2.0 wt % to 30 wt %, or 3.0 wt % to 20 wt %, relative to the total weight of the monolith in the catalyst bed. FIG.3shows an example of a portion of a monolith300that includes a catalyst system310deposited (or otherwise coated) on the surfaces of the monolith300. In the example shown inFIG.3, the portion of the monolith corresponds to a regular pattern of square cells that allow reactant gases (such as a reforming feed gas flow) to pass through the cells. In other aspects, any convenient type of cell shape can be used, such as round or hexagonal cells. The catalyst system310corresponds to a layer that includes catalyst312and a metal oxide support314that is coated on the surfaces of the cells of the monolith. Structure (Monolith) for Supporting Catalyst System One of the purposes of using a monolith or another supporting structure within a reforming environment is to increase the available surface area for holding a deposited catalyst/catalyst system. To achieve this, some monoliths correspond to a structure with a large plurality of cells or passages that allow gas flow through the monolith. Because each individual cell provides surface area for deposition of catalyst, including a large number of cells or passages per unit area can substantially increase the available surface area for catalyst. Generally, the monolith or other structure used to support the catalyst/catalyst system can be formed from a material is denoted by the formula (PQ). P can be at least one metal selected from the group consisting of Al, Si, Mg, Ca, Sr, Ba, K, Na, Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Fe, Ni, Co, Y, La, Ce, and mixtures thereof. Q is oxide. Thus, the monolith material (PQ) is a metal oxide. In some preferred aspects, the metal oxide can correspond to aluminum oxide (a.k.a. alumina), Al2O3. The preferred Al2O3in this invention is α-Al2O3. While α-Al2O3is the preferred crystalline phase, another phase containing sodium oxide (Na2O), which is sometimes an unavoidable impurity in α-Al2O3, could be also present, namely Na2O(Al2O3)11or NaAl5O8. Optionally, the monolith or structure material (PQ) can be α-Al2O3containing at least one additive oxide selected from the group consisting of SiO2, MgO, CaO, TiO2, Na2O, K2O, and mixtures thereof. The weight of additive oxide in the monolith materials composition can range from 0.1 wt % to 15 wt %, or 1.0 wt % to 10 wt %, or 2.0 wt % to 8.0 wt %, relative to the total weight of the monolith materials composition. As non-limiting illustrative examples, the monolith material (PQ) can be: i) 95 wt % α-Al2O3and 5 wt % SiO2, ii) 93 wt % α-Al2O3, 5 wt % SiO2and 2 wt % MgO, iii) 93 wt % α-Al2O3, 4 wt % SiO2, 2 wt % MgO and 1 wt % Na2O, and iv) 95 wt % α-Al2O3, 4 wt % SiO2and 1 wt. % TiO2. In other aspects, the monolith material (PQ) can be partially composed or substantially composed of non-alumina based oxides. As non-limiting illustrative examples, the monolith material (PQ) can be silica (SiO2), magnesia (MgO), ceria (CeO2), titania (TiO2), zirconia (ZrO2), cordierite (2MgO 2Al2O32SiO2), mullite (3Al2O32SiO2), aluminum titanate (Al2TiO5), magnesium aluminate (MgAl2O4), calcium-stabilized zirconia (CaO—ZrO2), magnesium-stabilized zirconia (MgO—ZrO2), yttria-stabilized zirconia (Y2O3—ZrO2), yttria (Y2O3), barium zirconate (BaZrO3), strontium zirconate (SrZrO3), and mixtures thereof. Still other examples of potential monolith materials include SiC, Si3N4, yttrium-stabilized zirconia, and Al2TiO5ceramics. It is noted that SiC and Si3N4do not follow the (PQ) structural formula. In some aspects, the monolith material (PQ) can further include supplementary components. Such supplementary components can facilitate easy extrusion and correspond to additional structural components within the monolith material composition. For example, the monolith material composition may further comprise one or more silicates comprising a metal selected from the group consisting of Al, Si, Ca, Mg, K, Na, Y, Zr, Hf, Ti, Cr, Mn, Fe, Ni, Co, and mixtures thereof. One example is bentonite, which is an aluminum phyllosilicate clay composed mostly of montmorillonite. The different types of bentonite are each named after the respective dominant element, such as potassium (K), sodium (Na), calcium (Ca), and aluminum (Al). For example, the chemical formula of sodium bentonite is Al2H2Na2O13Si4. Some hydroxyl ions (OH—) can be present in silicates, but under high temperature calcination and sintering conditions, such hydroxyl groups can be converted to oxide form. Yet another example is talc, a clay mineral composed of hydrated magnesium silicate with the chemical formula Mg3Si4O10(OH)2. Due to its nature of basal cleavage and uneven flat fracture, it is foliated with a two-dimensional plate form which is beneficial in extrusion of the monolith material. In various aspects, a monolith or other structure for providing a surface for the reforming catalyst system may be prepared by manufacturing techniques such as but not limited to conventional ceramic powder manufacturing and processing techniques, e.g., mixing, milling, degassing, kneading, pressing, extruding, casting, drying, calcining, and sintering. The starting materials can correspond to a suitable ceramic powder and an organic binder powder in a suitable volume ratio. Certain process steps may be controlled or adjusted to obtain the desired grain size and porosity range and performance properties, such as by inclusion of various manufacturing, property adjusting, and processing additives and agents as are generally known in the art. For example, the two or more types of oxide powders may be mixed in the presence of an organic binder and one or more appropriate solvents for a time sufficient to substantially disperse the powders in each other. As another example, precursors of the oxides present in a monolith may be dissolved in water at a desired ratio, spray dried, and calcined to make a mixed powder. Such precursors include (but are not limited to) chlorides, sulfates, nitrates, and mixtures thereof. The calcined powder can be further mixed in the presence of an organic binder and appropriate solvent(s) to make a mixed “dough”. Then, the mixed “dough” of materials can be placed in a die or form, extruded, dried or otherwise formed into a desired shape. The resulting “green body” can then be sintered at temperatures in the range of about 1200° C.˜1700° C. for at least ten minutes, such as from 10 minutes to 10 hours, or possibly from 10 minutes up to 48 hours or still longer. The sintering operation may be performed in an oxidizing atmosphere, reducing atmosphere, or inert atmosphere, and at ambient pressure or under vacuum. For example, the oxidizing atmosphere could be air or oxygen, the inert atmosphere could be argon, and a reducing atmosphere could be hydrogen, CO/CO2or H2/H2O mixtures. Thereafter, the sintered body is allowed to cool, typically to ambient conditions. The cooling rate may also be controlled to provide a desired set of grain and pore structures and performance properties in the particular component. In some aspects, the monolith material (PQ) can further include an intermediate bond layer. The intermediate bond layer can be applied on monolith surfaces prior to washcoat active materials comprising metal oxide support and catalytic metal. The intermediate bond layer provides a better adherence to the washcoated active material. The intermediate bond layer is a metal oxide, (M)xOy, wherein (M) is at least one metal selected from the group consisting of Al, Si, Mg, Ca, Sr, Ba, K, Na, Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Fe, Ni, Co, Y, La, Ce, and mixtures thereof. Aluminum oxide (a.k.a. alumina), Al2O3, is a preferred metal oxide for the bond layer. As an example of how to form an intermediate bond layer, the selected metal oxide, (M)xOy, can be dispersed in a solution to form a slurry. The slurry can then be washcoated on the monolith. The monolith washcoated with the selected metal oxide, (M)xOy, is dried and sintered at temperatures in the range of 1100° C.˜1600° C. to make the intermediate bonding layer. It has been discovered that limiting the maximum porosity in the final sintered body tends to effectively, if not actually, limit interconnectivity of the pore spaces with other pore spaces to an extent that increases or maximizes volumetric heat capacity of the sintered body. The porosity ranges for a monolith or other structure can depend upon the desired final component performance properties, but are within a range defined by one or more of the minimum porosity values and one or more of the maximum porosity values, or any set of values not expressly enumerated between the minimums and maximums. Examples of suitable porosity values are 0 vol % to 20 vol % porosity, or 0 vol % to 15 vol %, or 0 vol % to 10 vol %, or 0 vol % to 5 vol %. The sintered monolith and/or other formed ceramic structure can have any convenient shape suitable for use as a surface for receiving a catalyst or catalyst system. An example of a monolith can be an extruded honeycomb monolith. Honeycomb monoliths can be extruded structures that comprise many (e.g., a plurality, meaning more than one) small gas flow passages or conduits, arranged in parallel fashion with thin walls in between. A small reactor may include a single monolith, while a larger reactor can include a number of monoliths, while a still larger reactor may be substantially filled with an arrangement of many honeycomb monoliths. Each monolith may be formed by extruding monolith blocks with shaped (e.g., square, trigonal, or hexagonal) cross-section and two- or three-dimensionally stacking such blocks above, behind, and beside each other. Monoliths can be attractive as reactor internal structures because they provide high heat transfer capacity with minimum pressure drop. In some aspects, honeycomb monoliths can be characterized as having open frontal area (or geometric void volume) between 25% and 55%, and having conduit density between 50 and 2000 pores or cells per square inch (CPSI), or between 100 and 900 cells per square inch, or between 100 cells per square inch to 600 cells per square inch. For example, in one embodiment, the conduits may have a diameter/characteristic cell side length of only a few millimeters, such as on the order of roughly one millimeter. Reactor media components, such as the monoliths or alternative bed media, can provide for channels that include a packing with an average wetted surface area per unit volume that ranges from 50 ft−1to 3000 ft−1(˜0.16 km−1to ˜10 km−1), or from 100 ft−1to 2500 ft−1(˜0.32 km−1to ˜8.2 km−1), or from 200 ft−1to 2000 ft−1(˜0.65 km−1to ˜6.5 km−1), based upon the volume of the first reactor that is used to convey a reactant. These relatively high surface area per unit volume values can aid in achieving a relatively quick change in the temperature through the reactor, such as generally illustrated by the relatively steep slopes in the exemplary temperature gradient profile graphs shown inFIG.1(a)or1(b) ofFIG.1. Reactor media components can also provide for channels that include a packing that includes a high volumetric heat transfer coefficient (e.g., 0.02 cal/cm3s° C. or more, or 0.05 cal/cm3s° C. or more, or 0.10 cal/cal/cm3s° C. or more); that have low resistance to flow (low pressure drop); that have an operating temperature range consistent with the highest temperatures encountered during regeneration; that have high resistance to thermal shock; and/or that have high bulk heat capacity (e.g., 0.10 cal/cm3s° C. or more, or 0.20 cal/cm3s° C. or more). As with the high surface area values, these relatively high volumetric heat transfer coefficient values and/or other properties can aid in achieving a relatively quick change in the temperature through the reactor, such as generally illustrated by the relatively steep slopes in the exemplary temperature gradient profile graphs, such as inFIGS.1(a) and1(b)ofFIG.1. The cited values are averages based upon the volume of reactor used for conveyance of a reactant. In various aspects, adequate heat transfer rate can be characterized by a heat transfer parameter, ΔTHT, below 500° C., or below 100° C., or below 50° C. The parameter ΔTHT, as used herein, is the ratio of the bed-average volumetric heat transfer rate that is needed for recuperation, to the volumetric heat transfer coefficient of the bed, hv. The volumetric heat transfer rate (e.g. cal/cm3sec) that is sufficient for recuperation can be calculated as the product of the gas flow rate (e.g. g/sec) with the gas heat capacity (e.g. cal/g° C.) and desired end-to-end temperature change (excluding any reaction, e.g. ° C.), and then this quantity can be divided by the volume (e.g. cm3) of the reactor (or portion of a reactor) traversed by the gas. The volumetric heat transfer coefficient of the bed, hv, can typically be calculated as the product of an area-based coefficient (e.g. cal/cm2s° C.) and a specific surface area for heat transfer (av, e.g. cm2/cm3), often referred to as the wetted area of the packing. Process Example—Reverse Flow Reforming and Regeneration In various aspects, reforming of hydrocarbons can be performed under steam reforming conditions in the presence of H2O, under dry reforming conditions in the presence of CO2, or under conditions where both H2O and CO2are present in the reaction environment. As a general overview of operation during reforming in a swing reactor, such as a reverse flow reactor, a regeneration step or portion of a reaction cycle can be used to provide heat for the reactor. Reforming can then occur within the reactor during a reforming step or portion of the cycle, with the reforming reaction consuming heat provided during the reactor regeneration step. During reactor regeneration, fuel and an oxidant are introduced into the reactor from a regeneration end of the reactor. The bed and/or monoliths in the regeneration portion of the reactor can absorb heat, but typically do not include a catalyst for reforming. As the fuel and oxidant pass through the regeneration section, heat is transferred from the regeneration section to the fuel and oxidant. Combustion does not occur immediately, but instead the location of combustion is controlled to occur in a middle portion of the reactor. The flow of the reactants continues during the regeneration step, leading to additional transfer of the heat generated from combustion into the reforming end of the reactor. After a sufficient period of time, the combustion reaction is stopped. Any remaining combustion products and/or reactants can optionally be purged. The reforming step or portion of the reaction cycle can then start. The reactants for reforming can be introduced into the reforming end of the reactor, and thus flow in effectively the opposite direction relative to the flow during regeneration. The bed and/or monoliths in the reforming portion of the reactor can include a catalyst for reforming. In various aspects, at least a portion of the catalyst can correspond to a catalyst formed from a ceramic composition as described herein. As reforming occurs, the heat introduced into the reforming zone during combustion can be consumed by the endothermic reforming reaction. After exiting the reforming zone, the reforming products (and unreacted reactants) are no longer exposed to a reforming catalyst. As the reforming products pass through the regeneration zone, heat can be transferred from the products to the regeneration zone. After a sufficient period of time, the reforming process can be stopped, remaining reforming products can optionally be collected or purged from the reactor, and the cycle can start again with a regeneration step. The reforming reaction performed within the reactor can correspond reforming of methane and/or other hydrocarbons using steam reforming, in the presence of H2O; using dry reforming, in the presence of CO2, or using “bi” reforming in the presence of both H2O and CO2. Examples of stoichiometry for steam, dry, and “bi” reforming of methane are shown in equations (1)-(3). Dry Reforming: CH4+CO2=2CO+2H2(1) Steam Reforming: CH4+H2O=CO+3H2(2) Bi Reforming: 3CH4+2H2O+CO2=4CO+8H2. (3) As shown in equations (1)-(3), dry reforming can produce lower ratios of H2to CO than steam reforming. Reforming reactions performed with only steam can generally produce a ratio of H2to CO of around 3, such as 2.5 to 3.5. By contrast, reforming reactions performed in the presence of CO2can generate much lower ratios, possibly approaching a ratio of H2to CO of roughly 1.0 or even lower. By using a combination of CO2and H2O during reforming, the reforming reaction can potentially be controlled to generate a wide variety of H2to CO ratios in a resulting syngas. It is noted that the ratio of H2to CO in a synthesis gas can also be dependent on the water gas shift equilibrium. Although the above stoichiometry shows ratios of roughly 1 or roughly 3 for dry reforming and steam reforming, respectively, the equilibrium amounts of H2and CO in a synthesis gas can be different from the reaction stoichiometry. The equilibrium amounts can be determined based on the water gas shift equilibrium, which relates the concentrations of H2, CO, CO2and H2O based on the reaction: H2O+CO<=>H2+CO2. (4) Most reforming catalysts, such as rhodium and/or nickel, can also serve as water gas shift catalysts. Thus, if reaction environment for producing H2and CO also includes H2O and/or CO2, the initial stoichiometry from the reforming reaction may be altered based on the water gas shift equilibrium. This equilibrium is also temperature dependent, with higher temperatures favoring production of CO and H2O. It is noted that higher temperatures can also improve the rate for reaching equilibrium. As a result, the ability to perform a reforming reaction at elevated temperatures can potentially provide several benefits. For example, instead of performing steam reforming in an environment with excess H2O, CO2can be added to the reaction environment. This can allow for both a reduction in the ratio of H2to CO produced based on the dry reforming stoichiometry as well as a reduction in the ratio of H2to CO produced based on the water gas shift equilibrium. Alternatively, if a higher H2to CO ratio is desired, CO2can be removed from the environment, and the ratio of H2O to CH4(or other hydrocarbons) can be controlled to produce a desirable type of synthesis gas. This can potentially allow for generation of a synthesis gas having a H2to CO ratio of 0.1 to 15, or 0.1 to 3.0, or 0.5 to 5.0, or 1.0 to 10, by selecting appropriate amounts of feed components. The reforming reactions shown in equations (1)-(3) are endothermic reactions. One of the challenges in commercial scale reforming can be providing the heat for performing the reforming reaction in an efficient manner while reducing or minimizing introduction of additional components into the desired synthesis gas product. Cyclic reaction systems, such as reverse flow reactor systems, can provide heat in a desirable manner by having a cycle including a reforming step and a regeneration step. During the regeneration step, combustion can be performed within a selected area of the reactor. A gas flow during regeneration can assist with transferring this heat from the combustion zone toward additional portions of the reforming zone in the reactor. The reforming step within the cycle can be a separate step, so that incorporation of products from combustion into the reactants and/or products from reforming can be reduced or minimized. The reforming step can consume heat, which can reduce the temperature of the reforming zone. As the products from reforming pass through the reactor, the reforming products can pass through a second zone that lacks a reforming or water gas shift catalyst. This can allow the reaction products to cool prior to exiting the reactor. The heat transferred from the reforming products to the reactor can then be used to increase the temperature of the reactants for the next combustion or regeneration step. One common source for methane is natural gas. In some applications, natural gas, including associated hydrocarbon and impurity gases, may be used as a feed for the reforming reaction. The supplied natural gas also may be sweetened and/or dehydrated natural gas. Natural gas commonly includes various concentrations of associated gases, such as ethane and other alkanes, preferably in lesser concentrations than methane. The supplied natural gas may include impurities, such as H2S and nitrogen. More generally, the hydrocarbon feed for reforming can include any convenient combination of methane and/or other hydrocarbons. Optionally, the reforming feed may also include some hydrocarbonaceous compounds, such as alcohols or mercaptans, which are similar to hydrocarbons but include one or more heteroatoms different from carbon and hydrogen. In some aspects, an additional component present in the feed can correspond to impurities such as sulfur that can adsorb to the catalytic monolith during a step in a reaction cycle that has a reducing environment (such as a reforming step). Such impurities can be oxidized in a subsequent cycle to form sulfur oxides, which can then be reduced to release additional sulfur-containing components (or other impurity-containing components) into the reaction environment. In some aspects, the feed for reforming can include, relative to a total weight of hydrocarbons in the feed for reforming, 5 wt % or more of C2+compounds, such as ethane or propane, or 10 wt % or more, or 15 wt % or more, or 20 wt % or more, such as up to 50 wt % or possibly still higher. It is noted that nitrogen and/or other gases that are non-reactive in a combustion environment, such as H2O and CO2, may also be present in the feed for reforming. In aspects where the reformer corresponds to an on-board reforming environment, such non-reactive products can optionally be introduced into the feed, for example, based on recycle of an exhaust gas into the reformer. Additionally or alternately, the feed for reforming can include 40 wt % or more methane, or 60 wt % or more, or 80 wt % or more, or 95 wt % or more, such as having a feed that is substantially composed of methane (98 wt % or more). In aspects where the reforming corresponds to steam reforming, a molar ratio of steam molecules to carbon atoms in the feed can be 0.3 to 4.0. It is noted that methane has 1 carbon atom per molecule while ethane has 2 carbon atoms per molecule. In aspects where the reforming corresponds to dry reforming, a molar ratio of CO2molecules to carbon atoms in the feed can be 0.05 to 3.0. Within the reforming zone of a reverse flow reactor, the temperature can vary across the zone due to the nature of how heat is added to the reactor and/or due to the kinetics of the reforming reaction. The highest temperature portion of the zone can typically be found near a middle portion of the reactor. This middle portion can be referred to as a mixing zone where combustion is initiated during regeneration. At least a portion of the mixing zone can correspond to part of the reforming zone if a monolith with reforming catalyst extends into the mixing zone. As a result, the location where combustion is started during regeneration can typically be near to the end of the reforming zone within the reactor. Moving from the center of the reactor to the ends of the reactor, the temperature can decrease. As a result, the temperature at the beginning of the reforming zone (at the end of the reactor) can be cooler than the temperature at the end of the reforming zone (in the middle portion of the reactor). As the reforming reaction occurs, the temperature within the reforming zone can be reduced. The rate of reduction in temperature can be related to the kinetic factors of the amount of available hydrocarbons for reforming and/or the temperature at a given location within the reforming zone. As the reforming feed moves through the reforming zone, the reactants in the feed can be consumed, which can reduce the amount of reforming that occurs at downstream locations. However, the increase in the temperature of the reforming zone as the reactants move across the reforming zone can lead to an increased reaction rate. At roughly 500° C., the reaction rate for reforming can be sufficiently reduced that little or no additional reforming will occur. As a result, in some aspects as the reforming reaction progresses, the beginning portion of the reforming zone can cool sufficiently to effectively stop the reforming reaction within a portion of the reforming zone. This can move the location within the reactor where reforming begins to a location that is further downstream relative to the beginning of the reforming zone. When a sufficient portion of the reforming zone has a temperature below 500° C., or below 600° C., the reforming step within the reaction cycle can be stopped to allow for regeneration. Alternatively, based on the amount of heat introduced into the reactor during regeneration, the reforming portion of the reaction cycle can be stopped based on an amount of reaction time, so that the amount of heat consumed during reforming (plus heat lost to the environment) is roughly in balance with the amount of heat added during regeneration. After the reforming process is stopped, any remaining synthesis gas product still in the reactor can optionally be recovered prior to starting the regeneration step of the reaction cycle. The regeneration process can then be initiated. During regeneration, a fuel such as methane, natural gas, or H2, and oxygen can be introduced into the reactor and combusted. The location where the fuel and oxidant are allowed to mix can be controlled in any convenient manner, such as by introducing the fuel and oxidant via separate channels. By delaying combustion during regeneration until the reactants reach a central portion of the reactor, the non-reforming end of the reactor can be maintained at a cooler temperature. This can also result in a temperature peak in a middle portion of the reactor. The temperature peak can be located within a portion of the reactor that also includes the reforming catalyst. During a regeneration cycle, the temperature within the reforming reactor can be increased sufficiently to allow for the reforming during the reforming portion of the cycle. This can result in a peak temperature within the reactor of 1100° C. or more, or 1200° C. or more, or 1300° C. or more, or potentially a still higher temperature. The relative length of time and reactant flow rates for the reforming and regeneration portions of the process cycle can be selected to balance the heat provided during regeneration with the heat consumed during reforming. For example, one option can be to select a reforming step that has a similar length to the regeneration step. Based on the flow rate of hydrocarbons, H2O, and/or CO2during the reforming step, an endothermic heat demand for the reforming reaction can be determined. This heat demand can then be used to calculate a flow rate for combustion reactants during the regeneration step. Of course, in other aspects the balance of heat between reforming and regeneration can be determined in other manners, such as by determining desired flow rates for the reactants and then selecting cycle lengths so that the heat provided by regeneration balances with the heat consumed during reforming. In addition to providing heat, the reactor regeneration step during a reaction cycle can also allow for coke removal from the catalyst within the reforming zone. In various aspects, one or more types of catalyst regeneration can potentially occur during the regeneration step. One type of catalyst regeneration can correspond to removal of coke from the catalyst. During reforming, a portion of the hydrocarbons introduced into the reforming zone can form coke instead of forming CO or CO2. This coke can potentially block access to the catalytic sites (such as metal sites) of the catalyst. In some aspects, the rate of formation can be increased in portions of the reforming zone that are exposed to higher temperatures, such as portions of the reforming zone that are exposed to temperatures of 800° C. or more, or 900° C. or more, or 1000° C. or more. During a regeneration step, oxygen can be present as the temperature of the reforming zone is increased. At the temperatures achieved during regeneration, at least a portion of the coke generated during reforming can be removed as CO or CO2. Due to the variation in temperature across the reactor, several options can be used for characterizing the temperature within the reactor and/or within the reforming zone of the reactor. One option for characterizing the temperature can be based on an average bed or average monolith temperature within the reforming zone. In practical settings, determining a temperature within a reactor requires the presence of a measurement device, such as a thermocouple. Rather than attempting to measure temperatures within the reforming zone, an average (bed or monolith) temperature within the reforming zone can be defined based on an average of the temperature at the beginning of the reforming zone and a temperature at the end of the reforming zone. Another option can be to characterize the peak temperature within the reforming zone after a regeneration step in the reaction cycle. Generally, the peak temperature can occur at or near the end of the reforming zone, and may be dependent on the location where combustion is initiated in the reactor. Still another option can be to characterize the difference in temperature at a given location within the reaction zone at different times within a reaction cycle. For example, a temperature difference can be determined between the temperature at the end of the regeneration step and the temperature at the end of the reforming step. Such a temperature difference can be characterized at the location of peak temperature within the reactor, at the entrance to the reforming zone, at the exit from the reforming zone, or at any other convenient location. In various aspects, the reaction conditions for reforming hydrocarbons can include one or more of an average reforming zone temperature ranging from 400° C. to 1200° (or more); a peak temperature within the reforming zone of 800° C. to 1600° C.; a temperature difference at the location of peak temperature between the end of a regeneration step and the end of the subsequent reforming step of 25° C. or more, or 50° C. or more, or 100° C. or more, or 200° C. or more, such as up to 800° C. or possibly still higher; a temperature difference at the entrance to the reforming zone between the end of a regeneration step and the end of the subsequent reforming step of 25° C. or more, or 50° C. or more, or 100° C. or more, or 200° C. or more, such as up to 800° C. or possibly still higher; and/or a temperature difference at the exit from the reforming zone between the end of a regeneration step and the end of the subsequent reforming step of 25° C. or more, or 50° C. or more, or 100° C. or more, or 200° C. or more, such as up to 800° C. or possibly still higher. With regard to the average reforming zone temperature, in various aspects the average temperature for the reforming zone can be 500° C. to 1500° C., or 400° C. to 1200° C., or 800° C. to 1200° C., or 400° C. to 900° C., or 600° C. to 1100° C., or 500° C. to 1000° C. Additionally or alternately, with regard to the peak temperature for the reforming zone (likely corresponding to a location in the reforming zone close to the location for combustion of regeneration reactants), the peak temperature can be 800° C. to 1600° C., or 1000° C. to 1400° C., or 1200° C. to 1600° C., or 1200° C. to 1400° C. Additionally or alternately, the reaction conditions for reforming hydrocarbons can include a pressure of 0 psig to 1500 psig (10.3 MPa), or 0 psig to 1000 psig (6.9 MPa), or 0 psig to 550 psig (3.8 MPa); and a gas hourly space velocity of reforming reactants of 1000 hr−1to 50,000 hr−1. The space velocity corresponds to the volume of reactants relative to the volume of monolith per unit time. The volume of the monolith is defined as the volume of the monolith as if it was a solid cylinder. In some aspects, an advantage of operating the reforming reaction at elevated temperature can be the ability to convert substantially all of the methane and/or other hydrocarbons in a reforming feed. For example, for a reforming process where water is present in the reforming reaction environment (i.e., steam reforming or bi-reforming), the reaction conditions can be suitable for conversion of 10 wt % to 100 wt % of the methane in the reforming feed, or 20 wt % to 80 wt %, or 50 wt % to 100 wt %, or 80 wt % to 100 wt %, or 10 wt % to 98 wt %, or 50 wt % to 98 wt %. Additionally or alternately, the reaction conditions can be suitable for conversion of 10 wt % to 100 wt % of the hydrocarbons in the reforming feed, or 20 wt % to 80 wt %, or 50 wt % to 100 wt %, or 80 wt % to 100 wt %, or 10 wt % to 98 wt %, or 50 wt % to 98 wt % In other aspects, for a reforming process where carbon dioxide is present in the reforming reaction environment (i.e., dry reforming or bi-reforming), the reaction conditions can be suitable for conversion of 10 wt % to 100 wt % of the methane in the reforming feed, or 20 wt % to 80 wt %, or 50 wt % to 100 wt %, or 80 wt % to 100 wt %, or 10 wt % to 98 wt %, or 50 wt % to 98 wt %. Additionally or alternately, the reaction conditions can be suitable for conversion of 10 wt % to 100 wt % of the hydrocarbons in the reforming feed, or 20 wt % to 80 wt %, or 50 wt % to 100 wt %, or 80 wt % to 100 wt %, or 10 wt % to 98 wt %, or 50 wt % to 98 wt %. In some alternative aspects, the reforming reaction can be performed under dry reforming conditions, where the reforming is performed with CO2as a reagent but with a reduced or minimized amount of H2O in the reaction environment. In such alternative aspects, a goal of the reforming reaction can be to produce a synthesis gas with a H2to CO ratio of 1.0 or less. In some aspects, the temperature during reforming can correspond to the temperature ranges described for steam reforming. Optionally, in some aspects a dry reforming reaction can be performed at a lower temperature of between 500° C. to 700° C., or 500° C. to 600° C. In such aspects, the ratio of H2to CO can be 0.3 to 1.0, or 0.3 to 0.7, or 0.5 to 1.0. Performing the dry reforming reaction under these conditions can also lead to substantial coke production, which can require removal during regeneration in order to maintain catalytic activity. Example of Reverse Flow Reactor Configuration For endothermic reactions operated at elevated temperatures, such as hydrocarbon reforming, a reverse flow reactor can provide a suitable reaction environment for providing the heat for the endothermic reaction. In a reverse flow reactor, the heat needed for an endothermic reaction may be provided by creating a high-temperature heat bubble in the middle of the reactor. A two-step process can then be used wherein heat is (a) added to the reactor bed(s) or monolith(s) via in-situ combustion, and then (b) removed from the bed in-situ via an endothermic process, such as reforming, pyrolysis, or steam cracking. This type of configuration can provide the ability to consistently manage and confine the high temperature bubble in a reactor region(s) that can tolerate such conditions long term. A reverse flow reactor system can allow the primary endothermic and regeneration processes to be performed in a substantially continuous manner. A reverse flow reactor system can include first and second reactors, oriented in a series relationship with each other with respect to a common flow path, and optionally but preferably along a common axis. The common axis may be horizontal, vertical, or otherwise. During a regeneration step, reactants (e.g., fuel and oxygen) are permitted to combine or mix in a reaction zone to combust therein, in-situ, and create a high temperature zone or heat bubble inside a middle portion of the reactor system. The heat bubble can correspond to a temperature that is at least about the initial temperature for the endothermic reaction. Typically, the temperature of the heat bubble can be greater than the initial temperature for the endothermic reaction, as the temperature will decrease as heat is transferred from the heat bubble in a middle portion of the reactor toward the ends of the reactor. In some aspects, the combining can be enhanced by a reactant mixer that mixes the reactants to facilitate substantially complete combustion/reaction at the desired location, with the mixer optionally located between the first and second reactors. The combustion process can take place over a long enough duration that the flow of first and second reactants through the first reactor also serves to displace a substantial portion, (as desired) of the heat produced by the reaction (e.g., the heat bubble), into and at least partially through the second reactor, but preferably not all of the way through the second reactor to avoid waste of heat and overheating the second reactor. The flue gas may be exhausted through the second reactor, but preferably most of the heat is retained within the second reactor. The amount of heat displaced into the second reactor during the regeneration step can also be limited or determined by the desired exposure time or space velocity that the hydrocarbon feed gas will have in the endothermic reaction environment. After regeneration or heating the second reactor media (such as a phase stable monolith as described herein), in the next/reverse step or cycle, reactants for the endothermic reaction methane (and/or natural gas and/or another hydrocarbon) can be supplied or flowed through the second reactor, from the direction opposite the direction of flow during the heating step. For example, in a reforming process, methane (and/or natural gas and/or another hydrocarbon) can be supplied or flowed through the second reactor. The methane can contact the hot second reactor and mixer media, in the heat bubble region, to transfer the heat to the methane for reaction energy. For some aspects, the basic two-step asymmetric cycle of a reverse flow regenerative bed reactor system is depicted inFIGS.1(a) and1(b)ofFIG.1in terms of a reactor system having two zones/reactors; a first or recuperator/quenching zone (7) and a second or reaction zone (1). Both the reaction zone (1) and the recuperator zone (7) can contain regenerative monoliths and/or other regenerative structures. Regenerative monoliths or other regenerative structures, as used herein, comprise materials that are effective in storing and transferring heat as well as being effective for carrying out a chemical reaction. The regenerative monoliths and/or other structures can correspond to any convenient type of material that is suitable for storing heat, transferring heat, and catalyzing a reaction. Examples of structures can include bedding or packing material, ceramic beads or spheres, ceramic honeycomb materials, ceramic tubes, extruded monoliths, and the like, provided they are competent to maintain integrity, functionality, and withstand long term exposure to temperatures in excess of 1200° C., or in excess of 1400° C., or in excess of 1600° C., which can allow for some operating margin. To facilitate description ofFIG.1, the reactor is described herein with reference to a reforming reaction. It is understood that other convenient types of endothermic reactions can generally be performed using a reverse flow reactor, such as the reactor shown inFIG.1. As shown inFIG.1(a)ofFIG.1, at the beginning of the “reaction” step of the cycle, a secondary end5of the reaction zone1(a.k.a. herein as the second reactor) can be at an elevated temperature as compared to the primary end3of the reaction zone1, and at least a portion (including the first end9) of the recuperator or quench zone7(a.k.a. herein as the first reactor), can be at a lower temperature than the reaction zone1to provide a quenching effect for the resulting product. In an aspect where the reactors are used to perform reverse flow reforming, a methane-containing reactant feed (or other hydrocarbon-containing reactant feed) can be introduced via a conduit(s)15, into a primary end3of the reforming or reaction zone1. In various aspects, the hydrocarbon-containing reactant feed can also contain H2O, CO2, or a combination thereof. The feed stream from inlet(s)15can absorb heat from reaction zone1and endothermically react to produce the desired synthesis gas product. As this step proceeds, a shift in the temperature profile2, as indicated by the arrow, can be created based on the heat transfer properties of the system. When the ceramic catalyst monolith/other catalyst structure is designed with adequate heat transfer capability, this profile can have a relatively sharp temperature gradient, which gradient can move across the reaction zone1as the reforming step proceeds. In some aspects, a sharper temperature gradient profile can provide for improved control over reaction conditions. In aspects where another type of endothermic reaction is performed, a similar shift in temperature profile can occur, so that a temperature gradient moves across reaction zone1as the reaction step proceeds. The effluent from the reforming reaction, which can include unreacted feed components (hydrocarbons, H2O, CO2) as well as synthesis gas components, can exit the reaction zone1through a secondary end5at an elevated temperature and pass through the recuperator reactor7, entering through a second end11, and exiting at a first end9. The recuperator7can initially be at a lower temperature than the reaction zone1. As the products (and optionally unreacted feed) from the reforming reaction pass through the recuperator zone7, the gas can be quenched or cooled to a temperature approaching the temperature of the recuperator zone substantially at the first end9, which in some embodiments can be approximately the same temperature as the regeneration feed introduced via conduit19into the recuperator7during the second step of the cycle. As the reforming effluent is cooled in the recuperator zone7, a temperature gradient4can be created in the zone's regenerative bed(s) and can move across the recuperator zone7during this step. The quenching can heat the recuperator7, which can be cooled again in the second step to later provide another quenching service and to prevent the size and location of the heat bubble from growing progressively through the quench reactor7. After quenching, the reaction gas can exit the recuperator at9via conduit17and can be processed for separation and recovery of the various components. The second step of the cycle, referred to as the regeneration step, can then begin with reintroduction of the first and second regeneration reactants via conduit(s)19. The first and second reactants can pass separately through hot recuperator7toward the second end11of the recuperator7, where they can be combined for exothermic reaction or combustion in or near a central region13of the reactor system. An example of the regeneration step is illustrated inFIG.1(b)ofFIG.1. Regeneration can entail transferring recovered sensible heat from the recuperator zone7to the reaction zone1to thermally regenerate the reaction beds1for the subsequent reaction cycle. Regeneration gas/reactants can enter recuperator zone7, such as via conduit(s)19, and flow through the recuperator zone7and into the reaction zone1. In doing so, the temperature gradients6and8may move across the beds as illustrated by the arrows on the exemplary graphs inFIG.1(b), similar to but in opposite directions to the graphs of the temperature gradients developed during the reaction cycle inFIG.1(a)ofFIG.1. Fuel and oxidant reactants may combust at a region proximate to the interface13of the recuperator zone7and the reaction zone1. The heat recovered from the recuperator zone together with the heat of combustion can be transferred to the reaction zone, thermally regenerating the regenerative reaction monoliths and/or beds1disposed therein. In some aspects, several of the conduits within a channel may convey a mixture of first and second reactants, due at least in part to some mixing at the first end (17) of the first reactor. However, the numbers of conduits conveying combustible mixtures of first and second reactants can be sufficiently low such that the majority of the stoichiometrically reactable reactants will not react until after exiting the second end of the first reactor. The axial location of initiation of combustion or exothermic reaction within those conduits conveying a mixture of reactants can be controlled by a combination of temperature, time, and fluid dynamics. Fuel and oxygen usually require a temperature-dependent and mixture-dependent autoignition time to combust. Still though, some reaction may occur within an axial portion of the conduits conveying a mixture of reactants. However, this reaction can be acceptable because the number of channels having such reaction can be sufficiently small that there is only an acceptable or inconsequential level of effect upon the overall heat balance within the reactor. The design details of a particular reactor system can be selected so as to avoid mixing of reactants within the conduits as much as reasonably possible. FIG.2illustrates another exemplary reactor system that may be suitable in some applications for controlling and deferring the combustion of fuel and oxidant to achieve efficient regeneration heat.FIG.2depicts a single reactor system, operating in the regeneration cycle. The reactor system may be considered as comprising two reactor zones. The recuperator27can be the zone primarily where quenching takes place and provides substantially isolated flow paths or channels for transferring both of the quenching reaction gases through the reactor media, without incurring combustion until the gasses arrive proximate or within the reactor core13inFIG.1. The reformer2can be the reactor where regeneration heating and methane (and/or hydrocarbon) reformation primarily occurs, and may be considered as the second reactor for purposes herein. Although the first and second reactors in the reactor system are identified as separately distinguishable reactors, it is understood that the first and second reactors may be manufactured, provided, or otherwise combined into a common single reactor bed, whereby the reactor system might be described as comprising merely a single reactor that integrates both cycles within the reactor. The terms “first reactor” and “second reactor” can merely refer to the respective zones within the reactor system whereby each of the regeneration, reformation, quenching, etc., steps take place and do not require that separate components be utilized for the two reactors. However, various aspects can comprise a reactor system whereby the recuperator reactor includes conduits and channels as described herein, and the reformer reactor may similarly possess conduits. Additionally or alternately, some aspects may include a reformer reactor bed that is arranged different from and may even include different materials from, the recuperator reactor bed. As discussed previously, the first reactor or recuperator27can include various gas conduits28for separately channeling two or more gases following entry into a first end29of the recuperator27and through the regenerative bed(s) disposed therein. A first gas30can enter a first end of a plurality of flow conduits28. In addition to providing a flow channel, the conduits28can also comprise effective flow barriers (e.g., which effectively function such as conduit walls) to prevent cross flow or mixing between the first and second reactants and maintain a majority of the reactants effectively separated from each other until mixing is permitted. As discussed previously, each of the first and second channels can comprise multiple channels or flow paths. The first reactor may also comprise multiple substantially parallel flow segments, each comprising segregated first and second channels. In some aspects, the recuperator can be comprised of one or more extruded honeycomb monoliths, as described above. Each monolith may provide flow channel(s) (e.g., flow paths) for one of the first or second reactants. Each channel preferably includes a plurality of conduits. Alternatively, a monolith may comprise one or more channels for each reactant with one or more channels or groups of conduits dedicated to flowing one or more streams of a reactant, while the remaining portion of conduits flow one or more streams of the other reactant. It is recognized that at the interface between channels, a number of conduits may convey a mixture of first and second reactant, but this number of conduits is proportionately small. Alternative embodiments may use reactor media other than monoliths, such as whereby the channel conduits/flow paths may include a more tortuous pathways (e.g. convoluted, complex, winding and/or twisted but not linear or tubular), including but not limited to labyrinthine, variegated flow paths, conduits, tubes, slots, and/or a pore structure having channels through a portion(s) of the reactor and may include barrier portion, such as along an outer surface of a segment or within sub-segments, having substantially no effective permeability to gases, and/or other means suitable for preventing cross flow between the reactant gases and maintaining the first and second reactant gases substantially separated from each other while axially transiting the recuperator27. Such other types of reactor media can be suitable, so long as at least a portion of such media can be formed by sintering a ceramic catalytic composition as described herein, followed by exposing such media to reducing conditions to activate the catalyst. For such embodiments, the complex flow path may create a lengthened effective flow path, increased surface area, and improved heat transfer. Such design may be preferred for reactor embodiments having a relatively short axial length through the reactor. Axially longer reactor lengths may experience increased pressure drops through the reactor. However for such embodiments, the porous and/or permeable media may include, for example, at least one of a packed bed, an arrangement of tiles, a permeable solid media, a substantially honeycomb-type structure, a fibrous arrangement, and a mesh-type lattice structure. In some aspects, the reverse flow reactor can include some type of equipment or method to direct a flow stream of one of the reactants into a selected portion of the conduits. In the exemplary embodiment ofFIG.2, a gas distributor31can direct a second gas stream32to second gas stream channels that are substantially isolated from or not in fluid communication with the first gas channels, here illustrated as channels33. The result can be that at least a portion of gas stream33is kept separate from gas stream30during axial transit of the recuperator27. In some aspects, the regenerative bed(s) and/or monolith(s) of the recuperator zone can comprise channels having a gas or fluid barrier that isolates the first reactant channels from the second reactant channels. Thereby, both of the at least two reactant gases that transit the channel means may fully transit the regenerative bed(s), to quench the regenerative bed, absorb heat into the reactant gases, before combining to react with each other in the combustion zone. In various aspects, gases (including fluids)30and32can each comprise a component that reacts with a component in the other reactant30and32, to produce an exothermic reaction when combined. For example, each of the first and second reactant may comprise one of a fuel gas and an oxidant gas that combust or burn when combined with the other of the fuel and oxidant. By keeping the reactants substantially separated, the location of the heat release that occurs due to exothermic reaction can be controlled. In some aspects “substantially separated” can be defined to mean that at least 50 percent, or at least 75 percent, or at least 90 percent of the reactant having the smallest or limiting stoichiometrically reactable amount of reactant, as between the first and second reactant streams, has not become consumed by reaction by the point at which these gases have completed their axial transit of the recuperator27. In this manner, the majority of the first reactant30can be kept isolated from the majority of the second reactant32, and the majority of the heat release from the reaction of combining reactants30and32can take place after the reactants begin exiting the recuperator27. The reactants can be gases, but optionally some reactants may comprise a liquid, mixture, or vapor phase. The percent reaction for these regeneration streams is meant the percent of reaction that is possible based on the stoichiometry of the overall feed. For example, if gas30comprised 100 volumes of air (80 volumes N2and 20 Volumes O2), and gas32comprised 10 volumes of hydrogen, then the maximum stoichiometric reaction would be the combustion of 10 volumes of hydrogen (H2) with 5 volumes of oxygen (O2) to make 10 volumes of H2O. In this case, if 10 volumes of hydrogen were actually combusted in the recuperator zone (27), this would represent 100% reaction of the regeneration stream. This is despite the presence of residual un-reacted oxygen, because in this example the un-reacted oxygen was present in amounts above the stoichiometric requirement. Thus, in this example the hydrogen is the stoichiometrically limiting component. Using this definition, less than 50% reaction, or less than 25% reaction, or less than 10% reaction of the regeneration streams can occur during the axial transit of the recuperator (27). In various aspects, channels28and33can comprise ceramic (including zirconia), alumina, or other refractory material capable of withstanding temperatures exceeding 1200° C., or 1400° C., or 1600° C. Additionally or alternately, channels28and33can have a wetted area between 50 ft−1and 3000 ft−1, or between 100 ft−1and 2500 ft−1, or between 200 ft−1and 2000 ft−1. Referring again briefly toFIG.1, the reactor system can includes a first reactor7containing a first end9and a second end11, and a second reactor1containing a primary end3and a secondary end5. The embodiments illustrated inFIGS.1and2are merely simple illustrations provided for explanatory purposes only and are not intended to represent a comprehensive embodiment. Reference made to an “end” of a reactor merely refers to a distal portion of the reactor with respect to an axial mid-point of the reactor. Thus, to say that a gas enters or exits an “end” of the reactor, such as end9, means merely that the gas may enter or exit substantially at any of the various points along an axis between the respective end face of the reactor and a mid-point of the reactor, but more preferably closer to the end face than to the mid-point. Thereby, one or both of the first and second reactant gases could enter at the respective end face, while the other is supplied to that respective end of the reactor through slots or ports in the circumferential or perimeter outer surface on the respective end of the reactor. EXAMPLES In the Examples below, when monoliths are used, the monoliths used for supporting the catalyst systems corresponded to monoliths with 400 cpsi (cells per square inch) and an open frontal area of either 35% or 52%. Example 1—Phase Transitions for NiO/NiAl2O4 A sample of NiO and NiAl2O4was placed on a platinum substrate. The sample on the substrate was then used for in-situ characterization by powder X-ray diffraction (PXRD) while exposing the sample to reducing and oxidizing environments. This allowed for characterization of the crystal phases (Ni, NiO, NiAl2O4, and Al2O3) that were present in the sample after exposure to varying amounts of reducing and oxidizing conditions. The experiments in this example were conducted at 1300° C. The sample of NiO/NiAl2O4was placed on a Pt strip which was used to heat the sample. An atmosphere of H2, air, or N2was applied to the sample, according to the type of atmosphere (reducing, oxidizing, inert) that was used during a given time period. The NiO/NiAl2O4sample was first exposed to a reducing atmosphere (at 1300° C.) for a period of time, to allow for reduction of a substantial portion of NiO to Ni and NiAl2O4to Ni and Al2O3. The atmosphere was then purged with N2, followed by introduction of air (at 1300° C.) to allow for conversion of Ni back to NiO and then at least partially to NiAl2O4. FIG.4andFIG.5shows PXRD spectra taken at various times during the experiments.FIG.4shows PXRD spectra for the portion of the process corresponding to exposing the sample to a reducing atmosphere. As shown inFIG.4, prior to exposure to the reducing environment at 1300° C., the sample initially had a PXRD spectrum410that showed peaks for both NiO and NiAl2O4. The sample was then exposed to an H2atmosphere at 1300° C. for 13 minutes. Additional PXRD characterization was performed at 30 seconds, 1 minute, 5 minutes, and 13 minutes. After 30 seconds, the PXRD spectrum remained qualitatively similar to spectrum410. Spectrum420shows the spectrum obtained after 1 minute of exposure to the reducing atmosphere. As shown in spectrum420, the NiO peaks in the spectrum have disappeared, and a new peak corresponding to Ni is now visible. A peak for Al2O3was not quite visible yet at this time. After additional exposure to the reducing atmosphere to reach a total of 13 minutes, spectrum430was obtained. As shown in spectrum430, the only peak observable in this portion of the PXRD spectrum is the peak for Ni. (A peak for Al2O3was also observed.) All of the NiO and NiAl2O4has disappeared. Table 1 summarizes the results from the PXRD spectra obtained after 30 seconds, 1 minute, 5 minutes, and 13 minutes of exposure. Without being bound by any particular theory, it is noted that the NiO peaks in the PXRD spectrum disappeared first when exposed to a reducing atmosphere. The disappearance of the NiAl2O4peaks took longer, indicating that the conversion of NiO to metallic Ni occurs relatively quickly in comparison with the conversion of NiAl2O4to metallic Ni and Al2O3. TABLE 1PXRD Peak Formation and DisappearanceDuring Reduction of NiO/NiAl2O4H2Reduction TimePeak FormationPeak Disappearance30seconds——1minuteNiNiO5minutesNi (also smallNiO, NiAl2O4amount of α-Al2O3)13minutesNi, α-Al2O3NiO, NiAl2O4 FIG.5contains additional PXRD spectra. InFIG.5, spectra410corresponds to the spectrum for the NiO/NiAl2O4sample prior to exposure to the reducing atmosphere. Spectrum540corresponds to a spectrum for the sample after exposure of the sample to 13 minutes of a reducing atmosphere, purging with N2, and then exposure to air for 13 minutes. As shown in spectrum540, exposure of the sample to air for a sufficient amount of time resulted in a PXRD spectrum540that was substantially similar to the spectrum410. This demonstrates that the formation of Ni shown inFIG.4was substantially completely reversed after oxidation. FIG.5also includes a depiction of a proposed mechanism for the conversion of NiO and NiAl2O4to metallic Ni (row551), and then conversion of the metallic Ni back into NiO and NiAl2O4(row552). As depicted in row551, without being bound by any particular theory, it is believed that in a fully oxidized state, small domains of NiO are present on NiAl2O4. When exposed to reducing conditions, these domains of NiO are converted to Ni. After further exposure, the NiAl2O4can be converted to Ni and Al2O3. During both of these reduction processes, sintering can occur to increase the domain size of the resulting Ni. With regard to the Al2O3, it is believed that the initial phase for the alumina is θ-Al2O3. After additional exposure to heat (reducing atmosphere not required), this θ-Al2O3can be converted to α-Al2O3. When the sample is then exposed to an oxidizing atmosphere (row552), initially the Ni domains are at least partially converted to NiO. This may or may not result in a surface area change, but it is believed that any surface area change in the conversion to NiO is small relative to the subsequent surface area change. After conversion to NiO, the NiO is then combined with Al2O3to form spinel phase NiAl2O4. By definition, forming a crystalline spinel phase containing NiAl2O4requires dispersal of nickel away from larger domains and into the distributed crystalline NiAl2O4structure. Without being bound by any particular theory, it is believed that this formation of NiAl2O4is the mechanism that provides the “size-reversing” properties of the NiAl2O4system. To further illustrate the proposed mechanism,FIG.6shows SEM images from the sample after various exposures to the reducing atmosphere and then after full exposure of the reduced surface to the oxidizing atmosphere.FIG.6shows the time evolution of the Ni particle sizes of the sample after 30 seconds, 1 minute, 5 minutes, and 13 minutes of exposure to the reducing conditions at 1300° C. As shown inFIG.6, initially smaller size domains of Ni were formed, but the domains increased in size with increasing exposure to elevated temperature. However, after exposure of the sample to a sufficient time under oxidizing conditions, the domain size on the surface is reduced. Example 2—Temperature Dependence of Oxidation Two samples of NiO and α-Al2O3were prepare with a ratio of NiO to Al2O3of 1:1.5. One sample was calcined at 1200° C. for 4 hours. After calcination, the ratio of Al2O3to NiAl2O4was 3.60, indicating that less than 25% of the Al2O3was converted to NiAl2O4. A second sample was calcined at 1300° C. for 4 hours. After calcination, the ratio of Al2O3to NiAl2O4was 0.07, indicating substantially complete conversion to the spinel phase. This illustrates the strong temperature dependence for the reaction that converts NiO and Al2O3to the spinel phase. Based on this temperature dependence, little or no formation of spinel phase would occur under cyclic reforming conditions at temperatures below 1000° C. As a result, the size-reversing benefits of the NiAl2O4system would not be achieved under lower temperature conditions. Example 3A—NiO/Al2O3(NiAl2O4) α-Al2O3powder was dried in an oven overnight at 121° C. (˜250° F.). The dried α-Al2O3powder was then weighed, and mixed with the appropriate amount of NiO. This corresponded to a 1.5:1 Al:Ni mole ratio for the NiO/Al2O3catalyst system. It is noted that this catalyst system can form NiAl2O4in-situ after exposure to cyclic high temperature reforming conditions. The mixture was mixed in a Waring Blender. The blended material was then calcined at 1300° C. (2372° F.) in a Sentro Tech Furnace for 4 hours, using a ramp rate of 3° C./min, producing the desired NiO/Al2O3catalyst system. The calcined material was allowed to cool to room temperature. The weight percent of catalytic metal (Ni) in the NiO/Al2O3catalyst system was 38.8 wt % (relative to a weight of the catalyst system). The calcined NiO/Al2O3catalyst system was further milled in a liquid to prepare appropriate slurry and washcoated on monoliths having 93 wt % α-Al2O3, 5 wt % SiO2and 2 wt % MgO. The washcoated monoliths were further calcined at 1200° C. (2192° F.) for 2 hours to ensure complete adherence of the active material onto the monoliths. This corresponded to annealing at a temperature that was substantially similar to the peak temperature during the subsequent exposure of the monoliths to cyclic high temperature reforming conditions. The final washcoated monoliths were loaded at the highest temperature catalytic zone (e.g. 800˜1250° C.) in a pilot scale reverse flow reactor unit and exposed to various cyclic process conditions for about 750 hours.FIG.8shows a scanning electron microscope (SEM) image of the NiO and NiAl2O4materials that were present on the monoliths having 93 wt % α-Al2O3, 5 wt % SiO2and 2 wt % MgO after 750 hrs. During the exposure to the various cyclic process conditions, the NiAl2O4catalyst system provided steady and high methane conversion, good phase stability of the metal oxide support, insignificant sintering of active catalytic metals, and good adhesion to the monoliths. Examples 3B and 3C—NiAl2O4and Al2O3/NiAl2O4 Additional catalyst systems and corresponding washcoated monoliths were prepared according to the method in Example 3A, but with varying ratios of NiO to Al2O3. In Example 3B, the initial Al:Ni ratio was 2:1, resulting in a catalyst system with an Ni content of roughly 33.2 wt % (relative to the weight of the catalyst system). This roughly corresponds to the stoichiometric ratio for NiAl2O4. The catalyst system with the 2:1 ratio of Al:Ni was then washcoated on to a monolith with the same type of washcoat composition as the monolith in Example 3A, using a similar procedure. For Example 3C, the initial Al:Ni ratio was 3:1, so that excess Al2O3was present in the catalyst system. This resulted in forming a catalyst system with an Ni content of roughly 25.8 wt %. The catalyst system was then deposited as a washcoat on a monolith with the same type of washcoat composition as used in Examples 3A and 3B. Example 4—Rh/α-Al2O3 In this example, the catalyst system corresponds to Rh (Rh2O3) as catalytic metal (oxide) and α-Al2O3as the metal oxide support layer. α-Al2O3corresponds to a “corundum” type oxide. To make this catalyst system, α-Al2O3powder was dried in an oven overnight at 121° C. (˜250° F.). The dried α-Al2O3was then weighed, and small aliquot was taken in order to determine the absorption factor. A solution of rhodium (III) nitrate, Rh(NO3)3·nH2O, aqueous solution containing 9.77 wt % Rh at 27.4% solids content, was mixed with the appropriate amount of excess H2O (as necessary), and sprayed/impregnated onto the α-Al2O3. The material was then dried at 121° C. (250° F.) for at least 2 hrs. The dried material was then calcined at 500° C. (932° F.) in a Sentro Tech Furnace for 4 hours, using a ramp rate of 3° C./min, producing Rh2O3/α-Al2O3. The calcined material was allowed to cool to room temperature. The weight percent of catalytic metal Rh was roughly 4.0 wt %. The calcined Rh2O3/α-Al2O3catalyst system was further milled in a solution to prepare appropriate slurry and washcoated on monoliths having 93 wt % α-Al2O3, 5 wt % SiO2and 2 wt % MgO. The washcoated monoliths were further calcined at 500° C. (932° F.) for 2 hours to ensure complete adherence of the active material onto the monoliths. The final washcoated monoliths were loaded at the low temperature catalytic zone (e.g. 400˜1050° C.) in a pilot scale reverse flow reactor unit and exposed to various cyclic process conditions for about 750 hours. The desired Rh2O3/α-Al2O3catalyst system had steady high methane conversion, good phase stability of the metal oxide support, reduced or minimized sintering of active catalytic metals, and good adhesion to the monolith.FIG.9shows scanning electron microscope (SEM) images of an example of the Rh2O3/α-Al2O3catalyst system washcoated on a monolith having 93 wt % α-Al2O3, 5 wt % SiO2and 2 wt % MgO after 750 hrs. Examples 5A, 5B, and 5C—Catalyst Systems with Different Starting Alumina Phases Three different types of catalyst system washcoat preparations were used to prepare multi-zone catalyst systems for hydrocarbon reforming. In this Example, a multi-zone catalyst system was used, with two catalyst zones. A first zone (higher temperature) corresponded to a Ni-containing catalyst, while a second zone (lower temperature) corresponded to a Rh-containing catalyst. Two monoliths were used to fill the desired catalyst bed volume in each zone in the pilot scale reactor, so each catalyst system corresponded to a total of four monoliths. For Example 5A, the first two monoliths with the Ni-containing catalyst system was prepared according to Example 3A. Thus, the catalyst was NiO while the alumina phase in the first two monoliths in Example 5A was α-Al2O3. This type of catalyst system can result in in-situ formation of NiAl2O4after exposure to cyclic high temperature reforming conditions. A sufficient washcoat was applied so that the weight of the NiO/α-Al2O3catalyst system was roughly 5 wt % of the total weight of the washcoated monolith. For Example 5A, the second two monoliths with the Rh-containing catalyst system were prepared according to Example 4. Thus, the alumina phase in the second two monoliths in Example 5A was α-Al2O3. A sufficient washcoat was applied so that the weight of the Rh2O3/α-Al2O3catalyst system was roughly 4 wt % of the total weight of the washcoated monolith. For Examples 5B and 5C, instead of using catalyst systems comprised of phase stable materials, a washcoat containing higher surface-area material was deposited on a monolith. Thus, the washcoats used in Examples 5B and 5C did not include a thermally stable metal oxide support layer. In Example 5B, the catalyst system for the first two washcoats corresponded to θ-Al2O3doped with 4 wt % La, with 30% Ni as the catalyst, which was formed at 1200° C. This type of composition can be referred to as 30% Ni-4%-La-θ-Al2O3. This catalyst system corresponds to a molar ratio of Al:Ni of 3.7:1. It is noted that this ratio has a substantial amount of excess Al relative to the stoichiometric ratio for NiAl2O4. After depositing the washcoat, the first two monoliths in Example 5B were calcined at 500° C. For the second two monoliths in Example 5B, a washcoat of 5% Rh on 4%-La-γ-Al2O3was washcoated onto an alumina rich monolith. It is noted that the second two washcoats included γ-Al2O3rather than the θ-Al2O3of the first two washcoats. After depositing the washcoat, the second two monoliths in Example 5B were calcined at 500° C. In Example 5C, the first two washcoats and second two washcoats were prepared in a manner similar to Example 5B, but the calcination temperature of the first two monoliths was different. In Example 5C, a calcination temperature of 1200° C. was used after applying the washcoat (as compared to the 500° C. used for calcination in Example 5B). Example 6—Reforming with Monoliths with Different Starting Alumina Phases The washcoated monoliths described in Examples 5A, 5B, and 5C were used in a pilot scale reactor to investigate changes in catalytic activity over time and to determine the structural stability of the washcoated monoliths. The monoliths were used to perform steam reforming on a methane feed under cyclic high temperature conditions. The reaction conditions included a regeneration step and a reforming step. During the regeneration step, air was used to provide the source of oxygen for combustion of hydrocarbon fuel. The amount of air was sufficient to provide a 10% excess of O2relative to the amount of hydrocarbon fuel used for heating. The regeneration step during each cycle was performed for roughly 15 seconds at a pressure of 150 psig (˜1.0 MPa-g). The combustion during the regeneration step was performed to provide a temperature profile with a target peak temperature of 1150° C. at the end of the regeneration step/beginning of the reforming step. The reforming step during each cycle was performed for roughly 15 seconds at a pressure of 300 psig (˜2.1 MPa-g), with methane as the hydrocarbon for reforming. The molar ratio of H2O to CH4in the reforming step feed was 1.3. FIG.10shows the average conversion of methane during the reforming step (right plot) as the process is performed over a period of 4-6 weeks. Because of the impact of cooling in the reactor toward the end of a reforming step during a single cycle, the conversion rate at the end of the reforming step in each cycle is somewhat lower. This is illustrated by the difference in conversion when averaging over the first 10 seconds of each reforming step (left plot inFIG.10) versus averaging over the first 15 seconds of each reforming step (right plot inFIG.10). It is noted that a shorter reforming step could be used if it is desirable to maintain higher overall conversion for the full length of the reforming step. As shown in the right plot inFIG.10, both Example 5B and Example 5C initially provided higher conversion than Example 5A. This matches the expected effect of using the higher surface area phases of alumina as the support in Examples 5B and 5C. However, as shown in the right plot inFIG.10, the activity for conversion of the catalysts in Examples 5B and 5C is not stable. While the data is noisy, it is clear that the conversion activity for the catalyst in Example 5C starts to decline almost immediately after start of run. This activity decline continues until about 300 hours of performing the cyclic reaction. At that point, the reaction in Example 5C was stopped due to the rapid loss of activity that was occurring. Example 5B has a more gradual decline in catalytic activity, but Example 5B also undergoes a substantial drop in activity that started well before 400 hours of time on service. Between 400 hours and 800 hours, an alternative set of cyclic reforming conditions were used that are not plotted inFIG.10, but a similar drop in activity over time was observed. At 800 hours, the process conditions were restored to the initial reforming conditions. By 800 hours, the degradation of the catalyst appeared to stabilize, albeit at a significantly lower activity level than the catalyst of Example 5A. Again, this loss in activity is believed to be due to degradation of the catalyst system on the monolith in the reactor, resulting in loss of available surface area for the exposing the methane feed to the catalyst. In contrast to Example 5B and Example 5C, the right plot inFIG.10shows that the catalyst system in Example 5A maintained similar activity throughout the full length of the process run. Without being bound by any particular theory, it is believed that this is due to Example 5A corresponding to a) monoliths made from a material that is phase stable under the cyclic high temperature reforming conditions and b) the monoliths including an oxide support layer, so that any interaction between a catalyst metal (such as Ni) and an oxide material (such as Al2O3) occurs in the oxide support layer, and does not impact the structural integrity of the underlying monolith or other support structure. The left plot inFIG.10shows similar results. Again, Example 5C initially shows higher activity than Example 5A, but the activity for Example 5C started to decline almost immediately. Examples 5A and 5B initially had similar activity, but only Example 5A maintained that activity. As shown in both plots inFIG.10, Example 5A maintained substantially the same activity for a run length of 750 hours. In order to further characterize the results from performing cyclic reforming for the catalyst system and monolith from Example 5A, at the end of the run, alternating oxidation and reduction cycles were maintained while the peak temperature in the reactor was cooled to 1000° C. An N2flow was then used to cool the reactor down to room temperature. After cooling, additional characterization was then performed. After cooling, it was initially noted that for the first and second monolith of Example 5A, the catalyst system was difficult to remove from the underlying monolith. Due to structural breakdown, it is typically relatively easy to separate a catalyst or catalyst system from a monolith when the catalyst or catalyst system does not include a thermally stable metal oxide. However, it was unexpectedly found that by using a thermally phase stable support metal oxide in the catalyst system, strong adhesion of the catalyst system to the monolith could be maintained after extended exposure to the cyclic high temperature reforming environment. After removal, the catalyst system from Example 5A was characterized using X-ray diffraction (XRD). The XRD spectra indicated that all of the catalyst systems were in stable phases. For the first monolith, the catalyst system was primarily in a reduced state. This was indicated based on the presence of substantial peaks for Ni and α-Al2O3while little NiAl2O4was present. The catalyst system for the second monolith was more oxidized, with a mixture of Ni, NiO, α-Al2O3, and NiAl2O4being present. The XRD spectra for the third and fourth monoliths were similar to each other, with both showing primarily Rh and α-Al2O3. Additional microscopic analysis and elemental analysis of the catalyst systems was consistent with the XRD spectra. Example 7—Modification of Excess Oxygen in Regeneration Step Prior to removing the catalyst system from the first two monoliths of Example 5A, SEM micrographs were obtained of the surface of the catalyst system on the monoliths. The left SEM image inFIG.7is a representative image of the micrographs. As shown in Example 6, substantial sintering of the Ni on the surface of the monolith had occurred. It was noted in Example 6 that the first monolith was in a primarily reduced state, while the second monolith was in a partially reduced state. Without being bound by any particular theory, it is believed that the sintering observed in the left SEM image inFIG.6was due to incomplete oxidation of Ni metal during the regeneration step under the regeneration conditions for the process cycle used in Example 6. The first two monoliths in Example 5A included a total of 11.64 grams of Ni, or 0.198 moles of Ni. Thus, complete oxidation of the amount of Ni on the first two monoliths to form NiO (or NiAl2O4) would require 0.099 moles of O2. For complete reduction of all of the Ni on the first two monoliths (assuming the Ni was in the form of NiO or NiAl2O4), 0.198 moles of H2would be required. The cyclic reforming conditions included a combustion step and a reforming step. The combustion step was 15 seconds long at a pressure of 150 psig (˜1.0 MPa-g). Air was used to provide the oxidant. The flow rate of air during combustion was 6.77 standard cubic feet per minute, which corresponds to 0.424 moles of O2over the course of 15 seconds. This corresponded to 110% of the stoichiometric oxygen need for complete combustion of the fuel used during the combustion step, which means that (at most) 0.042 moles of excess oxygen were available for oxidation of any metallic Ni present in the reactor. It is noted that a portion of this excess oxygen could also potentially be consumed via combustion of coke that forms on interior surfaces of the reactor during reforming. The reforming step was 15 seconds long at 300 psig (˜2.1 MPa-g). Methane was used as the hydrocarbon feed at a flow rate of 2.0 scfm, which corresponded to 0.597 moles of CH4introduced into the reactor during the reforming step. As noted above, only 0.099 moles of H2would be needed to completely reduce the Ni in the reactor. This amount of H2would be generated at a CH4conversion level of only 11%. The reforming conditions were selected to provide roughly 90% or more conversion of the CH4. Thus, a substantial excess of H2was present during the reforming step relative to the amount of H2needed for converting NiO or NiAl2O4into metallic Ni. In Example 6, a 10% excess of O2was used relative to the stoichiometric amount for combustion (i.e., an excess molar amount of oxygen of 10%). Based on the total amount of Ni metal within the pilot scale reactor, the 10% excess molar amount of oxygen (relative to the stoichiometry for combustion) corresponded to roughly 42% of the amount of oxygen that would be needed to convert all Ni within the first two monoliths from Ni metal to NiO. Given the amount of hydrogen present within a reforming environment, it is believed that substantially all of the exposed Ni in the reactor was converted from NiO to Ni over time during the reforming steps of the reaction cycles, and then only a portion of the Ni metal was being converted back to NiO during the regeneration steps. After roughly 1 month of exposure to the cyclic reforming conditions, ex-situ SEM analysis of the hottest Ni-coated monolith showed significant sintering of the NiO/NiAl2O4material, as indicated by large regions of metallic Ni on top of Al2O3(i.e., Ni-deficient material) in the catalyst system layer. The first SEM micrograph shown inFIG.7provides an example of the large, sintered regions of metallic Ni. The large sintered areas of Ni metal and corresponding Ni-depleted regions of Al2O3in the catalyst system in the first SEM micrograph inFIG.7can be understood based on the relative reducing potential and oxidizing potential of the conditions during the reforming step of the reaction cycle (and/or other steps in the reaction cycle that correspond to a reducing environment). As detailed above, during the reforming step, a substantial excess of H2was available. By contrast, the excess oxygen during the combustion step corresponded to only enough oxygen for oxidation of 42% of the Ni in the reactor. Due to this disparity in the amount Ni that could be converted under the reducing conditions and the oxidation conditions in the reaction cycle, over time a large portion of the Ni in the reactor remained in the metallic (reduced) state during each cycle, thus providing long time periods for sintering to form larger domains of Ni. The first SEM micrograph inFIG.7is consistent with this analysis. In order to provide evidence that a high temperature oxidative process would allow for oxidation of Ni and redispersion to NiAl2O4, the monolith was exposed to calcination conditions at 1300° C. in the presence of air for 4 hours. The second SEM micrograph shown inFIG.7shows an example of the change in the structure of the catalyst system. As shown in the second SEM micrograph inFIG.7(and as confirmed by elemental analysis), Ni was oxidized to NiO. Additionally, the Al2O3matrix of the catalyst system was transformed into a mixture a mixture of NiO/NiAl2O4. This formation of NiAl2O4requires atomic redispersion of Ni, as indicated by the much broader distribution for the NiAl2O4in the second micrograph inFIG.7, as compared with the locations of the Ni regions in the first micrograph inFIG.7. To further test this hypothesis, additional reactor runs were performed using a second group of monoliths that were substantially the same in composition as the monoliths from Example 5A. The second group of monoliths were used to perform methane reforming under two sets of process conditions. First, the second group of monoliths was used to perform methane reforming under the same conditions used in Example 6. This corresponded to having a molar amount of excess O2used during the regeneration step of roughly 10% relative to the stoichiometric amount for combustion. After characterizing the amount of conversion using the conditions in Example 7, the process cycle was changed so that the molar amount of excess O2was roughly 30% during the regeneration step. During each process cycle, the temperature of the mid-point of the reactor was recorded along with the average methane conversion during the first 10 seconds.FIG.11shows the results from using 10% molar excess O2and 30% molar excess O2with the second group of monoliths. For comparison, similar data from Example 6A is also displayed inFIG.11. As shown inFIG.11, using 10% molar excess O2using the new monoliths resulted in substantially the same level of methane conversion, relative to the reactor mid-point temperature, as was observed in Example 6. However, a substantial increase in conversion was observed when the amount of O2in the regeneration step was increased to a molar excess of 30%. It is noted that a molar excess of roughly 24% would have provided the minimum stoichiometric amount of O2needed to oxidize all Ni in the reactor from Ni metal to NiO (or equivalently NiAl2O4). As shown inFIG.11, using a sufficient molar excess of O2to fully oxidize all Ni in the reactor resulted in a substantial activity improvement. This is consistent with the re-dispersion shown in the second SEM image inFIG.7. It is noted that the size-reversing benefits of conversion to the spinel phase (NiAl2O4) are further enhanced relative to a conventional cycle based on the nature of both sintering and formation of the spinel phase. Sintering is a relatively slow process that is accelerated at higher temperatures. When substantial amounts of Ni remain in the metallic state and then are exposed to the peak temperatures that occur during one or more steps of a reaction cycle for performing reforming, the amount of sintering is increased. However, conversion of NiO and Al2O3to spinel phase NiAl2O4also increases sharply with increased temperature. Thus, by providing sufficient oxygen to allow for substantially complete oxidation of Ni to NiO and/or NiAl2O4, the portions of a catalyst system that conventionally would be most susceptible to sintering can instead receive the highest amount of re-dispersion benefit from formation of the spinel phase NiAl2O4. Example 8—Ni/YSZ on Monolith Composed of NiAl2O4 An additional test run was performed in the pilot scale reactor where the first two monoliths were composed of NiAl2O4. For the first two monoliths, an intermediate bonding layer of Al2O3was provided on the NiAl2O4monoliths, followed by a Ni/YSZ catalyst system. The catalyst system washcoat corresponded to roughly 5 wt % of the total weight of the combined catalyst system and monolith. The third and fourth monoliths were similar to the third and fourth monoliths from Example 5A. Thus, the third and fourth monoliths included a washcoat of a Rh/α-Al2O3catalyst system on a monolith composed of α-Al2O3. The monoliths were then exposed to cyclic high temperature reforming conditions similar to those for Example 6.FIG.12shows the methane conversion during the course of the text run. As shown inFIG.12, the catalytic activity was initially good, indicating that Ni/YSZ in combination with Rh/α-Al2O3can be used effectively in multiple zones to achieve desirable reforming activity. However, after roughly 200 hours of exposure, the reforming activity dropped substantially. This is believed to be due to collapse of the first and second monoliths. The collapse of the first and second monoliths was visually confirmed after the test run was finished. Without being bound by any particular theory, it is believed that the cyclic oxidation and reduction environment caused rapid conversion of the underlying NiAl2O4monolith between states of NiAl2O4and (Ni+Al2O3). This cycling between states is believed to cause the structural breakdown of the monolith. Thus, although NiAl2O4can potentially be used to construct a monolith, the benefits of using NiAl2O4for reforming in a cyclic high temperature reforming environment cannot be realized over extended run lengths when NiAl2O4is used as a monolith material. Instead, as shown in Example 7, using NiAl2O4(preferably in the form NiO/NiAl2O4) as a catalyst system deposited on a phase stable monolith provides unexpectedly superior structural stability. It is noted that even after the collapse of monoliths1and2, substantial conversion of methane was still performed. This is believed to be due to the reforming activity provided by the Rh/α-Al2O3catalyst system on the α-Al2O3monoliths used for monoliths3and4. Additional Embodiments Embodiment 1. A method for reforming hydrocarbons, comprising: reacting a mixture comprising fuel and 0.1 vol % or more of O2under combustion conditions in a combustion zone within a reactor to heat one or more surfaces in a reaction zone to a regenerated surface temperature of 800° C. or more, the reaction zone comprising a catalyst system supported on one or more surfaces of a support structure, the catalyst system comprising M and Al2O3, where M is a metal from Groups 3 to 12 of the Periodic Table, the catalyst system optionally further comprising at least one of MO and MAl2O4; exposing the catalyst system to a gas flow to convert at least a portion of the M and Al2O3to MO, MAl2O4, or a combination thereof, the gas flow comprising 100 mol % to 250 mol % of a stoichiometric molar amount of O2for conversion of a molar amount of M in the catalyst system from metallic M to MO, MAl2O4, or a combination thereof exposing a reactant stream comprising a reformable hydrocarbon to the one or more surfaces in the reaction zone to increase the temperature of the reactant stream; and exposing the reactant stream to the catalyst system in the reaction zone at a temperature of 800° C. or more to form a product stream comprising H2and to convert at least a portion of the MO, MAl2O4, or a combination thereof to metallic M. Embodiment 2. The method of Embodiment 1, wherein M comprises Ni, and wherein the one or more surfaces are heated to a regenerated surface temperature of 1000° C. or more. Embodiment 3. The method of any of the above embodiments, wherein the 0.1 vol % or more of O2comprises an excess of O2of 20 mol % or more relative to the stoichiometric amount of O2for combustion of the fuel, and wherein the exposing the catalyst system to a gas flow comprises exposing the catalyst system to the 0.1 vol % or more of O2. Embodiment 4. The method of any of the above embodiments, wherein the 0.1 vol % or more of O2comprises an excess of O2of 30 mol % or more relative to the stoichiometric amount of O2for combustion of the fuel. Embodiment 5. The method of any of the above embodiments, wherein exposing the catalyst system to the gas flow comprises: after the reacting the mixture under combustion conditions, exposing the catalyst system to a catalyst regeneration flow comprising O2. Embodiment 6. The method of any of the above embodiments, wherein the combustion conditions comprise a combustion time, and wherein exposing the catalyst system to the gas flow comprises: periodically extending the combustion time so that the catalyst system is exposed to 100% or more of a stoichiometric amount for combustion of the fuel and 100% or more of a stoichiometric amount of conversion of the molar amount of M in the catalyst system from metallic M to MO, MAl2O4, or a combination thereof. Embodiment 7. The method of Embodiment 6, wherein periodically extending the combustion time comprises extending the combustion time at least once for every 10 instances of the reacting the mixture under combustion conditions. Embodiment 8. The method of any of the above embodiments, wherein the reacting the mixture, the exposing the catalyst system to the gas flow, the exposing reactant stream to the one or more surfaces in the reaction zone, and the exposing the reactant stream to the catalyst system comprise cyclic reforming conditions. Embodiment 9. The method of any of the above embodiments, wherein the reactor comprises a cyclic reaction environment, a reverse flow reactor, or a combination thereof. Embodiment 10. A method for reforming hydrocarbons in a cyclic reaction environment, comprising: reacting a mixture comprising fuel and O2under combustion conditions in a combustion zone within a reactor to heat one or more surfaces in a reaction zone to a regenerated surface temperature of 1000° C. or more, the mixture of fuel and O2comprising 120% or more of a stoichiometric molar amount of O2for combustion of the fuel, the reaction zone comprising a catalyst system supported on one or more surfaces of a support structure, the catalyst system comprising i) Ni and Al2O3, ii) NiO and Al2O3, iii) NiAl2O4, or iv) a combination of two or more of i), ii) and iii); exposing a reactant stream comprising a reformable hydrocarbon to the one or more surfaces in the reaction zone to increase the temperature of the reactant stream; and exposing the reactant stream to the catalyst system in the reaction zone at a temperature of 1000° C. or more to form a product stream comprising H2, a direction of flow for the reactant stream within the reaction zone being reversed relative to a direction of flow for the mixture. Embodiment 11. The method of Embodiment 10, wherein supporting the catalyst system on one or more surfaces of the support structure comprises: forming a washcoat suspension comprising the catalyst system; and exposing the one or more surfaces of the support structure to the washcoat suspension to support the catalyst system on the one or more surfaces of the support structure. Embodiment 12. The method of Embodiment 10, wherein the metal oxide support layer comprises one or more metal oxides, and wherein forming the catalyst system comprises mixing a powder comprising the catalyst with a powder comprising the one or more metal oxides of the metal oxide support layer, the powder comprising the one or more metal oxides of the metal oxide support layer having a surface area of 20 m2/g or less. Embodiment 13. The method of any of Embodiments 10 to 12, wherein the 0.1 vol % or more of O2comprises 125% to 200% of a stoichiometric molar amount of O2for combustion of the fuel. Embodiment 14. The method of any of the above embodiments, wherein the Al2O3comprises α-Al2O3. Embodiment 15. The method of any of the above embodiments, wherein the reacting the mixture comprises heating the one or more surfaces in the reaction zone to a temperature of 1200° C. or more. Additional Embodiment A. A method for reforming hydrocarbons, comprising: reacting a mixture comprising fuel and 0.1 vol % or more of O2under combustion conditions in a combustion zone within a reactor to heat one or more surfaces in a reaction zone to a regenerated surface temperature of 1000° C. or more, the reaction zone comprising a catalyst system supported on one or more surfaces of a support structure, the catalyst system comprising i) Ni and Al2O3, ii) NiO and Al2O3, iii) NiAl2O4, or iv) a combination of two or more of i), ii) and iii); exposing the catalyst system to a gas flow comprising 100 mol % to 250 mol % of a stoichiometric molar amount of O2for conversion of the molar amount of Ni in the catalyst system from metallic Ni to NiO, NiAl2O4, or a combination thereof; exposing a reactant stream comprising a reformable hydrocarbon to the one or more surfaces in the reaction zone to increase the temperature of the reactant stream; and exposing the reactant stream to the catalyst system in the reaction zone at a temperature of 1000° C. or more to form a product stream comprising H2. Additional Embodiment B. The method of claim10, wherein the combustion conditions comprise a combustion time, and wherein exposing the catalyst system to the gas flow comprises: periodically extending the combustion time so that the catalyst system is exposed to 100% or more of a stoichiometric amount for combustion of the fuel and 100% or more of a stoichiometric amount of conversion of the molar amount of Ni in the catalyst system from metallic Ni to NiO, NiAl2O4, or a combination thereof. Additional Embodiment C. The method of any of the above embodiments, wherein the 0.1 vol % or more of O2comprises an excess of O2of 30 mol % or more relative to the stoichiometric amount of O2for combustion of the fuel, and wherein the exposing the catalyst system to a gas flow comprises exposing the catalyst system to the 0.1 vol % or more of O2. While the present invention has been described and illustrated by reference to particular embodiments, those of ordinary skill in the art will appreciate that the invention lends itself to variations not necessarily illustrated herein. For this reason, then, reference should be made solely to the appended claims for purposes of determining the true scope of the present invention. | 135,290 |
11859134 | DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENT(S) Turning first toFIGS.1A-1E, a schematic diagram illustrates an exemplary embodiment of a method and apparatus for thermal processing of biomass. This example of an apparatus according to the present method involves the production of distillate oil for supply to an automobile. The present method involves the production of the base distillates for the manufacture of a low sulfur diesel fuel oil or other fuel distillate types such as kerosene that can be used to power automobiles, boats, planes, trains or power generation equipment. In various embodiments of the present method, heating can be performed at temperatures of between and inclusive of 200° C.-400° C. to both vaporize biomass and activate a catalyst for catalytical depolymerization of the biomass. In some embodiments, vapors from the biomass can be released into a slurry comprising carrier fluid and a catalyst such that the vapors can be dissolved into the liquid phase of the slurry and catalyst cracked. In some embodiments, the by-products of the catalyst cracking process can then be distilled and released from the liquid slurry phase as distillate vapor. In some embodiments, distillate vapor can be cooled, condensed, and used to produce a renewable fuel. Biomass A is obtained as waste product and can be a mixture of biological residues. Water content can be variable, where the present method involves processes specifically for the separation and recovery of both free and bound water from the reaction mixture. Biomass A can be of different compositions, based on source and difference in substance compositions of organic substances making up the biomass. The apparatus includes hopper2for accepting coarse to finely particulate biomass. Arranged on the bottom of the hopper is metering valve3which is connected to particle size reduction device4or from metering valve3along conduit8to pump6via conduit7. In some embodiments, metering valve3is a variable speed controlled rotary valve. In some embodiments, particle size reduction device4is an ultrasound particle size reduction unit. In some embodiments, pump6is air D driven educator or jet. Conduit8is used for when the biomass in its receiving state is finely ground and does not require particle size reduction. Medium to coarse particulate biomass A are reduced to a particle size using the particle size reduction device4to produce a finely ground particulate powder using conduit116. Arranged at particle size reduction device4is metering valve5, such as a variable speed controlled rotary valve, which is connected to slurrying device11, such as a jet mixer. Slurrying device11receives as a primary inlet flow carrier fluid C via conduit10, which can be a hydrocarbon-based oil, such as but not limited to motor oil, vegetable based oil, such as but not limited to canola oil, and/or an animal-derived fat such as, but not limited to, tallow/yellow grease, and as a secondary inlet flow ground biomass from metering valve5via conduit9. In some embodiments, the motor oil is used. Slurrying device11produces mixed slurry B known as the reaction mixture comprising carrier fluid and biomass. From slurrying device11along conduit15, the reaction mixture enters storage vessel16. In some embodiments, storage vessel16is a cone bottom circular steel tank. Alternatively, biomass A can enter vertical storage vessel13via conduit12. In some embodiments, vertical storage vessel13is a vertical circular storage silo. The reaction mixture can be produced by taking biomass A from the bottom of storage vessel13via conduit117, such that metering valve14meters biomass A via conduit117and18into blend vessel20which can be open or closed to the atmosphere and equipped with mixer21. In some embodiments, mixer21is a side entry fixed or swivel mechanical mixer. In some embodiments, metering valve14is a variable speed controlled rotary valve. In some embodiments, blend vessel20is a circular cone bottom steel tank. In some embodiments, mixer21is a side entry fixed or swivel mechanical mixer. Blend vessel20also receives via conduit19carrier fluid C, which can be warm or hot (85° C.). Mixer21mixes biomass A and carrier fluid C to produce reaction mixture E that is a homogeneous blend of biomass and carrier fluid. Alternatively blend vessel20can receive reaction mixture in the form of mixed slurry B from storage vessel16via conduit18where metering valve17meters mixed slurry B. In this case, carrier fluid C addition via conduit19can be optional. In some embodiments, metering valve17is a variable speed controlled rotary valve. Arranged at the bottom of blend vessel20is transport device22for transporting via conduit23reaction mixture E to heating device24. In some embodiments, transport device22is a feeder screw coupled with a slurry pump. In some embodiments, heating device24is a spiral heat exchanger. Heating device24indirectly heats reaction mixture E to temperatures up to 160° C. using as the heating medium steam or hot thermal fluid that enters and leaves the heating device via conduits25and26. From heating device24the hot reaction mixture enters heating vessel28via conduit27. In heating vessel28, the heating medium can be steam or hot thermal fluid which enters and leaves heating vessel28via conduits29and30, respectively. In some embodiments, heating vessel28is a jacketed vessel. Heating vessel28is equipped with mixer118, configured to continuously mix reaction mixture E while heating to vaporize and remove free water as a vapor via conduit33from reaction mixture E. Heating vessel28can be operated at atmospheric conditions or under a vacuum to reduce the boiling temperature for vaporization of the water. Arranged at the bottom of heating vessel28is transport device32for transporting via conduit31dehydrated reaction mixture F to both heating device43via conduit35and to blend vessel36equipped with mixer119via conduit34. In some embodiments, transport device32is a feeder screw coupled with a slurry pump. In some embodiments, heating device43is a spiral heat exchanger. In some embodiments, blend vessel36is a circular cone bottom steel tank that can be open or closed to the atmosphere. In some embodiments, mixer119, is a side entry fixed or swivel mechanical mixer. In blend vessel36, dehydrated reaction mixture F can be mixed with slurried catalyst O. Catalyst O can be delivered from vessel39to blend vessel36via conduit40. Vessel39can include hopper38with a mixer to slurry catalyst O. In some embodiments, the system utilizes a metering device, such as a belt conveyor with weigh scale. In some embodiments, vessel39can be configured to meter catalyst O in blend vessel36. Arranged at the bottom of blend vessel36is transport device42where catalyst slurry G is routed via conduit41from blend vessel36to transport device42. In some embodiments, transport device42is a feeder screw coupled with a slurry pump. From transport device42catalyst slurry G is routed via conduit44to heating device45configured to heat the catalyst slurry to temperatures below the activation temperature of the catalyst. In some embodiments, heating device45is a spiral heat exchanger. From heating device45, heated catalyst slurry G is routed via both conduit46to reactor47of the catalytic depolymerization unit and via conduit73to vaporizer device63. In some embodiments, vaporizer device63is a thermal desorption screw controllable to operate at temperatures from low to high temperature thermal desorption up to temperatures typical for pyrolysis (300° C. to 1000° C.). Heated dehydrated reaction mixture F and heated catalyst slurry G are injected at similar locations within reactor47, such that the blended temperature of the two mixtures can produce a catalytically active biomass slurry at a temperature that is above the catalyst activation temperature, immediately initiating the catalytic process. To supplement and make-up for heat loss associated with vapor H production, a slip stream of hot recovered carrier fluid N from the fractionation tower88is added via conduit56to reactor47. Vapors H produce from distillation and catalyst cracking of both the biomass, and to a lesser extent the carrier fluid are recovered in distillation tower48. Vapors H comprise both water and organic vapors typical of the distillates naphtha, kerosene and diesel. Vapors H recovered in distillation tower48are routed via conduit51into cooling device52. In some embodiments, cooling device52is an aerial cooler. In cooling device52, the vapors are condensed and allowed to gravity drain via conduit53into separator54. In some embodiments, separator54is a two-phase horizontal separator. Separator54can also receive condensed vapors via conduit72via cooling device71from the residual solids management system. Condensed vapors H comprising water and distillate are gravity separated in separator54, such that process water is removed via conduit121for disposal. Distillate is recovered and routed via conduit55into storage tank74. In some embodiments, storage tank74is a circular flat bottom steel tank operating closed to the atmosphere. From storage tank74, distillate to be desulfurized is routed via conduit75to heating device86using as a heating medium of steam or hot thermal fluid via conduit76. In some embodiment, heating device86is a plate and shell heat exchanger. Heating device86heats the distillate to temperatures up to 100° C., where it is routed via conduit87into a desulfurization device78. In some embodiments, desulfurization device78is a selective adsorption media desulfurization unit. Sulfur components associated with the distillate are adsorbed and removed via a selective medium. The selective adsorption media once exhausted can be heat treated to recover the sulfur thus regenerating the media for continued use. Desulfurized distillate recovered from desulfurization device78is routed via conduit79into cooling device80. In some embodiments, cooling device80is an aerial cooler. Cooling device80cools the desulfurized distillate to temperatures below 60° C., where it is routed via conduit81into storage tank82. In some embodiments, storage tank82is a circular flat bottom steel tank operating closed to the atmosphere. Arranged at the bottom of reactor47via conduit49is transport device50that routes residue I via conduit57into heating device58. In some embodiments, transport device50is a feeder screw coupled with a slurry pump. In some embodiments, heating device58is a spiral heat exchanger. Heated residue I is then delivered to separator60via conduit59. Residue I can include spent catalyst, carrier fluid, and residual biomass. Separator60separates light gases from the heated residue prior to gravity discharge via conduit62into vaporizer device63. In some embodiments, vaporizer device63is a thermal desorption screw controllable to operate at temperatures from low to high temperature thermal desorption up to temperatures typical for pyrolysis (300° C. to 1000° C.). Vaporizer63can optionally receive a catalyst slurry G via conduit73to form a catalytic reactive residue for enhanced distillate fuel production. Vaporizer63also receives a nitrogen gas P from a nitrogen gas production apparatus via conduit120to maintain an inert operating environment within the thermal screw to avoid or at least impede oxidation and degradation of the distillate vapor. Vapor H produced by distillation and catalyst cracking within vaporizer63can be due to indirect heating, which can be electric, steam or hot thermal fluid, singularly or in combination is then routed to either conduit65or conduit70. Conduit65route can be used to further catalyst crack the vapor using catalyst cracking vessel69. In some embodiments, catalyst cracking vessel69is a fixed bed of catalyst within a pressure vessel. Vapor H as flows through the fixed bed of catalyst is catalyst cracked to complement the quality of distillate produced by the present method. Catalyst cracked vapor Q is routed via conduit68into a cooling device71. In some embodiments, cooling device71is an aerial cooler. Alternatively vapor H recovered from vaporizer63can be routed directly to cooling device71. From cooling device71, the condensed distillate H is routed via conduit72to separator54. In some embodiments, ash residue R recovered from the outlet of vaporizer63is routed via conduit64into cooling device76such that the cooling medium, which can be return thermal fluid or glycol cooling water, is used to cool the ash residue R and to recover thermal energy prior to being routed via conduit66into a receiving bin67. In some embodiments, cooling device76is a jacketed thermal screw. Desulfurized distillate J is routed from storage tank82via conduit83to transport device84. In some embodiments, transport device84is a centrifugal pump. From transport device84desulfurized distillate J is routed via conduit85to heating device122. In some embodiments, heating device122is a furnace. In some embodiments, the heating device indirectly heats desulfurized distillate J to temperatures up to 400° C. From the heating device122the heated distillate is routed via conduit123into fractionation tower88. Fractionation tower88is configured to separate the distillate into the fractions naphtha, kerosene, diesel and carrier fluid. Naphtha distillate K production is promoted using reflux drum such that the gases are collected off the top of fractionation tower88and routed via conduit89to cooling device90. In some embodiments, cooling device90is an aerial cooler. In some embodiments, the condensed vapors are then routed via conduit91into separator92. Separator92releases non-condensable gases via conduit101which can be flared or used as fuel gas within the process. Naphtha distillate K that is condensed is routed via conduit93to transport device94which is configured to split the flow via the use of control valves to recycle a portion of Naphtha distillate K back to fractionation tower88via conduit95. In some embodiments, transport device94is a centrifugal pump. The remainder of the Naphtha distillate K is routed via conduit96to cooling device97for further cooling. From cooling device97the cooled distillate is routed into condenser99via conduit98. Conduit100is used to route the naphtha distillate K to its storage system. In some embodiments, cooling device97is an aerial cooler. Similarly, kerosene distillate L recovered from fractionation tower88is routed via conduit102to cooling device103. In some embodiments, cooling device103is an aerial cooler. From cooling device103the cooled distillate is routed into condenser105via conduit104. Conduit106is used to route the kerosene distillate L to its storage system. Similarly, diesel distillate M recovered from fractionation tower88is routed via conduit107to cooling device108. In some embodiments, cooling device108is an aerial cooler. From cooling device108the cooled distillate is routed into condenser110via conduit109. Conduit111is used to route the diesel distillate M to its storage system. Arranged at the bottom of fractionation tower88is the outlet for the recovery and recycling of the carrier fluid. This carrier fluid is recycled for slurrying, while a portion of the stream, recovered carrier fluid N, is recycled to reactor47for direct heating via conduit56. Another embodiment of an apparatus and method for thermal processing biomass is shown in the schematic diagram ofFIGS.2A-2E. In at least some embodiments, the described apparatus and method can use a carrier fluid derived from external renewable resources to produce a low carbon intensity renewable fuel. In some embodiments, the carrier fluid can be derived from mainly, if not entirely, renewable resources and replace oils such as petroleum, vegetable oil, and/or animal fat-based oils. Distillates produced from renewable resources, such as those produced by the processes and apparatuses described herein and/or acquired from an external source, can to be used as the carrier fluid to slurry biomass and other feedstocks, in lieu of other carrier fluids derived from petroleum-based oils or thermal fluids. In some embodiments, the process and apparatuses described herein can substitute or replace the petroleum-based carrier fluid with a renewable distillate as a slurring agent. In some embodiments, this can be achieved by using an initial charge of renewable diesel distillate obtained from an outside source to slurry the biomass for the production of a renewable distillate. In at least some of these embodiments, once the process produces sufficient volumes of renewable raw distillate, use of the outside source can be discontinued, and a portion of the raw renewable distillate generated by the process can be recycled for use as a carrier fluid. Renewable distillate P can be delivered to storage tank125via conduit124. In some embodiments, distillate P can be raw or treated distillate. In at least some embodiments, distillate P can be biomass made from biological waste materials derived from industrial operations including, but not limited to, wood residues, sawdust, cellulose from paper production, as well as other substances such as grains, straw, and/or corn. In other embodiments, distillate P can be a diesel and/or kerosene-based distillate. Distillate P can be delivered to blend vessel20via conduit127and used as a carrier fluid to slurry biomass A to produce reaction mixture E that can be a homogenous blend of biomass and carrier fluid. In some embodiments, addition of distillate P via conduit127can be optional. In some embodiments, once the present apparatus and method produce sufficient volumes of internal renewable distillates, the use of external renewable distillate P to slurry biomass A can be discontinued and replaced with renewable raw distillate R and/or renewable treated distillate S. In some embodiments, renewable raw distillate R from storage tank74can be recycled and delivered to storage tank125via conduit126. In some embodiments, raw distillate R can be subsequently used as a carrier fluid for biomass slurrying by delivering raw distillate R to blend vessel20via conduit127. In some embodiments, renewable treated distillate S from storage tank82can be recycled and delivered to storage tank125via conduit128. In some embodiments, treated distillate S is delivered following treatment for sulfur removal. In some embodiments, treated distillate S can be subsequently used as a carrier fluid for biomass slurrying by delivering treated distillate S to blend vessel20via conduit127. In some embodiments, combinations of distillate P, distillate R, and/or distillate S can be used as carrier fluid for biomass slurrying. In at least some embodiments, recycling distillate R and/or distillate S can create a diesel fuel made from close to, if not entirely, 100% renewable sources. In at least some embodiments, recycling distillate R and/or distillate S can reduce the amount of contaminates introduced during the production process. In some embodiments, the present method and apparatus can be used to generate a close to, if not an entirely 100% renewable, low carbon intensity fuel. Another embodiment of an apparatus and method for thermal processing biomass is shown in the schematic diagram ofFIGS.3A-3E. In at least some embodiments, the described apparatus and method can render unnecessary the use of a carrier fluid by employing a vaporizer to vaporize the biomass. In at least some of these embodiments, the method can include apparatuses with operating temperatures capable of directly vaporizing biomass and/or other feedstocks. In some embodiments, this can be achieved by operating a vaporizer at higher temperatures. In some embodiments, the vaporizer can be operated at temperatures of 200° C.-1200° C. In some embodiments, the vaporizer is operated in an inert environment. In some embodiments, an inert environment can be achieved using an inert gas, such as but not limited to nitrogen, as a blanketing gas. In some preferred embodiments, operating the vaporizer at temperatures greater than 600° C., under non-oxidizing conditions, can vaporize the biomass directly without the use of a carrier fluid. In some embodiments, the residual waste created by vaporization of biomass can be reduced. In some embodiments, the residual wastes include the hot ash underflow from the vaporizer. In some embodiments, direct vaporization of biomass and other feedstock can reduce operating costs. In some embodiments, the elimination of a carrier fluid provides significant operating cost savings. In some embodiments, biomass can be conveyed into a vaporizer via enclosed screw conveyors. In some embodiments, the biomass can be indirectly heated to pyrolysis temperatures. In some embodiments, the pyrolysis temperature is between and inclusive of 600° C.-1200° C. In some embodiments, use of pyrolysis temperatures results in vaporization of 45-85% of the biomass from a solid to a gaseous vapor. In some embodiments, the vapor can be conveyed into a reactor where the vapor is catalyst cracked using a fixed-bed reactor filled with catalyst. In some embodiments, the vapor can be catalyst cracked as the gaseous vapor flows through a fluidized bed, whereby the catalyst can be suspended in a gaseous stream comprising of an inert gas such as nitrogen. In some embodiments, a vaporizer can include a reactor whereby the biomass can be indirectly heated under a non-oxidizing environment using an inert gas such as nitrogen. In some embodiments, the vaporizer can be a thermal screw designed to operate at temperatures between and inclusive of 200° C.-1200° C. for continuous operation or as reactors with mixers equipped with heating jackets for batch operation. In some embodiments, the heating medium(s) for indirect heating of the biomass can be, among other things, hot combustion gases, steam and/or heat generated by the use of electric heating elements. In at least some embodiments, the heating medium(s) is/are capable of heating the biomass to temperatures of at least 1200° C. In some embodiments, vaporization of biomass can reduce the quantity of residual waste through the distillation and vaporization of the volatile organic fraction in the underflow produced by the catalytic depolymerization reactor, leaving the inserts and heavy distillate fractions for disposal. In some embodiments, the vaporizer at operating temperatures between and inclusive of 200° C.-1200° C. can vaporize residual biomass and carrier fluid. In some embodiments, in the absence of a catalyst, the vaporizer can be used to distill and vaporize oil and/or distillate-based carrier fluids such that they can be recovered and recycled, while simultaneously reducing the quality and volume of waste requiring third party disposal. In some embodiments, the vaporizer can be used to vaporize residual biomass that is not converted into a distillate vapor in the catalytic depolymerization step, increasing the overall yield of the process. In some embodiments, a catalyst can be added directly to the biomass, mixed with or without a carrier fluid. In some embodiments, the gaseous vapor produced in operation of the vaporizer can be treated with a catalyst using either a reactor with fixed bed of catalyst media or a fluidized bed whereby the catalyst is suspended using an inert gas for contact with the gaseous vapor. In some embodiments, the gaseous vapor can be optionally catalyst cracked for the production of distillate or alternatively cooled and condensed for the recovery of the carrier fluid, depending on the selected application and mode of operation. In some embodiments, biomass A can be delivered from storage vessel13and/or16to screw conveyor129via conduit130and then transferred to vaporizer63via conduit131. In at least some embodiments, use of conveyor129can be used to bypass apparatuses up to but not including separator54, thereby removing the need for a carrier fluid to treat biomass A. In some embodiments, vaporizer63can include electric heating elements. In some embodiments, the heating elements of vaporizer63can be mounted on the outer shell of vaporizer63to indirectly heat biomass A to temperatures up to and including 1200° C. In a least some embodiments, at temperatures between and inclusive of 200° C.-1200° C., biomass A can be converted from a solid to a vapor. In some embodiments, vaporization and catalyst cracking of biomass A can occur in vaporizer63. In at least some of these embodiments, vapor can be delivered via conduit65to catalyst cracking vessel69for further catalyst cracking. In some embodiments, a renewable distillate vapor can be produced via catalyst cracking the vapor in vaporizer63and/or catalyst cracking vessel69. In some embodiments, vaporizer63is configured to independently operate the process of receiving catalyst G via conduit73from the process of receiving biomass A via conduit131. In some embodiments, vaporization rather than carrier fluid treatment of biomass A can lower production costs. FIGS.4A-4Eillustrate an embodiment of an apparatus and method for thermal processing biomass that can desulfurize and treat carrier fluids to remove potential sulfur contaminants from carrier fluids. Sulfur and other contaminants can be present in carrier fluids derived from industrial waste processes such as used motor oil or other external distillates. In some embodiments, desulfurization of carrier fluids can be performed on external (carrier fluids from outside sources) or internal (carrier fluids derived from within the process) carrier fluids which can include recovered motor oil from the fractionation step and/or raw distillate recycled for use as a carrier fluid for the production of a renewable distillate. Desulfurized and treated carrier fluids can be used to slurry feedstock including biomass A. In some embodiments, desulfurization of carrier fluid can reduce the contaminant load on subsequent process components including desulfurization unit78. Desulfurization of carrier fluid can remove, or at least reduce, contaminates including but not limited to sulfur, metals, salts, aromatics, mercaptans, and suspended solids. In some embodiments, contaminates are recovered as a waste by-product sludge that can contain a range of solids from 10-35% by weight. In some embodiments, desulfurization of carrier fluids can involve steps of filtration, heating, chemical and/or clay treatment, sedimentation and/or neutralization, whereby the process steps can be conducted independently or in conjunction with other process steps depending on the type and level of contaminates to be removed. In some embodiments, chemical treatments can involve acids, bases, and/or caustic chemicals. In some embodiments, oil-based carrier fluids derived from industrial wastes can undergo filtration and heating to remove debris, water, suspended solids, and/or water. In some embodiments, oil-based carrier fluids derived from industrial wastes do not undergo filtration and heating to remove debris and can immediately undergo desulfurization and chemical treatment to remove contaminants including, but not limited to, metals, salts, acids, aromatics, asphaltenes, and sulfur. In some embodiments, treatment can involve acid or caustic treating through the mixing of sulfuric acid or caustic chemicals, including but not limited to sodium hydroxide, with the carrier fluid resulting in the partial or complete removal of unsaturated hydrocarbons, sulfur, nitrogen, oxygen compounds and resinous and asphaltic compounds. In at least some embodiments, treatment can improve the color, stability, odor and carbon residue of the oil. In some embodiments, mixing sulfuric acid and/or a caustic with oil-based carrier fluids can form a by-product of sludge that settles out of the oil. In some embodiments, the sludge can be gravity separated from its bulk fraction and centrifuged to produce a stackable waste the can be disposed. In at least some embodiments, the remaining slightly acidic oil can be either filtered and/or mixed with active fuller's earth (also known as clay) to remove mercaptans and additional sulfur. In some embodiments, when clay is mixed with the carrier fluid, impurities are gravity settled as a sludge, resulting in further contaminant reduction. In various embodiments of the method, treatment and desulfurization can be employed separately in the treatment of carrier fluids, including those derived from industrial processes, or used in conjunction with desulfurization by selective adsorption when treating raw distillates. In some embodiments, when used in conjunction with desulfurization by selective adsorption, the desulfurization and decontamination method can first remove the bulk of sulfur contamination and desulfurization by selective adsorption can then serve as a polishing step. In some embodiments, this multi-step process of desulfurization, decontamination by chemical treatment, and desulfurization by selective adsorption can reduce the sulfur content in the distillate to levels such that the distillate meets sulfur diesel fuel manufacturing standards. In some embodiments, desulfurization of carrier fluid lowers manufacturing costs by reducing the size and/or quantity of equipment and materials needed for desulfurization by selective adsorption process. In some embodiments, desulfurization and/or chemical treatment of carrier fluid to remove the bulk of the sulfur contamination from a carrier fluid and/or from a raw distillate can reduce the sulfur loading on the selective adsorption process. In at least some embodiments, this can result in longer run times and/or greater media service life of the selective adsorption process. Carrier fluid U can be imported into the process from external sources via conduit133and stored in storage tank132. In some embodiments, storage tank132can include a flat bottom circular tank equipped with or without an internal floating roof capable of reducing emissions from the tank. In some embodiments, storage tank132can include a heating element such as internal heating coil149that houses heating medium Z. Heating medium Z can be a fluid or gas distributed through heating coil149that functions to indirectly heat carrier fluid U in storage tank132. In some embodiments, storage tank132can be equipped with electric heater(s)152that function to directly heat carrier fluid U in storage tank132. Heating coil149and/or electric heaters152can be used to heat and maintain carrier fluid U at a temperature between and inclusive of 60° C.-90° C. In some preferred embodiments, the temperature of carrier fluid U prior to entry into conduit151is approximately 70° C. In some embodiments, storage tank132can receive recovered carrier fluid recycled from the process via conduit145, following water removal in heating vessel28. In some embodiments, carrier fluid is not heated in storage tank132and can be delivered to heat exchanger150via conduit151. In some embodiments, suitable heat exchangers can include those with a shell and tube, spiral, and/or double pipe design. In some embodiments, heat exchanger150can receive raw distillate from conduit142. Heat exchanger150can utilize a fluid or gas heating medium Z to heat raw distillate, external carrier fluid, and/or internal carrier fluid. In some embodiments, heat exchanger150can include in-line electric heaters. In some embodiments, heating medium Z and/or the electric heaters can be used to heat and maintain carrier fluid U at a temperature between and inclusive of 60° C.-90° C. In some embodiments, heated carrier fluid can be delivered to mix tank135via conduit133. Mix tank135can be comprised of single or multiple mix compartments equipped with mechanical mixer(s). In particular embodiments, mix tank135can include a first compartment with mechanical mixer143and a second compartment with mechanical mixer136. In some embodiments, as the carrier fluid passes from the first compartment to the second compartment, the rate and/or force of mixing can be reduced to allow solids and other precipitates and flocculates to settle via gravity sedimentation. In some embodiments, carrier fluid in mix tank135can be injected with chemicals and/or additives. In some embodiments, carrier fluid is injected with acid X via conduit146, base V via conduit140, and/or additive Y via conduit144. In some embodiments, acid X can be a strong acid such as, but not limited to, sulfuric acid with a concentration strength between and inclusive of 30%-90%. In some or the same embodiments, base V can be a strong base such as, but not limited to, soda ash, sodium hydroxide, or lime with a concentration strength between and inclusive of 30%-90%. In some embodiments, additive Y can be a clay that functions to trap and absorb contaminates as well as aid flocculation and sedimentation of the sludge. The type, concentration, and combination of acid X, base V, and additive Y can be selected based on the nature of contaminates to be removed from the carrier fluid. In some embodiments, contaminates in the carrier fluid can react with acid X, base V, and/or additive Y to produce a sludge containing a concentrated slurry. In some embodiments, the concentrated slurry is delivered from mix tank135to gravity sedimentation tank138via conduit137. In sedimentation tank138, solids in the concentrated slurry settle to the bottom of tank. In some embodiments, the sludge that settles to the bottom of sedimentation tank138can contain a range of solids from 10-35% by weight. In some embodiments, the sludge can be delivered to dewatering unit153via conduit139. In at least some embodiments, dewatering unit153functions to remove free liquid from the sludge to produce solid waste155that can be removed from the system via conduit154for disposal. In some embodiments, dewatering unit153can include a solid bowel centrifuge, dewatering screw conveyor, and/or dewatering press. In at least some embodiments, liquid removed from the sludge can removed via conduit156and then recycled into the process and/or disposed. In some embodiments, the supernatant W of the concentrated slurry can overflow via a weir within sedimentation tank138and then be delivered to reactor146via conduit141. In some embodiments, chemical additive AA can be injected into reactor146via conduit147and mixed with the incoming supernatant. In at least some embodiments, additive AA can be, among other things, soda ash, lime, sodium hydroxide or other suitable caustic chemicals that function to neutralize the desulfurized supernatant in reactor146. In some embodiments, the desulfurized and treated carrier fluid can be delivered to blend vessel20via conduit159to slurry biomass A or other feedstock. In a particular embodiment of the above-described apparatus and method, raw distillate can be desulfurized to reduce the sulfur and contaminate load of raw distillate. In some embodiments, such desulfurization can reduce the subsequent sulfur loading onto desulfurization device78. Distillate treated and recovered as described above can be delivered from reactor146to cooler158via conduit148. In some embodiments, cooler158can be an aerial cooler that functions to reduce the temperature of distillate to less than or equal to 45° C. In some embodiments, desulfurized and cooled distillate can be returned to the process via conduit157. In at least some embodiments, distillate passed through cooler158does not require additional sulfur-removal treatment. In some of these embodiments, the desulfurized and treated distillate can bypass desulfurization device78via conduit160. In some embodiments, such as when the distillate requires further sulfur removal, distillate can be delivered to heating device86via conduit75and desulfurization device78via conduit87. In some of these embodiments, desulfurization device78can act as a polishing step for sulfur removal. In some embodiments, carrier fluid C can be utilized and added to blend vessel28via conduit143. In some embodiments, carrier fluid C can be a renewable distillate. In at least some of these embodiments, carrier fluid C functions as a temporary distillate source until the process produces raw renewable distillate that can be recycled as carrier fluid. Such a starting distillate source can be used for producing low carbon intensity renewable fuel. In some embodiments, raw distillate can bypass desulfurization and be directly introduced as carrier fluid into blend vessel28via conduit143. The method described herein to desulfurize carrier fluid does not require every step depending on the application, source of the carrier fluid, and/or degree and nature of contaminates. The steps of the method can be conducted separately, in combination and suitable derivations thereof. For example, in some embodiments, the method can include treatment of carrier fluid with acid X, mixing in mix tank135, gravity sedimentation in tank138, and/or dewatering in unit153. Some embodiments of the apparatus and method can include treatment of carrier fluid with base V, mixing in mix tank135, gravity sedimentation in tank138, and/or dewatering in unit153. In some embodiments, treatment of carrier fluid using additive Y and neutralization using chemical AA can be optional. Some of the embodiments of the apparatus and method described herein can produce a distillate that can be subsequently used to manufacture renewable, low-sulfur diesel fuel and/or other fuel distillates including, but not limited to, kerosene that can be used to power automobiles, boats, planes, trains, and/or power generation equipment. Such embodiments can be used to produce naphtha which can be used in industrial applications including as diluent for heavy oil transportation. The present method can operate using feedstock materials other than biomass. Waste plastics typical of sorted municipal solid waste can also be substituted for biomass and found to produce distillate oil that can be used for fuel production. While particular elements, embodiments and applications of the present invention have been shown and described, it will be understood, that the invention is not limited thereto since modifications can be made by those skilled in the art without departing from the scope of the present disclosure, particularly in light of the foregoing teachings. | 38,449 |
11859135 | DETAILED DESCRIPTION OF THE INVENTION FIG.1shows the integration process for the production of liquid fuels. Unit1is a Solid Oxide Electrolyzer (SOE) or a Solid Oxide Electrolysis Cell (SOEC). Unit1is a high temperature electrolyzer that operates at temperatures from 550° C. to 900° C. The SOEC operates at ambient or near ambient pressure. Higher pressure operation is possible with improvements in the stack sealing. In an SOEC, electricity is fed to the system to drive electrochemical reactions. In this SOEC, the electricity used is low carbon electricity including electricity produced from wind, solar, nuclear, or hydro power. The SOEC comprises a cathode and an anode with an electrolyte. The reactants are fed to the SOE cathode, which is the electrode where reduction takes place. The anode is the electrode where oxygen is obtained. In the SOEC, water is split into hydrogen and oxygen ions at the cathode. The oxygen ions diffuse across a ceramic membrane, and oxygen is produced at the anode by combination of oxygen ions using heat and electrochemical gradients as driving force. See equation 1 below. When carbon dioxide is also supplied, co-electrolysis takes place, and the carbon dioxide is reduced and produces carbon monoxide at the cathode and oxygen at the anode. See equation 2 below. H2O(g)+2e-→H2+O2-ΔHO=241.57kJmolEq.1CO2+2e-→CO+O2-ΔHO=282.79kJmolEq.2 ΔH0is the energy (enthalpy) necessary for the reaction(s) at standard temperature of 25° C. Both reactions are endothermic and require the addition of energy to be completed. AH is adjusted for the operating temperature of the SOEC. It should be noted that Eq. 1 is for gaseous water or steam as the feedstock. If the water is in the liquid state, then ΔH0=285.84 kJ/mol. Eq. 3 shows the relationship between the enthalpy and the Gibbs Free Energy. The Gibbs free energy is the electrical work depending on the reversible potential between the cell electrodes (Erev, Eq. 4). Entropy is supplied in the form of heat. When the irreversible thermal losses are equivalent to the heat demand, the energy balance is obtained, and energy equilibrium takes the name of thermoneutral, Etn. ΔH=ΔG+TΔS Eq.3 ΔG=ΔH/zF ErevEq.4 Etn=ΔHzF=Erev+TΔSzFEq.5 The SOEC efficiency (Eq. 6) is calculated as the ratio between chemical energy exiting the system, in terms of enthalpy and the electrical energy fed from the outside (Ee). η=ΔHEeEq.6 Where Eeis the electrical energy input equal to the current (z * F) multiplied by the operating potential E. The efficiency is equal to 1 at the thermoneutral conditions where all electrical energy and relative heat losses are converted into chemical energy. As seen inFIG.1, the SOEC Unit1cathode is fed by Feed Stream1that consists of carbon dioxide. Feed stream1is mixed with a recycled Product Stream5that comprises water vapor (steam) produced as part of the heat management/steam system of Unit4, liquid fuel product reactor. This becomes the cathode feed stream. Additional steams can also be added to make the combined cathode feed stream. These additional streams may contain optional hydrogen. Hydrogen is useful as a co-feed to keep the cathode in reduction mode. Hydrogen can be recycled from the SOEC cathode product or can be produced in other areas of the facility including hydrogen that is produced in a hydrocarbon reforming unit from light hydrocarbons produced in unit4. Additionally, light hydrocarbons produced in the Unit4(LFP) can also be part of the cathode feed. The fuel electrode (cathode) material is a Ni doped YSZ. However, high steam partial pressures and low hydrogen partial pressures at the Ni-YSZ interface often causes oxidation of the nickel which results in catalyst degradation. The hydrogen in the cathode feedstock can aid in overcoming this problem. Instead of, or in addition to Ni-YSZ as a cathode material, there are perovskite-type lanthanum strontium manganese (LSM) and lanthanum strontium manganese chromate (LSCM) that can be used instead of or in combination with Ni-YSZ. On the anode side, the oxygen ions are produced in Eq. 1 and Eq. 2. In an SOEC, it is beneficial to use a sweep gas over the anode to sweep the anions (and oxygen gas) away from the anode. Feed stream2inFIG.1is the sweep gas stream that goes to the anode. The sweep gas can be chosen from several gases. The sweep gas can be chosen from air or steam. The Product Stream1is an oxygen rich gas with an oxygen percentage above the oxygen content of air when air is used as the sweep gas. The anode materials can be chosen from a number of materials including lanthanum strontium manganate (LSM), Gd-doped CeO2impregnated LSM, and neodymium nickelate or combinations thereof. The electrolyte used in the SOEC is a dense ionic conductor consisting of ZrO2doped with 8 mol % Y2O3 (also known as YSZ). Zirconia dioxide is used because of its high strength, high melting temperature (approximately 2700° C.) and excellent corrosion resistance. Other electrolyte materials include Scandia stabilized zirconia (ScSZ), ceria-based electrolytes or lanthanum gallate materials. All materials can be used separately or in combinations. Product stream2or the hot cathode product comprises hydrogen and carbon monoxide. The product stream leaves the cathode at the SOEC operating temperature of 550 to 900° C. The product stream can be used to heat the incoming cathode feed via a feed/product heat exchanger. The cathode feed stream comprises steam and carbon dioxide. Product stream1is the hot anode product stream that comprises oxygen or an oxygen-enriched sweep gas. The product stream leaves the cathode at the SOEC operating temperature of 550 to 900° C. The product stream can be used to heat the Feed Stream2or incoming anode feed via a feed/product heat exchanger. The cooled Product Stream2or cathode product comprises synthesis gas. The steam goes to Unit2or the syngas conditioning unit. The syngas conditioning block is actually a collection of a number of unit operations. It includes additional cooling, water removal, adjustment of the hydrogen to carbon monoxide ratio, carbon dioxide removal, and compression to raise the pressure to the Unit4(LFP reactor). This produces a Product Stream3that becomes the LFP Feed stream that comprises syngas with a hydrogen to carbon monoxide molar ratio of 1.5 to 2.5, or more preferably 2.0 to 2.2. Besides the hydrogen and carbon monoxide in product stream2, other components are likely in the stream including unreacted carbon dioxide, unreacted hydrogen, and water. Water is removed in a flash drum. The water removed becomes Product Stream7inFIG.1and can be optionally recycled to the liquid water that can be blended into Feed Stream3. If the hydrogen to carbon monoxide ratio in the syngas conditioning unit feed is greater than 2.2, excess hydrogen can be removed from the stream by any know hydrogen removal system such as pressure swing adsorption (PSA), hydrogen membranes, or other methods. The excess hydrogen containing stream can be recycled to the front end of the process to supplement the Unit1, and blended SOEC Cathode feed. If the hydrogen to carbon monoxide is less than 2.0, additional hydrogen can be produced by the water gas shift reaction (Eq. 7). H2O+CO↔H2+CO2Eq.7 If required to meet the syngas composition specification, a catalytic reactor can be used to convert some of the CO to additional hydrogen. Removal of carbon dioxide in the syngas is useful as carbon dioxide is a diluent in downstream processing. Carbon dioxide removal is done by any of a number of different processes including: 1) amine absorbers, 2) Rectisol or methanol absorbers, 3) Other physical absorbers like Solexol, 4) Cryogenic CO2fractionation. The captured and removed CO2will result in a CO2 stream that can be sent back to the front end of the process to add to the Feed Stream1. The operating pressure of the SOEC is near ambient pressure. Unit4, the LFP reactor operates at pressures of 250 to 450 psig. The syngas conditioning block, unit2, comprises a syngas compressor. This will be a multi-stage compressor system with interstage cooling. Unit2produces a Product Stream3that is the feed stream to Unit4. Unit4is the Liquid Fuel Production (LFP) reactor.FIG.2refers to the details of the LFP reactor. The LFP feed stream comprising hydrogen and carbon monoxide enter the LFP reactor. The LFP reactor is a multi-tubular fixed bed reactor that allows the Fischer-Tropsch (F-T) reaction of Eq. 8. nCO+(2n+1)H2→CnH2n+2+nH2OEq.8ΔH0=-165kJmolCO The F-T reaction is exothermic with a standard enthalpy of reaction released of 165 kJ/mol of CO converted. The high heat of reaction makes control of the F-T reactor temperature critical. If uncontrolled, the temperature rise in the reactor would result in higher methane production and higher catalyst deactivation. As such control of the temperature is important. Ideally the temperature of the F-T reactor is maintained at 200 to 240° C. The multi-tubular fixed bed reactor ofFIG.2aids in the control of the temperature of the F-T reactor. Syngas is fed to the reactor and is split to go through the tubular catalytic reactors (Unit6inFIG.2). The syngas reactor produces a mixture primarily consisting of alkanes, alkenes, and alcohols as shown by the F-T reaction (Eq. 8). The mixed hydrocarbons, Product Stream6, leaves the tubular reactor. Feed stream3, comprising liquid water, is fed to Unit3′(FIG.1). This stream can optionally include water that is separated out from Unit2, syngas conditioning unit. Unit3comprises water purification, treatment, and pumping. Purification can include reverse osmosis or other purification systems. Treatment of the water includes addition of chemicals, de-ionization, etc. The water is pumped to a pressure of 100 to 300 psig and preferably about 200 psig to produce product stream4. Pressurized and treated cooling water, Product Stream4, is fed into Unit5, the boiler portion of the multi-tubular reactor. The tubular catalytic reactors are surrounded by the Unit5boiler (FIG.2). The heat of reaction supplies the heat that allows the vaporization of the liquid water to steam. In some embodiments of the invention, a mixture of steam and liquid water are fed to Unit5that allows some of the liquid water to additional steam. In some embodiments, a mixture of steam and liquid water leave Unit5. A steam drum is used to separate the water and steam. The liquid water is recirculated as feed to the Unit5boiler. The steam produced in Unit5boiler is Product Stream5steam that is sent back to the front end and mixed with feed stream1and becomes feed to Unit1, SOEC. The ratio of liquid water to steam is typically about 0.5 but can range from 0.2 to 0.7. The hydrocarbon product from the LFP reactor is further processed downstream of the Unit4LFP reactor. Water is also produced in the F-T reaction (Eq. 8). Water can be removed from the F-T product stream by a flash drum. The recovered water can also be recycled back to the liquid water processing, Unit3, inFIG.1. The light hydrocarbons are also separated from the LFP hydrocarbon product which are primarily alkanes and alkenes with carbon numbers of one to five. The LFP reactor product that contains the desired C5-C23hydrocarbons can be further processed in a separation system. The separation system can include distillation. The desired C5-C23products can be used for gasoline blendstock, diesel fuel, jet fuel, or used as low-carbon chemicals that can displace chemicals derived from petroleum or natural gas. In one embodiment, the LFP product is sent to a series of fractionators are used to create a high cetane diesel fuel with an adjustable flash point between 32-50° C. (90-122° F.) and a stabilized naphtha (potentially a gasoline blend stock or chemical feedstock). A basic arrangement for these columns, which include:A) Wax Stripper—This unit uses steam to recover fuel-range components from the waxy material. The overhead fuel-range components and steam are sent to the Main Fractionator while the stripped wax is sent to heated storage for sales. The Wax Stripper is a column without a condenser or reboiler, operating at approximately 170° C. (340° F.) and with enough pressure, 2.75 barg (40 psig), for the overhead vapors to enter the Main Fractionator column.B) Main Fractionator—This column splits the raw fuel into naphtha and diesel range components to control the diesel flash point. This column includes a high pressure (HP) steam heated reboiler, and an external condenser with 3-phase separation for removing absorbed water and steam from the wax stripper feed.C) Optional Naphtha Stabilizer to control the Reid vapor pressure (RVP) to a spec of 8 psia. The stabilizer includes a low pressure (LP) steam reboiler, and an integrated knock-back, water-cooled condenser.D) Optional Diesel Cold-Flow/Kerosene vacuum column to adjust the diesel pour point for cold weather sales and/or produce a kerosene cut. The feed is heated to 300° C. (570° F.). The column is 20 stages with an overhead condenser pressure of 6 psia. The kerosene cut may be used as jet fuel component. Under certain conditions the kerosene cut may meet the ASTM specification (ASTM D7566) for use as a jet fuel. In one embodiment of the invention, the kerosene cut of the LFP product does not meet all the ASTM D7566 specification for use as jet fuel. Jet fuel may have a higher value than diesel fuel in certain circumstances. It may be necessary to hydroprocess the LFP product or a fraction of the LFP product such as LFP kerosene or LFP light diesel to meet the specification for use as jet fuel or sustainable aviation fuel (SAF). The hydroprocessing includes the hydroisomerization of the C9-C15alkanes produced in the LFP reactor. The LFP product or a fraction of the product is pressurized and mixed with a stream comprising hydrogen. The hydrogen can be produced from the electrolysis of water or from the reforming of natural gas or from the gasification of waste or biomass. It is preferred that the hydrogen is a low carbon hydrogen. The combined stream comprising hydrogen and at least a portion of the LFP product is then heated and fed to the hydroprocessing reactor. The hydroprocessing reactor operates at an elevated pressure of greater than 100 psig but generally less than 2000 psig. The hydroprocessing reactor operates at a temperature between 250° C. and 400° C. Effluent from the hydroprocessing reactor is cooled before entering the hot separator where gas and liquid are separated in hot and cold separators. The hydrocarbon products from the hot and cold separators are sent to the fractionation section where the light-ends and hydrocarbon products are separated. This fraction system may include a wax stripper and main fractionator as well as a naphtha stabilizer and a kerosene vacuum column. The fractionators are operated in a manner such that the kerosene stream will meet the specifications of ASTM D7566 and is useful as a Sustainable Aviation Fuel (SAF). The following examples illustrate some aspects of the invention. EXAMPLE 1 In this example, 1000 mol/s of liquid water and 500 mol/s of carbon dioxide are fed to an SOEC. 800 mol/s of hydrogen and 400 mol/s of carbon monoxide are produced. Using the heat requirements for the reactions of Eq. 1 and Eq. 2 with liquid water as a feedstock, a minimum electrical demand for the SOEC stack can be calculated. In this example, the minimum electrical requirement is 401.3 MW. EXAMPLE 2 In this example, 1000 mol/s of steam produced in the LFP reactor Unit5boiler are fed to the SOEC instead of the liquid water in example 1. 800 mol/s of hydrogen and 400 mol/s of carbon monoxide are produced in the SOEC. Using the heat requirements for the reactions of Eq. 1 and Eq. 2 with steam as a feedstock, a minimum electrical demand for the SOEC can be calculated. In this example, the minimum electrical requirement is 360.4 MW. Table 1 shows the improvement of Example 2 versus Example 1. The use of steam saves 40.9 MW of electricity and shows a 10.2% improvement in electricity required by the SOEC. The amount of steam that can be produced in the LFP reactor boiler drastically reduces the feed water required for the SOEC. Table 2 below highlights the overall process improvements. With a 50% single pass CO conversion in the LFP reactor, the amount of reaction heat released is equal to 33.0 MW. That amount of reaction heat can produce 745.4 mol/s of steam from water. The remaining 245.3 mol/s required by the SOEC can be partially supplied by recycle of unconverted steam. TABLE 1Comparison of Use of Liquid Water vs LFPSteamExample 1Example 2SOEC Feed StreamsLiquid water, mol/s10000Steam, mol/s01000Carbon Dioxide, mol/s500500SOEC Product StreamsHydrogen, mol/s800800Carbon Monoxide, mol/s400400Minimum Electricity Requirement, MW401.3360.4Electrical Use Improvement, MW40.9Percent Improvement10.2% TABLE 2Steam Production in LFP for Example 2Single pass F-T LFP CO Conversion50%Reaction Heat Produced, MW33.0Steam Production in LFP, mol/s745.4Makeup water required for SOEC, mol/s245.3Reduction in Water Consumption74.5% REFERENCES 1. G. Cinti, A. Baldinelli, A. Di Michele, and U. Desideri, Applied Energy 162 (2016) 308-320. “Integration of Solid Oxide Electrolyzer and Fischer-Tropsch: A sustainable pathway for synthetic fuel”.2. U.S. patent application Ser. No. 17/300,259.3. U.S. patent application Ser. No. 17/300,260.4. U.S. patent application Ser. No. 17/300,262.5. U.S. patent application Ser. No. 16/873,561. | 17,600 |
11859136 | DETAILED DESCRIPTION Various specific embodiments, versions and examples of the invention will now be described, including preferred embodiments and definitions that are adopted herein for purposes of understanding the claimed invention. While the following detailed description gives specific preferred embodiments, those skilled in the art will appreciate that these embodiments are exemplary only, and that the invention may be practiced in other ways. For purposes of determining infringement, the scope of the invention will refer to any one or more of the appended claims, including their equivalents, and elements or limitations that are equivalent to those that are recited. Any reference to the “invention” may refer to one or more, but not necessarily all, of the inventions defined by the claims. In this disclosure, a process is described as comprising at least one “step.” It should be understood that each step is an action or operation that may be carried out once or multiple times in the process, in a continuous or discontinuous fashion. Unless specified to the contrary or the context clearly indicates otherwise, multiple steps in a process may be conducted sequentially in the order as they are listed, with or without overlapping with one or more other steps, or in any other order, as the case may be. In addition, one or more or even all steps may be conducted simultaneously with regard to the same or different batch of material. For example, in a continuous process, while a first step in a process is being conducted with respect to a raw material just fed into the beginning of the process, a second step may be carried out simultaneously with respect to an intermediate material resulting from treating the raw materials fed into the process at an earlier time in the first step. Preferably, the steps are conducted in the order described. Unless otherwise indicated, all numbers indicating quantities in this disclosure are to be understood as being modified by the term “about” in all instances. It should also be understood that the precise numerical values used in the specification and claims constitute specific embodiments. Efforts have been made to ensure the accuracy of the data in the examples. However, it should be understood that any measured data inherently contains a certain level of error due to the limitation of the technique and/or equipment used for acquiring the measurement. Certain embodiments and features are described herein using a set of numerical upper limits and a set of numerical lower limits. It should be appreciated that ranges including the combination of any two values, e.g., the combination of any lower value with any upper value, the combination of any two lower values, and/or the combination of any two upper values are contemplated unless otherwise indicated. The indefinite article “a” or “an”, as used herein, means “at least one” unless specified to the contrary or the context clearly indicates otherwise. Thus, embodiments using “a reactor” or “a conversion zone” include embodiments where one, two or more reactors or conversion zones are used, unless specified to the contrary or the context clearly indicates that only one reactor or conversion zone is used. The term “hydrocarbon” means (i) any compound consisting of hydrogen and carbon atoms or (ii) any mixture of two or more such compounds in (i). The term “Cn hydrocarbon,” where n is a positive integer, means (i) any hydrocarbon compound comprising carbon atom(s) in its molecule at the total number of n, or (ii) any mixture of two or more such hydrocarbon compounds in (i). Thus, a C2 hydrocarbon can be ethane, ethylene, acetylene, or mixtures of at least two of these compounds at any proportion. A “Cm to Cn hydrocarbon” or “Cm-Cn hydrocarbon,” where m and n are positive integers and m<n, means any of Cm, Cm+1, Cm+2, . . . , Cn−1, Cn hydrocarbons, or any mixtures of two or more thereof. Thus, a “C2 to C3 hydrocarbon” or “C2-C3 hydrocarbon” can be any of ethane, ethylene, acetylene, propane, propene, propyne, propadiene, cyclopropane, and any mixtures of two or more thereof at any proportion between and among the components. A “saturated C2-C3 hydrocarbon” can be ethane, propane, cyclopropane, or any mixture thereof of two or more thereof at any proportion. A “Cn+ hydrocarbon” means (i) any hydrocarbon compound comprising carbon atom(s) in its molecule at the total number of at least n, or (ii) any mixture of two or more such hydrocarbon compounds in (i). A “Cn-hydrocarbon” means (i) any hydrocarbon compound comprising carbon atoms in its molecule at the total number of at most n, or (ii) any mixture of two or more such hydrocarbon compounds in (i). A “Cm hydrocarbon stream” means a hydrocarbon stream consisting essentially of Cm hydrocarbon(s). A “Cm−Cn hydrocarbon stream” means a hydrocarbon stream consisting essentially of Cm−Cn hydrocarbon(s). For the purposes of this disclosure, the nomenclature of elements is pursuant to the version of the Periodic Table of Elements (under the new notation) as provided in Hawley's Condensed Chemical Dictionary, 16thEd., John Wiley & Sons, Inc., (2016), Appendix V. For example, a Group 8 element includes Fe, a Group 9 element includes Co, and a group 10 element includes Ni. The term “metalloid”, as used herein, refers to the following elements: B, Si, Ge, As, Sb, Te, and At. In this disclosure, when a given element is indicated as present, it can be present in the elemental state or as any chemical compound thereof, unless it is specified otherwise or clearly indicated otherwise by the context. The term “alkane” means a saturated hydrocarbon. The term “cyclic alkane” means a saturated hydrocarbon comprising a cyclic carbon ring in the molecular structure thereof. An alkane can be linear, branched, or cyclic. The term “aromatic” is to be understood in accordance with its art-recognized scope, which includes alkyl substituted and unsubstituted mono- and polynuclear compounds. The term “rich” when used in phrases such as “X-rich” or “rich in X” means, with respect to an outgoing stream obtained from a device, e.g., a conversion zone, that the stream comprises material X at a concentration higher than in the feed material fed to the same device from which the stream is derived. The term “lean” when used in phrases such as “X-lean” or “lean in X” means, with respect to an outgoing stream obtained from a device, e.g., a conversion zone, that the stream comprises material X at a concentration lower than in the feed material fed to the same device from which the stream is derived. The term “mixed metal oxide” refers to a composition that includes oxygen atoms and at least two different metal atoms that are mixed on an atomic scale. For example, a “mixed Mg/Al metal oxide” has O, Mg, and Al atoms mixed on an atomic scale and is substantially the same as or identical to a composition obtained by calcining an Mg/Al hydrotalcite that has the general chemical formula [Mg(1-x)Alx(OH)2](Axnn-)·mH2O], where A is a counter anion of a negative charge n, x is in a range of from >0 to <1, and m is ≥0. A material consisting of nm sized MgO particles and nm sized Al2O3particles mixed together is not a mixed metal oxide because the Mg and Al atoms are not mixed on an atomic scale but are instead mixed on a nm scale. The term “selectivity” refers to the production (on a carbon mole basis) of a specified compound in a catalytic reaction. As an example, the phrase “an alkane hydrocarbon conversion reaction has a 100% selectivity for an olefin hydrocarbon” means that 100% of the alkane hydrocarbon (carbon mole basis) that is converted in the reaction is converted to the olefin hydrocarbon. When used in connection with a specified reactant, the term “conversion” means the amount of the reactant consumed in the reaction. For example, when the specified reactant is propane, 100% conversion means 100% of the propane is consumed in the reaction. Yield (carbon mole basis) is conversion times selectivity. Overview The hydrocarbon-containing feed can be or can include, but is not limited to, one or more alkane hydrocarbons, e.g., C2-C16linear or branched alkanes and/or C4-C16cyclic alkanes, and/or one or more alkyl aromatic hydrocarbons, e.g., C8-C16alkyl aromatics. In some embodiments, the hydrocarbon-containing feed can optionally include 0.1 vol % to 50 vol % of steam, based on a total volume of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. In other embodiments, the hydrocarbon-containing feed can include <0.1 vol % of steam or can be free of steam, based on the total volume of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. The hydrocarbon-containing feed can be contacted with a catalyst that includes a Group 8-10 element, e.g., Pt, disposed on a support to effect one or more of dehydrogenation, dehydroaromatization, and dehydrocyclization of at least a portion of the hydrocarbon-containing feed to produce a coked catalyst and an effluent that can include one or more upgraded hydrocarbons and molecular hydrogen. The one or more upgraded hydrocarbons can be or can include one or more dehydrogenated hydrocarbons, one or more dehydroaromatized hydrocarbons, one or more dehydrocyclized hydrocarbons, or a mixture thereof. The hydrocarbon-containing feed and catalyst can be contacted at a temperature in a range from 300° C. to 900° C. for a first time period of ≤3 hours, under a hydrocarbon partial pressure of at least 20 kPa-absolute, where the hydrocarbon partial pressure is the total partial pressure of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. The catalyst can include from 0.001 wt % to 6 wt % of the Group 8-10 element, e.g., Pt, based on the weight of the support. The support can be or can include, but is not limited to, a Group 2 element, a Group 4 element, a Group 12 element, an element having an atomic number of 21, 39, or 57-71, or a compound thereof. At least a portion of the coked catalyst can be contacted with one or more oxidants to effect combustion of at least a portion of the coke to produce a regenerated catalyst lean in coke and a combustion gas. In some embodiments the process can optionally include contacting at least a portion of the regenerated catalyst with a reducing gas to produce a regenerated and reduced catalyst. An additional quantity of the hydrocarbon-containing feed can be contacted with at least a portion of the regenerated catalyst and/or at least a portion of any regenerated and reduced catalyst to produce a re-coked catalyst and additional effluent. A cycle time from contacting the hydrocarbon-containing feed with the catalyst to contacting the additional quantity of the hydrocarbon-containing feed with the regenerated catalyst can be ≤5 hours. It has been surprisingly and unexpectedly discovered that the catalyst that includes a Group 8-10 element, e.g., Pt, disposed on the support can remain sufficiently active and stable after many cycles, e.g., at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 100 cycles, at least 125 cycles, at least 150 cycles, at least 175 cycles, or at least 200 cycles with each cycle time lasting for ≤5 hours, ≤4 hours, ≤3 hours, ≤2 hours, ≤1 hour, ≤50 minutes, ≤45 minutes, ≤30 minutes, ≤15 minutes, ≤10 minutes, ≤5 minutes, ≤1 minute, ≤30 seconds, or ≤10 seconds. In some embodiments, the cycle time can be from 5 seconds, 30 seconds, 1 minute or 5 minutes to 10 minutes, 20 minutes, 30 minutes, 45 minutes, 50 minutes, 70 minutes, 2 hours, 3 hours, 4 hours, or 5 hours. In some embodiments, after the catalyst performance stabilizes (sometimes the few first cycle can have a relatively poor or relatively good performance, but the performance can eventually stabilize), the process can produce a first upgraded hydrocarbon product yield, e.g., propylene when the hydrocarbon-containing feed includes propane, at an upgraded hydrocarbon selectivity, e.g., propylene, of ≥75%, ≥80%, ≥85%, or ≥90%, or >95% when initially contacted with the hydrocarbon-containing feed, and can have a second upgraded hydrocarbon product yield upon completion of the last cycle (at least 15 cycles total) that can be at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% of the first upgraded hydrocarbon product yield at an upgraded hydrocarbon selectivity, e.g., propylene, of ≥75%, ≥80%, ≥85%, or ≥90%, or >95%. Prior to this discovery, it was believed that catalysts having a Group 8-10 element, e.g., Pt, as the active component would not maintain sufficient activity and stability when subjected to so many short cycles with a simple oxidative regeneration that requires no addition of halogen. The first cycle begins upon contact of the catalyst with the hydrocarbon-containing feed, followed by contact with at least the oxidant to produce the regenerated catalyst or at least the oxidant and the optional reducing gas to produce the regenerated and reduced catalyst, and the first cycle ends upon contact of the regenerated catalyst or the regenerated and reduced catalyst with the additional quantity of the hydrocarbon-containing feed. The second and each subsequent cycle begins upon contact of the regenerated catalyst or the regenerated and reduced catalyst and the additional quantity of the hydrocarbon-containing feed and the second and each subsequent cycle ends upon contact of additional or subsequently regenerated catalyst or regenerated and reduced catalyst with the additional quantity of the hydrocarbon-containing feed. Furthermore, unprecedented propylene yields have been obtained via the processes and catalysts described herein. In some embodiments, when the hydrocarbon-containing feed includes propane and the upgraded hydrocarbon includes propylene, contacting the hydrocarbon-containing feed with the catalyst can produce a propylene yield of at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 55%, at least 57%, at least 60%, at least 62%, at least 63%, at least 64%, at least 65%, or at least 66% at apropylene selectivity of at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, when the hydrocarbon-containing feed includes propane and the upgraded hydrocarbon includes propylene, contacting the hydrocarbon-containing feed with the catalyst can produce a propylene yield of at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 55%, at least 57%, at least 60%, at least 62%, at least 63%, at least 64%, at least 65%, or at least 66% at a propylene selectivity of at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% for at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 100 cycles, at least 125 cycles, at least 150 cycles, at least 175 cycles, or at least 200 cycles. In other embodiments, when a hydrocarbon-containing feed includes at least 70 vol % of propane, based on a total volume of the hydrocarbon-containing feed, is contacted under a propane partial pressure of at least 20 kPa-absolute, a propylene yield of at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 55%, at least 57%, at least 60%, at least 62%, at least 63%, at least 64%, at least 65%, or at least 66% at a propylene selectivity of at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% can be obtained for at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 100 cycles, at least 125 cycles, at least 150 cycles, at least 175 cycles, or at least 200 cycles. It is believed that the propylene yield can be further increased to at least 67%, at least 68%, at least 70%, at least 72%, at least 75%, at least 77%, at least 80%, or at least 82% at a propylene selectivity of at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% for at least 15 cycles, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 100 cycles, at least 125 cycles, at least 150 cycles, at least 175 cycles, or at least 200 cycles by further optimizing the composition of the support and/or adjusting one or more process conditions. In some embodiments, the propylene yield can be obtained when the catalyst is contacted with the hydrocarbon feed at a temperature of at least 620° C., at least 630° C., at least 640° C., at least 650° C., at least 655° C., at least 660° C., at least 670° C., at least 680° C., at least 690° C., at least 700° C., or at least 750° C. for at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 100 cycles, at least 125 cycles, at least 150 cycles, at least 175 cycles, or at least 200 cycles. Such a high propylene yield under such processing conditions was not thought possible. Hydrocarbon Upgrading Process The hydrocarbon-containing feed and the catalyst can be contacted with one another within any suitable environment such as one or more reaction or conversion zones disposed within one or more reactors to produce the effluent and the coked catalyst. In some embodiments, the reaction or conversion zone can be disposed or otherwise located within one or more fixed bed reactors, one or more fluidized or moving bed reactors, one or more reverse flow reactors, or any combination thereof. The hydrocarbon-containing feed and catalyst can be contacted at a temperature in a range from 300° C., 350° C., 400° C., 450° C., 500° C., 550° C., 600° C., 620° C., 650° C., 660° C., 670° C., 680° C., 690° C., or 700° C. to 725° C., 750° C., 760° C., 780° C., 800° C., 825° C., 850° C., 875° C., or 900° C. In some embodiments, the hydrocarbon-containing feed and catalyst can be contacted at a temperature of at least 620° C., at least 650° C., at least 660° C., at least 670° C., at least 680° C., at least 690° C., or at least 700° C. to 725° C., 750° C., 760° C., 780° C., 800° C., 825° C., 850° C., 875° C., or 900° C. The hydrocarbon-containing feed can be introduced into the reaction or conversion zone and contacted with the catalyst therein for a time period of ≤3 hours, ≤2.5 hours, ≤2 hours, ≤1.5 hours, ≤1 hour, ≤45 minutes, ≤30 minutes, ≤20 minutes, ≤10 minutes, ≤5 minutes, ≤1 minute, ≤30 seconds, ≤10 seconds, ≤5 seconds, or ≤1 second or ≤0.5 second. In some embodiments, the hydrocarbon-containing feed can be contacted with the catalyst for a time period in a range from 0.1 seconds, 0.5 seconds, 0.7 seconds, 1 second, 30 second, 1 minute, 5 minutes, or 10 minutes to 30 minutes, 50 minutes, 70 minutes, 1.5 hours, 2 hours, or 3 hours. The hydrocarbon-containing feed and catalyst can be contacted under a hydrocarbon partial pressure of at least 20 kPa-absolute, where the hydrocarbon partial pressure is the total partial pressure of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. In some embodiments, the hydrocarbon partial pressure during contact of the hydrocarbon-containing feed and the catalyst can be in a range from 20 kPa-absolute, 50 kPa-absolute, 100 kPa-absolute, at least 150 kPa, at least 200 kPa 300 kPa-absolute, 500 kPa-absolute, 750 kPa-absolute, or 1,000 kPa-absolute to 1,500 kPa-absolute, 2,500 kPa-absolute, 4,000 kPa-absolute, 5,000 kPa-absolute, 7,000 kPa-absolute, 8,500 kPa-absolute, or 10,000 kPa-absolute, where the hydrocarbon partial pressure is the total partial pressure of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. In other embodiments, the hydrocarbon partial pressure during contact of the hydrocarbon-containing feed and the catalyst can be in a range from 20 kPa-absolute, 50 kPa-absolute, 100 kPa-absolute, 150 kPa-absolute, 200 kPa-absolute, 250 kPa-absolute, or 300 kPa-absolute to 500 kPa-absolute, 600 kPa-absolute, 700 kPa-absolute, 800 kPa-absolute, 900 kPa-absolute, or 1,000 kPa-absolute, where the hydrocarbon partial pressure is the total partial pressure of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. In some embodiments, the hydrocarbon-containing feed can include at least 60 vol %, at least 65 vol %, at least 70 vol %, at least 75 vol %, at least 80 vol %, at least 85 vol %, at least 90 vol %, at least 95 vol %, or at least 99 vol % of a single C2-C16alkane, e.g., propane, based on a total volume of the hydrocarbon-containing feed. The hydrocarbon-containing feed and catalyst can be contacted under a single C2-C16alkane, e.g., propane, pressure of at least 20 kPa-absolute, at least 50 kPa-absolute, at least 100 kPa-absolute, at least 150 kPa-absolute, at least 250 kPa-absolute, at least 300 kPa-absolute, at least 400 kPa-absolute, at least 500 kPa-absolute, or at least 1,000 kPa-absolute. The hydrocarbon-containing feed can be contacted with the catalyst within the reaction or conversion zone at any weight hourly space velocity (WHSV) effective for carrying out the upgrading process. In some embodiments, the WHSV can be 0.01 hr−1, 0.1 hr−1, 1 hr−1, 2 hr−1, 5 hr−1, 10 hr−1, 20 hr−1, 30 hr−1, or 50 hr−1to 100 hr−1, 250 hr−1, 500 hr−1, or 1,000 hr−1. In some embodiments, when the hydrocarbon upgrading process includes a fluidized or otherwise moving catalyst, a ratio of the catalyst to a combined amount of any C2-C16alkanes and any C8-C16alkyl aromatics can be in a range from 1, 3, 5, 10, 15, 20, 25, 30, or 40 to 50, 60, 70, 80, 90, 100, 110, 125, or 150 on a weight to weight basis. When the activity of the coked catalyst decreases below a desired minimum amount, the coked catalyst or at least a portion thereof can be contacted with the oxidant within the reaction or conversion zone or within a combustion zone that is separate and apart from the reaction or conversion zone, depending on the particular reactor configuration, to produce a regenerated catalyst. For example, regeneration of the catalyst can occur within the reaction or conversion zone when a fixed bed or reverse flow reactor is used, or within a separate combustion zone that can be separate and apart from the reaction or conversion zone when a fluidized bed reactor or other circulating or fluidized type reactor is used. Similarly, the optional reduction step can also occur within the reaction or conversion zone, within the combustion zone, and/or within a separate reduction zone. Accordingly, the hydrocarbon containing feed can be contacted with the catalyst to effect one or more of dehydrogenation, dehydroaromatization, and dehydrocyclization of at least a portion of the hydrocarbon-containing feed to produce a coked catalyst and a first effluent that includes the one or more upgraded hydrocarbons and molecular hydrogen in a cyclic type process such as those commonly employed in fixed bed and reverse flow reactors and/or a continuous type process commonly employed in fluidized bed reactors. The separation of the effluent that includes the upgraded hydrocarbon and molecular hydrogen from the coked catalyst, if needed, can be accomplished via one or more separators such as a cyclone separator. The oxidant can be or can include, but is not limited to, O2, O3, CO2, H2O, or a mixture thereof. In some embodiments, an amount of oxidant in excess of that needed to combust 100% of the coke on the catalyst can be used to increase the rate of coke removal from the catalyst, so that the time needed for coke removal can be reduced and lead to an increased yield in the upgraded product produced within a given period of time. The coked catalyst and oxidant can be contacted with one another at a temperature in a range from 300° C., 350° C., 400° C., 450° C., 500° C., 550° C., 600° C., 650° C., 700° C., 750° C., or 800° C. to 900° C., 950° C., 1,000° C., 1,050° C., or 1,100° C. to produce the regenerated catalyst. In some embodiments, the coked catalyst and oxidant can be contacted with one another at a temperature in a range from 500° C. to 1,100° C., 600° C. to 1,000° C., 650° C. to 950° C., 700° C. to 900° C., or 750° C. to 850° C. to produce the regenerated catalyst. The coked catalyst and oxidant can be contacted with one another for a time period of ≤2 hours, ≤1 hour, ≤30 minutes, ≤10 minutes, ≤5 minutes, ≤1 min, ≤30 seconds, ≤10 seconds, ≤5 seconds, or ≤1 second. For example, the coked catalyst and oxidant can be contacted with one another for a time period in a range from 2 seconds to 2 hours. In some embodiments, the coked catalyst and oxidant can be contacted for a time period sufficient to remove ≥50 wt %, ≥75 wt %, or ≥90 wt % or >99% of any coke disposed on the catalyst. In some embodiments, the time period the coked catalyst and oxidant contact one another can be less than the time period the catalyst contacts the hydrocarbon-containing feed to produce the effluent and the coked catalyst. For example, the time period the coked catalyst and oxidant contact one another can be at least 90%, at least 60%, at least 30%, or at least 10% less than the time period the catalyst contacts the hydrocarbon-containing feed to produce the effluent. In other embodiments, the time period the coked catalyst and oxidant contact one another can be greater than the time period the catalyst contacts the hydrocarbon-containing feed to produce the effluent and the coked catalyst. For example, the coked catalyst and oxidant contact one another can be at least 50%, at least 100%, at least 300%, at least 500%, at least 1,000%, at least 10,000%, at least 30,000%, at least 50,000%, at least 75,000%, at least 100,000%, at least 250,000%, at least 500,000%, at least 750,000%, at least 1,000,000%, at least 1,250,000%, at least 1,500,000%, or at least 1,800,000% greater than the time period the catalyst contacts the hydrocarbon-containing feed to produce the effluent. The coked catalyst and oxidant can be contacted with one another under an oxidant partial pressure in a range from 20 kPa-absolute, 50 kPa-absolute, 100 kPa-absolute, 300 kPa-absolute, 500 kPa-absolute, 750 kPa-absolute, or 1,000 kPa-absolute to 1,500 kPa-absolute, 2,500 kPa-absolute, 4,000 kPa-absolute, 5,000 kPa-absolute, 7,000 kPa-absolute, 8,500 kPa-absolute, or 10,000 kPa-absolute. In other embodiments, the oxidant partial pressure during contact with the coked catalyst can be in a range from 20 kPa-absolute, 50 kPa-absolute, 100 kPa-absolute, 150 kPa-absolute, 200 kPa-absolute, 250 kPa-absolute, or 300 kPa-absolute to 500 kPa-absolute, 600 kPa-absolute, 700 kPa-absolute, 800 kPa-absolute, 900 kPa-absolute, or 1,000 kPa-absolute to produce the regenerated catalyst. Without wishing to be bound by theory, it is believed that at least a portion of the Group 8-10 element, e.g., Pt, disposed on the coked catalyst can be agglomerated as compared to the catalyst prior to contact with the hydrocarbon-containing feed. It is believed that during combustion of at least a portion of the coke on the coked catalyst that at least a portion of the Group 8-10 element can be re-dispersed about the support. Re-dispersing at least a portion of any agglomerated Group 8-10 element can increase the activity and improve the stability of the catalyst over many cycles. In some embodiments, at least a portion of the Group 8-10 element, e.g., Pt, in the regenerated catalyst can be at a higher oxidized state as compared to the Group 8-10 element in the catalyst contacted with the hydrocarbon-containing feed and as compared to the Group 8-10 element in the coked catalyst. As such, as noted above, in some embodiments the process can optionally include contacting at least a portion of the regenerated catalyst with a reducing gas to produce a regenerated and reduced catalyst. Suitable reducing gases (reducing agent) can be or can include, but are not limited to, H2, CO, CH4, C2H6, C3H8, C2H4, C3H6, steam, or a mixture thereof. In some embodiments, the reducing agent can be mixed with an inert gas such as Ar, Ne, He, N2, CO2, H2O or a mixture thereof. In such embodiments, at least a portion of the Group 8-10 element in the regenerated and reduced catalyst can be reduced to a lower oxidation state, e.g., the elemental state, as compared to the Group 8-10 element in the regenerated catalyst. In this embodiment, the additional quantity of the hydrocarbon-containing feed can be contacted with at least a portion of the regenerated catalyst and/or at least a portion of the regenerated and reduced catalyst. In some embodiments, the regenerated catalyst and the reducing gas can be contacted at a temperature in a range from 300° C., 350° C., 400° C., 450° C., 500° C., 550° C., 600° C., 620° C., 650° C., or 670° C. to 720° C., 750° C., 800° C., or 900° C. The regenerated catalyst and the reducing gas can be contacted for a time period in a range from 1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, or 1 minute to 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes. The regenerated catalyst and reducing gas can be contacted at a reducing agent partial pressure of 20 kPa-absolute, 50 kPa-absolute, or 100 kPa-absolute, 300 kPa-absolute, 500 kPa-absolute, 750 kPa-absolute, or 1,000 kPa-absolute to 1,500 kPa-absolute, 2,500 kPa-absolute, 4,000 kPa-absolute, 5,000 kPa-absolute, 7,000 kPa-absolute, 8,500 kPa-absolute, or 10,000 kPa-absolute. In other embodiments, the reducing agent partial pressure during contact with the regenerated catalyst can be in a range from 20 kPa-absolute, 50 kPa-absolute, 100 kPa-absolute, 150 kPa-absolute, 200 kPa-absolute, 250 kPa-absolute, or 300 kPa-absolute to 500 kPa-absolute, 600 kPa-absolute, 700 kPa-absolute, 800 kPa-absolute, 900 kPa-absolute, or 1,000 kPa-absolute to produce the regenerated catalyst. At least a portion of the regenerated catalyst, the regenerated and reduced catalyst, new or fresh catalyst, or a mixture thereof can be contacted with an additional quantity of the hydrocarbon-containing feed within the reaction or conversion zone to produce additional effluent and additional coked catalyst. As noted above, the cycle time from the contacting the hydrocarbon-containing feed with the catalyst to the contacting the additional quantity of the hydrocarbon-containing feed with at least a portion of the regenerated catalyst, and/or the regenerated and reduced catalyst, and optionally with new or fresh catalyst can be ≤5 hours, e.g., ≤1 hour or ≤45 minutes. In some embodiments, one or more additional feeds, e.g., one or more sweep fluids, can be utilized between flows of the hydrocarbon-containing feed and the oxidant, between the oxidant and the optional reducing gas if used, between the oxidant and the additional hydrocarbon-containing feed, and/or between the reducing gas and the additional hydrocarbon-containing feed. The sweep fluid can, among other things, purge or otherwise urge undesired material from the reactors, such as non-combustible particulates including soot. In some embodiments, the additional feed(s) can be inert under the dehydrogenation, dehydroaromatization, and dehydrocyclization, combustion, and/or reducing conditions. Suitable sweep fluids can be of can include, N2, He, Ar, CO2, H2O, CO2, CH4, or a mixture thereof. In some embodiments, if the process utilizes a sweep fluid the duration or time period the sweep fluid is used can be in a range from 1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, or 1 minute to 10 minutes, 30 minutes, or 60 minutes. As noted above, the first cycle begins upon contact of the catalyst with the hydrocarbon-containing feed, followed by contact with at least the oxidant to produce the regenerated catalyst or at least the oxidant and the optional reducing gas to produce the regenerated and reduced catalyst, and the first cycle ends upon contact of the regenerated catalyst or the regenerated and reduced catalyst with the additional quantity of the hydrocarbon-containing feed. If any sweep fluid is utilized between flows of the hydrocarbon-containing feed and the oxidant, between the oxidant and the reducing gas (if used), between the oxidant and the additional quantity of the hydro-carbon containing feed, and/or between the reducing gas (if used) and the additional quantity of the hydrocarbon-containing feed is used, the period of time such sweep fluid is utilized would be included in the period included in the cycle time. As such, the cycle time from the contacting the hydrocarbon-containing feed with the catalyst in step (I) to the contacting the additional quantity of the hydrocarbon-containing feed with the regenerated catalyst in step (III) can be ≤5 hours. Systems suitable for carrying out the processes disclosed herein can include systems that are well-known in the art such as the fixed bed reactors disclosed in WO Publication No. WO2017078894; the fluidized riser reactors and/or downer reactors disclosed in U.S. Pat. Nos. 3,888,762; 7,102,050; 7,195,741; 7,122,160; and 8,653,317; and U.S. Patent Application Publication Nos. 2004/0082824; 2008/0194891; and the reverse flow reactors disclosed in U.S. Pat. No. 8,754,276; U.S. Patent Application Publication No. 2015/0065767; and WO Publication No. WO2013169461. Catalyst The catalyst can include 0.001 wt %, 0.002 wt %, 0.003 wt %, 0.004 wt %, 0.005 wt %, 0.006 wt %, 0.007 wt %, 0.008 wt %, 0.009 wt %, 0.01 wt %, 0.015 wt %, 0.02 wt %, 0.025 wt %, 0.03 wt %, 0.035 wt %, 0.04 wt %, 0.045 wt %, 0.05 wt %, 0.055 wt %, 0.06 wt %, 0.065 wt %, 0.07 wt %, 0.075 wt %, 0.08 wt %, 0.085 wt %, 0.09 wt %, 0.095 wt %, 0.1 wt %, 0.2 wt %, 0.3 wt %, 0.4 wt %, 0.5 wt %, 0.6 wt %, 0.7 wt %, 0.8 wt %, 0.9 wt %, or 1 wt % to 2 wt %, 3 wt %, 4 wt %, 5 wt %, or 6 wt % of the Group 8-10 element, based on the total weight of the support. In some embodiments, the catalyst can include ≤5.5 wt %, ≤4.5 wt %, ≤3.5 wt %, ≤2.5 wt %, ≤1.5 wt %, ≤1 wt %, ≤0.9 wt %, ≤0.8 wt %, ≤0.7 wt %, ≤0.6 wt %, ≤0.5 wt %, ≤0.4 wt %, ≤0.3 wt %, ≤0.2 wt %, ≤0.15 wt %, ≤0.1 wt %, ≤0.09 wt %, ≤0.08 wt %, ≤0.07 wt %, ≤0.06 wt %, ≤0.05 wt %, ≤0.04 wt %, ≤0.03 wt %, ≤0.02 wt %, ≤0.01 wt %, ≤0.009 wt %, ≤0.008 wt %, ≤0.007 wt %, ≤0.006 wt %, ≤0.005 wt %, ≤0.004 wt %, ≤0.003 wt %, or ≤0.002 wt % of the Group 8-10 element, based on the total weight of the support. In some embodiments, the catalyst can include >0.001, >0.003 wt %, >0.005 wt %, >0.007, >0.009 wt %, >0.01 wt %, >0.02 wt %, >0.04 wt %, >0.06 wt %, >0.08 wt %, >0.1 wt %, >0.13 wt %, >0.15 wt %, >0.17 wt %, >0.2 wt %, >0.2 wt %, >0.23, >0.25 wt %, >0.27 wt %, or >0.3 wt % and <0.5 wt %, <1 wt %, <2 wt %, <3 wt %, <4 wt %, <5 wt %, or <6 wt % of the Group 8-10 element, based on the total weight of the support. In other embodiments, the catalyst can include >0.025 wt %, ≥0.05 wt %, >0.1 wt %, >0.13 wt %, >0.15 wt %, >0.17 wt %, >0.2 wt %, >0.2 wt %, >0.23, >0.25 wt %, >0.27 wt %, or >0.3 wt % and <0.5 wt %, <1 wt %, <2 wt %, <3 wt %, <4 wt %, ≤5 wt %, or <6 wt % of the Group 8-10 element based on the total weight of the support. In some embodiments, the Group 8-10 element can be or can include, but is not limited to, Fe, Co, Ni, Ru, Pd, Os, Ir, Pt, a combination thereof, or a mixture thereof. In at least one embodiment, the Group 8-10 element can be or can include Pt. If two or more Group 8-10 elements are disposed on the inorganic support, the catalyst can include 0.001 wt %, 0.002 wt %, 0.003 wt %, 0.004 wt %, 0.005 wt %, 0.006 wt %, 0.007 wt %, 0.008 wt %, 0.009 wt %, 0.01 wt %, 0.015 wt %, 0.02 wt %, 0.025 wt %, 0.03 wt %, 0.035 wt %, 0.04 wt %, 0.045 wt %, 0.05 wt %, 0.055 wt %, 0.06 wt %, 0.065 wt %, 0.07 wt %, 0.075 wt %, 0.08 wt %, 0.085 wt %, 0.09 wt %, 0.095 wt %, 0.1 wt %, 0.2 wt %, 0.3 wt %, 0.4 wt %, 0.5 wt %, 0.6 wt %, 0.7 wt %, 0.8 wt %, 0.9 wt %, or 1 wt % to 2 wt %, 3 wt %, 4 wt %, 5 wt %, or 6 wt % of a combined amount of the two or more Group 8-10 elements disposed on the inorganic support, based on the weight of the total weight of the support. The support can be or can include, but is not limited to, one or more elements having an atomic number of 4, 12, 20-22, 30, 38-40, 48, or 56-71. Said another way, the support can be or can include one or more Group 2 elements, one or more Group 4 elements, one or more Group 12 elements, one or more elements having an atomic number of 21, 39, or 57-71, combinations thereof, or mixture thereof. In some embodiments, the Group 2 element, the Group 4 element, the Group 12 element, and/or the element having an atomic number of 21, 39, or 57-71 can be present in its elemental form. In other embodiments, the Group 2 element, the Group 4 element, the Group 12 element, and/or the element having an atomic number of 21, 39, or 57-71 can be present in the form of a compound. For example, the Group 2 element, the Group 4 element, the Group 12 element, and/or the element having an atomic number of 21, 39, or 57-71 can be present as an oxide, a phosphate, a halide, a halate, a sulfate, a sulfide, a borate, a nitride, a carbide, an aluminate, an aluminosilicate, a silicate, a carbonate, metaphosphate, a selenide, a tungstate, a molybdate, a chromite, a chromate, a dichromate, or a silicide. In some embodiments, a mixture of any two or more compounds that include the Group 2 element, the Group 4 element, the Group 12 element, and/or the element having an atomic number of 21, 39, or 57-71 can be present in different forms. For example, a first compound can be an oxide and a second compound can be an aluminate where the first compound and the second compound include the same or different Group 2 element, Group 4 element, Group 12 element, and/or element having an atomic number of 21, 39, or 57-71, with respect to one another. In some embodiments, the support can be or can include at least one of: w wt % of the one or more Group 2 elements, x wt % of the one or more Group 4 elements, y wt % of the one or more Group 12 elements, and z wt % of the one or more elements having an atomic number of 21, 39, or 57-71, based on the weight of the support, where w, x, y, and z are independently in a range from 0 to 100, and where w+x+y+z is ≤100. Any Group 2 element present in the support can be associated with a wt % m based on the weight of the support, any Group 4 element present in the support can be associated with a wt % n based on the weight of the support, any Group 12 element present in the support can be associated with a wt % p based on the weight of the support, and any element having an atomic number of 21, 39, or 57-71 present in the support can be associated with a wt % q based on the weight of the support, where m, n, p, and q can independently be a number that is in a range from 1 to 100. In some embodiments, m, n, p, and q can each be equal to 1, 2, 15, or 30, or m can be equal to 1, n can be equal to 15, p can be equal to 15, and q can be equal to 1. As used herein, “m” represents the minimum wt % of all Group 2 elements in the support, if none of the Group 4 elements, none of the Group 12 elements, and none of the elements having an atomic number of 21, 39, or 57-71 are present in the support. Similarly, as used herein, “n” represents the minimum wt % of all Group 4 elements in the support, if none of the Group 2 elements, none of the Group 12 elements, and none of the elements having an atomic number of 21, 39, or 57-71 are present in the support. Similarly, “p” represents the minimum wt % of all Group 12 elements in the support, if none of the Group 2 elements, none of the Group 4 elements, and none of the elements having an atomic number of 21, 39, or 57-71 are present in the support, Finally, “q” represents the minimum wt % of all elements having an atomic number of 21, 39, or 57-71 that are present in the support, if none of the Group 2 elements, none of the Group 4 elements, and none of the Group 12 elements are present in the support. In some embodiments, a sum of w/m+x/n+y/p+z/q can be at least 1, based on the weight of the support. In other embodiments, a sum of w/m+x/n+y/p+z/q can be at least 1, at least 2, at least 4, at least 6, at least 8, at least 12, at least 24, at least 48, or at least 60, based on the weight of the support. In other embodiments, a sum of w/m+x/n+y/p+z/q can be in a range from 1, 2, 3, 4, 5, 6, 7, or 8 to 10, 12, 16, 24, 30, 48, or 60. In other embodiments, a sum of w/m+x/n+y/p+z/q can be in a range from 1 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 12, 12 to 24, 24 to 48, or 48 to 60. As such, the m, n, p, and q not only specify the minimum amount of each group of elements present in the support when the other groups of elements are not present in the support, but also specify the minimum amount of each group of elements in the support when any one or more of the other groups of elements are also present in the support, which is explained by the following Example. In this Example: m=4, n=8, p=12, q=20. If none of the Group 4 elements, none of the Group 12 elements, and none of the elements having an atomic number of 21, 39, or 57-71 are present in the support, then the total amount of any Group 2 element(s) in the support has to be ≥4 wt %, i.e., w/m≥1. If none of the Group 2 elements, none of the Group 12 elements, and none of the elements having an atomic number of 21, 39, or 57-71 are present in the support, then the total amount of any Group 4 element(s) present in the support has to be ≥8 wt %, i.e., x/n≥1. If none of the Group 2 elements, none of the Group 4 elements, and none of the elements having an atomic number of 21, 39, or 57-71 are present in the support, then the total amount of any Group 12 element(s) present in the support has to be ≥12 wt %, i.e., y/p≥1. If none of the Group 2 elements, none of the Group 4 elements, and none of the Group 12 elements exist on the support, then the total amount of any element(s) having an atomic number of 21, 39, or 57-71 present in the support has to be ≥20 wt %, i.e., z/q≥1. If both Group 2 and 4 elements are present in the support and none of the Group 12 elements and none of the elements having an atomic number of 21, 39, or 57-71 are present in the support, then there is no need for the total amount of Group 2 element(s) to be ≥4 wt % since the Group 4 element(s) on the support share the role of the Group 2 element(s). Similarly, there is no need for the total amount of Group 4 element(s) to be ≥8 wt % since the Group 2 element(s) on the support share the role of the Group 4 element(s). Such an interchangeable relationship between the Group 2 and 4 elements is defined by m and n. Since m=4 and n=8, two mass units of the Group 4 element(s) interchanges one mass unit of the Group 2 element(s). For example, if the total amount of the Group 2 element(s) is w=1.1 wt % and the total amount of the Group 4 element(s) is x=4.3 wt %, then w/m+x/n=1.1/4+4.3/8=0.8125, which is <1, i.e., the total amount of the Group 2 and 4 elements is too little for the support to satisfy w/m+x/n+y/p+z/q is ≥1. In another example, if the total amount of the Group 2 element(s) is w=2.4 wt % and the total amount of the Group 4 element(s) is x=4.3 wt %, then w/m+x/n=2.4/4+4.3/8=1.1375, which is ≥1, such that the total amount of the Group 2 and Group 4 elements is sufficient to satisfy w/m+x/n+y/p+z/q is ≥1, despite that both w and x (2.4 and 4.3) are less than m and n (4 and 8), respectively. The same principle also applies to cases when the support includes at least one element from three of the group of elements, e.g., Group 2, Group 4, and Group 12, as well as when the support includes each group of elements, i.e., at least one Group 2 element, at least one Group 4 element, at least one Group 12 element, and at least one element having an atomic number of 21, 39, or 57-71. For example, if the support includes 0.5 wt % of Mg (Group 2 element), 2 wt % of Ca (Group 2 element), 4 wt % of Ce (atomic number of 58), 3 wt % of Zr (Group 4 element), and 6 wt % of Zn (Group 12 element), then the equation would be: (0.5+2)/4+4/20+3/8+6/12=1.7, which is ≥1. In summary, m, n, p, and q is the minimum amount of each Group of elements in the support when the other Groups of elements are not present in the support. The equation w/m+x/n+y/p+z/q≥1 defines how the 4 groups of elements can work together in the support. In some embodiments, m can be one often values selected from: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; n can be one of twelve values selected from: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24; p can be one of twelve values selected from: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24; and q can be one of twelve values selected from: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, and 40, where m, n, p, and q can be any combination such that there are 17,280 (10×12×12×12) distinct combinations. In other embodiments, m can be equal to 2, 7, 10, or 20, n can be 2, 10, 20, or 25, p can be 2, 10, 20, or 25, and q can be 2, 10, 30, or 40, where m, n, p, and q can be any combination such that there are 256 (4×4×4×4) distinct combinations. In some embodiments, m, n, p, and q can each be equal to 2, 10, 15, or 30. In other embodiments, m can be equal to 7, n can be equal to 10, p can be equal to 10, and q can be equal to 10. In other embodiments, m can be equal to 7, n can be equal to 20, p can be equal to 20, and q can be equal to 10. In other embodiments, m can be equal to 10, n can be equal to 20, p can be equal to 20, and q can be equal to 30. In other embodiments, m can be equal to 7, n can be equal to 10, p can be equal to 10, and q can be equal to 30. In some embodiments, w, x, y, and z can independently be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, where a sum of w, x, y, z is ≤100. In some embodiments, when the support includes the Group 2 element, a molar ratio of the Group 2 element to the Group 8-10 element can be in a range from 0.24, 0.5, 1, 10, 50, 100, 300, 450, 600, 800, 1,000, 1,200, 1,500, 1,700, or 2,000 to 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, or 900,000. In some embodiments, when the support includes the Group 4 element, a molar ratio of the Group 4 element to the Group 8-10 element can be in a range from 0.18, 0.3, 0.5, 1, 10, 50, 100, 200, 300, 400, 500, 600, 700, 810, 1,000, or 5,000 to 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, or 81,000. In some embodiments, when the support includes the Group 12 element, a molar ratio of the Group 12 element to the Group 8-10 element can be in a range from 0.29, 0.5, 1, 10, 50, or 100 to 200, 300, 400, 500, 590, 600, or 1,000 to 5,000, 10,000, 20,000, 30,000, 40,000, 50,000 or 59,000. In some embodiments, when the support includes the element having an atomic number of 21, 39, or 57-71, a molar ratio of the element having an atomic number of 21, 39, or 57-71 to the Group 8-10 element can be in a range from 0.19, 0.5, 1, 10, 50, 100, or 150 to 200, 250, 300, 350, 400, 438, 500, 750, or 1,000 to 5,000, 10,000, 20,000, 30,000, 40,000, or 43,800. In some embodiments, when the support includes two or more of the Group 2, 4, or 12 element and the element having an atomic number of 21, 39, or 57-71, a molar ratio of a combined amount of any Group 2 element, any Group 4 element, any Group 12 element, and any element having an atomic number of 21, 39, or 57-71 to the Group 8-10 element can be in a range from 0.18, 0.5, 1, 10, 50, 100, 300, 450, 600, 800, 1,000, 1,200, 1,500, 1,700, or 2,000 to 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, or 900,000. In some embodiments, the support can be or can include, but is not limited to, one or more of the following compounds: MguZn1-uO, where u is a positive number; ZnvAl2O3+v, where v is a positive number; MgwAl2O3+w, where w is a positive number; CaxAl2O3+z, where x is a positive number; SryAl2O3+y, where y is a positive number; BazAl2O3+z, where z is a positive number. BeO; MgO; CaO; BaO; SrO; BeCO3; MgCO3; CaCO3; SrCO3, BaCO3; ZrO2; ZrC; ZrN; ZrSiO4; CaZrO3; Ca7ZrAl6O18; TiO2; TiC; TiN; TiSiO4; CaTiO3; Ca7Al6O18; HfO2; HfC; HfN; HfSiO4; HfZrO3; Ca7HfAl6O18; ZnO; Zn3(PO4)2; Zn(ClO3)2; ZnSO4; B2O6Zn3; Zn3N2; ZnCO3; CeO2; Y2O3; La2O3; Sc2O3; Pr6O11; CePO4; CeZrO4; CeAlO3; BaCeO3; CePO4; Yttria-stabilized ZrO2; one or more magnesium chromates, one or more magnesium tungstates, one or more magnesium molybdates combinations thereof, and mixtures thereof. The MguZn1-uO, where u is a positive number, if present as the support or as a component of the support can have a molar ratio of Mg to Zn in a range from 1, 2, 3, or 6 to 12, 25, 50, or 100. The ZnvAl2O3+v, where v is a positive number, if present as the support or as a component of the support can have a molar ratio of Zn to Al in a range from 0.05, 0.3, or 0.6 to 0.9, 1.5, or 3 The MgwAl2O3+w, where w is a positive number, if present as the support or as a component of the support can have a molar ratio of Mg to Al in a range from 1, 2, 3, 4, or 5 to 6, 7, 8, 9, or 10. The CaxAl2O3+x, where x is a positive number, if present as the support or as a component of the support can have a molar ratio of Ca to Al in a range from 1:12, 1:4, 1:2, 2:3, 5:6, 1:1, 12:14, or 1.5:1. In some embodiments, the CaxAl2O3+xcan include tricalcium aluminate, dodecacalcium hepta-aluminate, moncalcium aluminate, moncalcium dialuminate, monocalcium hexa-aluminate, dicalcium aluminate, pentacalcium trialuminate, tetracalcium trialuminate, or any mixture thereof. The SryAl2O3+y, where y is a positive number, if present as the support or as a component of the support can have a molar ratio of Sr to Al in a range from 0.05, 0.3, or 0.6 to 0.9, 1.5, or 3. The BazAl2O3+z, where z is a positive number, if present as the support or as a component of the support can have a molar ratio of Ba to Al 0.05, 0.3, or 0.6 to 0.9, 1.5, or 3. In some embodiments, the support can also include, but is not limited to, at least one metal element and/or at least one metalloid element selected from Groups 5, 6, 7, 11, 13, 14, 15, and 16 and/or at least one compound thereof. If the support also includes a compound that includes the metal element and/or metalloid element selected from Groups 5, 6, 7, 11, 13, 14, 15, and 16, the compound can be present in the support as an oxide, a phosphate, a halide, a halate, a sulfate, a sulfide, a borate, a nitride, a carbide, an aluminate, an aluminosilicate, a silicate, a carbonate, metaphosphate, a selenide, a tungstate, a molybdate, a chromite, a chromate, a dichromate, or a silicide. In some embodiments, suitable compounds that include the metal element and/or metalloid element selected from Groups 5, 6, 7, 11, 13, 14, 15, and 16 can be or can include, but are not limited to, one or more of the following: B2O3, AlBO3, Al2O3, SiO2, SiC, Si3N4, an aluminosilicate, VO, V2O3, VO2, V2O5, Ga2O3, In2O3, Mn2O3, Mn3O4, MnO, one or more molybdenum oxides, one or more tungsten oxides, one or more zeolites, and mixtures and combinations thereof. In some embodiments, the support can include the Group 2 element and Al and can be in the form of a mixed Group 2 element/Al metal oxide that has O, Mg, and Al atoms mixed on an atomic scale. In some embodiments the support can be or can include the Group 2 element and Al in the form of an oxide or one or more oxides of the Group 2 element and Al2O3that can be mixed on a nm scale. In some embodiments, the support can be or can include an oxide of the Group 2 element, e.g., MgO, and Al2O3mixed on a nm scale. In some embodiments, the support can be or can include a first quantity of the Group 2 element and Al in the form of a mixed Group 2 element/Al metal oxide and a second quantity of the Group 2 element in the form of an oxide of the Group 2 element. In such embodiment, the mixed Group 2 element/Al metal oxide and the oxide of the Group 2 element can be mixed on the nm scale and the Group 2 element and Al in the mixed Group 2 element/Al metal oxide can be mixed on the atomic scale. In other embodiments, the support can be or can include a first quantity of the Group 2 element and a first quantity of Al in the form of a mixed Group 2 element/Al metal oxide, a second quantity of the Group 2 element in the form of an oxide of the Group 2 element, and a second quantity of Al in the form of Al2O3. In such embodiment, the mixed Group 2 element/Al metal oxide, the oxide of the Group 2 element, and the Al2O3can be mixed on a nm scale and the Group 2 element and Al in the mixed Group 2 element/Al metal oxide can be mixed on the atomic scale. In some embodiments, when the support includes the Group 2 element and Al, a weight ratio of the Group 2 element to the Al in the support can be in a range from 0.001, 0.005, 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.5, 0.7, or 1 to 3, 6, 12.5, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000. In some embodiments, when the support includes Al, the support can include Al in a range from 0.5 wt %, 1 wt %, 1.5 wt %, 2 wt %, 2.1 wt %, 2.3 wt %, 2.5 wt %, 2.7 wt %, 3 wt %, 4 wt %, 5 wt %, 6 wt %, 7 wt %, 8 wt %, 9 wt %, 10 wt %, or 11 wt % to 15 wt %, 20 wt %, 25 wt %, 30 wt %, 40 wt %, 45 wt %, or 50 wt %, based on the weight of the support. In some embodiments, the support can include ≥3 wt %, ≥6 wt %, ≥11 wt %, ≥15 wt %, ≥20 wt %, ≥25 wt %, >, 30 wt %, or ≥ of a Group 2 element based on the weight of the support. In some embodiments, the Group 2 element can be or can include, but is not limited to, Mg. In some embodiments, the support can be or can include, but is not limited to, calcined hydrotalcite. In some embodiments, the support can also include one or more promoters disposed thereon. The promoter can be or can include, but is not limited to, Sn, Ga, Zn, Ge, In, Re, Ag, Au, Cu, a combination thereof, or a mixture thereof. As such, the Zn, Ga, and/or In, if present as a component of the catalyst, can be present as a component of the support, as a promoter disposed on the support, or both as a component of the support and as a promoter disposed on the support. In some embodiments, the promoter can be associated with the Group 8-10 element, e.g., Pt. For example, the promoter and the Group 8-10 element disposed on the support can form Group-8-10 element-promoter clusters that can be dispersed on the support. The promoter, if present, can improve the selectivity/activity/longevity of the catalyst for a given upgraded hydrocarbon. In some embodiments, the addition of the promoter can improve the propylene selectivity of the catalyst when the hydrocarbon-containing feed includes propane. The catalyst can include the promoter in an amount of 0.01 wt %, 0.1 wt %, 0.2 wt %, 0.3 wt %, 0.4 wt %, 0.5 wt %, 0.6 wt %, 0.7 wt %, 0.8 wt %, 0.9 wt %, or 1 wt % to 3 wt %, 5 wt %, 7 wt %, or 10 wt %, based on the weight of the support. In some embodiments, the support can also include one or more alkali metal elements disposed on the support. The alkali metal element, if present, can be or can include, but is not limited to, Li, Na, K, Rb, Cs, a combination thereof, or a mixture thereof. In at least some embodiments, the alkali metal element ca be or can include K and/or Cs. The alkali metal element, if present, can improve the selectivity of the catalyst for a given upgraded hydrocarbon. The catalyst can include the alkali metal element in an amount 0.01 wt %, 0.1 wt %, 0.2 wt %, 0.3 wt %, 0.4 wt %, 0.5 wt %, 0.6 wt %, 0.7 wt %, 0.8 wt %, 0.9 wt %, or 1 wt % to 2 wt %, 3 wt %, 4 wt %, or 5 wt %, based on the weight of the support. The preparation of the support can be accomplished via any known process. For simplicity and ease of description, the preparation of a suitable support that includes a mixed oxide of magnesium and aluminum (Mg(Al)O or MgO/Al2O3) support will be described in more detail. Catalyst synthesis techniques are well-known and the following description is for illustrative purposes and not to be considered as limiting the synthesis of the support or the catalyst. In some embodiments, to make the MgO/Al2O3mixed oxide support, Mg and Al precursors such as Mg(NO3)2and Al(NO3)3can be mixed together, e.g., ball-milled, followed by calcination to produce the support. In another embodiment, the two precursors can be dissolved in H2O, stirred until dry (with heat optionally applied), followed by calcination to produce the support. In another embodiment, the two precursors can be dissolved in H2O, followed by the addition of a base and a carbonate, e.g., NaOH/Na2CO3to produce hydrotalcite, followed by calcination to produce the support. In another embodiment, a commercial ready MgO and Al2O3may be mixed and ball-milled. In another embodiment, the Mg(NO3)2precursor can be dissolved in H2O and the solution can be impregnated onto an existing support, e.g., an Al2O3support, that can be dried and calcined to produce the support. In another embodiment, Mg from Mg(NO3)2can be loaded onto an existing Al2O3support through ion adsorption, followed by liquid-solid separation, drying and calcination to produce the support. Without wishing to be bound by theory, it is believed that the inorganic support produced via any one of the above methods and/or other methods can include (i) the Mg and Al mixed together on the nm scale, (ii) the Mg and Al in the form of a mixed Mg/Al metal oxide, or (iii) a combination of (i) and (ii). Group 8-10 metals and any promoter and/or any alkali metal element may be loaded onto the mixed oxide support by any known technique. For example, one or more Group 8-10 element precursors, e.g., chloroplatinic acid, tetramineplatinum(II) nitrate, and/or tetramineplatinum(II) hydroxide, one or more promoter precursors (if used), e.g., a salt such as SnCl4and/or AgNO3, and one or more alkali metal element precursors (if used), e.g., KNO3, KCl, and/or NaCl, can be dissolved in water. In some embodiments, the Group 8-10 element precursor can be or can include, but is not limited to, chloroplatinic acid hexahydrate, tetraammineplatinum(II) nitrate, platinum(II) oxalate, platinum(II) acetylacetonate, platinum(II) bromide, platinum(II) iodide, platinum(II) chloride, platinum(IV) chloride, platinum(II)diammine dichloride, ammonium tetrachloroplatinate(II), tetraammineplatinum(II) chloride hydrate, tetraammineplatinum(II) hydroxide hydrate, iron nitrate, rhodium(III) nitrate, ruthenium(III) nitrate, cobalt(II) nitrate hexahydrate, nickel(II) nitrate hexahydrate, palladium(II) nitrate dihydrate, or any mixture thereof. In some embodiments, the promoter precursor can be or can include, but is not limited to, tin(II) oxide, tin(IV) oxide, tin(IV) chloride pentahydrate, tin(II) chloride dihydrate, tin citrate, tin sulfate, tin oxalate, tin(II) bromide, tin(IV) bromide, tin(II) acetylacetonate, tin(II) acetate, tin(IV) acetate, silver(I) nitrate, gold(III) nitrate, copper(II) nitrate, gallium(III) nitrate, or any mixture thereof. In some embodiments, the alkali metal element precursor can be or can include, but is not limited to, lithium nitrate, sodium nitrate, potassium nitrate, rubidium nitrate, cesium nitrate, or any mixture thereof. The solution can be impregnated onto the support, followed by drying and calcination to produce the catalyst. In some embodiments, the Group 8-10 element precursor and optionally the promoter precursor and/or the alkali metal element precursor can be loaded onto the support at the same time, or separately in a sequence separated by drying and/or calcination steps to produce the catalyst. In other embodiments, the Group 8-10 element and, optionally the promoter and/or alkali metal element, can be loaded onto the support by chemical vapor deposition, where the precursors are volatilized and deposited onto the support, followed by calcination to produce the catalyst. In other embodiments, the Group 8-10 element precursor and, optionally, the promoter precursor and/or alkali metal precursor, can be loaded onto the support through ion adsorption, followed by liquid-solid separation, drying and calcination to produce the catalyst. Optionally, the catalyst can also be synthesized using a one-pot synthesis method where the precursors of the support, Group 8-10 metal active phase and the promoters are all mixed together, dry or wet, with or without any other additives to aid the synthesis, followed by drying and calcination to produce the catalyst. In some embodiments, the drying or calcination may be carried out in an oxidative environment, or a reductive environment, or an inert environment, or a combination of two or more of the environments. In some embodiments, a suitable oxidative environment can be provided by air, enriched air, O2, O2diluted by one or more inert gases, O3, O3diluted by one or more inert gases, or any mixture thereof. In some embodiments, a suitable reductive environment can be provided by H2, CO, syngas, or any reductive gas diluted by one or more inert gases. In some embodiments, a suitable inert environment can be provided by steam, N2, Ar, He, or any mixture of the above. While drying/calcination is typically accompanied by the release of one or more volatiles, in some embodiments, the drying/calcination step can be preceded by an equilibration step where no release of volatiles is expected. Suitable processes that can be used to prepare the catalysts disclosed herein can include the processes described in U.S. Pat. Nos. 4,788,371; 4,962,265; 5,922,925; 8,653,317; EP Patent No. EP0098622; Journal of Catalysis 94 (1985), pp. 547-557; and/or Applied Catalysis 54 (1989), pp. 79-90. The as-synthesized catalyst, when examined under scanning electron microscope or transmission electron microscope, can appear as either primary particles, as agglomerates of primary particles, as aggregated primary particles, or a combination thereof. The primary particles in the as-synthesized catalyst, when examined under scanning electron microscope or transmission electron microscope, can have an average particle size, e.g., a diameter when spherical, in a range from 0.2 nm, 0.5 nm, 1 nm, 5 nm, 10 nm, 25 nm, 30 nm, 40 nm 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm to 1 μm, 10 μm, 25 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, or 500 μm. In some embodiments, the catalyst the discrete particles can have an average cross-sectional length of 0.2 nm to 500 μm, 0.5 nm to 300 μm, 1 nm to 200 μm, 2 nm to 100 μm, or 2 nm to 500 nm as measured by a transmission electron microscope. The catalyst can have a surface area in a range from 0.1 m2/g, 1 m2/g, 10 m2/g, or 100 m2/g to 500 m2/g, 800 m2/g, 1,000 m2/g, or 1,500 m2/g. The surface area of the catalyst can be measured according to the Brunauer-Emmett-Teller (BET) method using adsorption-desorption of nitrogen (temperature of liquid nitrogen, 77 K) with a Micromeritics 3 flex instrument after degassing of the powders for 4 hrs at 350° C. More information regarding the method can be found, for example, in “Characterization of Porous Solids and Powders: Surface Area, Pore Size and Density,” S. Lowell et al., Springer, 2004. In some embodiments, the support can be extruded or otherwise formed into any desired monolithic structure and the Group 8-10 element and any optional promoter and/or alkali metal element can be disposed thereon. Suitable monolithic structures can be or can include, but are not limited to, structures having a plurality of substantially parallel internal passages such as those in the form of a ceramic honeycomb. In some embodiments, the support can be in the form of beads, spheres, rings, toroidal shapes, irregular shapes, rods, cylinders, flakes, films, cubes, polygonal geometric shapes, sheets, fibers, coils, helices, meshes, sintered porous masses, granules, pellets, tablets, powders, particulates, extrudates, cloth or web form materials, honeycomb matrix monolith, including in comminuted or crushed forms, and the Group 8-10 element and any optional promoter and/or alkali metal element can be disposed thereon. The as-synthesized catalyst can be formulated into one or more appropriate forms for different short cycle (≤5 hours) hydrocarbon upgrading processes. Alternatively, the support can be formulated into appropriate forms for different short cycle hydrocarbon upgrading processes, before the addition of the Group 8-10 element and, any optional promoter and/or alkali metal element. During formulation, one or more binders and/or additives can be added to the catalyst and/or support to improve the chemical/physical properties of the catalyst. For example, spray-dried catalyst particles having an average cross-sectional area in a range from 40 μm to 80 μm are typically used in an FCC type fluid-bed reactor. To make spray-dried catalyst, the support/catalyst needs to be made into a slurry with binder/additive in the slurry before spray-drying and calcination. Hydrocarbon-Containing Feed The C2-C16alkanes can be or can include, but are not limited to, ethane, propane, n-butane, isobutane, n-pentane, isopentane, n-hexane, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, n-heptane, 2-methylhexane, 2,2,3-trimethylbutane, cyclopentane, cyclohexane, methylcyclopentane, ethylcyclopentane, n-propylcyclopentane, 1,3-dimethylcyclohexane, or a mixture thereof. For example, the hydrocarbon-containing feed can include propane, which can be dehydrogenated to produce propylene, and/or isobutane, which can be dehydrogenated to produce isobutylene. In another example, the hydrocarbon-containing feed can include liquid petroleum gas (LP gas), which can be in the gaseous phase when contacted with the catalyst. In some embodiments, the hydrocarbon in the hydrocarbon-containing feed can be composed of substantially a single alkane such as propane. In some embodiments, the hydrocarbon-containing feed can include ≥50 mol %, ≥75 mol %, ≥95 mol %, ≥98 mol %, or ≥99 mol % of a single C2-C16alkane, e.g., propane, based on a total weight of all hydrocarbons in the hydrocarbon-containing feed. In some embodiments, the hydrocarbon-containing feed can include at least 50 vol %, at least 55 vol %, at least 60 vol %, at least 65 vol %, at least 70 vol %, at least 75 vol %, at least 80 vol %, at least 85 vol %, at least 90 vol %, at least 95 vol %, at least 97 vol %, or at least 99 vol % of a single C2-C16alkane, e.g., propane, based on a total volume of the hydrocarbon-containing feed. The C8-C16alkyl aromatics can be or can include, but are not limited to, ethylbenzene, propylbenzene, butylbenzene, one or more ethyl toluenes, or a mixture thereof. In some embodiments, the hydrocarbon-containing feed can include ≥50 mol %, ≥75 mol %, ≥95 mol %, ≥98 mol %, or ≥99 mol % of a single C8-C16alkyl aromatic, e.g., ethylbenzene, based on a total weight of all hydrocarbons in the hydrocarbon-containing feed. In some embodiments, the ethylbenzene can be dehydrogenated to produce styrene. As such, in some embodiments, the processes disclosed herein can include propane dehydrogenation, butane dehydrogenation, isobutane dehydrogenation, pentane dehydrogenation, pentane dehydrocyclization to cyclopentadiene, naphtha reforming, ethylbenzene dehydrogenation, ethyltoluene dehydrogenation, and the like. In some embodiments, the hydrocarbon-containing feed can be diluted, e.g., with one or more diluents such as one or more inert gases. Suitable inert gases can be or can include, but are not limited to, Ar, Ne, He, N2, CO2, CH4, or a mixture thereof. If the hydrocarbon containing-feed includes a diluent, the hydrocarbon-containing feed can include 0.1 vol %, 0.5 vol %, 1 vol %, or 2 vol % to 3 vol %, 8 vol %, 16 vol %, or 32 vol % of the diluent, based on a total volume of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. In some embodiments, the hydrocarbon-containing feed can also include H2. In some embodiments, when the hydrocarbon-containing feed includes H2, a molar ratio of the H2to a combined amount of any C2-C16alkane and any C8-C16alkyl aromatic can be in a range from 0.1, 0.3, 0.5, 0.7, or 1 to 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the hydrocarbon-containing feed can be substantially free of any steam, e.g., <0.1 vol % of steam, based on a total volume of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. In other embodiments, the hydrocarbon-containing feed can include steam. For example, the hydrocarbon-containing feed can include 0.1 vol %, 0.3 vol %, 0.5 vol %, 0.7 vol %, 1 vol %, 3 vol %, or 5 vol % to 10 vol %, 15 vol %, 20 vol %, 25 vol %, 30 vol %, 35 vol %, 40 vol %, 45 vol %, or 50 vol % of steam, based on a total volume of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. In other embodiments, the hydrocarbon-containing feed can include ≤50 vol %, ≤45 vol %, ≤40 vol %, ≤35 vol %, ≤30 vol %, ≤25 vol %, ≤20 vol %, or ≤15 vol % of steam, based on a total volume of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. In other embodiments, the hydrocarbon-containing feed can include at least 1 vol %, at least 3 vol %, at least 5 vol %, at least 10 vol %, at least 15 vol %, at least 20 vol %, at least 25 vol %, or at least 30 vol % of steam, based on a total volume of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. In some embodiments, the hydrocarbon-containing feed can include sulfur. For example, the hydrocarbon-containing feed can include sulfur in a range from 0.5 ppm, 1 ppm, 5 ppm, 10 ppm, 20 ppm 30 ppm, 40 ppm, 50 ppm, 60 ppm, 70 ppm, or 80 ppm to 100 ppm, 150 ppm, 200 ppm, 300 ppm, 400 ppm, or 500 ppm. In other embodiments, the hydrocarbon-containing feed can include sulfur in a range from 1 ppm to 10 ppm, 10 ppm to 20 ppm, 20 ppm to 50 ppm, 50 ppm to 100 ppm, or 100 ppm to 500 ppm. The sulfur, if present in the hydrocarbon-containing feed, can be or can include, but is not limited to, H2S, dimethyl disulfide, as one or more mercaptans, or any mixture thereof. The hydrocarbon feed can be substantially free or free of molecular oxygen. In some embodiments, the hydrocarbon feed can include ≤5 mol %, ≤3 mol %, or ≤1 mol % of molecular oxygen (O2). It is believed that providing a hydrocarbon feed substantially-free of molecular oxygen substantially prevents oxidative coupling reactions that would otherwise consume at least a portion of the alkane and/or the alkyl aromatic in the hydrocarbon feed. Recovery and Use of the Upgraded Hydrocarbons The upgraded hydrocarbon can include at least one upgraded hydrocarbon, e.g., an olefin, water, unreacted hydrocarbons, unreacted molecular hydrogen, etc. The upgraded hydrocarbon can be recovered or otherwise obtained via any convenient process, e.g., by one or more conventional processes. One such process can include cooling the effluent to condense at least a portion of any water and any heavy hydrocarbon that may be present, leaving the olefin and any unreacted alkane or alkyl aromatic primarily in the vapor phase. Olefin and unreacted alkane or alkyl aromatic hydrocarbons can then be removed from the reaction product in one or more separator drums. For example, one or more splitters can be used to separate the dehydrogenated product from the unreacted hydrocarbon feed. In some embodiments, a recovered olefin, e.g., propylene, can be used for producing polymer, e.g., recovered propylene can be polymerized to produce polymer having segments or units derived from the recovered propylene such as polypropylene, ethylene-propylene copolymer, etc. Recovered isobutene can be used, e.g., for producing one or more of: an oxygenate such as methyl tert-butyl ether, fuel additives such as diisobutene, synthetic elastomeric polymer such as butyl rubber, etc. EXAMPLES The foregoing discussion can be further described with reference to the following non-limiting examples. The following process steps were performed on the catalysts used in most examples below. All experiments were carried out at ambient pressure, except for the few exceptions as noted in the examples below.1. A gas that included 10 vol % of O2in He, or air was passed through the catalyst at a regeneration temperature (Tregen) for a certain period of time (tregen) to regenerate the catalyst.2. Without changing the flow of the gas, the temperature within the reactor was changed from Tregento a reduction temperature (Tred).3. The system was flushed with He gas.4. A gas that included 10 vol % H2in Ar was passed through the catalyst at the Tredfor a certain period of time (tred).5. The system was flushed with He gas.6. The temperature within the reactor from was changed from Tredto a reaction temperature (Trxn) in the presence of the inert gas.7. A hydrocarbon-containing feed that included 90 vol % of C3H8in Ar or Kr or He at a flow rate (Frxn) was passed through the catalyst at the Trxnfor a certain period of time (trxn). In some examples, the hydrocarbon-containing feed was passed through a sparger immersed in deionized water kept at a temperature of T1, and then through a reflux with a carefully controlled temperature of T2before it was introduced into the reactor and reached the catalyst. When the sparger was used, the hydrocarbon feed included a certain amount of steam within the reactor, which is shown in the relevant tables below.8. The system was flushed with He gas.9. The gas that included 10 vol % of 02 in He, or air was again passed through the catalyst at Trxn, and the temperature within the reactor was changed from Trxnto Tregen. In certain examples, the catalyst reduction step was not carried out and the following steps were performed.1. The gas that included 10 vol % of O2in He or air was passed through the catalyst at the Tregenfor the tregen.2. Without changing the flow of the gas, the temperature within the reactor was changed from Tregento Trxn.3. The system was flushed with the inert gas (such as He).4. The hydrocarbon-containing feed that included 90 vol % of C3H8in Ar or Kr or He at a flow rate of Frxnwas passed through the catalyst at the Trxnfor the trxn. In some examples, the hydrocarbon-containing feed was passed through the sparger immersed in deionized water kept at the temperature of T1, and then through a reflux with carefully controlled temperature of T2before it was introduced into the reactor and reached the catalyst.5. The system was flushed with an inert gas (such as He).6. The gas that included 10 vol % of O2in He or air was again passed through the catalyst at Trxn, and the temperature within the reactor was changed from Trxnto Tregen. An AGILENT® microGC 490 was used to measure the composition of the reactor effluent every 1 minute to 1.5 minutes. The concentration of each component in the reactor effluent was then used to calculate the C3H6yield and selectivity. The C3H6yield and the selectivity at the beginning of trxnand at the end of trxnis denoted as Yini, Yend, Sini, and Send, respectively, and reported as percentages in the data tables below. For some experiments, repeated cycles were conducted to understand catalyst stability. The C3H6yield as reported in these examples are based on carbon only. In each example, a certain amount of the catalyst Mcatwas mixed with an appropriate amount of quartz/SiC diluent and loaded in a quartz reactor. The amount of diluent is determined so that the catalyst bed (catalyst+diluent) is largely isothermal during operation. The dead volume of the reactor was filled with quartz chips/rods. When the reaction temperature (Trxn) was ≥620° C., thermal cracking of propane/propylene became significant. Since thermal cracking of propane/propylene has a much higher selectivity to C1and C2hydrocarbons, the overall selectivity to C3H6is reduced. The amount of thermal cracking within the reactor is related to how much quartz/SiC diluent was added into the reactor and how well the dead volume within the reactor was reduced by the packing materials. Therefore, depending on how the reactor is packed in different experiments, the performance varies. As such, the experimental results shown in different tables are not necessarily comparable to one another. Examples 1-23, Catalyst 1 Catalyst 1: The catalyst used in Examples 1-23 (Exs. 1-23) was a Pt-based, Sn-containing catalyst supported on an Mg/Al mixed oxide support, crushed and sieved to 20-40 mesh particle size. Elemental analysis showed that the catalyst contained 0.48 wt % Pt, 1.25 wt % Sn, 67.93 wt % of Mg, and 29.23 wt % of Al, based on the total weight of the metal elements, with an Mg to Al molar ratio of about 2.58. Table 1 shows the experimental results for Examples 1-3. TABLE 1Ex. 1Ex. 2Ex. 3Catalyst111Mcat(g)111Trxn(° C.)620620620trxn(min)101010Frxn(sccm)222222Svol(%)NANANATred(° C.)620NA620tred(min)1NA5Tregen(° C.)620620620tregen(min)303030Cycles3511First cycleYini48.121.248.2Yend23.26.824Sini9896.498Send93.889.693.7 A comparison between Ex. 1 and Ex. 3 shows that the reduction of the catalyst in the presence of molecular hydrogen after the oxidative regeneration improve the propylene yield. Ex. 1 and Ex. 3 also show that the catalyst is not very sensitive to the duration of the reduction step (1 minute vs. 5 minutes) under the experimental conditions used for these examples. At other conditions, however, there might be an optimal duration for the reduction step to be carried out.FIG.1shows the catalyst stability results of a catalyst used in Examples 1-3 after having undergone 35 cycles (regeneration, reduction, and dehydrogenation) carried out under the same conditions used in Example 1. Table 2 shows the experimental results for Examples 4 and 5. The results in Table 2 show that the reduction step can be carried out at different temperatures (670° C. versus 750° C.). TABLE 2Ex. 4Ex. 5Catalyst11Mcat(g)0.7730.773Trxn(° C.)670670trxn(min)1010Frxn(sccm)1717Svol(vol %)1111Tred(° C.)670750tred(min)11Tregen(° C.)800800tregen(min)3030Cycles11First cycleYini63.161.9Yend61.761Sini86.787.7Send87.988.3 Table 3 shows the experimental results for Examples 6-10. Examples 6-10 were conducted by introducing a partial plug at the exhaust of the reactor so that as the hydrocarbon-containing feed passed through the reactor at room temperature, e.g., 25° C., the pressure indicator upstream of the reactor read 1.43 bara. During the experiment, the gas volumetric flow rate in the reactor was expected to increase due to steam addition, higher T and volume expansion of the flow due to propane dehydrogenation. Therefore, the pressure within the reactor should have been significantly higher than 1.43 bara. Unfortunately, the pressure during reactor could not be monitored due to equipment limitations. Experiments 8-10 show the effect of conducting the regeneration at different temperatures and durations. TABLE 3Ex. 6Ex. 7Ex. 8Ex. 9Ex. 10P (bara)1.431.431.431.431.43Catalyst11111Mcat(g)0.7730.7730.7730.7730.773Trxn(° C.)670670670670670trxn(min)1010101010Frxn(sccm)3434343434Svol(vol %)1111111111Tred(° C.)670660680670670tred(min)11111Tregen(° C.)800800800800900tregen(min)3030304530Cycles88177First cycleYini57.956.258.158.457.3Yend55.953.955.256.754.1Sini899186.28988.9Send89.691.78789.789.5Last cycleYini57.556.2NA58.5NAYend55.454.2NA57.1NASini88.991NA88.9NASend89.791.7NA89.7NA Table 4 shows the experimental results for Examples 11-14. The result sin Table 4 shown the effect space velocity had on the performance of the catalyst. TABLE 4Ex. 11Ex. 12Ex. 13Ex. 14Catalyst1111Mcat(g)0.1930.1930.1930.193Trxn(° C.)670670670700trxn(min)10101010Frxn(sccm)3417917Svol(vol %)11111111Tred(° C.)670670670670tred(min)1111Tregen(° C.)800800800800tregen(min)30303030Cycles1111First cycleYini54.159.360.658.5Yend4551.95644.4Sini95.292.889.686.3Send94.492.389.382.8 Table 5 shows the experimental results of Examples 15 and 16. Table 5 shows the effect of reduction in the presence of steam, respectively. TABLE 5Ex. 15Ex. 16Catalyst11Mcat(g)0.1930.193Trxn(° C.)670670trxn(min)1010Frxn(sccm)99Svol(vol %)1111Tred(° C.)670NAtred(min)1NATregen(° C.)800800tregen(min)3030Cycles11First cycleYini58.422.4Yend50.213.7Sini90.279.4Send89.768.7 Table 6 shows the results of Examples 17 and 18. Table 6 shows the effect of regeneration duration. TABLE 6Ex. 17Ex. 18Catalyst11Mcat(g)0.7730.773Trxn(° C.)670670trxn(min)1010Frxn(sccm)1717Svol(vol %)1111Tred(° C.)670670tred(min)11Tregen(° C.)800800tregen(min)3010Cycles11First cycleYini58.256.7Yend55.151.7Sini89.589.7Send8989.1 Table 7 shows the results of Examples 19-22. Table 7 shows the effect the amount steam in the hydrocarbon-containing feed has on the yield and selectivity. TABLE 7Ex. 19Ex. 20Ex. 21Ex. 22Catalyst1111Mcat(g)0.7730.7730.7730.773Trxn(° C.)670670650650trxn(min)10101010Frxn(sccm)17171717Svol(vol %)31111NATred(° C.)670670650650tred(min)1111Tregen(° C.)670670650650tregen(min)30303030Cycles1111First cycleYini54.958.556.852.1Yend49.955.455.322Sini90.790.493.690.8Send88.89093.684.7 In Ex. 23, the catalyst was subjected to 49 cycles total in the presence of about 11 vol % steam. The results of Ex. 23 are shown in Table 8. TABLE 8Ex. 23Catalyst1Mcat(g)0.773Trxn(° C.)670trxn(min)10Frxn(sccm)17Svol(vol %)11Tred(° C.)670tred(min)1Tregen(° C.)670tregen(min)30Cycles49First cycleYini56.5Yend51.6Sini89.8Send89+Last cycleYini57.6Yend52.4Sini89.8Send88.8 FIG.2shows the catalyst stability results of the catalyst used in Example 23 after having undergone 49 cycles (regeneration, reduction, and dehydrogenation) in the presence of steam. Example 24, Catalyst 2 The catalyst included 1 wt % of Pt and 3 wt % of Sn supported on CeO2, based on the weight of the CeO2. The CeO2support was made by calcining cerium (III) nitrate hexahydrate (Sigma-Aldrich 202991). The catalyst was made by incipient wetness impregnation of 3 g of CeO2with 0.788 g of 8 wt % chloroplatinic acid in water (Sigma Aldrich, 262587) and 0.266 g of tin (IV) chloride pentahydrate (Acros Organics 22369), followed by drying and calcination at 800° C. for 12 h. The data in Table 9 shows that catalyst 2 was stable over 42 cycles. TABLE 9Ex. 24Catalyst2Mcat(g)0.5Trxn(° C.)540trxn(min)10Frxn(sccm)12.3Svol(vol %)NATred(° C.)NAtred(min)NATregen(° C.)540tregen(min)10Cycles42First cycleYave15Save84.3Last cycleYave14.8Save89.7 Examples 25 and 26, Catalyst 3 The catalyst included 1 wt % of Pt and 2.7 wt % of Sn supported on Ceria-Zirconia, based on the weight of the Ceria-Zirconia. The Catalyst was made by incipient wetness impregnation of 16.5 g of Ceria-Zirconia (Sigma Aldrich 634174) with 0.44 g of chloroplatinic acid hexahydrate (BioXtra, P7082) and 1.33 g of tin (IV) chloride pentahydrate (Acros Organics 22369) dissolved in an appropriate amount of deionized water, followed by drying and calcination at 800° C. for 12 h. Table 10 shows the results of Examples 25 and 26. TABLE 10Ex. 25Ex. 26Catalyst33Mcat(g)0.4560.456Trxn(° C.)540580trxn(min)1010Frxn(sccm)1111Svol(vol %)NANATred(° C.)NANAtred(min)NANATregen(° C.)540580tregen(min)1010Cycles1012First cycleYini22.228.6Yend10.69.9Sini85.575.9Send91.391Last cycleYini21.428.8Yend11.710.4Sini86.276.9Send91.391.1 Examples 27-29, Catalyst 4 The catalyst included 1 wt % of Pt and 2.7 wt % of Sn supported on Y2O3, based on the weight of the Y2O3. The catalyst was made by incipient wetness impregnation of 4 g of Y2O3(US nano 3553) with 0.106 g of chloroplatinic acid hexahydrate (BioXtra, P7082) and 0.322 g of tin (IV) chloride pentahydrate (Acros Organics 22369) dissolved in an appropriate amount of deionized water, followed by drying and calcination at 800° C. for 12 h. Table 11 shows the results of Examples 27-29. TABLE 11Ex. 27Ex. 28Ex. 29Catalyst444Mcat(g)0.4560.4560.456Trxn(° C.)540540540trxn(min)101010Frxn(sccm)111111Svol(vol %)NANANATred(° C.)NANA540tred(min)NANA30Tregen(° C.)540540540tregen(min)102010Cycles2011First cycleYini22.723.223.9Yend14.91617.1Sini89.589.392.3Send949494.8Last cycleYini23.3NANAYend16.2NANASini90.5NANASend94NANA The data in Table 11 shows the performance of the catalyst was stable over 20 cycles. Examples 30-34, Catalyst 5 The catalyst included 1 wt % of Pt, 2.7 wt % of Sn supported on a CeO2and Al2O3support. The CeO2and Al2O3support was made by incipient wetness impregnation of 8.25 g of alumina (Sigma Aldrich 199443) with 5.67 g of cerium (III) nitrate hexahydrate (Sigma Aldrich 202991) dissolved in an appropriate amount of deionized water, followed by drying and calcination at 800° C. for 12 h. The catalyst was made by incipient wetness impregnation of the CeO2and Al2O3support with 0.22 g of chloroplatinic acid hexahydrate (BioXtra, P7082) and 0.67 g of tin (IV) chloride pentahydrate (Acros Organics 22369) dissolved in an appropriate amount of deionized water, followed by drying and calcination at 800° C. for 12 h. Table 13 shows the results of Examples 31-34. TABLE 13Ex. 31Ex. 32Ex. 33Ex. 34Catalyst5555Mcat(g)0.2280.2280.2280.228Trxn(° C.)620620620620trxn(min)10101010Frxn(sccm)17171717Svol(vol %)NA11NA11Tred(° C.)620NANA620tred(min)1NANA1Tregen(° C.)620620620620tregen(min)10101010Cycles1111First cycleYini27.825.89.233.5Yend24.620.93.129.2Sini91.590.989.392Send92.392.381.692.7 The data in Table 13 shows that both the co-addition of steam and catalyst pre-reduction helped to increase the yield and selectivity. Examples 35-38, Catalyst 6 The catalyst was 0.2 wt % of Pt, 0.2 wt % of Sn, and 0.67 wt % of K on high surface area ZrO2obtained from Alfa Aesar. Table 14 shows the results of Examples 35-38. TABLE 14Ex. 35Es. 36Ex. 37Ex. 38Catalyst6666Mcat(g)0.570.570.570.57Trxn(° C.)620620620620trxn(min)10101010Frxn(sccm)10101010Svol(vol %)11NANA1Tred(° C.)620NA620620tred(min)1NA11Tregen(° C.)800620620620tregen(min)30303030Cycles24111First cycleYini25.778.330.6Yend19.46.56.825.1Sini78.990.490.285.7Send78.490.690.284.2Last cycleYini24.7NANANAYend19.5NANANASini80.7NANANASend80.2NANANA The data in Table 14 shows that the catalyst was stable over 24 cycles and that the addition of steam significantly enhanced the yield. Catalyst Compositions 7-20 Catalyst Compositions 7-20 were prepared according to the following procedure. For each catalyst composition PURALOX® MG 80/150 (3 grams) (Sasol), which was a mixed Mg/Al metal oxide that contained 80 wt % of MgO and 20 wt % of Al2O3and had a surface area of 150 m2/g, was calcined under air at 550° C. for 3 hours to form a support. Solutions that contained a proper amount of tin (IV) chloride pentahydrate when used to make the catalyst composition (Acros Organics) and/or chloroplatinic acid when used to make the catalyst composition (Sigma Aldrich), and 1.8 ml of deionized water were prepared in small glass vials. The calcined PURALOX® MG 80/150 supports (2.3 grams) for each catalyst composition were impregnated with the corresponding solution. The impregnated materials were allowed to equilibrate in a closed container at room temperature (RT) for 24 hours, dried at 110° C. for 6 hours, and calcined at 800° C. for 12 hours. Table 15 shows the nominal Pt and Sn content of each catalyst composition based on the weight of the support. TABLE 15PtSnCatalyst(wt %)(wt %)70.4180.3190.21100.11110.051120.0251130.012511401150.10.5160.11170.12180.01250190.01250.5200.01252 Examples Using the Catalyst Compositions of Examples 7-20 Fixed bed experiments were conducted at approximately 100 kPa-absolute that used catalysts 7-14. A gas chromatograph (GC) was used to measure the composition of the reactor effluents. The concentrations of each component in the reactor effluents were then used to calculate the C3H6yield and selectivity. The C3H6yield and selectivity, as reported in these examples, were calculated on the carbon mole basis. In each example, 0.3 g of the catalyst composition was mixed with an appropriate amount of quartz diluent and loaded into a quartz reactor. The amount of diluent was determined so that the catalyst bed (catalyst+diluent) overlapped with the isothermal zone of the quartz reactor and the catalyst bed was largely isothermal during operation. The dead volume of the reactor was filled with quartz chips/rods. The C3H6yield and the selectivity at the beginning of trx˜ and at the end of trxnis denoted as Yini, Yend, Sini, and Send, respectively, and reported as percentages in Tables 5 and 6 below for catalysts 7-14. The process steps for catalysts 7-14 were as follows: 1. The system was flushed with an inert gas. 2. Dry air at a flow rate of 83.9 sccm was passed through a by-pass of the reaction zone, while an inert was passed through the reaction zone. The reaction zone was heated to a regeneration temperature of 800° C. 3. Dry air at a flow rate of 83.9 sccm was then passed through the reaction zone for 10 min to regenerate the catalyst. 4. The system was flushed with an inert gas. 5. A H2containing gas with 10 vol % H2and 90 vol % Ar at a flow rate of 46.6 sccm was passed through the by-pass of the reaction zone for a certain period of time, while an inert gas was passed through the reaction zone. This is then followed by flowing the H2containing gas through the reaction zone at 800° C. for 3 seconds. 6. The system was flushed with an inert gas. During this process, the temperature of the reaction zone was changed from 800° C. to a reaction temperature of 670° C. 7. A hydrocarbon-containing (HCgas) feed that included 81 vol % of C3H8, 9 vol % of inert gas (Ar or Kr) and 10 vol % of steam at a flow rate of 35.2 sccm was passed through the by-pass of the reaction zone for a certain period of time, while an inert gas was passed through the reaction zone. The hydrocarbon-containing feed was then passed through the reaction zone at 670° C. for 10 min. GC sampling of the reaction effluent started as soon as the feed was switched from the by-pass of the reaction zone to the reaction zone. The above process steps were repeated in cycles until stable performance was obtained. Tables 16 and 17 show that Catalyst 12 that contained only 0.025 wt % of Pt and 1 wt % of Sn had both a similar yield and a similar selectivity as compared to Catalyst 7 that contained 0.4 wt % of Pt and 1 wt % of Sn, which was surprising and unexpected. Catalyst 14 that did not include any Pt did not show an appreciable propylene yield. TABLE 16Catalyst 7Catalyst 8Catalyst 9Catalyst 10PerformanceYini61.761.760.763.7Yend55.255.754.256.7Sini97.397.297.097.1Send98.198.097.798.3 TABLE 17Catalyst 11Catalyst 12Catalyst 13Catalyst 14PerformanceYini62.462.056.72.0Yend57.254.645.71.7Sini96.797.396.964.2Send97.798.097.649.5 Catalyst compositions 15-20 were also tested using the same process steps 1-7 described above with regard to catalysts 7-14. Table 18 shows that the level of Sn should not be too low or too high for optimal propylene yield for the catalyst compositions that included 0.1 wt % of Pt based on the weight of the support. TABLE 18Catalyst 15Catalyst 10Catalyst 16Catalyst 170.5 wt % Sn1 wt % Sn1 wt % Sn2 wt % SnPerformanceYini58.463.763.456.5Yend49.556.755.547.7Sini96.997.197.297.8Send97.698.398.198.2 Table 19 shows that the level of Sn should not be too high or too low for optimal propylene yield for the catalyst compositions that included 0.0125 wt % of Pt based on the weight of the support. TABLE 19Catalyst 18Catalyst 19Catalyst 13Catalyst 200 wt % Sn0.5 wt % Sn1 wt % Sn2 wt % SnPerformanceYini2.64456.755.4Yend1.724.445.744.1Sini63.996.796.996.8Send61.195.697.697.6 Catalyst composition 12 that contained only 0.025 wt % of Pt and 1 wt % of Sn was also subjected to a longevity test using the same process steps 1-7 described above with regard to catalysts 7 to 14, except a flow rate of 17.6 sccm was used instead of 35.2 sccm in step 7.FIG.3shows that catalyst composition 12 maintained performance for 204 cycles (x-axis is time, y-axis is C3H6yield and selectivity to C3H6, both in carbon mole %). This disclosure can further include the following embodiments/aspects: E1. A process for upgrading a hydrocarbon, comprising: (I) contacting a hydrocarbon-containing feed with a catalyst comprising Pt disposed on a support to effect one or more of dehydrogenation, dehydroaromatization, and dehydrocyclization of at least a portion of the hydrocarbon-containing feed to produce a coked catalyst and an effluent comprising one or more upgraded hydrocarbons and molecular hydrogen, wherein: the hydrocarbon-containing feed comprises one or more of C2-C16linear or branched alkanes, or one or more of C4-C16cyclic alkanes, or one or more C8-C16alkyl aromatics, or a mixture thereof; the hydrocarbon-containing feed and catalyst are contacted at a temperature in a range from 300° C. to 900° C., for a time period of ≤3 hours, under a hydrocarbon partial pressure of at least 20 kPa-absolute, wherein the hydrocarbon partial pressure is the total partial pressure of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed; the catalyst comprises from 0.001 wt % to 6 wt % of Pt based on the weight of the support; and the one or more upgraded hydrocarbons comprise at least one of a dehydrogenated hydrocarbon, a dehydroaromatized hydrocarbon, and a dehydrocyclized hydrocarbon; (II) contacting at least a portion of the coked catalyst with an oxidant to effect combustion of at least a portion of the coke to produce a regenerated catalyst lean in coke and a combustion gas; and (III) contacting an additional quantity of the hydrocarbon-containing feed with at least a portion of the regenerated catalyst to produce a re-coked catalyst and additional effluent, wherein a cycle time from the contacting the hydrocarbon-containing feed with the catalyst in step (I) to the contacting the additional quantity of the hydrocarbon-containing feed with the regenerated catalyst in step (III) is ≤5 hours. E2. The process of E1, wherein in step (I), the hydrocarbon-containing feed and catalyst are contacted in the presence of steam at an amount from 0.1 vol % to 30 vol %, based on a total volume of any C2-C16alkanes, any C4-C16cyclic alkanes, and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. E3. The process of E1 or E2, wherein in step (I), the hydrocarbon-containing feed and the catalyst are contacted in the presence of steam at an amount from 1 vol % to 15 vol %, based on a total volume of any C2-C16alkanes, any C4-C16cyclic alkanes, and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. E4. The process of any of E1 to E3, wherein the coked catalyst comprises agglomerated Pt disposed on the support, and wherein at least a portion of the Pt agglomerated on the support is re-dispersed about the support during combustion of the coke in step (II). E5. The process of any of E1 to E4, wherein the hydrocarbon-containing feed comprises propane, wherein the upgraded hydrocarbon comprises propylene, and wherein contacting the hydrocarbon-containing feed with the catalyst in step (I) has a propylene yield of at least 52%, or at least 62%, or at least 72% at a propylene selectivity of ≥75%, ≥80%, ≥85%, or ≥90%, ≥95%. E6. The process of any of E1 to E5, wherein the hydrocarbon-containing feed comprises ≥70 vol % of propane, based on a total volume of the hydrocarbon-containing feed, wherein the hydrocarbon-containing feed and catalyst are contacted under a propane partial pressure of at least 40 kPa-absolute, and wherein contacting the hydrocarbon-containing feed with the catalyst in step (I) has a propylene yield of at least 52%, or at least 62%, or at least 72% at a propylene selectivity of ≥75%, ≥80%, ≥85%, or ≥90%, ≥95%. E7. The process of any of E1 to E6, wherein steps (I) to (III) are repeated for at least 15 cycles, wherein the catalyst has a first yield when initially contacted with the hydrocarbon-containing feed, and wherein the catalyst has a second activity upon completion of the fifteenth cycle that is at least 98% of the first yield. E8. The process of any of E1 to E7, wherein at least a portion of the Pt in the regenerated catalyst is at a higher oxidized state as compared to the Pt in the catalyst contacted with the hydrocarbon-containing feed; the process further comprising, after step (II) and before step (III), the following step: (IIa) contacting at least a portion of the regenerated catalyst with a reducing gas to produce a regenerated and reduced catalyst, wherein at a least a portion of the Pt in the regenerated and reduced catalyst is reduced to a lower oxidation state as compared to the Pt in the regenerated catalyst, and wherein the additional quantity of the hydrocarbon-containing feed is contacted with at least a portion of the regenerated and reduced catalyst. E9. The process of E8, wherein in step (IIa), the regenerated catalyst and reducing gas are contacted at a temperature in a range from 450° C. to 900° C., preferably 600° C. to 900° C., more preferably 620° C. to 800° C., more preferably 650° C. to 750° C., more preferably from 670° C. to 720° C. E10. The process of E8 or E9, wherein in step (IIa), the regenerated catalyst and reducing gas are contacted at a reducing agent partial pressure of 20 kPa-absolute to 10,000 kPa-absolute, or 50 kPa-absolute to 5,000 kPa-absolute, or 100 kPa-absolute to 1,000 kPa-absolute. E11. The process of any one of E8 to E10, wherein at least a portion of the Pt in the regenerated and reduced catalyst is in the elemental state. E12. The process of any of E1 to E11, wherein the hydrocarbon-containing feed further comprises an inert gas, e.g., Ar, Ne, He, N2, CH4, or a mixture thereof. E13. The process of any of E1 to E12, wherein in step (I), the hydrocarbon-containing feed and catalyst are contacted at a temperature in a range from 600° C. to 900° C., preferably from 600° C. to 800° C., more preferably from 650° C. to 750° C., more preferably from 670° C. to 720° C. E14. The process of any of E1 to E13, wherein in step (I), the hydrocarbon-containing feed and catalyst are contacted under a hydrocarbon partial pressure in a range from 20 kPa-absolute to 10,000 kPa-absolute, or 50 kPa-absolute to 5,000 kPa-absolute, or 100 kPa-absolute to 1,000 kPa-absolute. E15. The process of any of E1 to E14, wherein in step (II), the coked catalyst and oxidant are contacted at a temperature in a range from 600° C. to 1,100° C., preferably from 650° C. to 1,000° C., more preferably from 700° C. to 900° C., more preferably from 750° C. to 850° C. E16. The process of any of E1 to E15, wherein in step (II), the coked catalyst and oxidant are contacted under an oxidant partial pressure of 20 kPa-absolute to 10,000 kPa-absolute, or 50 kPa-absolute to 5,000 kPa-absolute, or 100 kPa-absolute to 1,000 kPa-absolute. E17. The process of any of E1 to E16, wherein the catalyst further comprises a promoter. E18. The process of E17, wherein the promoter comprises one or more of the following elements: Sn, Ga, Zn, Ge, In, Re, Ag, Au, Cu, a combination thereof, or a mixture thereof. E19. The process of E17 or 18, wherein the promoter is disposed on the support. E20. The process of any of E17 to E19, wherein the promoter is associated with the Pt. E21. The process of any of E17 to E20, wherein the promoter and the Pt form Pt-promoter clusters that are dispersed on the support. E22. The process of any of E17 to E21, wherein the catalyst comprises up to 10 wt % of the promoter based on the total weight of the support. E23. The process of any of E1 to E22, wherein the catalyst further comprises an alkali metal element disposed on the support. E24. The process of E17, wherein the alkali metal element comprises one or more of the following: Li, Na, K, Rb, Cs, a combination thereof, or a mixture thereof. E25. The process of E23 or 24, and wherein the catalyst comprises up to 5 wt % of the alkali metal element based on the total weight of the support. E26. The process of any of E1 to E25, wherein the support comprises at least one of: w wt % of one or more Group 2 elements, x wt % of one or more Group 4 elements, y wt % of one or more Group 12 elements, and z wt % of one or more elements having an atomic number of 21, 39, or 57-71 based on the weight of the support, wherein w, x, y, and z are independently in a range from 0 to 100, and wherein w+x+y+z≤100, wherein: any Group 2 element present is associated with a wt % m based on the weight of the support, any Group 4 element present is associated with a wt % n based on the weight of the support, any Group 12 element present is associated with a wt % p based on the weight of the support, and any element having an atomic number of 21, 39, or 57-71 present is associated with a wt % q based on the weight of the support, m, n, p, and q are independently a number that is in a range from 1 to 100, and wherein a sum of w/m+x/n+y/p+z/q is ≥1, based on the weight of the support. E27. The process of E26, wherein m, n, p, and q are each equal to 1, 2, 15, or 30, or wherein m=1, n=15, p=15, and q=1. E28. The process of E26 or 27, wherein a molar ratio of a combined amount of any Group 2 element, any Group 4 element, any Group 12 element, and any element having an atomic number of 21, 39, or 57-71 to the Pt is at least 0.18, 0.19, 0.24, or 0.29. E29. The process of any of E26 to E28, wherein the support further comprises at least one compound comprising at least one metal element or metalloid element selected from Groups 5, 6, 7, 11, 13, 14, 15, and 16. E30. The process of any one of E26 to E29, wherein at least a portion of any Group 2 element, any Group 4 element, any Group 12 element, and any element having an atomic number of 21, 39, or 57-71 present in the support is an oxide, a phosphate, a halide, a halate, a sulfate, a sulfide, a borate, a nitride, a carbide, an aluminate, an aluminosilicate, a silicate, a carbonate, metaphosphate, a selenide, a tungstate, a molybdate, a chromite, a chromate, a dichromate, or a silicide. E31. The process of any one of E26 to E30, wherein the support comprises one or more of the following: MguZn1-uO, where u is a positive number; ZnvAl2O3+v, where v is a positive number; MgwAl2O3+w, where w is a positive number; CaxAl2O3+x, where x is a positive number; SryAl2O3+y, where y is a positive number; BazAl2O3+z, where z is a positive number. BeO; MgO; CaO; BaO; SrO; BeCO3; MgCO3; CaCO3; SrCO3, BaCO3; ZrO2; ZrC; ZrN; ZrSiO4; CaZrO3; Ca7ZrAl6S18; TiO2; TiC; TiN; TiSiO4; CaTiO3; Ca7Al6O18; HfO2; HfC; HfN; HfSiO4; HfZrO3; Ca7HfAl6O18; ZnO; Zn3(PO4)2; Zn(ClO3)2; ZnSO4; B2O6Zn3; Zn3N2; ZnCO3; CeO2; Y2O3; La2O3; Sc2O3; Pr6O11; CePO4; CeZrO4; CeAlO3; BaCeO3; CePO4; Yttria-stabilized ZrO2; combinations thereof, and mixtures thereof. E32. The process of any of E26 to E31, wherein the support further comprises one or more of the following: B2O3, Al2O3, SiO2, SiC, Si3N4, an aluminosilicate, VO, V2O3, VO2, V2O5, Ga2O3, In2O3, Mn2O3, Mn3O4, MnO, one or more zeolites, and mixtures and combinations thereof. E33. The process of any of E1 to E32, wherein the cycle time is from 1 minute to 70 minutes, e.g., from 5 minutes to 45 minutes. E34. The process of any of E1 to E32, wherein the cycle time is from 5 minutes to 300 minutes, e.g., from 10 minutes to 50 minutes. E35. The process of any of E1 to E32, wherein the cycle time is from 0.1 seconds to 30 minutes, e.g., from 5 seconds to 10 minutes. E36. The process of any of E1 to E35, wherein the support is in the form of a plurality of primary particles comprising the Pt disposed thereon. E37. The process of any of E1 to E36, wherein the catalyst comprises primary particles having an average cross-sectional length of 0.2 nm to 500 μm, preferably 0.5 nm to 300 μm, more preferably 1 nm to 200 μm, more preferably 5 nm to 100 μm, and still more preferably 2 nm to 100 nm, as measured by a transmission electron microscope. E38. The process of any of E1 to E37, wherein the catalyst is in the form of a plurality of fluidized particles when contacted with the hydrocarbon-containing feed. E39. The process of any of E1 to E36, wherein the support is a monolithic structure comprising the Pt disposed thereon. E40. The process of any of E1 to E39, wherein the Pt is disposed on the support such that the Pt is the active component of the catalyst that effects the one or more of dehydrogenation, dehydroaromatization, and dehydrocyclization in step (I). E41. The process of any of E1 to E40, which is a fluid bed process, a fixed bed process, or a revers flow reactor process. E42. The process of any of E1 to E39, wherein the catalyst is in a fixed bed when contacted with the hydrocarbon-containing feed. E43. The process of any of E1 to E42, wherein the support has a surface area of 0.1 m2/g to 1,500 m2/g, preferably 1 m2/g to 1,000 m2/g, more preferably 10 m2/g to 800 m2/g, more preferably 100 m2/g to 500 m2/g. E44. The process of any of E1 or E4 to E43, wherein the hydrocarbon-containing feed is contacted with catalyst in the absence of any steam or in the presence of less than 0.1 vol % of steam based on a total volume of any C2-C16alkanes, any C4-C16cyclic alkanes, and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. E45. The process of any of E26 to E44, wherein the catalyst comprises the Group 3 element, and where the hydrocarbon-containing feed comprises 0.1 vol % to 50 vol % of steam, based on a total volume of any C2-C16alkanes, any C4-C16cyclic alkanes, and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. E46. A process for upgrading a hydrocarbon, comprising: (I) contacting a hydrocarbon-containing feed with a catalyst comprising a Group 8-10 element disposed on a support to effect one or more of dehydrogenation, dehydroaromatization, and dehydrocyclization of at least a portion of the hydrocarbon-containing feed to produce a coked catalyst and an effluent comprising one or more upgraded hydrocarbons and molecular hydrogen, wherein: the hydrocarbon-containing feed comprises one or more of C2-C16linear or branched alkanes, or one or more of C4-C16cyclic alkanes, or one or more of C8-C16alkyl aromatics, or a mixture thereof; the hydrocarbon-containing feed and catalyst are contacted at a temperature in a range from 300° C. to 900° C., for a time period of ≤3 hours, under a hydrocarbon partial pressure of at least 20 kPa-absolute, wherein the hydrocarbon partial pressure is the total partial pressure of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed; the one or more upgraded hydrocarbons comprise a dehydrogenated hydrocarbon, a dehydroaromatized hydrocarbon, a dehydrocyclized hydrocarbon, or a mixture thereof; the catalyst comprises from 0.001 wt % to 6 wt % of the Group 8-10 element based on the weight of the support, and wherein the support comprises: at least one of: w wt % of one or more Group 2 elements, x wt % of one or more Group 4 elements, y wt % of one or more Group 12 elements, and z wt % of one or more elements having an atomic number of 21, 39, or 57-71 based on the weight of the support, wherein w, x, y, and z are independently in a range from 0 to 100, and wherein w+x+y+z is ≤100, wherein: any Group 2 element present is associated with a wt % m based on the weight of the support, any Group 4 element present is associated with a wt % n based on the weight of the support, any Group 12 element present is associated with a wt % p based on the weight of the support, and any element having an atomic number of 21, 39, or 57-71 present is associated with a wt % q based on the weight of the support, m, n, p, and q are independently a number that is in a range from 1 to 100, and wherein a sum of w/m+x/n+y/p+z/q is ≥1, based on the weight of the support; (II) contacting at least a portion of the coked catalyst with an oxidant to effect combustion of at least a portion of the coke to produce a regenerated catalyst lean in coke and a combustion gas; and (III) contacting an additional quantity of the hydrocarbon-containing feed with at least a portion of the regenerated catalyst to produce a re-coked catalyst and additional effluent, wherein a cycle time from the contacting the hydrocarbon-containing feed with the catalyst in step (I) to the contacting the additional quantity of the hydrocarbon-containing feed with the regenerated catalyst in step (III) is ≤5 hours. E47. A process for upgrading a hydrocarbon, comprising: (I) contacting a hydrocarbon-containing feed with a catalyst comprising a Group 8-10 element or a compound thereof disposed on a support to effect one or more of dehydrogenation, dehydroaromatization, and dehydrocyclization of at least a portion of the hydrocarbon-containing feed to produce a coked catalyst and an effluent comprising one or more upgraded hydrocarbons and molecular hydrogen, wherein: the hydrocarbon-containing feed comprises one or more of C2-C16linear or branched alkanes, or one or more of C4-C16cyclic alkanes, or one or more of C8-C16alkyl aromatics, or a mixture thereof and 0.1 vol % to 50 vol % of steam, based on a total volume of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed; the hydrocarbon-containing feed and catalyst are contacted at a temperature in a range from 300° C. to 900° C., for a time period of ≤3 hours, under a hydrocarbon partial pressure of at least 20 kPa-absolute, wherein the hydrocarbon partial pressure is the total partial pressure of any C2-C16alkanes and any C8-C16alkyl aromatics in the hydrocarbon-containing feed; the catalyst comprises from 0.001 wt % to 6 wt % of the Group 8-10 element or a compound thereof based on the weight of the support, and wherein the upgraded hydrocarbon comprises a dehydrogenated hydrocarbon, a dehydroaromatized hydrocarbon, a dehydrocyclized hydrocarbon, or a mixture thereof; (II) contacting at least a portion of the coked catalyst with an oxidant to effect combustion of at least a portion of the coke to produce a regenerated catalyst lean in coke and a combustion gas; and (III) contacting an additional quantity of the hydrocarbon-containing feed with at least a portion of the regenerated catalyst to produce a re-coked catalyst and additional effluent, wherein a cycle time from the contacting the hydrocarbon-containing feed with the catalyst in step (I) to the contacting the additional quantity of the hydrocarbon-containing feed with the regenerated catalyst in step (III) is ≤5 hours. E48. The process of E47, wherein the hydrocarbon-containing feed comprises 1 vol % to 15 vol % of the steam. E49. The process of E47 or 48, wherein the support comprises: at least one of: w wt % of one or more Group 2 elements, x wt % of one or more Group 4 elements, y wt % of one or more Group 12 elements, and z wt % of one or more elements having an atomic number of 21, 39, or 57-71 based on the weight of the support, wherein w, x, y, and z are independently in a range from 0 to 100, and wherein w+x+y+z is ≤100, wherein: any Group 2 element present is associated with a wt % m based on the weight of the support, any Group 4 element present is associated with a wt % n based on the weight of the support, any group 12 element present is associated with a wt % p based on the weight of the support, and any element having an atomic number of 21, 39, or 57-71 present is associated with a wt % q based on the weight of the support, m, n, p, and q are independently a number that is in a range from 1 to 100, and wherein a sum of w/m+x/n+y/p+z/q is ≥1, based on the weight of the support E50. The process of E46 or E49, wherein m, n, p, and q are each equal to 1, 2, 15, or 30, or wherein m=1, n=15, p=15, and q=1. E51. The process of any of E46 to E50, wherein the hydrocarbon-containing feed comprises propane, wherein the upgraded hydrocarbon comprises propylene, and wherein contacting the hydrocarbon-containing feed with the catalyst in step (I) has a propylene yield of at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 55%, at least 57%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at or at least 66% at a propylene selectivity of ≥75%, ≥80%, ≥85%, or >90%, or ≥95% at a propylene selectivity of ≥75%, ≥80%, ≥85%, or ≥90%, or ≥95%. E52. The process of any of E46 to 51, wherein the hydrocarbon-containing feed comprises ≥51 vol % of propane, based on a total volume of the hydrocarbon-containing feed, wherein the hydrocarbon-containing feed and catalyst are contacted under a propane partial pressure of at least 20 kPa-absolute, and wherein contacting the hydrocarbon-containing feed with the catalyst in step (I) has a propylene yield of >52%, or >62%, or >72% at a propylene selectivity of ≥75%, ≥80%, ≥85%, or ≥90%, or >95%. E53. The process of any of E46 to E52, wherein steps (I) to (III) are repeated for at least 15 cycles, wherein the catalyst has a first yield of the upgraded hydrocarbon when initially contacted with the hydrocarbon-containing feed, and wherein the catalyst has a second yield of the upgraded hydrocarbon upon completion of the fifteenth cycle that is ≥95%, ≥97%, ≥98%, or ≥99% of the first yield. E54. The process of any of E46 to E53, further comprising, after step (II) and before step (III), the following step: (IIa) contacting at least a portion of the regenerated catalyst with a reducing gas to produce a regenerated and reduced catalyst, wherein the additional quantity of the hydrocarbon-containing feed is contacted with at least a portion of the regenerated and reduced catalyst. E55. The process of E54, wherein at least a portion of the Group 8-10 element in the regenerated catalyst is at a higher oxidized state as compared to the Group 8-10 element in the catalyst contacted with the hydrocarbon-containing feed, and wherein at a least a portion of the Group 8-10 element in the regenerated and reduced catalyst is reduced to a lower oxidation state as compared to the Group 8-10 element in the regenerated catalyst. E56. The process of E55, wherein at least a portion of the Group 8-10 element in the regenerated and reduced catalyst is in the elemental state. E57. The process of any one of E54 to E56, wherein in step (IIa), the regenerated catalyst and reducing gas are contacted at a temperature in a range from 450° C. to 900° C., preferably 600° C. to 900° C., more preferably 620° C. to 800° C., more preferably 650° C. to 750° C., more preferably from 670° C. to 720° C. E58. The process of any one of E54 to E57, wherein in step (IIa), the regenerated catalyst and reducing gas are contacted at a reducing agent partial pressure of 20 kPa-absolute to 10,000 kPa-absolute, or 50 kPa-absolute to 5,000 kPa-absolute, or 100 kPa-absolute to 1,000 kPa-absolute. E59. The process of any of E46 to E58, wherein the Group 8-10 element comprises Pt. E60. The process of any of E46 to E59, wherein the hydrocarbon-containing feed further comprises an inert gas, e.g., Ar, Ne, He, N2, CH4, and mixtures thereof. E61. The process of any of E46 to E60, wherein in step (I), the hydrocarbon-containing feed and catalyst are contacted at a temperature in a range from 650° C. to 900° C., more preferably from 650° C. to 800° C., preferably from 660° C. to 780° C., more preferably from 670° C. to 760° C. E62. The process of any of E46 to E61, wherein in step (I), the hydrocarbon-containing feed and catalyst are contacted under a hydrocarbon partial pressure in a range from 20 kPa-absolute to 10,000 kPa-absolute, or 50 kPa-absolute to 5,000 kPa-absolute, or 100 kPa-absolute to 1,000 kPa-absolute. E63. The process of any of E46 to E62, wherein the catalyst further comprises a promoter disposed on the support. E64. The process of E63, wherein the promoter comprises Sn, Ga, Zn, Ge, In, Re, Ag, Au, Cu, a compound thereof, or a mixture thereof. E65. The process of E63 or 64, wherein the promoter is associated with the Group 8-10 element. E66. The process of any of E63 to E65, wherein the promoter and the Group 8-10 element form Group 8-10 element/promoter clusters that are dispersed on the support. E67. The process of any of E63 to E66, wherein the catalyst comprises up to 10 wt % of the promoter based on the total weight of the support. E68. The process of any of E46 to E67, wherein the catalyst further comprises an alkali metal disposed on the support. E69. The process of E68, wherein the alkali metal comprises Li, Na, K, Rb, Cs, a combination thereof, or a mixture thereof. E70. The process of E68 or 69, wherein the catalyst comprises up to 5 wt % of the alkali metal based on the total weight of the support. E71. The process of any of E46 or E49 to E70, wherein at least a portion of any Group 2 element, any Group 4 element, any Group 12 element, and any element having an atomic number of 21, 39, or 57-71 present in the support is an oxide, a phosphate, a halide, a halate, a sulfate, a sulfide, a borate, a nitride, a carbide, an aluminate, an aluminosilicate, a silicate, a carbonate, metaphosphate, a selenide, a tungstate, a molybdate, a chromite, a chromate, a dichromate, or a silicide. E72. The process of any of E46 or E49 to E71, wherein a molar ratio of a combined amount of any Group 2 element, any Group 4 element, any Group 12 element, and any element having an atomic number of 21, 39, or 57-71 to the Pt is at least 0.18. E73. The process of any of E46 or E49 to E72, wherein the support comprises one or more of the following: MguZn1-uO, where u is a positive number; ZnvAl2O3+v, where v is a positive number; MgwAl2O3+w, where w is a positive number; CaxAl2O3+x, where x is a positive number; SryAl2O3+y, where y is a positive number; BazAl2O3+z, where z is a positive number. BeO; MgO; CaO; BaO; SrO; BeCO3; MgCO3; CaCO3; SrCO3, BaCO3; ZrO2; ZrC; ZrN; ZrSiO4; CaZrO3; Ca7ZrAl6O18; TiO2; TiC; TiN; TiSiO4; CaTiO3; Ca7Al6O18; HfO2; HfC; HfN; HfSiO4; HfZrO3; Ca7HfAl6O18; ZnO; Zn3(PO4)2; Zn(ClO3)2; ZnSO4; B2O6Zn3; Zn3N2; ZnCO3; CeO2; Y2O3; La2O3; Sc2O3; Pr6O11; CePO4; CeZrO4; CeAlO3; BaCeO3; CePO4; Yttria-stabilized ZrO2; combinations thereof, and mixtures thereof. E74. The process of any of E46 or E49 to E73, wherein the support further comprises one or more of the following: B2O3, Al2O3, SiO2, SiC, Si3N4, an aluminosilicate, VO, V2O3, VO2, V2O5, Ga2O3, In2O3, Mn2O3, Mn3O4, MnO, one or more zeolites, and mixtures and combinations thereof. E75. The process of any of E46 to E74, wherein the cycle time is from 1 minute to 70 minutes, e.g., from 5 minutes to 45 minutes. E76. The process of any of E46 to E75, wherein the cycle time is from 5 minutes to 300 minutes, e.g., from 10 minutes to 50 minutes. E77. The process of any of E46 to E75, wherein the cycle time is from 0.1 seconds to 30 minutes, e.g., from 5 seconds to 10 minutes. E78. The process of any of E46 to E77, wherein the support comprises a plurality of primary particles comprising the Group 8-10 element disposed thereon. E79. The process of any of E46 to E77, wherein the catalyst comprises primary particles, and wherein the primary particles have an average cross-sectional length of 0.2 nm to 500 μm, preferably 1 nm to 300 μm, more preferably 2 nm to 200 μm, more preferably 2 nm to 100 μm, still more preferably from 2 nm to 500 nm, still more preferably from 2 nm to 100 nm, as measured by a transmission electron microscope. E80. The process of any of E46 to E79, wherein the catalyst is in the form of a plurality of fluidized particles when contacted with the hydrocarbon-containing feed. E81. The process of any of E46 to E77, wherein the support is a monolithic structure comprising the Group 8-10 element disposed thereon. E82. The process of any of E46 to E81, wherein the Group 8-10 element comprises Pt, and wherein the Pt is disposed on the support such that the Pt is the active component of the catalyst that effects the one or more of dehydrogenation, dehydroaromatization, and dehydrocyclization in step (I). E83. The process of any of E46 to E82, which is a fluid bed process, a fixed bed process, or a reverse flow reactor process. E84. The process of any of E46 to E83, wherein the catalyst is in a fixed bed when contacted with the hydrocarbon-containing feed. E85. The process of any of E46 to E84, wherein the support has a surface area of 0.1 m2/g to 1,500 m2/g, preferably 1 m2/g to 1,000 m2/g, more preferably 10 m2/g to 800 m2/g, more preferably 100 m2/g to 500 m2/g. E86. The process of any of E46 or E49 to E85, wherein the catalyst comprises the Group 3 element, and where the hydrocarbon-containing feed comprises 0.1 vol % to 50 vol % of steam, based on a total volume of any C2-C16alkanes, any C4-C16cyclic alkanes, and any C8-C16alkyl aromatics in the hydrocarbon-containing feed. Various terms have been defined above. To the extent a term used in a claim is not defined above, it should be given the broadest definition persons in the pertinent art have given that term as reflected in at least one printed publication or issued patent. Furthermore, all patents, test procedures, and other documents cited in this application are fully incorporated by reference to the extent such disclosure is not inconsistent with this application and for all jurisdictions in which such incorporation is permitted. While the foregoing is directed to embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. | 122,501 |
11859137 | DETAILED DESCRIPTION Reference will now be made in detail to presently preferred compositions, embodiments and methods of the present invention, which constitute the best modes of practicing the invention presently known to the inventors. The Figures are not necessarily to scale. However, it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the invention and/or as a representative basis for teaching one skilled in the art to variously employ the present invention. It must also be noted that, as used in the specification and the appended claims, the singular form “a,” “an,” and “the” comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components. As used herein, the term “about” means that the amount or value in question may be the specific value designated or some other value in its neighborhood. Generally, the term “about” denoting a certain value is intended to denote a range within +/−5% of the value. As one example, the phrase “about 100” denotes a range of 100+/−5, i.e. the range from 95 to 105. Generally, when the term “about” is used, it can be expected that similar results or effects according to the invention can be obtained within a range of +/−5% of the indicated value. As used herein, the term “and/or” means that either all or only one of the elements of said group may be present. For example, “A and/or B” shall mean “only A, or only B, or both A and B”. In the ease of “only A”, the term also covers the possibility that B is absent, i.e. “only A, but not B”. It is also to be understood that this invention is not limited to the specific embodiments and methods described below, as specific components and/or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way. The term “comprising” is synonymous with “including,” “having,” “containing,” or “characterized by.” These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps. The phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. The phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. The phrase “composed of” means “including” or “consisting of.” Typically, this phrase is used to denote that an object is formed from a material. With respect to the terms “comprising,” “consisting of,” and “consisting essentially of,” where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms. The term “one or more” means “at least one” and the term “at least one” means “one or more.” The terms “one or more” and “at least one” include “plurality” as a subset. The term “substantially,” “generally,” or “about” may be used herein to describe disclosed or claimed embodiments. The term “substantially” may modify a value or relative characteristic disclosed or claimed in the present disclosure. In such instances, “substantially” may signify that the value or relative characteristic it modifies is within ±0%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% or 10% of the value or relative characteristic. It should also be appreciated that integer ranges explicitly include all intervening integers. For example, the integer range 1-10 explicitly includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Similarly, the range 1 to 100 includes 1, 2, 3, 4 . . . 97, 98, 99, 100. Similarly, when any range is called for, intervening numbers that are increments of the difference between the upper limit and the lower limit divided by 10 can be taken as alternative upper or lower limits. For example, if the range is 1.1. to 2.1 the following numbers 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 can be selected as lower or upper limits. In the examples set forth herein, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 50 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. In a refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 30 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. In another refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 10 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. The term “one or more” means “at least one” and the term “at least one” means “one or more.” The terms “one or more” and “at least one” include “plurality” and “multiple” as a subset. In a refinement, “one or more” includes “two or more.” ABBREVIATIONS “ag” means agricultural. “DHPO” means direct homogenous partial oxidation of methane to methanol. “DME” means dimethyl ether. “GLT” means gas-to-liquids. “KO” means knockout. “LNG” means liquified natural gas. “RNG” means renewable natural gas. VOC means volatile organic compounds. “WWTP” means wastewater treatment plant. FIG.1provides a schematic of an integrated system and method for converting biomass to renewable natural gas and then to methanol and other value-added products.FIG.2provides a schematic of an example of a site layout implementing the system ofFIG.1. Conversion system10includes a compressor12that receives gases (e.g., methane) from a source13. Examples of sources include landfills, methane-rich biomass gas products of an ag digester, biomass gasification-derived methane-rich gas, municipal waste, and products of a wastewater treatment plant. In a refinement, the gases from source13are renewable gas derived from gasification of biomass such as corn stover and other agricultural residues such as rice straw, wheat straw, sugarcane bagasse or municipal wastes. The gaseous product is purified in a series of purification stations to enhance the amount of methane that will be provided to a GTL plant. Knockout tank14is in fluid communication with compressor12receiving gas therefrom. H2S removal station16receives gas from knockout tank14and removes hydrogen sulfide. VOC station18acts on the output gas from H2S removal station16to remove volatile organic compounds. VOC station18includes a VOC removal lead component and a VOC removal lag component which operate in an alternating manner in which the lead component actively removes VOCs while the lag component is regenerating. The roles of the lead and lag components between these two components alternate. Scrubber20then acts on the output gas from VOC station18to remove carbon dioxide and potentially additional hydrogen sulfide. The output gas from scrubber20is then passed to an amine scrubber22that can remove amines and additional carbon dioxide. Alternative, amine scrubber can be replaced with pressure swing adsorption for removing carbon dioxide and/or a carbon dioxide-removing membrane. The output gas from scrubber20is then passed through molecular sieve system30to remove additional impurities. Scrubber20can be a SELEXOL® based scrubber that uses a Selexol solvent which is a mixture of the dimethyl ethers of polyethylene glycol to remove carbon dioxide and hydrogen sulfide. Still referring toFIG.1, the series of purification stations includes a nitrogen removal system32that receives output gas from the molecular sieve system and removes at least a portion of nitrogen gas therein. Nitrogen removal system32receives the output gas from molecular sieve system30and removes at least a portion of the nitrogen gas therein. The output gas33from nitrogen removal system32is received by reciprocating compressor34, which after compression to form output gas stream35is passed to GLT plant36. GTL plant then output a product blend40. The product blend40advantageously includes methanol and ethanol. In a refinement, the product blend can also include hydrogen (H2), acetone, dimethyl ether, isopropanol, acetic acid, formic acid, formaldehyde, dimethoxymethane, 1,1 dimethoxyethane, methyl formate, methyl acetate, and water. In another refinement, includes 0 to 15 mole percent acetone, 30 to 99 mole percent methanol, 0 to 20 mole percent ethanol, 0.0 to 10 mole percent isopropanol, 0 to 1 mole percent acetic acid, 0 to 1 mole percent formic acid, 0 to 15 mole percent formaldehyde, and 1 to 30 mole percent water. Advantageously, the integrated system has a carbon intensity that is less than +100 at its highest range depending on feedstock, and more typically +20 and typically, less than +15, with some feedstocks showing CI score less than −250 when using Ag digester dairy and pig farm gas.FIG.3illustrates the potential revenue flow for the system ofFIG.1from which a diversity of products can be produced, many of which are formed from GTL plant36. In a variation, GTL plant36can be the GLT system set forth in U.S. Pat. No. 9,255,051; the entire disclosure of which is hereby incorporated by reference. With reference toFIG.4, a schematic illustration of GLI plant for converting gas stream35to oxygenated hydrocarbons, e.g., methanol, ethanol, formaldehyde, acetaldehyde, and the like, is provided. In a refinement, the apparatus functions in a continuous manner when in operation. Homogeneous direct partial oxidation is performed in a reactor60, which is supplied with a hydrocarbon-containing gas62(and gas stream35) and an oxygen-containing gas64from oxygen supply38. In a refinement, the reaction is operated at pressures from about 450 to 1250 psia and temperatures from about 350 to 450° C. In particular, hydrocarbon-containing gas62and an oxygen-containing gas64react in a vessel to form a first product blend which is a blend (i.e., a mixture) of partially oxygenated compounds that include formaldehyde. In a refinement, the first product blend and/or output streams70,72include C1-10alcohols and/or C1-5aldehydes. In another refinement, the first product blend and/or output streams70,72include an alcohol selected from the group consisting of methanol, ethanol, propanols, butanols, pentanols, and combinations thereof, and/or aldehyde selected from the group consisting formaldehyde, acetaldehyde, propionaldehyde and combinations thereof. In another refinement, the first product blend and/or output streams70,72include an alcohol selected from the group consisting of methanol, ethanol, and combinations thereof and aldehyde selected from the group consisting formaldehyde, acetaldehyde, and combinations thereof. Examples of systems and methods of performing the partial oxidation as set forth in U.S. Pat. Nos. 8,293,186; 8,202,916; 8,193,254; 7,910,787; 7,687,669; 7,642,293; 7,879,296; 7,456,327; and 7,578,981; the entire disclosures of which are hereby incorporated by reference. In a refinement, the hydrocarbon-containing gas includes C1-10alkanes. In another refinement, the hydrocarbon-containing gas includes an alkane selected from the group consisting of methane, ethane, propanes, butanes, pentanes, and combinations thereof. In another refinement, the hydrocarbon-containing gas includes an alkane selected from the group consisting of methane, ethane, and combinations thereof. Examples of oxygen-containing gas include molecular oxygen, which may be in the form of concentrated oxygen or air. In a refinement, the oxygen-containing gas stream is made oxygen-rich (e.g., by passing air through a membrane to increase oxygen content). The low conversion and selectivity of homogeneous direct partial oxidation requires that a recycle loop is utilized to increase the overall carbon efficiency. Following partial oxidation reaction, the reactant stream is rapidly cooled in a series of heat exchangers74and76to prevent decomposition of the produced oxygenates. The heat energy transferred by exchanger76might optionally be used to provide energy that may be used in the creation of synthesis gas. After cooling, the liquids are separated from the gas stream as station102. The gas stream is then submitted to a separation process for removal of non-hydrocarbon fractions a station80, which may be performed via scrubbing, membrane separation, adsorption processes, cryogenic separations, or by purging a small gas fraction. If station80is a liquid scrubbing system, liquid products are sent to a flash drum82where dissolved gases are removed. Non-hydrocarbon gases84are removed from the recycle loop, and the hydrocarbon gases86are then recycled to combine with fresh hydrocarbon gas stream35(e.g., a purified methane-containing stream) from the system ofFIG.1, which has been pressurized to the pressure of the loop by compressor92. The stream composed of recycled hydrocarbons plus fresh methane gas is pressurized to make up for pressure losses in the recycle loop, preheated via the cross exchanger74and further by the preheater98, when necessary to meet the desired reaction conditions. Liquids generated by the gas-to-chemicals process are composed predominantly of alcohols and aldehydes (e.g., methanol, ethanol, and formaldehyde) as set forth above. The raw liquid stream100generated by the GTL process is generally composed of 50-70% alcohols and 5-20% aldehydes 15-30% water. Downstream processing of these liquids may include a number of different synthesis routes to higher-value chemicals and fuels, but a simple distillation of alcohols from aldehydes is performed in a simple fractional distillation column106in which alcohols are recovered in the distillate70and the aqueous aldehyde solution from the column bottoms72. The compositions of the streams84obtained from the separation of non-hydrocarbon gases from the recycle loop and from degassing the liquid mixture102may vary significantly depending on the separation methods employed in station104. Stream102would be typically be needed to regenerate a scrubbing fluid by liberating dissolved gasses such as carbon dioxide or carbon monoxide, which would be enriched in this stream. Stream102is composed predominantly of lighter hydrocarbons and carbon oxides (e.g., CO2and/or) which are soluble in the liquid solution but are vaporized when decreasing the pressure. Stream102may or may not be blended with stream84, depending on the needs of the synthesis gas reactor108. Stream84is a separated gas stream from station105such might be separated from a purge stream, membrane, cryogenic, or adsorption process. Although stream84would be enriched in non-hydrocarbon gasses, there would be some light alkanes present as well. A simple purge method in station104results in hydrocarbon fractions that may reach up to 70%, while selective removal techniques tend to preserve hydrocarbons in the recycle loop2. Stream84and102are blended to form stream110, which is rich in synthesis gas. Stream110goes through reactor108, which converts the hydrocarbon portion to synthesis gas in stream112. Stream112then goes on to react with liquid streams in reactor109(for example, output streams70or72). Stream72is the bottoms product of distillation column106and would contain low volatility, high boiling components such as formalin, heavy alcohols, and some acids. Stream70is the overhead from distillation column106and would be rich in the higher volatility low boiling components such as light alcohols. Streams112and said liquid product streams would then react to form oxygenates of a carbon number greater than that in the liquid reactant stream. Such oxygenates produced by reactor116might include esters such as formates and DMC, or carboxylic acids from a CO-rich synthesis gas in stream112. Higher alcohols and aldehydes from mixed alcohol synthesis, alcohol homologation, and aldehyde synthesis can form from a relatively hydrogen-rich synthesis gas in stream112. As mentioned, stream72contains aqueous formaldehyde, which is known to react with synthesis gas to form glycolic acid and glycolaldehyde. In another refinement, the synthetic gas is generated by a pyrolysis reaction or generated externally and blended with stream17. In a further refinement, the pyrolysis reaction generates light alkanes in addition to synthetic gas. Alternatively, stream112may react with itself in reactor116and form light alkanes (e.g., C1-4alkanes) for use as a feed gas to be blended with gas stream35. The light alkane product of this reaction would typically be rich in C2+ hydrocarbons, which are known to produce a distribution of alcohols with a higher molecular weight when compared to methane under homogenous partial oxidation conditions. Certain catalysts are also known to produce both alcohols and light alkanes. In addition, stream112may be blended with externally produced synthesis gas to produce a gas mixture in reactor116which can be utilized by reactor60. This feature allows for feedstock flexibility in the direct homogenous partial oxidation process. In another variation, the synthesis gas is generated in reactor108by implementing steam, dry, or tri-reforming reaction. In a refinement, the tri-reforming reaction is assisted by energy (e.g., it uses the heat) recovered from a heat exchanger76. In one embodiment, DHPO gas rejected by a DHPO recycle loop is used to produce syngas in reactor108. The syngas further reacts to produce both oxygenates and light alkanes in reactor116. The conversion may be effected using a suitable catalyst, for example, an actinide/lanthanide modified catalyst as described in U.S. Pat. No. 4,762,588; the entire disclosure of which is hereby incorporated by reference. DHPO Oxygenate products may be separated from light alkanes using any simple liquid separation system well-known in the art. The separated alkanes may then be blended with the feed gas in stream35following nitrogen removal, if necessary. In another embodiment, in a DHPO system comprising a synthesis gas, the gas may be separated in the recycle system using one or more membranes alkanes such as might be found with station104. Many membrane materials lack sufficient selectivity to completely separate non-hydrocarbon such as nitrogen and carbon dioxide from hydrocarbon streams. In this configuration, the light alkanes can be present in the permeate or retentate streams of the membrane. Using well known techniques, this stream would be converted into synthesis gas. Hydrogen and carbon dioxide may optionally be separated from this synthesis gas in stream112by a membrane or scrubbing system prior to reactor116to make a stream rich in CO, which could then be used in carbonylation and carbon insertion reactions in reactor116. The hydrogen may optionally be used to further reduce the carbonylated species. Alternatively, syngas is known to react directly with alcohols and form higher alcohols, esters, or aldehydes. In another embodiment, some of the light alkanes present in stream110may be thermally decomposed to provide hydrogen and carbon black in reactor108. This thermal decomposition may be assisted by a heat exchanger76. The carbon black could either be partially combusted in oxygen to yield pure carbon monoxide or reacted with the carbon dioxide to yield carbon monoxide. This pure carbon monoxide can then be used as a reactant in carbonylation or carbon insertion reactions in reactor116. The hydrogen may optionally be used to further reduce the carbonylated species present in stream120after reactor116. Further to the previous embodiment, an external carbon source may be utilized to react with carbon dioxide to yield carbon monoxide in either a catalytic or non-catalytic process assisted by heat recovered by heat exchanger76. The carbon monoxide may then be reacted with oxygenates in carbon insertion or carbonylation reactions in a manner consistent with the previous embodiment. In another variation, the carbon dioxide by-products produced above and be collected and converted to use products. An example of a plasma reactor system that can be used to process carbon dioxide is provided in U.S. provisional Pat. No. 63/177,040, filed Apr. 20, 2021; the entire disclosure of which is hereby incorporated by reference. Referring toFIG.5, a schematic of a multi-tubular plasma reactor system for CO2separation is provided. In a refinement, carbon dioxide by-products from the off gas lines of the VOC removal system and/or the amine scrubber22(or the PSA system or membrane) and/or the carbon dioxide output of the GTL system ofFIG.4are provided to a plasma reactor system120for highly economical and efficient production of high purity ethanol. Stilt referring toFIG.5, plasma reactor system130includes a plurality132of dielectric barrier discharge plasma reactors134iwhere i is an integer label for each reactor. Characteristically, the dielectric barrier discharge plasma reactors are adapted to receive a plasma feed gas from an industrial reactor and output ethanol. In a refinement, each dielectric barrier discharge plasma reactor can include 2-4 cm quartz or other inert reactor tubes of lengths between 5 and 50 cm. In a refinement, each DBD plasma reactor134iis packed with a transition metal oxide catalyst. The catalyst can include a transition metal, either supported or unsupported. The supports can include one metal oxide or a mixture of metal oxides. In a refinement, supports are oxides of p-block elements of the periodic table or hybrids such as zeolites, hydrotalcites or phosphor-silicates, activated carbon, and carbon nanotubes. Oxide supports may be acidic, neutral, or basic. Catalyst support with either oxygen storage capability or exhibiting redox property such as CeO2can also be used. Different loading of metal and supports weight ratios can be used for specific applications. In a refinement, the catalysts are promoted with metal promoters or unpromoted. In a refinement, a weight ratio of transition metal to support is from 0.1 to 100. A plasma in reactors can be thermally and/or non-thermally generated. Sources of power can be from both non-renewable or renewable sources such as methane, associated gases, nitrogen, carbon dioxide, wind, solar, hydro, nuclear, or a combination thereof. In a refinement, each plasma dielectric barrier discharge plasma reactors134iincludes a pair of electrodes136and138for generating the RF plasma. For the plasma generation, multiple electrodes made of conduction metals such as stainless steel and a negative power supply of 5-50 kV with a rectifier can be used. In refinement, plasma reactor system130includes furnace140for heating the plurality132of dielectric barrier discharge (DBD) plasma reactors134i. The reactors can be heated with clamp-shale furnace power with non-renewable or renewable electric sources. Alternatively, power produced at the site can also be used for heating the furnace. It should be appreciated that each reactor can be plasma generated with heating therein. For scale-up of the reactor configuration, a liner approach in parallel or series can be used that is already adapted in the industry. Therefore, a multi-tubular reactor system having 1-50 reactors is used for converting 60 m3/h (5.08×10−2MMSCFD) gas (e.g., CO2) can be converted to high purity ethanol. Each tube can be loaded with 1 to 10 g of the catalyst. The catalyst can be reduced with a gas comprising of pure hydrogen or hydrogen gas diluted in an inert gas such as Ar, N2, or He, which can be used. Also, other reducing gas such as CO may be used for the reduction of the catalyst prior to the plasma application. The rate of production of ethanol is about 1-10 μmol gcat−1h−1per tube. While exemplary embodiments are described above, it is not intended that these embodiments describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation, and it is understood that various changes may be made without departing from the spirit and scope of the invention. Additionally, the features of various implementing embodiments may be combined to form further embodiments of the invention. | 24,868 |
11859138 | DETAILED DESCRIPTION All numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and take into account experimental error and variations that would be expected by a person having ordinary skill in the art. Overview In various aspects, systems and methods are provided for performing hydrodeoxygenation of bio-derived feeds in a variety of settings, such as in a refinery setting or in a standalone plant, while maintaining the hydrodeoxygenation catalyst in a sulfided state. During hydrodeoxygenation, a hydrogen-containing stream is provided to the hydrodeoxygenation reactor as a hydrogen treat gas to provide hydrogen for the reaction. In some aspects, the hydrogen treat gas used for hydrodeoxygenation can be formed at least in part from hydrogen that has been used as a stripping gas for removing H2S from a rich amine stream. In such aspects, the hydrogen recycle loop for the hydroprocessing stage(s) that include the hydrodeoxygenation stage can be integrated with the amine absorber loop for one or more associated processes. In other aspects, H2S can be stripped using water vapor, and a resulting overhead H2S stream can be compressed prior to incorporation of the H2S into a hydrogen-containing stream. The rich amine stream can correspond to a rich amine stream from an amine absorber associated with an individual processing unit, such as a hydroprocessing reactor, or a rich amine stream from a centralized unit that performs amine capture of H2S from gases derived from multiple refinery processes. The resulting hydrogen-containing stream can include sufficient H2S to substantially maintain the catalyst in the hydrodeoxygenation stage in a sulfided state. Bio-derived fuels are a potential supplement or even alternative to conventional fuels. In order to be used in conventional fuel applications, however, bio-derived fuels typically need to be deoxygenated. While co-processing of substantial portions of mineral feeds (and or other non-bio-derived feeds) can be performed during hydrodeoxygenation, unless the mineral co-feed has no overlap in boiling range with the bio-derived feed, the resulting deoxygenated product corresponds to a mixture of bio-derived and non-bio-derived components. Increased flexibility can be achieved by performing hydrodeoxygenation on feedstreams that contain a reduced or minimized content of mineral petroleum, which can include processing substantially only bio-derived fractions in a hydrodeoxygenation process. This can allow for formation of a deoxygenated product that still corresponds to a substantially bio-derived fraction. Hydrodeoxygenation is a type of hydroprocessing. Generally, many types of hydrodeoxygenation catalysts can be similar in composition to hydrotreatment catalysts, although catalysts with activity for aromatic saturation, hydrocracking, and/or catalytic dewaxing can often also have activity for hydrodeoxygenation. As an example, hydrotreatment catalysts typically correspond to one or more types catalytic metals supported on an oxide support or other type of support. For such hydrotreatment catalysts, the catalytic metal can typically be present in one of two forms—an oxide form and a sulfided form. The oxide form of the metal is the stable phase of the metal in the presence of oxygen. However, the sulfide form of the metal is the form that actually provides catalytic activity for hydroprocessing. During hydroprocessing of conventional feeds, the sulfur content of the fresh portion of the feed can be sufficiently high (relative to the flow rates in the hydroprocessing reactor) and/or the oxygen content of the feed can be sufficiently low so that the catalyst remains in a sulfided state. In order to maintain a catalyst in a sulfided state, one option can be to maintain an H2S concentration in the gas phase portion of the hydroprocessing effluent can be roughly 100 vppm (volume parts per million) or more, such as up to 20,000 vppm. When the11I2S concentration in the hydroprocessing effluent is roughly 100 vppm or higher, or 200 vppm or higher, or 300 vppm or higher, such as up to 20,000 vppm, this indicates that sufficient H2S is present in the reaction environment to substantially maintain catalysts in a sulfided state. This sulfided state can be maintained even though water is present in the reaction environment due to hydrodeoxygenation of a bio-derived feed. However, when the H2S concentration drops below roughly 100 vppm while water is also present in the reaction environment, the equilibrium for the metals on the catalyst can be driven toward converting the metals to an oxidized state. Maintaining a gas phase concentration of H2S of roughly 100 vppm or higher (such as up to 20,000 vppm) can be dependent on several factors. Some factors can be related to the sulfur content of the feedstock that is being hydroprocessed as well as the severity of the hydroprocessing conditions. As organic sulfur is removed from a feed, the sulfur is converted to H2S under hydroprocessing conditions. If a feed has a relatively low sulfur content, little or no H2S can be generated regardless of reaction severity. On the other hand, with a relatively high sulfur content, conditions corresponding to a low percentage of desulfurization for a feed can still result in substantial formation of H2S. Other factors can be related to the flow rates within the hydroprocessing reactor. The H2S formed during desulfurization corresponds to a relatively small portion of the total gases within a hydroprocessing environment. Additionally, the gases within the hydroprocessing environment are continuously being replaced, based on the rate of addition of the hydrogen-containing treat gas into the hydroprocessing environment. Thus, as H2S is formed in the hydroprocessing environment at a rate that is proportional to the flow rate of liquid feed into the environment, H2S is removed at a rate that is proportional to the flow rate of treat gas into the environment. (Of course, based on mass balance considerations, the flow rate of liquids and gases into the hydroprocessing environment is roughly the same as the flow rate of liquids and gases out of the hydroprocessing environment.) In some aspects, the potential for a hydroprocessing environment to have an H2S content that is too low to maintain a catalyst in a sulfided state can be at least partially identified based on the sulfur content of the fresh feed into the environment. In this discussion, the fresh feed introduced into the hydroprocessing environment corresponds to feedstock that has not been recycled from a location downstream of the hydroprocessing reactor. In other words, any portion of a feedstock that corresponds to a recycled portion of the hydroprocessing effluent is considered recycled feed, and therefore is excluded from the definition of fresh feed. In various aspects, fresh feed includes both fresh bio-derived feed and fresh mineral feed. It is noted that both the fresh bio-derived feed and the fresh mineral feed can correspond to feedstock that has been processed in other reaction vessels prior to introduction to the hydroprocessing environment. For example, at least a portion of the fresh mineral feed can correspond to a cycle oil from a fluid catalytic cracking process, a product from a coker or other thermal cracking process, or another type of previously processed feedstock. Additionally or alternately, a fresh mineral feed can include at least a portion formed by distillation of a whole crude, partial crude, or crude oil fraction. With regard to fresh bio-derived feed, it is typical for raw biomass to undergo at least some processing, in order to form a liquid bio-derived feed that is suitable for introduction into a hydroprocessing reactor. When hydrodeoxygenating a bio-derived fraction either alone, with other co-feed(s) having a reduced or minimized sulfur content, and/or with a reduced or minimized amount of sulfur-containing mineral co-feed(s), the sulfur content of the fresh feed for hydrodeoxygenation can be 2000 wppm or less, or 1000 wppm or less, or 500 wppm or less, or 300 wppm or less, or 10 wppm or less, such as down to having substantially no sulfur content within detection limit (roughly 0 wppm). This sulfur content represents a weighted average of the sulfur contents of the various types of fresh feed included in the feedstock for hydroprocessing. It is noted that many types of bio-derived feeds can have sulfur contents of 50 wppm or less, including potentially having no sulfur content at all (i.e., sulfur content of 0 wppm). Thus, a feedstock could include a small portion of a relatively high sulfur content mineral fresh feed, or a more substantial portion of a lower sulfur content mineral fresh feed, while still having a weighted average sulfur content for the fresh feed of 2000 wppm or less, or 1000 wppm or less. Such feeds can often also have oxygen contents of substantially more than 1.0 wt %, such as 1.0 wt % to 20 wt %. For such feeds having substantial oxygen contents while having reduced or minimized sulfur content, the sulfided metals on the catalyst for hydrodeoxygenation can potentially be converted back into oxide form, thus reducing the activity of the catalyst. The above difficulties can be reduced or minimized by using an improved method to form a hydrogen-containing stream that also includes sufficient H2S to maintain a hydrodeoxygenation catalyst in a sulfided state. In various aspects, a hydrogen-containing stream that also contains H2S can be formed by using H2S from a rich amine stream derived from another location and/or reaction system. Optionally, the H2S can be added to the hydrogen-containing stream by using at least a portion of the hydrogen-containing stream as a stripping gas for separating the H2S from the rich amine. The resulting stripping gas containing both hydrogen and H2S can then be used as at least a portion of the hydrogen treat gas for hydrodeoxygenation of a bio-derived feedstock. In various aspects, incorporating H2S into the hydrogen treat gas can correspond to including 50 vppm to 10,000 vppm of H2S (or 100 vppm to 10,000 vppm, or 200 vppm to 10,000 vppm, or 1000 vppm to 10,000 vppm, or 50 vppm to 1000 vppm, or 100 vppm to 1000 vppm, or 200 vppm to 1000 vppm) in the hydrogen treat gas and/or the target value of desired sulfur to keep the catalyst in sulfided state. Because bio-derived feeds often have a relatively low sulfur content, the gas product portion of the effluent from hydrodeoxygenation can have a relatively low content of H2S. As a result, passing the gas product portion of a hydrodeoxygenation effluent through an amine absorber can tend to result in a CO2-enriched rich amine stream, where the molar ratio of H2S to amine is less than 0.25 while the molar ratio of CO2to amine is 0.20 or more. By contrast, in various aspects, the H2S for incorporation into a hydrogen treat gas can be derived from a rich amine stream that has a molar ratio of H2S to amine of 0.25 or more. In some aspects, incorporating H2S into the hydrogen treat gas can be accomplished by using a hydrogen-containing (H2) stream as a stripping gas for separating H2S from a rich amine stream. In such aspects, one potential source for a hydrogen-containing stream can be hydrogen recycled from the hydroprocessing of the bio-derived feed. This type of recycled hydrogen-containing stream can include CO2. Lean amine is used to absorb the CO2 in the recycle stream. The hydrogen-stream after contacting with the lean amine stream is then used as the medium for stripping the rich amine stream. In such aspects, using a recycled hydrogen-containing stream as part of the stripping gas can have the additional advantage of reducing or minimizing the CO2content of the stripping gas, while also adding H2S to the hydrogen-containing stream. This reduction in CO2content can reduce or minimize the amount of hydrogen purge that is needed in order to avoid build-up of CO2in the hydrogen recycle loop for the hydroprocessing reactor. In various aspects, incorporating H2S derived from a rich amine stream into the hydrogen treat gas can provide advantages over other methods for maintaining a hydroprocessing catalyst in a sulfided state. For example, one alternative option for introducing sulfur into a reaction system can be to use a sour hydrogen stream corresponding to the gas phase output from a hydrotreating reactor. During hydroprocessing, the hydrogen introduced into a reactor is typically provided in large excess relative to the stoichiometric need for performing the hydroprocessing. As a result, the gas phase effluent from a hydrotreatment stage can include sufficient hydrogen for hydrodeoxygenation while also including H2S generated during the hydrotreatment. However, such streams exiting from a hydrotreatment reactor also typically include other contaminants, such as NH3, that can be detrimental to catalyst performance. Another potential alternative for maintaining a catalyst in a sulfided state is to add a sulfur-containing agent to the feedstock. This type of strategy is used at catalyst startup for certain types of hydroprocessing catalysts are initially loaded into a reactor in metal oxide form. Examples of sulfur-containing agents include dimethyl disulfide (DMDS) or di-t-butyl polysulfide. Adding sulfur-containing agents to a feedstock can be effective for converting metal oxides on a catalyst into a sulfide form and/or maintaining a catalyst in a sulfide form. However, these types of components for adding sulfur to a feed can also present difficulties. For example, DMDS has a bad odor, a low flash point, and generally requires special handling. These issues are often managed on a temporary basis when using DMDS as part of the start-up procedure for a reactor. However, using DMDS long-term to maintain a catalyst in a sulfided state would likely require additional equipment to manage handling of the DMDS and to mitigate the odor. As another example, di-t-butyl polysulfide has low odor and is generally easier to handle than DMDS. However, di-t-butyl polysulfide can cause sulfur precipitation and/or line plugging within a reaction system. In some aspects, the methods described herein for adding H2S from a rich amine stream to a hydrogen-containing gas flow can be used to provide sulfur for a catalyst sulfidation procedure. In such aspects, incorporating H2S into the hydrogen treat gas can correspond to including 1.0 vol % to 10.0 vol % of H2S in the hydrogen treat gas and/or the target value of desired sulfur to achieve a sulfided state for a catalyst. Definitions In this discussion a “liquid product effluent” from a hydroprocessing reaction is defined as the portion of a hydroprocessing effluent that would be a liquid at 20° C.; and 100 kPa-a. In this discussion, a “gas product effluent” from a hydroprocessing reaction is defined as the portion of a hydroprocessing effluent that would be a gas at 20° C. and 100 kPa-a. It is noted that hydroprocessing is typically performed at temperatures substantially above 20° C., so that the “liquid product effluent” may be at least partially in the gas phase when formed. In this discussion, the sulfur content of a fraction can be determined according to ASTM D2622. The nitrogen content of a fraction can be determined according to ASTM D4629. The olefin content of a fraction can be determined by NMR. The oxygen content of a fraction can be determined by reductive pyrolysis. In this discussion, a non-hydrotreated feed or other fraction corresponds to a feed or other fraction that has not been exposed to 50 psia (˜345 kPa-a) or more of H2in the presence of a catalyst. References to a periodic table, such as references to the Group number of a metal, are defined herein as references to the current version of the IUPAC periodic table. Forming H2S-enriched Hydrogen-Containing Stream After addition of H2S to a hydrogen-containing stream, the resulting hydrogen-containing stream containing both hydrogen and H2S can include 50 vppm to 10,000 vppm of H2S, or 50 vppm to 3000 vppm, or 50 vppm to 1000 vppm, or 200 vppm to 10,000 vppm, or 200 vppm to 3000 vppm, or 200 vppm to 1000 vppm. In such aspects, the hydrogen content of the hydrogen-containing stream can be 75 vol % or more, or 80 vol % or more, or 85 vol % or more, or 90 vol % or more, such as up to 99.95 vol %. In some aspects, a rich amine stream can be heated prior to and/or during stripping in order to facilitate stripping of H2S from the amine stream. In other aspects, the stripping can be perthrmed at temperatures associated with the rich amine stream and/or the stripping stream without separate addition of heat. In various aspects, the temperature of the rich amine stream during contacting with a stripping medium for stripping of H2S can be 20° C. to 180° C., or 65° C. to 180° C., or 90° C. to 180° C., or 120° C. to 180° C., or 135° C. to 180° C., or 20° C. to 150° C., or 65° C. to 150° C., or 90° C. to 150° C., or 20° C. to 100° C. Optionally, the temperature of the rich amine stream during stripping to strip H2S can be varied based on a desired or target amount of H2S stripping. In such optional aspects, higher temperatures for the rich amine stream can allow for increased transfer of H2S into the stripping gas, while lower temperatures can reduce the transfer of H2S into the stripping gas. The stripping can be performed at a convenient pressure. In some aspects, the stripping can be performed at a pressure corresponding to a pressure that is used for forming the rich amine stream. In other aspects, the stripping can be performed at an elevated pressure, so that the resulting stripping gas plus H2S is at a pressure that can be introduced into a reactor. Depending on the aspect, the pressure during stripping can be 0.7 MPa-a to 8.0 MPa-a, or 1.4 MPa-a to 8.0 MPa-a, or 4.0 MPa-a to 8.0 MPa-a, or 0.7 MPa-a to 4.0 MPa-a, or 1.4 MPa-a to 4.0 MPa-a. In some aspects, stripping of a rich amine stream can be performed using steam. In such aspects, an H2S-containing stream can be formed from the overhead gas of a stripping tower. The H2S-containing stream can be compressed and then combined with a hydrogen-containing stream for use as at least a portion of a hydrogen treat gas for a hydroprocessing stage that performs hydrodeoxygenation. In other aspects, hydrogen can be used as the stripping gas for stripping a rich amine stream to form a hydrogen-containing stream that is enriched in H2S. In such aspects, prior to stripping, the hydrogen-containing stream used as the stripping gas can in have an H2S content of 0.10 vol % or less, or 0.05 vol % or less, or 0.02 vol % or less, such as down to having substantially no H2S content (0.01 vol % or less). A rich amine stream can generally be composed of mostly water. The H2S content of a rich amine stream can be defined based on the molar ratio of H2S to amine in the amine stream. It is noted that some H2S may remain with the amine stream after stripping. For example, a rich amine stream that contains H2S can have a molar ratio of H2S to amine of 0.25 or more, or 0.30 or more, such as up to 1.0. In various aspects, any convenient type of amine conventionally used in an acid gas absorber system can be used for a rich amine stream or lean amine stream. Examples of amines can include, but are not limited to, monoethanolamine (MEA), diethanolamine (DEA), methyldiethanolamine (MDEA), and diisopropanolamine (DTPA). Still another example is to use Flexsorb™, which corresponds to a mixture of sterically hindered amines. More generally, any amine (or combination of amines) that is suitable for use in an amine absorber can be used. Configuration Examples In some aspects, a hydrogen-containing stream can be used as a stripping gas for removing H2S from a rich amine stream. After use as a stripping gas, the hydrogen-containing stream, now enriched in H2S, can then be used as at least a portion of a hydrogen treat gas for hydrodeoxygenation of a feed including a bio-derived fraction. Optionally, additional hydrogen can be added to the hydrogen treat gas. FIG.1shows an example of a configuration for using a hydrogen-containing stream as a stripping gas for a rich amine stream. The configuration inFIG.1corresponds to a two-stage configuration for integrating amine stripping with a hydrogen recycle loop. In the configuration shown inFIG.1, the first stage of the system corresponds to an acid gas adsorber vessel110for treatment of a sour hydrogen-containing stream108. The sour hydrogen-containing stream108can correspond to any convenient type of hydrogen-containing stream that would be passed into a hydrogen recycle loop. In some aspects, the sour hydrogen-containing stream can correspond to a sour hydrogen-containing stream generated from the hydroprocessing stage that performs the hydrodeoxygenation of the bio-derived feed. In other aspects, the sour hydrogen-containing stream can correspond to a stream generated from any other convenient type of hydroprocessing stage. In still other aspects, the sour hydrogen-containing stream can correspond to a mixture of streams from various refinery processes. As an example, after performing hydroprocessing, separation(s) are usually performed to separate desired liquid products (i.e., products that are liquid at 20° C. and 100 kPa-a) from lower boiling products. Because hydroprocessing is often performed using a substantial excess of hydrogen in the treat gas, the lower boiling products can include a substantial amount of hydrogen. Additionally, the lower boiling products can include one or more types of contaminant gases (such as CO2, H2S, and/or NH3), as well as some C4−hydrocarbons. Due to the presence of the contaminant gases mixed with a substantial amount of hydrogen, such a stream of lower boiling products can be referred to as a sour hydrogen-containing stream. As an example, one option for separating liquid products from lower boiling products can be to use one or more gas-liquid separators that operate at different temperatures and/or pressures. Liquid products can be separated from lower boiling components, for example, in a low temperature, high pressure separator, although various combinations of separators at various temperatures and/or pressures could be used to generate such a stream including lower boiling components. In the example shown inFIG.1, the sour hydrogen-containing stream108is introduced into acid gas adsorber vessel110along with a lean amine stream123and an optional additional lean amine stream103(containing 0.10 vol % or less of H2S). In the configuration shown inFIG.1, the sour hydrogen-containing stream and the lean amine stream103are contacted in a counter-current manner. This can allow for adsorption of one or more contaminants from the sour hydrogen-containing stream108. This results in formation of a rich amine stream115and an overhead stream118that contains hydrogen with a reduced or minimized content of contaminants. As a result, the overhead stream118corresponds to a sweet hydrogen-containing stream. A portion116of overhead stream118(which corresponds to a sweet hydrogen stream) can then be used as a stripping gas for an amine stripper120. In the example of an amine stripper120shown inFIG.1, a rich amine stream125can be contacted in a counter-current manner with the hydrogen-containing stripping gas formed from portion116of overhead stream118. Optionally, the rich amine stream125can be heated (not shown) prior to contact with the hydrogen-containing stripping gas. This results in formation of the lean amine stream123and an overhead stream128that contains hydrogen enriched in H2S. The overhead stream128can be combined with the remainder (if any) of overhead stream118and then passed to a compressor (not shown) for use as at least a portion of the hydrogen treat gas for a hydrodeoxygenation process. To further assist with reducing or minimizing an entrainment of amine in the overhead stream128, and/or to assist with removal of any NH3that may have been present in the sow hydrogen-containing stream108, an optional water wash121can be added near the top of stripper120. In the configuration example shown inFIG.1, acid gas adsorber vessel110and amine stripper120correspond to separate vessels.FIG.2shows a type of configuration where the sour gas treatment and amine regeneration can be performed in a single vessel. InFIG.2, vessel230is used to perform the functions of both an acid gas adsorber and an amine stripper. In this type of configuration, vessel230receives four separate inputs. The inputs include a sour hydrogen-containing stream208, a lean amine stream203, a rich amine stream (that contains absorbed H2S)205, and a water wash201. In the configuration shown inFIG.2, the rich amine stream205at a location in the vessel230above the lean amine stream203. As sour hydrogen-containing stream208travels up in vessel230, the sour hydrogen-containing stream first contacts lean amine stream203. This allows for removal of at least a portion of any contaminants from the sour hydrogen-containing stream208. The hydrogen-containing stream can then contact the rich amine stream205, where H2S is transferred from the rich amine stream205to the hydrogen-containing stream. Optionally, the rich amine stream205can be heated (not shown) prior to contact with the hydrogen-containing stripping gas. This results in formation of an overhead stream238that corresponds to hydrogen enriched in H2S, as well as a rich amine stream235that contains CO2removed from the sour hydrogen-containing stream208. The (optional) water wash201can be used to reduce or minimize the potential for amine to be entrained in overhead stream238. The overhead stream can then be sent to a compressor for use as at least a portion of the hydrogen treat gas for a hydrodeoxygenation process. Another option for using a single vessel for both acid gas treatment and amine stripping is to use a vessel that includes a dividing wall.FIG.3shows an example of a configuration where a top dividing wall is included in the vessel, but the dividing wall extends for less than the full height of the vessel. InFIG.3, dividing wall340is used to separate the vessel330into at least two regions. Dividing wall340can divide vessel330in any convenient manner. In the example shown inFIG.3, dividing wall340divides the upper portion of vessel330into two separate regions. A first region342corresponds to a portion of the vessel330that serves as an acid gas adsorber. A second region347corresponds to a portion of the vessel330that serves as an amine stripper. Because dividing wall340does not match the full height of the vessel330, there is also a third common region349that is in fluid communication with both first region342and second region347. In a configuration such as the example shown inFIG.3, sour hydrogen stream308can be passed into vessel330at a location that is below the level of the bottom341of dividing wall340. This can allow portions of sour hydrogen stream308to enter both first region342and second region347. In the example shown inFIG.3, a greater portion of the sour hydrogen stream308can enter into first region342. During operation, portions of sour hydrogen stream308can travel upward on both sides of dividing wall340. In first region342, a portion of sour hydrogen stream308is contacted with lean amine stream303. This results in formation of a sweet hydrogen stream as a first overhead stream338. Rich amine containing adsorbed contaminants from the sour hydrogen stream308is also formed. In second region347, another portion of sour hydrogen stream308is contacted with a rich amine stream305that contains H2S. This results in formation of a second overhead stream348that is enriched in H2S. Optionally, the rich amine stream305can be heated (not shown) prior to contact with the sour hydrogen stream308. A second rich amine stream containing adsorbed CO2is also formed. The rich amine streams containing adsorbed CO2can exit the vessel330as a combined rich amine stream335. The first overhead stream338and second overhead stream348can be combined and passed to a compressor for use as at least a portion of a hydrogen treat gas for hydrodeoxygenation. A water wash301can be included in second region347. A water wash can also be included in the342region (not shown). In the example shown inFIG.3, the height of the dividing wall column340is less than the interior height of vessel330, so that a common volume349provides fluid communication between first region342and second region347. InFIG.4, still another type of configuration is shown where a dividing wall column440is used that matches the full interior height of the vessel430. InFIG.4, dividing wall440divides the interior of vessel430into a first region442and a second region447. However, because dividing wall440matches the full interior height of vessel430, there is substantially no fluid communication between first region442and second region447. Some of the input and flows inFIG.4can be similar to the input flows inFIG.3. Thus, sour hydrogen-containing stream408, lean amine stream403, rich amine stream405, and water wash401inFIG.4can be similar to sour hydrogen-containing stream308, lean amine stream303, rich amine stream305, and water wash301in inFIG.3. Stream405can be optionally heated (not shown inFIG.4). Additionally, overhead stream438and overhead stream448can be combined for subsequent compression and use as at least a part of the hydrogen treat gas for a hydrodeoxygenation process. However, because dividing wall440extends for the full height of the interior of vessel430, there are some differences. First, a separate hydrogen-containing stream458is introduced into second region447inFIG.4.FIG.4shows two examples of how the separate hydrogen-containing stream458can be provided. One option can be to use a portion468of overhead stream438. Another option can be to use a sweet or sour hydrogen-containing stream478generated by the hydroprocessing stage that performs the hydrodeoxygenation or hydroisomerization or a pure hydrogen stream. In this type of aspect, sour hydrogen-containing stream408can be from a different source than sour hydrogen-containing stream478. Additionally, in the configuration shown inFIG.4, separate rich amine streams465and475can be formed. Optionally, these streams can be combined to form a combined rich amine stream435. In the configurations shown inFIGS.1-4, the configurations correspond to flow schemes that can be used to generate a hydrogen stream that would need subsequent compression prior to use as a hydrogen treat gas. In other aspects, compression of the hydrogen can be performed prior to enriching the hydrogen treat gas with H2S.FIG.5shows an example of a configuration for forming an H2S-enriched hydrogen-containing stream after compression of the stream to the target pressure for use in hydroprocessing. In the example shown inFIG.5, hydrogen stream508is introduced into amine stripper510. Hydrogen stream508can correspond to fresh or make-up hydrogen, or can correspond to hydrogen that has already been sweetened and then compressed in a recycle loop. The hydrogen stream508is used to at least partially strip a rich amine505that includes H2S. Optionally, the rich amine stream505can be heated (not shown) prior to contact with the hydrogen stream508. This produces a lean amine513that can be sent to an amine absorber (such as a sour hydrogen stream adsorber) for further use. This also results in an overhead stream518that includes hydrogen and H2S. A water wash501can optionally also be included to reduce or minimize entrainment of amines in the overhead stream518. The configurations inFIGS.1-5all involve using a hydrogen-containing gas as a stripping medium fir a rich amine. Still other options for incorporating H2S into a hydrogen-containing gas can involve separately stripping a rich amine using a stripping gas such as steam, and then compressing the resulting stripped H2S so that the H2S can be added to a hydrogen-containing stream prior to introduction into a hydroprocessing stage.FIGS.6and7show examples of this type of configuration. InFIG.6, a stripping tower or vessel610can be used that includes a water wash601and a rich amine stream605(that contains H2S) as inputs. The stripping tower has an associated reboiler650. During operation, reboiler650can generate steam that travels up in the stripping vessel610. The reboiler also generates a lean amine stream653that can then be used in an amine absorber. The steam from reboiler650provides a counter-current flow for contact with water wash601and rich amine stream605. The steam provides energy for desorption of H2S from the rich amine stream. The portion of the steam that does not condense while traveling up through stripping vessel610can exit as part of overhead stream614, along with any H2S desorbed from the rich amine stream. The resulting overhead stream664can then be compressed660to allow the H2S to be combined with a hydrogen-containing stream and/or to allow the H2S to be introduced directly into a reactor that is part of a hydroprocessing stage that performs hydrodeoxygenation. Optionally, prior to and/or after compression660, water can be removed from overhead stream614. FIG.7shows an example of how a top dividing wall can allow a single stripping vessel to be used for generation of both sweetened hydrogen and a compressed H2S stream for addition to a hydroprocessing stage that performs hydrodeoxygenation. InFIG.7, stripping tower or vessel710includes a dividing wall740. This creates a first region743and a second region747on either side of the dividing wall, along with a common volume749below the bottom of the dividing wall. Optionally, an alternative to the configuration inFIG.7could be to have a dividing wall that extends the full height (not shown) of the stripping vessel710. This would require other changes in flows similar to the changes described in connection with changing from a configuration similar toFIG.3to a configuration similar toFIG.4. In the configuration shown inFIG.7, water wash701and a first rich amine stream705are introduced into first region743of stripping vessel710. First rich amine stream705corresponds to a rich amine stream from a part of the refinery different from the hydroprocessing stage that performs hydrodeoxygenation, so that first rich amine stream705corresponds to a stream that includes adsorbed H2S. A second rich amine stream775and a reflux771are introduced into second region747of stripping vessel710. The second rich amine stream775can correspond to, for example, the acid gas rich amine stream formed by the absorption of acid gases from a hydrogen-containing stream that is separated from the effluent of the hydroprocessing stage that performs hydrodeoxygenation. During operation, reboiler750can generate steam that passes through common region749and passes into both first region743and second region747. This can allow for stripping of both first rich amine stream705and second rich amine stream775. The dividing wall740allows the two different stripping products to for separate overhead streams714and774. Overhead stream714can correspond to an overhead stream that contains H2S. Overhead stream714can then be compressed760to allow the H2S to be combined with a hydrogen-containing stream and/or to allow the H2S to be introduced directly into a reactor that is part of a hydroprocessing stage that performs hydrodeoxygenation. Optionally, prior to and/or after compression760, water can be removed from overhead stream714. Overhead stream774can correspond to an overhead stream that includes H2S and CO2. Overhead stream774can be cooled782prior to passing into a gas-liquid separator780for separation of water (reflux)771from a remaining H2S and CO2-containing stream784. Feedstock for Hydrodeoxygenation In various aspects, a feedstock for hydrodeoxygenation can correspond to a feed derived from a biological source. In this discussion, a feed derived from a biological source refers to a feedstock derived from a biological raw material component, such as vegetable fats/oils or animal fats/oils, fish oils, pyrolysis oils, and algae lipids/oils, as well as components of such materials, and in some embodiments can specifically include one or more types of lipid compounds. Lipid compounds are typically biological compounds that are insoluble in water, but soluble in nonpolar (or fat) solvents. Non-limiting examples of such solvents include alcohols, ethers, chloroform, alkyl acetates, benzene, and combinations thereof. Examples of vegetable oils that can be used in accordance with this invention include, but are not limited to rapeseed (canola) oil, soybean oil, coconut oil, sunflower oil, palm oil, palm kernel oil, peanut oil, linseed oil, tall oil, corn oil, castor oil, jatropha oil, jojoba oil, olive oil, flaxseed oil, camelina oil, safflower oil, babassu oil, tallow oil and rice bran oil. Algae oils or lipids can typically be contained in algae in the form of membrane components, storage products, and/or metabolites. Certain algal strains, particularly microalgae such as diatoms and cyanobacteria, can contain proportionally high levels of lipids. Algal sources for the algae oils can contain varying amounts, e.g., from 2 wt % to 40 wt % of lipids, based on total weight of the biomass itself. Vegetable fats/oils, animal fats/oils, fish oils, pyrolysis oils, and/or algae lipds/oils as referred to herein can also include processed material. Non-limiting examples of processed vegetable, animal (including fish), and algae material include fatty acids and fatty acid alkyl esters. Alkyl esters typically include C1-C5alkyl esters of fatty acids. One or more of methyl, ethyl, and propyl esters are preferred. Other bio-derived feeds usable in the present invention can include any of those which comprise primarily triglycerides and free fatty acids (FFAs). The triglycerides and FFAs typically contain aliphatic hydrocarbon chains in their structure having from 8 to 36 carbons, preferably from 10 to 26 carbons, for example from 10 to 22 carbons or 14 to 22 carbons. Types of triglycerides can be determined according to their fatty acid constituents. The fatty acid constituents can be readily determined using Gas Chromatography (GC) analysis. This analysis involves extracting the fat or oil, saponifying (hydrolyzing) the fat or oil, preparing an alkyl (e.g., methyl) ester of the saponified fat or oil, and determining the type of (methyl) ester using GC analysis. In one embodiment, a majority (i.e., greater than 50%) of the triglyceride present in the lipid material can be comprised of C10to C26fatty acid constituents, based on total triglyceride present in the lipid material. Further, a triglyceride is a molecule having a structure corresponding to a reaction product of glycerol and three fatty acids. Although a triglyceride is described herein as having side chains corresponding to fatty acids, it should be understood that the fatty acid component does not necessarily contain a carboxylic acid hydrogen. Other types of feed that are derived from biological raw material components can include fatty acid esters, such as fatty acid alkyl esters (e.g., FAME and/or FAEE). A feed derived from a biological source can have a wide range of nitrogen and/or sulfur contents. For example, a feedstock based on a vegetable oil source can contain up to 300 wppm nitrogen. In contrast, a biomass based feedstream containing whole or ruptured algae can sometimes include a higher nitrogen content. Depending on the type of algae, the nitrogen content of an algae based feedstream can be at least 2 wt %, for example at least 3 wt %, at least 5 wt %, such as up to 10 wt % or possibly still higher. The sulfur content of a feed derived from a biological source can also vary. In some aspects, the sulfur content can be 500 wppm or less, or 200 wppm or less, or 100 wppm or less, or 50 wppm or less, such as down to being substantially free of sulfur (1.0 wppm or less). Aside from nitrogen and sulfur, oxygen can be another heteroatom component in feeds derived from a biological source. For example, a feed derived from a biological source, prior to hydrotreatment, can include 1.0 wt % to 20 wt % of oxygen, or 1.0 wt % to 15 Wt %, or 1.0 wt % to 10 wt %, or 3.0 wt % to 20 wt %, or 3.0 wt % to 15 wt %, or 3.0 wt % to 10 wt %, or 4.0 wt % to 15 wt %, or 4.0 wt % to 12 wt %. In some aspects, a portion of a mineral feedstock can be co-processed with a feed derived from a biological source. A mineral feedstock refers to a conventional feedstock, typically derived from crude oil and that has optionally been subjected to one or more separation and/or other refining processes. In one preferred embodiment, the mineral feedstock can be a petroleum feedstock boiling in the diesel range or above. Examples of suitable feedstocks can include, but are not limited to, virgin distillates, hydrotreated virgin distillates, kerosene, diesel boiling range feeds (such as hydrotreated diesel boiling range feeds), light cycle oils, atmospheric gasoils, and the like, and combinations thereof. The amount of fresh mineral feedstock blended with a feed derived from a biological source can correspond to 1.0 wt % to 50 wt % of the weight of the blended feedstock, or 1.0 wt % to 30 wt %, or 1.0 wt % to 20 wt %, or 10 wt % to 50 wt %, or 10 wt % to 30 wt %. Additionally or alternately, the amount of mineral feedstock blended with the bio-derived feed can be low enough so that the resulting blended or combined feed has a sulfur content of 1000 wppm or less, or 500 wppm or less, or 200 wppm or less. In various aspects, the amount of fresh bio-derived feed can correspond to 30 wt % to 99 wt % of the blended feedstock, or 50 wt % to 99 wt %, or 70 wt % to 99 wt %, or 30 wt % to 75 wt %. It is noted that the blended feedstock can optionally include a recycle portion. Mineral feedstocks for blending with a bio-derived can be relatively free of nitrogen (such as a previously hydrotreated feedstock) or can have a nitrogen content from about 1 wppm to about 2000 wppm nitrogen, for example from about 50 wppm to about 1500 wppm or from about 75 to about 1000 wppm. In some embodiments, the mineral feedstock can have a sulfur content from about 1 wppm to about 10,000 wppm sulfur, for example from about 10 wppm to about 5,000 wppm or from about 100 wppm to about 2,500 wppm. However, in various aspects, such mineral feedstocks can be combined with a bio-derived feed (and/or other feeds) so that the resulting combined feed has a sulfur content of 2000 wppm or less, or 1000 wppm or less, or 500 wppm or less, or 300 wppm or less, or 10 wppm or less, such as down to having substantially no sulfur content within detection limit (roughly 0 wppm). Additionally or alternately, the combined feed can have an oxygen content of 1.0 wt % or more, such as 1.0 wt % to 15 wt %. The content of sulfur, nitrogen, oxygen, and olefins in a feedstock created by blending two or more feedstocks can typically be determined using a weighted average based on the blended feeds. For example, a mineral feed and a bio-derived feed can be blended in a ratio of 20 wt % mineral feed and 80 wt % bio-derived feed. If the mineral feed has a sulfur content of about 1000 wppm, and the bio-derived feed has a sulfur content of about 10 wppm, the resulting blended feed could be expected to have a sulfur content of about 208 wppm. Hydroprocessing Stage that Performs Hydrodeoxygenation In various aspects, a feed having a low sulfur content and an oxygen content of 1.0 wt % or more can be exposed to hydroprocessing conditions in a hydroprocessing stage. The low sulfur content can correspond to a sulfur content of 2000 wppm or less, or 1000 wppm or less, or 500 wppm or less, or 300 wppm or less, or 10 wppm or less, such as down to having substantially no sulfur content within detection limit (roughly 0 wppm). A hydroprocessing stage can include one or more reactors, with each reactor optionally including one or more catalyst beds. The catalyst beds within a reactor can include similar catalysts or different catalysts, depending on the configuration. Exposing a feed having a low sulfur content and an oxygen content of 1.0 wt % or more to hydroprocessing conditions can result in hydrodeoxygenation of the feed. As an example, in some aspects a hydroprocessing stage can correspond to a stage for conversion of a feed including a substantial portion of vegetable oil into a renewable diesel fuel or fuel blending product. Such a feed can include 40 wt % or more of a bio-oil, or 60 wt % or more, or 80 wt % or more, such as up to being substantially composed of a bio-oil (99 wt % or more). Some types of bio-oil can correspond to soybean oil, canola oil, and/or other types of oils corresponding to a primary bio-oil product. In such aspects, the bio-oil can optionally have a triglyceride content of 40 wt % or more, or 60 wt % or more, or 80 wt % or more, such as up to being substantially composed of triglycerides. Other types of bio-oils can correspond to oils such as the corn oil that is formed as a secondary product during ethanol production from corn biomass. In this type of example, a hydroprocessing stage for conversion of vegetable oil into renewable diesel can involve two types of hydroprocessing. A first type of hydroprocessing can correspond to hydrodeoxygenation of the feed. After hydrodeoxygenation, additional cracking and/or catalytic dewaxing can be performed on the hydrodeoxygenated feed to improve one or more properties of the final fuel or fuel blending product. It is noted that both hydrodeoxygenation and the additional cracking and/or catalytic dewaxing can occur at the same time. However, due to the relatively rapid rate for hydrodeoxygenation under conditions suitable for cracking and/or dewaxing, at least a portion of the cracking and/or catalytic dewaxing can typically occur after hydrodeoxygenation has been substantially completed. Some examples of hydrodeoxygenation catalysts can correspond to hydrotreating catalysts. In some aspects, a catalyst can be used that includes a Group 6 metal on a support material, but less than 1.0 wt % of a Group 8 metal. In other aspects, conventional hydrotreating catalysts that include both a Group 6 metal and a Group 8 metal on a support material can be used. The at least one Group 6 metal, in oxide form, can typically be present in an amount ranging from 2.0 wt % to 40 wt %, relative to a total weight of the catalyst, or 6.0 wt % to 40 wt %, or 10 wt % to 30 wt %. When a Group 8-10 metal is also present, the at least one Group 8-10 metal, in oxide form, can typically be present in an amount ranging from 2.0 wt % to 40 wt %, preferably for supported catalysts from 2.0 wt % to 20 wt % or from 4.0 wt % to 15 wt %. The hydroprocessing catalyst can be provided in a reactor in one or more catalyst beds. For example, a convenient bed length in some reactors is a bed length of about 25 feet to 30 feet. Such a bed length reduces difficulties in a catalyst bed associated with poor flow patterns. Due to the heat release from the initial bed during olefin saturation and deoxygenation, it may be desirable to use a shorter catalyst bed as the initial bed, such as having a bed length of 10 feet to 25 feet. Typical effective conditions for processing a feedstock containing triglycerides, fatty acid alkyl esters, fatty acids, and/or fatty acid derivatives (and/or other oxygen-containing bio-derived feeds) to remove oxygen can include a hydrogen partial pressure of 200 psig (1.4 MPag) to 1200 psig (8.3 MPag). The hydrotreating conditions can also include a temperature, a hydrogen treat gas rate, and a liquid hourly space velocity (LHSV). Suitable effective temperatures can be from 230° C. to 375° C., or 250° C. to 350° C. The LHSV can be from 0.1 hr−1to 10 hr−1, or from 0.2 hr−1to 5.0 hr−1. The hydrogen treat gas rate can be any convenient value that provides sufficient hydrogen tier deoxygenation of a feedstock. Typical values can range from 2000 scf/B (˜340 Nm3/m3) to 20,000 scf/B (˜3400 Nm3/m3), or 5000 scf/B (˜840 Nm3/m3) to 20,000 scf/B (˜3400 Nm3/m3), or 8000 scf/B (˜1350 Nm3/m3) to 20,000 scf/B (˜3400 Nm3/m3) or possibly still higher. It is noted that the hydrogen consumption for fresh bio-derived feed can approach 2000 scf/B (˜340 Nm3/m3) or still higher values. One option for selecting a treat gas rate can be to select a rate based on the expected stoichiometric amount of hydrogen for complete deoxygenation and olefin saturation of the feedstock. In some aspects, the hydrogen treat gas rate can be selected based on a multiple of the stoichiometric hydrogen need, such as at least 1 times the hydrogen need, or at least 1.5 times the hydrogen need, or at least 2 times the hydrogen need, or 4 times the hydrogen need, such as up to 10 times the hydrogen need or possibly still higher. In other aspects where at least a portion of the gas phase deoxygenation effluent is recycled, any convenient amount of hydrogen relative to the stoichiometric need can be used. In various aspects, the hydrogen treat gas can be an H2S-enriched hydrogen treat gas as described herein with an H2S content of 50 vppm to 10,000 vppm. The hydrotreating conditions for can be suitable for reducing the oxygen content of the feed to 1.0 wt % or less, or 0.5 wt % or less, such as down to having substantially no oxygen (0.1 wt % or less). Although the stoichiometric hydrogen need is calculated based on complete deoxygenation, reducing the oxygen content to substantially zero is typically not required to allow further processing of the deoxygenated feed in conventional equipment. In some aspects, the hydrodeoxygenated effluent (or at least a portion thereof) can then be catalytically dewaxed in order to improve the cold flow properties of the distillate boiling range portion of the effluent. Fatty acid carbon chains often correspond to unbranched carbon chains. After deoxygenation, such unbranched carbon chains can often have relatively poor cold flow properties, such as relatively high pour points, cloud points, or cold filter plugging points. In applications where it is desired to use the distillate boiling range portion of the effluent as part of a diesel fuel, it can be desirable to expose a distillate boiling range product to a dewaxing catalyst under dewaxing conditions in order to improve the cold flow properties. Dewaxing catalysts can include molecular sieves such as crystalline aluminosilicates (zeolites). More generally, dewaxing catalysts can correspond to catalysts having a zeotype framework. The dewaxing catalyst can optionally be a supported catalyst, such as a catalyst including a zeotype framework and a binder material. In an embodiment, the zeotype framework can comprise, consist essentially of, or be ZSM-5, ZSM-22, ZSM-23, ZSM-35, ISM-48, zeolite Beta, or a combination thereof, for example ZSM-23 and/or ZSM-48, or ZSM-48 and/or zeolite Beta. Optionally but preferably, zeotype frameworks that are selective for dewaxing by isomerization as opposed to cracking can be used, such as ZSM-48, zeolite Beta, ZSM-23, or a combination thereof. Additionally or alternately, the zeotype framework can comprise, consist essentially of, or be a 10-member ring 1-D zeotype framework. Optionally but preferably, the dewaxing catalyst can include a binder for the zeotype framework, such as alumina, titania, silica, silica-alumina, zirconia, or a combination thereof, for example alumina and/or titania or silica and/or zirconia and/or titania. Aside from the zeotype framework(s) and optional binder, the dewaxing catalyst can also include at least one metal hydrogenation component, such as a Group 8-10 metal. Suitable Group 8-10 metals can include, but are not limited to, Pt, Pd, Ni, or a combination thereof. When a metal hydrogenation component is present, the dewaxing catalyst can include 0.1 wt % to 10 wt % of the Group 8-10 metal, or 0.1 wt % to 5.0 wt %, or 0.5 wt % to 10 wt %, or 0.5 wt % to 5.0 wt %, or 1.0 wt % to 10 wt %, or 1.0 wt % to 5.0 wt %. In some aspects, the dewaxing catalyst can include an additional Group 6 metal hydrogenation component, such as W and/or Mo. In such aspects, when a Group 6 metal is present, the dewaxing catalyst can include 0.5 wt % to 20 wt % of the Group 6 metal, or 0.5 wt % to 10 wt %, or 2.5 wt % to 20 wt %, or 2.5 wt % to 10 wt %. As one example, the dewaxing catalyst can include 0.1 wt % to 5.0 wt % Pt and/or Pd as the hydrogenation metal component. As another example, the dewaxing catalyst can include as the hydrogenation metal components Ni and W, Ni and Mo, or Ni and a combination of W and Mo. Catalytic dewaxing can be performed by exposing a feedstock to a dewaxing catalyst under effective (catalytic) dewaxing conditions. Dewaxing conditions can include temperatures of 550° F. (288° C.) to 840° F. (449° C.), hydrogen partial pressures of from 250 psig to 5000 psig (1.8 MPag to 34.6 MPag), and hydrogen treat gas rates of from 34 Nm3/m3to 1700 sm3/m3(˜200 SCF/B to ˜10,000 SCF/B). The liquid hourly space velocity (LHSV) of the feed relative to the dewaxing catalyst can be characterized can be from about 0.1 hr1to about 10 hr−1. Additionally or alternately, the hydrodeoxygenated feed can be exposed to conditions suitable for additional mild cracking of the hydrodeoxygenated feed. This can be performed, for example, in the presence of a conventional hydrotreating catalyst, aromatic saturation catalyst, and/or hydrocracking catalyst. Example—Hydrogen Stripping of Rich Amine Stream The stripping of a rich amine stream using hydrogen was modeled using ProMax® software. A configuration similar to the configuration shown inFIG.5was used to model the stripping process. For the model calculations, Table 1 shows the composition of the rich amine stream that was used. The amine used for the modeled rich amine stream was monoethanolamine (MEA). TABLE 1Composition of Rich Amine StreamComponentMole %H2S3CO20.05H20.01CH4TraceC2H6TraceMEA6.6H2O90.34NH3Trace In the model, pure hydrogen was used as the stripping medium. Water wash trays were included to prevent amine carryover into the overhead gas. Table 2 shows the composition (in mol %) of the various streams in the model of the amine stripping tower at steady state. The temperature of the rich amine stream was 120° F. (˜49° C.). The temperature of the stripping gas prior to contacting the rich amine stream was 130° F. (˜54° C.). The model included a rich amine flow rate of roughly 6 gallons per minute, in order to provide sufficient H2S for treating a fresh feedstock flow of roughly 10,000 barrels per day. TABLE 2Model Stream Compositions from H2Stripping of Rich AmineStream CompositionRichMakeupWaterH2S-Stripped(Mol %)AmineH2Washenriched H2AmineH2S3.0000.140.47CO20.05000.000320.027H20.01100099.150.072CH400000C2H600000MEA6.60003.98H2O90.3401000.7195.45 As shown in Table 2, using H2as a stripping gas allowed for formation of an H2S-enriched hydrogen stream that included 0.14 mol % (or roughly 0.14 vol %) of H2S. The stripping also reduced the molar ratio of H2S to MEA from 0.45 (in the rich amine stream) to 0.12 (in the stripped amine stream). Additional Embodiments Embodiment 1. A method for performing hydrodeoxygenation, comprising: stripping an amine-containing flow comprising a first molar ratio of H2S to amine of 0.25 or more with a stripping gas comprising 80 vol % or more of H2in a vessel to form an amine-containing flow comprising a second molar ratio of H2S to amine that is lower than the first molar ratio and a gas phase fraction comprising 80 vol % or more H2and 50 vppm or more of H2S; exposing a feedstock comprising at least a fresh feed portion to a sulfided hydroprocessing catalyst and a treat gas comprising at least a portion of the gas phase fraction under hydrodeoxygenation conditions to form a liquid product effluent comprising 0.5 wt % or less of oxygen and a gas product effluent comprising 100 vppm or more of H2S, the fresh feed portion comprising 1000 wppm or less of sulfur and 1.0 wt % or more of oxygen, the hydrodeoxygenation conditions comprising a flow of treat gas relative to a flow rate of the fresh feed of 500 SCF/bbl or higher (˜1350 Nm3/m3). Embodiment 2. A method for performing hydrodeoxygenation, comprising: stripping an amine-containing flow comprising a first molar ratio of H2S to amine of 0.25 or more with a stripping gas comprising steam in a vessel to form an amine-containing flow comprising a second molar ratio of H2S to amine that is lower than the first molar ratio and a gas phase fraction comprising H2S; compressing at least a portion of the gas phase fraction comprising H2S to form a compressed H2S fraction, the compressed H2S fraction comprising 1.0 vol % or more H2S; and exposing a feedstock comprising at least a fresh feed portion to a sulfided hydroprocessing catalyst and a treat gas comprising at least a portion of the compressed H2S fraction under hydrodeoxygenation conditions to form a liquid product effluent comprising 0.5 wt % or less of oxygen and a gas product effluent comprising 100 vppm or more of H2S, the fresh feed portion comprising 1.0 wt % or more of oxygen, the hydrodeoxygenation conditions comprising a flow of treat gas relative to a flow rate of the fresh feed of 5000 SCF/bbl or higher (˜1350 Nin3/m3), the treat gas comprising 80 vol % or more of H2and 50 vppm or more of H2S. Embodiment 3. The method of any of the above embodiments, wherein the fresh feed portion comprises 1000 wppm or less of sulfur. Embodiment 4. The method of any of the above embodiments, wherein the vessel comprises a dividing wall defining at least a first volume and a second volume within the vessel. Embodiment 5. The method of Embodiment 4, wherein the amine-containing flow comprising the first molar ratio of H2S to amine is introduced into the first volume, and wherein a second amine-containing flow is introduced into the second volume, the method optionally further comprising exposing at least a portion of the gas product effluent to an amine-containing stream in an amine absorber to form the second amine-containing flow. Embodiment 6. The method of Embodiment 4 or 5, wherein the dividing wall comprises a height that is less than an interior height of the vessel, the dividing wall further defining a common volume within the vessel. Embodiment 7. The method of any of the above embodiments, wherein the sulfided hydroprocessing catalyst comprises a hydrotreating catalyst, a dewaxing catalyst, a hydrocracking catalyst, an aromatic saturation catalyst, or a combination thereof. Embodiment 8. The method of any of the above embodiments, wherein the fresh feed portion comprises 50 wt % or more of a bio-derived fraction, or wherein the feedstock comprises 0.1 wt % to 50 wt % of a mineral feedstock, or a combination thereof. Embodiment 9. The method of any of the above embodiments, wherein the gas product effluent comprises an H2content of 75 vol % or more and a CO2content of 1.0 vol % to 20 vol %. Embodiment 10. The method of any of the above embodiments, wherein the treat gas comprises 100 vppm or more of H2S and 0.5 vol % or less of CO2. Embodiment 11. The method of any of the above embodiments, further comprising separating the liquid product effluent to form at least a recycle portion, the feedstock further comprising the recycle portion. Embodiment 12. The method of any of the above embodiments, wherein the fresh feed portion comprises 80 wt % or more of a bio-derived fraction. Embodiment 13. The method of any of the above embodiments, further comprising: stripping a third amine-containing flow comprising a third molar ratio of H2S to amine of 0.25 or more with a stripping gas comprising 80 vol % or more of H2in a vessel to form an amine-containing flow comprising a fourth molar ratio of H2S to amine that is lower than the third molar ratio and a sulfidation gas phase fraction comprising 80 vol % or more H2and 1.0 vol % or more of H2S; and exposing a hydroprocessing catalyst to sulfidation treat gas comprising at least a portion of the sulfidation gas phase fraction under sulfidation conditions to form the sulfided hydroprocessing catalyst. Embodiment 14. The method of any of the above embodiments, wherein the fresh feed portion comprises a non-hydrotreated feed portion. While the present invention has been described and illustrated by reference to particular embodiments, those of ordinary skill in the art will appreciate that the invention lends itself to variations not necessarily illustrated herein. For this reason, then, reference should be made solely to the appended claims for purposes of determining the true scope of the present invention. | 61,108 |
11859139 | DETAILED DESCRIPTION OF THE INVENTION The present invention relates a method for crude petroleum oil processing in a CDU to overcome the disadvantages of prior art processes. In order to describe the current invention, figures drawn in accordance with the existing design and preferred embodiments of the current invention are prepared. The same numeral is used in drawings to refer to the same or similar or stream or column elements. It is important to note that invention is not limited to the precise arrangements of apparatus shown in drawings. The reference toFIGS.1,2,3,4, and5are made to describe the present invention in detail. Referring toFIG.1, representing the method of prior art, conceptualized to construct a comparative example for establishing the basis for demonstrating the benefits of the present invention over the closest prior art for crude petroleum oil processing in CDU. Crude petroleum oil (1) is heated in a HEN1in the temperature range of 110-140° C. and fed to desalter (2), where it comes in contact with water to remove the water-soluble salt. Desalted Crude Oil (DCO) obtained from desalter is further heated in a HEN2in the temperature range of 160-230° C. using hot process streams. The partially vaporized crude oil (3) is fed to the vessel (4). The hydrocarbon vapour (7) from the vessel (4) is fed to flash zone (9) of the atmospheric distillation column (8). The liquid stream (5) from the vessel (4) is heated in the temperature range of 270-330° C. using a HEN3and further heated in a fired furnace (F1) in the temperature range of 360-390° C. The partially vaporized crude (6) is also fed to flash zone (9) of the atmospheric distillation column (8). Thus crude oil is separated into distillate products and long residue (18) in the Vacuum Distillation Column (VDC). The overhead vapour (15) is fed to vessel (V1) after its cooling in the condenser (E-1) in the temperature range of 85-125° C. The liquid from V1is used as reflux to ADC (8). The vapour stream (16) from V1is fed to the vessel (V2) after its cooling in a cooler (E-2) in the temperature range of 35-50° C. The unstabilized naphtha stream (17) from V2is routed to the distillation column (15) to produce the LPG and Light Naphtha (LN) product and noncondensed vapour stream (23). The heavy naphtha, kerosene, Light Gas Oil (LGO) distillate and Heavy Gas Oil (HGO) distillates from the different tray of distillation column (8) are routed to the side strippers11,12,13and14, respectively, to remove the lighter hydrocarbon for meeting the specifications of the final product. The heat of distillation column (8) having plural trays is removed by one or more pump-arounds (PA1, PA2and PA3) and condenser (E-1). The LR (18) from distillation column (8) along with furnace coil steam is heated in a furnace (F3) in the temperature range of 390-430° C. The partially vaporized long residue (19) is routed to the VDC for obtaining the distillate products (VD vapour, vacuum diesel, Light Vacuum Gas Oil-LVGO, Heavy Vacuum Gas Oil-HVGO) and Vacuum Residue (VR). VDC has plural trays and stripping steam at the bottom tray, equipped with pump-arounds for heat removal and vacuum generating device like ejector or vacuum pump (not shown in the figure) Referring toFIG.2, representing the method constructed in accordance with one of the embodiments of the present invention for crude petroleum oil processing in CDU to demonstrate the applicability and benefits of the present invention. This method has the same configuration and operation, as shown inFIG.1for generating the stream (3). The partially vaporized crude oil (3) along with hydrocarbon vapour (24) comprising of a lighter fraction of crude and natural gas stream (25) are fed to the two phases separating vessel (4), having plural trays. The hydrocarbon vapour (7) from the vessel (4) is further superheated in the temperature range of 300-370° C. using the combination of HEN4, and high-pressure steam heater (HPX), or furnace (F2) or separate vapour coils along with crude oil coils in the furnace (F1). The superheated vapour (7A) is injected into either the bottom tray of the stripping section (10) or a tray between the bottom tray and flash zone (9) of the atmospheric distillation column (8). The liquid stream (5) is heated in the temperature range of 270-330° C. by using a heat exchanger network (HEN3) and in the temperature range of 360-390° C. by the fired furnace (F1). The partially vaporized crude (6) is also fed to flash zone (9) of the atmospheric distillation column (8). Thus crude oil is separated into distillate products and long residue (18) in the ADC. The overhead vapour (15) is fed to vessel (V1) after its cooling in the condenser (E-1) in the temperature range of 85-125° C. The liquid from V1is used as reflux to ADC (8). The vapour stream (16) from V1and a water stream (16A) are mixed and fed to the vessel (V2) after cooling in a cooler (E-2) in the temperature range of 35-50° C. The vapour stream (20) from V2is compressed in the pressure range of 8-14 kg/cm2using the compressor (CP-1). Compressed stream is cooled in cooler (E-3) in the temperature range of 35-50° C. The cooled stream (20A) is routed to flash vessel (V3). The liquid stream (22) from V3and unstabilized naphtha stream (17) from V2are routed to the distillation column (15) to produce the LPG and Light Naphtha LN product and noncondensate vapour stream (23). The vapour stream (21) from V3and vapour stream (23) from distillation column (15) are mixed, and mixed stream (24) is routed to separating vessel (4) to generate the hydrocarbon vapour (7). The heavy naphtha and kerosene distillates from the different tray of ADC (8) are routed to the side strippers (11) and (12), respectively, to remove the lighter hydrocarbon for meeting the specifications of the final product. The LGO and HGO distillates are routed to the vessel (13A) and vessel (14A) through their respective valves. The vapour streams26and27from the vessel (13A) and vessel (14A) are condensed in cooler (E-4) and cooler (E-5), respectively, to generate condensed liquids that are returned back to the distillation column (8) using pumps (not shown in figure). The heat of distillation column (8) having plural trays is removed by one or more pump-arounds (Kero-PA, LGO-PA and HGO-PA). The downstream processing of the long residue stream (18) is the same as shown inFIG.1. Referring toFIG.3, representing another variation of a method as described inFIG.2, constructed in accordance with one of the embodiments of the present invention for crude petroleum oil processing in CDU. This method has the same configuration and operation, as shown inFIG.2, except that Light Gas Oil (LGO) and HGO distillates are heated in heat exchangers E-6and E-7, respectively, using either hot product and pump-around streams or the liquid stream (28) collected from the adjacent upper tray to flash zone (9) of ADC (8) or a stream (29) drawn from the long residue (18) before their routing to respective vessels13A and14A. Referring toFIG.4, representing another variation of a method as described inFIG.2, constructed in accordance with one of the embodiments of the present invention for crude petroleum oil processing in CDU to demonstrate new heating supplying mechanism in HGO reboiler stripper to meet the very high-temperature requirement. This method has the same configuration and operation, as shown inFIG.2, except that LGO distillate is routed to a reboiled side stripper (13B), which reboiler can use either hot product or hot pump around streams or HP steam, the liquid stream (28) collected from the adjacent upper tray to flash zone (9) of ADC (8) or a stream (29) drawn from the long residue (24) for supplying heating energy. The reboiled side stripper (13B) vapor (26) is routed to ADC without any cooling and pumping. HGO distillate is routed to reboiled side stripper (14B), which reboiler (E-8) uses either the liquid stream (27) collected from the adjacent upper tray to flash zone (9) of ADC (8) or a stream (29) drawn from the long residue (18) for supplying the heating energy at high temperature. The reboiled side stripper (14B) vapor (27) is routed to ADC without any cooling and pumping. Referring toFIG.5, representing another variation of a method as described inFIG.2, constructed in accordance with one of the embodiments of the present invention for crude petroleum oil processing in CDU. This method has the same configuration, operation and long residue processing, as shown inFIG.2, except that the vapour stream (20) from vessel (V2) is compressed in the pressure range of 6-12 kg/cm2using the compressor (CP-1). The compressed vapor is cooled in cooler (E-3) in the temperature range of 35-50° C. The cooled stream (20A) is routed to bottom tray of absorption tower (ABS) having plural trays. One part (stream,17A) of unstabilized naphtha stream (17) is routed to top tray of ABS. The liquid stream (22) from ABS and remaing part (stream,17B) of unstabilized naphtha stream (17) are routed to the distillation column (15) to produce the LPG and Light Naphtha LN product and noncondensate vapour stream (23). Steam (26) is injection at the bottom tray of ADC (8) to reduce the lighter vapor carryover with long residue stream (18) and VDC top vapor load to vacuum generating device. The present invention relates to a method for crude petroleum oil processing in the crude distillation unit, which is the starting point of the refining process of crude oil in a petroleum refinery. More particularly, the present invention relates to a novel crude petroleum oil processing scheme for CDU to eliminate the requirement of stripping steam in the Atmospheric Distillation Column (ADC) of CDU. This invention leads to a significant reduction in the operating cost and substantially ameliorates corrosion in the overhead section of the ADC. It is well known to one skilled in the art that the CDU has the highest throughput among all units in the refinery. The conventional processing of crude petroleum oil in CDU consumes enormous energy and stripping steam. A person skilled in the art knows that the quantitative requirement of energy and stripping steam for crude oil processing depends on the CDU design configuration, which, in turn, depends on the method used of crude petroleum oil processing in CDU. The person skilled in the art of crude distillation also understands that steam cost is much higher than fuel cost for the same thermal energy content in the refinery, since steam is produced by fuel with consequent energy efficiency losses in generation and use of steam. Moreover, there are corrosion and fouling issues in the Top Section and Overhead System (TSOVS) of ADC. The severities of these issues are typically higher for heavy and sour (Sulfur-rich) crude oils (referred to in the trade as “opportunity crudes”) and for crude oils that are difficult to desalt. However, the processing of such challenging crudes in CDU continues to increase due to their easier availability and lower cost. The primary sources for a corrosive environment in the TSOVS of ADC are aqueous corrosion due to Hydrogen Chloride (HCl), Hydrogen Chloride (HCl) and deposit corrosion due to ammonium and/or hydrochloride salts. The corrosion and fouling issues inside the ADC are caused by low-temperature reflux in conjunction with Hydrochloric Acid (HCl), ammonia (NH3) and amines present in the vapour. Cold reflux and cold reflux shocks cause localized water condensation inside the column. The first water droplets formed by localized condensation can be very acidic (very low pH) and can induce severe localized corrosion. Further, the dissolved HCl, NH3and amine in condensed water will lead to NH4Cl salts deposition as the water carrying these molecules vaporizes. These deposited salts remain dry and non-corrosive as long as the process temperature is sufficiently above the vapour's water dew point temperature. Thus, a positive temperature gap between the vapour's water dew point and reflux temperature (i.e. excess of reflux temperature relative to the vapour's water dew point) can help to mitigate corrosion and fouling issues in the TSOVS of ADC. In the existing methods for crude oil processing in CDU, high-pressure ADC operation is widely practiced in the industry. This facilitates the application of overhead systems consisting of two drums to provide the hot reflux to ADC to reduce the localized condensation, as compared to the alternative of a single drum overhead system that involves subcooled reflux to ADC. However, the temperature gap between vapour's water dew point and reflux temperature in the top section and overhead system of ADC tends to be negative even for two-drums based systems. This negative temperature gap can cause localized water condensation and thus lead to corrosion and fouling. Thus, there is always emphasis on developing new methods for crude petroleum oil processing in CDU, which can overcome the challenges of TSOVS temperature profiles in existing CDU designs. The novelty of the present invention resides in generating the lighter hydrocarbon vapour in the process, and its utilization as stripping media at ADC bottom and developing a new kind of processing scheme for processing the LGO and HGO distillates obtained from ADC to provide the stripping steam free ADC operation. The proposed method leads to a significant reduction in operating cost, GHG emissions and alleviates the severity of the corrosive environment compared to existing crude processing methods for crude petroleum oil processing in CDU. Further, the proposed method in the present invention increases the temperature gap between vapour's water dew point and operating temperature in the top section and overhead system of ADC without significant energy penalty or product quality loss. Thus, it avoids localized water condensation and alleviates the severity of the corrosive environment compared to existing crude processing methods for CDU. The temperature gap between vapour's water dew point and operating temperature inside the top section temperature of ADC in the existing stripping steam-based processing methods is increased by increasing the column pressure due to the use of the hot reflux. However, it would be apparent to one skilled in the art that such increases in the column pressure lead to lower relative volatility, higher fired furnace coil outlet temperature for specific crude vaporization and enhanced cracking tendency of crude oil in the system. In the present invention, the design temperature gap between reflux temperature and the vapour's water dew point temperature is significantly higher than that of existing crude oil processing methods. The present invention thus enables effective reduction of the ADC's pressure using a novel processing method, which can lead to a decrease in the Coil Outlet Temperature (COT) of the fired furnace to minimize the cracking tendency of crude oil in the fired furnace and ADC bottom section without compromising on the distillates yields and their quality. It can be understood by a person skilled in the art that the process of the present invention can increase the yield of atmospheric distillate without increasing the bottom stripping steam, i.e. without increasing the corrosion severity in the overhead system of ADC and without increasing the furnace coil outlet temperature, at the same time without increasing the cracking tendency of crude oil in the system. EXAMPLES The following three examples are given by way of illustration to substantiate the invention and, therefore, should not be construed to limit the scope of the invention. The properties of Iranian heavy crude used in these examples are given in Table 1. TABLE 1Iranian heavy Crude properties.InitialFinalCutStd LiquidtemperaturetemperatureYield ByDensityCrude(° C.)(° C.)Vol (%)(kg/m3)WholeIBPFBP100.0911.9CrudeCut1IBP404.1568.8Cut240.090.66.1693.9Cut390.6141.29.5738.0Cut4141.2191.89.3773.6Cut5191.8242.47.8813.2Cut6242.4292.98.0853.3Cut7292.9343.58.2891.8Cut8343.5394.18.0930.8Cut9394.1444.77.5969.7Cut10444.7495.36.81011.7Cut11495.3545.95.91051.5Cut12545.9596.55.01090.2Cut13596.5647.14.01131.3Cut14647.1697.63.11170.2Cut15697.6748.22.31209.2Cut16748.2798.81.61248.3Cut17798.8849.41.11287.3Cut18849.4900.00.71326.4Cut19900.0950.01.11359.1Cut20950.0FBP0.01395.5 Example 1 represents the base case (BC). This example is exemplary and constructed for establishing the basis to compare the quantitative advantages of the present invention. Example 2 represents the Proposed Case-1(PC-1). This example illustrates the validation of the proposed crude petroleum oil processing method in CDU to reduce the operating cost and increase the gap between vapour's water dew point and operating temperature. Example 3 represents the Proposed Case-2 (PC-2). This example illustrates the scope for reducing ADC overhead pressure and the fired furnace coil outlet temperature without compromising the quantity and quality of distillate products and still having a significant positive gap between vapour's water dew point and operating temperature to avoid corrosion in the overhead system of ADC. Example 1: The flow scheme shown inFIG.1is used for this example. The 1163.2 tonnes/hr crude containing 3.2 tonnes of water is heated to a temperature of 185° C. using Heat Exchanger Networks (HEN1and HEN2) and routed to a two-phase separating vessel (4) operating at 5 kg/cm2pressure to separate the vapour and liquid fractions. The liquid from the vessel (4) is preheated to a temperature of 385° C. using the (HEN4) and a fired furnace (F1). The partially vaporized liquid stream (6) and vapour stream (7) from the vessel (4) are routed to the flash zone (6th tray from bottom) of the ADC containing 58 trays and equipped with condensers, decanters and side strippers. The ADC was operated at top tray pressure of 3.7 kg/cm2a with condenser-E-1's pressure drop of 0.5 kg/cm2, condenser-E-2's pressure drop of 0.25 kg/cm2and column's pressure drop of 0.5 kg/cm.2 The vapour from the flash zone is fractionated into distillates vis-à-vis unstabilized Light Naphtha (LN), Heavy Naphtha (HN), kerosene, Light Gas Oil (LGO), and Heavy Gas Oil (HGO). Liquid falling from the flash zone is stripped out using the stripping steam (6A) at the bottom tray of ADC (8). The HN distillate is routed to a reboiled-side stripper (11), and Kerosene, LGO and HGO distillates are routed to their respective steam stripped side strippers12,13and14to remove the dissolved lighter fraction to meet the products ASTM D-86 distillation five volume percent point temperature. The stripping steam of 16.5 tonnes/hr is used at ADC's bottom. The kerosene, LGO and HGO pump arounds were used to remove the heat at different temperature level from the column. The unstabilized light naphtha (17) is processed in a distillation column (15) having 20 trays and 10 kg/cm2operating pressure to obtain the LPG and Light Naphtha (LN) products. The long residue (18) along with 5.0 tonne/hr furnace coil steam is heated in a fired furnace (F3) to the temperature value of 425° C. The partially vaporized crude (19) is processed in a Vacuum Distillation Column (VDC). VDC has 16 theoretical trays, top's pressure: 2.66 KPa, and bottom's pressure: 6.00 KPa. VDC produces the distillate products vis-á-vis vacuum diesel, Light Vacuum Gas Oil (LVGO), Heavy Vacuum Gas Oil (HVGO). The vacuum diesel, LVGO, HVGO products were withdrawn from the 15th9thand 6thtrays of VDC, having tray numbering from bottom to top. The 6.5 tonnes/hr stripping steam is used at the bottom of VDC. The details of pump-arounds and side strippers for ADC and pump-arounds for VDC are given in Table 2. TABLE 2Details of pump-arounds and side strippers usedPumpDrawReturnFlowTemperatureAroundtraytray[tonne/h]drop, ° C.ADC's Pump Around (PA.)Kerosene-PA3840630.0150.00LGO-PA2426580.0170.00HGO-PA1517370.0060.00VDC's Pump-around (PA)TOPPA1516342.058.0LVGOPA910461.065.4HVGOPA67817.053.0ADC's StrippersNo ofLiquidVapourStripping steam,trays/DrawReturnTonne/hr/reboilerStrippersEfficiencytraytrayduty (Mkcal/hr)HN reboiled6/0.548500.27StripperKerosene6/0.338405.41steam StripperLGO steam6/0.324262.42StripperHGO steam6/0.315171.20Stripper The flow rate of different products produced from ADC and VDC, for example-1, are given in Table 3.0. TABLE 3.0The flow rate of products produced from ADC and VDCProductProductFlowFlowrate,rate,ProductsTPHProductsTPHNon-condensate0.704VDC Top Vap2.43LPG16.00Vacuum Diesel (VD.)50.68Light Naphtha (LN)110.58Light Vacuum Gas Oil146.24Heavy Naphtha (HN)12.75(LVGO)Kerosene194.62Heavy Vacuum Gas154.72Light Gas Oil (LGO)112.27Oil (HVGO)Heavy Gas Oil (HGO)64.99Vacuum Slop Product15.00Long Residue (LR)648.69Vacuum Residue (VR)279.05 Further, it is known that one method of evaluating the quality of distillate products from the crude distillation unit is the measurement of ASTM D-86 temperatures corresponding to their 5 and 95 volume percent specification. The performance of a crude distillation is evaluated using the ASTM 5-95 gaps separation criteria. The details of 5 and 95 volume percent temperature for products streams and 5-95 gaps for adjacent products streams are given in Table 4.0. TABLE 4ASTM D-86 5 and 95 volume percent temperature and 5-95 gapsfor products streams95 volume %5 volume %ASTM 5-95 GapTemp.Temp.Temp.Stream° C.Stream° C.Stream° C.LN_95115.7HN-5120.2——HN_95160.0Kero_5148.9HN-LN4.5Kero_95240.0LGO_5223.8Kero-SN−11.0LGO_95320.0HGO_5286.1LGO-Kero−16.2HGO_95370.0LR_5319.4HGO-LGO−33.9VD_95362.0VD_5275.5LR-HGO−50.6LVGO_95480.0LVGO_5366.6LVGO-VD4.6HVGO_95580.0HVGO_5429.4HVGO-LVGO−50.6 Pinch analysis is a proven method for estimating the minimum thermal energy of the process without designing the heat exchanger networks. In present examples, pinch analysis is used to estimate the minimum thermal energy requirement of the processes used in all examples of the present invention. The enthalpy data, supply temperature and target temperatures for hot and cold streams for carrying out the pinch analysis were collected from a converged simulation model of process flow scheme used for example-1 (FIG.1). The delta min temperature of 20° C. was used in pinch analysis. The fuel Price of 5 $/MMBTU (Rs. 1389/Mkcal, 1$=70 rupees), operational hours of 8000, stripping steam price of Rs 1950/Tonne, furnace efficiency of 85% were used for estimating the operating cost of the process. The detail of operating energy requirement and operating cost is given in Table 5.0. TABLE 5.0Detail of operating energy requirement and operatingcost (TPH: Tonne/hr).Price/AnnumUtilityValues(Crores)Stripping steam (Strippers + bottom), TPH25.5339.83Min Hot utility (ADC furnaces duty), Mkcal/h62.2281.33VDC Furnace Duty, Mkcal/h35.5146.41Compressor duty, kW0.000.00Min Cold utility, Mkcal/h91.55#NeglectedTotal167.57#Neglected due to the much lower price of cold utility compared to hot utility. The gap between Reflux Temperature (RT) and Water Dew Point Temperature (WDPT) of vapour leaving the ADC's top tray can be used as indicators of the corrosive nature of the environment in the overhead systems. The positive difference will avoid the localized condensation of water in ADC's overhead system. In example-1, the values of the WDPT of vapour leaving the ADC's top tray and reflux temperatures are 111° C. and 103° C., respectively, for the specified ADC reflux drum pressure. This lead to a negative gap between RT and WDPT of vapor is −8.0° C. Example 2: This example of the present invention illustrates the effectiveness of a new method for crude petroleum oil processing in ADC to reduce the operating cost and increase the gap between vapour's water dew point and operating temperature to alleviate the corrosion of ADC's overhead system. The values of operating parameters such as crude flow rate, the temperature of crude oil to two-phase separating vessel (4), ADC top pressure, pressure drop across the ADC and condensers, number of trays in ADC, ADC's trays efficiency, crude entry location to ADC, pump-arounds draw and return stages, product's distillates draw stages, draw and return stages for strippers, number of trays and their efficacy in strippers, used in this example are same as used in Example 1. The flow scheme for this example is shown inFIG.2. The 1163.2 tonnes/hr crude containing 3.2 tonnes of dissolved water is heated to a temperature of 185° C. using Heat Exchanger Networks (HEN1and HEN2). The heated crude, 14.5 tonnes/hr of hydrocarbon vapour (24) generated in the process from the vessel (3), and 0.1 tonnes/hr of natural gas (NG) stream (25) having 95% methane and 5% ethane are routed to a two-phase separating vessel (4) having four trays and operating at 6 kg/cm2pressure to separate the vapour and liquid fractions. The liquid from the vessel (4) is preheated to a temperature of 385° C. using the (HEN4) and fired furnace (F1). The vapour stream (7) from the vessel (4) is heated to the temperature of 350° C. The partially vaporized liquid stream (6) is routed to the flash zone (6thtray from bottom) of the ADC, and the superheated heated vapour stream (7A) is routed to the stripping zone (10) of ADC (8). The ADC has 58 trays and equipped with condensers and decanters. The vapour from the flash zone is fractionated into distillates vis-à-vis unstabilized LN, HN, kerosene, LGO, and HGO. Liquid from the flash zone is stripped out using superheated hydrocarbon vapour (7B) at the bottom tray of ADC (8). The long residue (18) is obtained from the bottom of ADC (8). The HN distillate is routed to a reboiled side stripper (11), and kerosene distillate is routed to reboiled side stripper (12) to remove the dissolved lighter distillate from the product to meet the products ASTM D-86 distillation five volume percent point temperature. The LGO and HGO distillates are routed to the vessel (13A) and vessel (14A) through their respective pressure reducing valves. The vapour from the vessel (13A and14A) are condensed and returned back to ADC with the help of pumps. The vapour stream20from Vessel (V2) is compressed using a compressor (CP-1) to a pressure value of 10.0 kg/cm2and cooled to the temperature of ° C. using a cooler (E-3) before its routing to vessel (V3). The liquid stream (22) from V3and unstabilized light naphtha (17) from V2are feed to the distillation column (15), having 20 trays to produce LPG and light naphtha and vapour stream (23). The kerosene, LGO and HGO pump-arounds were used to remove the heat at different temperature level from the column. The downstream processing of long residue (18) in the VDC is the same as described in example-1. The details of pump-arounds and side strippers used in ADC and pump-arounds used in VDC to remove the heat from different trays are given in Table 6. TABLE 6details of pump-arounds and side strippers usedADC's Pump Around (PA.)PumpDrawReturnFlowTemperatureAroundtraytray[tonne/h]drop, ° C.Kerosene- PA3840699.9950.00LGO-PA2426370.0065.00HGO-PA1517385.0060.00VDC's Pump-around (PA)TOP-PA1516342.057.9LVGO-PA910461.065.5HVGO-PA67817.054.2ADC's StrippersNo of trays/LiquidVapourReboiler dutyStrippersEfficiencyDraw trayReturn tray(Mkcal/hr)HN reboiled6/0.548500.32StripperKero reboiled6/0.538404.12Stripper The flow rates of different product produced from ADC and VDC are given in Table 7.0. TABLE 7.0Flow rate of products produced from ADC and VDCProductProductFlowFlowrate,rate,ProductsTPHProductsTPHNon-condensate0.85VDC Top Vap8.55LPG16.00Vacuum Diesel (VD.)45.18Light Naphtha (LN)97.52Light Vacuum Gas Oil145.97Heavy Naphtha (HN.)22.42(LVGO)Kerosene187.92Heavy Vacuum Gas155.47Light Gas Oil (LGO)117.10Oil (HVGO)Heavy Gas Oil (HGO)68.90Vacuum Slop Product15.00Long Residue (LR)649.55Vacuum Residue (VR)279.30 The details of 5 and 95 volume percent temperature for products streams and 5-95 gaps for adjacent products streams are given in Table 8.0. TABLE 85 and 95 volume percent temperature and 5-95 gapsfor products streams.95 volume %5 volume %ASTM 5-95 GapTemp.Temp.Temp.Stream° C.Stream° C.Stream° C.LN_95113.1HN-5119.2——HN_95160.0Kero_5148.9HN-LN6.1Kero_95240.0LGO_5223.1Kero-SN−11.1LGO_95319.9HGO_5284.9LGO-Kero−16.9HGO_95369.9LR_5308.1HGO-LGO−35.0VD_95360.0VD_5271.5RCO-HGO−61.9LVGO_95480.0LVGO_5367.1LVGO-VD7.1HVGO_95580.0HVGO_5443.4HVGO-LVGO−36.6 For example, the operating energy requirement and operating cost, for example-2, is estimated using the same methodology and price values of different utilities as used in example-1. The price of 1 KWh electricity used in the example-2 is Rs. 5.0. The detail of operating energy requirement and operating cost for example-2 is given in Table 9. TABLE 9Detail of operating energy requirement and operatingcost (TPH: Tonne/hr).Price/AnnumUtilityValues(Crores)Stripping steam (Strippers + bottom), TPH0.000.00Min Hot utility (ADC furnaces duty), Mkcal/h62.9882.32VDC Furnace Duty, Mkcal/h32.4942.47Compressor duty, kWh664.972.66Min Cold utility, Mkcal/h66.16#NeglectedTotal127.45#Neglected due to the much lower price of cold utility compared to hot utility. In example-2, the values of the WDPT of vapour leaving the ADC's top tray and reflux temperature are 85.2° C. and 105° C., respectively, for the specified ADC reflux drum pressure as used in example 1. This leads to a positive gap of 19.8° C. between RT and WDPT. The operating cost of the crude oil processing method shown in example-2 constructed with the accordance of the present invention is 127.45 crores. Example 3: Example 3 (proposed case-2: PC-2) illustrates the scope for reducing ADC overhead pressure and the furnace coil outlet temperature without compromising on the quality of distillate products and still having the positive gap between vapour's water dew point and operating temperature compared to the negative gap in example-1 (base case). The values of operating parameters in this example such as crude flow rate, the temperature of crude to two-phase separating vessel (4), pressure drop across the ADC and condensers, number of trays in ADC, ADC's trays efficiency, crude entry location to ADC, pump arounds draw and return stages, product's distillates draw stage, draw and return stages for strippers, number of trays and their efficacy in strippers are same as used in Example-2. The flow scheme for this example is shown inFIG.3. The 1163.2 tonnes/hr desalted crude oil containing 3.2 tonnes of water is heated to 185° C. using Heat Exchanger Networks (HEN1and HEN2). The heated crude oil, 6.0 tonne/hr of light hydrocarbon vapour stream (24) generated in the process from the vessel (3), and 0.1 tonnes/hr of natural gas (NG) stream (25) having 95% methane and 5% ethane are routed to a two-phase separating vessel (4) having four trays and operating at 4.8 kg/cm2pressure to separate the vapour and liquid fractions. The liquid from the vessel (4) is preheated to a temperature of 369° C. using the (HEN4) and fired furnace (F1). The vapour stream (7) from the vessel (4) is heated to the temperature of 350° C. The partially vaporized liquid stream (6) is routed to the flash zone (6thtray from bottom) of the ADC, and the heated vapour stream (7A) is routed to the bottom tray of the stripping zone (10) of ADC (8). The ADC has 58 trays and equipped with condensers and decanters. The vapour from the flash zone is fractionated into distillates vis-à-vis unstabilized LN, HN, kerosene, LGO, and HGO. Liquid from the flash zone is stripped out using superheated hydrocarbon vapour (7B) at the bottom tray of ADC (8). The long residue (18) is obtained from the bottom of ADC (8). The HN distillate is routed to a reboiled side stripper (11), and kerosene distillate is routed to reboiled side stripper (12) to remove the dissolved lighter fraction from the product for meeting the products ASTM D-86 distillation five volume percent point temperature. The LGO and HGO distillates are routed to the vessel (13A) and vessel (14A) through their respective pressure reducing valves. The vapour streams from the vessel (13A and14A) are condensed and returned to ADC with the help of pumps. The vapour stream20from Vessel (V2) is compressed using a compressor (CP-1) to a pressure value of 10.0 kg/cm2and cooled to the temperature of 40° C. using a cooler (E-3) before its routing to vessel (V3). The liquid stream (22) from V3and unstabilized light naphtha (17) from V2are feed to the distillation column (15), having 20 trays to produce a vapour stream (23), LPG and LN. The kerosene, LGO and HGO pump-arounds were used to remove the heat at different temperature level from the column. The downstream processing of long residue (18) in the VDC is the same as used in example-1 and example-2. The details of pump-arounds and side strippers used in ADC and pump-arounds used in VDC to remove the heat from different trays are given in Table 10. TABLE 10details of pump-arounds and side strippers usedADC's Pump Around (PA.)PumpDrawReturnFlowTemperatureAroundtraytray[tonne/h]drop, ° C.Kerosene- PA3840780.0050.00LGO-PA2426400.0070.00HGO-PA1517300.0050.00VDC's Pump-around (PA)TOP-PA1516342.053.70LVGO-PA910461.065.46HVGO-PA67817.053.12ADC's StrippersNo of trays/LiquidVapourReboiler dutyStrippersEfficiencyDraw trayReturn tray(Mkcal/hr)HN reboiled6/0.548500.33StripperKero reboiled6/0.538402.66Stripper The flow rates of different products produced from ADC and VDC are given in Table 11. TABLE 11Flow rate of products produced from ADC and VDC.ProductProductFlowFlowrate,rate,ProductsTPHProductsTPHNon-condensate1.12VDC Top Vap8.48LPG16.01Vacuum Diesel (VD.)43.81Light Naphtha (LN)97.72Light Vacuum Gas Oil146.30Heavy Naphtha (HN. )24.67(LVGO)Kerosene187.81Heavy Vacuum Gas154.86Light Gas Oil (LGO)119.91Oil (HVGO)Heavy Gas Oil (HGO)65.36Vacuum Slop Product15.00Long Residue (LR)647.76Vacuum Residue (VR)279.20 The details of 5 and 95 volume percent temperature for products streams and 5-95 gaps for adjacent products streams are given in Table 12. TABLE 125 and 95 volume percent temperature and 5-95 gapsfor products streams.95 volume %5 volume %ASTM 5-95 GapTemp.Temp.Temp.Stream° C.Stream° C.Stream° C.LN_95112.70HN-5120.52——HN_95160.00Kero_5149.13HN-LN7.8Kero_95240.00LGO_5222.40Kero-SN−10.1LGO_95320.10HGO_5284.10LGO-Kero−17.6HGO_95370.10LR_5313.03HGO-LGO−36.0VD_95360.00VD_5282.44RCO-HGO−57.1LVGO_95480.00LVGO_5367.43LVGO-VD7.4HVGO_95580.00HVGO_5429.58HVGO-LVGO−50.4 The operating energy requirement and operating cost in example-3 is estimated using the same methodology and price values of different utilities as used in example-1 and 2. The detail of operating energy requirement and operating cost for example-3 is given in Table 13. TABLE 13Detail of operating energy requirement and operatingcost (TPH: Tonne/hr).Price/AnnumUtilityValues(Crores)Stripping steam (Strippers + bottom), TPH0.000.00Min Hot utility (ADC furnaces duty), Mkcal/h51.6167.47VDC Furnace Duty, Mkcal/h38.8550.78Compressor duty, kWh860.923.43Min Cold utility, Mkcal/h55.16#NeglectedTotal121.70#Neglected due to the much lower price of cold utility compared to hot utility. The values of WDPT of ADC's top vapour and reflux temperature are 79.5° C. and 92.1° C., respectively, even for the reduced ADC reflux drum pressure compared to example 1. This lead to the positive gap of 12.7° C. between RT and WDPT. The operating cost of the crude oil processing method shown in example-3 constructed with the accordance of the prior art is 121.70 crores. Example 4: Example 4 (proposed case-3: PC-3) illustrates the scope for reducing vapor stream (20) compression capital and energy cost compared to example 2. The flow scheme for this example is shown inFIG.5. The values of operating parameters in this example such as crude flow rate, the temperature of crude to two-phase separating vessel (4), pressure drop across the ADC and condensers, number of trays in ADC, ADC's trays efficiency, crude entry location to ADC, pump arounds draw and return stages, product's distillates draw stage, return stages for condensed vapor liquids and strippers vapor, number of trays and their efficacy in strippers, long residue processing in VDC are same as used in Example-2. The operation of crude processing is also same as used in example 2 except that the vapour stream (20) from vessel (V2) is compressed in the pressure range of 6-12 kg/cm2using the compressor (CP-1). The compressed vapor is cooled in cooler (E-3) in the temperature range of 35-50° C. The cooled stream (20A) is routed to bottom tray of absorption tower (ABS) having plural trays. One part (stream,17A) of unstabilized naphtha stream (17) is routed to top tray of ABS. The liquid stream (22) from ABS and remaining part (stream,17B) of unstabilized naphtha stream (17) are routed to the distillation column (15) to produce the LPG and Light Naphtha LN product and noncondensate vapour stream (23). The details of pump-arounds and side strippers used in ADC and pump-arounds used in VDC to remove the heat from different trays are given in Table 14. TABLE 14details of pump-arounds and side strippers usedADC's Pump Around (PA.)PumpDrawReturnFlowTemperatureAroundtraytray[tonne/h]drop, ° C.Kerosene- PA3840699.9950.00LGO-PA2426370.0065.00HGO-PA1517385.0060.00VDC's Pump-around (PA)TOP-PA1516342.057.9LVGO-PA910461.065.5HVGO-PA67817.054.2ADC's StrippersNo of trays/LiquidVapourReboiler dutyStrippersEfficiencyDraw trayReturn tray(Mkcal/hr)HN reboiled6/0.548500.54StripperKero reboiled6/0.538404.94Stripper The flow rates of different product produced from ADC and VDC are given in Table 7.0. TABLE 15.0Flow rate of products produced from ADC and VDCProductProductFlowFlowrate,rate,ProductsTPHProductsTPHNon-condensate0.68VDC Top Vap8.49LPG16.00Vacuum Diesel (VD.)43.63Light Naphtha (LN)96.18Light Vacuum Gas Oil146.16Heavy Naphtha (HN.)28.58(LVGO)155.46Kerosene183.00Heavy Vacuum Gas15.00Light Gas Oil (LGO)118.28Oil (HVGO)Heavy Gas Oil (HGO)69.41Vacuum Slop Product8.49Long Residue (LR)648.17Vacuum Residue (VR)43.63 The details of 5 and 95 volume percent temperature for products streams and 5-95 gaps for adjacent products streams are given in Table 16.0. TABLE 165 and 95 volume percent temperature and 5-95 gapsfor products streams.95 volume %5 volume %ASTM 5-95 GapTemp.Temp.Temp.Stream° C.Stream° C.Stream° C.LN_95123.5HN-5119.8——HN_95160.0Kero_5152.4HN-LN−3.7Kero_95240.0LGO_5223.5Kero-SN−9.1LGO_95319.9HGO_5285.1LGO-Kero−16.5HGO_95369.9LR_5309.60HGO-LGO−34.8VD_95197.7VD_5272.0RCO-HGO−60.3LVGO_95360.0LVGO_5366.9LVGO-VD6.9HVGO_95480.0HVGO_5443.2HVGO-LVGO−36.8 The operating energy requirement and operating cost, for example-3 is estimated using the same methodology and price values of different utilities as used in example-2. The detail of operating energy requirement and operating cost for example-3 is given in Table 17. TABLE 17Detail of operating energy requirement and operatingcost (TPH: Tonne/hr).Price/AnnumUtilityValues(Crores)Stripping steam (Strippers + bottom), TPH0.000.00Min Hot utility (ADC furnaces duty), Mkcal/h62.9582.99VDC Furnace Duty, Mkcal/h32.5242.51Compressor duty, kWh482.231.93Min Cold utility, Mkcal/h66.00#NeglectedTotal126.73#Neglected due to the much lower price of cold utility compared to hot utility. In example-4, the values of the WDPT of vapour leaving the ADC's top tray and reflux temperature are 84.1° C. and 102.8° C., respectively, for the specified ADC reflux drum pressure as used in example 2. This leads to a positive gap of 18.7° C. between RT and WDPT. The operating cost of the crude oil processing method proposed in example-2 constructed with the accordance of the present invention is 126.73 crores. Comparative results of example-1, example-2, example 3 and 4 indicate following observations:I. The values of the ASTM D-86 5 volume %, 95 volume % temperatures, ASTM D-86: 5-95 gap of distillate products, the flow rate of residue (Vacuum residue+Slop) are similar to example-1 (base case). This ensures that quality and total quantity of distillate products were not compromised in the present invention to reduce operating cost and the positive temperature gap between reflux temperature and vapour's water dew point to alleviate the corrosion in the overhead system ADC.II. The gap between reflux temperature and water dew point of vapour is −8.0 C for example-1, 19.8 for example-2, 12.7° C. for example-3 and 18.7° C. for example-4. This implies that a negative gap in example based on prior art processing scheme can lead to localized water condensation in the overhead system, which can dissolve HCl, NH3and amines; as a result, the chances of acid and salt deposition assisted corrosion in the overhead system of ADC can occur. Whereas, significant positive gap between reflux temperature and water dew point of vapour avoid the possibility of localised water condensation in present inventions schemes used in examples 2, 3 and 4.III. There is a scope of reduction in operating cost by 23.9% for the present invention's crude petroleum oil processing scheme compared to the prior art based scheme for example 2, 27.4% for example 3 and 24.4 for example 4.IV. Moreover, the coil outlet temperature in example-3 is reduced to 369° C. from 385° C. used in example-1 (BC) and example-2 (PC-1). This indicates that the implementation of this crude oil processing method of the present invention can help in reducing the Coil Outlet Temperature (COT) by 16° C. without compromising on the distillates yields, their quality. Lower COT will reduce the crude cracking tendency in the furnace and bottom section of ADC.V. The present invention also helps in reducing the ADC's pressure and still having the significant positive gap between reflux temperature and vapour water dew point, i.e. without increasing the intensity of corrosive environment in the overhead system of ADC. It can be noted that operating conditions (temperature, pressure, flow rate etc.) used in these studied examples were just a way of illustrating the present invention and, however, the invention is not limited to these operating conditions. ADVANTAGES OF INVENTION The utilization of the present invention has several distinct advantages over prior art crude oil processing methods.Energy savings by 12-18%, reduction in operating cost by 23-26% for crude processing compared to existing methods (base case).Increased temperature gap between vapour's water dew point and reflux temperature compared to existing methods (base case). The increased gap will avoid the chances of localized water condensation and help in alleviating the corrosive environment severity in the top section and overhead system of ADC (example 1 constructed with accordance to prior art: Negative (−8° C.), example 2 constructed with accordance to present invention: Positive (19.8° C.).Provides the flexibility to reduce ADC's operating pressure and still has a significant positive temperature gap between vapour's water dew point and reflux temperature. This leads to a significant reduction in the furnace Coil Outlet Temperature (COT), which helps in reducing the cracking tendency of crude oil in the furnace and ADC bottom section without compromising on ADC's distillate yield and products quality.Significant reduction in size and load of ADC's condenser due to stripping steam elimination from the ADC operation.Energy savings will also decrease GHG emissions from the crude unit to the environment and make the crude oil processing cleaner and greener. | 43,578 |
11859140 | For the purpose of describing the simplified schematic illustrations and descriptions of the relevant figures, the numerous valves, temperature sensors, electronic controllers and the like that may be employed and well known to those of ordinary skill in the art of certain chemical processing operations are not included. Further, accompanying components that are often included in typical chemical processing operations, such as air supplies, catalyst hoppers, and flue gas handling systems, are not depicted. Accompanying components that are in hydrocracking units, such as bleed streams, spent catalyst discharge subsystems, and catalyst replacement sub-systems are also not shown. It should be understood that these components are within the spirit and scope of the present embodiments disclosed. However, operational components, such as those described in the present disclosure, may be added to the embodiments described in this disclosure. It should further be noted that arrows in the drawings refer to process streams. However, the arrows may equivalently refer to transfer lines, which may serve to transfer process streams between two or more system components. Additionally, arrows that connect to system components define inlets or outlets in each given system component. The arrow direction corresponds generally with the major direction of movement of the materials of the stream contained within the physical transfer line signified by the arrow. Furthermore, arrows, which do not connect two or more system components, signify a product stream, which exits the depicted system, or a system inlet stream, which enters the depicted system. Product streams may be further processed in accompanying chemical processing systems or may be commercialized as end products. System inlet streams may be streams transferred from accompanying chemical processing systems or may be non-processed feedstock streams. Some arrows may represent recycle streams, which are effluent streams of system components that are recycled back into the system. However, it should be understood that any represented recycle stream, in some embodiments, may be replaced by a system inlet stream of the same material, and that a portion of a recycle stream may exit the system as a product. Additionally, arrows in the drawings may schematically depict process steps of transporting a stream from one system component to another system component. For example, an arrow from one system component pointing to another system component may represent “passing” a system component effluent to another system component, which may include the contents of a process stream “exiting” or being “removed” from one system component and “introducing” the contents of that product stream to another system component. It should be understood that according to the embodiments presented in the relevant figures, an arrow between two system components may signify that the stream is not processed between the two system components. In other embodiments, the stream signified by the arrow may have substantially the same composition throughout its transport between the two system components. Additionally, it should be understood that in embodiments, an arrow may represent that at least 75 wt. %, at least 90 wt. %, at least 95 wt. %, at least 99 wt. %, at least 99.9 wt. %, or even 100 wt. % of the stream is transported between the system components. As such, in embodiments, less than all of the stream signified by an arrow may be transported between the system components, such as if a slip stream is present. It should be understood that two or more process streams are “mixed” or “combined” when two or more lines intersect in the schematic flow diagrams of the relevant figures. Mixing or combining may also include mixing by directly introducing both streams into a like reactor, separation unit, or other system component. For example, it should be understood that when two streams are depicted as being combined directly prior to entering a separation unit or reactor, that in embodiments the streams could equivalently be introduced into the separation unit or reactor and be mixed in the reactor. Alternatively, when two streams are depicted to independently enter a system component, they may in embodiments be mixed together before entering that system component. Reference will now be made in greater detail to various embodiments, some embodiments of which are illustrated in the accompanying drawings. Whenever possible, the same reference numerals will be used throughout the drawings to refer to the same or similar parts. DETAILED DESCRIPTION Embodiments of the present disclosure are directed to processes and systems for hydrotreating and hydrocracking hydrocarbon oil and recycling the hydrotreating catalyst. As used herein, a “catalyst” refers to any substance that increases the rate of a specific chemical reaction. Catalysts described in this disclosure may be utilized to promote various reactions, such as, but not limited to, cracking (including aromatic cracking), demetalization, desulfurization, and denitrogenation. As used herein, “cracking” generally refers to a chemical reaction where carbon-carbon bonds are broken. For example, a molecule having carbon to carbon bonds is broken into more than one molecule by the breaking of one or more of the carbon to carbon bonds, or is converted from a compound which includes a cyclic moiety, such as a cycloalkane, cycloalkane, naphthalene, an aromatic or the like, to a compound which does not include a cyclic moiety or contains fewer cyclic moieties than prior to cracking. As used herein, the term “crude oil” is to be understood to mean a mixture of petroleum liquids, gases, or combinations of liquids and gases, including some impurities such as sulfur-containing compounds, nitrogen-containing compounds and metal compounds that have not undergone significant separation or reaction processes. Crude oils are distinguished from fractions of crude oil. As used herein, the crude oil may be a minimally treated crude oil to provide a crude oil feedstock having total metals (Nickel+Vanadium) content of less than 5 parts per million by weight (ppmw) and Conradson carbon residue of less than 5 wt. % Such minimally treated materials may be considered crude oils as described herein. As used herein, the term “hydrogen/oil ratio” or “hydrogen-to-oil ratio” or “hydrogen-to-hydrocarbon oil ratio” refers to a standard measure of the volume rate of hydrogen circulating through the reactor with respect to the volume of feed. The hydrogen/oil ratio may be determined by comparing the flow volume of a hydrogen stream and the flow volume of a hydrocarbon oil feed or the flow volume of a second hydrogen stream and the flow volume of a hydrocarbon product stream. As used herein, a “fixed-bed,” specifically in reference to reactors, refers to a catalyst bed inside a reactor that is not displaced by fluids entering and exiting the reactor, i.e., the catalysts remain in place. Also as used herein, a “moving-bed,” also specifically in reference to reactors, refers to a catalyst bed inside a reactor that is displaced by fluid entering and exiting the reactor, i.e., the catalysts do not remain in place. For moving-beds, catalyst may be carried with the fluid out of the reactor or the catalyst may not. Also for moving-beds, catalysts may be displaced such that they are suspended by the fluid as a fluidized bed. As used herein, a “reactor” refers to a vessel in which one or more chemical reactions may occur between one or more reactants optionally in the presence of one or more catalysts. For example, a reactor may include a tank or tubular reactor configured to operate as a batch reactor, a continuous stirred-tank reactor (CSTR), or a plug flow reactor. Exemplary reactors include packed bed reactors such as fixed-bed reactors, and fluidized bed reactors. One or more “reaction zones” may be disposed in a reactor. As used herein, a “reaction zone” refers to an area where a particular reaction takes place in a reactor. For example, a packed bed reactor with multiple catalyst beds may have multiple reaction zones, where each reaction zone is defined by the area of each catalyst bed. As used herein, the term “regenerated catalyst” or “regenerated hydrotreating catalyst” refers to catalyst that has been introduced to a cracking reaction zone and then regenerated in a regenerator to heat the catalyst to a greater temperature, oxidize and remove at least a portion of the coke from the catalyst to restore at least a portion of the catalytic activity of the catalyst, or both. The “regenerated catalyst” may have less coke, a greater temperature, or both compared to spent catalyst and may have greater catalytic activity compared to spent catalyst. The “regenerated catalyst” may have more coke and lower catalytic activity compared to fresh catalyst that has not passed through a cracking reaction zone and regenerator. As used herein, a “separation unit” or “separator” refers to any separation device that at least partially separates one or more chemicals that are mixed in a process stream from one another. For example, a separation unit may selectively separate differing chemical species, phases, or sized material from one another, forming one or more chemical fractions. Examples of separation units include, without limitation, distillation columns, flash drums, knock-out drums, knock-out pots, centrifuges, cyclones, filtration devices, traps, scrubbers, expansion devices, membranes, solvent extraction devices, and the like. It should be understood that separation processes described in this disclosure may not completely separate all of one chemical constituent from all of another chemical constituent. It should be understood that the separation processes described in this disclosure “at least partially” separate different chemical components from one another, and that even if not explicitly stated, it should be understood that separation may include only partial separation. As used herein, one or more chemical constituents may be “separated” from a process stream to form a new process stream. Generally, a process stream may enter a separation unit and be divided, or separated, into two or more process streams of desired composition. Further, in some separation processes, a “lower boiling point fraction” (sometimes referred to as a “light fraction” or “light fraction stream”) and a “higher boiling point fraction” (sometimes referred to as a “heavy fraction,” “heavy hydrocarbon fraction,” or “heavy hydrocarbon fraction stream”) may exit the separation unit, where, on average, the contents of the lower boiling point fraction stream have a lower boiling point than the higher boiling point fraction stream. Other streams may fall between the lower boiling point fraction and the higher boiling point fraction, such as a “medium boiling point fraction.” As used herein, the term “spent catalyst” or “spent hydrotreating catalyst” refers to catalyst that has been introduced to and passed through a cracking reaction zone to crack a crude oil, such as the higher boiling point fraction or the lower boiling point fraction for example, but has not been regenerated in the regenerator following introduction to the cracking reaction zone. The “spent catalyst” may have coke deposited on the catalyst and may include partially coked catalyst as well as fully coked catalysts. The amount of coke deposited on the “spent catalyst” may be greater than the amount of coke remaining on the regenerated catalyst following regeneration. It should further be understood that streams may be named for the components of the stream, and the component for which the stream is named may be the major component of the stream (such as comprising from 50 weight percent (wt. %), from 70 wt. %, from 90 wt. %, from 95 wt. %, from 99 wt. %, from 99.5 wt. %, or even from 99.9 wt. % of the contents of the stream to 100 wt. % of the contents of the stream). It should also be understood that components of a stream are disclosed as passing from one system component to another when a stream comprising that component is disclosed as passing from that system component to another. By way of non-limiting example, a referenced “hydrogen stream” passing from a first system component to a second system component should be understood to equivalently disclose “hydrogen” passing from a first system component to a second system component, and the like. Referring initially toFIG.1, an integrated system10for hydrotreating and hydrocracking hydrocarbon oil is illustrated. The integrated system10includes a moving bed hydrotreating reactor12, a hydrocracking reactor14, and a catalyst reclamation unit16. The catalyst reclamation unit16includes a stripper20and a catalyst regenerator22. As is meant to be shown inFIG.1, and in embodiments, the moving bed reactor12may be an ebullated-bed hydrotreating reactor. Now referring toFIG.2, an integrated system60for hydrotreating and hydrocracking hydrocarbon oil26is illustrated. The integrated system60may include any of the integrated systems10previously or hereinafter discussed as well as additional components. As is meant to be shown inFIG.2, and in embodiments, the moving bed reactor12may be a slurry-bed hydrotreating reactor. As used herein, “slurry-bed hydrotreating reactor” may also be referred to as a “slurry hydrotreating reactor.” Now referring toFIGS.1and2, the moving bed hydrotreating reactor12hydrotreats a hydrocarbon oil stream26and a hydrogen stream24with a hydrotreating catalyst to form a hydrocarbon product stream28and spent hydrotreating catalyst34. In embodiments, the hydrocarbon product stream28may include the hydrocarbon oil stream26and the hydrogen stream24. In embodiments, the hydrocarbon oil stream26and the hydrogen stream24may be mixed before entering the moving bed hydrotreating reactor12. In embodiments, the hydrotreating catalyst may be pre-loaded into the moving-bed hydrotreating reactor12. The hydrotreating catalyst may include an active-phase metal on a support. The active-phase metal may include nickel, molybdenum, tungsten, platinum, palladium, rhodium, ruthenium, gold, or combinations thereof. In embodiments, the support may include amorphous alumina, crystalline silica-alumina, alumina, silica, and combinations thereof. The hydrotreating catalyst may include MoNi on Al2O3, MoCO on Al2O3, MoS2, maghemite, Fe3O4, nickel, NiO, TiO2, ZrO2, CeO2, or combinations thereof. The hydrotreating catalyst may become spent hydrotreating catalyst34when coke produced as a byproduct of the hydrotreating reaction is deposited on the hydrotreating catalyst, thereby rendering the hydrotreating catalyst at least partially ineffective. The hydrotreating catalyst may also become spent hydrotreating catalyst34when at least part of the hydrocarbon oil stream26is adsorbed onto the hydrotreating catalyst, thereby rendering the hydrotreating catalyst at least partially ineffective. In embodiments, the moving-bed hydrotreating reactor12may have a temperature of from 370° C. to 500° C. The moving-bed hydrotreating reactor12may have a temperature of from 370° C. to 500° C., from 370° C. to 480° C., from 370° C. to 450° C., from 370° C. to 420° C., from 370° C. to 400° C., from 370° C. to 390° C., from 370° C. to 380° C., from 380° C. to 500° C., from 380° C. to 480° C., from 380° C. to 450° C., from 380° C. to 420° C., from 380° C. to 400° C., from 380° C. to 390° C., from 390° C. to 500° C., from 390° C. to 480° C., from 390° C. to 450° C., from 390° C. to 420° C., from 390° C. to 400° C., from 400° C. to 500° C., from 400° C. to 480° C., from 400° C. to 450° C., from 400° C. to 420° C., from 420° C. to 500° C., from 420° C. to 480° C., from 420° C. to 450° C., 450° C. to 500° C., from 450° C. to 480° C., or from 480° C. to 500° C. In embodiments, the moving-bed hydrotreating reactor12may have a pressure of from 12 MPa to 16 MPa. The moving-bed hydrotreating reactor12may have a pressure of from 12 MPa to 16 MPa, from 12 MPa to 15.5 MPa, from 12 MPa to 15 MPa, from 12 MPa to 14.5 MPa, from 12 MPa to 14 MPa, from 12 MPa to 13.5 MPa, from 12 MPa to 13 MPa, from 12 MPa to 12.5 MPa, from 12.5 MPa to 16 MPa, from 12.5 MPa to 15.5 MPa, from 12.5 MPa to 15 MPa, from 12.5 MPa to 14.5 MPa, from 12.5 MPa to 14 MPa, from 12.5 MPa to 13.5 MPa, from 12.5 MPa to 13 MPa, from 13 MPa to 16 MPa, from 13 MPa to 15.5 MPa, from 13 MPa to 15 MPa, from 13 MPa to 14.5 MPa, from 13 MPa to 14 MPa, from 13 MPa to 13.5 MPa, from 13.5 MPa to 16 MPa, from 13.5 MPa to 15.5 MPa, from 13.5 MPa to 15 MPa, from 13.5 MPa to 14.5 MPa, from 13.5 MPa to 14 MPa, from 14 MPa to 16 MPa, from 14 MPa to 15.5 MPa, from 14 MPa to 15 MPa, from 14 MPa to 14.5 MPa, from 14.5 MPa to 16 MPa, from 14.5 MPa to 15.5 MPa, from 14.5 MPa to 15 MPa, from 15 MPa to 16 MPa, from 15 MPa to 15.5 MPa, or from 15.5 MPa to 16 MPa. In embodiments, the moving-bed hydrotreating reactor12may have a liquid hourly space velocity of from 0.2 h−1to 0.7 h−1. The moving-bed hydrotreating reactor12may have a liquid hourly space velocity of from 0.2 h−1to 0.7 h−1, from 0.2 h−1to 0.6 h−1, from 0.2 h−1to 0.5 h−1, from 0.2 h−1to 0.4 h−1, from 0.2 h−1to 0.3 h−1, from 0.3 h−1to 0.7 h−1, from 0.3 h−1to 0.6 h−1, from 0.3 h−1to 0.5 h−1, from 0.3 h−1to 0.4 h−1, from 0.4 h−1to 0.7 h−1, from 0.4 h−1to 0.6 h−1, from 0.4 h−1to 0.5 h−1, from 0.5 h−1to 0.7 h−1, from 0.5 h−1to 0.6 h−1, or from 0.6 h−1to 0.7 h−1. Still referring toFIGS.1and2, and in embodiments, the moving-bed hydrotreating reactor12may have a ratio of hydrogen24to hydrocarbon oil stream26of from 800 L/L to 1200 L/L. The moving-bed hydrotreating reactor12may have a ratio of hydrogen24to hydrocarbon oil stream26of from 800 L/L to 1200 L/L, from 800 L/L to 1100 L/L, from 800 L/L to 1000 L/L, from 800 L/L to 900 L/L, from 900 L/L to 1200 L/L, from 900 L/L to 1100 L/L, from 900 L/L to 1000 L/L, from 1000 L/L to 1200 L/L, from 1000 L/L to 1100 L/L, from 1100 L/L to 1200 L/L, or from 1100 L/L to 1200 L/L. Referring again toFIG.1, and as previously mentioned, the moving-bed hydrotreating reactor12may be the ebullated-bed hydrotreating reactor. As described herein, “ebullated-bed reactors” are a type of fluidized bed reactor that utilizes ebullition, or bubbling, to achieve distribution of reactants and catalysts. Catalysts in the ebullated-bed reactor may remain suspended and held in a fluidized state through the upward lift of liquid reactant and gas. In one non-limiting example, the hydrogen24and hydrocarbon oil stream26may suspend the hydrotreating catalyst as a fluidized bed within the ebullated-bed hydrotreating reactor. In embodiments including the ebullated-bed hydrotreating reactor, the hydrocarbon oil stream26may exit at the top of the moving-bed hydrotreating reactor12. The spent hydrotreating catalyst34may exit at the bottom of the moving-bed hydrotreating reactor12after settling from the ebullated-bed. Additionally or alternatively, the spent hydrotreating catalyst34and at least some of the hydrotreating catalyst may be regularly withdrawn from the ebullated-bed hydrotreating reactor to control the level of catalyst activity. Fresh hydrotreating catalyst44, a regenerated hydrotreating catalyst40, or both may then be added on the top of the ebullated-bed to maintain constant catalyst activity. In one non-limiting example, 10% of the hydrotreating catalyst and spent hydrotreating catalyst34may be removed every 3 to 5 days and replaced. Still referring toFIG.1, and in embodiments, the hydrotreating catalyst may have an average particle size of from 500 μm to 1000 μm. The hydrotreating catalyst may have an average particle size of from 400 μm to 1100 μm, from 400 μm to 1000 μm, from 400 μm to 900 μm, from 400 μm to 800 μm, from 400 μm to 700 μm, from 400 μm to 600 μm, from 400 μm to 500 μm, from 500 μm to 1100 μm, from 500 μm to 1000 μm, from 500 μm to 900 μm, from 500 μm to 800 μm, from 500 μm to 700 μm, from 500 μm to 600 μm, from 600 μm to 1100 μm, from 600 μm to 1000 μm, from 600 μm to 900 μm, from 600 μm to 800 μm, from 600 μm to 700 μm, from 700 μm to 1100 μm, from 700 μm to 1000 μm, from 700 μm to 900 μm, from 700 μm to 800 μm, from 800 μm to 1100 μm, from 800 μm to 1000 μm, from 800 μm to 900 μm, from 900 μm to 1100 μm, from 900 μm to 1000 μm, and from 1000 μm to 1100 μm. Referring again toFIG.2, and as previously mentioned, the moving-bed hydrotreating reactor12may be the slurry-bed hydrotreating reactor. As described herein, “slurry-bed reactors” or “slurry reactors” are another type of fluidized bed reactor that suspends solid catalyst in a liquid reactant. Catalysts in the slurry-bed reactor may remain suspended and held in a fluidized state through the upward lift of the liquid reactant. After the liquid reactant is treated, the catalyst and liquid reactant may exit the slurry-bed hydrotreating reactor together, where they may be later separated. In one non-limiting example, the hydrocarbon oil stream26may suspend the hydrotreating catalyst within the slurry-bed hydrotreating reactor while the hydrocarbon oil stream26is hydrotreated. In embodiments including the slurry-bed hydrotreating reactor, the hydrogen stream24, the hydrocarbon oil stream26, the spent hydrotreating catalyst34, or combinations thereof may exit at the top of the moving-bed hydrotreating reactor12. It is contemplated that the hydrotreating catalyst used for the slurry-bed hydrotreating reactor will have a smaller average particle size than the hydrotreating catalysts used for the ebullated-bed hydrotreating reactor. It is further contemplated that the smaller average particle size is preferred because smaller catalyst particles may more easily pass through the various pumps and lines the catalyst particles encounter without plugging. Additionally, smaller catalyst particles may improve the catalyst efficiency and reaction performance of hydrotreating reactors due to a greater effective surface area for the particles. It is contemplated that the greater efficiency and reaction performance of the smaller particles may compensate for a shorter catalyst contact time in the slurry-bed hydrotreating reactor as compared to either the fixed-bed or ebullated-bed hydrotreating reactors. In embodiments, the hydrotreating catalyst may have an average particle size of from 0.01 μm to 10 μm. The hydrotreating catalyst may have an average particle size of from 0.01 μm to 10 μm, from 0.01 μm to 8 μm, 0.01 μm to 6 μm, 0.01 μm to 4 μm, 0.01 μm to 2 μm, 0.01 μm to 1 μm, 0.01 μm to 0.1 μm, 0.1 μm to 10 μm, from 0.1 μm to 8 μm, 0.1 μm to 6 μm, 0.1 μm to 4 μm, 0.1 μm to 2 μm, 0.1 μm to 1 μm, 1 μm to 10 μm, from 1 μm to 8 μm, 1 μm to 6 μm, 1 μm to 4 μm, 1 μm to 2 μm, 2 μm to 10 μm, from 2 μm to 8 μm, 2 μm to 6 μm, 2 μm to 4 μm, 4 μm to 10 μm, from 4 μm to 8 μm, 4 μm to 6 μm, 6 μm to 10 μm, from 6 μm to 8 μm, or from 8 μm to 10 μm. Referring again toFIGS.1and2, and in embodiments, fixed-bed reactors are not preferred in the integrated systems10and60because hydrotreating catalyst cannot be recycled continuously from the fixed-bed to maintain a constant catalyst activity within the moving-bed hydrotreating reactor12. To replace hydrotreating catalyst within the fixed-bed reactor, the fixed-bed reactor must be shut down. Consequently, as catalyst activity decreases, operating temperatures of the fixed-bed reactor must increase to maintain a desired conversation rate of >540° C. boiling point hydrocarbon fractions to <180° C. boiling point hydrocarbon fractions. The desired conversation rate may also include producing the hydrocarbon product stream28with less than 10 ppm nitrogen content. Catalyst deactivation rates for a fixed-bed reactor may necessitate increasing the operating temperature of the fixed-bed reactor from 0.5° C. to 1° C. per day to maintain the desired conversion rates. As operating temperatures in the fixed-bed reactor increase, particularly as operating temperatures approach and exceed 400° C., it is contemplated that more of the reactions within the moving-bed hydrotreating reactor12move to thermal cracking. As a result, more methane and coking on the hydrotreating catalysts are produced, increasing the hydrotreating catalyst deactivation rate. It is contemplated that the increased generation of methane in the fixed-bed reactor may negatively impacts yields that could be generated from steam cracking further downstream, as methane is inert in steam cracking. Further, it is contemplated that approximately 5 wt. % less of >540° C. boiling point hydrocarbon fractions, as compared to the embodiments described herein, may be converted to <180° C. boiling point hydrocarbon fractions in the fixed-bed reactor due to the previously discussed reasons. As operating temperatures in the fixed-bed reactor increase further, particularly as operating temperatures approach and exceed 440° C., it is contemplated that the fixed-bed reactor will need to be shut down to replace the hydrotreating catalysts. Continued operation of the fixed-bed reactor at these temperatures may result in undesirable loss of hydrotreating products to thermal cracking. In embodiments, using ebullated-bed hydrotreating reactors or slurry-bed hydrotreating reactors instead of fixed-bed reactors may maintain the desired conversion rates for 3 to 5 years before the reactors needs to be stopped to replace all of the used hydrotreating catalyst with new hydrotreating catalyst. In comparison, using fixed-bed reactors may maintain the desired conversion rates for 1 to 12 months before the fixed-bed system needs to be stopped to replace all of the used hydrotreating catalyst with the new hydrotreating catalyst. In embodiments, using ebullated-bed hydrotreating reactors or slurry-bed hydrotreating reactors instead of fixed-bed reactors may also allow operating temperatures to be maintained below 440° C. or below 400° C., which in turn may reduce the amount of reactions within the moving-bed hydrotreater12that move to thermal cracking. Still referring toFIGS.1and2, and in embodiments, the hydrocarbon oil stream26may include whole crude oil, topped crude oil, or a combination thereof. Whole crude oil may include crude oil as previously described. As described herein, “topped crude oil” is understood to mean a fraction of crude oil with boiling points less than 160° C. While the present description and examples may specify whole crude oil as the hydrocarbon oil stream26, it should be understood that the systems10and60, described with respect to the embodiments ofFIGS.1and2, may be applicable for the conversion of a wide variety of crude oils, which may be present in the hydrocarbon oil stream26. The hydrocarbon oil stream26may include one or more non-hydrocarbon constituents, such as one or more heavy metals, sulfur compounds, nitrogen compounds, inorganic components, or other non-hydrocarbon compounds. In embodiments, the hydrocarbon oil stream26may be a light crude oil, which includes crude oil having an American Petroleum Institute (API) gravity of greater than 35°, 36°, 37°, or 38°. In these embodiments, the light crude oil may also be categorized as a sour light crude oil, which includes crude oil having a sulfur content of less than 1.5 weight percent (wt. %), based on the total weight of the crude oil, such as less than or equal to 1.4 wt. %, 1.3 wt. %, 1.2 wt. %, 1.1 wt. %, or 1.0 wt. %. By way of non-limiting example, the hydrocarbon oil stream26may be Arab Light crude oil, which has an API gravity of approximately 330 and a sulfur content of approximately 1.77 wt. %. By way of another non-limiting example, the hydrocarbon oil stream26may be Arab Extra Light crude oil, which has an API gravity of approximately 390 and a sulfur content of approximately 1.1 wt. %. In embodiments, the hydrocarbon oil stream26may be a combination of crude oils, such as, for example, a combination of Arab Light crude oil and Arab Extra Light crude oil. It should be understood that, as used herein, the “hydrocarbon oil stream” may refer to crude oil, which has not been previously treated, separated, or otherwise refined. In embodiments, the hydrocarbon oil stream26may have a density lower than 0.89 g/mL. In embodiments, the hydrocarbon oil stream26may have a density of from 0.75 g/mL to 0.92 g/mL, from 0.75 g/mL to 0.89 g/mL, from 0.75 g/mL to 0.87 g/mL, from 0.75 g/mL to 0.84 g/mL, from 0.84 g/mL to 0.92 g/mL, from 0.84 g/mL to 0.89 g/mL, from 0.84 g/mL to 0.87 g/mL, from 0.87 g/mL to 0.92 g/mL, from 0.87 g/mL to 0.89 g/mL, or from 0.89 g/mL to 0.92 g/mL In embodiments, the hydrocarbon oil stream26may have 9.7 wt. %>540° C. boiling point hydrocarbon fractions. The hydrocarbon oil stream26may have 14.6 wt. %>540° C. boiling point hydrocarbon fractions. In embodiments, the hydrocarbon oil stream26may have from 1 wt. % to 20 wt. %, from 1 wt. % to 16 wt. %, from 1 wt. % to 14 wt. %, from 1 wt. % to 10 wt. % from 1 wt. % to 8 wt. %, from 1 wt. % to 4 wt. %, from 4 wt. % to 20 wt. %, from 4 wt. % to 16 wt. %, from 4 wt. % to 14 wt. %, from 4 wt. % to 10 wt. %, from 4 wt. % to 8 wt. %, from 8 wt. % to 20 wt. %, from 8 wt. % to 16 wt. %, from 8 wt. % to 14 wt. %, from 8 wt. % to 10 wt. %, from 10 wt. % to 20 wt. %, from 10 wt. % to 16 wt. %, from 10 wt. % to 14 wt. %, from 14 wt. % to 20 wt. %, from 14 wt. % to 16 wt. %, or from 16 wt. % to 20 wt. %>540° C. boiling point hydrocarbon fractions. In embodiments, the hydrocarbon oil stream26may have a nitrogen content of 844 ppm nitrogen. The hydrocarbon oil stream26may have a nitrogen content of 404 ppm nitrogen. In embodiments, the hydrocarbon oil stream26may have a nitrogen content of from 1 to 900 ppm, from 1 to 700 ppm, from 1 to 500 ppm, from 1 to 300 ppm, from 1 to 100 ppm, from 1 to 20 ppm, from 20 to 900 ppm, from 20 to 700 ppm, from 20 to 500 ppm, from 20 to 300 ppm, from 20 to 100 ppm, from 100 to 900 ppm, from 100 to 700 ppm, from 100 to 500 ppm, from 100 to 300 ppm, from 300 to 900 ppm, from 300 to 700 ppm, from 300 to 500 ppm, from 500 to 900 ppm, from 500 to 700 ppm, or from 700 to 900 ppm nitrogen. Still referring toFIGS.1and2, and in embodiments, the hydrocarbon product stream28may be mixed with a second hydrogen stream30to form a resulting mixture32. The hydrocracking reactor may crack the hydrocarbon product stream28, the second hydrogen stream30, or the resulting mixture32with a hydrocracking catalyst to form a hydrocracked hydrocarbon product stream48. In embodiments, the hydrocracking reactor14may also form a spent hydrocracking catalyst. As shown inFIG.1, the hydrocracking reactor14may be fluidly connected to the moving-bed hydrotreating reactor12. The hydrocracking catalyst may be pre-loaded into the hydrocracking reactor14. In embodiments, the hydrocracking catalyst may include an active metal and a support. The active metal may include NiMo, NiW, or combinations thereof. The support may include nano-sized zeolite, hierarchical zeolite, or combinations thereof. In embodiments, the hydrocracking reactor14may have a temperature of from 350° C. to 440° C. The hydrocracking reactor14may have a temperature of from 350° C. to 440° C., from 350° C. to 420° C., from 350° C. to 390° C., from 350° C. to 370° C., from 370° C. to 440° C., from 370° C. to 420° C., from 370° C. to 390° C., from 390° C. to 440° C., from 390° C. to 420° C., or from 420° C. to 440° C. In embodiments, the hydrocracking reactor14may have a pressure of from 13 MPa to 16 MPa. The hydrocracking reactor14may have a pressure of from 13 MPa to 16 MPa, from 13 MPa to 15.5 MPa, from 13 MPa to 15 MPa, from 13 MPa to 14.5 MPa, from 13 MPa to 14 MPa, from 13 MPa to 13.5 MPa, from 13.5 MPa to 16 MPa, from 13.5 MPa to 15.5 MPa, from 13.5 MPa to 15 MPa, from 13.5 MPa to 14.5 MPa, from 13.5 MPa to 14 MPa, from 14 MPa to 16 MPa, from 14 MPa to 15.5 MPa, from 14 MPa to 15 MPa, from 14 MPa to 14.5 MPa, from 14.5 MPa to 16 MPa, from 14.5 MPa to 15.5 MPa, from 14.5 MPa to 15 MPa, from 15 MPa to 16 MPa, from 15 MPa to 15.5 MPa, or from 15.5 MPa to 16 MPa. In embodiments, the hydrocracking reactor14may have a liquid hourly space velocity of from 0.5 h−1to 1.5 h−1. The hydrocracking reactor14may have a liquid hourly space velocity of from 0.5 h−1to 1.5 h−1, from 0.5 h−1to 1.3 h−1, from 0.5 h−1to 1.1 h−1, from 0.5 h−1to 0.9 h−1, from 0.5 h−1to 0.7 h−1, from 0.7 h−1to 1.5 h−1, from 0.7 h−1to 1.3 h−1, from 0.7 h−1to 1.1 h−1, from 0.7 h−1to 0.9 h−1, from 0.9 h−1to 1.5 h−1, from 0.9 h−1to 1.3 h−1, from 0.9 h−1to 1.1 h−1, from 1.1 h−1to 1.5 h−1, from 1.1 h−1to 1.3 h−1, or from 1.3 h−1to 1.5 h−1. In embodiments, the hydrocracking reactor14may have a ratio of the second hydrogen stream30to the hydrocarbon product stream28of from 1000 L/L to 2000 L/L. The hydrocracking reactor may have a ratio of the second hydrogen stream30to the hydrocarbon product stream28of from 1000 L/L to 1800 L/L, from 1000 L/L to 1600 L/L, from 1000 L/L to 1400 L/L, from 1000 L/L to 1200 L/L, from 1200 L/L to 1800 L/L, from 1200 L/L to 1600 L/L, from 1200 L/L to 1400 L/L, from 1400 L/L to 1800 L/L, from 1400 L/L to 1600 L/L, or from 1600 L/L to 1800 L/L. Referring again toFIG.2, and in embodiments, the integrated system60may additionally include a catalyst separator62. The catalyst separator62may be an oil-catalyst separator or a combination gas-oil-catalyst separator. The catalyst separator62may be fluidly connected to the moving-bed hydrotreating reactor12, the stripper20, and the hydrocracking reactor14. The catalyst separator62may separate the hydrocarbon product stream28from the spent hydrotreating catalyst34. Still referring toFIGS.1and2, and in embodiments, the integrated systems10and60may include a separator18. The separator18may be a gas-liquid separation unit. The separator18may be fluidly connected to the hydrocracking reactor14. The separator18may separate the hydrocracked hydrocarbon product stream48into a light fraction stream50and a heavy hydrocarbon fraction stream52. The light fraction stream50may include C1-C4hydrocarbons, hydrogen sulfide, hydrogen, ammonia, or combinations thereof. The heavy hydrocarbon fraction stream52may include C5+hydrocarbons. In embodiments, the integrated systems10and60may include a scrubbing unit fluidly connected to the hydrocracking reactor14. The scrubbing unit may separate the C1-C4hydrocarbons and hydrogen from the hydrogen sulfide and ammonia. The integrated systems10and60may also include a pressure swing adsorption (PSA) unit fluidly connected to the scrubbing unit. The PSA unit may separate the hydrogen from the C1-C4hydrocarbons to form recycled hydrogen. In embodiments, the PSA unit may also send the recycled hydrogen to be recycled in the integrated systems10and60as the hydrogen stream24or the second hydrogen stream30. (not shown) The integrated systems10and60may also include a methane cracking unit fluidly connected to the scrubbing unit. The methane cracking unit may crack the C1-C4hydrocarbons and separate methane from C2-C4hydrocarbons. The integrated systems10and60may also include a steam cracking unit fluidly connected to the separator18and scrubbing unit. The steam cracking unit may steam crack the C2-C4hydrocarbons and the C5+hydrocarbons to form steam cracked C2-C5+hydrocarbons. The integrated systems10and60may also include an aromatization process unit fluidly connected to the steam cracking unit. The aromatization process unit may separate aromatic hydrocarbons from the steam cracked C2-C5+hydrocarbons. (not shown) Still referring toFIGS.1and2, and as stated previously, the integrated systems10and60include a catalyst reclamation unit16. The catalyst reclamation unit16includes a stripper20and a catalyst regenerator22. The stripper20strips the spent hydrotreating catalyst34to form a stripped spent catalyst38. In embodiments, the stripper20may be a steam stripper. The steam stripper may strip the spent hydrotreating catalyst34with steam36. The steam may remove adsorbed oil from the spent hydrotreating catalyst34. In embodiments, and as shown inFIG.1, the stripper20may be fluidly connected to the hydrocracking reactor14. As shown inFIG.2, the stripper20may be fluidly connected to the catalyst separator62. Still referring toFIGS.1and2, the catalyst regenerator22regenerates the stripped hydrotreating catalyst38to form a regenerated hydrotreating catalyst40. The catalyst regenerator22is fluidly connected to the stripper20. The catalyst regenerator22may regenerate the stripped hydrotreating catalyst38by burning the deposited coke on the stripped hydrotreating catalyst38with a high temperature treatment, for example, heated air. The catalyst regenerator22and the heated air may have a temperature of from 500° C. to 700° C. The catalyst regenerator22and the heated air may have a temperature of from 500° C. to 700° C., from 500° C. to 650° C., from 500° C. to 600° C., from 500° C. to 550° C., from 550° C. to 700° C., from 550° C. to 650° C., from 550° C. to 600° C., from 600° C. to 700° C., from 600° C. to 650° C., or from 650° C. to 700° C. In embodiments, the regenerated hydrotreating catalyst40may be mixed with fresh hydrotreating catalyst44to produce a catalyst mixture46. In embodiments, the catalyst mixture46may have a ratio of from 20:1 to 1:20 regenerated hydrotreating catalyst40to fresh hydrotreating catalyst44. The catalyst mixture46may have a ratio of from 20:1, from 16:1, from 12:1, from 8:1, from 4:1, from 1:1, from 1:4, from 1:8, from 1:12, from 1:16, or from 1:20 regenerated hydrotreating catalyst40to fresh hydrotreating catalyst44. Referring again toFIG.1, and in embodiments, the catalyst regenerator22may be fluidly connected to the moving-bed hydrotreating reactor12. The regenerated hydrotreating catalyst40or catalyst mixture46may be recycled into the moving-bed hydrotreating reactor12. The regenerated hydrotreating catalyst40or catalyst mixture46may then be reused as the hydrotreating catalyst. By way of non-limiting example, the regenerated hydrotreating catalyst40or catalyst mixture46may be recycled into the moving-bed hydrotreating reactor12by adding it to an ebullated-bed of the ebullated-bed hydrotreating reactor or adding it to a slurry of the slurry-bed hydrotreating reactor. Referring again toFIG.2, and in embodiments, the catalyst reclamation unit16may additionally include a presulfiding unit64. The presulfiding unit64may be fluidly connected to the catalyst regenerator22and the moving-bed hydrotreating reactor12. The presulfiding unit64may presulfide the regenerated hydrotreating catalyst40or the catalyst mixture46to form a presulfided hydrotreating catalyst66. The presulfided hydrotreating catalyst66may then be recycled into the moving-bed hydrotreating reactor12to be reused and recycled as the hydrotreating catalyst. Referring again toFIGS.1and2, and in embodiments, at least part of the spent hydrotreating catalyst34, the stripped spent catalyst38, the regenerated hydrotreating catalyst40, the presulfided hydrotreating catalyst66, or combinations thereof may be removed from the integrated systems10and60as waste catalyst42for proper disposal. Waste catalyst42may be removed when the waste catalyst42is no longer effective as a hydrotreating catalyst even after being stripped, regenerated, presulfided, or combinations thereof. Still referring toFIGS.1and2, embodiments of the present disclosure also include integrated hydrotreating and hydrocracking processes. The processes may include any of the integrated systems10and60previously described. The process includes contacting the hydrocarbon oil stream26with the hydrogen stream24and the hydrotreating catalyst in the moving-bed hydrotreating reactor12, thereby producing the hydrocarbon product stream28and the spent hydrotreating catalyst34. The process further includes contacting the hydrocarbon product stream28with the second hydrogen stream30and the hydrocracking catalyst in the hydrocracking reactor14, thereby producing the hydrocracked hydrocarbon product stream48. In embodiments, and as previously mentioned, the hydrocracking reactor14may also produce the spent hydrocracking catalyst. The method further includes processing the spent hydrotreating catalyst34to produce regenerated hydrotreating catalyst40. The method further includes recycling the regenerated hydrotreating catalyst40to the moving-bed hydrotreating reactor12. In embodiments, the method may further include separating the hydrocarbon product stream28from the spent hydrotreating catalyst34. Processing the spent hydrotreating catalyst34to produce regenerated hydrotreating catalyst40may include stripping the spent hydrotreating catalyst34and regenerating the spent hydrotreating catalyst34. Stripping may occur in the stripper20with steam36, as previously described. Regenerating may occur in the catalyst regenerator22, as previously described. In embodiments, stripping and regenerating the spent hydrotreating catalyst34may at least partially remove coke deposited on the spent hydrotreating catalyst34, thereby producing the regenerated hydrotreating catalyst40. Still referring toFIGS.1and2, and in embodiments, the method may further include mixing the regenerated hydrotreating catalyst40with fresh hydrotreating catalyst44to produce the catalyst mixture46. As illustrated inFIG.2, the method may further include presulfiding the regenerated hydrotreating catalyst40or the catalyst mixture46to produce the presulfided hydrotreating catalyst66. Presulfiding may occur in the presulfiding unit64, as previously described. The method may then further include recycling the presulfided hydrotreating catalyst66to the moving-bed hydrotreating reactor12. In embodiments, the method may further include sending the hydrocracked hydrocarbon product stream48to the separator18. The method may then further include allowing the separator18to separate the hydrocracked hydrocarbon product stream48into the light fraction stream50and the heavy hydrocarbon fraction stream52. The method may then further include sending the light fraction stream50, the heavy hydrocarbon fraction stream52, or both to the scrubbing unit, the PSA unit, the methane cracking unit, the steam cracking unit, the aromatization process unit, or combinations thereof, as previously described in systems10and60. (not shown.) EXAMPLES The various embodiments of methods and systems for the conversion of a hydrocarbon oil will be further clarified by the following examples. The examples are illustrative in nature, and should not be understood to limit the subject matter of the present disclosure. Example 1 Arab light crude oil (AL) and Arab extra light crude oil (AXL) were processed in a pilot plant using the systems depicted inFIGS.1and2. The compositions of the Arab light crude oil and the Arab extra light crude oil used in the Examples are shown below in Table 1. The hydrotreating catalyst used in the examples was MoNi on Al2O3, particularly, 15 wt. % MoO3and 5 wt. % NiO by weight of the total hydrotreating catalyst. The hydrocracking catalyst used was Mo—Ni on mesoporous zeolite Y, particularly, 15 wt. % MoO3and 5 wt. % NiO by weight of the total hydrocracking catalyst. The mesoporous zeolite Y was commercialized CBV-760 obtained from Zeolyst™. TABLE 1Arab Light and Arab Extra Light CrudeOil CompositionsFeedAXLALDensity at 15.6° C.0.84120.8658Sulfur content, wt %0.6391.803Nitrogen content, ppm404844.1Percentage <540° C. boiling90.385.4point hydrocarbon fractionsPercentage >540° C. boiling9.714.6point hydrocarbon fractions Table 2 below illustrates the pilot plant testing results using Arab extra light crude oil as the feedstock. Performance was measured over 3-5 days to determine the initial deactivation rate of the catalysts and then extrapolated to determine when the hydrotreating catalyst would need to be replaced. Inventive Examples 1 and 2 were performed using a moving-bed hydrotreating reactor. Comparative Examples 1 and 2 were performed using a fixed-bed reactor. Inventive Example 1 and Comparative Example 1 did not include a hydrocracking reactor or hydrocracking catalysts. Inventive Example 2 and Comparative Example 2 did include a hydrocracking reactor and a hydrocracking catalyst. TABLE 2Inventive Example Testing with Arab Extra Light CrudeInventiveInventiveCompar-Compar-ExampleExampleativeativeExample #12Example 1Example 2Resulting Density0.79890.75620.81750.8077(g/mL)Sulfur Content282055100(ppm)Nitrogen Content1<1.01622(ppm)Percentage <180° C.40904060boiling pointhydrocarbonfractionsPercentage >540° C.0.702.83.1boiling pointhydrocarbonfractions Compared to Comparative Examples 1 and 2, which included a fixed-bed reactor, the Inventive Examples resulted in a lower nitrogen content and greater conversation of >540° C. boiling point hydrocarbon fractions to <180° C. boiling point hydrocarbon fractions. After adding in the hydrocracking reactor in Inventive Example 2, the conversation rate became even more significant with the >540° C. boiling point hydrocarbon fractions completely converted. Table 3 below illustrates the pilot plant testing results using Arab light crude oil as the feedstock. Performance was measured over 3-5 days to determine the initial deactivation rate of the catalysts and then extrapolated to determine when the hydrotreating catalyst would need to be replaced. Inventive Examples 3 and 4 were performed using the moving-bed hydrotreating reactor. Comparative Examples 3 and 4 were performed using the fixed-bed reactor. Inventive Example 3 and Comparative Example 3 did not include a hydrocracking reactor or hydrocracking catalysts. Inventive Example 4 and Comparative Example 4 did include a hydrocracking reactor and a hydrocracking catalyst. TABLE 3Inventive Example Results with Arab Light CrudeInventiveInventiveCompar-Compar-ExampleExampleativeativeExample #34Example 3Example 4Resulting Density0.81610.76660.81480.8192(g/mL)Sulfur Content26.2154(ppm)Nitrogen Content<1.0<1.0<1.0<1.0(ppm)Percentage <180° C.30602020boiling pointhydrocarbonfractionsPercentage >540° C.2.504.92.1boiling pointhydrocarbonfractions Similar to Table 2, the Inventive Examples resulted in a lower nitrogen content and greater conversation of >540° C. boiling point hydrocarbon fractions to <180° C. boiling point hydrocarbon fractions than the Comparative Examples. After adding in the hydrocracking reactor in Inventive Example 2, the conversation rate became even more significant, with the >540° C. boiling point hydrocarbon fractions completely converted. Based on the observed catalyst deactivation rate when using Arab extra light crude oil, the run length for Comparative Examples 1 and 2 is approximately 6 to 12 months. For Arab light crude oil, the run length for Comparative Examples 3 and 4 is approximately less than 6 months. For the Inventive Examples 1-4, the run length is approximately 3 to 5 years. As discussed previously, run time is the amount of time the system can operate before all of the hydrotreating catalysts need to be replaced with new hydrotreating catalyst. On average, the Inventive Examples resulted in naphtha yields increasing by 30 wt. % over the Comparative Examples. Additionally, the Inventive Examples resulted in diesel and vacuum gas oil yields of only 10 wt. % of the total hydrocarbon product. It is contemplated that the higher naphtha yields will increase olefin and aromatics yields in steam cracking. It is further contemplated that the higher naphtha yields will increase BTX yields as reforming feedstocks. The present application discloses several technical aspects. One aspect is an integrated hydrotreating and hydrocracking process comprising contacting a hydrocarbon oil stream with a hydrogen stream and a hydrotreating catalyst in a moving-bed hydrotreating reactor, thereby producing a hydrocarbon product stream and a spent hydrotreating catalyst; contacting the hydrocarbon product stream with a second hydrogen stream and a hydrocracking catalyst in a hydrocracking reactor, thereby producing a hydrocracked hydrocarbon product stream; processing the spent hydrotreating catalyst to produce regenerated hydrotreating catalyst; and recycling the regenerated hydrotreating catalyst to the moving-bed hydrotreating reactor. A second aspect includes any previous aspect, wherein the moving-bed hydrotreating reactor is an ebullated-bed hydrotreating reactor. A third aspect includes any previous aspect, wherein the hydrotreating catalyst has an average particle size from 500 μm to 1000 μm. A fourth aspect includes the first aspect, wherein the process further comprises separating the hydrocarbon product stream from the spent hydrotreating catalyst, and wherein the moving-bed hydrotreating reactor is a slurry-bed hydrotreating reactor. A fifth aspect includes the fourth aspect, wherein the hydrotreating catalyst has an average particle size from 0.01 μm to 10 μm. A sixth aspect includes any previous aspect, wherein processing the spent hydrotreating catalyst comprises stripping the spent hydrotreating catalyst; and regenerating the spent hydrotreating catalyst by thermal treatment, and wherein stripping and regenerating the spent hydrotreating catalyst at least partially removes coke deposited on the spent hydrotreating catalyst, thereby producing the regenerated hydrotreating catalyst. A seventh aspect includes the sixth aspect, wherein the process further comprises mixing the regenerated hydrotreating catalyst with fresh hydrotreating catalyst to produce a catalyst mixture; presulfiding the catalyst mixture to produce a presulfided hydrotreating catalyst; and recycling the presulfided hydrotreating catalyst to the moving-bed hydrotreating reactor. An eighth aspect includes any previous aspect, wherein the hydrocarbon oil stream comprises whole crude oil, topped crude oil, or both. A ninth aspect includes any previous aspect, wherein the hydrotreating catalyst comprises MoNi on Al2O3, MoCO on Al2O3, MoS2, maghemite, Fe3O4, nickel, NiO, TiO2, ZrO2, CeO2, or combinations thereof. A tenth aspect includes any previous aspect, wherein the hydrocracking catalyst comprises an active metal and a support, the active metal comprising NiMo, NiW, or a combination thereof, and the support comprising nano-sized zeolite, hierarchical zeolite, or a combination thereof. An eleventh aspect includes any previous aspect, wherein the moving-bed hydrotreating reactor has a temperature from 370° C. to 500° C., a pressure from 12 MPa to 16 MPa, or both. A twelfth aspect includes any previous aspect, wherein the moving-bed hydrotreating reactor has a liquid hourly space velocity from 0.2 h−1to 0.7 h−1and a ratio of hydrogen stream to hydrocarbon oil stream from 800 L/L to 1200 L/L. A thirteenth aspect includes any previous aspect, wherein the hydrocracking reactor has a temperature from 350° C. to 440° C., a pressure from 13 MPa to 16 MPa, or both. A fourteenth aspect includes any previous aspect, wherein the hydrocracking reactor has a liquid hourly space velocity from 0.5 h−1to 1.5 h−1and a ratio of hydrogen stream to the hydrocarbon product stream from 1000 L/L to 2000 L/L. A fifteenth aspect includes a system for hydrotreating and hydrocracking a hydrocarbon oil stream, the system comprising a moving-bed hydrotreating reactor configured to hydrotreat the hydrocarbon oil stream and a hydrogen stream with a hydrotreating catalyst to form a hydrocarbon product stream and spent hydrotreating catalyst; a hydrocracking reactor fluidly connected to the moving-bed hydrotreating reactor and configured to crack the hydrocarbon product stream and a second hydrogen stream to form a hydrocracked hydrocarbon product stream; a stripper fluidly connected to the moving-bed hydrotreating reactor and configured to strip the spent hydrotreating catalyst to form a stripped hydrotreating catalyst; and a catalyst regenerator fluidly connected to the stripper and the moving-bed hydrotreating reactor and configured to regenerate the stripped hydrotreating catalyst to form a regenerated hydrotreating catalyst. A sixteenth aspect includes the fifteenth aspect, wherein the moving-bed hydrotreating reactor is an ebullated-bed hydrotreating reactor; and the catalyst regenerator is configured to send the regenerated hydrotreating catalyst to the moving-bed hydrotreating reactor. A seventeenth aspect includes the fifteenth aspect, wherein the system further comprises a catalyst separator fluidly connected to the moving-bed hydrotreating reactor, the hydrocracking reactor, and the stripper, wherein the moving-bed hydrotreating reactor is a slurry-bed hydrotreating reactor; and the catalyst separator is configured to separate the hydrocarbon product stream from the spent hydrotreating catalyst. An eighteenth aspect includes the fifteenth through seventeenth aspects, wherein the system further comprises a presulfiding unit fluidly connected to the catalyst regenerator and the moving-bed hydrotreating reactor, wherein the presulfiding unit is configured to presulfide the regenerated hydrotreating catalyst to form a presulfided regenerated hydrotreating catalyst; and the presulfiding unit is configured to send the presulfided regenerated hydrotreating catalyst to the moving-bed hydrotreating reactor. A nineteenth aspect includes the fifteenth through eighteenth aspects, wherein the hydrocarbon oil stream comprises whole crude oil, topped crude oil, or both. A twentieth aspect includes the fifteenth aspect through nineteenth aspects, wherein the system further comprises a separator fluidly connected to the hydrocracking reactor and configured to separate the hydrocracked hydrocarbon product stream into a light fraction stream comprising C1-C4hydrocarbons, hydrogen sulfide, hydrogen, ammonia, or combinations thereof and a heavy hydrocarbon fraction stream comprising C5+hydrocarbons; a scrubbing unit fluidly connected to the separator and configured to separate the C1-C4hydrocarbons and hydrogen from the hydrogen sulfide and ammonia; a pressure swing adsorption (PSA) unit fluidly connected to the scrubbing unit and configured to separate the hydrogen from the C1-C4hydrocarbons; a methane cracking unit fluidly connected to the scrubbing unit and configured to crack the C1-C4hydrocarbons and separate methane from C2-C4hydrocarbons; a steam cracking unit fluidly connected to the separator and methane cracking unit and configured to steam crack the C2-C4hydrocarbons and the C5+hydrocarbons; and an aromatization process unit fluidly connected to the steam cracking unit and configured to separate aromatic hydrocarbons from the C2-C5+hydrocarbons. It is noted that recitations in the present disclosure of a component of the present disclosure being “operable” or “sufficient” in a particular way, to embody a particular property, or to function in a particular manner, are structural recitations, as opposed to recitations of intended use. More specifically, the references in the present disclosure to the manner in which a component is “operable” or “sufficient” denotes an existing physical condition of the component and, as such, is to be taken as a definite recitation of the structural characteristics of the component. It is also noted that terms like “preferably,” “commonly,” and “typically,” when utilized herein, are not utilized to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to identify particular aspects of an embodiment of the present disclosure or to emphasize alternative or additional features that may or may not be utilized in a particular embodiment of the present disclosure. It is noted that one or more of the following claims utilize the term “wherein” as a transitional phrase. For the purposes of defining the present invention, it is noted that this term is introduced in the claims as an open-ended transitional phrase that is used to introduce a recitation of a series of characteristics of the structure and should be interpreted in like manner as the more commonly used open-ended preamble term “comprising.” Having described the subject matter of the present disclosure in detail and by reference to specific embodiments, it is noted that the various details disclosed in the present disclosure should not be taken to imply that these details relate to elements that are essential components of the various embodiments described in the present disclosure. Further, it will be apparent that modifications and variations are possible without departing from the scope of the present disclosure, including, but not limited to, embodiments defined in the appended claims. The singular forms “a”, “an” and “the” include plural referents, unless the context clearly dictates otherwise. Throughout this disclosure ranges are provided. It is envisioned that each discrete value encompassed by the ranges are also included. Additionally, the ranges which may be formed by each discrete value encompassed by the explicitly disclosed ranges are equally envisioned. As used herein and in the appended claims, the words “comprise,” “has,” and “include” and all grammatical variations thereof are each intended to have an open, non-limiting meaning that does not exclude additional elements or steps. As used herein, terms such as “first” and “second” are arbitrarily assigned and are merely intended to differentiate between two or more instances or components. It is to be understood that the words “first” and “second” serve no other purpose and are not part of the name or description of the component, nor do they necessarily define a relative location, position, or order of the component. Furthermore, it is to be understood that the mere use of the term “first” and “second” does not require that there be any “third” component, although that possibility is contemplated under the scope of the present disclosure. | 58,184 |
11859141 | DETAILED DESCRIPTION OF THE INVENTION The inventors have determined that one drawback of the process100(and of similar processes that utilize solid additives such as catalysts, etc. to convert petroleum residues) is the loss of the value of the unconverted feedstock. While the process100is successful in converting a large portion (e.g., >95%) of the feedstock to higher value distillate streams, there is some value in the unconverted feedstock. For example, unconverted petroleum residue such as vacuum distillation residue can be processed and sold as asphalt. However, in the process100and similar processes that employ solid additives, the spent additive is integrated in the viscous unconverted feedstock (in the stream166), which prevents the further processing of the unconverted feedstock to generate a saleable product. As a result, the unconverted feedstock material and the spent additive are solidified and processed as a waste stream. Thus, the process100and similar processes result in not only the loss in value of the unconverted feedstock material but also the added cost of processing the unconverted material as a waste stream. Moreover, any value associated with the spent additive (either in the recovery of the additive itself or materials such as metals in the feedstock that are bound to the additive) is also lost when the stream166is handled as waste. To combat these problems, the inventors have conceived of a desolidification process that can be implemented within process100and other similar processes that involve the introduction of a solid additive into a feed stream. Referring toFIG.2, process100′ incorporates desolidification section200, which is positioned between the separator section130and the vacuum section158to remove the solids from the slurry stream156. The process100′ differs from the process100only in the addition of the desolidification section200and corresponding equipment and streams are labeled with the same designators even where the stream or equipment may differ in some respects as a result of the addition of the desolidification section200. The desolidification section200receives the slurry stream156from the separator section130. The slurry stream156is mixed with a solvent stream202, which enables the solids in the slurry stream156to be extracted as a solids stream204to generate a solids-free solvent and unconverted feedstock vacuum feed stream206. As a result of the addition of the desolidification section200, the vacuum section158bottoms stream208is free of solids. Consequently, the unconverted feedstock material in stream208is further processed in processing section210, such as an asphalt blending section, to generate a saleable product, and the waste processing section168is eliminated. Although the illustrated positioning of the desolidification section200upstream of the vacuum section158allows the vacuum section158to be designed to operate free of solids, in another embodiment, the desolidification section200could be positioned to receive the stream166that exits the bottom of the vacuum section158in the process100. FIG.3is a process flow diagram for one particular implementation of the desolidification section200in the process100′. The depicted process flow diagram shows a single process train in the desolidification section200. In some implementations, the desolidification section200may comprise one or more additional redundant parallel trains. In the illustrated embodiment, condensed vacuum gas oil is drawn from a chimney tray just below the top section of a vacuum tower304in the vacuum section158and is pumped to approximately 150 psig by vacuum gas oil pump314as vacuum gas oil stream302. A portion of the vacuum gas oil stream302is extracted for use as the solvent stream202that is provided to the desolidification section200. While the described embodiment employs vacuum gas oil as the solvent, other materials (such as diesel, kerosene, light vacuum gas oil, FCC light cycle oil, FCC heavy cycle oil, naptha, toluene, etc.) can also be employed as the solvent. The flow rate of the extracted stream306is regulated by a flow controller308to provide a consistent flow of vacuum gas oil solvent to be mixed with the slurry stream156. A first portion312of the extracted stream306, which has a temperature of approximately 190° C., can be routed through a heat exchanger310in which it is heated against the vacuum tower bottoms stream316, which is primarily unconverted feedstock material having a temperature of approximately 310° C. A second portion318of the extracted stream306is routed around the heat exchanger310and ties back into the first portion312at the exit of the heat exchanger310. The proportion of the extracted stream306that is routed around the heat exchanger310as the second portion318is regulated by the temperature controller320to keep the mixture of stream156and202below the bubble point. When lower boiling point solvents are used, the temperature of stream326must be reduced to maintain the mixture below the bubble point. A slurry mixture that contains spent additive solids and unconverted feedstock material is drawn off of the low pressure separator322in the separator section130as slurry stream156. The rate at which material is drawn off of the bottom of the low pressure separator322is regulated by the level controller324to maintain a constant level in the separator322. The slurry stream156is mixed with the solvent stream202to form the desolidification feed stream326. The streams202and156are mixed at a mixing tee or a mixing nozzle such as an orifice mixer to facilitate the integration of the streams. The warm solvent in the solvent stream202decreases the viscosity and density of the slurry stream156, which enables the spent additive solids to be more easily separated from the unconverted feedstock material in the desolidification section200. The warm solvent also helps dissolve heavy aromatics on the inside and outside of additive particles. The combination of the hot slurry stream156(which is still at an elevated temperature of approximately 400 to 425° C. due to its progression through the preheat section108and the reactor section106) and the solvent stream202(approximately 200° C.) results in a feed stream326temperature of between 250 and 300° C. This temperature range is appropriate for vacuum gas oil solvent, however, in other embodiments that employ lower density or vicosity solvents such as diesel, kerosene, light vacuum gas oil, or light cycle oil, the temperature of the feed stream326must be controlled to a level at which solvent flashing is avoided. Other solvents may be used to improve the solubility of the heavy oil in the solvent. The optimum temperature for any solvent will always be the temperature just below the bubble point of the mixture. A well-controlled operation should be able to maintain the temperature within 10 degrees of the optimum. The ratio of the solvent stream202flow rate to the slurry stream156flow rate will vary based upon the composition of the streams, but, in the illustrated embodiment, the flow rate of the solvent stream202is set by the flow controller308to achieve a target solvent-to-slurry mass flow ratio of approximately 7:4. In another embodiment, the flow rate of the slurry stream156is measured and the flow controller308setpoint is set by a ratio controller to maintain a fixed solvent-to-slurry mass flow ratio. The solvent-to-slurry mass flow ratio could change significantly depending on the solvent chosen. Increasing the ratio (i.e., increasing the solvent flow rate) reduces settling time in the vessel330, which can offset any improvement in separation that might be attained by the increased amount of solvent. Solvent-to-slurry mass flow ratios from 1.5:1 to 5:1 are envisioned. The desolidification feed stream326is routed to the mix drum328, which, in the illustrated embodiment, has a conical bottom and is mixed by an agitator (at minimum) to maintain a consistent mixture in which the spent additive solids are held in suspension. Recirculating pumps may also be required depending on the size of the drum to assist with the mixing. Such pumps would return a portion of stream334from drum330back to the mix drum. Internal baffles and other similar mixing improvement items can also be added to the mix drum328to improve mixing. In another embodiment, the mix drum328may be configured as a rotating disc column in which the solvent stream flows upward counter to the slurry stream156thru numerous stacked mixing chambers in series. The mix drum328is padded with fuel gas under control of the pressure controller330to maintain a mix drum pressure of approximately 30 psig. The mixture in the mix drum328flows either by gravity or a slight pressure differential to the solid separation drum330as suspension stream332. The solid separation drum330also has a conical bottom and is held in a liquid full state. In the solid separation drum330, the spent additive solids fall out of the suspension to the bottom of the drum330, which results in a substantially solid-free mixture of unconverted feedstock and solvent in the top section of the solid separation drum330. The solvent and unconverted feedstock liquid mixture is drawn off of the top section of the drum330as liquid stream334. The rate at which liquids are drawn off of the drum330is regulated by the level controller336to maintain a constant level in the mix drum328. In a preferred embodiment, the size of the mix drum328and the level controller336setpoint are determined to achieve a residence time in the mix drum of at least 10 minutes to allow time for the heavy oil inside the pores of the additive to be diluted and removed. Increased residence times will enable the removal of more materials from the additive. The inventors have observed that increased residence times of greater than 30 minutes result in increased capital costs but do not improve recovery significantly. The solid separation drum330is sized to achieve a residence time that enables the solids to fall out of the suspension to the bottom of the drum330. Larger particles settle in just a few minutes, and the smallest particles will likely not settle in any practical amount of time. There is therefore a tradeoff in capital cost (i.e., the size of the drum330) and the degree of solid settling. The inventors have determined that a residence time of 120 minutes (based on the total inlet volume flow) enables a recovery of a great degree of the solids that can be recovered practically, but again, the size of the separation drum330can be adjusted to attain the desired level of separation. The liquid stream334is routed to the vacuum column feed drum338, which also has a conical bottom. The vacuum column feed drum338is padded with fuel gas under control of the pressure controller356to maintain a pressure of approximately 15 psig. The unconverted feedstock and solvent mixture is pumped off by the feed pump340as stream342to the feed heater344at a rate regulated by the level controller346to maintain the level in the drum338. The stream342, which has a temperature of approximately 230 to 275° C., is heated to a temperature high enough to vaporize the solvent, approximately 370° C. in this scenario. The firing rate of the heater344is regulated by the temperature controller348to maintain the temperature of the stream206. The heated feed stream206is routed to the flash zone of the vacuum column304. In the vacuum column304, the lighter solvent goes overhead as a vapor stream and continues through the process100′ while the unconverted feedstock material, which is free of solids, exits the bottom of the vacuum column304and is further processed to generate a saleable product. The spent additive solids are removed from the solid separation drum330through the rotary valve350and routed to the solids-handling section354. The removal rate of the solids is regulated by level controller352to maintain the solid-liquid interface level in the drum330. FIG.4illustrates the solids-handling section354of the desolidification section200. The oil-wetted solids that are removed from the solid separation drum330through the rotary valve350are conveyed along the screw conveyor402into the solid rotary drier404. In the drier404, the solids are dried by a combination of indirect heating to approximately 450° C. and the introduction of stripping steam414. The stripping steam414may be saturated steam having a pressure between 50 and 100 psig, for example. The firing rate in the drier404is regulated by the temperature controller406to maintain the temperature in the drier404. The dried solid particles exit the drier404as dried solid stream408through the three flap solids valve410. The hydrocarbon vapors that are evaporated from the oil-wetted solids exit the drier404as vapor stream412, and they are either recycled into the process100′ or handled as waste. The hot, dried solids, which are at an elevated temperature of approximately 450° C., are routed to a rotary cooler416. In the rotary cooler416, the solids are exposed to a cooling nitrogen circulating stream418. The cooling solidifies any remaining hydrocarbons into a solid that can be handled without sticking. The cooled solids exit the rotary cooler416as cooled solid stream420through the three flap valve422and are routed to a roller crusher424. In the roller crusher424, the solids are again exposed to the cooling nitrogen stream418and are crushed into more uniformly-sized particles. The crushed particles are routed from the roller crusher424to the solid storage drum426as crushed solid stream428. In the storage drum426, the solid particles are once again exposed to the cooling nitrogen stream418to achieve a final temperature of approximately 80° C. The cooling nitrogen stream that exits the equipment (i.e., the rotating cooler416, the roller crusher424, and the storage drum426) is routed through a nitrogen cyclone430to remove any solid particles from the cooling stream. The nitrogen is then cooled in an air cooler432before being recirculated to the equipment by the nitrogen blower434. A nitrogen makeup stream replaces any nitrogen lost in the cooling process. Although the described embodiment of the solids handling section354employs nitrogen as a cooling medium, other types of cooling could also be used. For example, the dried solid particles in the stream408could alternatively be routed through a water bath to accomplish the necessary cooling. The solid particles in the storage drum426can be processed in different manners. In one embodiment, the solid particles may be disposed of as waste. In another embodiment, the solid particles may be further processed to extract any value in the additive itself. For example, if a metal additive is utilized in the process100′, the solids may be further processed to remove any impurities from the metal to recover the value of the metal additive. In yet another embodiment, the solid particles may be further processed to extract the value of compounds absorbed by the additive. For example, the additive may absorb valuable components such as metals from the feedstock stream and these valuable components may be extracted from the solids in the solid storage drum426through further processing. While the described desolidification process200utilizes vacuum gas oil as the solvent stream202, other solvents can also be employed. For example, diesel, kerosene, light vacuum gas oil, light cycle oil or other solvents that decrease the density and viscosity and improve solubility of the heavy oil in the slurry stream156can be used. As noted above, when different solvents are employed, the temperature of the combined solvent and hot slurry stream should be controlled to avoid solvent flashing. In another embodiment, the desolidification process may employ a multiple stage solid separation process. A portion of a desolidification process200′ that employs a multiple stage solid separation process is illustrated inFIG.5. As with the process200illustrated inFIG.3, the process200′ may include one or more additional redundant parallel trains. The desolidification process200′ is the same as the process200with the exception that the mix drum328and solid separation drum330are replaced with a series of mix drums328A/B/C and solid separation drums330A/B/C. As in the process200, the solid-containing slurry mixture156is mixed with a first solvent stream202A to form a feed stream326A. As described above, the solvent202A may be any solvent that decreases the viscosity of the slurry mixture156such that the solids can be separated out of the slurry. The combined feed stream326A is mixed in a first stage mix drum328A and the mixed suspension stream332A exiting the first stage mix drum328A is routed to the first stage solid separation drum330A, which is held in a liquid full state. Each of the mix drums328A/B/C is padded with fuel gas (not shown inFIG.5for purposes of clarity) in the same manner as is the mix drum328in process200to maintain a desired vessel pressure. The pressure setpoint is decreased from the first stage mix drum328A to a second stage mix drum328B to a third stage mix drum328C to provide the driving force for moving the liquid streams through the process200′. Liquid comprising the first solvent and the unconverted feedstock material is drawn from the top of the first stage solid separation drum330A as liquid stream502A at a rate that is regulated by the level controller336A to maintain a constant level in the mix drum328A. However, rather than routing the liquid stream502A to the vacuum feed drum338, the liquid stream502A is mixed with a second solvent202B, which may be the same as or a different solvent than the solvent202A, to form an intermediate stream326B. The stream326B is mixed in a second stage mix drum328B and the process flow continues in the same manner through a second stage solid separation drum330B to facilitate additional solid settling. Liquid is similarly drawn from the top of the second stage solid separation drum330B as liquid stream502B at a rate that is regulated by the level controller336B to maintain a constant level in the second stage mix drum328B. The liquid stream502B is mixed with a third solvent stream202C, which may again be the same as or a different solvent from the solvents202A and202B, to form intermediate stream326C. The process flow continues in the same manner through a third stage mix drum328C and a third stage solid separation drum330C until the liquid effluent in the stream502C is passed to the vacuum column feed drum338. Although process200′ illustrates a three-stage solid separation process, more or fewer stages could be utilized. Solids are drawn from the bottoms of the solid separation drums330A/B/C through rotary valves350A/B/C at a rate that is regulated by the level controllers252A/B/C to maintain the solid-liquid interface levels in the drums330. The extracted solids are routed to the solid handling section354in a similar manner as in the process200. The use of multiple stages enables the same or a higher degree of solid separation with smaller mix drums328and separation drums330than in the single stage arrangement. In one embodiment, a lower boiling point solvent is employed at each stage. For example, in one embodiment, vacuum gas oil is used as a first solvent202A, light cycle oil is used as a second solvent202B, and diesel is used as a third solvent202C. The disclosed desolidification process enables the isolation and extraction of solid additives from an unreacted petroleum residue stream in which the solids are integrated. Although described in the context of the process100, the desolidification process may be used in other similar processes (e.g., other slurry phase processes) that utilize solid additives (such as solid catalysts) in the conversion of petroleum residues. In a hydrocracking process that incorporates a solid additive into a petroleum residue feedstock, the desolidification process enables the recovery of the unreacted petroleum residue for conversion to a saleable product. As used herein, naptha and gasoline include petroleum distillates having a boiling point range of approximately 30° to 200° C.; kerosene includes petroleum distillates having a boiling point range of approximately 150° to 275° C.; diesel, distillate, and FCC light cycle oil include petroleum distillates having a boiling point range of approximately 150° to 375° C.; vacuum gas oil includes petroleum distillates having a boiling point range of approximately 350° to 625° C.; and asphalt or residue include petroleum products having a boiling point range of approximately 475° C. and higher, all according to the ASTM D86 standard. It will be understood that the disclosed embodiments are illustrative and not limiting. For example, the described process equipment, process conditions, instrumentation, and control schemes are provided as an illustration of one or more implementations of the disclosed desolidification process. Numerous modifications and variations could be made to the disclosed embodiments by those skilled in the art without departing from the scope of the invention set forth in the claims. | 21,260 |
11859142 | DETAILED DESCRIPTION Applicants disclose a process and an apparatus meeting the twin objective of producing high quality lubes while maximizing the naphtha production in the hydrocracking unit. Applicants' hydrocracking process maximizes naphtha production along with co-production of distillates and high quality UCO for base oil production through an integrated flow scheme shown in the FIGURE. Applicants' hydrocracking process uses both distillate selective catalysts and naphtha selective catalysts in the same hydrocracking unit shown in the FIGURE. Applicants' hydrocracking process not only maximizes naphtha production but also produces high quality UCO which can be used for base oil production along with distillates. Maximization of naphtha along with production of high quality base oil and distillates from the integrated hydrocracking unit with the same product qualities instead of using two separate units also reduces the CAPEX. The current hydrocracking process includes hydrocracking unit with two reactor stages upstream of a fractionation section involving two separate but integrated product strippers, and two separate but integrated product fractionation columns. The integrated hydrocracking unit is tailored to produce high quality UCO from the first stage as a base oil feedstock while making blended distillate products and high naphtha production from both reaction stages. The process and apparatus have been demonstrated in a pilot plant and is a viable option for a two stage hydrocracking unit to maximize naphtha production while producing high quality UCO for base oil production meeting the desired specifications. The following detailed description is merely exemplary in nature and is not intended to limit the various embodiments or the application and uses thereof. Furthermore, there is no intention to be bound by any theory presented in the preceding background or the following detailed description. The FIGURES have been simplified by the deletion of a large number of apparatuses customarily employed in a process of this nature, such as vessel internals, temperature and pressure controls systems, flow control valves, recycle pumps, etc. which are not specifically required to illustrate the performance of the process. Furthermore, the illustration of the current process in the embodiment of a specific drawing is not intended to limit the process to specific embodiments set out herein. As depicted, process flow lines in the FIGURES can be referred to, interchangeably, as, e.g., lines, pipes, branches, distributors, streams, effluents, feeds, products, portions, catalysts, withdrawals, recycles, suctions, and discharges. A hydrocracking process for maximization of naphtha while producing base oil is addressed with reference to a process and an apparatus100according to an embodiment as shown in the FIGURE. Referring to the FIGURE, the process and apparatus100comprise a hydrocracking unit101and a fractionation section105. A hydrocarbon feed stream in line102and a hydrogen stream in line336are fed to the hydrocracking unit101. The hydrocarbon feed stream in line102is the fresh feed to the hydrocracking unit101. In an exemplary embodiment, the hydrocracking unit101may be a two stage hydrocracking unit101comprising a first stage hydrocracking reactor130and a second stage hydrocracking reactor280. The hydrocracking unit101may also comprise a hydrotreating reactor124. In one aspect, the hydrocarbon feed stream in line102fed to the hydrocracking unit101may comprise a hydrocarbon stream having an initial boiling points (IBP) above about 288° C. (550° F.), such as atmospheric gas oils, vacuum gas oil (VGO) having T5 and T95 between about 315° C. (600° F.) and about 600° C. (1100° F.), deasphalted oil, coker distillates, straight run distillates, pyrolysis-derived oils, high boiling synthetic oils, cycle oils, hydrocracked feeds, catalytic cracker distillates, atmospheric residue having an IBP at or above about 343° C. (650° F.) and vacuum residue having an IBP above about 510° C. (950° F.). The hydrocarbon feed stream in line102may enter the hydrocracking unit101via a feed surge drum110. From the bottoms of the feed surge drum110, the feed flows in line112to the suction of a feed charge pump114to provide a pumped hydrocarbon feed stream in line116. The hydrogen stream in line336may be added to the pumped hydrocarbon feed stream in line116to provide a mixed feed stream in line118. The hydrogen stream in line336may be taken from a compressed hydrogen stream in line332as described hereinafter in detail. In an exemplary embodiment, the hydrogen stream in line336is a first hydrogen stream. In an aspect, the hydrogen stream in line336may join the pumped hydrocarbon feed stream in line116as the first hydrogen stream to provide the mixed feed stream in line118. The mixed feed stream in line118may be heated in a first stage feed heater120. A heated hydrocarbon feed stream in line122may be fed to the hydrotreating reactor124. Hydrotreating is a process wherein hydrocarbons are contacted with hydrogen in the presence of hydrotreating catalysts which are primarily active for the removal of heteroatoms, such as sulfur, nitrogen, oxygen and metals from the hydrocarbon feedstock. In hydrotreating, hydrocarbons with double and triple bonds such as olefins may be saturated. Aromatics may also be saturated. Some hydrotreating processes are specifically designed to saturate aromatics. In an exemplary embodiment, the hydrotreating reactor124may comprise a guard bed of hydrotreating catalyst followed by one or more beds of higher activity hydrotreating catalyst. The guard bed filters particulates and reacts with contaminants in the hydrocarbon feed stream such as metals like nickel, vanadium, silicon and arsenic which are detrimental to the higher activity hydrotreating catalyst. The guard bed may comprise material similar to the hydrotreating catalyst. Suitable hydrotreating catalysts for use in the present process may include any known conventional hydrotreating catalysts. The hydrotreating catalysts may comprise at least one Group VIII metal including iron, cobalt and nickel, or cobalt and/or nickel and at least one Group VI metal including molybdenum and tungsten, on a high surface area support material such as alumina. Other suitable hydrotreating catalysts may include zeolitic catalysts, as well as noble metal catalysts where the noble metal is selected from palladium and platinum. More than one type of hydrotreating catalyst may be used in the same hydrotreating reactor124. In an exemplary embodiment, the Group VIII metal may be present in an amount ranging from about 2 wt % to about 20 wt %, or from about 4 wt % to about 12 wt %. In another exemplary embodiment, the Group VI metal may be present in an amount ranging from about 1 wt % to about 25 wt %, or from about 2 wt % to about 25 wt %. The reaction conditions in the hydrotreating reactor124may include a temperature from about 290° C. (550° F.) to about 455° C. (850° F.), or from about 316° C. (600° F.) to about 427° C. (800° F.), a pressure from about 2.1 MPa (gauge) (300 psig), or from 4.1 MPa (gauge) (600 psig) to about 20.6 MPa (gauge) (3000 psig), or to about 12.4 MPa (gauge) (1800 psig), a liquid hourly space velocity of the hydrocarbon feed stream from about 0.1 hr−1, or from about 4 hr−1, to about 8 hr−1, or from about 1.5 hr−1to about 3.5 hr−1, and a hydrogen rate of about 168 Nm3/m3 (1,000 scf/bbl), to about 1,011 Nm3/m3 oil (6,000 scf/bbl), or from about 168 Nm3/m3 oil (1,000 scf/bbl) to about 674 Nm3/m3 oil (4,000 scf/bbl), with a hydrotreating catalyst or a combination of hydrotreating catalysts. Optionally, a first hydrogen manifold333amay provide a first supplemental hydrogen stream in between the hydrotreating catalyst beds. The hydrotreating reactor124provides a hydrotreated hydrocarbon feed stream that exits the hydrotreating reactor124in line126. The hydrogen gas laden with ammonia and hydrogen sulfide may be removed from the hydrotreated hydrocarbon feed stream in a separator, but the hydrotreated hydrocarbon feed stream in line126is typically fed directly to the first stage hydrocracking reactor130without separation. The hydrotreated hydrocarbon feed stream in line126may be passed to the first stage hydrocracking reactor130of the hydrocracking unit101. The hydrotreated hydrocarbon feed stream in line126is hydrocracked in the presence of the first hydrogen stream and a first hydrocracking catalyst to produce a hydrocracked effluent stream. The first stage hydrocracking reactor130may be a fixed bed reactor that comprises single or multiple catalyst beds, and various combinations of hydrotreating catalyst, and/or hydrocracking catalyst. The first stage hydrocracking reactor130may be operated in a continuous liquid phase in which the volume of the liquid hydrocarbon feed stream is greater than the volume of the hydrogen gas. The first stage hydrocracking reactor130may also be operated in a conventional continuous gas phase, a moving bed or a fluidized bed hydroprocessing reactor. The first stage hydrocracking reactor130may comprise a plurality of the first hydrocracking catalyst beds131. If the hydrocracking unit101does not include the hydrotreating reactor124, the first bed131in the first stage hydrocracking reactor130may include hydrotreating catalyst for the purpose of saturating, demetallizing, desulfurizing, deoxygenating or denitrogenating the hydrocarbon feed before it is hydrocracked with first hydrocracking catalyst in subsequent catalyst beds in the first stage hydrocracking reactor130. Otherwise, the first or an upstream bed in the first stage hydrocracking reactor130may comprise a hydrocracking catalyst bed. In an aspect, the first hydrocracking catalyst in beds131of the first stage hydrocracking reactor130may comprise a distillate selective catalyst to produce a first hydrocracked effluent stream. In an exemplary embodiment, when the preferred products are middle distillates, the first hydrocracking catalysts may utilize amorphous silica-alumina bases or low-level zeolite bases combined with one or more Group VIII or Group VIB metal hydrogenating components. In another exemplary embodiment, when the preferred products are in the gasoline boiling range, the first hydrocracking catalysts may comprise, in general, any crystalline zeolite cracking base upon which is deposited a minor proportion of a Group VIII metal hydrogenating component. Additional hydrogenating components may be selected from Group VIB for incorporation with the zeolite base. The zeolite cracking bases are sometimes referred to in the art as molecular sieves and are usually composed of silica, alumina and one or more exchangeable cations such as sodium, magnesium, calcium, rare earth metals, etc. They are further characterized by crystal pores of relatively uniform diameter between about 4 and about 14 Angstroms. It is preferred to employ zeolites having a relatively high silica/alumina mole ratio between about 3 and about 12. Suitable zeolites found in nature include, for example, mordenite, stilbite, heulandite, ferrierite, dachiardite, chabazite, erionite and faujasite. Suitable synthetic zeolites include, for example, the B, X, Y and L crystal types, e.g., synthetic faujasite and mordenite. The preferred zeolites are those having crystal pore diameters between about 8 and 12 Angstroms (10−10meters), wherein the silica/alumina mole ratio is about 4 to 6. One example of a zeolite falling in the preferred group is synthetic Y molecular sieve. The natural occurring zeolites are normally found in a sodium form, an alkaline earth metal form, or mixed forms. The synthetic zeolites are nearly always prepared first in the sodium form. In any case, for use as a cracking base it is preferred that most or all of the original zeolitic monovalent metals be ion-exchanged with a polyvalent metal and/or with an ammonium salt followed by heating to decompose the ammonium ions associated with the zeolite, leaving in their place hydrogen ions and/or exchange sites which have actually been decationized by further removal of water. Mixed polyvalent metal-hydrogen zeolites may be prepared by ion-exchanging first with an ammonium salt, then partially back exchanging with a polyvalent metal salt and then calcining. In some cases, as in the case of synthetic mordenite, the hydrogen forms can be prepared by direct acid treatment of the alkali metal zeolites. In one aspect, the preferred cracking bases are those which are at least about 10 wt %, and preferably at least about 20 wt %, metal-cation-deficient, based on the initial ion-exchange capacity. In another aspect, a desirable and stable class of zeolites is one wherein at least about 20 wt % of the ion exchange capacity is satisfied by hydrogen ions. In an embodiment, the active metals employed in the first hydrocracking catalysts of the present process as hydrogenation components are those of Group VIII, i.e., iron, cobalt, nickel, ruthenium, rhodium, palladium, osmium, iridium and platinum. In addition to these metals, other promoters may also be employed in conjunction therewith, including the metals of Group VIB, e.g., molybdenum and tungsten. The amount of hydrogenating metal in the catalyst can vary within wide ranges. In an aspect, any amount between about 0.05 wt % and about 35 wt % may be used. In the case of the noble metals such as platinum and palladium, the amount of such metals may range from about 0.05 wt % to about 2 wt % noble metal. One such method for incorporating the hydrogenation metal is to contact the base materials with an aqueous solution of a suitable compound of the desired metals. Following addition of the selected hydrogenation metal or metals, the resulting catalyst powder is then filtered, dried, pelleted with added lubricants, binders or the like if desired, and calcined in air at temperatures of, e.g., about 371° C. (700° F.) to about 648° C. (1200° F.) in order to activate the catalyst and decompose ammonium ions. Alternatively, the base component may first be pelleted, followed by the addition of the hydrogenating component and activation by calcining. The foregoing catalysts may be employed in undiluted form, or the powdered zeolite catalyst may be mixed and copelleted with other relatively less active catalysts, diluents or binders such as alumina, silica gel, silica-alumina co-gels, activated clays and the like in proportions ranging from about 5 to about 90 wt %. These diluents may be employed as such or they may contain a minor proportion of an added hydrogenating metal such as a Group VIB and/or Group VIII metal. Additional metal promoted hydrocracking catalysts may also be utilized in the present process which comprises, for example, aluminophosphate molecular sieves, crystalline chromosilicates and other crystalline silicates. In an embodiment, the hydrocracking conditions of the first stage hydrocracking reactor130may include a temperature from about 290° C. (550° F.) to about 468° C. (875° F.), or from about 343° C. (650° F.) to about 445° C. (833° F.). In another embodiment, the first stage hydrocracking reactor130may operate at a first hydrocracking pressure from about 4.8 MPa (gauge) (700 psig) to about 20.7 MPa (gauge) (3000 psig), a liquid hourly space velocity (LHSV) from about 0.4 hr−1to less than about 5 hr−1and a hydrogen rate of about 421 Nm3/m3(2,500 scf/bbl) to about 2,527 Nm3/m3oil (15,000 scf/bbl). The hydrocarbon feed stream in the hydrotreated hydrocarbon feed stream in line126is hydrocracked in the first stage hydrocracking reactor130operating at the first hydrocracking pressure in the presence of the first hydrogen stream passed in line336and the first hydrocracking catalyst to produce a first hydrocracked stream in line132. The hydrotreated hydrocarbon feed stream in line126may be first passed to the top catalyst bed of the first hydrocracking catalyst. Subsequent catalyst beds in the first stage hydrocracking reactor130may comprise hydrocracking catalyst over which additional hydrocracking occurs to the first hydrocracked stream. Optionally, a second hydrogen manifold333bmay deliver a second supplemental hydrogen streams to one, some or each of the catalyst beds at the interbed locations in the first stage hydrocracking reactor130. In an aspect, the supplemental hydrogen is added to each of the catalyst beds at an interstage location between adjacent beds, so supplemental hydrogen is mixed with hydroprocessed effluent exiting from the upstream catalyst bed before entering the downstream catalyst bed in the first stage hydrocracking reactor130. The first hydrocracked effluent stream in line132is withdrawn from the bottoms of the first stage hydrocracking reactor130. The first hydrocracked effluent stream in line132may be passed to the fractionation section105for separation. The fractionation section105may comprise one or more separators and fractionation columns in downstream communication with the hydrocracking unit101. The first hydrocracked effluent stream in line132may be separated in a hot separator of the fractionation section105to provide a vapor hydrocracked stream and a liquid hydrocracked stream. In an exemplary embodiment, the first hydrocracked effluent stream in line132is passed to a first stage hot separator140of the fractionation section105. In the first stage hot separator140, the first hydrocracked effluent stream in line132may be separated to provide a first hot separated vapor hydrocracked stream in line142and a first hot separated liquid hydrocracked stream in line149. The first hot separated vapor hydrocracked stream in line142is withdrawn from the overhead of the hot separator140. The first hot separated liquid hydrocracked stream in line149is withdrawn from the bottoms of the hot separator140. The first stage hot separator140may be in downstream communication with the hydrocracking unit101. The first stage hot separator140may operate at a bottoms temperature from about 177° C. (350° F.) to about 371° C. (700° F.) or from about 232° C. (450° F.) to about 315° C. (600° F.). In an aspect, the bottoms temperature of the first stage hot separator140may be reduced to minimize any UCO in the overhead. The first stage hot separator140may be operated at a slightly lower pressure than the first stage hydrocracking reactor130accounting for pressure drop through intervening equipment. The first stage hot separator140may be operated at an overhead pressure from about 3.4 MPa (gauge) (493 psig) and about 20.4 MPa (gauge) (2959 psig). The first hot separated vapor hydrocracked stream in line142from the overhead of the first stage hot separator140may have a temperature of the operating temperature of the first stage hot separator140. The first hot separated vapor hydrocracked stream in line142may be cooled before entering a cold separator145. As a consequence of the reactions taking place in the first stage hydrocracking reactor130wherein nitrogen, chlorine and sulfur are removed from the feed, ammonia and hydrogen sulfide are formed. At a characteristic sublimation temperature, ammonia and hydrogen sulfide will combine to form ammonium bisulfide and ammonia, and chlorine will combine to form ammonium chloride. Each compound has a characteristic sublimation temperature that may allow the compound to coat equipment, particularly heat exchange equipment, impairing its performance. To prevent such deposition of ammonium bisulfide or ammonium chloride salts in the first hot separated vapor hydrocracked stream in line142, a suitable amount of wash water in line143may be introduced into the first hot separated vapor hydrocracked stream in line142upstream of a cooler at a point in the first hot separated vapor hydrocracked stream in line142where the temperature is above the characteristic sublimation temperature of either compound. As described hereinafter in detail, a second hot separated vapor hydrocracked stream in line285may be combined with the first hot separated vapor hydrocracked stream in line142to provide a combined hot separated vapor hydrocracked stream in line144. The combined hot separated vapor hydrocracked stream in line144may be passed to the cold separator145to provide a cold separated vapor hydrocracked stream in line146and a cold separated liquid hydrocracked stream in line148. The cold separated vapor hydrocracked stream in line146is withdrawn from the overhead of the cold separator145. The cold separated liquid hydrocracked stream in line148is withdrawn from the bottoms of the cold separator145. The cold separated vapor hydrocracked stream in line146is a hydrogen-rich gas stream which can be recovered to be used as a hydrogen gas stream in the process. The cold separator145serves to separate hydrogen rich gas from the first hot separated vapor hydrocracked stream in line142and the second hot separated vapor hydrocracked stream in line285for recycle to the hydrocracking unit101in the cold separated vapor hydrocracked stream in line146. The cold separator145, therefore, is in downstream communication with the first hot separated vapor hydrocracked stream in line142of the first stage hot separator140and the hydrocracking unit101. The cold separator145may be operated at a bottoms temperature from about 38° C. (100° F.) to about 66° C. (150° F.), or from about 46° C. (115° F.) to about 63° C. (145° F.), and below the pressure of the first stage hydrocracking reactor130and the first stage hot separator150accounting for pressure drop through intervening equipment to keep hydrogen and light gases in the overhead and normally liquid hydrocarbons in the bottoms. The cold separator145may be operated at an overhead pressure between about 3 MPa (gauge) (435 psig) and about 20 MPa (gauge) (2,901 psig). The cold separator145may also have a boot for collecting an aqueous phase in line147. The cold separated liquid hydrocracked stream in line148may have a temperature of the operating temperature of the cold separator160. The cold separated liquid hydrocracked stream in line148may be fractionated. The cold separated vapor hydrocracked stream in line146is rich in hydrogen. Thus, hydrogen can be recovered from the cold separated vapor hydrocracked stream in line146. The cold separated vapor hydrocracked stream in line146may be passed through a trayed or packed recycle scrubbing column320where it is scrubbed by means of a scrubbing extraction liquid such as an aqueous solution fed by line321to remove acid gases including hydrogen sulfide and carbon dioxide by extracting them into the aqueous solution. In an exemplary embodiment, the aqueous solution in line321may include lean amines such as alkanolamines DEA, MEA, and MDEA. Other amines can also be used in place of or in addition to these amines. The lean amine fed by line321contacts the cold separated vapor hydrocracked stream in line146and absorbs acid gas contaminants such as hydrogen sulfide and carbon dioxide. The resultant “sweetened” cold gaseous stream is taken out from an overhead outlet of the recycle scrubber column320in a recycle scrubber overhead line322, and a rich amine is taken out from the bottoms at a bottom outlet of the recycle scrubber column320in a recycle scrubber bottoms line324. The spent scrubbing liquid from the bottoms in line324may be regenerated and recycled back to the recycle scrubbing column320in line321. The scrubbed hydrogen-rich stream emerges from the scrubber via the recycle scrubber overhead line322. The scrubbed hydrogen-rich stream in the recycle scrubber overhead line322may be compressed in a recycle compressor330to provide the compressed hydrogen stream in line332. The recycle scrubbing column320may be operated with a gas inlet temperature from about 38° C. (100° F.) and about 66° C. (150° F.) and an overhead pressure of about 3 MPa (gauge) (435 psig) to about 20 MPa (gauge) (2900 psig). Optionally, a supplemental hydrogen stream333may be separated from the compressed hydrogen stream in line332. The supplemental hydrogen stream333may be separated into the first supplemental hydrogen stream in line333a, the second supplemental hydrogen stream in line333b, and a third supplemental hydrogen stream in line333cwhich are passed to the hydrocracking unit101. The scrubbed hydrogen-rich stream in the recycle scrubber overhead line322may be provided with a make-up hydrogen stream in the make-up line335upstream or downstream of the compressor330. In exemplary embodiment, the make-up hydrogen stream in the make-up line335is combined with the scrubbed hydrogen-rich stream downstream of the compressor330. In another exemplary embodiment, the make-up hydrogen stream in the make-up line335is combined with a compressed hydrogen stream in line334after the separation of supplemental hydrogen stream333. In an aspect, the first hot separated liquid hydrocracked stream in line149may be let down in pressure and flashed in a first stage hot flash drum150to provide a first hot vapor hydrocracked stream in line152and a first hot liquid hydrocracked stream in line154. The light ends are separated in the first hot vapor hydrocracked stream in line152which may be withdrawn from the overhead of the first stage hot flash drum150. The first hot liquid hydrocracked stream in line154may be withdrawn from the bottoms of the first stage hot flash drum150. Accordingly, the first hot liquid hydrocracked stream in line154may be provided from the first stage hot separator140. The hot flash drum150may be in direct, downstream communication with the first hot separated liquid hydrocracked stream in line149and in downstream communication with the hydrocracking unit101. The light gases such as hydrogen sulfide may be stripped from the first hot liquid hydrocracked stream in line154in a stripper to provide a liquid hydrocracked stream. In an embodiment, the first hot liquid hydrocracked stream in line154may be stripped in a first stage stripper170to provide a liquid hydrocracked stream. The first stage stripper170may be in downstream communication with the first stage hot flash drum150and line154carrying the first hot liquid hydrocracked stream. The first hot flash drum150may be operated at the same bottoms temperature as the first stage hot separator140but at a lower overhead pressure of between about 1.4 MPa (gauge) (200 psig) and about 6.9 MPa (gauge) (1000 psig), suitably no more than about 3.8 MPa (gauge) (550 psig). The hot liquid hydrocracked stream in line154may be further fractionated in the fractionation section105. The first hot liquid hydrocracked stream in line154may have a temperature of the operating temperature of the first stage hot flash drum150. In an aspect, the cold separated liquid hydrocracked stream in line148may be directly fractionated. In a further aspect, the cold separated liquid hydrocracked stream in line148may be let down in pressure and flashed in a cold flash drum160to separate the cold separated liquid hydrocracked stream in line148. The cold flash drum160may be in direct downstream communication with the cold separated liquid hydrocracked stream in line148of the cold separator145and in downstream communication with the hydrocracking unit101. In a further aspect, the hot vapor hydrocracked stream in152may be fractionated in the fractionation section105. In a further aspect, the hot vapor hydrocracked stream in152may be cooled and also separated in the cold flash drum160. In an exemplary embodiment, the first hot vapor hydrocracked stream in152may be combined with a second hot vapor hydrocracked stream in line288to provide a combined hot vapor hydrocracked stream in line155. The combined hot vapor hydrocracked stream in line155may be passed to the cold flash drum160. The cold flash drum160may separate the cold separated liquid hydrocracked stream in line148and the combined hot vapor hydrocracked stream in line155to provide a cold vapor stream in line161and a cold liquid stream in line162. The cold vapor stream in line161may be withdrawn from an overhead of the cold flash drum160. The cold liquid stream in line162may be withdrawn from the bottoms of the cold flash drum160. In an embodiment, the cold separated liquid hydrocracked stream in line148and the combined hot vapor hydrocracked stream in line155may be combined to provide a combined separated stream in line156. The combined separated stream in line156may be separated in the cold flash drum160to provide the cold vapor stream in line161and the cold liquid stream in line162. In an aspect, light gases such as hydrogen sulfide may be stripped from the cold liquid stream in line162. In an embodiment, the cold liquid stream in line162may be stripped in the first stage stripper170to provide the liquid hydrocracked stream. The stripper170may be in downstream communication with the cold flash drum160and the cold liquid stream in line162. The cold flash drum160may be in downstream communication with the line148the line152, the line288and the hydrocracking unit101. The cold separated liquid hydrocracked stream in line148and the combined hot vapor hydrocracked stream in155may enter into the cold flash drum160either together or separately. The cold flash drum160may be operated at the same bottoms temperature as the cold separator145but typically at a lower overhead pressure of between about 1.4 MPa (gauge) (200 psig) and about 6.9 MPa (gauge) (1000 psig) or between about 3.0 MPa (gauge) (435 psig) and about 3.8 MPa (gauge) (550 psig). A flashed aqueous stream may be removed from a boot in the cold flash drum160in line163. The cold liquid stream in line162may have the same temperature as the operating temperature of the cold flash drum160. The fractionation section105may further include the first stage stripper170, a second stage stripper290, a first stage fractionation column180, and a second stage fractionation column310. In an embodiment, the first stage stripper170may be a vessel that contains a top cold stripper170aand a bottom hot stripper170bwith a wall that isolates each of the stripping columns from the other. In an exemplary embodiment, the cold liquid stream in line162may be stripped in the cold stripper170aand the first hot liquid hydrocracked stream in line154may be stripped in the hot stripper170bof the stripping column170to provide the liquid hydrocracked stream. As described herein after in detail, an overhead vapor hydrocracked stream in line292may also be passed along with the cold liquid stream in line162to the cold stripper170aof the first stage stripper170. In an exemplary embodiment, the overhead vapor hydrocracked stream in line292may be combined with the cold liquid stream in line162and passed to the cold stripper170ain line164. The first hot liquid hydrocracked stream in line154may be passed to the hot stripper170bof the first stage stripper170. The cold liquid stream in line162may be fed to the first stage stripper170at a location above an entry point of the first hot liquid hydrocracked stream in line154to the first stage stripper170. The cold liquid stream in line162, the overhead vapor hydrocracked stream in line292, and the hot liquid hydrocracked stream in line154may be stripped of gases in the first stage stripper170with a stripping media which is an inert gas such as steam to provide a gaseous stream of naphtha, hydrogen, hydrogen sulfide, steam and other gases in a vapor hydrocracked stream in line171. A stripping media such as medium pressure steam in line165may be provided to the hot stripper170b. The steam in line165is optionally a medium pressure steam and a steam at any suitable pressure may be provided to the hot stripper170b. The stripping media to the cold stripper170amay be provided with the overhead vapor hydrocracked stream in line292. Alternatively, a stripping media such as medium pressure steam in line168may also be injected into the cold stripper170a. The steam in line168is optionally a medium pressure steam and steam at any suitable pressure may be injected into the cold stripper170a. In an exemplary embodiment, the cold liquid stream in line162and the overhead vapor hydrocracked stream in line292may be stripped of gases in the cold stripper170ato provide the vapor hydrocracked stream in line171and a first liquid hydrocracked stream in line177. In another exemplary embodiment, the first hot liquid hydrocracked stream in line154may be stripped of gases/vapors in the hot stripper170bto provide a second liquid hydrocracked stream in line178. The stripped gases/vapors may be withdrawn in line167from the overhead of the hot stripper170b. The stripped gases/vapors in line167may be passed to the cold stripper170a. In an exemplary embodiment, the stripped gases/vapors in line167may be passed to the cold stripper170ain line164along with the cold liquid stream in line162and the overhead vapor hydrocracked stream in line292. The vapor hydrocracked stream in line171may be withdrawn from the overhead of the first stage stripper170. The vapor hydrocracked stream in line171may be condensed and separated in an overhead receiver172. A sour off gas stream in line173may be withdrawn from the overhead receiver172. Unstabilized liquid naphtha from the bottoms of the receiver172may be split in a reflux portion refluxed to the top of the cold stripper170ain a reflux line176and a first stage overhead naphtha stream in line175which may be further recovered or processed as described later in detail. In an aspect, the first stage overhead naphtha stream in line175may be passed to a debutanizer column240to separate LPG from light naphtha. The cold stripper170amay be operated with a bottoms temperature between about 149° C. (300° F.) and about 288° C. (550° F.), or a bottoms temperature of no more than about 260° C. (500° F.), and an overhead pressure of about 0.35 MPa (gauge) (50 psig), or an overhead pressure of no less than about 0.70 MPa (gauge) (100 psig), to no more than about 2.0 MPa (gauge) (290 psig). The temperature in the overhead receiver172may range from about 38° C. (100° F.) to about 66° C. (150° F.) and the pressure may be same as in the overhead of the cold stripper170a. The hot stripper170bmay be operated with a bottoms temperature between about 160° C. (320° F.) and about 360° C. (680° F.) and an overhead pressure of about 0.35 MPa (gauge) (50 psig), or an overhead pressure of about 0.70 MPa (gauge) (100 psig), to about 2.0 MPa (gauge) (292 psig). The first liquid hydrocracked stream in line177and the second liquid hydrocracked stream in line178may be withdrawn from the bottoms of the cold stripper170aand the bottoms of the hot stripper170brespectively. The first liquid hydrocracked stream in line177and the second liquid hydrocracked stream in line178may be fractionated in the first stage fractionation column180. In an embodiment, the second liquid hydrocracked stream in line178may be heated in a fractionation column feed heater260to provide a heated second liquid hydrocracked stream in line262. The heated second liquid hydrocracked stream in line262may be fractionated in the first stage fractionation column180. The first stage fractionation column180may be in a downstream communication with the first stage stripper170. Low pressure steam in line179may be injected into the first stage fractionation column180. The steam in line179is optionally a low pressure steam and steam at any suitable pressure may be injected to the first stage fractionation column180. As described later in detail, a combined side draw stream in line319may also be passed to the first stage fractionation column180. In an exemplary embodiment, the combined side draw stream in line319may be passed to the first stage fractionation column180at a location above the second liquid hydrocracked stream in line262and below the first liquid hydrocracked stream in line177. The first stage fractionation column180separates the first liquid hydrocracked stream in line177, the combined side draw stream in line319, and the heated second liquid hydrocracked stream in line262into different product streams. In an embodiment, the first liquid hydrocracked stream in line177, the combined side draw stream in line319, and the heated second liquid hydrocracked stream in line262may be fractionated in the first stage fractionation column180to provide a naphtha stream, a kerosene stream, a diesel stream and a first unconverted oil (UCO) stream. In an exemplary embodiment, the first liquid hydrocracked stream in line177, the combined side draw stream in line319, and the heated second liquid hydrocracked stream in line262are fractionated in the first stage fractionation column180to provide the fractionation column overhead stream in line181, a first side draw stream comprising naphtha stream in line182, a second side draw stream comprising kerosene stream in line183, and a third side draw stream comprising the diesel stream in line184, and the first unconverted oil (UCO) stream in line186. The fractionated overhead stream in line181may be further processed to provide a light naphtha stream. In accordance with the present process, the first stage fractionation column180is a fractionation column180with multiple side cut stripping columns. In an exemplary embodiment, the first stage fractionation column180may comprise a first side cut stripping column210, a second side cut stripping column220, and a third side cut stripping column230. Alternatively, the first side cut stripping column210, a second side cut stripping column220, and a third side cut stripping column230may be housed in a single vessel and optionally separated from each other by walls. The first stage fractionation column180also separates light naphtha range hydrocarbons from heavy naphtha range hydrocarbons. In accordance with the present process, the light naphtha range hydrocarbons may be separated in the fractionation column overhead stream in line181and the heavy naphtha range hydrocarbons may be separated in the first side draw stream in line182. Heavy naphtha may be separated from the first side draw stream in the first side cut stripping column210. In an exemplary embodiment, the first side cut stripping column210is a heavy naphtha stripping column. The first side draw stream comprising heavy naphtha in line182may be fed to the first side cut stripping column210. The first side draw stream comprising heavy naphtha in line182may be stripped of gases in the first side cut stripping column210to provide a gaseous stream having a lower amount of heavy naphtha than the first side draw stream in line182. The gaseous stream may be withdrawn from the overhead of the first side cut stripping column210and passed to the first stage fractionation column180for further recovery. A heavy naphtha stream in line214may be withdrawn from the bottoms of the first side cut stripping column210. A boilup stream of the first side cut stripping column210in the reboil line215is returned to the first side cut stripping column210after reboiling. The second side draw stream comprising kerosene in line183may be fed to the second side cut stripping column220. In an exemplary embodiment, the second side cut stripping column220is a kerosene stripping column220. In the second side cut stripping column220, the second side draw stream in line183may be stripped of gases to separate a kerosene stream. A gaseous stream comprising a lower amount of kerosene than the second side draw stream in line183may be withdrawn in line222from the overhead of the second side cut stripping column220and passed to the first stage fractionation column180for further recovery. The kerosene stream may be withdrawn in line224from the bottoms of the second side cut stripping column220. Also, a boilup stream of the second side cut stripping column220in the reboil line225is returned to the second side cut stripping column220after reboiling. In an embodiment, a kerosene yield ranging from about 10 wt % to about 40 wt % of the fresh feed in line102may be obtained in the kerosene stream in line224in accordance with the current process. In an aspect of the present disclosure, a portion of the kerosene stream in line226may be withdrawn and recycled to the hydrocracking unit101. In an exemplary embodiment, the portion of the kerosene stream in line226recycled to the hydrocracking unit101may range from about 0 wt % to about 100 wt % of the kerosene stream in line224. In another exemplary embodiment, the portion of the kerosene stream in line226recycled to the hydrocracking unit101may range from about 40 wt % to about 80 wt % of the kerosene stream in line224. In yet another exemplary embodiment, the portion of the kerosene stream in line226recycled to the hydrocracking unit101may range from about 65 wt % to about 85 wt % of the kerosene stream in line224. A kerosene product stream may be withdrawn in line228. In another aspect, the kerosene stream in line228is a first stage kerosene product stream. In an exemplary embodiment, the first stage kerosene product stream in line228may range from about 100 wt % to about 0 wt % of the kerosene stream in line224. In another exemplary embodiment, the first stage kerosene product stream in line228may range from about 60 wt % to about 20 wt % of the kerosene stream in line224. In yet another exemplary embodiment, the first stage kerosene product stream in line228may range from about 35 wt % to about 15 wt % of the kerosene stream in line224. The third side draw stream comprising the diesel stream in line184may be passed to the third side cut stripping column230. In an exemplary embodiment, the third side cut stripping column230is a diesel stripping column220. A low pressure steam in line231may also be provided to the third side cut stripping column230. The steam in line231is optionally a low pressure steam and steam at any suitable pressure may be provided to the third side cut stripping column230. The third side draw stream in line184may be stripped of gases in the third side cut stripping column230to separate a diesel stream. A gaseous stream comprising a lower amount of diesel than the third side draw stream in line184may be withdrawn in line232from the overhead of the third side cut stripping column230and passed to the first stage fractionation column180for further recovery. The diesel stream may be withdrawn in line234from the bottoms of the third side cut stripping column230. In an embodiment, a diesel yield ranging from about 10 wt % to about 50 wt % of the fresh feed in line102may be obtained in the diesel stream in line234in accordance with the current process. In an aspect of the present disclosure, a portion of the diesel stream in line236may be withdrawn and recycled to the hydrocracking unit101. In an exemplary embodiment, the portion of the diesel stream in line236recycled to the hydrocracking unit101may range from about 0 wt % to about 100 wt % of the diesel stream in line234. In another exemplary embodiment, the portion of the diesel stream in line236recycled to the hydrocracking unit101may range from about 55 wt % to about 95 wt % of the diesel stream in line234. In yet another exemplary embodiment, the portion of the diesel stream in line236recycled to the hydrocracking unit101may range from about 65 wt % to about 85 wt % of the diesel stream in line234. A diesel product stream may be withdrawn in line238. In another aspect, the diesel stream in line223is a first stage diesel product stream. In an exemplary embodiment, the first stage diesel product stream in line238may range from about 100 wt % to about 0 wt % of the kerosene stream in line234. In another exemplary embodiment, the first stage diesel product stream in line238may range from about 45 wt % to about 5 wt % of the kerosene stream in line234. In yet another exemplary embodiment, the first stage diesel product stream in line238may range from about 35 wt % to about 15 wt % of the kerosene stream in line234. An unconverted oil (UCO) stream in line186may be withdrawn from the bottoms of the first stage fractionation column180. In accordance with the present process, unconverted oil stream in line186is a first UCO stream obtained from the first stage of the hydrocracking unit101. The quality of the first UCO stream in line186inter alia depends upon the catalyst of the first stage hydrocracking reactor130and the conversion rate in the first stage hydrocracking reactor130. Applicants have found that running the first stage hydrocracking reactor130with the distillate selective catalyst and at a conversion rate of from about 50% to about 80% or from about 60% to about 70% provides a high quality first UCO stream in line186. In an aspect of the present process, the high quality first UCO stream in line186may be characterized by a viscosity index (VI) in the range from about 100 to about 150 or from about 105 to about 140. The high quality first UCO stream in line186may be used for base oil production. In an embodiment, a UCO yield ranging from about 10 wt % to about 50 wt % of the fresh feed or form about 20 wt % to about 45 wt % of the fresh feed in line102may be obtained in the high quality first UCO stream in line186in accordance with the current process. A required amount of UCO may be taken from the first UCO stream in line186to downstream base oil unit for the production of lube base oils. In an aspect, a portion of the first UCO stream in line186may be recycled to the hydrocracking unit101in line188. In an exemplary embodiment, the portion of the first UCO stream in line188recycled to the hydrocracking unit101may range from about 0 wt % to about 75 wt % of the UCO stream in line186. In another exemplary embodiment, the portion of the first UCO stream in line188recycled to the hydrocracking unit101may range from about 20 wt % to about 60 wt % of the UCO stream in line186. In yet another exemplary embodiment, the portion of the first UCO stream in line188recycled to the hydrocracking unit101may range from about 30 wt % to about 50 wt % of the UCO stream in line186. A remaining portion of the first UCO stream may be withdrawn in line187and passed to the base oil unit for the production of lube base oils. In an exemplary embodiment, the remaining portion of the first UCO stream in line187for producing the base oils may range from about 100 wt % to about 25 wt % of the UCO stream in line186. In another exemplary embodiment, the remaining portion of the first UCO stream in line187for producing the base oils may range from about 80 wt % to about 40 wt % of the UCO stream in line186. In yet another exemplary embodiment, the remaining portion of the first UCO stream in line187for producing the base oils may range from about 70 wt % to about 50 wt % of the UCO stream in line186. In accordance with an aspect of the present process, the fractionated overhead stream in line181is further processed to provide the naphtha stream. The fractionated overhead stream in line181may be condensed and separated in a receiver190with a portion of a condensed liquid in line192being refluxed back to the first stage fractionation column180in a reflux line194. A net portion of the condensed liquid192may be further processed or recovered as naphtha product stream in line196. In an aspect, the first stage fractionation column180may be a totally condensing column that does not produce an off-gas stream. In an embodiment, the condensed liquid stream in line196along with the first stage overhead naphtha stream in line175and a second stage overhead naphtha stream in line317may be passed to the debutanizer column240to recover naphtha product stream. In a non-limiting aspect, any suitable method may be employed to recover naphtha product stream from the condensed liquid in line196, the first stage overhead naphtha stream in line175and the second stage overhead naphtha stream in line317. In an exemplary embodiment, the condensed liquid in line196, the first stage overhead naphtha stream in line175and the second stage overhead naphtha stream in line317may be combined to provide a net overhead stream in line198. The net overhead stream in line198may be passed to the debutanizer column240to recover naphtha. The debutanizer column240may separate the condensed liquid in line196, the first stage overhead naphtha stream in line175and the second stage overhead naphtha stream in line317to provide a debutanizer overhead stream comprising LPG and a debutanized bottoms stream comprising light naphtha. An overhead stream in line241from the debutanizer column240may be cooled and separated in a receiver242to provide an overhead gas stream comprising C2 and lighter gases in a debutanizer off-gas stream in a debutanizer off-gas line243and a debutanizer liquid overhead stream comprising LPG in line244. A portion of the debutanizer liquid overhead stream in line244may be recycled to the debutanizer column240in a debutanizer reflux line246. A net debutanizer overhead liquid stream comprising LPG is withdrawn in a net debutanizer overhead liquid stream in line245. A debutanized boilup stream in debutanized reboil line249may be returned to the debutanizer column240after reboiling. A net debutanized bottoms stream in line248is withdrawn from the debutanizer column240. The debutanizer column240may be operated at a bottoms temperature between about 121° C. (250° F.) and about 177° C. (350° F.) and an overhead pressure between about 690 kPa (100 psi) and about 1379 kPa (200 psi). The net debutanized bottoms stream in line248comprises more light naphtha than in the net debutanizer overhead liquid stream comprising LPG in the net debutanizer overhead liquid stream in line245. The net debutanizer overhead liquid stream comprising LPG in the net debutanizer overhead liquid stream in line245may comprise between about 10 mol % and about 30 mol % propane and between about 60 mol % and about 90 mol % butane. The net debutanized bottoms stream in line248may be passed to a naphtha splitter column250to separate light naphtha from heavy naphtha. The naphtha splitter column250separates the net debutanized bottoms stream in line248to provide an overhead stream comprising light naphtha in line251and a net bottoms stream comprising heavy naphtha in line258. The overhead stream comprising light naphtha in line251from the naphtha splitter column250may be cooled and separated in a receiver252to provide an overhead gas stream comprising lighter gases in an off-gas stream in an off-gas line253and an overhead liquid stream comprising light naphtha in line254. In an embodiment, the naphtha splitter column250may be a totally condensing column and does not produce the off-gas stream253. A portion of the overhead liquid stream in line254may be recycled back to the naphtha splitter column250in a reflux line256. The light naphtha stream is withdrawn in a net overhead liquid line255. A boilup stream in a reboil line259may be returned to the naphtha splitter column250after reboiling. The net bottoms stream in line258may passed to a sulfur guard bed217to remove sulfur. In an aspect, the heavy naphtha stream in line214may be passed to the sulfur guard bed217along with the net bottoms stream in line258to remove sulfur and provide a desulfurized heavy naphtha stream in line218. In exemplary embodiment, the heavy naphtha stream in line214may be combined with the net bottoms stream in line258and passed to the sulfur guard bed217in line216. Referring back to the first stage fractionation column180, the portion of the kerosene stream in line226, the portion of the diesel stream in line236, and the portion of the first UCO stream in line188may be recycled to the hydrocracking unit101. In an aspect, one, two or all of the portion of the kerosene stream in line226, the portion of the diesel stream in line236, and the portion of the first UCO stream in line188may be recycled to a second stage hydrocracking reactor280of the hydrocracking unit101to provide a second unconverted oil stream. In an exemplary embodiment, one, two or all of the portion of the kerosene stream in line226, the portion of the diesel stream in line236, and the portion of the first UCO stream in line188may be combined to provide a recycle stream in line239. The recycle stream in line239may be recycled to the hydrocracking unit101. As described hereinafter in detail, a second unconverted oil stream in line314may also be recycled to the hydrocracking unit101along with the recycle stream in line239. In an exemplary embodiment, second unconverted oil stream in line314may be combined with the recycle stream in line239to provide a combined recycle stream in line264which may be recycled to the hydrocracking unit101. The present process provides a unique scheme that employs both distillate and naphtha selective catalysts in the same hydrocracking unit in an integrated manner in such a way that not only naphtha production can be maximized but also high quality UCO can be produced for base oil production along with distillates. In accordance with applicants' process, the hydrocracking unit101may comprise a multistage hydrocracking unit101having a first stage hydrocracking reactor130and a second stage hydrocracking reactor280. The applicants' process comprise using a distillate selective catalyst in the first stage hydrocracking reactor130and a naphtha selective catalyst in the second stage hydrocracking reactor280to maximize naphtha production while producing base oil. The current process may also include more than two hydrocracking reactors with distillate selective catalyst and naphtha selective catalyst. Kerosene and distillate production in lines226and236from the integrated scheme of theFIG.1nexcess of what is required for fuels requirements may be recirculated back to the second stage hydrocracking reactor280to maximize naphtha production. The present process comprises recycling the portion of the kerosene stream in line226, the portion of the diesel stream in line236, and the portion of the first UCO stream in line188to the second stage hydrocracking reactor280of the hydrocracking unit101. Referring back to the FIGURE, the combined recycle stream in line264may be recycled to the second stage hydrocracking reactor280. The combined recycle stream in line264may enter the second stage hydrocracking reactor280via a recycle surge drum265. From the bottoms of the recycle surge drum265, the combined recycle stream flows in line266to the suction of a recycle charge pump267to provide a pumped combined recycle stream in line268. The pumped combined recycle stream in line268may be heated up by heat exchange with a second hydrocracked effluent stream in line282. After the heat exchange, a hydrogen stream in line338may be added to the pumped combined recycle stream in line268to provide a mixed recycle stream in line272. The hydrogen stream in line338may also be taken from the compressed hydrogen stream in line332. In an exemplary embodiment, the hydrogen stream in line338is a second hydrogen stream. In an aspect, the hydrogen stream in line338may join the pumped combined recycle stream in line268as the second hydrogen stream to provide the mixed recycle stream in line272. The mixed recycle stream in line272may be heated in a second stage feed heater274to provide a heated recycle stream in line276that may be fed to the second stage hydrocracking reactor280. The second stage hydrocracking reactor280may comprise a plurality of second hydrocracking catalyst beds281. In an aspect, the second hydrocracking catalyst in beds281of the second stage hydrocracking reactor280may comprise a naphtha selective catalyst to produce a second hydrocracked effluent stream. Suitable second hydrocracking catalyst may comprise one or more of the hydrocracking catalysts as described earlier for the first hydrocracking catalyst. Compared to the distillate selective catalyst of the first stage hydrocracking reactor130, the naphtha selective catalyst of the second stage hydrocracking reactor280may be characterized by a higher activity. The higher activity the naphtha selective catalyst of the second stage hydrocracking reactor280may be due to a higher zeolite content and/or a higher metals contents in the naphtha selective catalyst as compared to the distillate selective catalyst. Optionally, a third hydrogen manifold333cmay provide a third set of supplemental hydrogen streams to some or all of the catalyst beds281at the interbed locations in the second stage hydrocracking reactor280. In the second stage hydrocracking reactor280, the combined recycle stream comprising the recycle stream in line239and the the second unconverted oil stream in line314is hydrocracked in the presence of the naphtha selective catalyst and the second hydrogen stream to produce a second hydrocracked effluent stream in line282. The second hydrocracked effluent stream in line282is withdrawn from the bottoms of the second stage hydrocracking reactor280. The second hydrocracked effluent stream in line282may be passed to the fractionation section105for separation after heat exchange with the combined recycle stream in line268. In an embodiment, the hydrocracking conditions of the second stage hydrocracking reactor280may include a temperature from about 290° C. (550° F.) to about 468° C. (875° F.), or from about 343° C. (650° F.) to about 445° C. (833° F.). In another embodiment, the second stage hydrocracking reactor280may operate at a first hydrocracking pressure from about 4.8 MPa (gauge) (700 psig) to about 20.7 MPa (gauge) (3000 psig), a liquid hourly space velocity (LHSV) from about 0.4 hr−1to less than about 5 hr−1and a hydrogen rate of about 421 Nm3/m3(2,500 scf/bbl) to about 2,527 Nm3/m3oil (15,000 scf/bbl). The second hydrocracked effluent stream in line282may be separated in a hot separator of the fractionation section105to provide a vapor hydrocracked stream and a liquid hydrocracked stream. In an aspect, the second hydrocracked effluent stream in line282is passed to a second stage hot separator284of the fractionation section105. In the second stage hot separator284, the second hydrocracked effluent stream in line282may be separated to provide the second hot separated vapor hydrocracked stream in line285and a second hot separated liquid hydrocracked stream in line286. The second hot separated vapor hydrocracked stream in line285may be withdrawn from the overhead of the second stage hot separator284. The second hot separated vapor hydrocracked stream in line285may be combined with the first hot separated vapor hydrocracked stream in line142and passed to the cold separator145in the combined hot separated vapor hydrocracked stream in line144. The second stage hot separator284may operate at a bottoms temperature from about 177° C. (350° F.) to about 371° C. (700° F.) or from about 232° C. (450° F.) to about 315° C. (600° F.). In an aspect, the temperature of the second stage hot separator284may be reduced to minimize any UCO in the overhead. The second stage hot separator284may be operated at a slightly lower pressure than the second stage hydrocracking reactor280accounting for pressure drop through intervening equipment. The second stage hot separator284may be operated at an overhead pressure from about 3.4 MPa (gauge) (493 psig) and about 20.4 MPa (gauge) (2959 psig). The second hot separated vapor hydrocracked stream in line285from the overhead of the second stage hot separator284may have a temperature of the operating temperature of the second stage hot separator282. In an aspect, the second hot separated liquid hydrocracked stream in line286may be let down in pressure and flashed in a second stage hot flash drum287to provide the second hot vapor hydrocracked stream in line288and a second hot liquid hydrocracked stream in line289. The light ends get separated in the second hot vapor hydrocracked stream in line288which may be withdrawn from the overhead of the second stage hot flash drum287. The second hot vapor hydrocracked stream in line288may be combined with the first hot vapor hydrocracked stream in152and passed to the cold flash drum160in the combined hot vapor hydrocracked stream in line155. The second hot liquid hydrocracked stream in line289may be withdrawn from the bottoms of the second stage hot flash drum287. Accordingly, the second hot liquid hydrocracked stream in line289may be provided from the second stage hot separator284. The second hot flash drum287may be in direct, downstream communication with the second hot separated liquid hydrocracked stream in line286and in downstream communication with the hydrocracking unit101. The light gases such as hydrogen sulfide may be stripped from the second hot liquid hydrocracked stream in line289in a stripper to provide a liquid hydrocracked stream. In an embodiment, the second hot liquid hydrocracked stream in in line289may be fractionated in a second stage fractionation column310of the fractionation section105to provide the second stage overhead naphtha stream in line317, a second stage kerosene stream, a second stage diesel stream, and the second unconverted oil stream in line314. The second stage fractionation column310may be in downstream communication with the second stage hot flash drum287and the second hot liquid hydrocracked stream in line289. A stripping media such as low pressure steam in line297may be provided to the second stage fractionation column310for stripping the light materials from the heated second hot liquid hydrocracked stream in line296. The steam in line297is optionally a low pressure steam and steam at any suitable pressure may be provided to the second stage fractionation column310. The second hot flash drum287may be operated at the same bottoms temperature as the second hot separator284but at a lower overhead pressure of between about 1.4 MPa (gauge) (200 psig) and about 6.9 MPa (gauge) (1000 psig), suitably no more than about 3.8 MPa (gauge) (550 psig). The second hot liquid hydrocracked stream in line289may have a temperature of the operating temperature of the second stage hot flash drum287. The second hot liquid hydrocracked stream in line289may be passed to the stripping column290. A stripping media such as medium pressure steam in line291may be provided to the stripping column290. The steam in line291is optionally a medium pressure steam and steam at any suitable pressure may be provided to the stripping column290. In the stripping column290, the second hot liquid hydrocracked stream in line289may be stripped to provide the overhead vapor hydrocracked stream in line292and a stripped second hot liquid hydrocracked stream in line294. The overhead vapor hydrocracked stream in line292may be passed in line164along with the cold liquid stream in line162to the cold stripper column170aof the first stage stripper170. The stripped second hot liquid hydrocracked stream in line294may be passed to the second stage fractionation column310. In an exemplary embodiment, the stripped second hot liquid hydrocracked stream in line294may be heated in a feed heater295to provide a heated second hot liquid hydrocracked stream in line296. The heated second hot liquid hydrocracked stream in line296may be fractionated in the second stage fractionation column310. The second stage fractionation column310separates the heated second hot liquid hydrocracked stream in line296to provide a fractionator overhead stream comprising naphtha in line312and a bottoms stream comprising UCO in line311. Compared to high quality first UCO stream186obtained from the first stage of the hydrocracking unit101, the UCO obtained from the second stage fractionation column310may require heavy polynuclear aromatic (HPNA) removal. In an aspect, a UCO drag stream, which may be managed for HPNA removal, may be withdrawn in line313from the bottoms stream comprising UCO in line311. The stripping media in line297provided to the second stage fractionation column310strips the light material and helps in HPNA removal. In an exemplary embodiment, the UCO drag stream comprising HPNA's in line313may range from about 0 wt % to about 5 wt % of the fresh feed stream102or from about 0.1 wt % to about 2 wt % of the fresh feed stream102or from about 0.2 wt % to about 1 wt % of the fresh feed stream102. The remaining bottoms stream may be recycled to the hydrocracking unit101to maximize overall unit conversion and naphtha production. In another aspect, the remaining bottoms stream is withdrawn as the second unconverted oil stream in line314and may be recycled to the second stage hydrocracking reactor280. In the second stage fractionation column310, a second stage diesel stream and/or a second stage kerosene stream may also be separated from the heated second hot liquid hydrocracked stream in line296. In an exemplary embodiment, the second stage diesel stream and/or the second stage kerosene stream from the second stage fractionation column310may be withdrawn in the combined side draw stream in line319. The combined side draw stream in line319may be passed to the first stage fractionation column180along with the first liquid hydrocracked stream in line177, and the heated second liquid hydrocracked stream in line262. The fractionator overhead stream comprising naphtha in line312may be cooled and separated in a receiver315to provide a condensed liquid overhead stream in line316. A portion of the condensed liquid overhead stream in line316may be recycled to the second stage fractionation column310in a reflux line318. A net condensed liquid overhead stream is withdrawn as a second stage overhead naphtha stream in line317from the second stage fractionation column310. The second stage overhead naphtha stream in line317may be passed to the debutanizer column240. The present process also provides integration of the first and the second stage in a manner to maximize the recovery of desired hydrocarbons and minimizing the equipment in the hydrocracking unit101. As shown in the FIGURE, the second hot separated vapor hydrocracked stream in line285from the second stage hot separator284is mixed with first hot separated vapor hydrocracked stream in line142from the first stage hot separator140to minimize reactor section equipment. Similarly hot flash vapor from both stages i.e. the first hot vapor hydrocracked stream in152and the second hot vapor hydrocracked stream in288are combined and processed together. Second stage hot flash liquid i.e. the second hot liquid hydrocracked stream in line289is then independently stripped of dissolved light components. Any of the above lines, conduits, units, devices, vessels, surrounding environments, zones or similar may be equipped with one or more monitoring components including sensors, measurement devices, data capture devices or data transmission devices. Signals, process or status measurements, and data from monitoring components may be used to monitor conditions in, around, and on process equipment. Signals, measurements, and/or data generated or recorded by monitoring components may be collected, processed, and/or transmitted through one or more networks or connections that may be private or public, general or specific, direct or indirect, wired or wireless, encrypted or not encrypted, and/or combination(s) thereof; the specification is not intended to be limiting in this respect. Further, the FIGURES show one or more exemplary sensors such as 11, 21, 31, 41, and 51 located on one or more conduits. Nevertheless, there may be sensors present on every stream so that the corresponding parameter(s) can be controlled accordingly. Signals, measurements, and/or data generated or recorded by monitoring components may be transmitted to one or more computing devices or systems. Computing devices or systems may include at least one processor and memory storing computer-readable instructions that, when executed by the at least one processor, cause the one or more computing devices to perform a process that may include one or more steps. For example, the one or more computing devices may be configured to receive, from one or more monitoring component, data related to at least one piece of equipment associated with the process. The one or more computing devices or systems may be configured to analyze the data. Based on analyzing the data, the one or more computing devices or systems may be configured to determine one or more recommended adjustments to one or more parameters of one or more processes described herein. The one or more computing devices or systems may be configured to transmit encrypted or unencrypted data that includes the one or more recommended adjustments to the one or more parameters of the one or more processes described herein. Specific Embodiments While the following is described in conjunction with specific embodiments, it will be understood that this description is intended to illustrate and not limit the scope of the preceding description and the appended claims. A first embodiment of the present disclosure is a hydrocracking process for maximization of naphtha while producing base oil comprising hydrocracking a hydrocarbon feed stream in a hydrocracking unit in the presence of a hydrogen stream and a hydrocracking catalyst to produce a hydrocracked effluent stream; separating the hydrocracked effluent stream in a separator to provide a vapor hydrocracked stream and a liquid hydrocracked stream; fractionating the liquid hydrocracked stream to provide a naphtha stream, a kerosene stream, a diesel stream and a first unconverted oil stream; recycling a recycle stream comprising one, two or all of a portion of the kerosene stream, a portion of the diesel stream, and a portion of the first unconverted oil stream, to the hydrocracking unit to provide a second unconverted oil stream; and withdrawing a remaining portion of the first unconverted oil stream for base oil production. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein the separating step further comprises stripping a hot liquid hydrocracked stream and a cold liquid stream to provide the liquid hydrocracked stream. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein the hydrocracking unit is a two stage hydrocracking unit comprising a first stage hydrocracking reactor in which the hydrocarbon feed stream is hydrocracked and a second stage hydrocracking reactor to which the recycle stream is recycled. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein the separating step further comprises passing the hydrocracked effluent stream to a hot separator to provide a hot separated vapor hydrocracked stream and a hot separated liquid hydrocracked stream; and separating the hot separated liquid hydrocracked stream to provide a hot vapor hydrocracked stream and the hot liquid hydrocracked stream. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein separating the hot separated liquid hydrocracked stream comprises passing the hot separated vapor hydrocracked stream to a cold separator to provide a cold separated vapor hydrocracked stream and a cold separated liquid hydrocracked stream; separating the cold separated liquid hydrocracked stream and the hot vapor hydrocracked stream to provide a cold vapor stream and the cold liquid stream; stripping the cold liquid stream and the hot liquid hydrocracked stream to provide the liquid hydrocracked stream; and fractionating the liquid hydrocracked stream to provide the naphtha stream, the kerosene stream, the diesel stream and the first unconverted oil stream. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein the stripping step further comprises fractionating the liquid hydrocracked stream in a fractionation column to provide a first side draw stream comprising a naphtha stream, a second side draw stream comprising the kerosene stream and a third side draw stream comprising the diesel stream; and optionally stripping the first side draw stream, the second side draw stream and the third side draw stream to provide a stripped naphtha stream, the kerosene stream and the diesel stream. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein the first stage hydrocracking reactor comprises distillate selective catalyst and the second stage hydrocracking reactor comprises naphtha selective catalyst. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, further comprising hydrocracking the hydrocarbon feed stream in the first stage hydrocracking reactor in the presence of a first hydrogen stream and a distillate selective catalyst to produce a first hydrocracked effluent stream; passing the first hydrocracked effluent stream to a first stage hot separator to provide a first hot separated vapor hydrocracked stream and a first hot separated liquid hydrocracked stream; passing the first hot separated vapor hydrocracked stream and a second hot separated vapor hydrocracked stream to the cold separator to provide the cold separated vapor hydrocracked stream and the cold separated liquid hydrocracked stream; separating the first hot separated liquid hydrocracked stream in a first stage hot flash drum to provide a first hot vapor hydrocracked stream and a first hot liquid hydrocracked stream; passing the first hot vapor hydrocracked stream, the first cold separated liquid hydrocracked stream, and a second hot vapor hydrocracked stream to the cold flash drum to provide a cold vapor stream and the cold liquid stream; and stripping the cold liquid stream, an overhead vapor hydrocracked stream, and the first hot liquid hydrocracked stream to provide the liquid hydrocracked stream. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein the stripping step comprises passing the cold liquid stream and the overhead vapor hydrocracked stream to a first stage stripper, wherein the first stage stripper comprises a cold stripper and a hot stripper; stripping the cold liquid stream and the overhead vapor hydrocracked stream in the cold stripper to provide a first liquid hydrocracked stream and a first stage overhead naphtha stream; and stripping the first hot liquid hydrocracked stream in the hot stripper to provide a second liquid hydrocracked stream; the stripping steps further comprise passing the first liquid hydrocracked stream and the second liquid hydrocracked stream to a first stage fractionation column, wherein the first liquid hydrocracked stream is passed at a location above the second liquid hydrocracked stream into the first stage fractionation column; passing a combined side draw stream to the first stage fractionation column at a location above the second liquid hydrocracked stream and below the first liquid hydrocracked stream in the first stage fractionation column; and fractionating the first liquid hydrocracked stream, the second liquid hydrocracked stream, and the combined side draw stream in the first stage fractionation column to provide the naphtha stream, the kerosene stream, the diesel stream and the first unconverted oil stream. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, further comprising hydrocracking the recycle stream and the second unconverted oil stream in the second stage hydrocracking reactor in the presence of the naphtha selective catalyst and a second hydrogen stream to produce a second hydrocracked effluent stream; passing the second hydrocracked effluent stream to a second stage hot separator to provide a second hot separated vapor hydrocracked stream and a second hot separated liquid hydrocracked stream; separating the second hot separated liquid hydrocracked stream in a second stage hot flash drum to provide a second hot vapor hydrocracked stream and a second hot liquid hydrocracked stream; and fractionating the second hot liquid hydrocracked stream in a second stage fractionation column to provide a second stage overhead naphtha stream, a second stage kerosene stream or a second stage diesel stream, and the second unconverted oil stream. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein fractionating the second hot liquid hydrocracked stream further comprises combining the second unconverted oil stream with the recycle stream to provide a combined recycle stream; and recycling the combined recycle stream to the second stage hydrocracking reactor An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein the second stage kerosene stream and the second stage diesel stream are withdrawn as the combined side draw stream from the second stage fractionation column. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein fractionating the second hot liquid hydrocracked stream further comprises stripping the second hot liquid hydrocracked stream to provide the overhead vapor hydrocracked stream and a stripped second hot liquid hydrocracked stream; and fractionating the stripped second hot liquid hydrocracked stream in the second stage fractionation column to provide the second stage overhead naphtha stream, the second stage kerosene stream or the second stage diesel stream, and the second unconverted oil stream. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein the second unconverted oil stream has a viscosity index of about 100 to about 150. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein the portion of the first unconverted oil stream recycled to the hydrocracking reactor ranges from about 0 wt % to about 75 wt % of the first unconverted oil stream. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the first embodiment in this paragraph, wherein fractionating the stripped second hot liquid hydrocracked stream further comprises passing the second stage overhead naphtha stream, the first stage overhead naphtha stream and the naphtha stream to a debutanizer column to provide an overhead stream comprising LPG and a bottoms stream comprising naphtha; passing the bottoms stream to a naphtha splitter column to provide an overhead stream comprising light naphtha and a splitter bottoms stream comprising heavy naphtha; and combining the splitter bottoms stream with the stripped naphtha stream to provide a heavy naphtha stream. A second embodiment of the present disclosure is a hydrocracking process for maximization of naphtha while producing base oil, comprising hydrocracking a hydrocarbon feed stream in a first stage hydrocracking reactor in the presence of a first hydrogen stream and a distillate selective catalyst to produce a first hydrocracked effluent stream; separating the first hydrocracked effluent stream to provide a vapor hydrocracked stream and a liquid hydrocracked stream; fractionating the liquid hydrocracked stream to provide a naphtha stream, a kerosene stream, a diesel stream and a first unconverted oil stream; recycling a portion of the kerosene stream, a portion of the diesel stream, and a portion of the first unconverted oil stream, to a second stage hydrocracking reactor; hydrocracking a second unconverted oil stream and a recycle stream comprising a portion of the kerosene stream, a portion of the diesel stream, and a portion of the first unconverted oil stream in the second stage hydrocracking reactor in the presence of the naphtha selective catalyst and a second hydrogen stream to produce a second hydrocracked effluent stream; separating the second hydrocracked effluent stream to provide the second unconverted oil stream; and withdrawing a remaining portion of the first unconverted oil stream for base oil production. An embodiment of the present disclosure is one, any or all of prior embodiments in this paragraph up through the second embodiment in this paragraph, wherein the first hydrocracked effluent stream is separated in a first stage hot separator and the second hydrocracked effluent stream is separated in a second stage hot separator. A third embodiment of the present disclosure is a hydrocracking process for maximization of naphtha while producing base oil, comprising hydrocracking a hydrocarbon feed stream in a hydrocracking unit to produce a first hydrocracked effluent stream; passing the first hydrocracked effluent stream to a first stage hot separator to provide a first hot separated vapor hydrocracked stream and a first hot separated liquid hydrocracked stream; passing the first hot separated liquid hydrocracked stream to a first stage hot flash drum to provide a first hot vapor hydrocracked stream and a first hot liquid hydrocracked stream; passing the first hot vapor hydrocracked stream, a second hot vapor hydrocracked stream and a cold separated liquid hydrocracked stream to a cold flash drum to provide a cold liquid stream; fractionating the cold liquid stream and the first hot liquid hydrocracked stream to provide a naphtha stream, a kerosene stream, a diesel stream and a first unconverted oil stream; hydrocracking a second unconverted oil stream and a recycle stream comprising a portion of the kerosene stream, a portion of the diesel stream, and a portion of the first unconverted oil stream in the hydrocracking unit to produce a second hydrocracked effluent stream; passing the second hydrocracked effluent stream to a second stage hot separator to provide the second hot separated vapor hydrocracked stream and a second hot separated liquid hydrocracked stream; passing the second hot separated liquid hydrocracked stream to a second stage hot flash drum to provide the second hot vapor hydrocracked stream and a second hot liquid hydrocracked stream; fractionating the second hot liquid hydrocracked stream to provide the second unconverted oil stream; and withdrawing a remaining portion of the first unconverted oil stream for base oil production. Without further elaboration, it is believed that using the preceding description that one skilled in the art can utilize the present disclosure to its fullest extent and easily ascertain the essential characteristics of this disclosure, without departing from the spirit and scope thereof, to make various changes and modifications of the present disclosure and to adapt it to various usages and conditions. The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limiting the remainder of the disclosure in any way whatsoever, and that it is intended to cover various modifications and equivalent arrangements included within the scope of the appended claims. In the foregoing, all temperatures are set forth in degrees Celsius and, all parts and percentages are by weight, unless otherwise indicated. | 83,981 |
11859143 | DETAILED DESCRIPTION OF THE INVENTION Jet fuel or aviation fuel is fuel aimed for use in aircrafts powered by gas-turbine engines. Jet fuel needs to fulfil certain physical properties in order to be classified as jet fuel. The standards for definition of jet fuel include at least DEF STAN 91-091 (2018), ASTM D1655-19 (JetA-1) and ASTM D7566-19. One of the most important properties for jet fuel is the freezing point. The freezing point is a measurement of the temperature at which visible solid fuel wax particles disappear on warming a composition, which has been cooled to a temperature where visible particles occur. The freezing point of a Jet A (ASTM D1655-19) standard jet fuel must be at least −40° C. and for a Jet A-1 fuel at least −47° C. Density is another important property for any fuel but especially for jet fuel. A low freezing point of a hydrocarbon composition is typically associated with hydrocarbons with lower carbon number. Hydrocarbons with lower carbon number also have a lower density. A short chain length paraffin therefore has a lower density compared to a paraffin with longer chain length. However, the freezing point is lower for short chain paraffins. Surprisingly, it has now been achieved a hydrocarbon composition with a high density and a low freezing point that satisfy the jet fuel specification. This is achieved setting certain cut-off points for the distillation curve of the hydrocarbon composition. An embodiment of the current invention is therefore a hydrocarbon composition comprising isomerised paraffins with certain cut-off points for the distillation curve and high density. The freezing point of the hydrocarbon composition fulfils the jet fuel specification of equal to or lower than −40° C. With a hydrocarbon composition is hereby meant a composition comprising mainly hydrocarbons i.e. organic molecules containing only carbon and hydrogen atoms. The hydrocarbon composition can contain minor amounts of molecules containing heteroatoms such as sulphur. The hydrocarbon composition according to the invention is useful as a fuel component, especially as a jet fuel component. The hydrocarbon composition according to the current invention comprised isomerised paraffins (or i-paraffins or iso-paraffins). With isomerised paraffins is hereby meant paraffins with one or more side chain. The side chains are typical in form of methyl, ethyl and propyl substituents and can be situated anywhere on the paraffin chain. The isomerised paraffins can be produced by isomerisation of normal paraffins (n-paraffins). The isomerised paraffins can be from any source. Non-limiting examples of sources for the isomerised paraffins are hydrocarbons produced by hydrodeoxygenation of fatty acids or hydrocarbons produced in a Fischer-Tropsh process. The invention relates to a hydrocarbon composition, which has a T10 (° C.) cut-off temperature from 185° C. to 205° C. The T90 (° C.) cut-off temperature of the composition is from 270° C. to 295° C. and the final boiling point (° C.) is from 275° C. to 300° C. The distillation conditions and properties of the collected fraction vary with the process used for producing the isomerised paraffins and the renewable source used. The person skilled in the art is well familiar with various distillation and fractionation processes and can optimize the conditions needed to obtain the hydrocarbon composition according to the invention. The hydrocarbon composition according to the invention has a density from 768.0 kg/m3to 772.0 kg/m3. In another embodiment of the invention the density of the hydrocarbon composition is from 770.0 kg/m3to 772.0 kg/m3, and in yet another embodiment from 771.0 kg/m3to 772.0 kg/m3. The density ranges shall be interpreted to include the density equal to the endpoints of the ranges. It should be noted that even a small increase in the density of the hydrocarbon composition is significant. A higher density of the composition means there is more energy and higher heat value (caloric value) per volume. This is significant especially in fuel applications, since the volume of the fuel tanks are always limited. In addition, higher density also gives benefits when the hydrocarbon composition is blended with other components. For example, the minimum density for jet fuel is 775 kg/m3(ASTM D7566) and if the density of the renewable component is higher then there is more flexibility for the density of the petroleum based jet fuel component. The density of the hydrocarbon composition can be measured using any standardised method for measuring density of a hydrocarbon fuel composition, such as ASTM D4052. In one embodiment the invention the hydrocarbon composition comprises hydrocarbons with an average carbon number from 14.3 to 15.1. In another embodiment the average carbon number of the hydrocarbons in the composition is from 14.5 to 15.1 and in yet another embodiment from 14.7 to 15.0. The carbon number ranges shall be interpreted to include hydrocarbons with carbon number equal to the endpoints of the ranges. Average carbon number for the hydrocarbons in the hydrocarbon composition is measured using a gas chromatography (GC) method. The conditions for the GC method are listed below in table 1. TABLE 1GC settings for determination of carbon numberof hydrocarbonsGCInjectionsplit/splitless-injectorSplit 80:1 (injection volume 0.2 μL)ColumnDB ™-5 (length 30 m, i.d. 0.25 mm,phase thickness 0.25 μm)Carrier gasHeDetectorFID (flame ionization detector)GC30° C. (2 min)-5° C./min-300° C.program(30 min), constant flow 1.1 mL/min It was surprisingly found that a hydrocarbon composition with high carbon number and high density could be achieved without sacrificing the freezing point of the hydrocarbon composition. Higher carbon numbers typically mean lower freezing points. According to an embodiment of the invention the amounts of hydrocarbons in the hydrocarbon composition having a carbon number from 14 to 17 is at least 60 wt-% of the whole hydrocarbon content. According to an embodiment of the invention the hydrocarbon composition comprises isomerised paraffins over 90 wt-%, preferably over 92 wt-% and most preferably over 95 wt-% as calculated from the total paraffinic content of the hydrocarbon composition. The isomerised paraffins are mainly mono-, di or tri-isomerised, but some paraffins can have even more side chains. The isomerised paraffins can be methyl-, ethyl- or propyl-substituted. The hydrocarbons composition according to the invention is a mixture of various isomerised paraffins. It should be noted that the carbon number does not vary with the degree of isomerisation or the type of side chain. The numbers of carbons in the paraffin remains the same. The high degree of isomerisation enables the unique properties of the hydrocarbon composition of the invention. Typically, a higher degree of isomerisation means lower freezing points. However, the isomerisation degree in itself does not sufficiently explain the low freezing point and high density of the hydrocarbon composition of the invention. In one aspect of the invention the hydrocarbon composition has a freezing point of −40° C., or preferably −43° C. or below. Typically, the lowest freezing point can be −60° C. The lowest measurable freezing point is −80° C. It is required that the freezing point of a composition to be used as a jet fuel is −40° C. or lower. The freezing point of aviation fuel is measured according to the standard IP529. It is obviously highly crucial that a jet fuel remains pumpable in all possible conditions to ensure that the aircraft gas-turbine engine is fully functional. Especially, when a jet fuel or jet fuel component is produced from a biological or renewable source it can sometimes be difficult to reach the low freezing points required. This is especially difficult in paraffinic renewable fuel components, where the overall yield is important. In one aspect of the invention the hydrocarbon composition is produced from a renewable source (renewable raw material). Here, the term renewable source or renewable raw material is meant to include feedstocks other than those obtained from petroleum crude oil (fossil-based oil or petroleum based oil). The renewable source that can be used in the present invention include, but is not limited to, bio oils and fats from plants and/or animals and/or fish and/or insects, and from processes utilizing microbes, such as algae, bacteria, yeasts and moulds, and suitable are also compounds derived from said fats and oils and mixtures thereof. The species yielding the bio oils or fats may be natural or genetically engineered. The bio oils and fats may be virgin oils and fats or recycled oils and fats. Suitable bio oils containing fatty acids and/or fatty acid esters and/or fatty acid derivatives are wood-based and other plant-based and vegetable-based fats and oils such as rapeseed oil, colza oil, canola oil, tall oil, jatropha seed oil, sun-flower oil, soybean oil, hempseed oil, olive oil, linseed oil, mustard oil, palm oil, pea-nut oil, castor oil, coconut oil, as well as fats contained in plants bred by means of gene manipulation, animal-based fats such as lard, tallow, train oil, and fats contained in milk, as well as recycled fats of the food industry and mixtures of the above, as well as fats and oils originating from processes utilizing microbes, such as algae, bacteria, yeasts and moulds. The renewable source also includes recyclable waste oils and fats or residues of recyclable waste oils and fats. Bio oil and fat suitable as fresh feed may comprise C12-C24 fatty acids, derivatives thereof such as anhydrides or esters of fatty acids as well as triglycerides and diglycerides of fatty acids or combinations of thereof. Fatty acids or fatty acid derivatives, such as esters may be produced via hydrolysis of bio oils or by their fractionalization or transesterification reactions of triglycerides or microbiological processes utilizing microbes. The isomerised paraffins of the hydrocarbon composition according to the current invention can be produced by any suitable method. In one embodiment the paraffins are produced from renewable oil, such as vegetable oil or animal fat, which is subjected to a deoxygenation process for removal of heteroatoms, mainly oxygen from the renewable oil. In a preferred embodiment, the deoxygenation treatment, to which the renewable raw material is subjected, is hydrotreatment. Preferably, the renewable raw material is subjected to hydrodeoxygenation (HDO) which preferably uses an HDO catalyst. Catalytic HDO is the most common way of removing oxygen and has been extensively studied and optimized. However, the present invention is not limited thereto. As the HDO catalyst, an HDO catalyst comprising hydrogenation metal supported on a carrier may be used. Examples include an HDO catalyst comprising a hydrogenation metal selected from a group consisting of Pd, Pt, Ni, Co, Mo, Ru, Rh, W or a combination of these. Alumina or silica is suited as a carrier, among others. The hydrodeoxygenation step may, for example, be conducted at a temperature of 100-500° C. and at a pressure of 10-150 bar (absolute). In an embodiment, the isomerised paraffins component is produced through Fischer-Tropsch process starting from gasification of biomass. This synthesis route is generally also called BTL, or biomass to liquid. It is well established in the literature that biomass, such as lignocellulosic material, can be gasified using oxygen or air in high temperature to yield a gas mixture of hydrogen and carbon monoxide (syngas). After purification of the gas, it can be used as feedstock for a Fischer-Tropsch synthesis route. In the Fischer-Tropsch synthesis paraffins are produced from syngas. The Fischer-Tropsch paraffins range from gaseous component to waxy paraffins and middle distillate boiling range paraffins can be obtained by distillation from the product. The n-paraffins formed either through hydrotreating renewable oils or Fischer-Tropsch method need to be subjected to a further isomerisation treatment. The isomerisation treatment causes branching of hydrocarbon chains, i.e. isomerisation, of the hydrotreated raw material. Branching of hydrocarbon chains improves cold properties, i.e. the isomeric composition formed by the isomerisation treatment has better cold properties compared to the hydrotreated raw material. Better cold properties refer to a lower temperature value of a freezing point. The isomeric hydrocarbons, or isomerised paraffins, formed by the isomerisation treatment may have one or more side chains, or branches. The isomerisation step may be carried out in the presence of an isomerisation catalyst, and optionally in the presence of hydrogen added to the isomerisation process. Suitable isomerisation catalysts contain a molecular sieve and/or a metal selected from Group VIII of the periodic table and optionally a carrier. Preferably, the isomerisation catalyst contains SAPO-11, or SAPO-41, or ZSM-22, or ZSM-23, or fernerite, and Pt, Pd, or Ni, and Al2O3, or SiO2. Typical isomerisation catalysts are, for example, Pt/SAPO-11/Al2O3, Pt/ZSM-22/Al2O3, Pt/ZSM-23/Al2O3, and Pt/SAPO-11/SiO2. The catalysts may be used alone or in combination. The presence of added hydrogen is particularly preferable to reduce catalyst deactivation. In a preferred embodiment, the isomerisation catalyst is a noble metal bifunctional catalyst, such as Pt-SAPO and/or Pt-ZSM-catalyst, which is used in combination with hydrogen. The isomerisation step may, for example, be conducted at a temperature of 200-500° C., preferably 280-400° C., and at a pressure of 5-150 bar, preferably 10-130 bar, more preferably 30-100 bar (absolute). The isomerisation step may comprise further intermediate steps such as a purification step and a fractionation step. The isomerisation may be performed e.g. at 300° C. to 350° C. In an embodiment of the invention the isomerised paraffins formed in the isomerisation process need to be fractionated in order to get a hydrocarbon composition according to the invention. Fractionation of the isomerised paraffins is not necessary if the formed isomerised paraffins fulfils the requirements of the hydrocarbon composition according to the invention. The fractionation can be performed using any suitable method and is not limited to distillation. Distillation is the most commonly used method for separating various fractions from hydrocarbon compositions and is also suitable here. According to another aspect the invention also relates to a fuel or a fuel component comprising a hydrocarbons composition according to the invention. In another aspect of the invention the fuel or fuel component is a jet fuel or jet fuel component. In one aspect the invention concerns a jet fuel containing a hydrocarbon component according to the invention in a content up to 50 vol. % of the jet fuel and the balance being petroleum based jet fuel. Preferably the jet fuel contains a hydrocarbon component according to the invention in a concentration from 3 vol. % to 50 vol. %, more preferably from 5 vol. % to 45 vol. % and even more preferably from vol. % to 30 vol. %. The balance in the jet fuel according to the invention being petroleum based jet fuel. With the term “petroleum based jet fuel” is meant any conventional jet fuel or aviation fuel produced from petroleum or crude oil that fulfils at least one specification for jet fuels. Specifications for jet fuel or aviation fuel include but are not limited to Jet A, Jet A-1 (DEF STAN 91-91, ASTM D1655) and various military standards (JP-1 to JP-8). According to another aspect the invention also relates to a method to produce a hydrocarbon composition according to the invention. The method to produce the hydrocarbon composition comprises the following method steps:providing a renewable feedstock comprising fatty acids,deoxygenating the feedstock to produce paraffins,subjecting the produced paraffins to an isomerisation step to produce isomerised paraffins, andfractionating the produced isomerised paraffins to obtain a hydrocarbon composition according to the invention. In an embodiment the fractionation comprises fractionating the produced isomeric paraffins according to the invention as one single fraction with yield of at least 20 wt-%, preferably with yield of at least 30 wt-%, most preferably with yield of at least 40 wt-%. EXAMPLES Example 1 (Comparative) A renewable paraffinic product was produced by heavily cracking hydrodeoxygenation and isomerisation of feedstock mixture of vegetable and animal fat origin. This product was analysed using various analysis methods (Table 2). TABLE 2Analysed renewable paraffinic product.AnalysisMethodUnitValueFreezing pointIP529° C.−42.0DensityASTMkg/m3753.0D4052Weighted averageNM490—12.0carbon number% carbon number 14-17NM490wt-%30.5T10 (° C.) cut-off temperatureASTM D86° C.168.5T90 (° C.) cut-off temperatureASTM D86° C.245.5Final boiling pointASTM D86° C.256.0 The analysed product in Table 2 fulfils the freezing point of jet fuel specification, but the freezing point is not exceptionally low. Example 2 (Comparative) A renewable paraffinic product was produced by hydrodeoxygenation and isomerisation of feedstock mixture of vegetable and animal fat origin. This product was analysed using various analysis methods (Table 3). TABLE 3Analysed renewable paraffinic product.AnalysisMethodUnitValueFreezing pointIP529° C.−41.0DensityASTMkg/m3774.1D4052Weighted averageNM490—15.6carbon number% carbon number 14-17NM490wt-%55.8T10 (° C.) cut-off temperatureASTM D86° C.210.0T90 (° C.) cut-off temperatureASTM D86° C.289.0Final boiling pointASTM D86° C.308.2 The analysed product in Table 3 fulfils the freezing point of jet fuel specification, but the freezing point is not exceptionally low. Example 3 A renewable paraffinic product produced by hydrodeoxygenation and isomerisation of feedstock mixture of vegetable and animal fat origin in Example 2 is further directed to a fractionation unit. In the fractionation unit, the renewable paraffinic product is divided into two fractions. Lighter of the fractions containing 80 wt-% of the original renewable paraffinic product is re-analysed using various analysis methods (Table 4). TABLE 4Analysed renewable paraffinic product.AnalysisMethodUnitValueFreezing pointIP529° C.−49.1DensityASTMkg/m3771.6D4052Weighted averageNM490—15.0carbon number% carbon number 14-17NM490wt-%68.5T10 (° C.) cut-off temperatureASTM D86° C.198.6T90 (° C.) cut-off temperatureASTM D86° C.280.3Final boiling pointASTM D86° C.287.5 This analysed product fulfils all requirements of a high-quality renewable aviation fuels. From the analysis results it can be seen that when the density of the paraffinic product is below 772 kg/m3(measured 771.6 kg/m3) the freezing point drops significantly to −49.1° C., compared to the product of comparative example 2. Example 4 Another renewable paraffinic product produced by hydrodeoxygenation and isomerisation of another feedstock mixture of vegetable and animal fat origin is further directed to a fractionation unit. In the fractionation unit, the renewable paraffinic product is divided into two fractions. Lighter of the fractions containing 80 wt-% of the original renewable paraffinic product is re-analysed using various analysis methods (Table 5). TABLE 5Analysed renewable paraffinic product.AnalysisMethodUnitValueFreezing pointIP529° C.−50.9DensityASTMkg/m3770.1D4052Weighted averageNM490—14.7carbon number% carbon number 14-17NM490wt-%73.6T10 (° C.) cut-off temperatureASTM D86° C.191.9T90 (° C.) cut-off temperatureASTM D86° C.276.6Final boiling pointASTM D86° C.283.1 This product also fulfils all requirements of a high-quality renewable aviation fuels. From the analysis results it can be seen that despite the fact that the density of the paraffinic composition is over 768 kg/m3(measured 770.1 kg/m3) the freezing point (measured −50.9° C.) is significantly lower than the freezing point of the product of comparative example 1. It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described above but may vary within the scope of the claims. | 20,396 |
11859144 | DETAILED DESCRIPTION Various changes and various embodiments may be made in the present disclosure, such that specific embodiments are illustrated and described in detail in the detailed description. It should be understood, however, that it is not intended to limit the present disclosure to the particular disclosed forms, but includes all modifications, equivalents, and alternatives falling within the sprit and technical scope of the present disclosure. The terminology used in the present disclosure is merely for the purpose of describing particular embodiments, and is not intended to limit the present disclosure. The singular forms may include plural forms, unless the phrases clearly indicate the opposite. In the present disclosure, it should be understood that the term “comprising”, “having”, or the like specifies the presence of the characteristic, integer, step, operation, component, part, or a combination thereof described in the specification, and does not exclude the presence or addition possibility of one or more other characteristics, integers, steps, operations, components, parts or combinations thereof in advance. First, a configuration of an operating guide system1000of a coal gasification plant according to an exemplary embodiment will be described with reference to FIG.1. The operating guide system100includes a fuel determiner110, an operating condition deriver120, a start guide generator130, a performance analyzer140, an operation guide generator150, an action guide generator160, an operation recorder170, a stop guide generator180, and a storage200. The fuel determiner110determines gasification suitability of an analysis target fuel as selected by a user before the plant is started, where gasification suitability includes basic suitability and suitability of slag behavior. To do so, the fuel determiner110first determines the basic suitability and the suitability of slag behavior based on whether the analysis target fuel satisfies a predetermined reference value of the basic attributes and a predetermined reference value of the slag attributes. Here, the basic attributes may include coal heating amount (MJ/kg, MAF), volatile matter (wt %, MF), Cl+F (wt %, AR), Fe2O3 (wt %, ash), Na2O+K2O (wt %, ash), S/A ratio (w/w, ash), and ash (wt %, ME); and the slag attributes may include coal conversion temperature (° C.), total slag thickness (mm), liquidus slag thickness (mm), average slag viscosity (poise), and muffle length versus slag thickness (%). In an exemplary embodiment, when the analysis target fuel contains coal and flux, the fuel determiner110may determine the gasification suitability based on a flux input ratio input by the user. In another exemplary embodiment, when the primary coal and at least one target coal added to the primary coal are selected by the user, the fuel determiner110determines the gasification suitability while increasing a mixing ratio of the target coal at a predetermined ratio (e.g., 10 wt %). Particularly, the fuel determiner110may output by classifying whether the analysis target fuel is coal which may be used without flux or mixed coal, coal which may be used when the flux is input, coal which requires the mixed coal, or coal which is not suitable for gasification based on the aforementioned determination results of the gasification suitability. When the fuel is selected, the operating condition deriver120derives operating conditions. The derived operating conditions include upper limit and lower limit temperatures of the slag and upper limit and lower limit temperatures of the gasifier. The operating condition deriver120derives the slag temperature limits by analyzing the crystal phase and the solid-liquid equilibrium temperature of the slag based on a composition ratio of components containing SiO2, Al2O3, CaO, and FeO among the slag composition of the selected fuel, and derives the gasifier temperature limits based on the derived slag temperature limits. The start guide generator130derives a start-up table including supply fuel input amount and main control factor state values for each step of the start-up process including coal burner stages of start, ready, and load up when the plant is started. At this time, the start guide generator130may derive the supply fuel input amount and the control factor state values for each step of the start-up process in the reverse order of the start-up process based on the supply fuel input amount of 100% load. In addition, the start guide generator130may provide a guide based on the starting of the plant by outputting the derived supply fuel input amount and control factor state values for each step of the start-up process. The performance analyzer140basically performs the performance analysis of the plant including gasifier performance and synthesis gas cooler performance during the operation of the plant. The operation guide generator150provides an operating guide indicating control values for operating the plant based on the performance analyzed by the performance analyzer140. When the occurrence of an abnormal situation is predicted in the plant based on the performance analysis, the action guide generator160provides an action guide indicating control values capable of controlling the plant so that the predicted abnormal situation is eliminated. The operation recorder170may continuously store the control values based on the operation and action of the plant based on the user input, and provide the stored control values as the operating history. When the stop event occurs during the operation of the plant, the stop guide generator180provides a stop guide indicating control values for each step of a plurality of steps which stop the plant. The storage200stores various data as a database according to an exemplary embodiment. Next, an operating guide method of the coal gasification plant according to an exemplary embodiment will be described with reference toFIG.2. Referring toFIG.2, the operating guide system determines fuels such as coal and mixed coal in S100before the plant is started, and derives operating conditions after determining the gasification suitability for the determined fuel. Next, the operating guide system derives a start-up table based on the operating conditions derived in S200while the plant is started, and provides a guide based on the starting of the plant. Subsequently, after the plant is started, the operating guide system analyzes the performance of the plant including the gasifier in S300during operation, provides the guide necessary for the operation and control based on the analyzed performance, and provides the guide for the alarm and action by the occurrence of the abnormal situation or the like as necessary. Meanwhile, the operating guide system provides the guide necessary for stopping the plant in S400, when the operation is stopped. The step S100of FIG. will be described with reference toFIG.3. Referring toFIG.3, the fuel determiner110may select an analysis target fuel based on the user input in S110. At this time, the user may select a coal which may be used without flux or mixed coal, select coal and flux, or select mixed coal. Next, the fuel determiner110determines the gasification suitability for the analysis target fuel selected by the user in S120. The gasification suitability includes basic suitability and suitability of slag behavior. That is, the fuel determiner110determines the basic suitability and the suitability of slag behavior of the analysis target fuel based on whether the analysis target fuel satisfies the predetermined reference value of basic attributes (physical properties) and the predetermined reference value of slag attributes in S120. The basic attributes include, for example, coal heating amount (MJ/kg, MAF), volatile matter (wt %, MF), Cl+F (wt %, AR), Fe2O3(wt %, ash), Na2O+K2O (wt %, ash), S/A ratio (w/w, ash), ash (wt %, MF), and the like. In addition, the slag attributes include, for example, coal conversion temperature (° C.), slag250viscosity (poise) temperature (° C.), total slag thickness (mm), liquidus slag thickness (mm), average slag viscosity (poise), muffle length versus slag thickness (%), and the like. At this time, when the user selects coal and flux, the fuel determiner110determines the basic suitability of the coal and the suitability of the slag behavior based on the flux input ratio input by the user. In addition, in the case of mixed coal, when the primary coal selected by the user and at least one target coal added to the primary coal are selected, the fuel determiner110may analyze the mixed coal while increasing the mixing ratio of the target coal at a predetermined ratio (e.g., 10 wt %). Subsequently, the fuel determiner110outputs the determination results of the gasification suitability on a screen in S130. Here, the determination results of the fuel suitability based on the basic attributes and the slag attributes output by classifying whether it is fuel which may be used without flux or mixed coal, fuel which may be used when the flux is input, fuel which requires the mixed coal, or fuel which is not suitable for the gasification. For example, the determination results of the fuel suitability may be output by being classified as good, caution, or bad. Here, the good classification indicates that the analysis target fuel may be used for the corresponding plant, and the bad classification indicates that it is the fuel which is not suitable for the gasification. In addition, the caution classification indicates a state where the necessary flux input amount exceeds the flux input upper limit value of the corresponding plant or additional mixed coal is needed. The user may finally select the fuel based on the determination results of the gasification suitability. Otherwise, the steps S110to S130ofFIG.3are repeatedly performed. When the fuel is finally selected in S140, the step100proceeds to S150. The operating condition deriver120derives the optimum operating conditions of the gasifier of the fuel selected in S150. The optimum operating conditions of the gasifier include slag temperature limits, gasifier upper limit, lower limit, and proper operating temperatures, operation window, ash addition input and circulation rate, flux input rate and the type (limestone or silica), and the like. At this time, the operating condition deriver120may derive the slag temperature limits by analyzing the crystal phase and the solid-liquid equilibrium temperature of the slag based on the composition ratio of the components containing SiO2, Al2O3, CaO, and FeO among the slag composition of the selected fuel. In addition, the operating condition deriver120derives the upper limit, lower limit, and proper operating temperatures of the gasifier based on the slag temperature limits. Particularly, when operating the gasifier with the selected fuel, the operating condition deriver120may derive by analyzing that the slag mixed with the ash and flux of the fuel changes to liquidus slag at what temperature (° C.), and that the slag changes to what structure when the crystallization progresses to the solid. Accordingly, the operating condition deriver120may derive a change in the average viscosity of the liquidus flow slag based on the internal temperature of the gasifier, and derive the operation window of the gasifier for the selected fuel based on the above. Then, the operating condition deriver120outputs the gasifier optimum operating conditions previously derived through the screen in S160. The step S200ofFIG.2will be described with reference toFIG.4. Referring toFIG.4, the start guide generator130generates a start-up table based on the gasifier optimum operating conditions of the selected fuel previously derived in S210. To start the plant, the start guide generator130performs the start-up process composed of three step which includes coal burner stages of start, ready, and load up. The start-up table includes the supply fuel input amount and main control factor state values for each step of the coal burner stage. Specifically, the start-up table includes 1) the flux type and flow rate ratio considering operating temperature and type of coal; 2) the optimum supply fuel flow rate considering operating temperature and capacity; 3) the gasifier and post facility performance; and 4) the optimal feedstock flow rate and control factor values based on each of step of the coal burner stages of the start-up process. That is, the start guide generator130calculates the supply fuel flow rate and gasification performance necessary for each load-up step from starting the fuel and oxygen flow rate, which are necessary for operating a first coal burner, and gasification performance prediction values based on the previously derived gasifier optimum operating conditions of the selected fuel to the 100% operating load, and generates the start-up table based on the above. The start guide generator130derives the supply fuel input amount and the control factor state values for each step of the coal burner stages of the start-up in the reverse order (i.e., load up, ready, and start) of the start-up process based on the supply fuel input amount of 100% load. That is, the start guide generator130first determines the optimum supply fuel input amount of the load for each step by reducing the input amount based on the optimum supply fuel input amount of the 100% load which is derived through the gasifier performance prediction. In addition, the start guide generator130determines the oxygen input amount of the load for each step by reducing the oxygen input amount in a simple proportional formula based on the oxygen input amount of the 100% load. In addition, the start guide generator130determines the operating temperature for each load to be equal to the operating temperature of the 100% load, and also determines the steam/O2ratio and the flux/coal ratio for each load to be equal to the ratio of the 100% load. The start guide generator130provides the guide based on the starting of the plant by outputting the supply fuel input amount and the control factor state values for each step of the coal burner stages of the start-up based on the start-up table in S220. The step S300will be described with reference toFIG.5. The plant (gasification plant) according to an exemplary embodiment produces the synthesis gas by gasifying coal. That is, coal, flux, oxygen, steam, and nitrogen for transport are supplied to four coal burners installed under the gasifier during the operation of the plant, thereby producing the synthesis gas through pyrolysis-combustion-gasification processes. In an exemplary embodiment, a control is needed to set the operation window and to adjust an O2/coal ratio, steam/O2ratio, and the like so that during the operation of the plant, cold gas efficiency at which the coal energy is converted into the synthesis energy is increased, a carbon conversion rate is increased, and the molten slag is discharged smoothly. To this end, the operation guide generator150analyzes the plant performance in real time during the operation of the plant in S310. The plant performance analysis includes gasifier performance analysis and synthesis gas cooler performance analysis. The gasifier performance analysis includes gasification reaction analysis and internal heat transfer and heat balance analyses for the gasifier. The gasification reaction analysis uses an equilibrium reaction analysis model based on Gibbs energy minimization. At this time, the operation guide generator150determines the fraction set of the product in which the total Gibbs energy becomes a minimum at given temperature and pressure conditions based on the product in the gasification reaction analysis. The set of the product includes {CO, CO2, H2, H2O, H2S, COS, NH3, HCl, CH4, HCN, N2, Ar, C, S, Cl, O2} and calculates a total of sixteen types of product concentrations. The operation guide generator150calculates the heat input to the gasifier, the output heat, and the lost heat through the gasifier heat balance and heat transfer analyses. The synthesis gas cooler performance analysis includes outlet temperature prediction together with the synthesis gas cooler performance analysis. The operation guide generator150calculates synthesis gas temperature and Heat Duty for each heat exchange section of the synthesis gas cooler through the synthesis gas cooler performance analysis. In S320, it is determined whether the occurrence of an abnormal situation is predicted based on the plant performance analysis result. As the determination result in the S320, when the occurrence of the abnormal situation is not predicted, the step S300proceeds to S330, and when the occurrence of the abnormal situation is predicted, the step S300proceeds to S340. When the occurrence of the abnormal situation is not predicted, the operation guide generator150provides the operating guide based on the plant performance analysis in S330. The operating guide indicates control values capable of operating the plant so that cold gas efficiency of the plant is increased, a carbon conversion rate is increased, and the molten slag is discharged smoothly based on the real-time plant performance analysis. The operating control values indicate the supply amounts and operating temperatures of coal, flux, oxygen, steam, and nitrogen for transport. When the occurrence of the abnormal situation is predicted, the action guide generator160provides the action guide indicating the necessary action in response to the predicted abnormal situation in S340. The action guide indicates action control values which allow the abnormal situation to be eliminated, and the action control values include the supply amounts and operating temperatures of the coal, flux, oxygen, steam, and nitrogen for transport. Accordingly, the user may operate the plant based on the operating guide or the action guide. Meanwhile, the operation recorder170continuously stores the control values based on the operation and action of the plant based on the user input, and provides the stored control values as the operation history in S350. The step S400ofFIG.2will be described with reference toFIG.6. The stop guide generator170determines whether a plant stop event occurs in S410. The plant stop event may be an automatic stop based on an emergency situation or made by the user input. When the stop event occurs, the stop guide generator170provides a stop guide for stopping the plant in S420. The stop guide includes control values inevitably controlled by the user for each step of a plurality of steps which stop the plant. FIG.7illustrates a computing apparatus TN100according to an exemplary embodiment. The operating guide system1000of the coal gasification plant may employ the computing apparatus TN100ofFIG.7to execute the method ofFIG.2. In an exemplary embodiment ofFIG.7, the computing apparatus TN100may include at least one processor TN110, a transceiver TN120, and a memory TN130. The computing apparatus TN100may further include a storage device TN140, an input interface device TN150, an output interface device TN160, and the like. The components included in the computing apparatus TN100may be connected by a bus TN170to communicate with each other. The processor TN110may execute a program command stored in at least one of the memory TN130and the storage device TN140. The processor TN110may include one or more of a central processing unit (CPU), a graphics processing unit (GPU), and a dedicated processor to execute methods according to an exemplary embodiment. The processor TN110may be configured to implement the procedure, function, method, and the like described in connection with exemplary embodiments. The processor TN110may control each component of the computing apparatus TN100. Each of the memory TN130and the storage device TN140may store various information related to an operation of the processor TN110. Each of the memory TN130and the storage device TN140may include at least one of a volatile storage medium and a nonvolatile storage medium, and the memory TN130may include at least one of a read only memory (ROM) and a random access memory (RAM). The transceiver TN120may transmit and receive a wired signal or a wireless signal. The transceiver TN120may be connected to a network to perform communication. Meanwhile, the methods according to the aforementioned exemplary embodiment may be implemented in a program form readable through computer means to be recorded on a computer-readable recording medium. Here, the recording medium may include program commands, data files, data structures, and the like alone or in combination. The program commands recorded on the recording medium may be those specially designed and configured for the present disclosure or may also be known and available to those skilled in computer software. For example, the recording medium may be magnetic media such as hard disks, floppy disks, and magnetic tapes; optical media such as CD-ROMs and DVDs; magnetic-optical media such as floptical disks; and hardware devices specifically configured to store and perform the program commands, such as ROMs, RAMs, and flash memories. Examples of the program commands may include high-level language wiring executable by a computer using an interpreter, as well as machine language wiring such as those produced by a compiler. Such hardware devices may be configured to operate as one or more software modules to perform the operations of the present disclosure, and vice versa. As described above, although an exemplary embodiment has been described, those skilled in the art may modify and change variously by adding, changing, deleting, or the like the components without departing from the spirit of the present disclosure described in the claims, and this will also be included within the scope of the present disclosure. | 21,987 |
11859145 | DETAILED DESCRIPTION OF THE EMBODIMENTS In the following detailed description, embodiments are described in sufficient detail to enable those skilled in the art to practice them, and it is to be understood that other embodiments may be used and that chemical and procedural changes may be made without departing from the spirit and scope of the present subject matter. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of embodiments of the present invention is defined only by the appended claims. The term “biomass” as used herein, refers in general to organic matter harvested or collected from a renewable biological resource as a source of energy and bioproducts. The renewable biological resource can include plant materials, animal materials, and/or materials produced biologically. The term “biomass” is not considered to include fossil fuels, which are not renewable. The term “plant biomass” or “ligno-cellulosic biomass (LCB)” as used herein is intended to refer to any plant-derived organic matter containing cellulose and/or hemicellulose as its primary carbohydrates (woody or non-woody) available for producing energy on a renewable basis and bioproducts. Plant biomass can include, but is not limited to, agricultural residues such as corn stover, wheat straw, rice straw, sugar cane bagasse, sorghum, and the like. Plant biomass can also include agricultural residues and forest residues that are dedicated for bioenergy purposes, such as residues of grasses and trees. Plant biomass further includes, but is not limited to, “woody biomass”, i.e., woody energy crops, wood wastes and residues such as trees, including fruit trees, such as fruit-bearing trees, (e.g., apple trees, orange trees, and the like), softwood forest thinnings, barky wastes, sawdust, paper and pulp industry waste streams, wood fiber, and the like. Additionally, perennial grass crops, such as various prairie grasses, including prairie cord grass, switchgrass,Miscanthus, big bluestem, little bluestem, side oats grama, and the like, have potential to be produced large-scale as additional plant biomass sources. For urban areas, potential plant biomass feedstock includes yard waste (e.g., grass clippings, leaves, tree clippings, brush, etc.) and vegetable processing waste. Plant biomass is known to be the most prevalent form of carbohydrate available in nature and corn stover is currently the largest source of readily available plant biomass in the United States. When describing the various embodiments and used without a qualifier, the term “biomass” is intended to refer to “plant biomass,” i.e., lignocellulosic biomass (LCB) containing plant cell wall polysaccharides. The term “biofuel” as used herein, refers to any renewable solid, liquid, or gaseous fuel produced biologically and/or chemically, for example, those derived from biomass. Most biofuels are originally derived from biological processes such as the photosynthesis process and can therefore be considered a solar or chemical energy source. Some types of biofuels, such as some types of biodiesel, can be derived from animal fats. Other biofuels, such as natural polymers (e.g., chitin or certain sources of microbial cellulose), are not synthesized during photosynthesis, but can nonetheless be considered a biofuel because they are biodegradable. There are generally considered to be three types of biofuels derived from biomass synthesized during photosynthesis, namely, agricultural biofuels (defined below), municipal solid waste biofuels (residential and light commercial garbage or refuse, with most of the recyclable materials such as glass and metal removed) and forestry biofuels (e.g., trees, waste or byproduct streams from wood products, wood fiber, pulp and paper industries). Biofuels produced from biomass not synthesized during photosynthesis include, but are not limited to, those derived from chitin, which is a chemically modified form of cellulose known as an N-acetyl glucosamine polymer. Chitin is a significant component of the waste produced by the aquaculture industry because it comprises the shells of seafood. The term “agricultural biofuel”, as used herein, refers to a biofuel derived from agricultural crops, lignocellulosic crop residues, grain processing facility wastes (e.g., wheat/oat hulls, corn/bean fines, out-of-specification materials, etc.), livestock production facility waste (e.g., manure, carcasses, etc.), livestock processing facility waste (e.g., undesirable parts, cleansing streams, contaminated materials, etc.), food processing facility waste (e.g., separated waste streams such as grease, fat, stems, shells, intermediate process residue, rinse/cleansing streams, etc.), value-added agricultural facility byproducts (e.g., distiller's wet grain (DWG) and syrup from ethanol production facilities, etc.), and the like. Examples of livestock industries include, but are not limited to, beef, pork, turkey, chicken, egg, and dairy facilities. Examples of agricultural crops include, but are not limited to, any type of non-woody plant (e.g., cotton), grains such as corn, wheat, soybeans, sorghum, barley, oats, rye, and the like, herbs (e.g., peanuts), short rotation herbaceous crops such as switchgrass, alfalfa, and so forth. The term “pretreatment step” as used herein, refers to any step intended to alter biomass so it can be more efficiently and economically converted to reactive intermediate chemical compounds such as sugars, organic acids, etc., which can then be further processed to a variety of end products such as ethanol, isobutanol, long chain alkanes, etc. Pretreatment can reduce the degree of crystallinity of a polymeric substrate, reduce the interference of lignin with biomass conversion, and hydrolyze some of the structural carbohydrates, thus increasing their enzymatic digestibility and accelerating the degradation of biomass to useful products. Pretreatment methods can utilize acids of varying concentrations, including dilute acid pretreatments, concentrated acid pretreatments (using, for example, sulfuric acids, hydrochloric acids, organic acids, and the like) and/or pretreatments with alkali such as ammonia and/or ammonium hydroxide and/or calcium hydroxide and/or sodium hydroxide and/or lime, and the like, and/or oxidative pretreatments using oxidants such as air, oxygen, hydrogen peroxide, organic peroxide, ozone, and the like. Pretreatment methods can additionally or alternatively utilize hydrothermal treatments including water, heat, steam, or pressurized steam pretreatments, including, but not limited to, hydro-thermolysis pretreatment and liquid hot water pretreatment, further including, for example, acid catalyzed steam explosion pretreatment (e.g., SO2catalyzed). Pretreatment can occur or be deployed in various types of containers, reactors (e.g., batch, counter-current, and the like), pipes, flow through cells, and the like. Many pretreatment methods will cause the partial or full solubilization and/or destabilization of lignin and/or hydrolysis of hemicellulose to pentose sugars. Further examples of pretreatment include, but are not limited wet oxidation, organosolv pretreatment and mechanical extrusion. The term “metal-ligand complex” as used herein refers to a metal complex containing one or more metal-coordinating ligands and one or more metal atoms which are in a state of interaction with each other. Such interactions include various types of forces and bonds, which include, but are not limited to, ionic bonds, covalent bonds, and van der Waals forces. The term “metal-ligand complex” and “ligand-metal complex” can be used interchangeably. The term “metal-coordinating ligand” as used herein refers to a ligand, such as an ion, a molecule, or the like, that can interact with the metal portion of a metal-ligand complex. When used without qualification, the term “ligand” is intended to refer to a “metal-coordinating ligand.” The term “copper-coordinating ligand” as used herein refers to a metal coordinating ligand capable of interacting with copper atoms or copper ions. The term “single-ligand metal complex” as used herein refers to a metal-ligand complex containing only one ligand that coordinates with, i.e., interacts with metal atoms or metal ions. The term “multi-ligand metal complex” as used herein refers to a metal-ligand complex containing more than one ligand that coordinates with, i.e., interacts with metal atom or metal ions. The term “toxicity” as used herein refers to ions, molecules, and metal-ligand complexes present in the process streams during biomass conversion and cellulosic biofuel production that negatively impact the yield of the products. The term “slow add” as used herein refers to a gradual rate of addition of a reagent to a reaction vessel. The gradual rate can be continuous or discontinuous, i.e., it may include intermittent periods of no reagent being added. A “slow add” is in contrast to a batch method of adding a reagent, in which all the desired reagent is added to the reactive vessel at once. The term “state of interaction” as used herein refers to an interaction between a ligand and a metal or between a metal and multiple ligands. Such an interaction can include various types of forces and bonds, which include, but are not limited to, ionic bonds, covalent bonds, and van der Waals forces. The term “alkaline oxidative pretreatment” as used herein relates to a pretreatment of plant biomass in an alkaline environment with one or more oxidants. which can include, but are not limited to, hydrogen peroxide, oxygen, ozone, hydroperoxide anion, superoxide radical, hydroxyl radical, and peroxy acids (e.g., peracetic acid, peroxymonosulfuric acid, peroxyphosphoric acid, and meta-chloroperoxybenzoic acid). See, for example, Liu, et al.,Coupling alkaline pre-extraction with alkaline-oxidative post-treatment of corn stover to enhance enzymatic hydrolysis and fermentability, Biotechnology for Biofuels, 2014, 7:48, which describes example conditions for alkaline oxidative pretreatment. An alkaline oxidative pretreatment which uses hydrogen peroxide as one of the oxidants is to be distinguished from a conventional “alkaline hydrogen peroxide” (AHP) pretreatment which is a one-step catalytic pretreatment process which requires much higher oxidant loadings. See, for example, Biotechnol Bioeng 1984, 26:46-52; Biotechnol Bioeng 1984, 26:628-631; Biotechnol Bioeng 1985, 27:225-231; Science 1985, 230:820-822 and Biotechnol Biofuels 2011, 4:16, which describe conventional AHP with much higher oxidant loadings. The term “ss-AHP/O” process, “ss-Cu-AHP/O” process or “ss-dual oxidant” process as used herein relates to a single-stage alkaline oxidative pretreatment with at least two oxidants. The terms may be used interchangeably. While the present description is in the context of hydrogen peroxide and oxygen as the two oxidants, it will be understood that other oxidants may also be used in the oxidative treatment. Nearly all forms of lignocellulosic biomass, i.e., plant biomass, such as monocots, comprise three primary chemical fractions: hemicellulose, cellulose, and lignin. Lignin, which is a polymer of phenolic molecules, provides structural integrity to plants, and is difficult to hydrolyze. As such, after sugars in the biomass have been fermented to a bioproduct, such as alcohol, lignin remains as residual material, i.e., a non-easily digestible portion. Cellulosic biofuel production from lignocellulosic biomass has gained considerable momentum due to both environmental and social sustainability benefits. However, the technology is not yet fully commercialized. One issue impeding cellulosic biofuel production using the sugar platform is the hydrolysis-resistant nature of certain components in the lignocellulosic biomass. Cellulose and hemicelluloses in plant cell walls exist in complex structures within the residual material. Hemicellulose is a polymer of short, highly branched chains of mostly five-carbon pentose sugars (xylose and arabinose), and to a lesser extent six-carbon hexose sugars (galactose, glucose and mannose). Because of its branched structure, hemicellulose is amorphous and relatively easy to hydrolyze into its individual constituent sugars by enzyme or dilute acid treatment. Cellulose is a linear polymer comprising of β(1,4) linked D-glucose in plant cell wall, much like starch with a linear/branched polymer comprising of α(1,4) linked D-glucose, which is the primary substrate of corn grain in dry grind and wet mill ethanol plants. However, unlike starch, the glucose sugars of cellulose are strung together by β-glycosidic linkages, which allow cellulose to form closely associated linear chains. Because of the high degree of hydrogen bonding that can occur between cellulose chains, cellulose forms a rigid crystalline structure that is highly stable and much more resistant to hydrolysis by chemical or enzymatic attack than starch or hemicellulose polymers. Although hemicellulose sugars represent the “low-hanging” fruit for conversion to a biofuel, the substantially higher content of cellulose represents the greater potential for maximizing biofuel yields, on a per ton basis of plant biomass. Lignocellulose can also be characterized as a highly heterogeneous composite material comprised of multiple cell wall biopolymers (cellulose, heteropolysaccharides including hemicelluloses and pectins, and lignins) associated primarily by non-covalent interactions which are assembled into cell walls with composition and properties varying by cell and tissue type. These components are interconnected through a variety of covalent and non-covalent interactions, giving rise to a highly organized network which is assembled in a tightly controlled sequence during plant growth. This heterogeneous higher order structure of the cell wall impacts the cell wall's response to deconstruction and conversion. Plant cell walls exhibit substantial heterogeneity in both content and distribution of the inorganic elements which also have implications for biomass conversion processes. This includes differences between content and distribution of inorganics in disparate plant taxa, differences between related species, within a single species as a function of its phenotype and environment, and even between tissues in a single plant. Inorganic elements in plants are known to be responsible for diverse roles, including maintenance of ionic equilibrium in cells (e.g., K) and storage (e.g., Fe in ferritin), which, despite being localized in plastids, is water extractable. A subset of the inorganic elements in plants is strongly associated with the cell wall. These elements are more resistant to aqueous extraction and include inorganic elements that may have structural roles, including, but not limited to, Ca and B ionic cross-links in pectic polysaccharides, calcium oxalate raphide crystals in some grasses, and Si in the cell walls of grasses which can comprise a significant fraction of the mass of a plant. Another class of role of cell-wall associated inorganic elements are metal co-factors in enzymes (e.g., Zn, Fe, Mn, Cu). Redox-active metals, such as Cu, Mn, Fe, can exist in multiple oxidation states in vivo and are often involved in reactions involving electron transfer. Specifically, Fe in plants is associated with Fe-heme proteins and iron-sulfur (Fe—S) clusters in proteins, such as ferredoxins, which function as electron carriers in the photosynthetic electron transport chain. Cu in plants has diverse roles as a structural element in regulatory proteins, in photosynthetic electron transport, mitochondrial respiration, and Fe mobilization, among others. Metals may also be associated with metallothioneins (MTs) and phytochelatins (PCs), which are cysteine-rich polypeptides involved in either ameliorating the toxicity or controlling homeostasis of metals such as Fe, Ni, Cd, Zn, and Cu by coordination by thiols. In addition to its involvement with enzymes associated with the shikimic acid pathway and lignin biosynthesis, Mn is contained in a metallo-oxo cluster containing 4 Mn ions at differing oxidation states in the oxygen-evolving complex of photosystem II. There are differences in the strength and nature of association of cell wall-associated metals. Specifically, Mn may be strongly associated with the cell wall and be present in “organic chelates” or “bound to lignin.” Alkali delignified hardwoods are known to have differences in the extractability of cell wall-associated Mn versus Fe using chelating compounds. Mg, which is a component of chlorophyll, is useful for photosynthesis and protein synthesis, although a portion of Mg may be bound to pectin or precipitated as salts in the vacuole, while the remainder is extractable with water. During either oxidative delignification or biomass conversion processes where oxygen may be present, cell wall-associated transition metals can catalyze the formation of reactive oxygen species through Fenton chemistry. This catalytic activation of oxygen by transition metals has been shown to contribute to the oxidative scission of polysaccharides during alkaline-oxidative bleaching or delignification using H2O2or O2. As a result, precautions are taken during these processes through chelation and washing steps to remove metals and addition Mg salts and silicates to complex transition metals during these unit operations. Therefore, a pretreatment process is typically used to alter and expand the cell wall matrix, to hydrolyze the hemicelluloses, and to alter the hemicelluloses. Pretreatment disrupts the non-easily digestible portion of lignocellulosic biomass, e.g., cellulose and lignin, thus improving its accessibility. After pretreatment, much of the biomass becomes easily digestible, while a portion remains non-easily digestible. Ultimately, the pretreatment process makes the cellulose more accessible (during a subsequent hydrolysis process, such as with lytic enzymes) for conversion of the lignocellulose polysaccharides (e.g., cellulose and hemicellulose) to monomeric sugars, which can be transformed to target products via catalytic conversion or microbial fermentation. However, enzymatic hydrolysis of lignocellulose polysaccharides is usually hindered by the natural resistance of plant cell wall against deconstruction. To overcome this resistance, pretreatment processes of biomass feedstock have been developed and employed. Biomass pretreatment modifies cell wall structure and renders the biomass more digestible by enzymes. A wide range of pretreatments are known, but few pretreatment methods have been identified as effective for biomass feedstocks, such as woody biomass, which are highly resistant to enzymatic hydrolysis. For example, enzymatic hydrolysis of hybrid poplar wood usually produces sugars at only 5 to 30% of the theoretical maximum yield. As noted above, such resistance involves the structural rigidity of the plant cell wall, the crystallinity of cellulose, and the presence of lignin, which remains as a residual material. However, in the embodiments described herein, the alkaline pretreatments not only solubilize the lignin, it is expected that they also produce a lignin that closely resemble native lignin, such that less than 35% of the α-carbon of the solubilized lignin is oxidized from a hydroxyl to a carbonyl. Lignin is known to be useful in a variety of applications including, but not limited to, carbon fiber composites, bio-oil, resins, adhesive binders and coating, plastics, paints, enriching soil organic carbon, fertilizer, rubbers and elastomers, paints, antimicrobial agents, and slow nitrogen release fertilizer, and the like, and can be a substitute for polymers produced using crude oil. One current source of lignin in the market is produced from sulfite (or sulfonate) based paper/pulp mills, a kraft pulping process, and the like. Most such mills currently burn the lignin to recover energy and to reduce the environmental impact of discharge. Very few sulfite mills currently process the lignosulfonates from sulfite spent liquors. Additionally, the quality and quantity of lignin obtained via currently known methods are inadequate for most applications. As such, methods to fractionate and convert lignin into value-added products are needed. Known methods for pretreating plant biomass are typically performed under elevated pressures and temperatures (above room temperature). Such methods include hot water and steam treatments, ammonia treatments and sulfite treatments. Other pretreatment methods utilize an oxidant-based pretreatment, such as the alkaline oxidative pretreatment (AOP) process or a conventional alkaline hydrogen peroxide known by those skilled in the art. Yet other methods include catalytic processes. Catalytic approaches to plant cell wall deconstruction and conversion of insoluble biomass rely on homogeneous catalysts to allow the catalyst to diffuse through nano-scale pores within the cell walls to perform the desired reactions. Heterogeneous catalysis is known to be inefficient unless the cell walls are solubilized in expensive solvents such as ionic liquids. Homogeneous catalysts are used in many applications, such as homogeneous copper catalysts used for atom transfer radical polymerization where catalyst removal to prevent contamination of the product adds cost to the process. Table 1 (below) summarizes some known pretreatment methods and the pretreatment method described herein. TABLE 1Pre-extractionBaseNameamountPretreatmentTemp.Other cond.ReferenceSingle stageReference-None10% H2O2R.T.2 mM 2,2′-Li, Z., Chen, C. H.,catalyticCu-AHPbipyridine,Hegg, E. L., & Hodge,pretreatmentprocess1 mMD. B. (2013).process(AOP)CuSO4Biotechnology forBiofuels, 6, 119.Two stageCu-AHP10%10% H2O2,R.T.2 mM 2,2′-Bhalla, A., Bansal, N.,One oxidant,processNaOHbipyridine,Stoklosa, R. J.,Metal-single1 mMFountain, M., Ralph, J.,ligand catalystCuSO4Hodge, D. B., & Hegg,E. L. (2016).Biotechnology forbiofuels, 9(1), 1-10.Two stageCu-AHP/O10%H2O2(2-120° C. pre-1 mM 2,2′-Yuan, Z., Klinger, G.2 oxidantprocessNaOH,10%)extractionbipyridine,E., Nikafshar, S., Cui,120° C.O2(25-50and 80° C.1 mMY., Fang, Z., Alherech,psi)oxidativeCuSO4M., . . . & Hegg, E. L.10% NaOH,post(2021). ACS80° C.treatmentSustainable Chemistry& Engineering, 9(3),1118-1127.Single-stagess-Cu-NoneH2O2(2-80-100° C.1 mM 2,2′-(described herein)2 oxidantAHP/O)10%)bipyridine,O2(100-3001 mMpsi)CuSO415% NaOH Use of a single ligand copper complex as a catalyst in combination with an alkaline oxidative pretreatment (AOP) process with one oxidant is known. See, for example, Li et al.,Rapid and Effective Oxidative Pretreatment of Woody Biomass at Mild Reaction Conditions and Low Oxidant LoadingsBiotechnol Biofuels 6(1), 119 (2013), and Li, et al., Catalysis with CuII(bpy) Improves Alkaline Hydrogen Peroxide Pretreatment. Biotechnol Bioeng. 110(4):1078-1086 (2013), each of which is incorporated by reference in its entirety. However, the amount of oxidant required in such processes is high, such as at least 10% of the weight of the biomass to be treated. Additionally, to achieve suitable pretreatment results, the amount of metal utilized in a single-ligand copper complex is high (e.g., more than 50 μmol of metal complexes per gram of biomass to be pretreated). Use of such high levels of a metal can pose toxicity issues in subsequent processes (e.g., fermentation). Moreover, use of such high amounts of metals and oxidants can be cost prohibitive. A sequential two-stage, dual oxidant pretreatment process has been described, for example, in U.S. patent application Ser. No. 16/903,598. In the two-stage pretreatment process as shown inFIG.1A, the plant biomass is treated with alkaline, e.g., 10% NaOH in the first stage. The alkaline treated plant biomass is filtered and then the solids are subject to a second stage of pretreatment with two oxidants, e.g., pressurized oxygen and hydrogen peroxide, in an alkaline environment. The two stage, dual oxidant (Cu-AHP/O) process can include the use of about 10% NaOH in the first stage. After filtering, the solids are pretreated in the second stage with another addition of 10% NaOH, about 2% hydrogen peroxide and about 50 psi of oxygen. In various embodiments, a single-stage, alkaline oxidative pretreatment process with two or more oxidants is described herein (ss-dual oxidant process). In one embodiment, the single stage alkaline oxidative pretreatment process with two oxidants can be a single stage O2-enhanced alkaline hydrogen peroxide (ss-Cu-AHP/O) pretreatment process that improves the economics of the oxidative lignocellulosic-to-biofuel technique. In one embodiment, the alkaline pre-extraction step prior to the addition of oxidants is eliminated resulting in a one-stage pretreatment process as shown inFIG.1B. This process leads to a reduction in the loading of expensive chemical inputs in conjunction with an increase in the recovery of biomass polymers as well as significantly streamlining the process steps by elimination of the alkaline pre-extraction step. In one embodiment, the ss-dual oxidant process uses sodium hydroxide as the base, a metal-ligand complex, e.g., a copper-ligand complex, hydrogen peroxide in combination with pressurized oxygen as the oxidants. In one embodiment, a portion of the hydrogen peroxide generally used in a single oxidant process can be replaced with the molecular oxygen. Reduction of the hydrogen peroxide in the oxidative pretreatment process can lead to a cost savings. In one embodiment, in the dual oxidant process, the hydrogen peroxide and oxygen are present simultaneously in the oxidative pretreatment process. In various embodiments, the ligand metal complexes with one or more ligands with two or more oxidants can be used in the single-stage alkaline oxidative pretreatment process of plant biomass. In various embodiments, the ss-dual oxidant process described herein combines the alkaline pre-extraction first stage and the alkaline dual oxidant oxidative pretreatment second stage into a one-stage alkaline oxidative pretreatment process. In one embodiment, the present description includes a single-stage metal catalyzed alkaline oxidative pretreatment using two oxidants, e.g., both O2and H2O2, as co-oxidants to improve the enzymatic digestibility of the biomass as well as recovering high quality lignin to be converted into aromatic monomers such as vanillin, syringaldehyde, p-hydroxybenzoic, vanillic acid, and syringic acid. The ss-Cu-AHP/O process can improve the recovery of biopolymers, including both polysaccharides and lignin by eliminating the alkaline pre-extraction stage of the two-stage process. In one embodiment, the process described herein can increase the O2pressure and/or H2O2loading during the alkaline oxidative pretreatment. This can eliminate the initial alkaline pre-extraction stage while maintaining high sugar yields and native-like lignin stream amenable to depolymerization into monomers or formulation of lignin-based polyurethanes. In one embodiment, this single stage strategy can utilize 25% less NaOH than a two-stage process and has the potential to improve the life cycle analysis (LCA) and simplify biomass handling and the entire plant biomass pretreatment process, thereby further reducing the minimum fuel selling price (MFSP). In various embodiments, oxidants useful in an alkaline oxidative pretreatment process include, but are not limited to, air, oxygen, hydrogen peroxide, ozone, persulfate, percarbonate, sodium peroxide and combinations thereof. In one embodiment, the oxygen can be pressurized oxygen that is added or provided to the reaction in amounts greater than the amount of oxygen present in the atmospheric. In one embodiment, the process includes the addition of at least two oxidants to the reaction mixture containing the plant biomass for pretreatment. In one embodiment, one or more oxidants are combined with the other reactants at a low weight percent (%) loading based on original biomass (w/w), i.e., loading of no more than 15% w/w based on original biomass. In one embodiment, the H2O2loading is less than 10% w/w, such as less than 5% w/w based on original biomass. In one embodiment, the H2O2loading ranges from about 1% to about 15%, such as about 1% to about 10%, such as about 1% to 5% or less, including any range there between based on original biomass w/w. Such loadings are lower than conventional H2O2loadings which can be as high as 200%. In various embodiments, the loading of hydrogen peroxide in the ss-AHP/O process can be less than about 10% w/w (H2O2/original biomass), or less than about 8% w/w, or less than about 5% w/w, or less than about 4% w/w, or less than about 3% w/w, or less than about 2% w/w, or less than about 1% w/w. In one embodiment, the loading of hydrogen peroxide in the ss-AHP/O process is about 2% w/w. In various embodiments, the amount of oxygen used in the ss-AHP/O process is greater than about 50 psi, or greater than about 100 psi, or greater than about 250 psi, or greater than about 300 psi, or greater than about 400 psi. In various embodiments, the amount of oxygen used to enhance the ss-AHP/O process is less than about 500 psi, or less than about 400 psi, or less than about 350 psi, or less than about 300 psi. In one embodiment, the amount of oxygen used to enhance the ss-AHP/O process is about 300 psi. In one embodiment, the oxidants used in the ss-AHP/O process included hydrogen peroxide at about 2% w/w and oxygen at about 300 psi. Variations of this combination of concentrations may be used as described above and are within the scope of this description. In various embodiments, a single-ligand or a multi-ligand metal complex can be used in the ss-AHP/O process described herein. In one embodiment, a single-ligand metal complex may be used. In another embodiment, a multi-ligand metal complex may be used. In one embodiment, the metal-coordinating ligands includes 2,2′-bipyridine. Other metal-coordinating ligand, including, but not limited to nitrogen-donating ligands such as pyridine, 1,10-phenanthroline, and ethylenediamene, and ligands containing both a nitrogen donor and a carboxylate group such as the amino acids including histidine or glycine may also be used. In one embodiment, the catalytic metal element(s) (i.e., metal or metals) in the catalyst can include, but are not limited to, aluminum, zinc, nickel, magnesium, manganese, iron, copper cobalt and/or vanadium in various oxidation states. In various embodiments, the catalytic metal element is a metal ion(s). The metal ion(s) is redox active. The metal ions can be oxidized and/or reduced. In one embodiment, the elements include, but are not limited to, iron (e.g., Fe(II), Fe(III)), copper (e.g., Cu(I), Cu(II)), cobalt (e.g., Co(III), Co(VI)), and/or vanadium (e.g., V(II), V(III), V(IV), V(V)). By substituting an amount of the 2,2′-bypyridine (bpy) with other, lower costs ligands, substantial savings can be achieved. In one embodiment, about 1 weight/weight (w/w) % up to about 99% or higher, such as 100% of bpy is substituted, such as about 10 to about 90%, such as about 20 to about 80%, such as about 35% to about 60%, including any range there between. In the various embodiments described herein, the multi-ligand metal complexes have low production costs. In one embodiment, substitution of bpy with other metal coordinating ligands provides a savings on the order of 10-fold or more, such as a savings of about 20 to 30 times the cost of using bpy alone. In various embodiments, use of a multi-ligand metal complex in the ss-AHP/O process allows for a reduction in the amount of metals used in the process and also a reduction in the amount of oxidant. The multi-ligand metal complex can be, for example, a multi-ligand copper complex. The copper complex can be, for example, copper(II) 2,2′-bipyridine complex (Cu(bpy)) modified to contain at least one additional metal-coordinating ligand, such as pyridine; 1,10-phenanthroline; ethylenediamine; histidine; and/or glycine. While not wishing to be bound by this proposed theory, both the single- and multi-ligand metal complexes are thought to function as suitable catalysts for lignocellulosic biomass (i.e., cause sufficient catalyst sorption into the biomass) due to the ability of the cationic metal, such as copper, to interact with (e.g., bond with) charged anionic groups, such as deprotonated phenolic hydroxyls in lignin, carboxylate groups in lignin, and/or uronic acids in pectins and hemicelluloses. In one embodiment, use of the multi-ligand metal complex as a catalyst during an oxidative pretreatment may allow the pretreatment process to proceed significantly faster (e.g., at least two times as fast) as compared with an oxidative pretreatment performed using a conventional single-ligand metal complex as a catalyst. In various embodiments, the ligand metal complexes can include any of the metals described herein. In one embodiment, the ligand metal complexes can be copper ligand complexes. The ligand can be any ligand as described herein. In various embodiments, the ligand metal complexes can include a single ligand. In various embodiments, the ligand metal complexes can be multi-ligand complexes as described herein. In one embodiment, the ligand metal complex is a Cu(bpy) complex. In various embodiments, the amount of ligand metal complex, e.g., Cu(bpy) complex, is less than about 2 mM, or less than about 1.5 mM, or less than about 1 mM, or less than about 0.8 mM, or less than about 0.5 mM. In one embodiment, the ligand metal complex is about 1 mM. The amount of the ligand metal complex is reduced by at least about 25%, or by at least about 50% or by at least about 75% compared to oxidative pretreatment process with one oxidant. Use of the multi-ligand metal complexes described herein also reduces the amount of metal, such as copper, used in the process as compared to a single-ligand metal complex, such as a single ligand copper complex, by at least 50%, or at least 40%, or at least 30% or at least 20% or at least 10% or at least 5% or lower, including any range therein. Use of a reduced amount of metal not only reduces toxicity levels, but further reduces costs. Use of the multi-ligand metal complex may reduce the amount of oxidant, such as hydrogen peroxide and/or oxygen, used in the oxidative pretreatment by at least 90%, by at least 80%, by at least 70%, by at least 60%, by at least 50%, or at least 40%, or at least 30% or at least 20% or at least 10% or at least 5% or lower, including any range therein. Use of a reduced amount of oxidant further reduces costs. In one embodiment, the pH of the plant biomass being pretreated is adjusted to increase the number of deprotonated groups. In one embodiment, the pH of the pretreated biomass is, or the biomass is pH adjusted to achieve, a neutral pH during the pretreatment process. In one embodiment, the pH is adjusted to achieve an alkaline pH to deprotonate the phenolic groups in lignin and to increase lignin solubility. In one embodiment, the pH is adjusted to at least 11, such as at least 11.5, including any value in between. In some embodiments, elevation of the pH is achieved with bases such as ammonia and/or ammonia derivatives, such as amines, in which copper is stabilized in solution in the form of a complex ion. In one embodiment, the pH is adjusted via addition of a base, which can react with lignin and cause depolymerization and/or solubilization, i.e., helps the plant cell wall to become degraded and/or destroyed, thus reducing resistance to subsequent hydrolysis. In various embodiments, the ss-AHP/O process described herein includes the addition of base only once in the pretreatment process. In contrast, the two-stage process includes the addition of base in each of the steps. Advantageously, the total amount of base added in the ss-AHP/O process can be less than the amount of base added in the combined two stage process. In various embodiments, bases used in the ss-AHP/O include sodium hydroxide, ammonia, potassium hydroxide, and sodium carbonate. In various embodiments, the amount of base added to the pretreatment process is less than about 30 percent w/w based on the dry weight of the original biomass. In one embodiment, the amount of base is less than about 25 percent, or less than about 20 percent, or less than about 15 percent based on the dry weight of the original biomass. In various embodiments, the amount of base added to the pretreatment process is at least about 10 percent based on the dry weight of the original biomass. In one embodiment, the amount of base added in the ss-AHP/O can be about 15% of sodium hydroxide or less, w/w of the dry weight of the original biomass. In contrast, the two stage process can use about 10% of sodium hydroxide, w/w of the dry weight of the original biomass, in each of the two steps, resulting in the use of about 20% of sodium hydroxide total, w/w of the dry weight of the original biomass. In one embodiment, the oxidants used in the ss-AHP/O process included hydrogen peroxide at about 2% w/w of the plant biomass and oxygen at about 300 psi and the amount of base used is at about 15% w/w of the original plant biomass. In one embodiment, the base is sodium hydroxide. Variations of this combination of concentrations may be used as described above and are within the scope of this description. In various embodiments, the temperature at which the ss-AHP/O process is conducted can be increased to improve the economics of the pretreatment process. In various embodiments, the ss-AHP/O process is conducted at a temperature of about 60° C. or greater, or about 70° C. or greater, or about 80° C. or greater, or about 90° C. or greater, or about 100° C. or greater. In various embodiments, the ss-AHP/O process is conducted at a temperature of about 140° C. or less, or about 130° C. or less, or about 120° C. or less, or about 110° C. or less, or about 100° C. or less, or about 90° C. or less, or about 80° C. or less, or about 70° C. or less. In one embodiment, the ss-AHP/O process is conducted at a temperature of about 80° C. In various embodiments, the ss-AHP/O process is conducted for about 48 hours or less. In one embodiment, the ss-AHP/O process is conducted for about 36 hours or less, or about 24 hours or less, or about 18 hours or less, or about 12 hours or less, or about 9 hours or less, or about 6 hours or less. In one embodiment, the ss-AHP/O pretreatment process can be conducted at about 80° C. for about 3 hours to about 24 hours, or for about 8 hours to about 16 hours, or for about 10 hours to about 14 hours. Variation of these combinations of temperature and incubation time may be used and are within the scope of this description. Any suitable plant biomass can be used. In one embodiment, the plant biomass contains transition metals. Use of a plant biomass containing more than trace amounts of one or more transition metals results in further cost savings, as a reduced amount of catalyst is needed to affect the same or substantially the same results. Examples of plant biomass containing more than trace amounts of transition metals include, but are not limited to, hardwoods of the genusPopulus(e.g., various types of poplar including hybrid poplar, hybrid aspen, western balsam poplar, and the like), birch (including silver birch and the like), maple (including sugar maple and the like), further including grasses (including, but not limited to, corn, switchgrass, sorghum,miscanthus) and gymnosperms, which are also referred to as conifers and softwoods (including, but not limited to, the genus ofPinus, such asPinus resinosa, i.e., red pine). In one embodiment, the plant biomass contains one or more transition metals that are redox-active, including, but not limited to, Fe, Mn, Cr, Co, Ni, Cu, Mo, Pd, Ru, Re, Pt, Pd, Os, Jr and combinations thereof. In various embodiments, the biomass may be subjected to a cycle of hydrolysis (e.g., enzymatic, acid, etc.) using any conventional methods known in the art. In one embodiment, a reduced amount of enzymes is used, as compared to hydrolysis of conventionally catalyzed pretreated biomass. In various embodiments, use of a ss-AHP/O process, as described herein provides improved downstream bioproduct yields, such as sugar yields and lignin yields, as compared to yields obtained in the two-stage, two oxidant process as shown, for example, below in the Examples. In various embodiments, the amount of solubilized lignin from the plant biomass can be similar or improved relative to a two-stage, dual oxidant process. In one embodiment, the amount of solubilized lignin in the ss-AHP/O process was at least 50%, or at least 60%, or at least 70%, or at least 75%, or at least 80% of the lignin in the plant biomass. In various embodiments, the overall sugar release following the enzymatic hydrolysis can be similar or improved relative to a two-stage, dual oxidant process. In one embodiment, in the ss-AHP/O process, the overall glucose yield was at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, or 100% of glucan in the plant biomass. In one embodiment, in the ss-AHP/O process, the overall xylose yield was at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, or 100% of xylose in the plant biomass. In one embodiment, yields may be improved compared to a two-stage, two oxidant process by at least about 5% or higher, such as at least about 10% or higher, at least about 20% or higher, at least about 30% or higher, at least about 40% or higher, at least about 50% or higher, up to two or three times higher, including any range there between. In one embodiment, the use of ss-AHP/O process can result in using lower amounts of enzymes for enzymatic hydrolysis of the catalytically pretreated biomass than the catalytically treated biomass generated from a two-stage, two oxidant process. In various embodiments, the amount of enzyme used for hydrolysis is less than about 50 mg protein/g glucan, or less than about 40 mg protein/g glucan, or less than about 30 mg protein/g glucan, or less than about 20 mg protein/g glucan, or less than about 15 mg protein/g glucan, or less than about 10 mg protein/g glucan. In various embodiments, the amount of enzyme used for hydrolysis is more than about 5 mg protein/g glucan, or more than about 10 mg protein/g glucan, or more than about 15 mg protein/g glucan, or more than about 20 mg protein/g glucan, or more than about 25 mg protein/g glucan. In one embodiment, the amount of enzyme used for hydrolysis is about 15 mg protein/g glucan. In various embodiments, the overall monomeric sugar yields following enzymatic hydrolysis in the ss-AHP/O process increased compared to a two-stage, two oxidant process. In various embodiments, the amount of xylose yield (based on the initial sugar composition in the plant biomass) is more than about 70%, or more than about 80%, or more than about 90%, or more than about 95%, or about 100%. In various embodiments, the amount of xylose yield (based on the initial sugar composition in the plant biomass) is about 100% or less, or about 95% or less, or about 90% or less, or about 85% or less. In various embodiments, the amount of glucose yield (based on the initial sugar composition in the plant biomass) is more than about 70%, or more than about 80%, or more than about 90%, or more than about 95%, or about 100%. In various embodiments, the amount of glucose yield (based on the initial sugar composition) is about 100% or less, or about 95% or less, or about 90% or less, or about 85% or less. In one embodiment, the amount of glucose yield is about 93% and the amount of xylose yield is about 100% (based on the initial sugar composition in the plant biomass). Use of the metal-ligand complexes in a ss-AHP/O process described herein also reduces the amount of metal, such as copper, and the amount of ligand, e.g. bpy, used in the process as compared to a two-stage, two oxidant process, by at least 75%, or by at least 50%, or at least 40%, or at least 30% or at least 20% or at least 10% or at least 5% or lower, including any range therein. Use of a reduced amount of metal and ligand can reduce costs and can reduce toxicity. Use of a ligand-metal complex in a ss-AHP/O process may reduce the amount of the one of the more expensive oxidants, e.g. hydrogen peroxide, used compared to other oxidative treatments by at least 80%, or by at least 70%, or by at least 60%, or by at least 50%, or by at least 40%, or by at least 30% or by at least 20% or by at least 10% or by at least 5% or lower, including any range therein. Use of a reduced amount of oxidant further reduces costs. Use of alkaline, e.g., sodium hydroxide, in a single stage dual oxidant process, e.g., ss-AHP/O process, may reduce the amount of alkaline used in the biomass pretreatment process compared to the two stage, two oxidant process by at least about 50%, or at least about 40%, or at least about 30%, or at least about 25%, or at least about 20%, or at least about 15%, or at least about 10%. Use of a reduced amount of alkaline further reduces costs. An additional benefit relates to reduced microbial toxicity. Microbial toxicity is characterized by the final growth of yeast cells during yeast fermentation, and/or the growth rate of yeast during fermentation, and/or the length of the lag phase during fermentation. Such toxicity is caused by metal ions and other chemicals present in the processing stream, including the metals present in the multi-ligand catalyst and metal elements present in the plant biomass itself. Since the various embodiments allow for a reduced amount of metal as compared to conventional processes, the yeast used downstream is less adversely affected down to minimally adversely affected. As such, in one embodiment, the multi-ligand complexes have minimal microbial toxicity towards yeast fermentation (i.e., less than 50% reduction in final growth of yeast cells, as quantified with optical density). In various embodiments, hydrolysis may optionally be followed by or integrated with either fermentation or sugar catalytic conversion of sugars to bioproducts, such as biofuels, biochemicals and biopolymers. In one embodiment, such yields may be improved by at least 5% or higher, such as at least 10%, at least 20%, at least 30%, at least 40% at least 50% or higher, up to two or three times higher, including any range there between. In various embodiments, the use of two oxidants in the single-stage oxidative pretreatment process can result in favorable technoeconomic analysis (TEA) of the pretreatment of biomass to generate biofuels. In various embodiments, the improved TEA can result from conducting the pretreatment process in a single stage, conducting the ss-AHP/O process at a higher temperature, using two oxidants, hydrogen peroxide and oxygen, in conjunction with a single-ligand metal complex, e.g. Cu(bpy), reducing the amount of ligand used in the oxidative pretreatment process, reducing the load of the enzyme in the enzymatic hydrolysis with improved yield of monomeric sugars and/or a combination of these factors. In one embodiment, improved TEA can result from conducting the pretreatment of the biomass in a single stage without a separate alkaline pre-extraction step, conducting the ss-AHP/O pretreatment process at about 80° C., using hydrogen peroxide and oxygen as oxidants in conjunction with Cu(bpy), and reducing the amount of the ligand to 1 mM, reducing the amount of hydrogen peroxide to 2% w/w (H2O2/original biomass) with oxygen at about 300 psi, reducing the load of the enzyme to 15 mg protein/g glucan in the enzymatic hydrolysis leading to an improved yield of monomeric sugars, e.g. about 93% glucose and about 100% xylose (based on the initial sugar composition in the plant biomass). TEA indicates that the improved conditions can reduce the minimum fuel selling price (MFSP) compared to a two-stage, two oxidant process (Cu-AHP/O) by more than about 20%, or by more than about 30%, or by more than about 40%, or by more than about 50%, or by more than about 60%. In one embodiment, TEA indicates that the improved conditions can reduce the MFSP by about 30% to about 40% compared to a two-stage, two oxidant process using H2O2. TEA indicates that the improved conditions can reduce the minimum fuel selling price (MFSP) compared to a one-stage one oxidant process (conventional-AHP) by more than about 20%, or by more than about 30%, or by more than about 40%, or by more than about 50%, or by more than about 60%, or by more than about 70%, or by more than about 80%, or by more than about 90%. In one embodiment, TEA indicates that the improved conditions can reduce the MFSP by about 30% to about 40% compared to a one-stage one oxidant process using H2O2. In one embodiment, the process can further include recovery and reuse of the ligand metal complex, including recovery of the metal itself. Conventional technologies for metal removal (e.g., copper) from wastewater streams are based on ion exchange, precipitation/co-precipitation plus filtration, and membrane separation. Additionally, lignocellulose such as waste biomass or biomass fractions, such as lignin, have been proposed as biosorbant materials in the treatment of wastewater to remove heavy metals, including copper. Cationic metals can sorb to charged anionic groups such as deprotonated phenolic hydroxyls in lignin or carboxylate groups in lignin or uronic acids in pectins and hemicellulose and are known to be strongly affected by pH with more deprotonated groups at elevated pH. In one embodiment, catalyst sorption to biomass is strongly pH-dependent with near-complete catalyst adsorption to biomass at alkaline pH and substantial desorption at neutral to acidic pH. In one embodiment, pH is adjusted to recover the multi-ligand metal complex. In one embodiment, untreated plant biomass is used as an adsorbent to both recover the catalyst and impregnate the catalyst into the untreated plant biomass (such as woody biomass, including, but not limited to, poplar, hybrid polar, and other trees). In one embodiment, any conventional method is used to recover the catalyst from either the unhydrolyzed pretreated biomass (often referred to as “pretreatment liquor”) and/or the clarified (cell-free) stillage following fermentation and distillation. Such methods include, but are not limited to flocculation, precipitation, and filtration using a polyanionic flocculant (e.g., Betz-Dearborn MR2405 or Ondeo-Nalco 8702) which is commercially employed to remove heavy metals during wastewater treatment. Such methods can further include recovery by adsorption to a commercial ion exchange resin (e.g., Amberlyst™ 40Wet) which is used industrially to recover and recycle copper catalyst used in the production of adipic acid. In one embodiment, the catalyst is recovered and recycled. In embodiments which include a sugar conversion step, recovery and reuse of the ligand metal complex provides the additional benefit of further reducing toxicity during subsequent sugar conversion steps. Recovering and recycling the ligand metal complex further helps to reduce costs. In one embodiment, the process may produce monomeric aromatic compounds, such as, syringic acid, vanillin, syringaldehyde acid, vanillic acid. Such aromatic compounds are useful in a number of applications, such as food additives, polymer precursors, and several types of chemicals. In one embodiment, the process may produce aliphatic acids, including, but not limited to formic acid, oxalic acid, acetic acid, lactic acid, succinic acid, azaleic acid. Such aromatic compounds are useful in a number of applications, such as food additives, polymer precursors, and fine chemicals. The various embodiments will be further described by reference to the following examples, which are offered to further illustrate various embodiments. It should be understood, however, that many variations and modifications may be made while remaining within the scope of the various embodiments. Example 1 Materials—The hybrid poplar (Populus nigravar.charkoviensisxcaudinacv. NE-19), obtained from the University of Wisconsin Arlington Agricultural Research Station, was harvested in 2012. The air-dried wood logs were chipped and hammer-milled (HammerHead, Muson Co., Inc. USA) to pass through a 5-mm screen. The milled biomass was stored in airtight bags prior to use. The chemical composition analysis of the biomass was 45.5% glucan, 15.8% xylan (sum of xylan, galactan, and manan), 22.3% Klason lignin, 9.5% acid soluble lignin, 3.2% extractives, and 0.85% ash. The enzyme cocktails Cellic CTec3 (197.3 mg/g) and HTec3 (170.5 mg/g) were kindly provided by Novozymes A/S (Bagsværd, Denmark). All other chemicals were reagent grade and purchased from Fisher Scientific (USA) unless otherwise noted. Single-stage Cu-AHP pretreatment—The single stage O2-enhanced Cu-AHP pretreatment was conducted using a 100-mL stainless steel Parr reactor (Parr Instruments Company, Moline, IL, USA) at a biomass loading of 10% (w/v) (based on the weight of original biomass). The oxidative pretreatment was performed with several fixed conditions, including 1 mM CuSO4(0.159% w/w, based on original biomass) and 1 mM 2,2′-bipyridine (bpy) (0.156% w/w, based on original biomass). For each experiment, 5 g (dry basis) of hybrid poplar, 49.7 mL of a NaOH aqueous solution (including deionized water, 2.5 or 3.75 mL of 5 M NaOH, catalyst, and H2O2) were incubated at 200 rpm for 6-24 h. The reactions were performed at 80-110° C. with the H2O2loading of 2-8% (w/w) (based on the dry weight of original biomass) and the O2pressure of 100-300 psi (689-2068 kPa). After reaction, the reactor was quenched in an ice/water bath and depressurized at room temperature. Then, the solid fraction was separated from the liquor via filtration. The solid was washed with deionized water and stored at 4° C. for compositional analysis and enzymatic hydrolysis. The liquid phase was subjected to lignin precipitation by acidification with 72% (w/w) H2SO4. After reducing the pH to 2.0, the precipitate was recovered by centrifugation (10 min at 11269×g). After washing, the precipitate (lignin) was frozen at −80° C., lyophilized, and stored in the dark at 4° C. Enzymatic hydrolysis—Enzymatic hydrolysis was performed using 15-mL Falcon tubes at 5% (w/v) solid loading (based on the weight of original, untreated biomass) and 50° C. for 72 h in 50 mM sodium citrate buffer (pH 5) with orbital shaking at 80 rpm (C24KC Incubator Shaker, New Brunswick Scientific, NJ, USA). The enzyme loading was 15 mg protein/g glucan (based on initial glucan content) using an enzyme cocktail consisting of CTec3 and HTec3 at a protein ratio of 1:1. Following enzymatic hydrolysis, the reaction mixture was centrifuged for 10 min at 3075×g to separate liquid phase. The concentration of glucose and xylose released into solution was measured using a high-performance liquid chromatography (HPLC) system (Agilent 1260 Series) following a National Renewable Energy Laboratory (NREL) protocol. Chemical composition analysis—The moisture content of the biomass was determined by drying at 105±2° C. to a constant weight. Before and after the pretreatment, the chemical composition of biomass was measured following an NREL two-stage hydrolysis protocol. In brief, the air-dried biomass was ground with a Wiley Mill to pass through 20 mesh screen. A sample (0.1 g) of the ground material was digested by the two-step H2SO4hydrolysis protocol. After hydrolysis, the acid-insoluble lignin (Klason lignin) was separated by filtration, dried at 105±2° C., and weighed. The content of carbohydrates was quantified by an HPLC system (Agilent 1260 Series equipped with an infinity refractive index detector) fitted with a Bio-Rad Aminex HPX-87H column (Bio-Rad Laboratories, USA) using 5.0 mM sulfuric acid as the mobile phase with a flow rate of 0.6 mL min−1and an operation temperature of 65° C. The xylose content reported is the combination of xylose, mannose, and galactose because the HPX-87H column cannot separate these three sugars. Sugar quantification was accomplished by comparing the peak area to a standard curve prepared using pure glucose and xylose. Lignin characterization—The hydroxyl content of the lignin samples was measured using phosphorous-31 nuclear magnetic resonance (31P NMR) spectroscopy. Approximately 40 mg of the dry lignin was dissolved in 325 μL of a mixture of anhydrous pyridine and CDCl3(volume ratio of 1.6:1) and 300 μL anhydrous dimethylformamide (DMF). After completely dissolving the lignin, 100 μL cyclohexanol (stock concentration of 22 mg/mL) in anhydrous pyridine and CDCl3(volume ratio of 1.6:1) was added into the solution as an internal standard. Chromium (III) acetylacetonate (50 μL of a 5.6 mg/mL stock solution) in anhydrous pyridine and CDCl3(volume ratio of 1.6:1) was added into the mixture as a relaxation reagent. Phosphorylation of the lignin hydroxyl groups was initiated by the addition of 100 μL of the phosphitylation reagent 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane. Analysis of the solution was performed using an Agilent DDR2 500 MHz NMR spectrometer (relaxation delay of 5 s and 128 scans) equipped with a 7600AS autosampler and VnmrJ 3.2A software. After obtaining the31P NMR spectra, the hydroxyl content of the lignin sample was calculated based on the ratio of the cyclohexanol internal standard (145.3.1-144.9 ppm) peak areas to the sample peak areas as described by Brandt et al. To evaluate the appropriateness of using the lignin in polyurethane formulations, the reactivity of the lignin towards isocyanate (a key reagent that reacts with polyols in the manufacture of polyurethane) was tested. The reactivity of the lignin was determined using a titration method following a modified version of the American Society for Testing and Materials standard ASTM-D5155-14. Briefly, 1.0 g of methylene diphenyl diisocyanate (MDI) and 1.0 g of oven-dried lignin were dissolved in 6.0 g of DMF (dried with 4 A molecular sieves, Fisher Scientific) and heated at 50° C. for 60 min. Then, 0.5 g of the solution was added to 25 mL of a dibutylamine solution (2 M) in toluene and mixed at 23° C. for 15 min at 150 rpm, followed by the addition of 110 mL of 2-propanol. After mixing, the solution was titrated with 1 M HCl to pH 4.2. A separate experiment was performed without the addition of the lignin-isocyanate sample as the control. Polyethylene glycol 400 (PEG 400) was used as a reference petroleum-based polyol. The amount of free isocyanate (unreacted isocyanate) was calculated according to equation (1): %NCO=4.202(V1-V2)×Mm(1) where % NCO is the fraction of free (unreacted) isocyanate, V1is the volume of HCl required to reach pH 4.2 for the control sample (mL), V2is the volume of HCl required to reach pH 4.2 for lignin-isocyanate sample (mL), M is the molarity of HCl, and m is the weight (g) of the lignin/isocyanate sample added to the analysis mixture. 1H-13C-gradient heteronuclear single quantum coherence (HSQC) spectra were recorded on a 500 MHz Bruker NMR spectrometer equipped with a 5 mm iProbe (BBO probe) at room temperature using pulse sequence “hsqcedetgpsisp2.3”. Spectra were recorded with spectral widths of 8013 Hz (1H) and 20 kHz (13C) with an acquisition time of 63.9 ms (F2, 512 complex points for1H) and 63.9 ms (F1, 1024 increments for the13C dimension) and 48 scans were taken per increment using a delay of 1.5 s. Depolymerization of lignin—Lignin oxidation was conducted using the lignin oxidation and depolymerization (LOAD) process described in Alherech, M. et al.ACS Cent. Sci.2021, vol. 11, pp. 1831-1837, incorporated herein by reference. Briefly, to a hollowed, 100 mm tall, 26 mm O.D., and 24 mm I.D. PTFE vial were added a 1.5 mm×7.9 mm PTFE coated stir bar, 50 mg of lignin, 10 mL 2 M aqueous sodium hydroxide, and 3.3 mg CuSO4·5 H2O. The solution was stirred at room temperature until the lignin dissolved while 115 mL of water as a heating medium was added to a 1-L, stainless steel Parr reactor. The PTFE tube containing the reaction contents was placed in a 1-L stainless steel vessel. The Pan vessel was wrapped with a heating mantle, affixed to a stir plate, sealed with a lid bearing a pressure gauge and thermocouple, then protected with a blast shield. The stirring was turned on and the reactor was pressurized to 25 bar with air. The heating mantle and thermocouple were connected to a Parr 4838 Reaction Controller tuned to a 175° C. set point and turned on to heat the reactions. After 45 minutes, when the reaction reached 160° C., the heating was turned off and the reactor was submerged in a bucket of ice. When the reaction temperature fell to below 45° C., the pressure was released and the reactor was opened. The contents of the PTFE tube were acidified with concentrated HCl until the solution became cloudy and was extracted with ethyl acetate (3×5 mL). The ethyl acetate solutions were combined and concentrated by rotary evaporation until a dark residue remained. Results and Discussion Alkaline-oxidative pretreatment condition investigation—To examine the performance of the single-stage alkaline-oxidative pretreatment, reactions with original biomass were performed. The first series of reactions were performed with 8% H2O2and 300 psi O2.FIG.2shows the effect of reaction temperature and time on the solubilization of lignin. The base case (FIG.2) was the biomass pretreated following the two-stage alkaline pre-extraction (120° C., 10% NaOH, 1 h)/alkaline-oxidative pretreatment (80° C., 10% NaOH, 1 mM Cu, 1 mM 2,2′-bipyridine, 2% H2O2, 50 psi O2, 12 h) process (Yuan et al., 2021). As shown, with increasing the pretreatment severity, the amount of solubilized lignin increased. Under the most severe conditions investigated (100° C., 24 h), about 80% of original lignin was solubilized, which was even higher than the two-stage pretreatment process (base case, −75%). The solubilization of such high amount of lignin has the potential to generate biomass with high enzymatic digestibility. FIGS.3A-3Bshows the overall sugar release following enzymatic hydrolysis. As shown inFIG.3A, when increasing the single-stage alkaline-oxidative pretreatment severity, the overall glucose yield (˜95%) was similar to that of the two-stage alkaline pre-extraction/alkaline-oxidative pretreatment process. Also, the xylose yield was close to 100% (based on initial xylan content). Based on the results shown inFIGS.2,3A and3B, the pretreatment conditions under temperature of 100° C. and time of 24 h were selected for the following study. Since the H2O2loading (8% w/w) and O2pressure (300 psi) were high under the preliminary screening conditions (FIGS.2,3A and3B), the possibility of reducing these inputs during the single-stage alkaline-oxidative pretreatment was investigated.FIG.4shows the solubilized lignin under various studied conditions with reduced H2O2loading and O2pressure. As shown inFIG.4, when reducing the pretreatment severity, the lignin solubilization decreased. However, when performing the Cu-AHP pretreatment with 4% H2O2and 300 psig O2pressure, the lignin solubilization was still around 80% (based on initial lignin). To further reduce the O2pressure (from 300 psig to 200 psig) while maintaining the solubilization of such high amount of lignin, the temperature of the pretreatment needed to be increased to 110° C. FIGS.5A-5Bshow the overall sugar yields following enzymatic hydrolysis of the pretreated biomass. As shown, the overall glucose yields (FIG.5A) could still reach −95% (based on initial glucan content) when reducing both the H2O2loading (from 8% to 4%) and the O2pressure (from 300 psig to 200 psig). Technoeconomic Analysis (TEA) of the Single-Stage Cu-AHP Pretreatment TEA analysis was conducted under different conditions as indicated below in Table 2 and Table 3. 5 mm NE-19 poplar was used as the raw biomass for all the runs in the Experiment series #1-9. Experiment Series 1: #1-#9 Pretreatment conditions: one-stage O2—Cu-AHP pretreatment at some fixed conditions: 15% (w/w) NaOH loading, 8% (w/w) H2O2, 1 mM Cu(bpy), 300 psi O2(Table 2). The time and/or temperature of the pretreatment process was varied in the various runs. Enzymatic hydrolysis: 15 mg protein/g glucan, 72 h, 50° C., pH 5. Table 2 andFIG.6shows the sugar hydrolysis yields and technoeconomic analysis results for a number of conditions (Experiment series 1: #1-#9). Table 2 also shows sugar yields following enzymatic hydrolysis of O2—Cu-AHP pretreated poplar under different conditions. TABLE 2H2O2O2TemperatureloadingpressureTimeSugar yields %)aRun #(° C.)(%)(psi)(h)GlucoseXylose1808300652.3 ± 0.853.9 ± 1.228083001257.4 ± 1.161.9 ± 1.238083002460.8 ± 1.263.0 ± 0.94908300664.4 ± 1.469.6 ± 1.459083001267.1 ± 1.369.1 ± 1.169083002479.7 ± 1.178.9 ± 0.971008300670.9 ± 0.868.4 ± 0.8810083001278.6 ± 0.878.0 ± 0.8910083002482.8 ± 1.078.2 ± 0.9abased on original sugar composition Table 2 shows that higher sugar yields can be obtained at higher temperatures and incubation times.FIG.6shows that the MFSP can be reduced with a process using higher temperature and shorter reaction times. At 12 hours and 100° C.,FIG.6also illustrates (bars (a) and (b) that the MFSP can be even lower based on different assumptions for lignin. The value of acid insoluble lignin, the value of lignin being used as polyurethanes and the value of lignin being used as aromatic monomers can lead to lower MFSP. Experiment Series 2: #10-#18 This is a set of one-stage O2—Cu-AHP pretreatment process (no alkaline pre-extraction stage). Fixed experimental conditions: 15% (w/w) NaOH loading, 1 mM Cu(bpy), 24 h. Experiments were performed in triplicate. Enzymatic hydrolysis: 15 mg protein/g glucan, 72 h, 50° C., pH 5. TEA of samples #10-18 is shown inFIG.7and Table 3. Conditions are as indicated for #10-18. Assumption: precipitated lignin is sold as polyol replacement at $0.8/kg. Costs of the samples #10-18. O2pressure was taken into consideration. Table 3 shows sugar yields following enzymatic hydrolysis of one-stage O2—Cu-AHP pretreated poplar under different conditions. TABLE 3H2O2O2RunTemperatureloadingpressureSugar yields %)a#(° C.)(%)(psi)GlucoseXylose10100820082.2 ± 1.375.1 ± 0.911100810078.6 ± 1.172.4 ± 0.812100430079.9 ± 1.269.9 ± 0.513100420079.2 ± 1.270.5 ± 0.414100410077.8 ± 1.471.8 ± 1.315100230079.1 ± 0.970.9 ± 1.216100210076.9 ± 0.770.6 ± 0.817110430083.4 ± 0.973.7 ± 1.418110420081.5 ± 1.571.3 ± 0.6abased on original sugar composition The oxygen pressure, the temperature and H2O2loading were varied and the effect of MFSP was calculated.FIG.7shows the effect of MFSP under different conditions. The MFSP is the lowest for run #16. The sugar yields are greater for runs #17 and #18 than for #16 with slight increases in MFSP. FIG.8is a plot of the effect of pretreatment temperature, H2O2loading, and O2pressure during the Cu-AHP pretreatment on pretreatment capital costs (CAPEX) and operating costs (OPEX) (bars with (x) inFIG.8) for experiments #10-18. These results show that the contributions of low CAPEX and OPEX for run #16, coupled with high sugar yields, are strong contributors to the low in MFSP inFIG.7. All publications, patents and patent documents are incorporated by reference herein, as though individually incorporated by reference, each in their entirety, as though individually incorporated by reference. In the case of any inconsistencies, the present disclosure, including any definitions therein, will prevail. Although specific embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that any procedure that is calculated to achieve the same purpose may be substituted for the specific embodiments shown. For example, although the process has been discussed using particular types of plant biomass, any type of plant biomass, such as grasses, rice straw and the like, for example, may be used. Additionally, although the process has been discussed using primarily copper as the metal in the multi-ligand metal catalyst, other metals, such as iron, in various oxidation states, for example, may be used. This application is intended to cover any adaptations or variations of the present subject matter. Therefore, it is manifestly intended that embodiments of this invention be limited only by the claims and the equivalents thereof. | 68,966 |
11859146 | DESCRIPTION OF THE INVENTION The present disclosure relates to a compression ignition engine fuel which comprises about 95.0% to about 99.9% by weight ammonia and about 0.01% to about 5.0% by weight of a fuel ignition enhancement compound consisting of an alkyl nitrate or a mixture of alkyl nitrates. In one embodiment, the fuel comprises about 0.05% to about 2.0% by weight of said compound. In another embodiment, the fuel comprises about 0.1% to about 0.8% by weight of said compound. In one embodiment, the fuel of the invention consists of ammonia and said compound (and in this case, the amount of compound in the fuel is at least 0.1% by weight). In another embodiment, when the sum of the amount of ammonia and the amount of compound is not equal to 100% by weight, the fuel may contain one or more other additives to complete the fuel to 100%, such as additives with preservative, anti-corrosion or detergent functions. Said compound added to the ammonia is selected from one or more linear, branched or cyclic alkyl nitrates. Said compound is more particularly selected from linear alkyl nitrates having 4 to 36, advantageously 4 to 24 carbon atoms, branched alkyl nitrates having 4 to 36, advantageously 4 to 24 carbon atoms, cyclic alkyl nitrates (or cycloalkyl nitrates) having 5 to 18 carbon atoms, and mixtures thereof. In one embodiment, said compound is selected from 2-ethylhexyl nitrate, cyclohexyl nitrate, dodecyl nitrate, n-nonyl nitrate, 2-tetradecyl-1-octadecyl nitrate, hexyl nitrate, 2-octyl nitrate, isononyl nitrate, 2-propylheptyl nitrate, a mixture of C9to C13branched alky nitrates, and mixtures thereof. In one embodiment, the alkyl nitrate is 2-ethylhexyl nitrate alone or in admixture with one or more other alkyl nitrates as defined above, advantageously the alkyl nitrate is-2-ethylhexyl nitrate alone. Mixtures of C9to C13branched alkyl nitrates can be synthesized from the corresponding mixtures of branched C9to C13 alcohols, for example the alcohols available under the tradename Exxal™ from Exxon. According to an embodiment, the compound consisting of an alkyl nitrate or a mixture of alkyl nitrates is mixed with liquefied ammonia (under pressure) in a tank which feeds an engine, to obtain the fuel according to the invention. According to one embodiment, said compound and the liquefied ammonia are stored separately, and brought into the presence of each other in an injector, thus forming the fuel according to the invention, before it is fed into the combustion chamber of the engine. According to one embodiment, said compound is stored separately from the ammonia and is co-injected with the liquefied or gaseous ammonia to form the fuel according to the invention in a premix chamber of the engine. The present disclosure also relates to the use of an alkyl nitrate or a mixture of alkyl nitrates, in the proportions defined above, as an ignition enhancer for an ammonia-based fuel. The invention is illustrated by the following illustrative examples. EXAMPLES The ignition delay improvement of liquid ammonia was measured under test conditions equivalent to those described in the scientific article “Ignition delay times of NH3/DME blends at high pressure and low DME fraction: RCM experiments and simulations” (Combustion and Flame, Volume 227, May 2021, Pages 120-134). The test laboratory engine is a fast compression machine equivalent to the one described in this scientific article. It is a fast compression machine for measuring the auto-ignition time of a mixture. This machine allows compressing in a very short time the mixture in order to obtain preset pressure and temperature conditions. The liquids are admitted into the tank through a different orifice than the gas inlet. The liquid quantities are measured with a syringe and a precision balance. The ignition delay dAI is defined according to the following formula in which Pc is the pressure applied to the injected fuel: dAI=t(dPdtmax)-t(Pc) Example 1 The ignition delay was determined as a function of the injection temperature (between 950K and 1100K) at a pressure Pc of 40 bar of a fuel consisting of 99.6% by weight ammonia and 0.4% by weight EHN and for a mixture richness of 1 with air. The given reference points of the ignition delay of ammonia alone (FIG.2, left curve) are from the above mentioned article. A significant reduction of about a factor of 10 in fuel ignition delay compared to ammonia alone is observed inFIG.2(right curve). This reduction in ignition delay with the fuel of the invention compared to ammonia alone is greater the lower the temperature. Example 2 The ignition delay of a fuel consisting of 99.8% by weight of ammonia and 0.2% by weight of EHN was determined as a function of the injection temperature (between 925K and 1000K) at a pressure Pc of 30 bars and for a mixture richness of 1.5 with air. The ignition delays of the fuel are less than 800 ms (FIG.3) whereas under these test conditions ammonia alone does not ignite. Example 3 The ignition delay of a fuel consisting of either ammonia alone, or 99.9% by weight ammonia and 0.1% by weight EHN, or 98.0% by weight ammonia and 2.0% by weight EHN, was determined at 3 temperatures (1000K, 1050K and 1100K), at a pressure Pc of 43.4 bar, for a mixture richness of 0.35 with air. For all three temperatures, the ignition delays of the fuels are lower than those of ammonia alone (FIG.4). The effectiveness of EHN addition on ignition delay compared to ammonia alone (as already observed in Example 1) is greater the lower the temperature. An optimum weight ratio of 0.25% EHN is substantially achieved by extrapolating the curves under these three temperature conditions. These examples show that the use of alkyl nitrate(s) in very low weight percentage can significantly improve the ignition delay of an ammonia-based fuel. There was no reason to believe that additives known to increase the cetane number of a diesel or biodiesel hydrocarbon could be used so effectively, in very small quantities, to improve the ignition of ammonia. Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps. In addition to the various embodiments depicted and claimed, the disclosed subject matter is also directed to other embodiments having any other possible combination of the features disclosed and claimed herein. As such, the particular features presented herein can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter includes any suitable combination of the features disclosed herein. Thus, the foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed. It will be apparent to those skilled in the art that various modifications and variations can be made in the device, method, and system of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Thus, it is intended that the disclosed subject matter include modifications and variations that are within the scope of the appended claims and their equivalents. For any patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of all of which are incorporated herein by reference in their entireties for all purposes. | 8,588 |
11859147 | DETAILED DESCRIPTION OF THE INVENTION The present invention describes a HyRICH (hydrogen enrichment) process for HCNG (hydrogen mixed/enriched compressed natural gas) production. The process also presents quick start up and shut down solution and also eliminates the need of high temperature operation as evident in reported prior art (partial reforming, methanation or solid oxide electrolyzer cell (SOEC) based co-electrolysis). The process also presents flexibility not only in handling wider turn down ratios but also in level of hydrogen concentration in CNG, by optimizing the operating potential besides offering flexibility regarding the source of hydrogen (like water, methanol, ethanol etc.) by varying the operating conditions. In one typical mode of operation (with water as hydrogen feedstock), this technique also generates pure oxygen which can be utilized for various other applications. With the current configuration, the need of separate low/high pressure gas mixing arrangement (as in the case of separate hydrogen generation, compression, storage and mixing with CNG) is eliminated thus making the process more compact and energy efficient. The present invention gives a single stage process for HCNG production. It eliminates the necessity of additional mixing device for hydrogen and gaseous fuel thus making the system more energy efficient and responsive. The process is carried out at low temperature and offers wide range of turn down ratio. The process also presents flexibility not only in handling wider turn down ratios but also in level of hydrogen concentration in CNG. The solution in the present process also generates pure oxygen, which can be used for other applications. Carbon monoxide (CO) is a known impurity present in natural gas (NG) which poisons the catalyst of electrochemical device, resulting in higher energy losses to maintain same hydrogen concentration in product gas throughput. The present invention addressed the above issue in an efficient way by modifying the gas distribution material and flow design in HCNG generation unit. The process of producing hydrogen enriched compressed natural gas (HCNG) consists of storage arrangement for base gas (CNG) (01) and hydrogen source (02) as shown in FIG. (1). CNG may contain contaminant such as sulfur and CO, which needs to be removed through suitable purification processes like desulfurization and CO-methanation/PSA/electrochemical oxidation respectively (02). The adsorbents normally used in adsorption desulfurization processes are natural or synthetic zeolites, activated carbons, and metal oxides. These materials can have crystalline (zeolites) and/or amorphous structures (activated carbons) at both the macro and nanoscale, but they can be further modified to alter their physicochemical properties, thereby upgrading their adsorption capacity toward target molecule. Presently, the Cu(i)-based adsorbents (zeolites or activated carbon) are mainly used for CO adsorption using PSA (pressure swing adsorption). Whereas Ni/ZrO2and Ru/TiO2were the most effective catalysts for complete removal of CO through the methanation. The present process presents flexibility of HCNG generation with or without CO in the feed gas thus the CO pre-treatment unit may also be eliminated. FIG. (1) discloses the schematic representation of device and method for generation of hydrogen enriched/mixed gases. CNG is preheated to desired process operating temperature in heat exchanger (03) through compressor waste heat. The flow rate of preheated CNG can be controlled through control valves (10and11) to allow partial or complete bypass in HCNG generation unit (04) depending on degree of hydrogen enrichment in HCNG production. Water is preheated to process operating temperature (70-80° C.) through heat exchanger (08) by utilizing the heat rejected by compressor coolant stream. However, the preheating stage for both gas feed and water is optional and can be omitted during system start-up. The flow rate of hydrogen feedstock to zone-A of HCNG generation unit (04) can be controlled through control valve (12). The present invention process consists of HCNG generation unit (04) having two zones, one for hydrogen source (Zone-A) and other for HCNG generation reaction (Zone-B), in a single step. Hydrogen feedstock is oxidized at Zone-A, whereas hydrogen generation and homogeneous mixing with CNG occurs at Zone-B. Zone-B of HCNG generation unit consists of perforated catalyst protective sheets (PCPS) which prevents catalyst poisoning issue from impurities present in the feed (especially CO). PCPS is highly electrically conductive and designed with optimized number of opening for hydrogen diffusion. HCNG generation without PCPS results in higher energy losses due to CO poisoning of catalyst as shown in FIG. (2). Hydrogen generation unit (HCNG) may consist of single unit or combinations of multiple units. In FIG. (3), each individual unit is connected in common header where CNG gas stream is distributed among all units of Zone-B equally. Each Zone-B is also consisting of optimized micro channels configurations to ensure uniform mixing and homogeneous gas compositions of HCNG. The structure enables the efficient arrangement of numerous channels and allows uniform gas distribution and mixing in each of channels. During start-up phase, localized hydrogen pressure at catalyst-PCPS interface is maintained higher than pressure prevailing PCPS-microchannel interface by applying DC Power Source13. Until then control valve-10is maintained in OFF position to avoid catalyst contamination by any gas impurity. CNG or any other gas for hydrogen enrichment is then passed through heat exchanger (HEX03) to control valve-10into zone-B of HCNG generation unit04. Control valve10is maintained in ON position during subsequent operation. CNG gas stream is prevented from poisoning catalyst surface of Zone-B due to the maintained pressure difference. Hydrogen enriched gas is then passed through moisture eliminator14to buffer vessel15which is further compressed in compression unit05and stored in storage unit06. Operating pressure of HCNG generation unit (04) is maintained at 1-20 barg and temperature (25-80° C.). The two zones are separated by ion conductive polymer with suitable catalyst (such as Pt, Au, Ir etc.) to enhance the rate of hydrogen generation reaction. Since the feed gas is preheated to operating temperature of 70-80° C., molecular collision between Hydrogen and CNG is greatly enhanced in the microchannels. Turbulent mixing of HCNG is further enhanced due to recombination of multiple gas streams from various microchannels of several units mixing together in the return header. FIG. (4) depicts one such design for PCPS. The perforation position and size is determined by availability of mixing length, localized pressure distribution of gas stream in microchannel and degree of mixing in microchannel configuration. The high heat transfer coefficient in the channels ensures instantaneous heat absorption/rejection from heat transfer media due to excess enthalpy of hydrogen-NG mixing. Effective % RH (Relative Humidity) of HCNG stream at the outlet of HCNG generation unit (08) is lower than the conventional saturated Hydrogen stream in PEM (Proton exchange membrane) water electrolyser. Thus, better water management enhances operation efficiency. The process is a single stage process which means the desired concentration of H2can be achieved in a single step without any further need of thermal/electrochemical conversion. There are no different stages of process. The process is continuous and desired product is delivered at the end of first step itself. Localized hydrogen pressure at catalyst sites is greater than partial pressure of gas on the other side of PCPS, thereafter, a positive flux of hydrogen is maintained from catalyst sites into microchannels. PCPS acts a diaphragm in between catalyst and microchannels. The perforations in PCPS allows hydrogen molecules to pass through them freely. The reaction in Zone-A is can be summarized as below: For Water based feed: H2O⇌2H++12O2+2e*(1) Whereas the reaction in Zone-B is as given below: 2H++2e−+CH4⇄H2+CH4(2) The rate of hydrogen enrichment can be controlled by the applied potential across two zones through DC source (13), thereby giving the user flexibility of deciding the degree of hydrogen enrichment in CNG, without changing any other process parameter, through feed forward mechanism. The HCNG generation unit (04) can operate intermittently or continuously to generate HCNG depending on downstream requirement. The process also offers flexibility of changing the fuel (like LPG and biogas) to be enriched, without any need of changing the device or catalyst. The resultant HCNG generated from Zone-B is stored in buffer tank (15) which is compressed in compression unit (05) to the suitable pressure. HCNG is then stored in storage vessel (06) before being dispensed/transported for further application. The compressor unit can be deployed with suitable coolant for interstage cooling to maintain near ambient discharge temperature of the compressed stream. Heat integration of the coolant stream can be done with CNG and water feed to HyRICH for better system efficiency. During the operation, pure oxygen is also generated from Zone-A (for the case of water-based operation mode) which can be further utilized for other application. The complete operation can be regulated by pre-defined logic-based controller so as to ensure same level of hydrogen enrichment for variable fuel flow rate or variable hydrogen enrichment of fuel with same flow rate or combination of thereof. The generated oxygen can be utilized for various medical or industrial applications. For on-board deployment of HyRICH for automotive applications, generated oxygen can be utilized for making oxygenated gas stream to improve fuel economy. The HyRICH system pressure can be adjusted to make it suitable for integration with gas trunkline, for distribution of hydrogen enriched fuel. In an aspect of the present invention, the presentation invention discloses a single stage process for generation of hydrogen enriched gas, wherein the process comprising: a) routing hydrogen source through heat exchanger HEX08to Zone-A of hydrogen enriched gas generation unit04; b) maintaining localized hydrogen pressure at catalyst-PCPS (perforated catalyst protective sheets) interface higher than pressure prevailing PCPS-microchannel interface; c) passing feed gas for hydrogen enrichment through heat exchanger HEX03to Zone-B of hydrogen enriched gas generation unit04. In an aspect of the present invention, the presentation invention discloses a single stage process for generation of hydrogen enriched gas, wherein the process comprising: a) routing hydrogen source through heat exchanger HEX08to Zone-A of hydrogen enriched gas generation unit04; b) maintaining localized hydrogen pressure at catalyst-PCPS (perforated catalyst protective sheets) interface higher than pressure prevailing PCPS-microchannel interface by applying DC Power Source13and maintaining Control valve-10in OFF position to avoid catalyst contamination by any gas impurity; c) passing feed gas for hydrogen enrichment through heat exchanger HEX03to the control valve-10into Zone-B of hydrogen enriched gas generation unit04and maintaining the control valve10in ON position during subsequent operation; d) preventing poisoning of catalyst surface of Zone-B with feed gas stream due to the maintained difference in the localized hydrogen pressure; e) passing the hydrogen enriched CNG generated in step c) through moisture eliminator14to buffer vessel15which is further compressed in hydrogen enriched gas compression unit05and stored in storage unit06. In an aspect of the present invention, the presentation invention discloses a single stage process for generation of hydrogen enriched compressed natural gas (HCNG), wherein the process comprising: a) routing hydrogen source through heat exchanger HEX08to Zone-A of HCNG generation unit04; b) maintaining localized hydrogen pressure at catalyst-PCPS (perforated catalyst protective sheets) interface higher than pressure prevailing PCPS-microchannel interface by applying DC Power Source13and maintaining Control valve-10in OFF position to avoid catalyst contamination by any gas impurity; c) passing CNG or any other gas for hydrogen enrichment through heat exchanger HEX03to the control valve-10into Zone-B of HCNG generation unit04and maintaining the control valve10in ON position during subsequent operation; d) preventing poisoning of catalyst surface of Zone-B with CNG gas stream due to the maintained difference in the localized hydrogen pressure; e) passing the hydrogen enriched CNG or any other gas through moisture eliminator14to buffer vessel15which is further compressed in HCNG compression unit05and stored in storage unit06. In an embodiment of the present invention, the ion conductive polymer is tetrafluoroethylene-perfluoro-3,6-dioxa-4-methyl-7-octenesulfonic acid copolymer. In one another aspect of the present invention, the present invention discloses a single stage process for generation of hydrogen enriched gas, wherein the process comprising: a) routing hydrogen source through heat exchanger HEX08to Zone-A of hydrogen enriched gas generation unit04; b) maintaining localized hydrogen pressure at catalyst-PCPS (perforated catalyst protective sheets) interface higher than pressure prevailing PCPS-microchannel interface; c) passing feed gas for hydrogen enrichment through heat exchanger HEX03to Zone-B of hydrogen enriched gas generation unit04. In an embodiment of the present invention, the hydrogen enriched gas generated in step c) is passed through moisture eliminator14to buffer vessel15which is further compressed in hydrogen enriched gas compression unit05and stored in storage unit06. In an embodiment of the present invention, the hydrogen enriched gas generation unit04consists of a single unit or a combination of multiple units. In an embodiment of the present invention, the process in the hydrogen enriched gas generation unit04is operated at a pressure in the range of 1-20 barg and at a temperature in the range of 25-80° C. In an embodiment of the present invention, in step b) the localized hydrogen pressure at catalyst-PCPS interface higher than pressure prevailing PCPS-microchannel interface is maintained by applying DC Power Source13. In an embodiment of the present invention, the feed gas is selected from the group consisting of CNG, LPG and biogas with or without CO impurity. In an embodiment of the present invention, the hydrogen enriched gas generation unit04is with ion conducting electrolyte and electrodes; and the electrodes are selected from the group consisting of noble metals, transition metals and any combinations thereof. In an embodiment of the present invention, the noble metals are selected from the group comprising of Pt, Pd, Ru, Rh, Ir, Au, and Ag; and the transition metals are selected from the group comprising of Mo, Cu, Ni, Mg, Co, Cr, Sn, and W. In an embodiment of the present invention, the ion conductive electrolyte is a solid or liquid electrolyte. In an embodiment of the present invention, the catalyst-PCPS interface is with catalyst protective sheet between catalyst layer and flow channels in Zone-B to elude CO contamination. In an embodiment of the present invention, the catalyst protective sheet is having high electrical conductivity, corrosion resistance, and optimized size and positions of perforations. In an embodiment of the present invention, the catalyst protective sheet is made of carbon allotropes, Al, Cu, Au, Ag, Fe, Cr, or any combinations thereof and the carbon allotrope is selected from graphite, graphene and CNT (carbon nanotubes). In an embodiment of the present invention, catalyst surface of the Zone-B is prevented from poisoning with feed gas stream due to the maintained difference in the localized hydrogen pressure. In an embodiment of the present invention, the hydrogen enriched gas generation unit04is with feedback or feed forward control mechanism to ensure desired hydrogen enrichment in CNG. In an embodiment of the present invention, the hydrogen enriched gas generation unit04operates on voltage source not limited to any DC power source or energy converter for mobility and stationary applications. In an embodiment of the present invention, the process generates a source of pure oxygen, and the process utilizes the pure oxygen generated during operation for making an oxygenated gas stream in downstream of Zone-B with or without hydrogen. In an embodiment of the present invention, the process is having arrangement to control temperature of the hydrogen enriched gas generation unit04for hydrogen enriched gas generation. In an embodiment of the present invention, the hydrogen enriched gas generation unit04is for in-situ measuring of hydrogen percentage in hydrogen mixed gas. In an embodiment of the present invention, the noble metals are selected from the group consisting of Pt, Pd, Ru, Rh, Ir, Au, and Ag; and the transition metals are selected from the group consisting of Mo, Cu, Ni, Mg, Co, Cr, Sn, and W. The technical advantages offered by the present invention are:Single stage process (HyRICH process) for HCNG or hydrogen enriched (0.1-99.9%) gas production;High tolerance to presence of impurity in natural gas or in any gas stream (mainly CO);Zero carbon footprint process;Energy efficient process as operated at low temperature;Fast start-up and shut down as required;Wide turn-down ratio (practically from 0 to 1 in lowest time);Elimination of need of a separate high pressure gas mixing arrangement;Flexibility of selection of gaseous fuel for Hydrogen enrichment;Co-generation of pure oxygen gas;Improved fuel economy with oxygen supported combustion (vs conventional air combustion) in end application when used on board;Single step in-situ enrichment process; andIntegration can be done with gas trunk line for city gas distribution of hydrogen blended gaseous fuels. EXAMPLES Example 1: FIG. (2) graphically illustrates hydrogen enrichment of NG. The graph shows performance comparison of HCNG generation unit with and without unit PCPS. The PCPS is made of copper with optimized number of holes. The ion conducting membrane between zone-A and zone-B is Nafion™-117 (Tetrafluoroethylene-perfluoro-3,6-dioxa-4-methyl-7-octenesulfonic acid copolymer). The membrane is coated with IrO2(2 mg/cm2) and 40% Pt/C (0.2 mg/cm2) as zone-A and zone-B catalysts respectively with active area of 25 cm2. Water is used as the source of hydrogen. Flow rates of water and gas were maintained at 0.05 LPM and 0.32 NLPM respectively. The operating temperature and pressure of HCNG generation unit were 50° C. and 1.5 barg respectively. Hydrogen enrichment level in final mixture was maintained at 18% (v/v). FIG. (2) reveals that PCPS prevents catalyst contamination (due to CO) by maintaining same overpotential throughout the operation as against HCNG generation without PCPS which records increase in overpotential requirement. | 19,244 |
11859148 | DETAILED DISCLOSURE Aspects according to the present technology are described hereinafter. Various modifications, adaptations or variations of such exemplary aspects described herein may become apparent to those skilled in the art as such are disclosed. It will be understood that all such modifications, adaptations or variations that rely on the teachings of the present technology, and through which these teachings have been advanced in the art, are considered to be within the scope and spirit of the disclosed technology. The disclosed technology provides a lubricating oil composition comprising:a) an oil of lubricating viscosity; andb) one or more N-aralkyl α-carbonyl functional amine(s) additive(s) in an effective amount to increase TBN, reduce SAPS, mitigate corrosion and seals compatibility in an internal combustion engine. The N-aralkyl α-carbonyl functional amine additive of the disclosed technology will typically be presented in a lubricant or lubricant formulation, one component of which is an oil of lubricating viscosity. The oil of lubricating viscosity, also referred to as a base oil, may be selected from any of the base oils in Groups I-V of the American Petroleum Institute (API) Base Oil Interchangeability Guidelines. Oil of Lubricating Viscosity The oils of lubricating viscosity of can include, for example, natural and synthetic oils, oil derived from hydrocracking, hydrogenation, and hydrofinishing, unrefined, refined and re-refined oils and mixtures thereof. Oils of lubricating viscosity may also be defined as specified in the American Petroleum Institute (API) Base Oil Interchangeability Guidelines. Unrefined oils are those obtained directly from a natural or synthetic source generally without (or with little) further purification treatment. Refined oils are similar to the unrefined oils except they have been further treated in one or more purification steps to improve one or more properties. Purification techniques are known in the art and include solvent extraction, secondary distillation, acid or base extraction, filtration, percolation and the like. Re-refined oils are also known as reclaimed or reprocessed oils and are obtained by processes similar to those used to obtain refined oils and often are additionally processed by techniques directed to removal of spent additives and oil breakdown products. Natural oils useful in making the inventive lubricants include animal oils, vegetable oils (e.g., castor oil,), mineral lubricating oils such as liquid petroleum oils and solvent-treated or acid-treated mineral lubricating oils of the paraffinic, naphthenic or mixed paraffinic-naphthenic types and oils derived from coal or shale or mixtures thereof. Synthetic lubricating oils are useful and include hydrocarbon oils such as polymerised and interpolymerised olefins (e.g., polybutylenes, poly-propylenes, propyleneisobutylene copolymers); poly(1-hexenes), poly(1-octenes), poly(1-decenes), and mixtures thereof; alkyl-benzenes (e.g., dodecylbenzenes, tetradecylbenzenes, dinonylbenzenes, di-(2-ethylhexyl)-benzenes); polyphenyls (e.g., biphenyls, terphenyls, alkylated polyphenyls); diphenyl alkanes, alkylated diphenyl alkanes, alkylated diphenyl ethers and alkylated diphenyl sulphides and the derivatives, analogs and homologs thereof or mixtures thereof. Other synthetic lubricating oils include polyol esters (such as Priolube® 3970), diesters, liquid esters of phosphorus-containing acids (e.g., tricresyl phosphate, trioctyl phosphate, and the diethyl ester of decane phosphonic acid), or polymeric tetrahydrofurans. Synthetic oils may be produced by Fischer-Tropsch reactions and typically may be hydroisomerised Fischer-Tropsch hydrocarbons or waxes. In one aspect, oils may be prepared by a Fischer-Tropsch gas-to-liquid synthetic procedure as well as other gas-to-liquid oils. Oils of lubricating viscosity may also be defined as specified in the American Petroleum Institute (API) Base Oil Interchangeability Guidelines. The five base oil groups are as follows: Group I (sulfur content >0.03 wt. %, and/or <90 wt. % saturates, viscosity index 80-120); Group II (sulphur content≤0.03 wt. %, and ≥90 wt. % saturates, viscosity index 80-120); Group III (sulphur content≤0.03 wt. %, and ≥0.90 wt. % saturates, viscosity index≥120); Group IV (all polyalphaolefins (PAOs)); and Group V (all others not included in Groups I, II, III, or IV). The oil of lubricating viscosity comprises an API Group I, Group II, Group III, Group IV, Group V oil or mixtures thereof. Often the oil of lubricating viscosity is an API Group I, Group II, Group III, Group IV oil or mixtures thereof. Alternatively, the oil of lubricating viscosity is often an API Group II, Group III or Group IV oil or mixtures thereof. In some aspects, the oil of lubricating viscosity used in the described lubricant compositions includes a Group III base oil. The lubricating oil compositions of the disclosed technology comprise a major amount of oil of lubricating viscosity and a minor amount of one or more N-aralkyl α-carbonyl functional amine(s). The amount of the oil of lubricating viscosity present is typically the balance remaining after subtracting from 100 wt. % the sum of the amount of the additive(s), including the one or more N-aralkyl α-carbonyl functional amine(s) as described hereinbelow. Basic Ashless Additive A primary additive contained in the lubricating oil compositions of the disclosed technology is a basic ashless additive selected form a N-aralkyl α-carbonyl functional amine. By N-aralkyl α-carbonyl functional amine is meant that the α-carbon atom relative to the carbonyl group is situated between and covalently bonded to the amine nitrogen atom and the carbonyl group of the carbonyl functional moiety. Here and throughout the specification the term “aryl” refers to an unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl, anthryl, and phenanthryl) which condensed rings may or may not be aromatic. In one aspect, the aryl group contains from 6 to 14 annular carbon atoms. The term “aryl” also includes aromatic compounds that include alkyl, alkenyl, amino, hydroxyl, alkoxy, and halo substituents. Aryl is inclusive of “heteroaryl” which refers to an unsaturated aromatic carbocyclic group having from 2 to 10 annular carbon atoms and at least one annular heteroatom, including but not limited to heteroatoms such as nitrogen, oxygen and sulfur. A heteroaryl group may have a single ring (e.g., pyridyl, furyl) or multiple condensed rings (e.g., indolizinyl, benzothienyl) which condensed rings may or may not be aromatic. The term “aralkyl” refers to a moiety in which an aryl or heteroaryl substituent is attached to a divalent alkylene moiety and wherein the alkylene moiety is attached to the parent structure through an amine nitrogen through an alkylene moiety. The alkylene moiety can be substituted or unsubstituted. When substituted the substituent(s) is selected from a C1-C24, or a C1-C10, or a C1to C8, or a C1to C5, or a C1to C5hydrocarbyl group. In one aspect, the divalent alkylene moiety is a substituted or unsubstituted methylene group which is between and directly bonded to the aryl substituent and the amine nitrogen. In one aspect, the substituent on the alkylene residue is an alkyl group containing 1 to 5 carbon atoms. In one aspect, the substituent is methyl, ethyl, propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, pentyl, neo-pentyl, hexyl, and iso-octyl, 2-ethylhexyl. In one aspect, the aralkyl substituent is a benzyl group wherein the benzylic carbon atom can be substituted or unsubstituted. In one aspect, the benzylic carbon atom is mono-substituted with an alkyl group containing 1 to 5 carbon atoms. In one aspect, the benzylic carbon atom can be di-substituted with an alkyl group containing 1 to 5 carbon atoms, wherein the substituents can be the same or different. In one aspect, the benzylic carbon atom is substituted with at least one alkyl group independently selected from methyl, ethyl, propyl, butyl, pentyl and combinations thereof. Here and throughout the specification the term hydrocarbyl” is used in its ordinary sense, which is well-known to those skilled in the art. Specifically, it refers to a group having a carbon atom directly attached to the remainder of the molecule and having predominantly hydrocarbon character. In one aspect, hydrocarbyl includes aliphatic (e.g., alkyl, alkenyl, alkynyl, and aryl), alicyclic (e.g., cycloalkyl, cycloalkenyl), as well as cyclic groups wherein the ring is completed through another portion of the molecule (e.g., two substituents together form an alicyclic moiety). In one aspect, the hydrocarbyl includes hydrocarbon moieties containing 1 to 24, or 1 to 10, or 1 to 8, or 1 to 5, or 1 to 3 carbon atoms. When the hydrocarbon moiety is aryl it contains 6 to 14 annular carbon atoms and is as defined above. The hydrocarbon moieties can be substituted or unsubstituted. Substituents include alkyl, alkenyl, amino, hydroxyl, alkoxy, and halo groups. The term “alkyl” refers to and includes saturated linear, branched, or cyclic hydrocarbon structures and combinations thereof. Alkyl groups are those having 1 to 24, or 1 to 10, or 1 to 8, or 1 to 5, or 1 to 3 carbon atoms. When an alkyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described, for example, “butyl” is meant to include n-butyl, sec-butyl, iso-butyl, tert-butyl and cyclobutyl; “propyl” includes n-propyl, iso-propyl and cyclopropyl. This term is exemplified by groups such as methyl, ethyl, propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, pentyl, neo-pentyl, hexyl, iso-octyl, 2-ethylhexyl, and the like. Cycloalkyl is a subset of alkyl and can consist of one ring, such as cyclohexyl, or multiple rings, such as adamantyl. A cycloalkyl comprising more than one ring may be fused, spiro or bridged, or combinations thereof. In one aspect, cycloalkyl is a saturated cyclic hydrocarbon having from 3 to 7 annular carbon atoms. Examples of cycloalkyl groups include adamantyl, decahydronaphthalenyl, cyclopropyl, cyclobutyl, cyclopentyl cyclohexyl, and the like. The term “alkenyl” refers to an unsaturated hydrocarbon group having at least one site of olefinic unsaturation (i.e., having at least one carbon-carbon double bond). In one aspect, the alkenyl group contains from 2 to 24, or 2 to 10, or 2 to 8, or 2 to 5, or 2 to 3 carbon atoms. Examples of alkenyl groups include but are not limited to ethyanyl propenyl, octenyl, nonenyl, and oleoyl. The term “alkynyl” refers to an unsaturated hydrocarbon group having at least one site of acetylinic unsaturation (i.e., having at least one carbon-carbon triple bond). In one aspect the alkynyl group contains from 2 to 24, or 2 to 10, or 2 to 8, or 2 to 5, or 2 to 3 carbon atoms. Examples of alkynyl groups include but are not limited to ethynyl, propynyl, and butynyl. In one aspect, the N-aralkyl α-carbonyl functional amine of the disclosed technology can be generally represented by schematic structure (I): wherein aryl represents an aromatic ring or fused aromatic ring system containing 6 to 14 carbon atoms, or a heteroaryl group having from 2 to 10 annular carbon atoms and at least one annular heteroatom selected from nitrogen, oxygen and sulfur; the benzylic carbon atom and the α-carbon atom, independent of the other, optionally contain mono- or di-substitution by a substituent independently selected from C1-C24, or a C1-C10, or a C1to C8, or a C1to C5, or a C1to C3hydrocarbyl group. In one aspect, the hydrocarbyl group is selected from methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, pentyl, neo-pentyl, 4-methyl-2-pentyl, hexyl, cyclohexyl, iso-octyl, 2-ethylhexyl, phenyl and combinations thereof; A represents oxygen or substituted nitrogen; and R6represents a hydrocarbyl group, excluding hydrogen. By carbonyl functional moiety is meant that the carbonyl group taken together with A and R6represents an ester (A=O) or an amide (A=substituted nitrogen) group. The carbonyl functional moiety cannot represent an acid (R6≠H). In one aspect, R6represents a C1-C24, or a C1-C10, or a C1to C8, or a C1to C5, or a C1to C3hydrocarbyl group. In one aspect, R6does not represent an aromatic group. In one aspect, R6is selected from methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, pentyl, neo-pentyl, 4-methyl-2-pentyl, hexyl, cyclohexyl, iso-octyl, and 2-ethylhexyl. In one aspect, the N-aralkyl α-carbonyl functional amine of the disclosed technology can be generally represented by schematic structure (II): wherein R, R1, R2, R3and R4independently represent hydrogen or a substituted or unsubstituted hydrocarbyl group containing 1 to 24 carbon atoms, or 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 5 carbon atoms, or 1 to 3 carbon atoms; A is selected from O and NR5, wherein R5represents hydrogen or a hydrocarbyl group containing 1 to 24, or 1 to 10, or 1 to 8, or 1 to 5, or 1 to 3 carbon atoms carbon atoms; and R6represents a substituted or unsubstituted hydrocarbyl group containing 1 to 24 carbon atoms, or 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 5 carbon atoms, or 1 to 3 carbon atoms, subject to the proviso that R6is not aromatic or heterocyclic; and wherein any of two of R1, R2, R3and R4taken together with the carbon atom to which they are attached form a 5 or 6 membered carbocylic ring. In one aspect, the hydrocarbyl group defined under R, R1, R2, R3, R4and R5is independently selected from hydrogen, substituted and unsubstituted C1-C10alkyl, substituted and unsubstituted C2-C10alkenyl, and substituted and unsubstituted C6-C14aryl; and the hydrocarbyl group defined under R6is independently selected from substituted and unsubstituted C1-C10alkyl, substituted and unsubstituted C2-C10alkenyl (R6does not represent hydrogen), wherein when R, R1, R2, R3, R4, R5and R6are substituted said substituent(s), if present, is selected from C1-C5alkyl, C1-C10hydroxyalkyl, C1-C10alkoxy, amino, hydroxyl, halo (i.e., Br, Cl, F, and I), and combinations thereof. In one aspect, the hydrocarbyl group defined under R, R1, R2, R3, R4and R5is independently selected from hydrogen, methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl, iso-butyl, pentyl, neo-pentyl, 4-methyl-2-pentyl, hexyl, 2-ethylhexyl and phenyl; and the hydrocarbyl group defined under R6is selected from methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl, iso-butyl, pentyl, neo-pentyl, 4-methyl-2-pentyl, hexyl, and 2-ethylhexyl. In one aspect, A is O; R1and R2are independently selected from hydrogen, methyl and phenyl; R3and R4are independently selected from hydrogen and methyl; and R6is selected from a C1to C8alkyl group. The N-aralkyl α-carbonyl functional amines of the disclosed technology can be prepared by the reaction of a N-arylalkyl amine with an hydrocarbyl α-halo alkanoate to form the ester as illustrated in the following reaction scheme. wherein aryl, R1, R2, R3, R4, R6, and A are as previously defined, and X represents a halo substituent selected from Br, Cl, F, and I. In one aspect, the N-arylalkyl amine reactant is a primary amine. In one aspect, N-aralkyl α-carbonyl ester amine of the disclosed technology can be prepared by the reaction of benzyl amine with an alkyl α-halo ethanoate as illustrated by the reaction scheme below: wherein R, R1, R2, R3, R4, and R6are as previously defined. The reaction conditions to prepare the basic ashless additive compounds of the disclosed technology may vary depending on the starting materials employed and can be determined by the person of ordinary skill in the art. In one aspect, the reaction can be conducted in a suitable organic solvent or diluent as would be readily known by one of ordinary skill in the art, such as, for example, an alcohol or acetonitrile. In one aspect the reaction can be conducted in suitable alcohol solvents including methanol, ethanol, isopropanol, and tert-butanol. In one aspect, the reaction can be conducted in a mineral oil, such as, for example, a hydrocarbon base oil selected from an API Group I to Group V base oil, and mixtures thereof. In one aspect, the amount of solvent or diluent that can be used in the reaction can range from about 5 to about 80 wt. %, or from about 10 to about 70 wt. %, or from about 15 to about 60 wt. %, or from about 20 to 50 wt. %, or from about 25 to 40 wt. % based on the weight of the total reaction mixture. The relative molar amounts of the arylalkyl amine reactant to the alkyl α-halo alkanoate reactant can range from approximately 0.5:1 to about 1:0.5, or a slight molar excess of one reactant or the other. In one aspect, the molar ratio of the arylalkyl amine to the alkyl α-halo alkanoate can range from about 0.8 to about 1 or from about 0.9 to about 1.2, or about 1 to about 1. In one aspect, an acid scavenger, such as, for example, potassium carbonate and sodium carbonate can be employed in the reaction medium. The acid scavenger should be a stronger base than the reaction product to insure against salt formation in the product. In one aspect, a Group IA metal halide, such as, for example, potassium iodide can be employed in the reaction medium to facilitate halogen exchange in the reaction. The reaction can be conducted under an inert atmosphere (e.g., nitrogen gas) at a temperature ranging from ambient room temperature (approximately 18 to 25° C.) to about 85° C., or from about 30 to about 80, or from about 60 to about 75° C. The reaction time can vary depending on the reaction temperature and the reactivity of the starting materials. In one aspect, the reaction time can range from about 5 hours to about 90 hours, or from about 8 to about 60 hours, or from about 10 to about 50 hours, or from about 14 to about 30 hours. At the end of the reaction, the reaction product can be isolated and purified by conventional means known in the art. In one aspect, the amount (treat rate) of the basic ashless N-aralkyl α-carbonyl functional amine component in the oil of lubricating viscosity of the disclosed technology can range from about 0.1 to about 6 wt. %, or from about 0.2 to about 4 wt. %, or from about 0.25 to about 2 wt. %, or from about 0.3 to about 1 wt. %, based on the weight of the total lubricating composition. The material can also be employed in a concentrate form, alone or with other additives and a lesser amount of oil. In a concentrate, the amount of material may be two to ten times the above concentration amounts. The concentrate can be used as a post-treatment additive to maintain TBN between scheduled drain intervals. In a lubricant, the amount of the basic ashless N-aralkyl α-carbonyl functional amine may be suitable to provide at least 0.3, or 0.5, or 0.7, or 1.0, or 1.2, or 1.5 TBN to the lubricant, and in some aspects, up to 3, or 4, or 5 TBN as measured by ASTM D4739. In one aspect, the basic ashless N-aralkyl α-carbonyl functional amine delivers from about 0.5 to about 8, or from about 0.7 to about 7, or from about 0.7 to about 5, or from about 0.8 to about 4, or from about 0.8 to about 2.5, or from about 0.8 to about 1.5 mg KOH/g of ashless TBN as measured by ASTM D4739. The increase in TBN is determined relative to an identical composition in the absence of N-aralkyl α-carbonyl functional amine. The term TBN as used herein denotes the total base number in mg of KOH/gram of sample as measured by ASTM D2896 or ASTM D4739. In certain aspects, a lubricant employing the present technology may have an entire TBN, from all sources, of at least 5 or at least 6, 7, 8, 9, or 10, and may have a TBN of up to (or less than) 25, 20, or 15. In certain aspects, a lubricant employing the present technology may have a sulfated ash content of less than 1.5 or less than 1.3 or 1.0 or 0.8 percent (as measured by ASTM D874) or may be at least 0.05 or 0.1 percent. In addition to the disclosed basic ashless N-aralkyl α-carbonyl functional amine materials, the lubricating oil composition can optionally comprise other performance additives as well. The other performance additives can comprise at least one of detergents, metal deactivators, dispersants, viscosity modifiers, friction modifiers, anti-wear agents, corrosion inhibitors, dispersant viscosity modifiers, extreme pressure agents, anti-scuffing agents, antioxidants, foam inhibitors, demulsifiers, pour point depressants, seal swelling agents, color stabilizers and mixtures thereof. Typically, fully-formulated lubricating oil will contain one or more of these performance additives. The performance additives are not necessarily limited to the additives discussed below. Detergents Detergents are typically overbased materials, otherwise referred to as overbased or superbased salts, which are generally homogeneous Newtonian systems having a metal content in excess of that which would be present for neutralization according to the stoichiometry of the metal and the detergent anion. The amount of excess metal is commonly expressed in terms of metal ratio, that is, the ratio of the total equivalents of the metal to the equivalents of the acidic organic compound. Overbased materials are prepared by reacting an acidic material (such as carbon dioxide) with an acidic organic compound, an inert reaction medium (e.g., mineral oil), a stoichiometric excess of a metal base or a quaternary ammonium base, and a promoter such as a phenol or alcohol. The acidic organic material will normally have a sufficient number of carbon atoms, to provide oil-solubility. Overbased detergents can be characterized their TBN, the amount of strong acid needed to neutralize all of the material's basicity, which may be expressed as mg KOH per gram of sample. Since overbased detergents are commonly provided in a form which contains diluent oil, for the purpose of this document, TBN is to be recalculated (when referring to a detergent or specific additive) to an oil-free basis. Some useful detergents may have a TBN of 100 to 800, or 150 to 750, or, 400 to 700. The metal compounds useful in making the basic metal salts are generally any Group 1 or Group 2 metal compounds (CAS version of the Periodic Table of the Elements). Examples include alkali metals such as sodium, potassium, lithium, copper, magnesium, calcium, barium, zinc, and cadmium. In one aspect, the metals are sodium, magnesium, or calcium. The anionic portion of the salt can be hydroxide, oxide, carbonate, borate, or nitrate. The lubricant compositions of the present technology can contain one or more of the following overbased detergents. In one aspect, the lubricant can contain an overbased sulfonate detergent. Suitable sulfonic acids include sulfonic and thiosulfonic acids, including mono or polynuclear aromatic or cyclo-aliphatic compounds. Certain oil-soluble sulfonates can be represented by R10-T(SO3−)aor R11(SO3−)b, where a and b are each at least one; T is a cyclic nucleus such as benzene or toluene; R10is an aliphatic group such as alkyl, alkenyl, alkoxy, or alkoxyalkyl; (R10)-T typically contains a total of at least 15 carbon atoms; and R3is an aliphatic hydrocarbyl group typically containing at least 15 carbon atoms. The groups T, R10, and R11can also contain other inorganic or organic substituents. In one aspect, the sulfonate detergent may be a predominantly linear alkylbenzenesulfonate detergent having a metal ratio of at least 8 as described in paragraphs [0026] to [0037] of U.S. Pat. No. 7,407,919. In some aspects, the linear alkyl group may be attached to the benzene ring anywhere along the linear chain of the alkyl group, but often in the 2, 3 or 4 position of the linear chain, and in some instances predominantly in the 2 position. Another overbased material is an overbased phenate detergent. The phenols useful in making phenate detergents can be represented by (R15)a—Ar—(OH)b, wherein R15is an aliphatic hydrocarbyl group of 4 to 400, or 6 to 80, or 6 to 30, or 8 to 25, or 8 to 15 carbon atoms; Ar is an aromatic group such as benzene, toluene or naphthalene; a and b are each at least one, the sum of a and b being up to the number of displaceable hydrogens on the aromatic nucleus of Ar, such as 1 to 4 or 1 to 2. There is typically an average of at least 8 aliphatic carbon atoms provided by the R15groups for each phenol compound. Phenate detergents are also sometimes provided as sulfur-bridged species. In one aspect, the overbased material is an overbased saligenin detergent. Overbased saligenin detergents are commonly overbased magnesium salts which are based on saligenin derivatives. A general example of such a saligenin derivative can be represented by formula (III): wherein Z is —CHO or —CH2OH, Y is —CH2— or —CH2OCH2—, and the —CHO groups typically comprise at least 10 mole percent of the Z and Y groups; M is hydrogen, ammonium, or a valence of a metal ion (that is, if M is multivalent, one of the valences is satisfied by the illustrated structure and other valences are satisfied by other species such as anions or by another instance of the same structure), R17is a hydrocarbyl group of 1 to 60 carbon atoms, m is 0 to typically 10, and each p is independently 0, 1, 2, or 3, provided that at least one aromatic ring contains an R17substituent and that the total number of carbon atoms in all R17groups is at least 7. When m is 1 or greater, one of the Z groups can be hydrogen. In one aspect, M is a valence of a Mg ion or a mixture of Mg and hydrogen. Saligenin detergents are disclosed in greater detail in U.S. Pat. No. 6,310,009, with special reference to their methods of synthesis (column 8 and Example 1) and preferred amounts of the various species of Z and Y (column 6). Salixarate detergents are overbased materials that can be represented by a compound comprising at least one unit represented by formula (IV) or formula (V): wherein each end of the compound represented by formula (IV) and formula (V) has a terminal group represented by formula (VI) and formula (VII): wherein such groups being linked by divalent bridging groups A, which may be the same or different. In formulae (IV) to (VII) R20is hydrogen, a hydrocarbyl group, or a valence of a metal ion or an ammonium ion; R25is hydroxyl or a hydrocarbyl group, and j is 0, 1, or 2; R23is hydrogen, a hydrocarbyl group, or a hetero-substituted hydrocarbyl group; either R21is hydroxyl and R22and R24are independently either hydrogen, a hydrocarbyl group, or hetero-substituted hydrocarbyl group, or else R22and R24are both hydroxyl and R21is hydrogen, a hydrocarbyl group, or a hetero-substituted hydrocarbyl group; provided that at least one of R21, R22, R23and R24is hydrocarbyl containing at least 8 carbon atoms; and wherein the molecules on average contain at least one of unit (IV) or (VI) and at least one of unit (V) or (VII) and the ratio of the total number of units (IV) and (VI) to the total number of units of (V) and (VII) in the composition is 0.1:1 to 2:1. The divalent bridging group “A”, which may be the same or different in each occurrence, includes —CH2— and —CH2OCH2—, either of which may be derived from formaldehyde or a formaldehyde equivalent (e.g., paraform, formalin). Salixarate derivatives and methods of their preparation are described in greater detail in U.S. Pat. No. 6,200,936 and PCT Publication WO 01/56968. It is believed that the salixarate derivatives have a predominantly linear, rather than macrocyclic, structure, although both structures are intended to be encompassed by the term “salixarate”. Glyoxylate detergents are similar overbased materials which are based on an anionic group which, in one aspect, can have a structure represented by the formula (VIII): wherein R30is independently an alkyl group containing at least 4 or 8 carbon atoms, provided that the total number of carbon atoms in all R30substitutents is at least 12 or 16 or 24. Alternatively, each R30substituent can be an olefin polymer substituent. The acidic material upon from which the overbased glyoxylate detergent is prepared may be a condensation product of a hydroxyaromatic material such as a hydrocarbyl-substituted phenol with a carboxylic reactant such as glyoxylic acid or another omega-oxoalkanoic acid. Overbased glyoxylic detergents and their methods of preparation are disclosed in greater detail in U.S. Pat. No. 6,310,011 and references cited therein. The overbased detergent can also be an overbased salicylate, e.g., an alkali metal or alkaline earth metal or ammonium salt of a substituted salicylic acid. The salicylic acids may be hydrocarbyl-substituted wherein each substituent contains an average of at least 8 carbon atoms per substituent and 1 to 3 substituents per molecule. The substituents can be polyalkene substituents. In one aspect, the hydrocarbyl substituent group contains 7 to 300 carbon atoms and can be an alkyl group having a molecular weight of 150 to 2000. Overbased salicylate detergents and their methods of preparation are disclosed in U.S. Pat. Nos. 4,719,023 and 3,372,116. Other overbased detergents can include overbased detergents having a Mannich base structure, as disclosed in U.S. Pat. No. 6,569,818. In certain aspects, the hydrocarbyl substituents on hydroxy-substituted aromatic rings in the above detergents (e.g., phenate, saligenin, salixarate, glyoxylate, or salicylate) are free of or substantially free of C12aliphatic hydrocarbyl groups (e.g., less than 1%, 0.1%, or 0.01% by weight of the substituents are C12aliphatic hydrocarbyl groups). In some aspects, such hydrocarbyl substituents contain at least 14 or at least 18 carbon atoms. The amount of the overbased detergent, in the formulations of the present technology, is typically at least 0.6 weight percent on an oil-free basis, or 0.7 to 5 weight percent, or 1 to 3 weight percent. Either a single detergent or multiple detergents can be present. The amount of overbased detergent can also be represented by the amount of metal, specifically alkaline earth metal, delivered to the lubricating composition by the detergent. In one aspect, the overbased detergent is present in an amount to deliver 500 ppm to 3000 ppm, or 800 to 2400 ppm by weight alkaline earth metal to the composition, or combinations of alkaline earth metals. The overbased detergent may be present in an amount to deliver 1000 ppm to 2500 ppm calcium to the composition, or in an amount to deliver 400 ppm to 2500 ppm magnesium to the composition, or combinations thereof. In one embodiment, the lubricating composition comprises at least 400 ppm magnesium and no more than 1500 ppm calcium from overbased detergents. The amount of overbased detergent can also be represented by the amount of sulfated ash delivered to the lubricating composition by the detergent. In one aspect, the one or more overbased detergents are present in an amount to deliver 0.05 weight percent to 1.2 weight percent, or 0.25 to 0.85 weight percent, or 0.15 to 0.5 weight percent sulfated ash to the lubricating composition. In one aspect, the overbased detergent is present in an amount to deliver less than 1 weight percent, or less than 0.75 weight percent, or less than 0.45 weight percent sulfated ash to the lubricant composition. Dispersants Dispersants are well-known in the field of lubricants and include primarily what is known as ashless dispersants and polymeric dispersants. Ashless dispersants are so-called because, as supplied, they do not contain metal and thus do not normally contribute to sulfated ash when added to a lubricant. However, they may, of course, interact with ambient metals once they are added to a lubricant which includes metal-containing species. Ashless dispersants are characterized by a polar group attached to a relatively high molecular weight hydrocarbon chain. Typical ashless dispersants include N-substituted long-chain alkenyl succinimides, having a variety of chemical structures including those conforming to formula (IX): wherein in one aspect, each R35is independently an alkyl group, and in another aspect, a polyisobutylene group with a molecular weight (Mn) of 500-5000 based on the polyisobutylene precursor, and R36are alkylene groups, commonly ethylene (C2H4) groups. Such molecules are commonly derived from reaction of an alkenyl acylating agent with a polyamine, and a wide variety of linkages between the two moieties is possible beside the simple imide structure shown above, including a variety of amides and quaternary ammonium salts. In the above structure, the amine portion is shown as an alkylene polyamine, although other aliphatic and aromatic mono- and polyamines may also be used. Also, a variety of modes of linkage of the R35groups onto the imide structure are possible, including various cyclic linkages. The ratio of the carbonyl groups of the acylating agent to the nitrogen atoms of the amine may be 1:0.5 to 1:3, and in other instances 1:1 to 1:2.75 or 1:1.5 to 1:2.5. Succinimide dispersants are more fully described in U.S. Pat. Nos. 4,234,435 and 3,172,892 and in EP 0355895. Another class of ashless dispersant is high molecular weight esters. These materials are similar to the above-described succinimides except that they may be prepared by reaction of a hydrocarbyl acylating agent and a polyhydric aliphatic alcohol such as glycerol, pentaerythritol, or sorbitol. Such materials are described in more detail in U.S. Pat. No. 3,381,022. Another class of ashless dispersant is Mannich bases. These are materials which are formed by the condensation of a higher molecular weight, alkyl substituted phenol, an alkylene polyamine, and an aldehyde such as formaldehyde. Such materials may have general structure (X): wherein R38is an alkylene group, e.g., an ethylene group (—CH2CH2—); and R39is a hydrocarbyl substituent having from about 40 to about 20,000 carbon atoms, or from about 80 to about 250 carbon atoms. In one aspect, R39is selected from polyisobutyl and polypropyl substitutents derived from the alkylation of the phenol moiety with polybutylenes or polypropylenes. The foregoing Mannich base dispersants described in more detail in U.S. Pat. No. 3,634,515. Other dispersants include polymeric dispersant additives, which are generally hydrocarbon-based polymers which contain polar functionality to impart dispersancy characteristics to the polymer. Dispersants can also be post-treated by reaction with any of a variety of agents. Among these are urea, thiourea, dimercaptothiadiazoles, carbon disulfide, aldehydes, ketones, carboxylic acids, hydrocarbon-substituted succinic anhydrides, nitriles, epoxides, boron compounds, and phosphorus compounds. References detailing such treatment are disclosed in U.S. Pat. No. 4,654,403. The amount of the dispersant in a fully formulated lubricant of the present technology may be at least 0.1% of the lubricant composition, or at least 0.3 wt. %, or 0.5 wt. %, or 1 wt. %, and in certain aspects, at most 9 wt. %, or 8 wt. %, or 6 wt. %, or 4 wt. %, or 3 wt. %, or 2 wt. %, based on the weight of the total composition. Viscosity Modifiers Another performance additive component that can be employed in the lubricant of the disclosed technology is a viscosity modifier. Viscosity modifiers (VM) and dispersant viscosity modifiers (DVM) are well known. Examples of VMs and DVMs may include polymethacrylates, polyacrylates, polyolefins, hydrogenated vinyl aromatic-diene copolymers (e.g., styrene-butadiene, styrene-isoprene), styrene-maleic ester copolymers, and similar polymeric substances including homopolymers, copolymers, and graft copolymers. The DVM may comprise a nitrogen-containing methacrylate polymer, for example, a nitrogen-containing methacrylate polymer derived from methyl methacrylate and dimethylaminopropyl amine. Examples of commercially available VMs, DVMs and their chemical types may include the following: polyisobutylenes (such as Indopol™ from BP Amoco or Parapol™ from ExxonMobil); olefin copolymers (such as Lubrizol™ 7060, 7065, and 7067 from Lubrizol and Lucant™ HC-2000L and HC-600 from Mitsui); hydrogenated styrene-diene copolymers (such as Shellvis™ 40 and 50, from Shell and LZ® 7308, and 7318 from Lubrizol); styrene/maleate copolymers, which are dispersant copolymers (such as LZ® 3702 and 3715 from Lubrizol); polymethacrylates, some of which have dispersant properties (such as those in the Viscoplex™ series from RohMax, the Hitec™ series of viscosity index improvers from Afton, and LZ® 7702, LZ® 7727, LZ® 7725 and LZ® 7720C from Lubrizol); olefin-graft-polymethacrylate polymers (such as Viscoplex™ 2-500 and 2-600 from RohMax); and hydrogenated polyisoprene star polymers (such as Shellvis™ 200 and 260, from Shell). Viscosity modifiers that may be used are described in U.S. Pat. Nos. 5,157,088, 5,256,752 and 5,395,539. The VMs and/or DVMs may be used in the functional fluid at a concentration of up to 20 wt. % by weight. Concentrations of 1 to 12 wt. %, or 3 to 10 wt. %, based on the weight of the total lubricant composition may be employed. Antioxidants Another performance additive component that can be employed in the lubricant of the disclosed technology is an antioxidant. Antioxidants encompass phenolic antioxidants, which may be hindered phenolic antioxidants, one or both ortho positions on a phenolic ring being occupied by bulky groups such as t-butyl. The para position may also be occupied by a hydrocarbyl group or a group bridging two aromatic rings. In certain aspects, the para position is occupied by an ester-containing group, such as, for example, an antioxidant of the formula (XI): wherein R40is a hydrocarbyl group such as an alkyl group containing, e.g., 1 to 18, or 2 to 12, or 2 to 8, or 2 to 6 carbon atoms; and t-alkyl can be a t-butyl moiety. Such antioxidants are described in greater detail in U.S. Pat. No. 6,559,105. Antioxidants also include aromatic amines. In one aspect, an aromatic amine antioxidant can comprise an alkylated diphenylamine such as nonylated diphenylamine or a mixture of a di-nonylated and a mono-nonylated diphenylamine, or an alkylated phenylnaphthylamine, or mixtures thereof. Antioxidants also include sulfurized olefins such as mono- or disulfides or mixtures thereof. These materials generally have sulfide linkages of 1 to 10 sulfur atoms, e.g., 1 to 4, or 1 or 2. Materials which can be sulfurized to form the sulfurized organic compositions of the present technology include oils, fatty acids and esters, olefins and polyolefins made thereof, terpenes, or Diels-Alder adducts. Details of methods of preparing some such sulfurized materials can be found in U.S. Pat. Nos. 3,471,404 and 4,191,659. Molybdenum compounds can also serve as antioxidants, and these materials can also serve in various other functions, such as antiwear agents or friction modifiers. U.S. Pat. No. 4,285,822 discloses lubricating oil compositions containing a molybdenum- and sulfur-containing composition prepared by combining a polar solvent, an acidic molybdenum compound and an oil-soluble basic nitrogen compound to form a molybdenum-containing complex and contacting the complex with carbon disulfide to form the molybdenum- and sulfur-containing composition. Other materials that may serve as antioxidants include titanium compounds. U.S. Pat. No. 7,727,943 discloses a variety of titanium compounds, including titanium alkoxides and titanated dispersants, which materials may also impart improvements in deposit control and filterability. Other titanium compounds include titanium carboxylates such as neodecanoate. Typical amounts of antioxidants will, of course, depend on the specific antioxidant and its individual effectiveness, but illustrative total amounts can range from about 0.01 to about 5 wt. %, or from about 0.15 to about 4.5 wt. %, or from about 0.2 to about 4 wt. %, based on the weight of the total composition. Anti-Wear Agents The lubricant compositions of the disclosed technology can also contain anti-wear agent. In one aspect the anti-wear agent is a metal salt of a phosphorus acid of the formula (XII): [(R43O)(R44O)P(═S)(—S)]n-M (XII) wherein R43and R44are, independently, hydrocarbyl groups containing 3 to 30 carbon atoms, and can be obtained by heating phosphorus pentasulfide (P2S5) and an alcohol or phenol to form an O,O-dihydrocarbyl phosphorodithioic acid. The alcohol which reacts to provide the R43and R44groups may be a mixture of alcohols, for instance, a mixture of isopropanol and 4-methyl-2-pentanol, and in some aspects, a mixture of a secondary alcohol and a primary alcohol, such as isopropanol and 2-ethylhexanol. The resulting acid may be reacted with a basic metal compound to form the salt. The metal M, having a valence n, generally is aluminum, lead, tin, manganese, cobalt, nickel, zinc, or copper, and in many cases, zinc, to form zinc dialkyldithiophosphates (ZDP). Such materials are well-known and readily available to those skilled in the art of lubricant formulation. Suitable variations to provide good phosphorus retention in an engine are disclosed, for instance, in U.S. Pat. No. 7,772,171. Examples of materials that may serve as anti-wear agents include phosphorus-containing antiwear/extreme pressure agents such as metal thiophosphates as described above, phosphoric acid esters and salts thereof, phosphorus-containing carboxylic acids, esters, ethers, and amides; and phosphites. In certain aspects, a phosphorus antiwear agent may be present in an amount to deliver from about 0.01 to about 0.2, or from about 0.015 to about 0.15, or from about 0.02 to about 0.1, or from about 0.025 to about 0.08 percent phosphorus. Often the antiwear agent is a zinc dialkyldithiophosphate (ZDP). For a typical ZDP, which may contain 11 percent P (calculated on an oil free basis), suitable amounts may include from about 0.09 to about 0.82 percent. Non-phosphorus-containing anti-wear agents include borate esters (including borated epoxides), dithiocarbamate compounds, molybdenum-containing compounds, and sulfurized olefins. Other materials that may be used as anti-wear agents include tartrate esters, tartramides, and tartrimides. Examples include oleyl tartrimide (the imide formed from oleylamine and tartaric acid) and oleyl diesters (from, e.g., mixed C12-C16alcohols). Other related materials that may be useful include esters, amides, and imides of other hydroxy-carboxylic acids in general, including hydroxy-polycarboxylic acids, for instance, acids such as tartaric acid, citric acid, lactic acid, glycolic acid, hydroxy-propionic acid, hydroxyglutaric acid, and mixtures thereof. These materials may also impart additional functionality to a lubricant beyond antiwear performance. These materials are described in greater detail in U.S. Pat. No. 7,651,987 and PCT Publication WO WO2010/077630. Such derivatives of (or compounds derived from) a hydroxy-carboxylic acid, if present, may typically be present in the lubricating composition in an amount of from about 0.1 weight % to about 5 wt. %, or from about 0.2 to about 3 wt. %, based on the weight of the total composition. The amount of each chemical component described herein is presented exclusive of any solvent or diluent oil, which may be customarily present in the commercial material, that is, on an active chemical basis, unless otherwise indicated. However, unless otherwise indicated, each chemical or composition referred to herein should be interpreted as being a commercial grade material which may contain the isomers, by-products, derivatives, and other such materials which are normally understood to be present in the commercial grade product. These additional performance additives may be present in the overall lubricant composition from about 0 or about 0.1 to about 30 wt. %, or from about 1 to about 20 wt. %, or from about 3 to about 20 wt. %, or from about 5 to about 18 wt. %, or from about 8 to about 15 wt. %, or from about 10 to about 12 wt. %, based on the weight of the total composition. The oil of lubricating viscosity will in some aspects make up the balance of the composition, and/or may be present from about 66 to about 99.9 wt. %, or about 99.8 wt. %, or from about 78 to about 98.9 wt. %, or from about 78.5 to about 94.5 wt. %, or from about 78.9 to about 89.1 wt. %, or from about 83.9 to about 89.1 wt. %, or about 85 wt. %, based on the weight of the total composition. In different aspects, the lubricating composition can have a composition as described in the following table. Aspects (wt %)AdditiveABCCorrosion Additive0.01 to0.2 to0.35 to421.5Antiwear Agents0.15 to0.2 to0.5 to642Ashless Antioxidants1.2 to1.2 to2 to755Metal Detergents0.2 to0.2 to0.5 to842Polyolefin Dispersants0.8 to1 to1.5 to864Viscosity Modifier0 or0.5 to0.8 to0.1 to42.54.5Dispersant Viscosity0 or0 or0.5 toModifier0.1 to0.1 to1.64.52.5Friction Modifier0 or0.05 to0.1 to0.05 to324Any Other Performance0 or0 or0 orAdditive0.05 to0.05 to0.05 to1086Oil of Lubricating ViscosityBalance to 100% The lubricating composition of the disclosed technology may be utilized in an internal combustion engine. The engine components may have a surface of steel or aluminum (typically a surface of steel) and may also be coated for example with a diamond-like carbon (DLC) coating. An aluminum surface may be comprised of an aluminum alloy that may be a eutectic or hyper-eutectic aluminum alloy (such as those derived from aluminum silicates, aluminum oxides, or other ceramic materials). The aluminum surface may be present on a cylinder bore, cylinder block, or piston ring having an aluminum alloy, or aluminum composite. The internal combustion engine may be fitted with an emission control system or a turbocharger. Examples of the emission control system include diesel particulate filters (DPF), or systems employing selective catalytic reduction (SCR). The internal combustion engine may or may not have an Exhaust Gas Recirculation system. In one aspect, the internal combustion engine may be a diesel fueled engine (typically a heavy-duty diesel engine), a gasoline fueled engine, a natural gas fueled engine or a mixed gasoline/alcohol fueled engine. The engine may be a spark ignited engine and or a compression ignited engine. The internal combustion engine may be a 2-stroke or 4-stroke engine. Suitable internal combustion engines include marine diesel engines, aviation piston engines, low-load diesel engines, and gasoline fueled automobile and truck engines. The internal combustion engine described herein is distinct from a gas turbine. In an internal combustion engine, individual combustion events translate from a linear reciprocating force into a rotational torque through the rod and crankshaft. In contrast, in a gas turbine (which may also be referred to as a jet engine) a continuous combustion process generates a rotational torque continuously without translation and can also develop thrust at the exhaust outlet. These differences in operation conditions of a gas turbine and internal combustion engine result in different operating environments and stresses. The lubricant composition for an internal combustion engine may be suitable for any engine lubricant irrespective of the sulfur, phosphorus or sulfated ash (ASTM D-874) content. In one aspect, the sulfur content of the engine oil of lubricating viscosity can be 1 wt. % or less, or 0.8 wt. % or less, or 0.5 wt. % or less, or 0.3 wt. % or less, based on the total weight of the engine oil composition. In one aspect, the sulfur content can be in the range of 0.001 wt. % to 0.5 wt. %, or 0.01 wt. % to 0.3 wt. %, based on the total weight of the engine oil composition. In one aspect, the phosphorus content is 0 wt. %, or 0.2 wt. % or less, or 0.12 wt. % or less, or 0.1 wt. % or less, or 0.085 wt. % or less, or 0.08 wt. % or less, or 0.06 wt. % or less, 0.055 wt. % or less, or 0.05 wt. % or less, based on the total weight of the engine oil composition. In one aspect, the phosphorus content is 0 ppm, or can range from 100 ppm to 1000 ppm, or 200 ppm to 600 ppm, based on the total weight of the engine oil composition. In one aspect, the total sulfated ash content can be 2 wt. % or less, or 1.5 wt. % or less, or 1.1 wt. % or less, or 1 wt. % or less, or 0.8 wt. % or less, or 0.5 wt. % or less, or 0.4 wt. % or less, based on the total weight of the engine oil composition. In one aspect, the sulfated ash content may be 0.05 to 0.9 wt. %, or 0.1 wt. % to 0.2 wt. % or up to 0.45 wt. %, based on the total weight of the engine oil composition. In one aspect, the lubricating composition is characterized as having at least one of (i) a sulfur content of about 0.5 wt. % or less, or 0.4 wt. % or less, (ii) a phosphorus content of about 0.1 wt. % or less, and (iii) a sulfated ash content of about 1.5 wt. % or less, or combinations thereof. In one aspect, the lubricating composition comprises less than about 1.5 wt. % unreacted polyisobutene, or less than about 1.25 wt. %, or less than about 1.0 wt. %. In some aspects, the lubricant composition is an engine oil composition for a turbocharged direct injection (TDI) engine. The disclosed technology also provides a method of reducing deposits and mitigating seals degradation in an internal combustion engine comprising:(1) supplying to the engine a lubricant composition comprising:a) an oil of lubricating viscosity; andb) a basic ashless additive selected form a N-aralkyl α-carbonyl functional amine of the formula: wherein R, R1, R2, R3, R4, R5, and A are as previously defined; and(2) operating the engine. In some aspects, the engine is a turbocharged direct injection (TDI) engine. The following examples provide illustrations of the disclosed technology. Unless otherwise specified the amounts of components set forth in the Examples below are given in weight percent based on the weight of the total composition. These examples are non-exhaustive and are not intended to limit the scope of the present technology. EXAMPLES Example A (Comparative) To a 2 L round bottom flask equipped with overhead stirrer, thermocouple, N2inlet and water-cooled condenser was added ethanol (1000 ml) followed by alpha-methylbenzylamine (131.3 g) and the mixture was stirred. To this was added ethyl acrylate (108.5 g) over a period of 4 hours. The reaction was allowed to exotherm. The reaction mixture was then stirred for an additional 3 hours before being left to stand overnight. The reaction mixture was then concentrated under reduced pressure to remove ethanol. The resulting liquid was then vacuum stripped at 110° C. at 0 to 20 mbar pressure. This yielded 226.6 g of ethyl 3-((1-phenylethyl)amino)propanoate. Example B (Comparative) To a 3 L jacketed vessel equipped with overhead stirrer, thermocouple, N2inlet and water-cooled condenser was added acetonitrile (1500 ml), alpha-methylbenzylamine (100 g), potassium carbonate (250.9 g), potassium iodide (24.7 g). To the stirred mixture was added ethyl chloroacetate (197.1 g) over a period of 1 hour and the reaction was stirred at room temperature for 2 hours. The reaction was heated to 50° C. and held for 5 hours, then heated to 70° C. and stirred for 7 hours. The reaction mixture was cooled to room temperature and filtered. The filtrates were concentrated under reduced pressure and filtered once more. This yielded 188 g of diethyl 2,2′-((1-phenylethyl)azanediyl)diacetate. Example C N-benzylglycine ethyl ester was purchased commercially from Sigma-Aldrich, Inc. Example D To a 3 L jacketed vessel equipped with overhead stirrer, thermocouple, N2inlet and water-cooled condenser was added ethanol (2000 ml), alpha-methylbenzylamine (250 g), and sodium carbonate (240.5 g) and the mixture was stirred. Ethyl chloroacetate (252.8 g) was added dropwise to the reaction mixture over a period of 30 minutes. The stirred reaction mixture was heated to 65° C. and held at this temperature for approximately 18 hours. The reaction mixture was cooled to room temperature and filtered, the precipitate was washed with cold ethanol. The filtrates were concentrated under reduced pressure and the resulting slurry was taken up in hexane (500 ml) and filtered. The filtrate was again concentrated under reduced pressure. The material was finally stripped at 90° C. (80 mbar) for 1 hour. This yielded 316.1 g of the product, ethyl (1-phenylethyl)glycinate. Example E To a 3 L jacketed vessel equipped with overhead stirrer, thermocouple, N2inlet and water-cooled condenser was added ethanol (1200 ml), alpha-methylbenzylamine (196.6 g), and sodium carbonate (214.9 g) and the mixture was stirred. Ethyl 2-Bromopropionate (293.7 g) was added to the mixture and the stirred reaction mixture was heated to 65° C. and held at this temperature for 6 hours. The reaction mixture was cooled to room temperature and filtered, the precipitate was washed with cold ethanol. The filtrate was concentrated under reduced pressure and was then filtered a second time. The material (filtrate) was vacuum stripped at 100° C. (0-10 mbar). The residue was re-dissolved in dichloromethane (500 ml), washed with 1M NaOH (2×250 ml) and water (2×250 ml), the organics were dried with magnesium sulfate and the solvent removed under reduced pressure. This yielded ethyl (1-phenylethyl)alaninate, 267.6 g. Example F To a 2 L jacketed vessel equipped with overhead stirrer, thermocouple, N2inlet and water-cooled condenser was added ethanol (800 ml), benzylamine (103.4 g), and sodium carbonate (127.4 g) and the mixture was stirred. Ethyl 2-bromopropionate (174.7 g) was added to the mixture and the stirred reaction mixture was heated to 65° C. and held at this temperature for approximately 18 hours. The reaction mixture was cooled to room temperature and filtered, the precipitate was washed with cold ethanol. The filtrate was concentrated under reduced pressure and then filtered a second time. This yielded ethyl benzylalaninate, 157.2 g. Example G To a 2 L jacketed vessel equipped with overhead stirrer, thermocouple, N2inlet and water-cooled condenser was added ethanol (750 ml), cumylamine (94.6 g), and sodium carbonate (92.7 g) and the mixture was stirred. Ethyl Chloroacetate (85.7 g) was added to the reaction mixture and the stirred reaction mixture was heated to 65° C. and held at this temperature for a total of 26 hours. The reaction mixture was cooled to room temperature and 2-ethyl-1-hexanol was added (500 ml) and a Dean-Stark trap, the reaction mixture was then heated to 80° C. and for 2 hours, allowing the initial reaction solvent (ethanol) to distill into the Dean-Stark. A further 150 ml of 2-ethylhexanol was added and the reaction mixture was heated to 100° C. for 7 hours. The reaction mixture was cooled, filtered and the filtrates were concentrated under reduced pressure. This yielded Ethyl (2-phenylpropan-2-yl)glycinate, (46.9 g). Example H To a 3 L jacketed vessel equipped with overhead stirrer, thermocouple, N2inlet and water-cooled condenser was added acetonitrile (1200 ml), benzylamine (145.3 g), potassium carbonate (224.8 g), potassium iodide (22.5 g). To the stirred mixture was added ethyl alpha-bromoisobutyrate (264.4) and the reaction was stirred at room temperature for 30 minutes. The reaction was heated to 75° C. and held for approximately 84 hours. The reaction was cooled to room temperature and filtered. The filtrates were concentrated under reduced pressure and the concentrate was filtered again. Finally, the liquid was vacuum stripped at 110° C. at 0 to 20 mbar pressure. This yielded 230.2 g of ethyl 2-(benzylamino)-2-methylpropanoate. Lubricating Compositions and Test Data. A series of 15 W-40 engine lubricants in Group II base oils of lubricating viscosity were prepared containing the basic amine additives described above as well as conventional additives including polyisobutenyl succinimide dispersants, polymeric viscosity modifier, overbased detergents, antioxidants (combination of phenolic ester and diarylamine), zinc dialkyldithiophosphate (ZDDP), as well as other conventional performance additives as follows (Table 1). The calcium, magnesium, phosphorus, zinc and ash contents of each of the examples are also presented in the table in part to show that each example has a similar amount of these materials and so provide a proper comparison between the comparative and illustrative examples of the present technology. TABLE 1(Lubricating Compositions)EX1EX2EX3EX4EX5EX6EX7EX8Group II BaseBalance to 100%OilExample A0.37Example B0.53Example D0.37Example E0.4Example F0.37Example G0.4Example H0.4Boron-Free3.43.43.43.43.43.43.43.4PIBSuccinimide2Borated PIB1.01.01.01.01.01.01.01.0Succinimide3Overbased1.061.061.061.061.061.061.061.06CalciumSulfonate4Calcium0.250.250.250.250.250.250.250.25Sulfurized-Phenate5Calcium0.40.40.40.40.40.40.40.4SalixarateC3/C61.01.01.01.01.01.01.01.0SecondaryZDDPAshless2.552.552.552.552.552.552.552.55Antioxidant6Soot0.50.50.50.50.50.50.50.5Dispersant7Ethylene-0.480.480.480.480.480.480.480.48PropyleneCopolymerOther0.660.660.660.660.660.660.660.66Additives8TBN (D4739)6.57.36.47.47.57.57.37.5TBN (D2896)9.210.310.210.210.110.210.310.0Calcium (ppm)22302126214421262050205221052022Phosphorus11671086111611011074108010791024(ppm)Zinc (PPM)135712831279125212491257118312191All treat rates are oil free, unless otherwise indicated2Combination of conventional (chlorine process) and thermal ene polyisobutenyl succinimide dispersants, prepared with a mixture of aliphatic and aromatic polyamines3Boron-containing polyisobutenyl succinimide dispersant4Combination of overbased calcium alkylbenzene sulfonate detergents (TBN of 170 and 500 mg KOH/g)5Overbased calcium sulfur-coupled phenate detergent (TBN 400 mg KOH/g)6Combination of sulfurized olefins, alkylated diarylamine compounds and hindered phenol ester compounds7Ethylene-propylene copolymers functionalized with a mixture of aromatic amines and aromaticpolyamines8Other additives include pourpoint depressant, corrosion inhibitor, and anti-foam agent The engine lubricating compositions formulated in Table 1 are evaluated in bench and engine tests designed to assess the ability of the lubricant to prevent corrosion and mitigate seals degradation. The lubricating compositions are further tested to evaluate the ability to prevent or reduce deposit formation, provide cleanliness, improve oxidation stability and reduce or prevent acid-mediated wear or degradation of the lubricant. The lubricant samples are subjected to industry standard deposit and oxidation tests such as Komatsu Hot Tube (KHT), Pressure Differential Scanning calorimetry (PDSC) (e.g. L85-99), MHT TEOST (ASTM D7097), and TEOST 33C (ASTM D6335). The lubricant compositions are subjected to industry standard seals and corrosion bench tests. The lubricant samples were subjected to a 168 hour, 150° C. fluorocarbon seal compatibility test. Seal materials (“MB”—Mercedes Benz seals) DBL6674-FKM are evaluated before and after immersion in the lubricants under the stated conditions. The lubricants were also subjected to a corrosion test according to ASTM D6594. The compositions and results are summarized below in Table 2. TABLE 2(Corrosion and Seals Evaluation)EX1EX2EX3EX4EX5EX6EX7EX8Fluorelastomer SealsRupture elongation change (%)−42−64−56−52−49−55−57−51Tensile Strength change (%)−41−50−59−49−41−47−44−45High Temperature CorrosionCopper (ppm)4.95.43.82.32.72.71.63.9Lead (ppm)2.33.10.50.10.10.20.22.7Copper Rating11a21a21a21a21a21a21a21a21Copper Strip Corrosion Test (ASTM D130)2ASTM D130 Visual Rating: Class 1, Designation—Slight Tarnish, Description—Light Orange, Almost the Same as a Freshly Polished Strip (1a) The data indicates that the lubricant compositions containing the amine additive of the present technology provide TBN by both strong (D4739) and weak (D2896) titrants while maintaining strong corrosion resistance. Each of the documents referred to above is incorporated herein by reference, including any prior applications, whether or not specifically listed above, from which priority is claimed. The mention of any document is not an admission that such document qualifies as prior art or constitutes the general knowledge of the skilled person in any jurisdiction. Except in the Examples, or where otherwise explicitly indicated, all numerical quantities in this description specifying amounts of materials, reaction conditions, molecular weights, number of carbon atoms, and the like, are to be understood as modified by the word “about.” It is to be understood that the upper and lower amount, range, and ratio limits set forth herein may be independently combined. Similarly, the ranges and amounts for each element of the disclosed technology can be used together with ranges or amounts for any of the other elements. As used herein, the expression “consisting essentially of” permits the inclusion of substances that do not materially affect the basic and novel characteristics of the composition under consideration. | 61,129 |
11859149 | DETAILED DESCRIPTION OF THE INVENTION It will be understood that various components used, essential as well as optional and customary, may react under conditions of formulation, storage or use and that the invention also provides the product obtainable or obtained as a result of any such reaction. Further, it is understood that any upper and lower quantity, range and ratio limits set forth herein may be independently combined. Also, it will be understood that the preferred features of each aspect of the present invention are regarded as preferred features of every other aspect of the present invention. Accordingly, preferred and more preferred features of one aspect of the present invention may be independently combined with other preferred and/or more preferred features of the same aspect or different aspects of the present invention. The importance of nitrogen dioxide-initiated degradation in fresh lubricant at elevated temperature has recently been reported by the applicant in the Paper cited as Coultas, D. R. “The Role of NOx in Engine Lubricant Oxidation” SAE Technical Paper 2020-0101427, 2020. doi:10.4271/2020-01-1427. This paper notes in its introduction that “The principal mechanism by which NOx degrades the lubricant is through its involvement in free-radical nitro-oxidation reactions.” The equations which follow show that nitrogen dioxide initiates the process via abstraction of a proton from liquid hydrocarbon species, setting in motion a sequence of reactions involving other species and leading to chemical degradation of the hydrocarbonaceous liquid. Nitrogen dioxide also features prominently further down this degradation pathway, by reacting with RO. radicals to form hydrocarbonaceous nitrate esters of the formula RONO2. These accumulate in the lubricant, forming a reservoir of nitrate esters. At higher operating temperatures, these nitrate esters increasingly dissociate to release the captured RO radicals, leading to the characteristic nitrate ester “volcano curve” pictured in FIG. 14 of this Paper. This rapid dissociation of nitrate esters into free radicals accelerates the chemical breakdown of the hydrocarbonaceous species in the liquid. This plurality of reactions involving nitrogen dioxide, including both initial proton abstraction and the dissociation of subsequently formed nitrate esters, is herein referred to as “nitration” of the hydrocarbonaceous liquid. The initiation of this nitration reaction pathway through proton abstraction by nitrogen dioxide, and the formation and dissociation of a reservoir of nitrate esters in the further action of nitrogen dioxide, have been determined by the applicant to be a function of elevated bulk liquid temperature. The initiation of the nitration reaction sequence is underway at 60° C., and grows at higher temperatures of 80° C. and above. The formation of nitrate ester builds significantly in the range of 110 to 180° C., and from 130° C. the dissociation rate of nitrate esters increases. In the temperature range of 110 to 160° C., the production and dissociation of nitrate ester is most pronounced and leads to more chemical degradation of the hydrocarbonaceous liquid. The trend to higher bulk liquid (sump) temperatures in modern engine lubricants (to temperatures of 130° C. and higher) thus increases the practical consequences of nitrogen dioxide contamination, and renders the lubricants of these engines more susceptible to this form of degradation. Without being bound to a particular theory, the applicant believes from technical investigations that the ionic liquid deployed in this invention has a particularly advantageous affinity for nitrogen dioxide which leads to its deactivation when present as a contaminant in hydrocarbonaceous liquids. Consequently, the nitrogen dioxide is inhibited from reacting with hydrocarbonaceous liquid species and initiating degradation via proton abstraction to begin the nitration reaction pathway. The nitrogen dioxide is further inhibited from reacting to form the nitrate esters that produces the volcano curve at higher temperatures and its eruption of radicals that leads to further degradation. In particular, as detailed herein, the applicant has demonstrated the affinity of the ionic liquid deployed in this invention for nitrogen dioxide and shown it to be superior to other ionic liquids from the prior art. The applicant has also demonstrated the correspondingly improved ability of this invention to inhibit nitration of hydrocarbonaceous liquids under service conditions subject to elevated temperatures, and to inhibit the growth in bulk liquid acidity over time. The applicant has also found the ionic liquid of the present invention to be particularly advantageous for reducing friction and/or wear of mechanical systems that are lubricated by hydrocarbonaceous liquids, as evidenced by its ability to reduce the friction coefficient of the lubricant, or improve its resistance to mechanical wear on the contact surfaces of the hardware it services. This benefit of the ionic liquid can be deployed independently, but is more advantageously employed in combination with the ionic liquid's ability to deactivate nitrogen dioxide contamination and so inhibit the nitration and consequent chemical degradation of the hydrocarbonaceous liquid. The ionic liquid of the invention thus offers advantages over previous ionic liquid additives in providing control of nitration and consequent degradation in a hydrocarbonaceous liquid through the deactivation of nitrogen dioxide contamination, whilst also providing improved mechanical properties to the hydrocarbonaceous liquid in the form of reduced friction and/or wear on the contact surfaces of the hardware serviced by the liquid. This combination of features is believed to result from the selection of cation and anion comprising the ionic liquid of the present invention, which combine to provide an improved balance of properties to the ionic liquid. The related benefits in service conditions for the ionic liquid deployed in the present invention are demonstrated in the worked examples later in this specification. The Ionic Liquid of the First Aspect of the Invention An ionic liquid is conventionally understood as an ionic compound, composed of one or more cation-anion pairs, which exists in liquid physical form at industrially useful temperatures. The present invention provides a defined ionic liquid composed of: (i) one or more nitrogen-free organic cations each comprising a central atom or ring system bearing the cationic charge and multiple pendant hydrocarbyl substituents, and (ii) one or more halogen- and boron-free organic anions each comprising an aromatic ring bearing a carboxylate functional group and a further heteroatom-containing functional group, these functional groups being conjugated with the aromatic ring and this conjugated system bearing the anionic charge, and the aromatic ring additionally bearing one or more hydrocarbyl substituents. The one or more cations (i) carry the cationic (positive) charge and comprise multiple hydrocarbyl substituents providing organophilic character to the ionic liquid, enabling it to mix readily with hydrocarbonaceous bulk liquid. In this specification the term “hydrocarbyl substituents” refer to groups which contain hydrogen and carbon atoms and are each bonded to the remainder of the compound directly via a carbon atom. The group may contain one or more atoms other than carbon and hydrogen (i.e., heteroatoms) provided they do not affect the essentially hydrocarbyl nature of the group, namely oxygen, nitrogen and sulfur atoms; such groups include amino, alkoxyl, mercapto, alkylmercapto, nitro, nitroso, and sulfoxy. Preferably, however, the hydrocarbyl group consists essentially of, and more preferably consists of, hydrogen and carbon atoms unless specified otherwise. Preferably, the hydrocarbyl group is or comprises an aliphatic hydrocarbyl group. The term “hydrocarbyl” encompasses the term “alkyl” as conventionally used herein. Preferably, the term “alkyl” means a radical of carbon and hydrogen (such as a C1 to C30, such as a C4 to C20 group). Alkyl groups in a compound are typically bonded to the compound directly via a carbon atom. Unless otherwise specified, alkyl groups may be linear (i.e., unbranched) or branched, be cyclic, acyclic or part cyclic/acyclic. The alkyl group may comprise a linear or branched acyclic alkyl group. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, neo-pentyl, hexyl, heptyl, octyl, dimethyl hexyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl and triacontyl. Substituted alkyl groups are alkyl groups where a hydrogen or carbon has been replaced with a heteroatom (i.e., not H or C) or heteroatom-containing group. The term “substituted” generally means that a hydrogen has been replaced with a carbon or heteroatom-containing group. Each cation (i) of the ionic liquid is nitrogen-free. The ionic liquids of this composition have been found to be advantageous in the present invention. It is preferred that each cation (i) of the ionic liquid consists of a tetra-hydrocarbyl substituted central atom or ring system bearing the cationic charge. Most preferably, each cation (i) of the ionic liquid is a phosphorus-containing cation. In this embodiment, it is preferred that each cation (i) is an alkyl substituted phosphonium cation, ideally a tetra-alkyl substituted phosphonium cation. The alkyl groups suitable as substituents for such phosphonium cations include those straight- or branched-chain alkyl groups containing 1 to 28 carbon atoms, such as 4 to 28 carbon atoms, preferably 6 to 28 carbon atoms, more preferably 6 to 14 carbon atoms. Particularly suitable alkyl substituents for such phosphonium cations include hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl groups, and especially where n-alkyl groups. Preferably at least one of the alkyl substituents contains at least 10 carbon atoms and is selected from the above examples. Most preferably, each cation (i) is a trihexyltetradecyl phosphonium cation, i.e., a cation carrying three hexyl and one tetradecyl groups as substituents, these substituents preferably being linear alkyl groups. Such an anion is sometimes known in the industry by the shorthand term ‘P66614’ wherein the numbers relate the carbon numbers (6,6,6,14) of the three hexyl and one tetradecyl groups respectively. The one or more anions (ii) comprise an aromatic ring bearing at least two substituent functional groups containing heteroatoms, these functional groups being conjugated with the aromatic ring and at least one of them being a carboxylate group, this conjugated system bearing the anionic (negative) charge. In this specification, the term “conjugated” is used in its conventional chemical sense to mean these substituent functional groups are bonded directly to the aromatic ring, wherein one or more p orbitals of one or more atoms comprised within each of these functional groups link to the p orbitals of the adjacent aromatic ring to participate in the delocalised electron cloud of the aromatic ring. It is believed that anions of this configuration have a particular affinity for nitrogen dioxide, and are able to bind to it in such a way that its reactivity towards hydrocarbonaceous compounds is significantly reduced. The aromatic ring is composed of carbon and optionally one or more heteroatoms such as nitrogen or oxygen. However, it is preferred that each anion (ii) of the ionic liquid is nitrogen-free or sulfur-free or both. Such ionic liquids have been found to be more advantageous in the present invention, and cannot make a contribution to nitrogen and/or sulfur oxide(s) formation in environments where a proportion of the ionic liquid will be consumed by combustion, for example in engine lubricant environments. The aromatic ring of each anion (ii) of the ionic liquid bears a carboxylate group and a further heteroatom-containing functional group bonded directly to the aromatic ring, this system bearing the anionic charge. It is preferred that the heteroatom(s) in both these functional groups consist of oxygen atoms. These functional groups are more preferably positioned on adjacent ring carbon atoms in ‘ortho’ configuration to each other on the aromatic ring. In this respect, it is highly preferred that each anion (ii) is a disubstituted benzene ring bearing a carboxylate group and a second hetero-atom-containing functional group containing only oxygen as the heteroatom, these two groups preferably being positioned in ‘ortho’ configuration to each other on the aromatic ring. It is preferred that the second functional group is a hydroxyl group, giving rise to a hydroxybenzoate anion (ii). Most preferably the one or more anions (ii) of the ionic liquid are one or more salicylate anions, i.e., anions formed from the deprotonation of salicylic acid. The aromatic ring of each anion (ii) of the ionic liquid additionally bears one or more hydrocarbyl substituents. These substituents provide additional organophilic character to the ionic liquid, enabling it to mix more readily with hydrocarbonaceous bulk liquid. The hydrocarbyl substituent(s) of the anion are as previously defined. Preferably, these substituent(s) are alkyl substituents. Suitable alkyl groups include those straight- or branched-chain alkyl groups containing 6 or more carbon atoms, preferably 6 to 28 carbon atoms, more preferably 6 to 14 carbon atoms. Particularly suitable alkyl substituents include hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl and octadecyl groups, and especially where n-alkyl groups. The aromatic ring of anion (ii) may bear a single alkyl substituent or multiple alkyl substituents. The consequent ionic liquid may be composed of a mixture of anions (ii) differing in their number and/or position of alkyl substituents, which are preferably selected from the above-specified alkyl substituents. Preferably at least one of the alkyl substituents contains at least 10 carbon atoms and is selected from the above examples. More preferably, the aromatic ring of each anion (ii) of the ionic liquid bears one or more straight- or branched-chain alkyl substituents having more than 10 carbon atoms. In the anion, it is particularly preferred that one or more, and preferably all, anions (ii) are hydrocarbyl-substituted hydroxybenzoates of the structure: wherein R is a linear or branched hydrocarbyl group, and more preferably an alkyl group as defined above, including straight- or branched-chain alkyl groups. There may be more than one R group attached to the benzene ring. The carboxylate group and hydroxyl group are conjugated to the aromatic ring, and this system bears the negative (anionic) charge. The carboxylate group can be in the ortho, meta or para position with respect to the hydroxyl group; the ortho position is preferred. The R group can be in the ortho, meta or para position with respect to the hydroxyl group. In this embodiment of the anion, one or more (and preferably all) anions (ii) of the ionic liquid are most preferably one or more alkyl-substituted salicylate anions, wherein the alkyl substituent(s) of each anion are independently selected from alkyl groups containing from 12 to 24 carbon atoms; and more preferably from dodecyl, tetradecyl, hexadecyl and octadecyl groups. Such hydroxybenzoate and salicylate anions are typically prepared via the carboxylation, by the Kolbe-Schmitt process, of phenoxides, and in that case, will generally be obtained (normally in a diluent) in admixture with uncarboxylated phenol. In particular, ionic liquids are preferred in which each cation (i) is nitrogen-free and consists of a tetra-hydrocarbyl substituted central atom or ring system bearing the cationic charge, and each anion (ii) bears two substituent functional groups containing heteroatoms, being a carboxylate group and a further heteroatom-containing functional group as hereinbefore described. It is more preferred that the heteroatom(s) in both these functional groups consist of oxygen atoms. These functional groups are most preferably positioned on adjacent ring carbon atoms in ‘ortho’ configuration to each other on the aromatic ring. The preferred embodiments described hereinbefore for each such cation (i) and anion (ii) are particularly useful in combination. In all the preferred ionic liquids, and especially the ionic liquids of the three preceding paragraphs, each cation (i) is most preferably an alkyl substituted phosphonium cation, ideally a tetra-alkyl substituted phosphonium cation as hereinbefore described. The trihexyltetradecyl-phosphonium cation (P66614 cation) is most preferred. Each anion is most preferably an alkyl-substituted salicylate anion, wherein the alkyl substituent(s) of each anion are independently selected from alkyl groups containing from 12 to 24 carbon atoms; and more preferably from dodecyl, tetradecyl, hexadecyl and octadecyl groups. The ionic liquid of all aspects of the invention may be prepared by synthetic routes known in the art, chosen by the skilled person according to conventional synthesis criteria with regard to suitability for the desired cation-anion combination. Thus, the cation (i) can be formed from the cation—halide complex of the desire cation (ii), such as the preferred phosphonium cation, which is then subjected to anion exchange in a suitable solvent with the precursor of the desired anion. An anion exchange resin may be employed to promote the exchange. The solvent is then stripped and the ionic liquid recovered. Examples of synthetic methods for ionic liquids are provided in US-A-2008/0251759 and in the worked examples detailed later in this specification. In addition, the individual cations and anions or precursors thereto are available as items of chemical commerce. Without being bound to a particular theory, the applicant believes that the particular advantages of the ionic liquid of this invention in deactivating the degradative effects of nitrogen dioxide arises from the ionic liquid's composition and elucidated mechanism of action, with both anion and cation combining to play advantageous roles. Firstly, the functionalised aromatic anion (ii) in the ionic liquid ion-pair is particularly highly capable of interacting with nitrogen dioxide molecules, effectively removing them from reactive circulation within the hydrocarbonaceous liquid. Consequently, the initial deprotonation of hydrocarbonaceous components in the bulk liquid is inhibited, and the nitration reaction sequence and formation of nitrate esters is likewise inhibited, resulting in a slower degradation of the bulk liquid over time. Secondly, it is postulated that nitric acid formed in situ from the oxidation of some bound nitrogen dioxide is captured by the associated cation of the ionic liquid. This nitric acid loses its acidic proton to the negatively-charged anion—nitrogen dioxide complex, resulting in the formation of an ion-pair comprising the ionic liquid cation and nitrate anion, and a further complex between the protonated anion and remaining bound nitrogen dioxide. This sequence effectively also locks away the nitric acid from reactive circulation within the hydrocarbonaceous liquid. As a result, the build-up of acid over time in the hydrocarbonaceous liquid is also slower, and the ionic liquid helps to contain acid-mediated oxidation and acidic attack of the hydrocarbonaceous liquid and the underlying hardware. In this way, the cation and anion of the ionic liquid act in combination to inhibit the degradative consequences of nitrogen dioxide contamination of the hydrocarbonaceous liquid and prolong service life. The applicant further believes that the reduction in friction and/or wear attributable to the ionic liquid of the invention arises from its particular composition, which imparts advantageous protection to the contact surfaces of the mechanism concerned. The Additive Composition of the Second Aspect of the Invention The second aspect of the invention is an additive composition for a hydrocarbonaceous liquid, comprising the ionic liquid of the first aspect, a carrier liquid and, optionally, further additives. This additive composition is preferably in the form of a concentrate, allowing the ionic liquid to be added to the hydrocarbonaceous liquid without the need to introduce large quantities of excess carrier liquid. It is desirable to prepare one or more additive concentrates according to the second aspect, comprising the ionic liquid in a carrier liquid (being a diluent or solvent mutually compatible with both the ionic liquid and the hydrocarbonaceous liquid), to enable easier mixing or blending, whereby other additives can also be added simultaneously or sequentially to the concentrate (such concentrates sometimes being referred to as additive packages). Where an additive concentrate is used in the second aspect, it may contain from 5 to 25 mass %, preferably 5 to 22 mass %, typically 10 to 20 mass % of the ionic liquid, the remainder of the concentrate being solvent or diluent. The additive composition (and preferably the additive concentrate composition) may comprise further additives as a convenient way of incorporating multiple additives simultaneously into the hydrocarbonaceous liquid. Such further additives can have various properties and purposes depending on the needs of the service liquid in question. Where an additive concentrate of the second aspect is used, it may contain from 5 to 25 mass %, preferably 5 to 22 mass %, typically 10 to 20 mass % of the ionic liquid, the remainder of the concentrate being solvent or diluent. In particular, where the hydrocarbonaceous liquid is a lubricating oil or power transmission oil, particularly an engine lubricating oil, a variety of further additives may be incorporated to enhance other characteristics of the oil, which may comprise one or more phosphorus-containing compounds; dispersants; metal detergents; anti-wear agents; friction modifiers, viscosity modifiers; anti-oxidants; and other co-additives, provided they are different from essential ionic liquids hereinbefore described. These are discussed in more detail below. Suitable phosphorus-containing compounds include dihydrocarbyl dithiophosphate metal salts, which are frequently used as antiwear agents. The metal is preferably zinc, but may be an alkali or alkaline earth metal, or aluminum, lead, tin, molybdenum, manganese, nickel or copper. The zinc salts are most commonly used in lubricating oil in amounts of 0.1 to 10, preferably 0.2 to 2 mass %, based upon the total weight of the lubricating oil composition. They may be prepared in accordance with known techniques by first forming a dihydrocarbyl dithiophosphoric acid (DDPA), usually by reaction of one or more alcohol or a phenol with P2S5, and then neutralizing the formed DDPA with a zinc compound. For example, a dithiophosphoric acid may be made by reacting mixtures of primary and secondary alcohols. Alternatively, multiple dithiophosphoric acids can be prepared where the hydrocarbyl groups on one are entirely secondary in character and the hydrocarbyl groups on the others are entirely primary in character. To make the zinc salt, any basic or neutral zinc compound could be used but the oxides, hydroxides and carbonates are most generally employed. Commercial additives frequently contain an excess of zinc due to the use of an excess of the basic zinc compound in the neutralization reaction. The preferred zinc dihydrocarbyl dithiophosphates are oil-soluble salts of dihydrocarbyl dithiophosphoric acids and may be represented by the following formula: wherein R and R′ may be the same or different hydrocarbyl radicals containing from 1 to 18, preferably 2 to 12, carbon atoms and including radicals such as alkyl, alkenyl, aryl, arylalkyl, alkaryl and cycloaliphatic radicals. Particularly preferred as R and R′ groups in this context are alkyl groups of 2 to 8 carbon atoms. Thus, the radicals may, for example, be ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, amyl, n-hexyl, i-hexyl, n-octyl, decyl, dodecyl, octadecyl, 2-ethylhexyl, phenyl, butylphenyl, cyclohexyl, methylcyclopentyl, propenyl, butenyl. In order to obtain oil solubility, the total number of carbon atoms (i.e., R and R′) in the dithiophosphoric acid will generally be 5 or greater. The zinc dihydrocarbyl dithiophosphate (ZDDP) can therefore comprise zinc dialkyl dithiophosphates. Additive concentrates of the present invention for lubricants may have a phosphorus content of no greater than about 0.08 mass % (800 ppm). Preferably, in the practice of the present invention, ZDDP is used in an amount close or equal to the maximum amount allowed, preferably in an amount that provides a phosphorus content within 100 ppm of the maximum allowable amount of phosphorus. Thus, resulting lubricating oil compositions preferably contain ZDDP or other zinc-phosphorus compounds, in an amount introducing from 0.01 to 0.08 mass % of phosphorus, such as from 0.04 to 0.08 mass % of phosphorus, preferably, from 0.05 to 0.08 mass % of phosphorus, based on the total mass of the lubricating oil composition. A dispersant is an additive whose primary function is to hold oil-insoluble contaminations in suspension, thereby passivating them and reducing deposition on surfaces. For example, a dispersant maintains in suspension oil-insoluble substances that result from oxidation during use, thus preventing solids flocculation and precipitation or deposition on hardware parts. Dispersants in this invention are preferably “ashless”, being non-metallic organic materials that form substantially no ash on combustion, in contrast to metal-containing and hence ash-forming materials. They comprise a long hydrocarbon chain with a polar head, the polarity being derived from inclusion of preferably an oxygen, phosphorus or nitrogen atom. The hydrocarbon is an oleophilic group that confers oil-solubility, having, for example 40 to 500 carbon atoms, such as 60 to 250 carbon atoms. Thus, ashless dispersants may comprise an oil-soluble polymeric backbone. The hydrocarbon portion of the dispersant may have a number average molecular weight (Mn) of from 800 to 5,000 g/mol, such as from 900 to 3000 g/mol. A preferred class of olefin polymers is constituted by polybutenes, specifically polyisobutenes (PIB) or poly-n-butenes, such as may be prepared by polymerization of a C4 refinery stream. Dispersants include, for example, derivatives of long chain hydrocarbon-substituted carboxylic acids, examples being derivatives of high molecular weight hydrocarbyl-substituted succinic acid. Typically, a hydrocarbon polymeric material, such as polyisobutylene, is reacted with an acylating group (such as maleic acid or anhydride) to form a hydrocarbon-substituted succinic acid (succinate). A noteworthy group of dispersants is constituted by hydrocarbon-substituted succinimides, made, for example, by reacting the above acids (or derivatives) with a nitrogen-containing compound, advantageously a polyalkylene polyamine, such as a polyethylene polyamine. Particularly preferred are the reaction products of polyalkylene polyamines with alkenyl succinic anhydrides, such as described in U.S. Pat. Nos. 3,202,678; 3,154,560; 3,172,892; 3,024,195; 3,024,237, 3,219,666; and 3,216,936, that may be post-treated to improve their properties, such as borated (as described in U.S. Pat. Nos. 3,087,936 and 3,254,025), fluorinated or oxylated. For example, boration may be accomplished by treating an acyl nitrogen-containing dispersant with a boron compound selected from boron oxide, boron halides, boron acids and esters of boron acids. Preferably, the dispersant, if present, is a succinimide-dispersant derived from a polyisobutene of number average molecular weight in the range of 800 to 5000 g/mol, such as 1000 to 3000 g/mol, preferably 1500 to 2500 g/mol, and of moderate functionality. The succinimide is preferably derived from highly reactive polyisobutene. Another example of dispersant type that may be used is a linked aromatic compound such as described in EP-A-2 090 642. Combinations of borated and non-borated succinimide are useful herein. Combinations of one or more (such as two or more) higher Mn succinimides (Mn of 1500 g/mol or more, such as 2000 g/mol or more) and one or more (such as two or more) lower Mn (Mn less than 1500 g/mol, such as less than 1200 g/mol) succinimides are useful herein, where the combinations may optionally contain one, two, three or more borated succinimides. A detergent is an additive that reduces formation of deposits, for example high-temperature varnish and lacquer deposits; it normally has acid-neutralising properties and is capable of keeping finely divided solids in suspension. Most detergents are based on metal “soaps”, that is metal salts of acidic organic compounds. Detergents generally comprise a polar head with a long hydrophobic tail, the polar head comprising the metal salt of the acidic organic compound. The salts may contain a substantially stoichiometric amount of the metal when they are usually described as normal or neutral salts and would typically have a total base number or “TBN” at 100% active mass (as may be measured by ASTM D2896) of from 0 to 150 mg KOH/g, such as 10 to 80 mg KOH/g. Large amounts of a metal base can be included by reaction of an excess of a metal compound, such as an oxide or hydroxide, with an acidic gas such as carbon dioxide. The resulting overbased detergent comprises neutralised detergent as an outer layer of a metal base (e.g., carbonate) micelle. Such overbased detergents may have a total base number (TBN) at 100% active mass of more than 150 mg KOH/g, such as 200 mg KOH/g or greater, such as such as 250 mg KOH/g or greater and typically of from 200 to 800 mg KOH/g, 225 to 700 mg KOH/g, such as 250 to 650 mg KOH/g, or 300 to 600 mg KOH/g, such as 150 to 650 mg KOH/g, preferably from 200 to 500 or more. Suitably, detergents that may be used include oil-soluble neutral and overbased sulfonates, phenates, sulfurised phenates, thiophosphonates, salicylates and naphthenates and other oil-soluble carboxylates of a metal, particularly alkali metal or alkaline earth metals, e.g., Na, K, Li, Ca and Mg. The most commonly used metals are Ca and Mg, which may both be present in detergents used particularly in lubricating compositions, and mixtures of Ca and/or Mg with Na. Detergents may be used in various combinations. Preferably, the detergent additive(s) useful in the present invention comprises calcium and/or magnesium metal salts. The detergent may a calcium and or magnesium carboxylate (e.g., salicylates), sulfonate, or phenate detergent. More preferably, the detergents additives are selected from magnesium salicylate, calcium salicylate, magnesium sulfonate, calcium sulfonate, magnesium phenate, calcium phenate, and hybrid detergents comprising two, three, four or more of more of these detergents and/or combinations thereof. The magnesium detergent provides the lubricating composition thereof with from 200-4000 ppm of magnesium atoms, suitably from 200-2000 ppm, from 300 to 1500 or from 450-1200 ppm of magnesium atoms (ASTM D5185). Calcium detergent is typically present in amount sufficient to provide at least 500 ppm, preferably at least 750 more preferably at least 900 ppm atomic calcium to the lubricating oil composition (ASTM D5185). If present, any calcium detergent is suitably present in amount sufficient to provide no more than 4000 ppm, preferably no more than 3000, more preferably no more than 2000 ppm atomic calcium to the lubricating oil composition (ASTM D5185). If present, any calcium detergent is suitably present in amount sufficient to provide at from 500-4000 ppm, preferably from 750-3000 ppm more preferably from 900-2000 ppm atomic calcium to the lubricating oil composition (ASTM D5185). The detergent composition may comprise (or consist of) a combination of one or more magnesium sulfonate detergents and one or more calcium salicylate detergents. The combination of one or more magnesium sulfonate detergents and one or more calcium salicylate detergents provides the lubricating composition thereof with: 1) from 200-4000 ppm of magnesium atoms, suitably from 200-2000 ppm, from 300 to 1500 or from 450-1200 ppm of magnesium atoms (ASTM D5185), and 2) at least 500 ppm, preferably at least 750 more preferably at least 900 ppm of atomic calcium, such as from 500-4000 ppm, preferably from 750-3000 ppm, more preferably from 900-2000 ppm atomic calcium (ASTM D5185). Additional additives may be incorporated into the additive concentrates of the invention to enable particular performance requirements to be met. Examples of such additives which may be included in lubricating oil compositions of the present invention are friction modifiers, viscosity modifiers, metal rust inhibitors, viscosity index improvers, corrosion inhibitors, oxidation inhibitors, anti-foaming agents, anti-wear agents and pour point depressants. Friction modifiers (and, also in engine lubricants, fuel economy agents) that are compatible with the other ingredients of hydrocarbonaceous liquid may be included in the lubricating oil composition. Examples of such materials include glyceryl monoesters of higher fatty acids, for example, glyceryl mono-oleate; esters of long chain polycarboxylic acids with diols, for example, the butane diol ester of a dimerized unsaturated fatty acid; and alkoxylated alkyl-substituted mono-amines, diamines and alkyl ether amines, for example, ethoxylated tallow amine and ethoxylated tallow ether amine. Other known friction modifiers comprise oil-soluble organo-molybdenum compounds. Such organo-molybdenum friction modifiers also provide antioxidant and antiwear credits to a lubricating oil composition. Examples of such oil-soluble organo-molybdenum compounds include dithiocarbamates, dithiophosphates, dithiophosphinates, xanthates, thioxanthates, sulfides, and the like, and mixtures thereof. Particularly preferred are molybdenum dithiocarbamates, dialkyldithiophosphates, alkyl xanthates and alkylthioxanthates. Additionally, the molybdenum compound may be an acidic molybdenum compound. These compounds will react with a basic nitrogen compound as measured by ASTM test D-664 or D-2896 titration procedure and are typically hexavalent. Included are molybdic acid, ammonium molybdate, sodium molybdate, potassium molybdate, and other alkali metal molybdates and other molybdenum salts, e.g., hydrogen sodium molybdate, MoOCl4, MoO2Br2, Mo2O3Cl6, molybdenum trioxide or similar acidic molybdenum compounds. Among the molybdenum compounds useful in the compositions of this invention are organo-molybdenum compounds of the formulae: Mo(R″OCS2)4and Mo(R″SCS2)4 wherein R″ is an organo group selected from the group consisting of alkyl, aryl, aralkyl and alkoxyalkyl, generally of from 1 to 30 carbon atoms, and preferably 2 to 12 carbon atoms and most preferably alkyl of 2 to 12 carbon atoms. Especially preferred are the dialkyldithiocarbamates of molybdenum. Another group of organo-molybdenum compounds useful as further additives in this invention are trinuclear molybdenum compounds, especially those of the formula Mo3SkAnDz and mixtures thereof wherein the A are independently selected ligands having organo groups with a sufficient number of carbon atoms to render the compound soluble or dispersible in the oil, n is from 1 to 4, k varies from 4 to 7, D is selected from the group of neutral electron donating compounds such as water, amines, alcohols, phosphines, and ethers, and z ranges from 0 to 5 and includes non-stoichiometric values. At least 21 carbon atoms should be present among all the ligand organo groups, such as at least 25, at least 30, or at least 35, carbon atoms. Where the hydrocarbonaceous liquid is a lubricating oil, it preferably contains at least 10 ppm, at least 30 ppm, at least 40 ppm and more preferably at least 50 ppm molybdenum. Suitably, such lubricating oil compositions contain no more than 1000 ppm, no more than 750 ppm or no more than 500 ppm of molybdenum. Lubricating oil compositions useful in the present invention preferably contain from 10 to 1000, such as 30 to 750 or 40 to 500, ppm of molybdenum (measured as atoms of molybdenum). The viscosity index of the hydrocarbonaceous liquid, and especially lubricating oils, may be increased or improved by incorporating therein certain polymeric materials that function as viscosity modifiers (VM) or viscosity index improvers (VII). Generally, polymeric materials useful as viscosity modifiers are those having number average molecular weights (Mn) of from 5,000 to 250,000, preferably from 15,000 to 200,000, more preferably from 20,000 to 150,000. These viscosity modifiers can be grafted with grafting materials such as, for example, maleic anhydride, and the grafted material can be reacted with, for example, amines, amides, nitrogen-containing heterocyclic compounds or alcohol, to form multifunctional viscosity modifiers (dispersant-viscosity modifiers). Polymers prepared with diolefins will contain ethylenic unsaturation, and such polymers are preferably hydrogenated. When the polymer is hydrogenated, the hydrogenation may be accomplished using any of the techniques known in the prior art. For example, the hydrogenation may be accomplished such that both ethylenic and aromatic unsaturation is converted (saturated) using methods such as those taught, for example, in U.S. Pat. Nos. 3,113,986 and 3,700,633 or the hydrogenation may be accomplished selectively such that a significant portion of the ethylenic unsaturation is converted while little or no aromatic unsaturation is converted as taught, for example, in U.S. Pat. Nos. 3,634,595; 3,670,054; 3,700,633 and Re 27,145. Any of these methods can also be used to hydrogenate polymers containing only ethylenic unsaturation and which are free of aromatic unsaturation. Pour point depressants (PPDs) lower the lowest temperature at which the bulk liquid flows and may also be present, especially in lubricating oils. PPDs can be grafted with grafting materials such as, for example, maleic anhydride, and the grafted material can be reacted with, for example, amines, amides, nitrogen-containing heterocyclic compounds or alcohol, to form multifunctional additives. In the present invention it may be advantageous to include a co-additive which maintains the stability of the viscosity of the blend. Thus, although polar group-containing additives achieve a suitably low viscosity in the pre-blending stage, it has been observed that some compositions increase in viscosity when stored for prolonged periods. Additives which are effective in controlling this viscosity increase include the long chain hydrocarbons functionalized by reaction with mono- or dicarboxylic acids or anhydrides which are used in the preparation of the ashless dispersants as hereinbefore disclosed. The Hydrocarbonaceous Liquid Composition of the Third Aspect of the Invention In a third aspect, the invention provides a hydrocarbonaceous liquid comprising the ionic liquid of the first aspect, or additive concentrate of the second aspect, in an amount of up to 5.0% by weight of ionic liquid, per weight of hydrocarbonaceous liquid. The hydrocarbonaceous liquid deployed in this aspect of the invention is a liquid suitable for service at bulk liquid temperatures of between 60 and 180° C. and being free of aged components and nitrogen dioxide contamination prior to service. Such service liquids are used in a variety of applications, including industrial and automotive oils and power transmission fluids, such as engine lubricating oils. The hydrocarbonaceous liquid is preferably a lubricating oil for a mechanical device. More preferably, the hydrocarbonaceous liquid is a crankcase lubricating oil for an internal combustion engine, and is subjected in service to nitrogen dioxide contamination originating from exhaust gas, which gas becomes entrained in the lubricant via the effects of blow-by gas into the crankcase and direct contact on the engine cylinder walls. Most preferably, this crankcase lubricating oil is one periodically or continuously to bulk liquid temperatures in the crankcase of between 110 and 160° C. It is important to obtaining the benefits of the invention that, prior to service, the hydrocarbonaceous liquid be initially free of nitrogen dioxide contamination and also of the aged liquid components that arise during service from oxidative or other chemical breakdown, in order not to seed the liquid with significant quantities of reactive chemical species that can offer an alternative or complementary degradative pathway to nitrogen-dioxide initiated nitration. Thus, the hydrocarbonaceous liquid should be freshly prepared and not have been in prior service; and prior to being placed into the service environment should not be pre-mixed or diluted with a proportion of aged liquid that has been in prior use or exposed to nitrogen dioxide contamination. It is also important that the ionic liquid is added prior to service and the resulting onset of elevated temperatures and nitrogen dioxide contamination, to maximise its nitration-inhibiting effect and not allow nitrogen dioxide concentration in the bulk liquid to build unhindered. The hydrocarbonaceous liquid used as the bulk service liquid may be derived from petroleum or synthetic sources, or from the processing of renewable materials, such as biomaterials. Where the hydrocarbonaceous liquid is a petroleum oil, and especially a lubricating oil, such oils range in viscosity from light distillate mineral oils to heavy lubricating oils such as gasoline engine oils, mineral lubricating oils and heavy-duty diesel oils. Generally, the kinematic viscosity of the oil ranges from about 2 mm2/sec (centistokes) to about 40 mm2/sec, especially from about 3 mm2/sec to about 20 mm2/sec, most preferably from about 9 mm2/sec to about 17 mm2/sec, measured at 100° C. (ASTM D445-19a). Suitable oils, especially as lubricating oils, include natural oils such as animal oils and vegetable oils (e.g., castor oil, lard oil); liquid petroleum oils and hydrorefined, solvent-treated or acid-treated mineral oils of the paraffinic, naphthenic and mixed paraffinic-naphthenic types. Oils of lubricating viscosity derived from coal or shale also serve as useful bulk oils. Synthetic oils, and especially synthetic lubricating oils, include hydrocarbon oils and halo-substituted hydrocarbon oils retaining hydrocarbonaceous character, such as polymerized and copolymerized olefins (e.g., ethylene-propylene copolymers, polybutylene homo- and copolymers, polypropylene homo and copolymers, propylene-isobutylene copolymers, chlorinated polybutylenes, poly(1-hexenes), poly(1-octenes), poly-n-decenes (such as decene homopolymers or copolymers of decene and one or more of C8 to C20 alkenes, other than decene, such as octene, nonene, undecene, dodecene, tetradecene and the like); alkylbenzenes (e.g., dodecylbenzenes, tetradecylbenzenes, dinonylbenzenes, di(2-ethylhexyl)benzenes); polyphenyls (e.g., biphenyls, terphenyls, alkylated polyphenols); and alkylated diphenyl ethers and alkylated diphenyl sulfides and derivative, analogs and homologs thereof. Also useful are synthetic oils derived from a gas to liquid process from Fischer-Tropsch synthesized hydrocarbons, which are commonly referred to as gas to liquid, or “GTL” base oils. Esters are useful as synthetic oils having hydrocarbonaceous character, and include those made from C5 to C12 monocarboxylic acids and polyols and polyol esters such as neopentyl glycol, trimethylolpropane, pentaerythritol, dipentaerythritol and tripentaerythritol. Where the hydrocarbonaceous liquid is a lubricating oil, it may comprise a Group I, Group II, Group III, Group IV or Group V base stock or blend of the aforementioned base stocks. Preferably, the lubricating oil is a Group II, Group III, Group IV or Group V base stock, or a mixture thereof, such as a mixture of a Group I base stock and one or more a Group II, Group III, Group IV or Group V base stock. Definitions for these base stocks and base oils are found in the American Petroleum Institute (API) publication Engine Oil Licensing and Certification System, (“ELOCS”) Industry Services Department, Fourteenth Edition, December 1996, Addendum 1, December 1998. The base stock, or base stock blend preferably has a saturate content of at least 65%, more preferably at least 75%, such as at least 85%. Preferably, the base stock or base stock blend is a Group III or higher base stock or mixture thereof, or a mixture of a Group II base stock and a Group III or higher base stock or mixture thereof. Most preferably, the base stock, or base stock blend, has a saturate content of greater than 90%. Preferably, the oil or oil blend will have a sulfur content of less than 1 mass %, preferably less than 0.6 mass %, most preferably less than 0.4 mass %, such as less than 0.3 mass % (as determined as indicated in API EOLCS). Group III base stock has been found to provide a wear credit relative to Group I base stock and therefore, in one preferred embodiment, at least 30 mass %, preferably at least 50 mass %, more preferably at least 80 mass % of the lubricating oil is Group III base stock. Preferably the volatility of the lubricating oil or oil blend, as measured by the Noack test (ASTM D5800), is less than or equal to 30 mass %, such as less than about 25 mass %, preferably less than or equal to 20 mass %, more preferably less than or equal to 15 mass %, most preferably less than or equal 13 mass %. Preferably, the viscosity index (VI) of the oil or oil blend is at least 85, preferably at least 100, most preferably from about 105 to 140 (ASTM D 2270). In this aspect of the invention, the ionic liquid can be added to the hydrocarbonaceous liquid by physical mixing or blending techniques known in the art. It may be desirable, although not essential, to prepare one or more additive concentrates according to the second aspect, comprising the ionic liquid in a carrier liquid (being a diluent or solvent mutually compatible with both the ionic liquid and the hydrocarbonaceous liquid), to enable easier mixing or blending, whereby other additives can also be added simultaneously to the concentrate, and hence to the oil, to form the lubricating oil composition (such concentrates sometimes being referred to as additive packages). The ionic liquid may be added to an additive concentrate prior to the concentrate being combined with a hydrocarbonaceous liquid or may be added to a combination of additive concentrate and hydrocarbonaceous liquid. The ionic liquid may be added to an additive package prior to the package being combined with a hydrocarbonaceous liquid or may be added to a combination of additive package and hydrocarbonaceous liquid. Where an additive concentrate of the second aspect is used, it may contain from 5 to 25 mass %, preferably 5 to 22 mass %, typically 10 to 20 mass %, based upon the weight of the concentrate of the ionic liquid, the remainder of the concentrate being solvent or diluent. Where an additive package is used, it may contain from 5 to 25 mass %, preferably 5 to 22 mass %, typically 10 to 20 mass %, based upon the weight of the concentrate of the ionic liquid, the remainder of the package being other additives (such as dispersant, detergent, etc.), solvent or diluent. When hydrocarbonaceous liquids contain one or more of the above-mentioned further additives in addition to the ionic liquid, each further additive is typically blended into the bulk liquid in an amount that enables the additive to provide its desired function. Representative effective amounts of such further additives, when used in hydrocarbonaceous liquids which are crankcase lubricants, are listed below. All the values listed (with the exception of detergent values since the detergents are used in the form of colloidal dispersants in an oil) are stated as mass percent active ingredient (A.I.). These amounts of further additives are used in combination with the amount of ionic liquid hereinbefore described. MASS %MASS %ADDITIVE(Broad)(Preferred)Dispersant0.1-201-8Metal Detergents0.1-150.2-9Corrosion Inhibitor0-50-1.5Metal dihydrocarbyl dithiophosphate0.1-60.1-4Antioxidant0-50.01-2.5Pour Point Depressant0.01-50.01-1.5Antifoaming Agent0-50.001-0.15Friction Modifier0-50-1.5Viscosity Modifier0.01-100.25-3Ionic Liquid0.1 to 5.00.1 to 3Hydrocarbonaceous liquid (basestock)BalanceBalance The Use of the Fourth Aspect of the Invention In a fourth aspect, the invention provides the use of the ionic liquid of the first aspect, or of the additive concentrate of the second aspect, as an additive for a hydrocarbonaceous liquid to chemically deactivate nitrogen dioxide entrained within the hydrocarbonaceous liquid. In the use of the fourth aspect, the ionic liquid consequently inhibits the formation of hydrocarbonaceous nitrate esters and prolongs the service life of the hydrocarbonaceous liquid. In this use, the effectiveness of the ionic liquid in inhibiting the nitration reactions initiated by the nitrogen dioxide on hydrocarbonaceous compounds at elevated temperatures leads to the slower onset of degradation in the bulk liquid by this chemical pathway, prolonging its service life. The ionic liquid firstly acts through inhibiting the proton abstraction by nitrogen dioxide which initiates nitration of the bulk liquid, slowing the initial formation of free radicals which feeds other chemical reactions further along the pathway and delaying the onset of significant degradation. The ionic liquid further acts later in the pathway by inhibiting the formation of hydrocarbonaceous nitrate esters from the reaction of nitrogen dioxide with subsequent RO radicals, resulting in a smaller accumulation of these reactive compounds within the bulk liquid. As a result, the bulk liquid is exposed to lower concentrations of released RO radicals at elevated temperatures, especially those service temperatures rising (continuously or periodically) above 110° C., where the rate of dissociation of these nitrate esters greatly increases and results in escalating, more severe degradation of the bulk liquid. The amount of ionic liquid effective to inhibit nitration in the use can be arrived at by routine testing under conditions reproducing or simulating nitrogen dioxide contamination at the elevated service temperatures experienced in the system in question. In a preferred aspect of the use, the chemical degradation inhibited by the ionic liquid is that resulting from the decomposition of hydrocarbonaceous nitrate esters formed in service by the nitration of the hydrocarbonaceous liquid by nitrogen dioxide at bulk liquid temperatures of between 60 and 180° C., wherein the ionic liquid is used to inhibit the formation of hydrocarbonaceous nitrate esters in that service. In this way, the accumulation of a reservoir of reactive hydrocarbonaceous nitrate esters at elevated service temperatures is directly inhibited, and degradation is better limited. In a more preferred aspect of the use, the chemical degradation inhibited by the ionic liquid is that resulting from the decomposition of the hydrocarbonaceous nitrate esters due to the hydrocarbonaceous liquid being periodically or continuously subjected in service to bulk liquid temperatures of between 110 and 160° C., wherein the ionic liquid is used to inhibit the formation of hydrocarbonaceous nitrate esters in that service. In this way, the more rapid, severe degradation that occurs in service at higher elevated temperatures is directly inhibited. In these embodiments of the invention, the level of nitrate ester formation in the bulk liquid can be determined spectroscopically by observing the growth in the infra-red peak height associated with nitrate ester over time in the bulk liquid under suitable test conditions. This spectroscopic approach allows the determination of the amount of ionic liquid required to inhibit the formation of nitrate esters in the bulk liquid. The inhibition of hydrocarbonaceous nitrate ester formation in service is determined by the observance of a lower nitrate ester peak height in the bulk liquid in the presence of the ionic liquid, as measured by infrared spectroscopy according to DIN 51 453 or ASTM D8048-20 (in the event of conflict between DIN 51 453 and ASTM D8048-20, DIN 51 453 shall control), under like conditions of service and nitrogen dioxide contamination. According to the DIN method, the height of a single infrared absorption frequency at 1630 cm-1 is measured above a straight-line baseline defined by the absorption at 1615 and 1645 cm-1. The higher the peak height, the more nitrate ester is present in the bulk liquid. Measurement of a series of samples taken over time also allows the change in peak height to be followed as the level of nitrate ester in the service liquid changes over time. According to the ASTM D8048-20 Standard test method, oxidation and nitration peak heights are measured by first subtracting the fresh oil infrared spectrum. The baseline is defined by absorption between 1950 cm-1 and 1850 cm-1 with highest peak in the range 1740 cm-1 to 1700 cm-1 used for oxidation and 1640 cm-1 to 1620 cm-1 for nitration. Determining the amount of reduction or limitation of nitrate ester formation in a lubricating oil composition is determined by the observance of a lower (by at least 10%, such by at least 20%, such as by at least 30%, such as by at least 40%, such as by at least 50%, such as by 100%) nitrate ester peak height in the presence of the lubricating oil composition containing ionic liquid (as compared to the nitrate ester peak of the same lubricating oil composition where the ionic liquid is replaced with an ionic liquid having the same cation, but hexanoate as the anion in the same proportions), as measured by infrared spectroscopy according to DIN 51 453 or ASTM D8048-20, under like conditions of service and nitrogen dioxide contamination, provided that in the event of conflicting results between DIN 51 453 and ASTM D8048-20, DIN 51 453 shall control. In normal circumstances, however, the amount of ionic liquid added to thereafter inhibit the nitration of the hydrocarbonaceous liquid in service at bulk liquid temperatures of 60° C. or more, such as 110° C. or more, such as between 60 and 180° C. (such as from 60 to 180° C., such as 60 to 160° C., such as 110 to 160° C., such as 130 to 160° C.), in the presence of nitrogen dioxide contamination, is in the range of 0.1 to 5.0% by weight, per weight of hydrocarbonaceous liquid; and preferably 0.5 to 4.0% by weight, per weight of hydrocarbonaceous liquid. More preferably, the ionic liquid is added in an amount in the range of 1.0 to 3.5% by weight, per weight of hydrocarbonaceous liquid; and most preferably in the range of 1.0 to 3.0% by weight, per weight of hydrocarbonaceous liquid. The hydrocarbonaceous liquid deployed in the method of the invention is a liquid suitable for service at bulk liquid temperatures of 60° C. or more, such as 110° C. or more, such as between 60 and 180° C. (such as from 60 to 180° C., such as 60 to 160° C., such as 110 to 160° C., such as 130 to 160° C.) and being free of aged components and nitrogen dioxide contamination prior to service (or substantially free, e.g., less than 5 ppm, of aged components and less than 10 ppm, of nitrogen dioxide contamination). Such service liquids are used in a variety of applications, including industrial and automotive oils and power transmission fluids, such as engine lubricating oils. In the use the hydrocarbonaceous liquid is preferably a lubricating oil for a mechanical device. More preferably in the use, the hydrocarbonaceous liquid is a crankcase lubricating oil for an internal combustion engine, and is subjected in service to nitrogen dioxide contamination originating from exhaust gas, which gas becomes entrained in the lubricant via the effects of blow-by gas into the crankcase and direct contact on the engine cylinder walls. Most preferably, this crankcase lubricating oil is one periodically or continuously subjected to bulk liquid temperatures in the crankcase of between 110 and 160° C. It is important to obtaining the benefits of the use that, prior to service, the hydrocarbonaceous liquid be initially free of nitrogen dioxide contamination and also be initially free of the aged liquid components that arise during service from oxidative or other chemical breakdown, in order not to seed the liquid with significant quantities of reactive chemical species that can offer an alternative or complementary degradative pathway to nitrogen-dioxide initiated nitration. Thus, preferably the hydrocarbonaceous liquid should be freshly prepared and not have been in prior service; and prior to being placed into the service environment should not be pre-mixed or diluted with a proportion of aged liquid that has been in prior use or exposed to nitrogen dioxide contamination. Alternately, prior to service, the hydrocarbonaceous liquid may be initially substantially free of nitrogen dioxide contamination (10 ppm or less, such as 5 ppm or less, such as 0 ppm) and also substantially free of the aged liquid components (10 ppm or less, such as 5 ppm or less, such as 0 ppm) that arise during service from oxidative or other chemical breakdown (or substantially free, e.g., less than 0.0001-mass % of aged components and less than 10 ppm, of nitrogen dioxide contamination). It is also important that the ionic liquid is added prior to service and the resulting onset of elevated temperatures and nitrogen dioxide contamination, to maximise its nitration-inhibiting effect and not allow nitrogen dioxide concentration in the bulk liquid to build unhindered. In this use aspect, the ionic liquid can be added to the hydrocarbonaceous liquid by physical mixing or blending techniques known in the art. It may be desirable, although not essential, to prepare one or more additive concentrates under the second aspect comprising the ionic liquid in a carrier liquid (being a diluent or solvent mutually compatible with both the ionic liquid and the hydrocarbonaceous liquid), to enable easier mixing or blending, whereby other additives can also be added simultaneously to the concentrate, and hence to the oil, to form the lubricating oil composition (such concentrates sometimes being referred to as additive packages). Where an additive concentrate is used, it may contain from 5 to 25 mass %, preferably 5 to 22 mass %, typically 10 to 20 mass % of the ionic liquid, the remainder of the concentrate being solvent or diluent. The advantageous nature of the use in chemically deactivating nitrogen dioxide entrained in the hydrocarbonaceous liquid, thereby limiting its chemical degradation due to nitration, is demonstrated hereinafter in the worked examples of the invention. The Use of the Fifth Aspect of the Invention In a fifth aspect, the invention provides the use of the ionic liquid of the first aspect, or of the additive concentrate of the second aspect, as an additive for a hydrocarbonaceous liquid lubricant to reduce the friction coefficient of the lubricant, or improve its resistance to mechanical wear, or both. In the use of the fifth aspect, it is preferred that the ionic liquid is also used to chemically deactivate nitrogen dioxide entrained within the hydrocarbonaceous lubricant, and more preferably to also inhibit the rise in total acid number, as measured according to ASTM D664, in the hydrocarbonaceous lubricant in service, as described under the fourth aspect of the invention. The ionic liquids and hydrocarbonaceous liquids that are suitable and preferred in the fifth aspect of the invention are those already described in this specification. The amount of ionic liquid effective to reduce the friction coefficient, or improve its resistance to mechanical wear, or both can be determined through the use of industry-recognised friction and wear tests, by comparing the effect of ionic liquid addition on the baseline performance of the hydrocarbonaceous liquid lubricant in question. In normal circumstances, however, the amount of ionic liquid used to friction or wear or both of the hydrocarbonaceous liquid lubricant in service is in the range of 0.1-5.0% by weight, per weight of hydrocarbonaceous liquid; and preferably 0.5 to 4.0% by weight, per weight of hydrocarbonaceous liquid. More preferably, the ionic liquid is used in an amount in the range of 1.0 to 3.5% by weight, per weight of hydrocarbonaceous liquid; and most preferably in the range of 1.0 to 3.0% by weight, per weight of hydrocarbonaceous liquid. The amount of ionic liquid effective to inhibit nitration in the preferred embodiment of this use of the invention can be arrived at by routine testing under conditions reproducing or simulating nitrogen dioxide contamination at the elevated service temperatures experienced in the system in question. In a preferred aspect of this use, the chemical degradation inhibited by the ionic liquid is that resulting from the decomposition of hydrocarbonaceous nitrate esters formed in service by the nitration of the hydrocarbonaceous liquid by nitrogen dioxide at bulk liquid temperatures of between 60 and 180° C., and the ionic liquid inhibits the formation of hydrocarbonaceous nitrate esters in that service. In this way, the accumulation of a reservoir of reactive hydrocarbonaceous nitrate esters at elevated service temperatures is directly inhibited, and degradation is better limited. In a more preferred aspect of this use, the chemical degradation inhibited by the ionic liquid is that resulting from the decomposition of the hydrocarbonaceous nitrate esters due to the hydrocarbonaceous liquid being periodically or continuously subjected in service to bulk liquid temperatures of between 110 and 160° C., and the ionic liquid inhibits the formation of hydrocarbonaceous nitrate esters in that service. In this way, the more rapid, severe degradation that occurs in service at higher elevated temperatures is directly inhibited. In these use embodiments of the invention, the level of nitrate ester formation in the bulk liquid can be determined spectroscopically by observing the growth in the infra-red peak height associated with nitrate ester over time in the bulk liquid under suitable test conditions. This spectroscopic approach allows the observation of the effect of ionic liquid to inhibit the formation of nitrate esters in the bulk liquid. The inhibition of hydrocarbonaceous nitrate ester formation in service is determined by the observance of a lower nitrate ester peak height in the bulk liquid in the presence of the ionic liquid, as measured by infrared spectroscopy according to DIN 51 453, under like conditions of service and nitrogen dioxide contamination. According to this DIN method, the height of a single infrared absorption frequency at 1630 cm-1 is measured above a straight-line baseline defined by the absorption at 1615 and 1645 cm-1. The higher the peak height, the more nitrate ester is present in the bulk liquid. Measurement of a series of samples taken over time also allows the change in peak height to be followed as the level of nitrate ester in the service liquid changes over time. In normal circumstances, however, the amount of ionic liquid used to inhibit the nitration of the hydrocarbonaceous liquid in service at bulk liquid temperatures of between 60 and 180° C., in the presence of nitrogen dioxide contamination, is in the range of 0.1-5.0% by weight, per weight of hydrocarbonaceous liquid; and preferably 0.5 to 4.0% by weight, per weight of hydrocarbonaceous liquid. More preferably, the ionic liquid is used in an amount in the range of 1.0 to 3.5% by weight, per weight of hydrocarbonaceous liquid; and most preferably in the range of 1.0 to 3.0% by weight, per weight of hydrocarbonaceous liquid. The Method of the Sixth Aspect of the Invention In a sixth aspect, the invention provides a method of prolonging the service life of a hydrocarbonaceous liquid lubricant exposed to nitrogen dioxide contamination in service, comprising the addition thereto prior to service of the ionic liquid of the first aspect, or the additive concentrate of the second aspect, in an amount effective to thereafter reduce the friction coefficient of the lubricant or improve its resistance to mechanical wear, or to chemically deactivate nitrogen dioxide entrained within the hydrocarbonaceous liquid lubricant and consequently inhibit the formation of hydrocarbonaceous nitrate esters therein, or to both. In the method of the sixth aspect, it is preferred that the ionic liquid or additive concentrate is effective both to chemically deactivate nitrogen dioxide entrained within the lubricant, and to reduce the friction coefficient of the lubricant and improve its resistance to mechanical wear. Most preferably, the uses of the fourth and fifth aspects of the invention and the method of the sixth aspect, are directed to limiting the chemical degradation, and friction and/or wear, of hydrocarbonaceous liquids that are engine lubricating oils. These oils are exposed to nitrogen dioxide contamination in service, due to the presence of exhaust gas blow-by from the combustion chamber past the piston rings into the crankcase. Such oils, also termed crankcase oils, operate at bulk liquid temperatures wherein the nitration pathway to oil degradation is significant, especially when the oil is fresh and aged oil components have not appreciably formed by other mechanisms. Hotter-running engines are particularly susceptible to such degradation, especially those experiencing temperature regimes or cycles in the bulk crankcase oil of between 110 and 160° C., and in particular between 130 and 160° C. Definitions For purposes of this specification and all claims to this invention, the following words and expressions, if and when used, have the meanings ascribed below. For purposes herein, the new numbering scheme for the Periodic Table of the Elements is referred to as set out in CHEMICAL AND ENGINEERING NEWS, 63(5), 27 (1985). Alkali metals are Group 1 metals (e.g., Li, Na, K, etc.). Alkaline earth metals are Group 2 metals (e.g., Mg, Ca, Ba, etc.) The term “comprising” or any cognate word specifies the presence of stated features, steps, or integers or components, but does not preclude the presence or addition of one or more other features, steps, integers, components or groups thereof. The expressions “consists of” or “consists essentially of” or cognates may be embraced within “comprises” or cognates, wherein “consists essentially of” permits inclusion of substances not materially affecting the characteristics of the composition to which it applies. The term “mass %” means mass percent of a component, based upon the mass of the composition as measured in grams, unless otherwise indicated, and is alternately referred to as weight percent (“weight %”, “wt %” or “% w/w”). The term “absent” or “free” as it relates to components included within the lubricating oil compositions described herein and the claims thereto means that the particular component is present at 0 wt %, based upon the weight of the lubricating oil composition, or if present in the lubricating oil composition the component is present at levels that do not impact the lubricating oil composition properties, such as less than 10 ppm, or less than 1 ppm or less than 0.001 ppm. The term “absent” or “free” as it relates to amounts of aged components and nitrogen dioxide contamination means levels that do not impact the lubricating oil composition properties, such as less than 10 ppm, or less than 1 ppm or less than 0.001 ppm. Unless otherwise indicated, all percentages reported are mass % on an active ingredient basis, i.e., without regard to carrier or diluent oil, unless otherwise stated. This invention further relates to: 1. An ionic liquid composed of: (i) one or more nitrogen-free organic cations each comprising a central atom or ring system bearing the cationic charge and multiple pendant hydrocarbyl substituents, and (ii) one or more halogen- and boron-free organic anions each comprising an aromatic ring bearing a carboxylate functional group and a further heteroatom-containing functional group, these functional groups being conjugated with the aromatic ring and this conjugated system bearing the anionic charge, and the aromatic ring additionally bearing one or more hydrocarbyl substituents. 2. The ionic liquid of paragraph 1 wherein each cation (i) of the ionic liquid consists of a tetra-hydrocarbyl substituted central atom or ring system bearing the cationic charge. 3. The ionic liquid of paragraph 1 or paragraph 2, wherein each cation (i) of the ionic liquid is a tetra-alkyl substituted phosphonium cation. 4. The ionic liquid of paragraph 3, wherein each cation (i) is a trihexyltetradecyl phosphonium cation. 5. The ionic liquid of any preceding paragraph 1 to 4, wherein each anion (ii) of the ionic liquid is nitrogen-free. 6. The ionic liquid of any preceding paragraph 1 to 5, wherein the one or more hydrocarbyl substituents on the aromatic ring of each anion (ii) of the ionic liquid are one or more straight- or branched-chain alkyl substituents. 7. The ionic liquid of paragraph 5, or of paragraph 6 when read with paragraph 5, wherein the one or more anions (ii) of the ionic liquid are one or more alkyl-substituted salicylate anions, wherein the alkyl substituent(s) of each anion are independently selected from alkyl groups containing from 12 to 24 carbon atoms. 8. An additive concentrate for hydrocarbonaceous liquids, comprising the ionic liquid of any preceding paragraph and a compatible carrier liquid therefor. 9. A hydrocarbonaceous liquid comprising the ionic liquid or additive concentrate of any preceding paragraph in an amount of up to 5.0% by weight of ionic liquid, per weight of hydrocarbonaceous liquid. 10. The additive concentrate of paragraph 8, or hydrocarbonaceous liquid of paragraph 9, further comprising one or more performance-enhancing additives, preferably which comprise one or more of: phosphorus-containing compounds; dispersants; metal detergents; anti-wear agents; friction modifiers, viscosity modifiers, anti-oxidants; metal rust inhibitors, viscosity index improvers, corrosion inhibitors, anti-foaming agents, and pour point depressants. 11. The use of the ionic liquid of any of paragraphs 1 to 7, or of the additive concentrate of paragraph 8 or paragraph 10, as an additive for a hydrocarbonaceous liquid to chemically deactivate nitrogen dioxide entrained within the hydrocarbonaceous liquid. 12. The use of paragraph 11, wherein the ionic liquid consequently inhibits the formation of hydrocarbonaceous nitrate esters and prolongs the service life of the hydrocarbonaceous liquid. 13. The use of the ionic liquid of any of paragraphs 1 to 7, or of the additive concentrate of paragraph 8 or paragraph 10, as an additive for a hydrocarbonaceous liquid lubricant to reduce the friction coefficient of the lubricant, or improve its resistance to mechanical wear, or both. 14. The use of paragraph 13, wherein the ionic liquid also chemically deactivates nitrogen dioxide entrained within the hydrocarbonaceous lubricant. 15. The use of paragraph 14, wherein the ionic liquid inhibits the rise in total acid number, as measured according to ASTM D664, in the hydrocarbonaceous lubricant in service. 16. A method of prolonging the service life of a hydrocarbonaceous liquid lubricant exposed to nitrogen dioxide contamination in service, comprising the addition thereto prior to service of the ionic liquid of any of paragraphs 1 to 8, or the additive concentrate of paragraphs 8 or 10, in an amount effective to thereafter reduce the friction coefficient of the lubricant or improve its resistance to mechanical wear, or to chemically deactivate nitrogen dioxide entrained within the hydrocarbonaceous liquid lubricant and consequently inhibit the formation of hydrocarbonaceous nitrate esters therein, or to both. 17. The method of paragraph 16, wherein the ionic liquid or additive concentrate is effective both to chemically deactivate nitrogen dioxide entrained within the lubricant, and to reduce the friction coefficient of the lubricant and improve its resistance to mechanical wear. 18. A method of prolonging the service life of a hydrocarbonaceous liquid lubricant exposed to nitrogen dioxide contamination in service, comprising the addition thereto prior to service of the ionic liquid of any of paragraphs 1 to 7 or the concentrate of paragraph 8 or 10, in an amount effective to thereafter to have 1, 2, or 3 of the following effects: 1) to reduce the friction coefficient of the lubricant, 2) to improve its resistance to mechanical wear, and 3) to chemically deactivate nitrogen dioxide entrained within the hydrocarbonaceous liquid lubricant and consequently inhibit the formation of hydrocarbonaceous nitrate esters therein. 19. The method of paragraph 18, wherein the ionic liquid is effective: to chemically deactivate nitrogen dioxide entrained within the lubricant, to reduce the friction coefficient of the lubricant, and to improve its resistance to mechanical wear. EXAMPLES The practice and advantages of the present invention are now illustrated by way of examples. For purposes of this invention and the claims thereto, determining the amount of reduction or limitation of nitrate ester formation in a lubricating oil composition is determined by the observance of a lower (such as by at least 10%, such by at least 20%, such as by at least 30%, such as by at least 40%, such as by at least 50%, such as by 100%) nitrate ester peak height in the presence of the lubricating oil composition containing ionic liquid (as compared to the nitrate ester peak of the same lubricating oil composition where the ionic liquid is replaced with an ionic liquid having the same cation, but hexanoate as the anion in the same proportions), as measured by infrared spectroscopy according to DIN 51 453 or ASTM D8048-20, under like conditions of service and nitrogen dioxide contamination, provided that in the event of conflicting results between DIN 51 453 and ASTM D8048-20, DIN 51 453 shall control. Preparatory Examples—Preparation of Ionic Liquids Ionic liquids were synthesised using the following method deploying an ion-exchange resin. Example 1: [P66614][Alkyl-Salicylate] (Example of the Invention) [P66614][Alkyl-Salicylate] was produced using a two-step synthesis method starting from commercially available trihexyltetradecylphosphonium chloride, [P66614]Cl (CYPHOS IL-101, >95%, CAS: 258864-54-9). In the first step, [P66614][OH] was synthesized from [P66614]Cl using a commercially available basic anion exchange resin (Amberlite IRN-78, OH-form resin, CAS: 11128-95-3). [P66614]Cl (100 g, 0.193 mol) was added to a 2 L round-bottom flask and diluted with absolute ethanol (900 mL, 19.5 mol, CAS: 64-17-5). To this, 100 g of the ion exchange resin was added, and the mixture was stirred for 5 hours at 22° C. The resin was then filtered off, and 100 g of fresh resin was added. This step was repeated three times, or until a negative silver halide test was observed, indicating complete ion exchange. The silver halide test was carried out as follows: a small aliquot (0.2 mL) of the reaction mixture was transferred to a 2 mL vial, and diluted with 1 mL absolute ethanol. 2-3 drops of HNO3 were added to acidify the solution, and 2-3 drops of a saturated aqueous solution of AgNO3 (≥99 wt. %, Sigma-Aldrich, CAS: 7761-88-8) was subsequently added. Complete ion exchange was indicated when a transparent solution with no precipitate was observed. In the second step, the concentration of [P66614][OH] in ethanol was determined using 1H NMR. This was followed by the dropwise equimolar addition, dissolved in 100 mL ethanol, of commercially available alkyl-salicylic acid from Infineum UK Ltd, being a mono-alkyl salicylic acid mixture bearing alkyl substituents of 14 and 16 carbon atoms. The acid number of the alkyl-salicylic acid (0.00261 g H+/mol) was used to calculate the amount of acid required (equimolar—equating to 73.96 g of alkyl salicylic acid) for the neutralisation reaction with [P66614][OH], and this mixture was subsequently stirred overnight at 22° C. The solution was then dried under rotary evaporation and subsequently in vacuo (10-3 Pa) at 50° C. for a minimum of 96 h, to obtain the dry pure ionic liquid. Following drying the ionic liquid material was characterised via NMR: [P66614][Alkyl-Salicylate]: 1H NMR (500 MHz, DMSO-d6): δ (ppm)=0.69-0.88 (s), 1.04-1.29 (m), 1.37 (m), 1.46 (m), 2.15 (m), 2.29 (s), 3.34 (s), 3.43 (m), 4.36 (s), 6.49 (m), 6.72 (m), 6.93 (m), 7.18 (m), 7.25 (m), 7.41 (s), 7.47 (m), 7.65 (s), 7.70 (s), 8.16 (s), 9.07 (s), 9.11 (s), 9.15 (s). A further sample of [P66614][Alkyl-Salicylate] was prepared by the following scaled up procedure. [P66614]Cl (808 g, 1.56 mol) was charged into a 5 L glass reactor and diluted with absolute ethanol (770 mL, 13.2 mol). To this solution was dosed a pre-prepared solution of KOH (87.3 g, 1.56 mol) in absolute ethanol (770 mL, 13.2 mol) over 28 minutes using a water bath to limit the exotherm to 23° C. The mixture was aged for between 90 and 250 min and then blended with celite filter aid (164 g, 20 mass %) and filtered to remove KCl, rinsing the filter cake with absolute ethanol (160 mL, 2.74 mol). The filtrate was transferred to a clean 5 L glass reactor and treated with Amberlite ion exchange resin TRN-78 (400 g, 50 mass %) for 30-70 min and then separated by filtration, rinsing the resin with absolute ethanol (2×160 mL, 2×2.74 mol). The filtrate was transferred to a clean 5 L glass reactor, into which was dosed an equimolar amount of the same alkyl-salicylic acid as a xylene solution over 33 min using a water bath to limit the exotherm to 28° C. The mixture was aged for 16 hours and then the volatile Example 2: [P66614][Hexanoate] (Comparative Example) [P66614][Hexanoate] was synthesised via the procedure used for [P66614][Alkyl-Salicylate] in Example 1.1. [P66614][OH] was firstly prepared from [P66614]Cl (100 g, 0.193 mol). Equimolar addition of hexanoic acid (≥99 wt. %, CAS: 142-62-1) in place of salicylic acid in the second step (22.4 g, 0.193 mol) was used to produce the desired ionic liquid, followed by drying. Example 3: [P66614][NTf2] (Comparative Example) Trihexyltetradecylphosphonium chloride, [P66614]Cl (100 g, 0.193 mol) was dissolved in a minimum amount of dichloromethane (≥99%, CAS: 75-09-2), in a 1 L round-bottom flask. To this, an aqueous solution of commercially available LiNTf2 (55.3 g, 0.193 mol; 99 wt. %, CAS: 90076-65-6) was added dropwise. The reaction mixture was stirred for 12 h at 22° C., forming a biphasic solution. The organic layer was extracted and washed with ultrapure water five times to remove the LiCl by-product, and until a negative halide test was observed. The solution was then dried under rotary evaporation and subsequently in vacuo (10-3 Pa) at 50° C. for a minimum of 96 hours, to obtain dry pure trihexyltetradecylphosphonium bis(trifluoromethanesulfonyl)imide, [P66614][NTf2], determined by NMR as follows: [P66614][NTf2]: 1H NMR (500 MHz, CDCl3): δ (ppm)=0.88 (m, 12H, CH3-(P)) 1.23-1.29 (m, 32H, —CH2-(P)), 1.46 (m, 16H, —CH2-(P)), 2.08 (m, 8H, —CH2-(P)); 13C NMR (126 MHz, CDCl3): δ (ppm)=13.85, 14.12, 18.56, 18.94, 21.55, 22.28, 22.69, 28.80, 29.25, 29.36, 29.49, 29.65, 30.17, 30.52, 30.89, 31.92, 118.62, 121.17. The ionic liquids prepared by these syntheses were used in the further examples below. Worked Example 1: Mechanistic Evaluation of the Ionic Liquid of the Invention To evaluate the effectiveness and mechanism of the ionic liquid of the invention, the onset and progress of nitration in a hydrocarbonaceous liquid subject to nitrogen dioxide contamination can be observed and measured using infrared spectroscopy. Monitoring the progressing nitration of the hydrocarbonaceous liquid involves taking periodic samples of the liquid in use under real or simulated service conditions, and following the evolution of the fingerprint nitration peak height on the infrared spectrum. The rate of increase of the nitration peak height provides information on the rate of chemical degradation due to nitration and build-up of the nitrate ester reservoir in the bulk liquid. According to the DIN 51453 peak height method [Standard DIN 51453 (2004-10): Testing of lubricants—Determination of the oxidation and nitration of used motor oils—Infrared spectrometric method], the height of a single infrared absorption frequency at 1630 cm-1 attributable to forming hydrocarbonaceous nitrate ester is measured above a straight-line baseline defined by the absorptions at 1615 and 1645 cm-1. The higher the peak height, the more hydrocarbonaceous nitrate ester is present in the bulk liquid. The above DIN method also provides for monitoring of the progress of conventional oxidation of the bulk liquid via the measurement of peak height at 1710 cm-1 attributable to carbonyl moieties (ketones, aldehydes, esters and carboxylic acids) formed as a result of oxidation. This peak height is measured relative to a straight-line baseline defined by absorptions at 1970 and 1650 cm-1. Again the rate of increase of peak height provides information on the rate of chemical oxidation in the bulk liquid. According to ASTM D8048-20 Standard test method for evaluation of diesel engine oils in Volvo (Mack) T-13 diesel engines, oxidation and nitration peak heights are measured by first subtracting the fresh oil infrared spectrum. The baseline is defined by absorption between 1950 cm-1 and 1850 cm-1 with highest peak in the range 1740 cm-1 to 1700 cm-1 used for oxidation and 1640 cm-1 to 1620 cm-1 for nitration. Samples of hydrocarbonaceous liquid being tested under service conditions can be measured via the above methods, and allow the reporting of the effect of different ionic liquids present in the hydrocarbonaceous liquid on the progress, and/or level of inhibition, of degradation due to nitration and due to oxidation. Anion Contribution Towards Inhibiting Degradation Caused by Nitration The DIN 51453 method was used to illustrate the contribution of the anion of the ionic liquid in the performance of the present invention. Testing was conducted on a freshly prepared lubricating oil as bulk hydrocarbonaceous liquid, this composition containing a conventional package of commercial additives. To this starting composition was added 2% by mass, per mass of the oil, of the ionic liquid Example 1 of this invention, being composed of the tetraalkylphosphonium cation “P66614” and an alkyl salicylate anion. A comparative test sample was prepared from the same starting oil composition by adding 2% by mass, per mass of oil, of an ionic liquid composed of Example 3, having the same P66614 cation but an NTf2 anion [Trihexyltetradecylphosphonium bis(trifluoromethanesulfonyl)imide]. This comparative ionic liquid thus contains an anion of the type favoured in US-A-2010/0187481 for conventional antioxidancy. The starting oil composition was also used as a control run to set the baseline offered by a commercial formulated oil. The test samples were subjected to a laboratory simulation of service conditions as an engine lubricant, in which the oil was exposed to sump operating temperatures and exposed to a source of nitrogen dioxide to mimic contamination in service. This simulation comprises a three-necked 250 mL conical flask fitted with a glycol condenser and heated on an electrical hot-plate. Gas containing 766 ppm N02 in air is bubbled through 250 g of the test lubricant at a rate of 10 litres per minute. A sintered glass frit is used to disperse the gas in the oil. The gas flow rate is regulated using a mass flow controller. The third neck is used to introduce a thermocouple which feeds-back to the hotplate to maintain constant temperature. The test samples were each run for 96 hours at 130° C., and the nitration and oxidation peak heights determined at the end of the test by the above DIN 51453 method. The results for the two samples containing ionic liquid were then compared with the control oil formulation, and the impact of their respective ionic liquids reported as percentage reductions in nitration and oxidation peak height against the control. Results peak height % reductionvs control2% treat rate by mass of ionic liquidOxidationNitrationExample 3 - [P66614][Ntf2]5943Example 1 - [P66614][Alkyl-Salicylate]7070 The presence of the P66614 alkyl-salicylate ionic liquid of the invention resulted in substantially greater reduction in nitration peak height than the comparative ionic liquid with identical cation, but anion not according to the present invention. These results support the differential effect of anion composition in the ionic liquid, and demonstrate the significant advantage provided by the anion defined in the present invention for deactivating nitrogen dioxide entrained in the bulk liquid. Whilst the present invention also showed a substantial reduction in oxidation peak height, the oxidation results showed less differentiation between the two ionic liquid samples, supporting the existence of different chemical pathways to nitration and classical oxidation of the lubricant. The differential benefit for the present invention towards nitration indicates its higher selectivity for inhibiting the nitration pathway and greater suitability for controlling the effect of nitrogen dioxide contamination under service. Cation Contribution Towards Inhibiting Degradation Caused by Nitration The DIN 51453 method and laboratory test method of the above anion comparison was also used to illustrate the contribution of the cation of the ionic liquid in the performance of the present invention. Testing was again conducted on the freshly prepared formulated lubricating oil as bulk hydrocarbonaceous liquid containing a conventional package of commercial additives. To this starting composition was added 2% by mass, per mass of the oil, of ionic liquid Example 1 of this invention, being composed of the tetraalkylphosphonium cation “P66614” and an alkyl salicylate anion. However, the comparative test sample was prepared from the same starting oil composition by adding the alkyl salicylic acid from which the ionic liquid had been prepared, in an amount equivalent to the amount of anion in the ionic liquid sample. Thus, in this case, the same aromatic ring structure was added to the oil, in the same amount, but the cation was omitted. The starting oil composition was again used as a control. The test samples were subjected to the same laboratory simulation of service conditions as an engine lubricant, in which the oil was exposed to sump operating temperatures and exposed to a source of nitrogen dioxide to mimic contamination in service. The test samples were each run for 96 hours at 130° C., and the nitration and oxidation peak heights determined at the end of the test by the above DIN method. The results for the two samples were then compared with the control oil, and their impact reported as percentage reductions in nitration and oxidation peak height against the control: peak height % reductionvs control2% treat rate by mass of ionic liquidOxidationNitrationExample 1.2 - [P66614][Alkyl-Salicylate]70700.84% Alkyl-Salicylic acid3427 The results demonstrate that whilst alkyl-salicylic acid itself brought about some reduction in nitration, the ionic liquid was a more potent inhibitor of nitration. This performance advantage was much more apparent for nitration than for oxidation. The full nitration-inhibiting effect of the ionic liquid of the present invention is therefore attributable to the ion-pair combination in the ionic liquid, which co-operate to deactivate nitrogen dioxide present in the bulk liquid. Further investigation of the mechanism of this combination effect was carried out in the same laboratory simulation test using the same freshly prepared lubricating oil composition, this time treated with the ionic liquid P66614 Cl. This comparative ionic liquid did not give as much reduction in nitration peak height as the alkyl salicylate example of the invention, but nevertheless still reduced the nitration level by over 60% as compared to the control lacking this ionic liquid. Compositional analysis of the bulk oil composition at the end of the test showed a decrease in chloride concentration in the oil over the course of the test; and a gas purge through the end-of-test bulk oil and into silver nitrate solution confirmed the formation of hydrochloric acid during the test. Thus, the P66614 cation is considered too complex with nitric acid formed in situ from a proportion of the nitrogen dioxide, this complex rearranging to the P66614-nitrate ion pair and releasing HCl. In this way, the cation of the ionic liquid also serves to lock away some nitrogen dioxide in a deactivated form, reducing the effective contaminant level and slowing the resulting degradation. In the practice of the present invention, the advantages of the defined ionic liquid thus result from the co-operative effect of the defined anion's particularly high affinity for sequestering away nitrogen dioxide, coupled with the ability of the associated cation to form a stable complex with nitrate ions formed in situ from a proportion of the nitrogen dioxide, which further reduces the available nitrogen dioxide concentration within the bulk liquid. This combined effect produces particularly good inhibition of the nitration, and hence degradation, caused by nitrogen dioxide in the bulk liquid. This effect likewise provides for slower increases in total acid number in the bulk liquid, and reduces the potential for the consequences of nitration and acidification, such as bulk liquid viscosity growth. Worked Example 2: Performance of the Invention in Controlling Degradation Under Service Conditions The advantageous nature of the present invention is illustrated by testing under real service conditions. For these purposes, an engine lubricating oil was used as the hydrocarbonaceous liquid and the service environment chosen was the ASTM D8048-20 Standard test method for evaluation of diesel engine oils in Volvo (Mack) T-13 diesel engines. The test uses a 2010 Volvo/Mack D13/MP8, 505BHP, 13 L in-line six cylinder diesel engine with electronically controlled fuel injection, with six electronic unit injectors, VGT (variable geometry turbocharger), and cooled EGR (exhaust gas recirculation). It is a 360 hour test run at at 1500 RPM steady state conditions producing approximately 2200 Nm torque and 130° C. oil temperature with 19-20% EGR. The principal aim is to evaluate the oxidation stability performance of engine oils at an elevated oil temperature using ULSD (ultra-low sulfur diesel) fuel. The test appears in the following industry specification for oil quality: Mack EOS-4.5, Volvo EOS-4.5, Renault RLD-4, API CK-4 and FA-4The T13 engine test was chosen in view of its known-in-the-art characteristics of high operating temperatures and representative engine-out NOx emissions. The engine (crankcase) lubricating oil of the T13 test is thus exposed in service to higher bulk temperatures in the sump and to nitrogen dioxide contamination via direct entrainment in the lubricant draining down from the cylinder walls, and exhaust gas blowby past the piston rings into the crankcase. The T13 test provides an endurance test for the lubricant under conditions that promote chemical degradation due to nitration initiated by nitrogen dioxide contamination. To increase the endurance element of the test further, its normal duration of 360 hours was extended to 400 hours in some cases below. Periodically during the test, the oil is sampled and nitration and oxidation peak heights measured by infrared spectroscopy using the ASTM D8048-20 Mack (Volvo) T13 oxidation method described in application 3 above. The rise in total acid number (TAN ASTM D445) during the test and the increase in viscosity (ASTM D445) of the oil at 40° C. and 100° C. during the test were also measured. Three T13 tests were conducted to compare the effects of different additives to controlling chemical degradation due to nitration and ultimately oxidation. In each case, the same freshly prepared starting lubricating oil composition was used, being a conventional lubricant base oil base-stock containing a standard commercial package of additives. To this starting composition was added one further material in each test, and the effects of these materials compared. In Oil 1 (comparative), the further material was a comparative ionic liquid from the prior art, being composed of the tetra-alkylphosphonium cation “P66614” and a hexanoate anion, i.e., having an anion of the structure YCOO(−) wherein Y is C6 alkyl. This ionic liquid was used at the treat rate of 2% by mass, per mass of lubricating oil composition, and was produced in preparative Example 2 as hereinbefore described. In Oil 2 (comparative), the further material was a commercial antioxidant additive composed of a hindered phenolic compound. This material is known to be an effective control on conventional free-radical based oxidation processes. In Oil 3 (invention), the further material was an ionic liquid from the present invention, being composed of the tetraalkylphosphonium cation “P66614” and an alkyl salicylate anion. This ionic liquid was used in the lubricating oil composition at equimolar concentration to the P66614-hexanoate ionic liquid used in the first case, approximating to a treat rate of 2.8% by mass, per mass of lubricating oil composition. This ionic liquid was produced by the scaled-up process in preparative Example 1 as hereinbefore described. The results over the course of the T13 tests are shown graphically inFIGS.1,2and3for nitration, oxidation and increase in kinematic viscosity at 100° C. respectively. InFIG.1, all three test compositions showed an increase in nitration peak height as the test progressed, with some nitration occurring due to the contamination by nitrogen dioxide. However, Oil 2 containing the conventional antioxidant generally showed the fastest growth in nitration peak height, which accelerated from the 300 hour point of its test. This test run was accordingly stopped at the normal 360 hour point, with the nitration peak height at over 40. The progress of nitration was generally slower with the ionic liquid-treated Oils 1 and 3, however the nitration rate with hexanoate-based ionic liquid (Oil 1) also increased after the 200 hour mark, and by 360 hours had exceeded 30 on nitration peak height. In contrast, the alkyl salicylate-based ionic liquid (Oil 3) retained a slow and steady gradient throughout the 360 hours normal duration, and by that point had only just exceeded 20 on nitration peak height, less than half the nitration of the conventional anti-oxidant Oil 2, and substantially less than Oil 1. By 400 hours the nitration level of Oil 3 was still significantly less than that of Oil 1. Thus, in the real service conditions of the engine, under hot sump temperatures and in the presence of nitrogen dioxide contamination, the present invention showed substantially improved ability to inhibit nitration over the conventional antioxidant additive solution. It also showed significantly better performance than an analogous alkyl-carboxylate ionic liquid, demonstrating the benefit arising from its different composition. LikewiseFIG.2shows that both the conventional antioxidant solution (Oil 2) and the hexanoate-based ionic liquid (Oil 1) showed rapid increase in oxidation towards the end of the test, as the oils lost their oxidation control and oxidation peak height rose sharply. In contrast, Oil 3 retained excellent oxidation control right through to the 360 hour mark, and by 400 hours was still showing significantly lower oxidation than either comparative oil. The slower growth in nitration peak height and consequently oxidation peak height exhibited by Oil 3 likewise demonstrates the greater efficacy of the present invention to inhibit the chemical degradation of the bulk liquid (lubricating oil) caused by nitrogen dioxide contamination during service. The slower growth in nitration peak height over time records a slower build-up of nitrate esters in the bulk liquid and, consequently, a slower onset of chemical degradation due to nitration, allowing the liquid to remain in service for longer. The rise in kinematic viscosity (as measured by ASTM D445) over the course of the tests shown inFIG.3also diverged between the two ionic liquids. The kinematic viscosity of Oil 1 rose steeply towards the end of the test, as this oil lost its control of the degradative processes. In contrast, Oil 3 of the invention maintained an essentially flat viscosity for the whole duration of the test. The higher initial viscosity of the ionic-liquid treated oils in these tests results from the direct viscosity effect of the addition of the ionic liquid without adjustment to the underlying oil composition, in order to avoid introducing other variables, and would be formulated out in practice of the invention by viscometric adjustments to the underlying oil composition. The invention also showed improved TAN control over the analogous hexanoate-based ionic liquid. At the end of the test, Oil 3 had a TAN of only 2.8 at the normal end of test point of 360 hours, and a TAN of 4.2 at the end of the 400 hours extended test; whereas by 360 hours the TAN of Oil 1 had already risen to 8.32, so this test was not extended further. Thus, the present invention (Oil 3) also provided advantages in terms of both viscosity control and total acid number control, providing formulating benefits to the user. Worked Example 3: Performance of the Ionic Liquid in Reducing Friction and Wear The ability of the ionic liquid of the invention to lower the coefficient of friction and to reduce wear on contact surfaces lubricated by hydrocarbonaceous liquids was demonstrated using the following industrial tests. Coefficient of friction and wear scar volume were both measured using the High Frequency Reciprocating Rig (HFRR) test, in which a steel plate is reciprocated against a steel ball under standard conditions whilst immersed in the liquid lubricant. In this test, a commercially available HFRR machine was used with test disc plates of 10 mm SAEAMS 6440 steel (AISO 52100/535A99) with a surface finish of <0.2 μm Ra and a hardness of 190-210 Hv30, and 6.00 mm test balls (grade 28 per ISO 3290) of SAE-AMS 6440 steel with a roughness of <0.5 μm and a hardness of 58-66 on the Rockwell “C” scale. The test was run according to the following profile: the test specimen was subjected to a series of increasing temperature steps of 40, 60, 80, 100, 120 and 140° C. At each step, the specimen was held constant at that temperature for 1 minute, and a reciprocating cycle then run at that stabilised temperature for 5 minutes using a 400 g load, frequency of 40 Hz and stroke length of 1000 um, and 5 seconds output interval. The sample temperature was then raised to the next step, and the reciprocating cycle repeated, until all temperature steps were completed. Coefficient of friction was measured during the test, and the wear scar volume determined at end of test. Coefficient of friction was also measured using the TE-77 test method, for which the test specimens were cut from an uncoated steel top-ring and steel liner used in the DD13 engine test. Temperature was fixed at 150° C. throughout the test, and the frequency of reciprocation was fixed at 10 Hz throughout the test. The test profile involved running in for 5 minutes at 20 N load, followed by progressive load increase from 20 N to 200N at a rate of 1.5 N/min (a step time approximately 2 hours), followed by a constant load of 200 N for a further 1 hour. The wear scar volume is also measured using the MTM-R test. In this test, commercially available MTM equipment was used with test discs of 46 mm AISI 52100 steel with a surface finish of <0.02 μm Ra and a hardness of 720-780 Hv, and test balls of 19.05 mm AISI 52100 steel with a surface finish of <0.02 μm Ra and a hardness of 800-920 Hv. The test profile involved a stroke length of 4 mm and maximum force (Fmax) of 20N at position 5. In the ball on disc reciprocating step, the ball speed was 350 m/s at a frequency of 10 Hz and test temperature stabilised at 100° C. The step duration was 45 min and log interval 10 s. Tests according to the above methods were conducted on a hydrocarbonaceous lubricating engine oil containing a typical package of commercial oil additives (the “baseline” oil formulation), with and without the presence of Example 1 of the present invention in the amount of 2% by weight, per weight of the baseline oil formulation. These comparisons allowed the effect of the ionic liquid to be seen directly. The results are shown inFIGS.5to8inclusive. InFIGS.4and5, the results show how the introduction of ionic liquid Example 1 resulted in a lower coefficient of friction over the HFRR and TE-77 tests. In the TE-77, the friction coefficient achieved in the presence of Example 1 ran lower than the baseline oil formulation throughout the test sequence. In the HFRR test, the coefficients of friction in the presence and absence of Example 1 diverged over the course of the test, with the ionic liquid enabling a constant friction level to be maintained over time. InFIGS.6and7, the presence of Example 1 resulted in lower wear scar volumes in both the MTM-R and HFRR tests respectively, showing the ionic liquid of the invention also to be effective in reducing wear on the contact surfaces lubricated by the oil. A follow-up fuel economy test conducted in an M276 motored engine rig, using the ionic liquid of Example 1 in the amount of 1% by weight, per weight of the lubricating oil demonstrated a reduction in torque of 0.5 and 0.7% at medium, high and extra-high temperatures, confirming the ability of the ionic liquid to provide a frictional advantage that translates into a measurable benefit in engine performance. In the above examples, the ionic liquid of the invention is thus shown to have a combination of performance benefits when employed as an additive for hydrocarbonaceous liquids, reducing the chemical degradation of the hydrocarbonaceous liquid by inhibiting the formation of hydrocarbonaceous nitrate esters arising from nitrogen dioxide contamination, and serving to reduce friction and/or wear between contact surfaces lubricated by the hydrocarbonaceous liquid. Consequent benefits are also seen, such as inhibiting the viscosity or acid number increase in service and improved fuel economy. All documents described herein are incorporated by reference herein, including any priority documents and/or testing procedures, to the extent they are not inconsistent with this text. As is apparent from the foregoing general description and the specific embodiments, while forms of the invention have been illustrated and described, various modifications can be made without departing from the spirit and scope of the invention. Accordingly, it is not intended that the invention be limited thereby. The term “comprising” specifies the presence of stated features, steps, integers or components, but does not preclude the presence or addition of one or more other features, steps, integers, components or groups thereof. Likewise, the term “comprising” is considered synonymous with the term “including.” Likewise, whenever a composition, an element, or a group of elements is preceded with the transitional phrase “comprising,” it is understood that we also contemplate the same composition or group of elements with transitional phrases “consisting essentially of,” “consisting of,” “selected from the group of consisting of,” or “is” preceding the recitation of the composition, element, or elements and vice versa. Further, when a range is stated as between A and B, the range includes endpoints A and B, thus “between A and B” is synonymous with “from A to B.” | 104,962 |
11859150 | EXPERIMENTAL SECTION The examples that follow illustrate the invention without limiting it. 1. Synthesis of Random Copolymers A1 Bearing a Diol Function 1.1: Starting with a Monomer Bearing a Diol Function In one embodiment, the random copolymers A1 of the invention are obtained according to reaction scheme 11 below: The copolymer obtained after removing the RAFT chain end contains, inter alia, styrene as comonomer and the thiocarbonylthio residue was removed, for example by converting it into a thioether. 1.1.1. Synthesis of the Monomer M1 Bearing a Diol Function The synthesis of a methacrylate monomer bearing a diol function is performed in three steps (steps 1, 2 and 3 of reaction scheme 11) according to the protocol below: First Step: 42.1 g (314 mmol) of 1,2,6-hexanetriol (1,2,6-HexTri) are placed in a 1 L round-bottomed flask. 5.88 g of molecular sieves (4 Å) are added, followed by 570 mL of acetone. 5.01 g (26.3 mmol) of para-toluenesulfonic acid (pTSA) are then slowly added. The reaction medium is stirred for 24 hours at room temperature. 4.48 g (53.3 mmol) of NaHCO3are then added. The reaction medium is stirred for 3 hours at room temperature before being filtered. The filtrate is then concentrated under vacuum using a rotary evaporator until a suspension of white crystals is obtained. 500 mL of water are then added to this suspension. The solution thus obtained is extracted with 4×300 mL of dichloromethane. The organic phases are combined and dried over MgSO4. The solvent is then totally evaporated off under vacuum at 25° C. using a rotary evaporator. Second Step: 5.01 g (28.8 mmol) of the product thus obtained are placed in a 1 L round-bottomed flask. 4.13 g (31.9 mmol) of DIPEA and 37.9 mg (0.31 mmol) of DMAP are then placed in the flask, followed by 5.34 g (34.6 mmol) of methacrylic anhydride. The flask is then stirred at room temperature for 24 hours. 0.95 g of methanol (29.7 mmol) is then added to the solution and the flask is stirred for a further 1 hour. The product is then dissolved in 40 mL of hexane. The organic phase is then washed successively with 25 mL of water, 3×25 mL of aqueous 0.5 M hydrochloric acid solution, 3×25 mL of aqueous 0.5 M NaOH solution and again with 25 mL of water. The organic phase is dried over MgSO4, filtered and then concentrated under vacuum using a rotary evaporator to give a pale yellow liquid. Third Step: 17.23 g (71.2 mmol) of the product thus obtained are placed in a 1 L round-bottomed flask. 90 mL of water and 90 mL of acetonitrile are then placed in the flask, followed by 59.1 mL (159 mmol) of acetic acid. The flask is then stirred for 24 hours at 30° C. while a gentle stream of nitrogen is bubbled through to force the removal of the acetone. The solution thus obtained is extracted with 6×30 mL of ethyl acetate. The organic phase is washed successively with 5×30 mL of aqueous 0.5 M NaOH solution and then 3×30 mL of water. The organic phase is then dried over MgSO4, filtered and then concentrated under vacuum using a rotary evaporator to give a pale yellow liquid, the characteristics of which are as follows: 1H NMR (400 MHz, CDCl3) δ: 6.02 (singlet, 1H), 5.49 (singlet, 1H), 4.08 (triplet, J=6.4 Hz, 1H), 3.65-3.58 (multiplet, 1H), 3.57-3.50 (multiplet, 3H), 3.35 (doublet of doublets, J=7.6 Hz and J=11.2 Hz, 1H), 1.86 (doublet of doublets, J=1.2 Hz and J=1.6 Hz, 3H), 1.69-1.31 (multiplet. 6H). 1.1.2. Synthesis of Methacrylate Copolymers Bearing Diol Functions with Removal of the RAFT Chain End The synthesis of methacrylate copolymers bearing diol functions is performed in two steps (steps 4 and 5 of reaction scheme 11):Copolymerization of two alkyl methacrylate monomers with a methacrylate monomer bearing a diol function and a styrene monomer;Removal of the RAFT chain end (aminolysis of the thiocarbonylthio residue to a thiol followed by Michael addition of the thiol with an alkyl acrylate). 1.1.2.1 Synthesis of the Copolymer A-1a More specifically, the synthesis of the copolymer A-1a is performed according to the following protocol: First Step: 12.56 g (37.1 mmol) of stearyl methacrytate (StMA), 12.59 g (49.5 mmol) of lauryl methacrylate (LMA), 2.57 g (24.7 mmol) of styrene (Sty), 2.54 g (12.4 mmol) of methacrylate bearing a diol function obtained according to the protocol described in section 1.1.1, 82.5 mg (0.30 mmol) of cumyl dithiobenzoate, 15 mg (0.09 mmol) of azobisisobutyronitrile (AIBN) and 30 mL of anisole are placed in a 250 mL Schlenk tube. The reaction medium is stirred and degassed for 30 minutes by bubbling nitrogen through, and is then maintained at 65° C. for a period of 24 hours. Second Step: After 24 hours of polymerization, the Schlenk tube is placed in an ice bath to stop the polymerization, and 30 mL of dimethytformamide (DMF) and 0.4 mL of n-butylamine (4 mmol) are added to the solution without degassing the medium. 15 hours later, 3 mL (21 mmol) of butyl acrylate are added. 16 hours later, the polymer isolated by 3 successive precipitations in methanol, filtering and drying under vacuum at 50° C. overnight. A copolymer is thus obtained with a number-average molar mass (Mn) of 53 000 g/mol, a polydispersity index (Ip) of 1.19 and a number-average degree of polymerization (DPn) of 253. These values are obtained, respectively, by size exclusion chromatography using tetrahydrofuran as eluent and poly(methyl methacrylate) calibration and by monitoring the monomer conversion during the copolymerization. A poly(alkyl methacrylate-co-alkyldiol methacrylate-co-styrene) copolymer A-1a containing about 10 mol % of diol monomer units M1 (obtained according to the protocol described in section 1.1.1) is obtained. 1.1.2.2 Synthesis of the Copolymer A-1c The synthesis of the copolymer A-1c is performed according to the following protocol: 12.51 g (36.9 mmol) of stearyl methacrylate (StMA), 12.65 g (49.7 mmol) of lauryl methacrylate (LMA), 2.58 g (24.7 mmol) of styrene, 2.51 g (12.4 mmol) of methacrylate bearing a diol function obtained according to the protocol described in section 1.1.1, 15.3 mg (0.06 mmol) of cumyl dithiobenzoate, 4.6 mg (0.03 mmol) of AIBN and 3.2 mL of anisole are placed in a 100 mL Schlenk tube. The reaction medium is stirred and degassed for 30 minutes by bubbling nitrogen through, and is then maintained at 65° C. for a period of 24 hours. After 24 hours of polymerization, the Schlenk tube is placed in an ice bath to stop the polymerization, and 20 mL of DMF, 30 mL of tetrahydrofuran (THF) and 0.27 mL of n-butylamine (2.7 mmol) are added to the solution. 16 hours later, 4 mL (28 mmol) of butyl acrylate are added. 24 hours later, the polymer is isolated by 3 successive precipitations in methanol and drying under vacuum at 50° C. overnight. A copolymer is thus obtained with a number-average molar mass (Mn) of 154 000 g/mol, a polydispersity index (Ip) of 1.23 and a number-average degree of polymerization (DPn) of 893. These values are obtained, respectively, by size exclusion chromatography using THF as eluent and poly(methyl methacrylate) calibration and by monitoring the monomer conversion during the copolymerization. A poly(alkyl methacrylate-co-alkyldiol methacrylate-co-styrene) copolymer A-1c containing about 9 mol % of diol monomer units is obtained. 1.1.3. Synthesis of Methacrylate Copolymers Bearing Diol Functions without RAFT Chain Removal The synthesis of the methacrylate copolymers bearing diol functions and not having improved properties when compared with the copolymers of the prior art is performed in a single step which consists of the copolymerization of two alkyl methacrylate monomers with a methacrylate monomer bearing a diol function. The term “methacrylate copolymers not having improved properties when compared with the copolymers of the prior art” means methacrylate copolymers bearing diol functions not containing any styrene monomer and always bearing the thiocarbonylthio residue at the chain end. This copolymer is representative of methacrylate copolymers bearing diol functions obtained by following the protocol described in patent application WO 2015/110642 (experimental section § 1.). More specifically, the synthesis of the copolymer A-1b is performed according to the following protocol: First Step: 13.38 g (39.5 mmol) of stearyl methacrylate (StMA), 12.58 g (49.4 mmol) of lauryl methacrylate (LMA), 2.01 g (9.9 mmol) of methacrylate bearing a diol function obtained according to the protocol described in section 1.1.1, 93.7 mg (0.34 mmol) of cumyl dithiobenzoate, 12.4 mg (0.08 mmol) of azobisisobutyronitrile (AIBN) and 28 mL of anisole are placed in a 250 mL Schlenk tube. The reaction medium is stirred and degassed for 30 minutes by bubbling nitrogen through, and is then maintained at 65° C. for a period of 18 hours 30 minutes. The polymer is then isolated by 3 successive precipitations in methanol, filtering and drying under vacuum at 50° C. overnight. A copolymer is thus obtained with a number-average molar mass (Mn) of 56 700 g/mol, a polydispersity index (Ip) of 1.21 and a number-average degree of polymerization (DP) of 253. These values are obtained, respectively, by size exclusion chromatography using tetrahydrofuran as eluent and poly(methyl methacrylate) calibration and by monitoring the monomer conversion during the copolymerization. A poly(alkyl methacrylate-co-alkyldiol methacrylate) copolymer A-1b containing about 10 mol % of diol monomer units M1 is obtained. 2. Synthesis of the Poly(Alkyl Methacrylate-Co-Boronic Ester Monomer) Copolymer This synthesis is performed according to the protocol described in patent application WO 2016/113229 (experimental section § 2.). 3. Rheological Studies 3.1 Ingredients for the Formulation of Compositions A to F Lubricant Base Oil The lubricant base oil used in the test compositions is an oil from group III of the API classification, sold by SK under the name Yubase 4. It has the following characteristics:Its kinematic viscosity at 40° C. measured according to the standard ASTM D445 is 19.57 cSt;Its kinematic viscosity measured at 100° C. according to the standard ASTM D445 is 4.23 cSt;Its viscosity index measured according to the standard ASTM D2270 is 122;Its Noack volatility, as a weight percentage, measured according to the standard DIN 51581 is 15;Its flash point in degrees Celsius measured according to the standard ASTM D92 is 230° C.;Its pour point in degrees Celsius measured according to the standard ASTM D97 is −15° C.Polydiol Random Copolymer A-1a (According to 61.1.2) This copolymer comprises 10 mol % of monomer bearing a diol function and 24 mol % of styrene monomer. The mean side chain length is 13.5 carbon atoms. Its number-average molar mass is 53 000 g/mol. Its polydispersity index is 1.19. Its number-average degree of polymerization (DPn) is 253. The number-average molar mass and the polydispersity index are measured by size exclusion chromatography measurement using poly(methyl methacrylate) calibration. This copolymer is obtained by performing the protocol described in section 1.1.2.1 above. Polydiol Random Copolymer A-1c (According to 61.1.2) This copolymer comprises 9 mol % of monomer bearing a diol function and 26 mol % of styrene monomer. The mean side chain length is 13.5 carbon atoms. Its number-average molar mass is 154 000 g/mol. Its polydispersity index is 1.23. Its number-average degree of polymerization (DPn) is 893. The number-average molar mass and the polydispersity index are measured by size exclusion chromatography measurement using poly(methyl methacrylate) calibration. This copolymer is obtained by performing the protocol described in section 1.1.2.2 above. Boronic Ester Random Copolymer A-2: This copolymer comprises 5 mol % of monomers bearing boronic ester functions. The mean side chain length is 12 carbon atoms. Its number-average molar mass is 39 000 g/mol. Its polydispersity index is 1.41. Its number-average degree of polymerization (DPn) is 192. Its number-average molar mass and the polydispersity index are measured by size exclusion chromatography measurement using poly(methyl methacrylate) calibration. This copolymer is obtained by performing the protocol described in section 2 above.Polydiol Random Copolymer A-1b (According to 1.1.2) This copolymer comprises 10 mol % of monomer bearing a diol (and does not contain any styrene). The mean side chain length is 13.8 carbon atoms. Its number-average molar mass is 56 700 g/mol. Its polydispersity index is 1.21. Its number-average degree of polymerization (DPn) is 253. The number-average molar mass and the polydispersity index are measured by size exclusion chromatography measurement using poly(methyl methacrylate) calibration. This copolymer is obtained by performing the protocol described in section 1.1.3 above. 3.2 Formulation of Compositions for the Viscosity Study Composition A (Comparative) is Obtained in the Following Manner: It contains a solution containing 4.20% by mass of a polymethacrylate polymer in a lubricant base oil from group III of the API classification. The polymer has a number-average molar mass (Mn) equal to 106 000 g/mol, a polydispersity index (Ip) equal to 3.06, a number-average degree of polymerization of 466 and the mean side chain length is 14 carbon atoms. This polymethacrylate is used as viscosity-index-enhancing additive. 4.95 g of a formulation with a mass concentration of 42% of this polymethacrylate in a group III base oil and 44.6 g of group III base oil are placed in a flask. The solution thus obtained is stirred at 90° C. until the polymethacrylate has fully dissolved. A solution containing 4.20% by mass of this polymethacrylate is obtained. This composition is used as reference for the viscosity study. It represents the rheological behavior of commercial lubricant compositions. Composition B (Comparative) is Obtained in the Following Manner: 6.52 g of polydiol copolymer A-1a (according to § 1.1.1) and 58.68 g of a group III base oil are placed in a flask. The solution thus obtained is stirred at room temperature until the polydiol A-1a has fully dissolved. A solution containing 10% by mass of polydiol copolymer A-1a is obtained. 4.20 g of this solution of polydiol A-1a at 10% by mass in the group III base oil are mixed with 2.80 g of this same base oil. The solution thus obtained is stirred at room temperature for 5 minutes. A solution containing 6% by mass of polydiol copolymer A-1a is obtained. Composition C (Comparative) is Obtained in the Following Manner: 7.33 g of poly(boronic ester) copolymer A-2 and 65.97 g of a group III base oil are placed in a flask. The solution thus obtained is stirred at room temperature until the poly(boronic ester) A-2 has fully dissolved. A solution containing 10% by mass of poly(boronic ester) copolymer A-2 is obtained. 4.20 g of this solution of poly(boronic ester) A-2 at 10% by mass in the group III base oil are mixed with 2.80 g of this same base oil. The solution thus obtained is stirred at room temperature for 5 minutes. A solution containing 6% by mass of poly(boronic ester) copolymer A-2 is obtained. Composition D (Comparative) is Obtained in the Following Manner. 4.10 g of polydiol copolymer A-1b (according to § 1.1.2) and 36.90 g of a group III base oil are placed in a flask. The solution thus obtained is stirred at room temperature until the polydiol A-1b has fully dissolved. A solution containing 10% by mass of polydiol copolymer A-1b is obtained. 4.20 g of this solution of polydiol A-1b at 10% by mass in the group III base oil are mixed with 2.80 g of this same base oil. The solution thus obtained is stirred at room temperature for 5 minutes. A solution containing 6% by mass of polydiol copolymer A-1b is obtained. Composition E (According to the Invention) is Obtained in the Following Manner: 2.80 g of the solution containing 10% by mass of polydiol A-1a prepared previously and 1.4 g of group III base oil are placed in a flask. 2.80 g of the solution containing 10% by mass of poly(boronic ester) A-2 prepared previously are added to this solution. The solution thus obtained is stirred at room temperature for 5 minutes. A solution containing 4% by mass of polydiol copolymer A-1a and 4% by mass of poly(boronic ester) copolymer A-2 is obtained. Composition F (Comparative) is Obtained in the Following Manner 2.80 g of the solution containing 10% by mass of polydiol A-1b prepared previously and 1.40 g of group III base oil are placed in a flask. 2.80 g of the solution containing 10% by mass of poly(boronic ester) A-2 prepared previously are added to this solution. The solution thus obtained is stirred at room temperature for 5 minutes. A solution containing 4% by mass of polydiol copolymer A-1b and 4% by mass of poly(boronic ester) copolymer A-2 is obtained. Composition G (According to the Invention) is Obtained in the Following Manner. 1.05 g of the solution containing 10% by mass of polydiol A-1c prepared previously and 5.25 g of group III base oil are placed in a flask. 0.70 g of the solution containing 10% by mass of poly(boronic ester) A-2 prepared previously are added to this solution. The solution thus obtained is stirred at room temperature for 5 minutes. A solution containing 1.5% by mass of polydiol copolymer A-1c and 1% by mass of poly(boronic ester) copolymer A-2 is obtained. 3.3 Apparatus and Protocol for Measuring the Viscosity The rheological studies were performed using a Couette MCR 501 controlled stress rheometer from the company Anton Paar. In the case of the polymer formulations which do not form gels in a group III base oil over the temperature range of the study (compositions A to F), the rheology measurements were performed using a cylindrical geometry of reference DG 26.7. The viscosity was measured as a function of the shear rate for a temperature range extending from 10° C. to 150′C. For each temperature, the viscosity of the system was measured as a function of the shear rate from 1 to 100 s−1. The measurements of the viscosity as a function of the shear rate at T=10° C., 50° C., 70° C., 110° C., 130° C. and 150° C. were performed (going from 10° C. to 150° C.). A mean viscosity was then calculated for each temperature using the measurement points located on the same plateau. The relative viscosity calculated according to the following formula (ηrelative=ηsolutionηbaseoil) was chosen to represent the change in viscosity of the system as a function of the temperature, since this magnitude directly reflects the compensation for the natural viscosity loss of a group III base oil of the polymer systems studied. 3.4 Rheological Results Obtained The relative viscosity of compositions A, E and F was studied for a temperature range extending from 10° to 150° C. whereas that of compositions B, C and D was studied between 10° C. and 110° C. In the cases where the viscosity was not perfectly constant with the shear rate, the viscosity of the solution was calculated by taking the mean of the viscosities obtained on all the shear rates. The relative viscosity of these compositions is illustrated inFIGS.6,7and8. Copolymer A-1a alone in composition B does not allow a significant compensation for the natural viscosity loss of the group III base oil (FIG.6). This is likewise the case for the poly(boronic ester) copolymer A-2 when it is used alone in composition C or else for the polydiol copolymer A-1b when it is used alone in composition D (FIG.6). When the polydiol random copolymer A-1a and the poly(boronic ester) copolymer A-2 are present together in the same lubricant composition (composition E), compensation for the natural viscosity loss of the group III base oil which is greater than that which results from the addition of the polymer methacrylate polymer to the group III base oil (composition A) at 150° C. is observed (FIG.7). At the same time, composition E shows a lower relative viscosity than composition A (reference polymethacrylate) at 10° C. (FIG.7). The relative viscosity values are also represented for three successive cycles of heating-cooling between 10′C to 150° C. (E-1, E-2 and E-3). These values change slightly in the course of the 3 cycles, but still give an increase in the relative viscosity of about 1 between 10° C. and 150° C., reflecting the great compensation for the natural viscosity loss of the group III base oil over this temperature range (FIG.8). When the polydiol random copolymer A-1b and the poly(boronic ester) copolymer A-2 are present together in the same lubricant composition (composition F), a slight compensation for the natural viscosity loss of the group III base oil is observed (FIG.7). This compensation is lower than in the case of composition E. The relative viscosity values are also represented for the first three successive cycles from 10° C. to 150° C. (F-1, F-2 and F-3). During the first cycle, composition F gives relative viscosity values that are virtually identical to those of composition E from 10° C. to 110° C. On the other hand, when the temperature reaches 130° C. and then 150° C. during the first heating cycle, the relative viscosity drops substantially (FIG.8). The next two cycles (F-2, F-3) give comparable relative viscosities and an increase in the relative viscosity when the temperature increases (FIG.8). For these two cycles an increase in relative viscosity of less than 0.5 is reached between 10° C. and 150° C. This result shows that composition F appears to be degraded after its first passage beyond 110° C. The effect of this degradation is to reduce the compensation for the natural viscosity loss of the oil obtained with this composition (up to 110° C. in the first cycle). The change in composition of the polydiol (addition of styrene and removal of the RAFT chain end) thus made it possible to maintain the rheological properties of composition E for several cycles above 110° C. The relative viscosity values for formulation G are represented for three successive cycles of heating-cooling between 10° C. to 150° C. (G-1, G-2 and G-3) inFIG.11. These values change slightly in the course of the three cycles, but still give an increase in the relative viscosity of about 0.55 between 10° C. and 150° C., reflecting the great compensation for the natural viscosity loss of the group III base oil over this temperature range. Furthermore, irrespective of the cycle, the composition gives a relative viscosity ranging from about 1.3 at 10° C. to about 1.85 at 150° C. Composition G thus appears to be more stable than composition F for this study. 4. Thermogravimetric Studies 4.1 Apparatus and Protocols for Thermogravimetric Analysis (TGA) The thermogravimetric studies were performed using a TG 209 F1 thermogravimetric analyzer from the company Netzsch. The experiments were performed under a stream of 20 mL/minute of dinitrogen. 15 to 30 mg of polymers are placed in an aluminum crucible before each analysis. The isotherms were applied for 20 hours at 150° C., whereas the ramps were applied from 25° C. to 600° C. at a heating rate of 10° C./minute. 4.2 TGA Results The thermal stability of the polydiol A-1a, of the polydiol A-1c and of the polydiol A-1b was studied under a dinitrogen atmosphere via two different protocols. Firstly, the polydiols were subjected to a temperature ramp from 25° C. to 600° C. so as to observe the change in mass of the samples as a function of the temperature (FIG.9). Secondly, the polydiols were subjected to an isotherm at 150° C. for 20 hours so as to the change in mass as a function of time under these conditions (FIG.10). During the temperature ramp from 25° C. to 600° C., the polydiol A-1a loses 1% of its mass at 290° C. and 5% of its mass at 335° C. and the polydiol A-1c loses 1% of its mass at 240° C. and 5% of its mass at 310° C., whereas the polydiol A-1b loses 1% of its mass at 220° C. and 5% of its mass at 290° C. (FIG.9). Above 320° C., the three polydiols show a very rapid loss of mass leading to total degradation of these polymers at 450° C. Initiation of the loss of mass thus takes place at lower temperatures for the polydiol A-1b than for the polydiol A-1a and the polydiol A-1c. Even during the isotherm of 20 hours at 150° C., the polydiol A-1a loses 0.8% of its mass after 2 hours and 1.1% of its mass after 20 hours and the polydiol A-1c loses 0.4% of its mass after 2 hours and 0.4% of its mass after 20 hours, whereas the polydiol A-1b loses 1% of its mass after 2 hours and 3.3% of its mass after 20 hours (FIG.10). It is probable that the loss of mass which takes place at the start of the isotherm is attributable to the loss of water which has been adsorbed onto the polymers. This result shows that the polydiol A-1b has a greater loss of mass than the polydiol A-1a and the polydiol A-1c during this isotherm. Furthermore, a constant rate of loss of mass appears to become established after 4 hours of isotherm for the polydiol A-1a and after 7 hours of isotherm for the polydiol A-1b. The polydiol A-1a has a loss of mass of 0.009%/hour, whereas the polydiol A-1b reaches a rate of 0.037%/hour (FIG.10). The polydiol A-1c does not show any significant loss of mass after 3 hours of isotherm at 150° C. This measurement indicates that the polydiol A-1 b degrades more rapidly than the polydiol A-1a and the polydiol A-1c under these conditions. | 25,421 |
11859151 | EXAMPLES The invention will now be described in further detail by way of the following examples, wherein the abbreviations have the usual meaning in the art, the temperatures are indicated in degrees centigrade (° C.); the NMR spectral data were recorded in CDCl3(if not stated otherwise) with a 360 or 400 MHz machine for1H and13C, the chemical shifts δ are indicated in ppm with respect to TMS as standard, the coupling constants J are expressed in Hz. Example 1 Synthesis of compounds of formula (I) 2-(hex-5-en-1-yl)cyclopentan-1-one Step 1: ethyl 1-(hex-5-en-1-yl)-2-oxocyclopentane-1-carboxylate: To a solution of ethyl 2-oxocyclopentane-1-carboxylate (56 mL, 375 mmol, 1 equiv.) in acetone (871 mL) at r.t. was rapidly added potassium carbonate (118 g, 845 mmol, 2.25 equiv.) and potassium iodide (20 g, 120 mmol, 0.32 equiv.). After stirring for 10 min, a solution of 6-bromohex-1-ene (51 mL, 381 mmol, 1.01 equiv.) in acetone (232 ml) was added and the reaction was refluxed for 19 h. Diethyl ether (900 mL) was added, the mixture was filtered on a Celite pad and the solvent was evaporated. The residue was diluted with ether, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo to afford ethyl 1-(hex-5-en-1-yl)-2-oxocyclopentane-1-carboxylate as an oil (93.3 g, 91% purity, 95% yield). 1H NMR: 1.25 (t, J=7.1 Hz, 3H), 1.27-1.42 (m, 4H), 1.53-1.59 (m, 1H), 1.86-2.07 (m, 6H), 2.21-2.28 (m, 1H), 2.37-2.44 (m, 1H), 2.50-2.56 (m, 1H), 4.11-4.21 (m, 2H), 4.92-5.01 (m, 2H), 5.73-5.82 (m, 1H). 13C NMR: 215.0 (s), 171.1 (s), 138.6 (d), 114.5 (t), 61.3 (t), 60.5 (s), 38.0 (t), 33.7 (t), 33.4 (t), 32.7 (t), 29.1 (t), 24.3 (t), 19.6 (t), 14.1 (q). Step 2: 2-(hex-5-en-)-yl)cyclopentan-1-one: To a solution of the keto-ester of step 1 (93.3 g, 91% purity, 356 mmol, 1 equiv.) in methanol (860 mL) at r.t. was added a 6 M aqueous HCl solution (428 mL, 2.57 mol, 7.2 equiv.) dropwise. The reaction was refluxed for 6 days. Diethyl ether was added and the aqueous layer was extracted with ether twice. The combined organic extracts were washed sequentially with water, a saturated solution of sodium bicarbonate, water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by distillation on a Vigreux column (92-93° C., 1.5 mbar) to afford the desired ketone as an oil (42.0 g, 71% yield). 1H NMR: 1.21-1.44 (m, 5H), 1.46-1.56 (m, 1H), 1.71-1.83 (m, 2H), 1.96-2.15 (m, 5H), 2.18-2.33 (m, 2H), 4.91-5.02 (m, 2H), 5.74-5.85 (m, 1H). 13C NMR: 221.5 (s), 138.9 (d), 114.4 (t), 49.1 (d), 38.2 (t), 33.6 (t), 29.6 (t), 29.5 (t), 28.9 (t), 27.0 (t), 20.8 (t).2-(hept-6-en-1-yl)cyclopentan-1-one Step 1: ethyl 1-(hept-6-en-1-yl)-2-oxocyclopentane-1-carboxylate: To a solution of ethyl 2-oxocyclopentane-1-carboxylate (3.56 mL, 26.7 mmol, 1 equiv.) in acetone (62 mL) at r.t. was rapidly added potassium carbonate (8.43 g, 60.4 mmol, 2.25 equiv.) and potassium iodide (1.43 g, 8.55 mmol, 0.32 equiv.). After stirring for 10 min, a solution of 7-bromohept-1-ene (4.23 mL, 26.9 mmol, 1.01 equiv.) in acetone (17 ml) was added and the reaction was refluxed for 23 h. Diethyl ether (100 mL) was added, the mixture was filtered on a Celite pad and the solvent was evaporated. The residue was diluted with ether, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo to afford ethyl 1-(hept-6-en-1-yl)-2-oxocyclopentane-1-carboxylate as an oil (6.37 g, 88% purity, 92% yield). Step 2: 2-(hept-6-en-1-yl)cyclopentan-1-one: To a solution of the keto-ester of step 1 (6.64 g, 88% purity, 24.5 mmol, 1 equiv.) in methanol (59 mL) at r.t. was added a 6 M aqueous HCl solution (29.4 mL, 177 mol, 7.2 equiv.) dropwise. The reaction was refluxed for 5 days. Diethyl ether was added and the aqueous layer was extracted with ether twice. The combined organic extracts were washed sequentially with water, a saturated solution of sodium bicarbonate, water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (Heptane/AcOEt 95:5) and bulb-to-bulb distillation (115° C., 0.8-0.9 mbar) to afford the desired ketone as an oil (3.06 g, 68 % yield). 1H NMR (CDCl3, 400 MHz): 1.22-1.41 (m, 7H), 1.48-1.55 (m, 1H), 1.73-1.81 (m, 2H), 1.97-2.13 (m, 5H), 2.19-2.23 (s, 1H), 2.27-2.31 (m, 1H), 4.95-5.00 (m, 2H), 5.76-5.83 (m, 1H). 13C NMR (CDCl3, 100 MHz): 221.6 (s), 139.0 (d), 114.3 (t), 49.1 (d), 38.2 (t), 33.7 (t), 29.7 (t), 29.6 (t), 29.1 (t), 28.7 (t), 27.4 (t), 20.8 (t). Example 2 Preparation of a Perfuming Composition A perfuming composition was prepared by admixing the following ingredients: IngredientParts by weightAmyl acetate40Ethyl acetate20Hexyl acetate200Isobutyl acetate60Benzyl acetate80Geranyl acetate160Linalyl acetate400(Z)-3-hexen-1-ol acetate60Styrallyl acetate8010%* Methylbutyric acid40Benzoic aldehyde4010%* methyl anthranilate40Carbinol butyrate80(Z)-3-hexen-1-ol butyrate20Ethyl caproate40Hexyl caproate80(Z)-3-hexen-1-ol caproate20Lemon160Maltol8010%* Damascenon40γ-n-decalactone8001%* Dimethylsulfid40Dodecalactone800Ethylvanillin1003-(4-Methoxyphenyl)-2-methylpropanal80Gamma hexalactone40Gamma jasmolactone10Gamma nonalactone40Gamma undecalactone600Geraniol400Habanolide ®1800Hedione ®2300Helvetolide ®380Ethyl isobutyrate40Limonene160Linalol1600Menthol1010%* Methylisopropylthiazol20Ocimene4010%* Orange aldehyde801%*, (3Z)-1-(2-butenyloxy)-3-Hexene80Linalyl ether8010%* (2E,6Z)-2,6-nonadienal40(Z)-3-hexen-1-ol dist160Hexyl salicylate800(Z)-3-hexen-1-ol salicylate100Terpineol600Verdox480Beta ionone2009920*in dipropyleneglycol1pentadecenolide; origin: Firmenich SA, Geneva, Switzerland2Methyl dihydrojasmonate; origin: Firmenich SA, Geneva, Switzerland3(1S,1′R)-2-[1-(3′,3′-dimethyl-1′-cyclohexyl)ethoxy]-2-methylpropyl propanoate; origin: Firmenich SA, Geneva, Switzerland42-tert-butyl-1-cyclohexyl acetate; origin: International Flavors & Fragrances, USA The addition of 80 parts by weight of 2-(hex-5-en-1-yl)cyclopentan-1-one to the above-described composition imparted to the latter a fruity-exotic (mango and passionfruit like) connotation with a slightly acidulous facet which makes the fragrance more juicy and natural. When, instead of the invention's compound, 2-heptylcyclopentan-1-one (Fleuramone) was added, there is little effect observed in the above-described composition. The above-described composition becomes just slightly more floral (jasmine like). When, instead of the invention's compound, 2-pentylcyclopentan-1-one (Delphone) was added, the perfumery composition becomes much greener (celery like) and more floral (jasmine like). Example 3 Preparation of a Liquid Detergent Comprising the Invention's Compound TABLE 1Composition of the liquid detergent formulationConcentrationIngredients[wt %]Sodium C14-17 Alkyl Sec Sulfonate1)7Fatty acids, C12-18 and C18-unsaturated2)7.5C12/14 fatty alcohol polyglycol ether with177 mol EO3)Triethanolamine7.5Propylene Glycol11Citric acid6.5Potassium Hydroxyde9.5Properase L4)0.2Puradax EG L4)0.2Purastar ST L4)0.2Acrylates/Steareth-20 Methacrylate6structuring Crosspolymer5)Deionized Water27.41)Hostapur SAS 60; Origin: Clariant2)Edenor K 12-18; Origin: Cognis3)Genapol LA 070; Origin: Clariant4)Origin: Genencor International5)Aculyn 88; Origin: Dow Chemical The liquid detergent was prepared by adding 1.5% by weight, relative to the total weight of the liquid detergent, of the invention's composition of example 2 into the unperfumed liquid detergent formulation of Table 1 under gentle shaking. Example 4 Preparation of a Fabric Softener Comprising the Invention's Compound TABLE 2Composition of the softener formulationIngredientConcentration [wt %]Methyl bis[ethyl (tallowate)]-2-12.20hydroxyethyl ammonium methylsulfate1)1,2-benzisothiazolin-3-one2)0.04CaCl2(10% aqueous solution)0.40Water87.361)Stepantex VL90 A Diester Quat; Origin: Stepan2)Proxel GXL; Origin: Arch The softener was prepared by weighting Methyl bis[ethyl (tallowate)]-2-hydroxyethyl ammonium methyl sulfate which was heated at 65° C. Then Water and 1,2-benzisothiazolin-3-one were placed in the reactor and were heated at 65° C. under stirring. To the above mixture was added Methyl bis[ethyl (tallowate)]-2-hydroxyethyl ammonium methyl sulfate. The mixture was stirred 15 minuted and CaCl2was added. Then 0.5 to 2% by weight, relative to the total weight of the softener, of the invention's composition of example 2 was added. The mixture was stirred 15 minutes and was cooled down to room temperature under stirring (viscosity measure: result 35+/−5 mPas. (shear rate 106 sec-1)). Example 5 Preparation of a Transparent Isotropic Shampoo Comprising the Invention's Composition TABLE 3Composition of the transparent isotropic shampoo formulationConcentrationPhasesIngredients[wt %]AWater deionized44.4Polyquaternium-101)0.3Glycerin 85%2)1DMDM Hydantoin3)0.2BSodium Laureth Sulfate4)28Cocamidopropyl Betaine5)3.2Disodium Cocoamphodiacetate6)4Ethoxy (20) Stearyl Alcohol6)1CSodium Laureth Sulfate4)3Glyceryl Laureate7)0.2DWater deionized1Sodium Methylparaben8)0.1ESodium Chloride 10% aqueous sol.15Citric acid 10% aqueous sol. till pH 5.5-6q.s.1)Ucare Polymer JR-400, Origin: Noveon2)Origin: Schweizerhall3)Glydant, Origin: Lonza4)Texapon NSO IS, Origin: Cognis5)Tego Betain F 50, Origin: Evonik6)Amphotensid GB 2009, Origin: Zschimmer & Schwarz7)Monomuls 90 L-12, Origin: Gruenau8)Nipagin Monosodium, Origin: NIPA The shampoo was prepared by dispersed in water Polyquatemium-10. The remaining ingredients of phase A were mixed separately by addition of one after the other while mixing well after each adjunction. This pre-mix was added to the Polyquaternium-10 dispersion and mixed for another 5 min. Then, the premixed phase B and the premixed Phase C were added (Monomuls 90L-12 was heated to melt in Texapon NSO IS) while agitating. Phase D and Phase E were added while agitating. PH was adjusted with citric acid solution till pH: 5.5-6.0 leading to an unperfumed shampoo formulae. The perfumed shampoo was prepared by adding 0.4 to 0.8% by weight, relative to the total weight of the shampoo, of the invention's composition of example 2 into the unperfumed shampoo formulation of Table 3 under gentle shaking. Example 6 Preparation of a Structured Shower Gel Comprising the Invention's Composition TABLE 4Composition of the shower gel formulationIngredientsAmount (% wt)WATER deionised49.350Tetrasodium EDTA1)0.050Acrylates Copolymer2)6.000Sodium C12-C15 Pareth Sulfate3)35.000Sodium Hydroxide 20% aqueous solution1.000Cocamidopropyl Betaine4)8.000Methylchloroisothiazolinone and0.100Methylisothiazolinone5)Citric Acid (40%)0.5001)EDETA B POWDER; trademark and origin: BASF2)CARBOPOL AQUA SF-1 POLYMER; trademark and origin: NOVEON3)ZETESOL AO 328 U; trademark and origin: ZSCHIMMER & SCHWARZ4)TEGO-BETAIN F 50; trademark and origin: GOLDSCHMIDT5)KATHON CG; trademark and origin: ROHM & HASS The shower gel was prepared by adding 0.5 to 1.5% by weight, relative to the total weight of the shower gel, of the invention's composition of example 2 into the unperfumed shower gel formulation of Table 4 under gentle shaking. Example 7 Preparation of a Transparent Shower Gel Comprising the Invention's Composition TABLE 5Composition of the transparent shower gel formulationIngredientsConcentration (% wt)WATER deionized52.40Tetrasodium EDTA1)0.10Sodium Benzoate0.50Propylene Glycol2.00Sodium C12-C15 Pareth Sulfate2)35.00Cocamidopropyl Betaine3)8.00Polyquaternium-74)0.20Citric Acid (40%)1.00Sodium Chloride0.801)EDETA B POWDER; trademark and origin: BASF2)ZETESOL AO 328 U; trademark and origin: ZSCHIMMER & SCHWARZ3)TEGO-BETAIN F 50; trademark and origin: GOLDSCHMIDT4)MERQUAT 550; trademark and origin: LUBRIZOL The transparent shower gel was prepared by adding 0.5 to 1.5% by weight, relative to the total weight of the shower gel, of the invention's composition of example 2 into the unperfumed shower gel formulation of Table 5 under gentle shaking. Example 8 Preparation of a Milky Shower Gel Comprising the Invention's Composition TABLE 6Composition of the milky shower gel formulationConcentrationIngredients(% wt)WATER deionized50.950Tetrasodium EDTA1)0.050Sodium Benzoate0.500Glycerin 86%3.500Sodium Laureth Sulfate2)27.000Polyquaternium-73)1.000Coco-Betaine4)6.000PEG-120 Methyl Glucose trioleate5)1.000Citric Acid (40%)1.000Glycol Distearate & Laureth-4 &3.000Cocamidopropyl Betaine6)Sodium Chloride 20%5.000PEG-40 Hydrogenated Castor Oil7)1.0001)EDETA B POWDER; trademark and origin: BASF2)Texapon NSO IS; trademark and origin: COGNIS3)MERQUAT 550; trademark and origin: LUBRIZOL4)DEHYTON AB-30; trademark and origin: COGNIS5)GLUCAMATE LT; trademark and origin: LUBRIZOL6)EUPERLAN PK 3000 AM; trademark and origin: COGNIS7)CREMOPHOR RH 40; trademark and origin: BASF The transparent shower gel was prepared by adding 0.5 to 1.5% by weight, relative to the total weight of the shower gel, of the invention's composition of example 2 into the unperfumed shower gel formulation of Table 6 under gentle shaking. Example 9 Preparation of a Pearly Shampoo Comprising the Invention's Composition TABLE 7Composition of the pearly isotropic shampoo formulationConcentrationPhasesIngredients(% wt)AWater deionized45.97Tetrasodium EDTA1)0.05Guar Hydroxypropyltrimonium Chloride2)0.05Polyquaternium-103)0.075BNaOH 10% aqueous sol.0.3CAmmonium Lauryl Sulfate4)34Ammonium Laureth Sulfate5)9.25Cocamidopropyl Betaine6)2Dimethicone (&) C12-13 Pareth-4 (&)2.5C12-13 Pareth-23 (&) Salicylic Acid7)DCetyl Alcohol8)1.2Cocamide MEA9)1.5Glycol Distearate10)2EMethylchloroisothiazolinone &0.1Methylisothiazolinone11)D-Panthenol 75%12)0.1Water deionized0.3FSodium Chloride 25% aqueous sol.0.61)EDETA B Powder, Origin: BASF2)Jaguar C14 S, Origin: Rhodia3)Ucare Polymer JR-400, Origin: Noveon4)Sulfetal LA B-E, Origin: Zschimmer & Schwarz5)Zetesol LA, Origin: Zschimmer & Schwarz6)Tego Betain F 50, Origin: Evonik7)Xiameter MEM-1691, Origin: Dow Corning8)Lanette 16, Origin: BASF9)Comperlan 100, Origin: Cognis10)Cutina AGS, Origin: Cognis11)Kathon CG, Origin: Rohm & Haas12)D-Panthenol, Origin: Roche The shampoo was prepared by dispersed in water and Tetrasodium EDTA, Guar Hydroxypropyltrimonium Chloride and Polyquaternium-10. NaOH 10% solution (Phase B) was added once Phase A was homogeneous. Then, the premixed Phase C was added. and mixture was heated to 75° C. Phase D ingredients were added and mixed till homogeneous. The mixture was cooled down. At 45° C., Phase E ingredients were added while mixing. Final viscosity was adjusted with 25% NaCl solution and pH of 5.5-6 was adjusted with 10% NaOH solution. The perfumed pearly shampoo was prepared by adding 0.4 to 0.8% by weight, relative to the total weight of the shampoo, of the invention's composition of example 2 into the unperfumed shampoo formulation of Table 7 under gentle shaking. Example 10 Preparation of a Structured Shower Gel Comprising the Invention's Composition TABLE 8Composition of the milky shower gel formulationIngredientsAmount (% wt)WATER deionised49.350Tetrasodium EDTA1)0.050Acrylates Copolymer2)6.000Sodium C12-C15 Pareth Sulfate3)35.000Sodium Hydroxide 20% aqueous solution1.000Cocamidopropyl Betaine4)8.000Methylchloroisothiazolinone and0.100Methylisothiazolinone5)Citric Acid (40%)0.5006)EDETA B POWDER; trademark and origin: BASF7)CARBOPOL AQUA SF-1 POLYMER; trademark and origin: NOVEON8)ZETESOL AO 328 U; trademark and origin: ZSCHIMMER & SCHWARZ9)TEGO-BETAIN F 50; trademark and origin: GOLDSCHMIDT10)KATHON CG; tradeark and origin: ROHM & HASS The transparent shower gel was prepared by adding 0.5 to 1.5% by weight, relative to the total weight of the shower gel, of the invention's composition of example 2 into the unperfumed shower gel formulation of Table 8 under gentle shaking. Example 11 Preparation of a Eau De Toilette Comprising the Invention's Compound The eau de toilette was prepared by adding 5 to 20% by weight, relative to the total weight of the eau de toilette, of the invention's composition of example 2 into ethanol under gentle shaking. | 16,171 |
11859152 | DETAILED DESCRIPTION OF THE INVENTION Mode for Carrying Out the Invention Embodiments of the present invention are described below in detail. Definitions Unless otherwise specified in the present specification, the definitions and examples described in this paragraph are followed. The singular form includes the plural form and “one” or “that” means “at least one”. An element of a concept can be expressed by a plurality of species, and when the amount (for example, mass % or mol %) is described, it means sum of the plurality of species. “And/or” includes a combination of all elements and also includes single use of the element. When a numerical range is indicated using “to” or “-”, it includes both endpoints and units thereof are common. For example, 5 to 25 mol % means 5 mol % or more and 25 mol % or less. The descriptions such as “Cx-y”, “Cx-Cy” and “Cx” mean the number of carbons in a molecule or substituent. For example, C1-6alkyl means an alkyl chain having 1 or more and 6 or less carbons (methyl, ethyl, propyl, butyl, pentyl, hexyl etc.). When polymer has a plural types of repeating units, these repeating units copolymerize. These copolymerization may be any of alternating copolymerization, random copolymerization, block copolymerization, graft copolymerization, or a mixture thereof. When polymer or resin is represented by a structural formula, n, m or the like that is attached next to parentheses indicate the number of repetitions. Celsius is used as the temperature unit. For example, 20 degrees means 20 degrees Celsius. The additive refers to a compound itself having a function thereof (for example, in the case of a base generator, the compound itself that generates a base). An aspect in which the compound is dissolved or dispersed in a solvent and added to the composition is also possible. As one embodiment of the present invention, it is preferable that such a solvent is contained in the composition according to the present invention as the solvent (C) or an other component. Substrate Pattern Filling Composition The substrate pattern filling composition according to the present invention comprises a certain first solute (A), a second solute (B) and a solvent (C). Furthermore, it comprises other components, if needed. In the present invention, the substrate pattern means a pattern formed by processing a substrate, and does not include a pattern formed from an other film or layer on the substrate. For example, an aspect in which a resist pattern is formed on a bare wafer is not included in the substrate pattern of the present invention. The substrate pattern filling composition means a composition that is filled (being overflowed is accepted) between the patterns of a substrate pattern, and an aspect in which a film is formed after that is more preferable. Each component is described below. First Solute (A) and Second Solute (B) The first solute (A) comprises at least any one of an amino group, a hydroxy group or a carbonyl group. Provided that the first solute has at most one hydroxy group per molecule. The second solute (B) comprises at least any one of an amino group, a hydroxy group or a carbonyl group. Provided that the second solute has at most one hydroxy group per molecule. The first solute (A) and the second solute (B) are different substances. Preferably, the first solute (A) and/or the second solute (B) each independently comprises a 5-membered or 6-membered hydrocarbon ring or heterocyclic ring. As an aspect of the present invention, when the film formed from the substrate pattern filling composition is later vaporized, it is preferable that the second solute (B) vaporizes prior to the first solute (A). As an aspect of the present invention, the substrate pattern filling composition according to the present invention is filled in a substrate pattern to form a film. It is preferable that the solvent (C) is vaporized first, the solid components form a film, and then the solid components are vaporized to remove the film. It is more preferable that the first solute (A) and the second solute (B) are vaporized independently as the solid components. A preferred aspect of the vaporization is sublimation. Preferably, the sublimation is that a portion of a solid component changes directly from the solid phase to the gas phase. More preferably, the sublimation is that substantially all of a solid component changes directly from the solid phase to the gas phase. Further, as another embodiment, the first solute (A) and/or the second solute (B) are substances having a sublimation point that each changes from the solid phase to the gas phase at room temperature without passing through the liquid phase. In another preferable embodiment, the first solute (A) and/or the second solute (B) can be a substance that changes from the solid phase to the gas phase through the liquid phase when heated at normal pressure, has a melting point, and gradually sublimates below the melting point. Preferably, when removing the film formed from the substrate pattern filling composition, heating and/or pressure reduction are not performed. The heating referred to here is more preferably 70° C. or higher, further preferably 80° C. or higher, further more preferably 90° C. or higher, and the upper limit is more preferably 200° C. or lower, further preferably 170° C. or lower, further more preferably 150° C. or lower. The reduced pressure referred to here is more preferably 80 kPa or lower, further preferably 50 kPa or less, further more preferably 20 kPa or lower, and the lower limit is more preferably 1 kPa or higher, further preferably 5 kPa or higher, further more preferably 10 kPa or higher. Further, it is also an advantage of the present invention that when the above-described film is removed in the present invention, the cooling step as described in Patent Document 1 is not essential. As another aspect of the present invention, it is also an advantage of the present invention that gas blowing is not essential for removal of the film. The gas referred here includes air, Ar and nitrogen gas, and for example, using a gas of which humidity and oxygen concentration are reduced is included. In order to make the substrate pattern clean, from the viewpoint of reducing the amount remaining in the substrate pattern, it is desirable that the first solute (A) and the second solute (B) are substances that are easily vaporized. In order to further reduce the remaining amount of solid components having such characteristics, it is also possible to add a heating step. In one embodiment of the present invention, it is possible to heat when the film formed from the substrate pattern filling composition is removed, and the conditions therefor can be 35 to 150° C. (more preferably 35 to 120° C., further preferably 40 to 110° C., and further more preferably 40 to 100° C.) and 10 to 180 seconds (more preferably 10 to 120 seconds, and further preferably 10 to 90 seconds). In one aspect of the present invention, the amino group and/or the carbonyl group in the first solute (A) and/or the second solute (B) are each independently a part of the ring in the hydrocarbon ring or the heterocyclic ring, and the hydroxy group is directly added to the ring in the hydrocarbon ring or the heterocyclic ring. That is, the compound having a carboxyl group does not fall under the first solute (A) and the second solute (B) in this embodiment. Preferably, the first solute (A) and/or the second solute (B) each independently has a cage-shaped steric mother structure. As an example of the compound having the cage-shaped steric structure, 1,4-diazabiccyclo[2.2.2]-octane (hereinafter, DABCO) is included. The advantage is that the bulkiness can be suppressed as compared with its molecular weight. As another aspect, an aspect in which each independently in the first solute (A) and/or the second solute (B) the amino group is directly added to the ring is also suitable. For example, 1-adamantanamine has a cage-shaped steric mother structure, and the amino group is added not to a part of the ring but directly to the ring. In a preferred aspect of the present invention, the first solute (A) and/or the second solute (B) each independently has 1 to 5 (more preferably 1 to 4, further preferably 2 to 4) amino groups, 1 to 3 (more preferably 1 to 2) carbonyl groups, and/or one hydroxy group per molecule. The amino group also includes an aspect in which bonding hands of a nitrogen atom are used for a double bond as in C═N— (imino group). The number of amino groups is counted by the number of nitrogen atoms present in one molecule. An embodiment having any one kind of an amino group, a carbonyl group and a hydroxy group in one molecule is a preferred aspect of the present invention. As another aspect, it is also preferable to have a carbonyl group and an amino group in one molecule. As one aspect of the present invention, the molecular weight of the first solute (A) and/or the second solute (B) are each independently 80 to 300 (preferably 90 to 200). Although not to be bound by theory, it can be considered that if the molecular weight is too large, energy is required at the time of vaporization, which is not suitable for the method according to the present invention. As a preferred aspect of the present invention, the sum of the mass of the first solute (A) and the mass of the second solute (B) is 1 to 40 mass % (more preferably 1 to 30 mass %, and further preferably 2 to 20 mass %) based on the mass of the substrate pattern filling composition. Although not to be bound by theory, it is considered that if the amount of the solute is too small, film formation becomes difficult and the effect of suppressing the collapse of the substrate pattern is reduced. Preferably, the mass ratio of the first solute(A):the second solute (B) is 99:1 to 1:99 (more preferably 95:5 to 5:95, further preferably 90:10 to 10:90, and further more preferably 80:20 to 20:80). In another preferred aspect of the present invention, the mass ratio of the second solute (B) to the first solute (A) is 0.5 to 20 (more preferably 1 to 20, and further preferably 5 to 20). As one aspect of the present invention, the first solute (A) is represented by the formula (A): wherein, Cy11and Cy12are each independently a saturated or unsaturated hydrocarbon ring or a heterocyclic ring. Preferably both Cy11and Cy12are saturated or unsaturated hydrocarbon rings or heterocyclic rings, and more preferably both Cy11and Cy12are saturated hydrocarbon rings or heterocyclic rings. The heterocyclic ring mentioned here can be a heterocyclic ring resulted by replacing Cn1that forms the ring. Cn1is each carbon, n1 is an integer of 10 to 19 (that is, C10, C11, . . . C19). The remaining bonding hand of Cn1is bonded with H. Cn1can be each independently replaced with —Cn1Rn1—, —Cn1Rn1Rn1′—, —Cn1(OH)—, —Cn1(═O)—, —Nn1H— and/or —Nn1Rn1—. Provided that at least one Cn1is replaced with at least any one of the above. It goes without saying that elements that do not exist are excluded from this proviso. For example, in the case of n11=n12=0, at least any one of C10to C14is replaced. It is preferred that adjacent Cn1are not replaced at the same time. Rn1and Rn1′are each independently C1-5alkyl (preferably C1-4, more preferably C1-3), —NH2and/or C1-5aminoalkyl (preferably C1-4, more preferably C1-3, and further preferably C1), and Rn1and/or Rn1′can be combined with another Rn1. Rn1′and/or Cn1to form a ring. An aspect in which Rn1and Rn1′are combined with another Rn1, Rn1′and/or Cn1to form a ring is preferred. n11, n12and n13are each independently 0 or 1. Preferably, n11=0. Preferably, n12=1. Preferably, n13=1. As one aspect of the present invention, the second solute (B) is represented by the formula (B): The definitions, examples, and descriptions of Cy21, Cy22, Rn2, Rn2, n21, n22and n23are each independently the same as those of Cy11, Cy12, Rn1, Rn1′, n11, n12and n13. The definition, example and description of Cn2are each independently the same as those of Cn1. n2is an integer of 20 to 29 (that is, C20, C21, . . . C29). Examples and descriptions of n2 (20 to 29) each independently correspond to those of n1 (10 to 19). As one aspect of the present invention, the following compound can be represented by the formula (A). In this case, Cy11is a saturated six-membered hydrocarbon ring, and n11=0 and n12=1. C12is replaced with —C12R12R12′—. R12is methyl (C1alkyl) and R12is isopropyl (C3alkyl). R12′is combined with C15to form a ring. C13is replaced with —C13(═O)—, and C14is replaced with —C14(═O)—. The following compound has two carbonyl groups per molecule. As a whole, the following compound has a cage-shaped steric mother structure. As one aspect of the present invention, the following compound can be represented by the formula (B). In this case, Cy21is a saturated six-membered hydrocarbon ring, and n21=0 and n22=1. C20is replaced with —N20R20—, C22is replaced with —N22R22—, and C24is replaced with —N24R24—. R20is aminomethyl (C1), and R22and R24are methyl (C1). R20, R22and R24are bonded to form a ring. The following compound has 4 amino groups per molecule. As a whole, the following compound has a cage-shaped steric mother structure. As one aspect of the present invention, the following compound can be represented by the formula (A). In this case, Cy11is a saturated six-membered hydrocarbon ring (which becomes a heterocyclic ring by the subsequent replacement). n11=0 and n12=1. C12is replaced with —C12R12R12′—, C14is replaced with —C14R14—, and C10is replaced with —C10R10—. R12is —NH2, R12′is ethyl (C2), and R14and R10are methyl (C1). R12, R14and R10are bonded to form a ring. The following compound has one amino group per molecule. As a whole, the following compound has a cage-shaped steric mother structure. As one aspect of the present invention, the following compound can be represented by the formula (B). In this case, Cy21is an unsaturated six-membered hydrocarbon ring (phenyl), and Cy22is a saturated five-membered hydrocarbon ring (which becomes a heterocyclic ring by the subsequent replacement). n21=n22=1 and n23=0. C26is replaced with —C26(═O)—, and C28is replaced with —C28(═O)—. The following compound has two carbonyl groups per molecule. As a whole, the following compound can be planarly described of its structural formula and has no cage-shaped steric mother structure. Although the scope of the present invention is not limited, exemplified embodiments of the first solute (A) and/or the second solute (B) respectively include the followings. That is, these are each independently any of phthalic anhydride, caffeine, melamine, 1,4-benzoquinone, camphor, hexamethylenetetramine, hexahydro-1,3,5-trimethyl-1,3,5-triazine, 1-adamantanol, 1,4-diazabicyclo[2.2.2]octane, borneol, (−)-borneol, (+)-isoborneol, 1,2-cyclohexanedione, 1,3-cyclohexanedione, 1,4-cyclohexanedione, 3-methyl-1,2-cyclopentanedione, (+)-camphorquinone, (−)-camphorquinone, (+)-camphorquinone or 1-adamantaneamine. Although the scope of the present invention is not limited, exemplified embodiments of the first solute (A) and/or the second solute (B) include the followings: The first solute (A) consists of a single type of compound and is not expressed by a plurality of types. For example, an aspect in which phthalic anhydride and caffeine are simultaneously contained in the composition as the first solute (A) is outside the scope of the present invention. In addition, an aspect containing in the composition, phthalic anhydride as the first solute (A) and caffeine as the second solute (B), can be included in the scope of the composition of the present invention. However, the optical isomers among those included in the exemplified embodiments can be used as a mixture. The same applies to the second solute (B) as well as the third solute (D) and the fourth solute (E), which are described later. The first solute (A) and/or the second solute (B) do not exclude being mixed with trace impurities. For example, when the first solute (A) is phthalic anhydride, 2 mass % or less (preferably 1 mass % or less, more preferably 0.1 mass % or less, and further preferably 0.01 mass % or less) of impurities (other than phthalic anhydride) is accepted to be present, based on the total amount of the first solute (A). Solvent (C) The substrate pattern filling composition according to the present invention comprises a solvent (C). It is preferred that the solvent (C) comprises an organic solvent. As one embodiment of the present invention, the solvent (C) has volatility. It is preferred that the solvent (C) is more easily vaporized as compared with water. As one aspect of the present invention, a solvent that is vaporized by spin drying is preferable. For example, the solvent (C) has a boiling point at 1 atm of preferably 50 to 200° C., more preferably 60 to 170° C., and further preferably 70 to 150° C. It is allowable that the solvent (C) contains a small amount of pure water. It is a preferred embodiment of the present invention that no pure water is contained (0 mass %). In the present specification, the pure water is preferably deionized water. As a preferred aspect of the present invention, components (including additives) contained in the substrate pattern filling composition are dissolved in the solvent (C). The substrate pattern filling composition taking this aspect is considered to have good embedding properties and/or film uniformity. Examples of the organic solvent include alcohols such as methanol (MeOH), ethanol (EtOH) and isopropanol (IPA); alkanes such as hexane, heptane and octane; ethers such as ethyl butyl ether, dibutyl ether, and tetrahydrofuran (THF); lactic acid esters such as methyl lactate and ethyl lactate (EL); aromatic hydrocarbons such as benzene, toluene and xylene; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, 2-heptanone, cyclopentanone and cyclohexanone; amides such as N,N-dimethylacetamide and N-methylpyrrolidone; and lactones such as γ-butyrolactone. The above ethers can include, besides the above-described, ethylene glycol monoalkyl ethers such as ethylene glycol monomethyl ether and ethylene glycol monoethyl ether; ethylene glycol monoalkyl ether acetates such as ethylene glycol monomethyl ether acetate and ethylene glycol monoethyl ether acetate; propylene glycol monoalkyl ethers such as propylene glycol monomethyl ether (PGME) and propylene glycol monoethyl ether (PGEE); and propylene glycol monoalkyl ether acetates such as propylene glycol monomethyl ether acetate (PGMEA) and propylene glycol monoethyl ether acetate. These organic solvents can be used alone or in a mixture of any two or more of these. As a preferred aspect, the organic solvent contained in the solvent (C) is selected from MeOH, EtOH, IPA, THF, PGEE, benzene, acetone, methyl isobutyl ketone, cyclopentanone, cyclohexanone, and any combination thereof. The organic solvent contained in the solvent (C) is selected more preferably from MeOH, EtOH, IPA, PGEE, acetone and any combination thereof, and further preferably from MeOH, EtOH, IPA and PGEE. When the organic solvent is used as a combination of two kinds, the volume ratio is preferably 20:80 to 80:20, more preferably 30:70 to 70:30, and further preferably 40:60 to 60:40. As one aspect of the present invention, the mass of the solvent (C) is 30 to 99 mass % (preferably 50 to 95 mass %, more preferably 80 to 95 mass %, further preferably 85 to 95 mass %) based on the mass of the substrate pattern filling composition. In one aspect of the present invention, bpA, bpBand bpC, which are the boiling points under 1 atm respectively of the first solute (A), the second solute (B) and the solvent (C), satisfy bpA>bpB>bpC. Further, vpA, vpBand vpC, which are the saturated vapor pressures at 25° C. under 1 atm respectively of the first solute (A), the second solute (B) and the solvent (C), satisfy vpA<vpB<vpC. Although not to be bound by theory, it is considered that using a composition that satisfies such relations, when the composition is applied to the substrate pattern, the solvent (C) is volatilized to form a film made of solid components, and then the second solute (B) and the first solute (A) are vaporized in this order. In addition, these state changes mean the tendency as a whole, and it is not necessary to be completely separated and a part thereof can be overlapped. An aspect in which the substance first vaporized takes heat of vaporization and this enable stepwise vaporization as a whole is also possible. It is considered that the second solute (B) is first vaporized from inside the film, thereby avoiding rapid disappearance of the film and reducing interaction with the substrate pattern. Further, it is considered that in the film after the second solute (B) is vaporized, a low-density film of the first solute (A) remains. It is considered that since this low-density film has a low density, the force applied to the substrate pattern during vaporization is reduced. Therefore, it is preferably a component that vaporizes when left to stand for 180 seconds at normal temperature (20 to 27° C., preferably 23 to 25° C.). bpAand/or bpBare preferably 100 to 300° C., and more preferably 150 to 295° C. bpCis preferably 50 to 170° C., more preferably 50 to 150° C., and further preferably 60 to 140° C. Third Solute (D) and (E) Fourth Solute The substrate pattern filling composition according to the present invention can further comprises a third solute (D). Also, it can further comprise a fourth solute (E). These remain as solid components in the film formed from the substrate pattern filling composition filled in the substrate pattern. They each independently vaporize from this film. Exemplified embodiments of the third solute (D) and the fourth solute (E) are the same as the exemplified embodiments of the first solute (A) and/or the second solute (B). bpDand bpE, which are the boiling points under 1 atm respectively of the third solute (D) and the fourth solute (E), and vpDand vpE, which are the saturated vapor pressures at 25° C. and under 1 atm respectively of the third solute (D) and the fourth solute (E), preferably satisfy bpC<bpE<bpD<bpB<bpA, and it is also preferable to satisfy vpA<vpB<vpD<vpE<vpC. Although not to be bound by theory, it is considered that by satisfying such relations, the fourth solute (E), the third solute (D), the second solute (B) and the first solute (A) are vaporized in this order from the film formed from the composition. Preferably, the mass ratio of the first solute(A):the third solute (D) is 99:1 to 1:99 (more preferably 95:5 to 5:95, further preferably 90:10 to 10:90, and further more preferably 80:20 to 20:80). Preferably, the mass ratio of the first solute(A):the fourth solute (E) is 99:1 to 1:99 (more preferably 95:5 to 5:95, further preferably 90:10 to 10:90, and further more preferably 80:20 to 20:80). Other Additive (F) The substrate pattern filling composition according to the present invention can further comprise an other additive (F). The other additive (F) comprises a surfactant, an antibacterial agent, a bactericidal agent, an antiseptic agent, an antifungal agent, an acid, and/or a base. The other additive (F) is preferably highly volatile. It is desirable that the other additive (F) is vaporized at the time of vaporization of the first solute (A) and the second solute (B), which are solid components, in the process or vaporized before and after their vaporization. Compared with the sum of the mass of the first solute (A) and the mass of the second solute (B), the other additive (F) is 0 to 20 mass % (preferably 0 to 10 mass %, and more preferably 0 to 5 mass %). It is also a preferred embodiment of the present invention that no other additive (F) is contained (0 mass %). Surfactants that can be contained in the other additive (F) can be expected to improve coating properties. As the surfactant, any one can be used. Examples of the surfactant that can be used in the present invention include an anionic surfactant (F-1), a cationic surfactant (F-2) or a nonionic surfactant (F-3), and more particularly (F-1): alkyl sulfonate, alkyl benzene sulfonic acid and alkyl benzene sulfonate, (F-2): lauryl pyridinium chloride and lauryl methyl ammonium chloride, and (F-3): polyoxyethylene octyl ether, polyoxyethylene lauryl ether and polyoxyethylene acetylenic glycol ether are preferred. These surfactants, for example, nonionic alkyl ether-based surfactant manufactured by Nippon Nyukazai etc. as an example of nonionic surfactant, are commercially available. Further, the substrate pattern filling composition according to the present invention can comprise an antibacterial agent, a bactericidal agent, an antiseptic agent, and/or an antifungal agent as the other additive (F). These agents are used to prevent bacteria or fungi from propagating in an aged substrate pattern filling composition. Examples of these include alcohols such as phenoxyethanol and isothiazolone. Bestcide (trade name) commercially available from Nippon Soda Co., Ltd. is an effective antiseptic agent, antifungal agent and bactericidal agent. Typically, these agents do not affect the performance of the substrate pattern filling composition and the content thereof is usually 1 mass % or less, preferably 0.1 mass % or less, and more preferably 0.001 mass % or less, based on the total mass of the substrate pattern filling composition. Further, the substrate pattern filling composition according to the present invention can comprise an acid and/or a base as the other additive (F). The acid or base is used to adjust pH of the treatment liquid or improve solubility of each component. Although following is described to ensure clarity, in one substrate pattern filling composition, the other additive (F) is a compound different from the components (A) to (E). The acid or base used can be freely selected within a range not impairing the effects of the present invention, and examples thereof include carboxylic acids, amines and ammonium salts. These include fatty acids, aromatic carboxylic acids, primary amines, secondary amines, tertiary amines, ammonium compounds, and these can be substituted with any substituent. More particularly, formic acid, acetic acid, propionic acid, benzoic acid, phthalic acid, salicylic acid, lactic acid, malic acid, citric acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, aconitic acid, glutaric acid, adipic acid, monoethanolamine, diethanolamine, triethanolamine, triisopropanolamine, and tetramethyl-ammonium are included. Method for Forming a Substrate Pattern The method for forming a substrate pattern according to one embodiment of the present invention is described below with reference to the drawings. The method for forming a substrate pattern can be freely selected from known methods such as dry etching. Such a method for forming a substrate pattern is also described, for example, in Non-Patent Document 1 etc. In the method for forming a substrate pattern of the present invention, various pretreatments can be combined. FIG.1(a)shows a state in which a coated carbon film (also referred to as “coated C film” or “Spin On Carbon film”) layer2is formed on a substrate1, a silicon-containing anti-reflective coating (also referred to as “Si-ARC”) layer3is formed thereon, and a resist pattern4is formed thereon. The substrate used is not particularly limited, and examples thereof include a semiconductor substrate (for example, a silicon wafer etc.), a glass substrate of LCD and PDP etc., and the like. A conductive film, a wiring, a semiconductor, or the like can be formed on the substrate. The coated carbon film can be formed by applying with a conventionally known method such as spin coating, and performing pre-baking. Alternatively, the film can be formed also by CVD (chemical vapor deposition) method or ALD (atomic layer deposition) method. The silicon-containing anti-reflective coating layer can be formed by applying with spin coating, and performing pre-baking. By such a silicon-containing anti-reflective coating layer, the sectional shape and the exposure margin can be improved. Further, since the silicon-containing anti-reflective coating layer is used as an etching mask, for example, one having etching resistance is preferable. The resist pattern can be formed by combining known methods. For example, it is described in Patent Document 3. The process of etching the underlying film (interlayer) using the resist pattern as a mask and using the resultant as a mask can be performed stepwise, or the substrate can be directly etched using the resist pattern as a mask. The etching of the interlayer can be performed by either of dry etching or wet etching. FIG.1(b)shows a state in which the substrate is subjected to dry etching to form a gap5. The gas type for dry etching is not particularly limited, but a chlorofluorocarbon-based gas is usually used. After dry etching, a residue6(debris) remains between the patterns. FIG.1(c)shows a state in which the substrate pattern is cleaned with a cleaning liquid7. As the method for applying the cleaning liquid7, known methods can be used, and examples thereof include coating, dropping and immersing. Any combination of these can be used. The residue8is removed by the cleaning process. FIG.1(d)shows a state in which the substrate pattern filling composition 9 of the present invention is filled in the substrate pattern. The composition of the present invention is applied (preferably coated, dropped, or immersed) in a state that the liquid ofFIG.1(c)is remained. The coating method is not particularly limited, and coating can be performed, for example, by a method of dropping the composition on the surface of the substrate to spread it while rotating the substrate at 1 to 500 rpm, a method of dropping the composition on the surface of the substrate while the substrate is stationary and then rotating the substrate at 1 to 500 rpm to spread the composition, a method of immersing the substrate, or supplying by spraying or blasting. Among these, the method of dropping the composition on the surface of the substrate to spread it while rotating the substrate at 1 to 500 rpm, and the method of dropping the composition on the surface of the substrate while the substrate is stationary and then rotating the substrate at 1 to 500 rpm to spread the composition are preferred. In this regard, at least a part of the above-described liquid is replaced, and the composition is filled in the substrate pattern. In order to sufficiently exhibit the effects of the present invention, it is preferable that the replacement is sufficiently performed. After the above process, the substrate can be rotated at faster than 500 rpm and 5,000 rpm or less. By this rotation, the excess organic solvent and water in the present composition disappear from the substrate, but at least a part of the first solute (A) and the second solute (B) remains. It is considered that pattern collapse can be prevented because the composition does not all disappear from between the patterns. Here, one embodiment of the present invention includes the following. In the method for forming a substrate pattern of the present invention, the previously formed substrate pattern is cleaned with a cleaning liquid before applying (preferably coating) the substrate pattern filling composition and the liquid present on the substrate can be replaced with the substrate pattern filling composition of the present invention. The previously formed substrate pattern is in a state before the cleaning treatment is performed, and a residue (debris or the like) sometimes remains on the pattern surface. The liquid present on the substrate is, for example, a cleaning liquid. The cleaning can be performed in multiple steps, and for example, after applying the cleaning liquid (acid, alkali, etc.) that dissolves and removes inorganic substances, a cleaning liquid (deionized water, organic solvent) that is highly compatible with the substrate pattern filling composition of the present invention can be applied. The replacement of the liquid present on the substrate means replacing the liquid present before the substrate pattern filling composition is applied. Preferably, the liquid remaining in the substrate pattern is sufficiently replaced. FIG.1(e)shows a state in which the filled composition is removed to form a pattern10. Removal of the composition can be performed by heating, decompression, air drying, standing, or a combination thereof. Any of these removal methods can be used as long as the pattern shape is not impaired. The heating time is not particularly limited, but is preferably 10 to 180 seconds, more preferably 10 to 120 seconds, and further preferably 10 to 90 seconds. The time of pressure reduction is not particularly limited, but is preferably 0.5 to 60 minutes, and more preferably 0.5 to 10 minutes. Conditions of the pressure reduction can be controlled using a desiccator or oil pump. Further, removal of the composition by air drying can be performed by holding the pattern in the airflow. In this regard, the airflow can be either due to positive pressure or due to negative pressure. In particular, an airflow can be generated by blowing gas. In such a case, the gas used is not particularly limited, and air or the like can be used; however, an inert gas is preferably used. In particular, it is preferable to use argon gas, nitrogen gas, or the like. The airflow velocity is not particularly limited, and is appropriately selected so that removal of the composition is performed. In the above removal of the composition, the humidity of the gas forming the atmosphere or airflow is preferably low, and the humidity can be set, for example, 10% or less, preferably 5% or less, more preferably 1% or less, and particularly preferably 0.1% or less. The method for forming a substrate pattern of the present invention can suppress the collapse ratio even with respect to a fine pattern. For example, a pillar (cylinder) whose middle part is thinner than the bottom part and/or the top part is easy to collapse, but even such a pillar pattern structure can be cleaned while suppressing the collapse ratio. The pattern of the line space structure which is a wall structure is considered to be harder to collapse than the pillar pattern, but the collapse ratio can be further lowered using the method for forming a substrate pattern of the present invention. Here, as shown inFIG.1(e), the line width of the pattern formed on the substrate is x, and the length in the depth direction is y. The aspect ratio of the pattern is represented by y/x. In a pattern to which the present invention can be effectively applied, y is 0.01 to 6 μm or less, preferably 0.05 to 5 μm, and more preferably 0.1 to 3 μm. The aspect ratio is preferably 5 to 25, and more preferably 15 to 22. Substrate In the present invention, the substrate includes a semiconductor wafer, a glass substrate for a liquid crystal display device, a glass substrate for an organic EL display device, a glass substrate for a plasma display, a substrate for an optical disk, a substrate for a magnetic disk, a substrate for a magneto-optical disk, a glass substrate for a photomask, and a substrate for a solar cell. The substrate can be either a non-processed substrate (for example, a bare wafer) or a processed substrate (for example, a pattern substrate). The substrate can be configured by laminating a plurality of layers. Preferably, the surface of the substrate is a semiconductor. The semiconductor can be composed either of an oxide, a nitride, a metal or a combination of any of these. Further, the surface of the substrate is preferably selected from the group consisting of Si, Ge, SiGe, Si3N4, TaN, SiO2, TiO2, Al2O3, SiON, HfO2, T2O5, HfSiO4, Y2O3, GaN, TiN, TaN, Si3N4, NbN, Cu, Ta, W, Hf, and Al Device A device can be manufactured by further processing the substrate according to the present invention. As the device, a semiconductor device, a liquid crystal display device, an organic EL display device, a plasma display device, and a solar cell device are included. The device is preferably the semiconductor. Known methods can be used for these processing. After forming a device, if necessary, the substrate can be cut into chips, connected to a lead frame, and packaged with resin. An example of the packaged one is the semiconductor. The present invention is described below with reference to various examples. The aspect of the present invention is not limited to these examples. Preparation Example 1 of Example Composition 1 1-adamantanamine as the first solute and camphor as the second solute are added to an IPA solvent so that each is 10 mass %. The vessel is capped and stirring is performed overnight to obtain a solution. It can be visually confirmed that the solutes are dissolved. The solution is filtered through a filter having a pore size of 0.1 μm to obtain Example Composition 1. Preparation Examples 2 to 21 of Example Compositions 2 to 21, and Comparative Preparation Examples 1 to 8 of Comparative Example Compositions 1 to 8 Example Compositions 2 to 21 and Comparative Example Compositions 1 to 8 are prepared as in Preparation Example 1, except that the solutes, amount thereof or solvent are changed as shown in Table 1. As to each, it can be visually confirmed that the solutes are dissolved after stirring. Evaluation of Sublimability A 300 mm bare silicon wafer is put into a coater developer RF3 (SOKUDO). 10 cc of each composition is dropped on the wafer transferred to the coater cup and spin-coated at 1,500 rpm for 20 seconds. Thereafter, the wafer is left to stand in the coater cup and visually observed setting 120 seconds as the upper limit. The temperature in the coater cup is about 21 to 23° C. Sublimability of each sample is evaluated according to the following evaluation criteria. The results are described in Table 1. A: Although a film based on the solid components in the composition is formed, it is confirmed that the film vaporizes and disappears within 120 seconds. B: Although a film based on the solid components in the composition is formed, it is confirmed that the film does not disappear even after 120 seconds. The sample wafers evaluated as B are further heated (100° C. for 90 seconds) with a hot plate and visually observed. Sublimability is evaluated according to the following criteria. B1: It is confirmed that the film disappears after heating. B2: It is confirmed that the film does not disappear after heating and remains. TABLE 1The first soluteThe second soluteSolventSublimabilityExampleComposition 11-adamantanaminecamphor (10)IPAAComposition 21-adamantanaminecamphor (9.5)IPAAComposition 3DABCO (10)1,4-benzoquinone (10)IPAAComposition 41-adamantanamine1,4-benzoquinone (10)MeOHAComposition 5DABCO (10)borneol (10)IPAAComposition 61-adamantanamineborneol (10)IPAAComposition 7DABCO (10)1-adamantanamineIPAAComposition 81-adamantanamine1-adamantanamineIPAAComposition 9borneol (10)camphor (10)IPAAComposition 10DABCO (10)1-adamantanamineIPAAComposition 11camphor (10)1,4-benzoquinone (10)IPAAComposition 12DABCO (10)camphor (10)PGEEAComposition 13hexamethylenetetramine1,4-benzoquinone (10)EtOHB1(10)Composition 14hexamethylenetetramineborneol (10)IPAB1(10)Composition 15hexamethylenetetramine1-adamantanamineIPAB1(10)(10)Composition 161-adamantanol (10)camphor (10)IPAB1Composition 171-adamantanol (10)1,4-benzoquinone (10)IPAB1Composition 181-adamantanol (10)3-methyl-1,2-IPAB1cyclopentanedione (10)Composition 19DABCO (10)hexamethylenetetramineIPAB1(10)Composition 20borneol (10)1-adamantanol (10)IPAB1Composition 21hexamethylenetetraminecamphor (10)PGEEB1(10)ComparativeComposition 1DABCO (20)—PGEEAExampleComposition 21-adamantanamine—IPAAComposition 3borneol (20)—IPAAComposition 41-adamantanamine—EtOHAComposition 51,4-benzoquinone (20)—MeOHAComposition 6hexamethylenetetramine—PGEEB1(20)Composition 7trimethylolethane (20)—IPAB2Composition 8phthalic acid (20)—EtOHB1 The FIGURES in parentheses in the above table mean mass % of the solute based on the whole composition. Hereinafter, the compositions are divided into (Group A): those, of which sublimability is evaluated to be A and (Group B): those, of which sublimability is evaluated to be B (B1, B2), and they are evaluated. (Group A) Evaluation of Remaining Film The sample after the above evaluation of sublimability is used. The film thickness on the wafer is measured with an ellipsometer M-2000 (J. A. Woollam). In the ellipsometer measurement, constructing a two-layer model in which the remaining film derived from the present test and the natural oxide film are overlapped, only film thickness of the remaining film is calculated. The remaining film of each sample is evaluated according to the following evaluation criteria. The results are described in Table 2. A: The film thickness of the remaining film is less than 1 nm. B: The film thickness of the remaining film is 1 nm or more. Or, the measurement beam is scattered by the crystal grains and the film thickness of the remaining film cannot be measured. (Group A) Evaluation of Collapse Ratio A 300 mm silicon wafer (provided by Interuniversity Microelectronics Centre (imec)) with a pillar pattern that is patterned is used. The pillar (cylinder) has a diameter of the top part of about 31 nm, a diameter of the bottom part of about 67 nm, a height of about 590 nm, and a pillar pattern with a pitch of 80 nm is formed on the entire surface of the wafer. In order to evaluate each composition, the above wafer is cut into about 5 cm square. The cut wafer is set on a spin coater MS-A150 (Mikasa). 2 cc of each composition is dropped on the wafer and spin-coated at 1,000 rpm for 20 seconds. Immediately, the wafer is taken out and left on a laboratory desk in a clean room for about 120 seconds. The temperature of the clean room is controlled at normal temperature (about 23° C.). After the above treatment, each wafer is observed from the upper surface with SEM (SU8200, Hitachi High-Technologies). The collapse ratio is calculated by dividing the area of the part where the pillar pattern is collapsed by the total area that is observed. The results are described in Table 2. A: The collapse ratio is less than 5%. B: The collapse ratio is 5% or more. Total Evaluation The cases that the remaining film and the collapse ratio are both A are regarded as good. Others are regarded as insufficient. The results are described in Table 2. TABLE 2RemainingCollapseTotalThe first soluteThe second soluteSolventfilmratioevaluationExampleComposition 11-adamantanaminecamphor (10)IPAAAgoodComposition 21-adamantanaminecamphor (9.5)IPAAAgood(0.5)Composition 3DABCO (10)1,4-benzoquinoneIPAAAgood(10)Composition 41-adamantanamine1,4-benzoquinoneMeOHAAgood(10)(10)Composition 5DABCO (10)borneol (10)IPAAAgoodComposition 61-adamantanamineborneol (10)IPAAAgood(10)Composition 7DABCO (10)1-adamantanamineIPAAAgood(10)Composition 81-adamantanamine1-adamantanamineIPAAAgood(10)(10)Composition 9borneol (10)camphor (10)IPAAAgoodComposition 10DABCO (10)1-adamantanamineIPAAAgood(10)Composition 11camphor (10)1,4-benzoquinoneIPAAAgood(10)Composition 12DABCO (10)camphor (10)PGEEAAgoodComparativeComposition 1DABCO (20)—PGEEABinsufficientexampleComposition 21-adamantanamine—IPAABinsufficient(20)Composition 3borneol (20)—IPAABinsufficientComposition 41-adamantanamine—EtOHABinsufficient(20)Composition 51,4-benzoquinone—MeOHABinsufficient(20) The FIGURES in parentheses in the above table mean mass % of the solute based on the whole composition. (Group B) Evaluation of Remaining Film by Sublimation The sample after the above evaluation of sublimability is used. The measuring method and evaluation criteria are the same as in the above (Group A) evaluation of the remaining film. The results are described in Table 3. (Group B) Evaluation of Collapse Ratio by Sublimation A 300 mm silicon wafer (imec) with a pillar pattern that is patterned, which is used in the above (Group A) evaluation of the collapse ratio, is used. In order to evaluate each composition, the above wafer is cut into about 5 cm square. The cut wafer is set on a spin coater MS-A150. 2 cc of each composition is dropped on the wafer and spin-coated at 1,000 rpm for 20 seconds. Immediately, the wafer is taken out and heated on a hot plate at 100° C. for 90 seconds. After this step, the measuring methods and evaluation criteria are the same as in the above evaluation of the collapse ratio. The results are described in Table 3. Total Evaluation The cases that the remaining film and the collapse ratio are both A are regarded as good. Others are regarded as insufficient. The results are described in Table 3. TABLE 3RemainingCollapseTotalThe first soluteThe second soluteSolventfilmratioevaluationExampleComposition 13hexamethylenetetramine1,4-benzoquinoneEtOHAAgood(10)(10)Composition 14hexamethylenetetramineborneol (10)IPAAAgood(10)Composition 15hexamethylenetetramine1-adamantanamineIPAAAgood(10)(10)Composition 161-adamantanol (10)camphor (10)IPAAAgoodComposition 171-adamantanol (10)1,4-benzoquinoneIPAAAgood(10)Composition 181-adamantanol (10)3-methyl-1,2-IPAAAgoodcyclopentanedione(10)Composition 19DABCO (10)hexamethylenetetramineIPAAAgood(10)Composition 20borneol (10)1-adamantanol (10)IPAAAgoodComposition 21hexamethylenetetraminecamphor (10)PGEEAAgood(10)ComparativeComposition 6hexamethylenetetramine—PGEEABinsufficientexample(20)Composition 7trimethylolethane (20)—IPABBinsufficientComposition 8phthalic acid (20)—EtOHBBinsufficient The FIGURES in parentheses in the above table mean mass % of the solute based on the whole composition. EXPLANATION OF SYMBOLS 1. substrate2. coated carbon film layer3. silicon-containing anti-reflective coating layer4. resist pattern5. gap6. residue7. cleaning liquid8. residue9. substrate pattern filling composition according to the present invention10. pattern | 46,214 |
11859153 | DETAILED DESCRIPTION Exemplary embodiments disclosed in the disclosure are described in more detail with reference to drawings. Although the exemplary embodiments of the disclosure are shown in the drawings, it should be understood that the disclosure may be implemented in various forms and should not be limited by the specific embodiments described here. On the contrary, these embodiments are provided for more fully understanding of the disclosure, and to completely convey a scope disclosed by the disclosure to a person skilled in the art. In the drawings, the sizes of a layer, a region, and an element and their relative sizes may be magnified for clarity. The same reference sign represents the same element throughout. It should be understood that while the element or the layer is referred to as being “on . . . ”, “adjacent to . . . ”, “connected to . . . ” or “coupled to . . . ” other elements or layers, it may be directly on the other elements or layers, adjacent to, connected or coupled to the other elements or layers, or an intermediate element or layer may be present. In contrast, while the element is referred to as being “directly on . . . ”, “directly adjacent to . . . ”, “directly connected to . . . ” or “directly coupled to . . . ” other elements or layers, the intermediate element or layer is not present. It should be understood that although terms “first”, “second”, “third” and the like may be used to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Therefore, without departing from the teaching of the disclosure, a first element, component, region, layer, or section discussed below may be represented as a second element, component, region, layer or section. While the second element, component, region, layer, or section is discussed, it does not mean that the first element, component, region, layer or section is necessarily present in the disclosure. Spatial relation terms, such as “under . . . ”, “below . . . ”, “lower”, “underneath . . . ”, “above . . . ”, “upper” and the like, may be used here for conveniently describing a relationship between one element or feature shown in the drawings and other elements or features. It should be understood that in addition to orientations shown in the drawings, the spatial relation terms are intended to further include the different orientations of a device in use and operation. For example, if the device in the drawings is turned over, then the elements or the features described as “below” or “underneath” or “under” other elements may be oriented “on” the other elements or features. Therefore, the exemplary terms “below . . . ” and “under . . . ” may include two orientations of up and down. The device may be otherwise oriented (rotated by 90 degrees or other orientations) and the spatial relation terms used here are interpreted accordingly. The terms used here are only intended to describe the specific embodiments and are not limitations to the disclosure. As used herein, singular forms of “a”, “an” and “said/the” are also intended to include plural forms, unless otherwise clearly indicated in the context. It should also be understood that terms “composing” and/or “including”, while used in the description, demonstrate the presence of the described features, integers, steps, operations, elements and/or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups. As used herein, a term “and/or” includes any and all combinations of related items listed. The embodiments of the disclosure provide a method for cleaning a substrate. With reference toFIG.1, the method includes the following steps. At step101, the substrate is exposed to a cleaning agent, to remove impurities located on a surface of the substrate. At step102, the substrate is exposed to a dewetting chemical agent in a liquid phase, to remove the cleaning agent on the surface of the substrate. At step103, the dewetting chemical agent in the liquid phase remaining on the surface of the substrate is solidified, to obtain the dewetting chemical agent in a solid phase. At step104, the dewetting chemical agent in the solid phase is sublimated and removed. As shown inFIG.2, due to the limitations of the polishing process or device design, the surface of the substrate is often not smooth, but distributed with some fine structures, such as high aspect ratio structures (such as pillars), spaces and the like. Therefore, the cleaning agent, after cleaning, may remain in some tiny spaces or between adjacent high aspect ratio structures on the surface of the substrate, and generate capillary force between the adjacent structures. The capillary force will damage the surface structure of the substrate during subsequent drying processes, and also prevent the removal of residual cleaning agent (as shown in a) ofFIG.2. Combined with b) ofFIG.2, it can be seen that in the embodiments of the disclosure, the dewetting chemical agent in the liquid phase is removed by sublimating after solidified. The solidified dewetting chemical agent can eliminate the vapor/liquid interface, so that the surface tension is approach to zero. Therefore, the capillary forces are eliminated in the subsequent sublimation and removal process, reducing the damage of capillary forces to the substrate structure, and achieving the effect of nondestructive cleaning. Meanwhile, because the influence of the capillary forces is eliminated, the residue of the cleaning agent and impurities on the surface of the substrate can be greatly reduced. The method for cleaning a substrate provided by the embodiments of the disclosure will be described in detail below with reference toFIG.3AtoFIG.3B. As shown in a) ofFIG.3A, a substrate201to be cleaned is provided, and may have some impurities on the surface thereof. The impurities may be dirt, foreign matter, moisture, or may be sacrificial materials or mask materials from previous processes, such as residues of dry etching. In some embodiments, the surface of the substrate includes a space structure having an aspect ratio greater than or equal to 8. In practice, the substrate may be a wafer, the space structure may correspond to an isolation region (e.g. shallow trench isolation (STI) region) or a high aspect ratio feature (e.g. features used in forming capacitors, transistors, and other electrical components). The space structure may have a high aspect ratio (HAR), and the HAR may be 15 to 1, 30 to 1, or higher. In some embodiments, the space structure also includes a structure with a half pitch less than 19 nm. Next, step101is performed. The substrate is exposed to a cleaning agent to remove impurities located on the surface of the substrate, as shown in b) and c) inFIG.3A. In some embodiments, the cleaning agent is miscible with the impurities. In the practices, the cleaning agent may include, for example, but is not limited to, deionized water (DI water), aqueous ammonium hydroxide solution, aqueous hydrochloric acid solution, aqueous hydrogen peroxide solution, and the like. In some embodiments, the expose of the substrate to the cleaning agent may include the following operations. The substrate is controlled to spin at a third speed, and then a first cleaning agent is sprayed on the surface of the substrate. The substrate is controlled to spin at a fourth speed, and then a second cleaning agent is sprayed on the surface of the substrate to remove the first cleaning agent located on the surface of the substrate. In view of the above, first, the first cleaning agent is used for primary cleaning to remove most of the impurities dissolved in the first cleaning agent, and then, the second cleaning agent is used for secondary cleaning to remove the first cleaning agent and the impurities dissolved in the second cleaning agent, so that the effective removal of impurities is ensured and the cleanliness is improved through multiple cleanings. Specifically, as shown in b) ofFIG.3A, the substrate is controlled to spin at the third speed204, and the first cleaning agent203is sprayed on the surface of the substrate. In the practices, the third speed may be in a range of 500 to 1200 rpm (revolutions per minute). The first cleaning agent may be a wet chemical agent to remove impurities on the surface of the substrate. Next, as shown in c) ofFIG.3A, the substrate is controlled to spin at the fourth speed206, and the second cleaning agent205is sprayed on the surface of the substrate, to remove the first cleaning agent203on the surface of the substrate. In the practices, the third speed may be in range of 500 to 1200 rpm (revolutions per minute), and the second cleaning agent may be deionized water. Next, step102is performed. As shown in d) and e) inFIG.3A, the substrate is exposed to the dewetting chemical agent in a liquid phase207to remove the cleaning agent on the surface of the substrate. In some embodiments, in the method, the dewetting chemical agent in the liquid phase has the saturated vapor pressure greater than 4.0 KPa at room temperature, the miscibility of the dewetting chemical agent in the liquid phase with the cleaning agent is greater than 70%, and the dewetting chemical agent in the liquid phase has the evaporation rate greater than 1. In the practices, the cleaning agent and the dewetting chemical agent may be deionized water and acetone, respectively. Thus, it is facilitated to mix the dewetting chemical agent in the liquid phase and the cleaning agent, and to accelerate the evaporation ratio. The cleaning agent can be replaced by the dewetting chemical agent in the liquid phase more easily, thus improving the cleaning efficiency. In some embodiments, the dewetting chemical agent in the liquid phase includes acetone or isopropanol. Both acetone and isopropanol have lower surface tension, which can reduce the risk of structural deformation or collapse of the surface of the substrate during evaporation. In some embodiments, the expose of the substrate to the dewetting chemical agent in the liquid phase to remove the cleaning agent on the surface of the substrate may include the following operations. The substrate is controlled to spin at a first speed, and the dewetting chemical agent in the liquid phase is sprayed on the surface of the substrate to dissolve the cleaning agent remaining on the surface of the substrate in the dewetting chemical agent in the liquid phase. The substrate is controlled to spin at a second speed to remove part of the dewetting chemical agent in the liquid phase on the surface of the substrate, in which the second speed is less than the first speed. The cleaning agent is replaced by the dewetting chemical agent in the liquid phase through high-speed spin, and then the substrate is spinning at a lower speed, so that only part of the dewetting chemical agent in the liquid phase remains on the surface of the substrate, thereby reducing the sample amount in subsequent freeze-drying, and improving the cleaning efficiency. Specifically, as shown in d) ofFIG.3A, the substrate is controlled to spin at the first speed208, and the dewetting chemical agent in the liquid phase207is sprayed on the surface of the substrate to dissolve the cleaning agent remaining on the surface of the substrate in the dewetting chemical agent in the liquid phase. In the practices, the first speed may be in a range of 500 to 1200 rpm (revolutions per minute), the cleaning agent may be deionized water, and the dewetting chemical agent may be acetone. Next, as shown in e) ofFIG.3A, the substrate is controlled to spin at the second speed210to remove part of the dewetting chemical agent in the liquid phase on the surface of the substrate, in which the second speed is less than the first speed. Thus, a residual dewetting chemical agent in the liquid phase209on the surface of the substrate is obtained. In the practices, the second speed may be in a range of 5 to 50 rpm (rpm) and the dewetting chemical agent may be acetone. Next, step103is performed. As shown in f) and g) ofFIG.3B, the residual dewetting chemical agent in the liquid phase209on the surface of said substrate is solidified to obtain the dewetting chemical agent in a solid phase211. In the practices, the dewetting chemical agent in the liquid phase can be solidified by physical reaction or chemical reaction. In some embodiments, the dewetting chemical agent in the liquid phase is exposed to a reactive gas, in which the dewetting chemical agent in the liquid phase includes acetic acid and the reactive gas includes ammonia. For example, acetic acid reacts with ammonia to form volatile solid ammonium salt. In some embodiments, the solidification of the residual dewetting chemical agent in the liquid phase209on the surface of the substrate to obtain the dewetting chemical agent in the solid phase211includes the following operations. The temperature and/or pressure is controlled to solidify the residual dewetting chemical agent in the liquid phase209on the surface of the substrate into the dewetting chemical agent in a solid phase. By controlling the temperature and/or pressure, the dewetting chemical agent is solidified via physical reaction, which is environmentally friendly and can avoid introducing intermediates to contaminate the substrate. In some embodiments, the solidification of the residual dewetting chemical agent in the liquid phase209on the surface of the substrate to obtain the dewetting chemical agent in the solid phase211includes the following operations. The substrate is controlled to spin at the fifth speed212, and the temperature and/or pressure of the chamber is controlled such that the temperature of the chamber is below the freezing point of the dewetting chemical agent in a liquid phase under the pressure of the chamber, to solidify the residual dewetting chemical agent in the liquid phase on the substrate into the dewetting chemical agent in the solid phase. In the practices, the fifth speed of the substrate is controlled in a range of 5 to 50 rpm (revolutions per minute), the temperature of the chamber is controlled in a range of −120 to −40° C., and the pressure of the chamber is controlled from 0.1 to 100 mTorr (millitorr). If the pressure or temperature of the chamber is too high, it is not conducive to the solidification of the dewetting chemical agent in the liquid phase; while if the temperature and pressure are too low, it will require more severe process requirements and increase energy consumption. In other embodiments, the chamber or the wafer stage can be precooled first, and thus when the substrate is placed on the wafer stage, the dewetting chemical agent in the liquid phase can be quickly cooled to −30° C., thereby freezing the dewetting chemical agent. Because the dewetting chemical agents are volatile, rapid cooling can prevent the substrate from being affected by capillary force during volatilization of the dewetting chemical agent. In the practices, the dewetting chemical agent is frozen within no more than 30 seconds and the pressure of the chamber is reduced within no more than 20 seconds. In other embodiments, the rapid cooling can be achieved by contacting the substrate with liquid nitrogen. In some embodiments, in the method, the density value of the dewetting chemical agent in the liquid phase is less than the density value of the dewetting chemical agent in the solid phase. In this way, during solidification, the structure of the surface of the substrate would not be destroyed due to the increase in volume during phase transition of the dewetting chemical agent. Finally, step104is performed. As shown in h) and i) ofFIG.3B, the dewetting chemical agent in the solid phase211is sublimated and removed. It should be noted that, the term “sublimation” refers to the conversion of a dewetting chemical agent in solid phase into gas phase. The conversion may be caused by chemical reaction, for example, the dewetting chemical agent in the solid phase is decomposed into one or more gases. In the practices, when the dewetting chemical agent in the solid phase is an ammonium salt, the dewetting chemical agent in the solid phase may be decomposed, for example, by heating ammonium salt crystals. In other embodiments, the method may further include an operation of mechanical removing part of the dewetting chemical agent in the solid phase, before sublimating and removing the dewetting chemical agent in the solid phase. In the practices, the part of the dewetting chemical agent in the solid phase can be removed by external force or vibration. Specifically, it can be directly grasped by a mechanical arm or directly purged by an air gun. In this way, the cleaning efficiency can be improved and the energy consumption can be reduced. In some embodiments, the sublimation and removal of the dewetting chemical agent in the solid phase211includes the following operations. A temperature and/or pressure is controlled to sublimate the dewetting chemical agent in solid phase into a dewetting chemical agent in gas form, and the dewetting chemical agent in gas form is vented out. By controlling the temperature and/or pressure, the dewetting chemical agent in the solid phase is sublimated by physical reaction, which is environmentally friendly and can avoid introducing intermediates to contaminate the substrate. In some embodiments, as shown in h) and i) ofFIG.3B, the removal of the dewetting chemical agent in the solid phase211includes the following operations. The substrate is controlled to spin at a sixth speed213, and the temperature and/or pressure of the chamber is controlled such that the temperature of the chamber is higher than the sublimation point of the dewetting chemical agent in the solid phase under the pressure of the chamber, to sublimate the dewetting chemical agent in the solid phase into the dewetting chemical agent in a gas form. In the practices, the sixth speed of the substrate is controlled in a range of 100 to 1000 rpm (revolutions per minute) and the temperature of the chamber is controlled in a range of 20 to 50° C. If the temperature of the chamber is too low, it is not conducive to the sublimation of the dewetting chemical agent in solid phase, while if the temperature is too high, the energy consumption would be increased. Specifically, as shown in h), i) ofFIG.3B, the dewetting chemical agent in solid phase can be directly heated into the chemical agent in gas phase without melting into the dewetting chemical agent in liquid phase, by a heating unit214located in the wafer stage below the substrate and a heating lamp (not shown in the figure) above the substrate. Then, the temperature of the chamber is raised for a secondary drying, while keeping the pressure of the chamber at a lower pressure, the dewetting chemical agent in gas phase is obtained, and the sublimated dewetting chemical agent in gas phase is directly removed. In other embodiments, the secondary drying is performed under a temperature higher than the triple point temperature of the dewetting chemical agent, and multiple dryings can ensure the complete removal of the dewetting chemical agent in solid phase. For example, the dewetting chemical agent in gas phase can be removed by a dry vacuum pump. In the practices, the atmosphere of the chamber is nitrogen, argon, helium or other inert gases, which can prevent the substrate from being contaminated, make the heat distribution in the chamber uniform and improve the heat transfer rate. In some embodiments, as shown in i) ofFIG.3B, the method further includes, after removing the dewetting chemical agent in gas phase, an operation of exposing the substrate to an inert gas to place the substrate at atmospheric pressure, in which the substrate is purged along a center of the substrate toward edge with the inert gas. In the practices, the pressure of the chamber is restored to atmospheric pressure by supplying the chamber with the inert gas, and the inert gas purges the surface of the substrate from the center to the edge of the substrate, to ensure that no dry chemical are remained. The inert gas may be nitrogen or noble gases, such as argon and helium. When the chamber is brought to atmospheric pressure by the inert gas, the substrate can be cleaned again. The residual dewetting chemical agent on the surface of the substrate can be removed by direct purging, and the cleaning efficiency can also be improved by purging from the center to the edge. On the other hand, the inert gas is not easy to react with other substances in the substrate or in the environment of the chamber, so it does not cause a secondary contamination. In some embodiments, the first speed208, the third speed204, and the fourth speed206are greater than the sixth speed213, and the sixth speed213is greater than the second speed210and the fifth speed212. For example, the first speed, the third speed and the fourth speed may be in a range of 500 to 1200 rpm (revolutions per minute), the sixth speed may be in a range of 100 to 1000 rpm (revolutions per minute), and the second speed and the fifth speed may be in a range of 5 to 50 rpm (revolutions per minute). Relatively fast spinning speed can improve the cleaning efficiency, and relatively slow spinning speed can ensure that the structure of the surface of the substrate is not damaged during cleaning. In some embodiments, the method further includes, after removing the dewetting chemical agent in solid phase, an operation that the substrate is exposed to a cleaning gas for plasma ashing. The cleaning gas includes H2N2gas. When H2N2gas is used as a cleaning gas, H from H2N2de-chains the carbonized to shorter chain, so as to volatile easier. Furthermore, hydrogen reacts with carbon, hydrogen and oxygen (C, H, O) of the residual dewetting chemical agent to form volatile substances. The dewetting chemical agent will be completely removed after the ashing process, which increases the cleanliness of the substrate and is beneficial to the performance of the semiconductor device. The H2N2gas contains 96% N2, and has a similar function as N2on ashing process enhancement. In the practices, the ashing process may be performed under the following parameters: the temperature of the ashing chamber of 220 to 280° C., the radio frequency power of 2000 to 5000 W, the pressure of the ashing chamber of 50 to 1500 mTorr (millitorr), and the ashing time of 5 to 300 seconds. In some embodiments, the H2N2gas has a purity of at least 98%. There is no extra oxide layer on the surface of the substrate after ashing with pure H2N2, and no intermediates are formed, so the cleanliness of the substrate is improved. In the practices, the flow rate of the H2N2gas may be 5000 to 15000 sccm (standard ml/min). Embodiments of the disclosure also provide a system for cleaning a substrate. As shown inFIG.4, the system includes: a chamber501for receiving and processing the substrate201; a wafer stage503for supporting and clamping the substrate201in the chamber; a nozzle505for at least providing a dewetting chemical agent in a liquid phase to the surface of the substrate; a vacuum pump507for controlling a pressure of the chamber; a temperature controller509for controlling a temperature of the substrate and/or the chamber; an atmosphere control system511for exhausting a gas from the chamber or supplying a gas into the chamber; a controller513in communication with the vacuum pump507, the temperature controller509and the atmosphere control system511, and configured to solidify the dewetting chemical agent in the liquid phase to into a dewetting chemical agent in solid phase and configured to remove the dewetting chemical agent in the solid phase. In the practices, various different devices, such as a dry vacuum pump, a mechanical pump, a cryopump, and/or a turbomolecular pump can be used as the vacuum pump to reduce the pressure of the chamber. The atmosphere control system may be configured to supply an inert gas, such as nitrogen, argon, helium, or the like, to the chamber at a controlled flow rate to maintain the pressure of the chamber, or configured to directly purge the substrate with the inert gas. In the practices, the temperature controller may include a refrigerator system having one or more refrigerators or thermoelectric units. The cooling and/or heating of the substrate may be accomplished by the refrigerator system having one or more refrigerators or thermoelectric units. In the practices, the controller may be an electronic device having various integrated circuits, logics, memories, and/or software to receive instructions, issue instructions, control operations, etc. The controller513may be connected to one or more sensors that monitor operating parameters (such as temperature, pressure, etc.) of the chamber501. A temperature controller509may be provided as needed to control the temperature of the substrate and/or the chamber. An atmosphere control system may be provided as needed to exhaust a gas from the chamber or supply a gas into the chamber. A vacuum pump may be provided as needed to control the pressure of the chamber. In some embodiments, the controller configured to solidify the dewetting chemical agent in the liquid phase to obtain the dewetting chemical agent in the solid phase is operated to control a temperature and/or pressure of the chamber to bring the temperature of the chamber lower than a freezing point of the dewetting chemical agent in the liquid phase under the pressure of the chamber, to solidify the dewetting chemical agent in the liquid phase into the dewetting chemical agent in the solid phase. In some embodiments, the controller configured to remove the dewetting chemical agent in the solid phase is operated to control the temperature and/or pressure of the chamber, to bring the temperature of the chamber higher than a sublimation point of the dewetting chemical agent in the solid phase under the pressure of the chamber, to sublimate the dewetting chemical agent in the solid phase into the dewetting chemical agent in a gas phase and remove the dewetting chemical agent in the gas phase. In this way, the dewetting chemical agent in the liquid phase is removed by sublimating after solidified. The solidified dewetting chemical agent can eliminate the vapor/liquid interface, so that the surface tension is approach to zero. Therefore, the capillary forces are eliminated in the subsequent sublimation and removal process, reducing the damage of the capillary forces to the substrate structure and achieving the effect of nondestructive cleaning. In some embodiments, as shown inFIG.4, the system includes the controller in communication with the wafer stage503and the nozzle505, and the controller is further configured to control the wafer stage to spin at the first speed, control the nozzle to provide the dewetting chemical agent in the liquid phase to the surface of the substrate, and to control the wafer stage to spin at the second speed less than the first speed. The cleaning agent is replaced by the dewetting chemical agent in the liquid phase through high-speed spin, and then the substrate is spinning at a lower speed, so that only part of the dewetting chemical agent in the liquid phase remains on the surface of the substrate, thereby reducing the sample amount in the subsequent freeze-drying, and improving the cleaning efficiency. To sum up, in the disclosure, the dewetting chemical agent in the liquid phase is removed by sublimating after solidified. The solidified dewetting chemical agent can eliminate the vapor/liquid interface, so that the surface tension is approach to zero. Therefore, the capillary forces are eliminated in the subsequent sublimation and removal process, reducing the damage of the capillary forces to the substrate structure and achieving the effect of nondestructive cleaning. Meanwhile, because the influence of capillary force is eliminated, the residue of the cleaning agent and impurities on the surface of the substrate can be greatly reduced. It should be noted that, the technical features in the technical solutions recorded by the embodiments can be arbitrarily combined without conflict. Those skilled in the art can change the sequence of operations of the formation method described above, without departing from the scope of protection of the disclosure. The above mentioned is only the preferred embodiments of the disclosure, and is not intended to limit the scope of protection of the disclosure. Any modification, equivalent replacement and improvement made within the spirit and principles of the disclosure shall be included in the scope of protection of the disclosure. | 29,071 |
11859154 | DETAILED DESCRIPTION OF THE INVENTION The following detailed description is merely exemplary in nature and is not intended to limit the unit dose pack, or the method for producing or using the same. Furthermore, there is no intention to be bound by any theory presented in the preceding background or the following detailed description. The term “about” as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is ±10%. Thus, “about ten” means 9 to 11. All numbers in this description indicating amounts, ratios of materials, physical properties of materials, and/or use are to be understood as modified by the word “about,” except as otherwise explicitly indicated. A unit dose pack includes a wash composition encapsulated within a film, where the film may be transparent or colored. The wash composition includes a surfactant, water, non-aqueous solvents, and other components. One of the non-aqueous solvents is a poloxamer, and more particularly a reverse poloxamer. The use of a reverse poloxamer in appropriate concentrations with other solvents reduces or eliminates efflorescence in the film while maintaining good film haptics, such as minimal weight gain of the film when exposed to the wash composition. Without being bound by theory, it is believed that the reverse poloxamer slows the rate of diffusion of salts (such as sodium) from the liquid portion of the unit dose into the polyvinyl alcohol film. It is believed that the reverse poloxamers have a slower rate of diffusion than traditional solvents such as propylene glycol and glycerin. Therefore, the use of a reverse poloxamer is believed to delay or prevent efflorescence of the film, but no literature has been found to support or refute this non-binding hypothesis. A unit dose pack is formed by encapsulating a wash composition within a container, where the container includes a film. In some embodiments, the film forms one half or more of the container, where the container may also include dyes, print, or other components in some embodiments. The film is water soluble such that the film will completely dissolve when an exterior of the film is exposed to water, such as in a washing machine typically used for laundry. When the film dissolves, the container ruptures and the contents are released. As used herein, “water soluble” means at least 2 grams of the solute (the film in one example) will dissolve in 5 liters of water, for a solubility of at least 0.4 grams per liter (gift at a temperature of 25 degrees Celsius (° C.) unless otherwise specified. Suitable films for packaging are rapidly and completely soluble in water at temperatures of about 5° C. or greater. The film is desirably strong, flexible, shock resistant, transparent, and non-tacky during storage at both high and low temperatures and high and low humidity's. In an exemplary embodiment, the film is initially formed from polyvinyl acetate, and at least a portion of the acetate functional groups are hydrolyzed to produce alcohol groups. Therefore, the film includes polyvinyl alcohol (PVOH), and may include a higher concentration of PVOH than polyvinyl acetate. Such films are commercially available with various levels of hydrolysis, and thus various concentrations of PVOH. In an exemplary embodiment the film initially has about 85 percent of the acetate groups hydrolyzed to alcohol groups, but other percentages of hydrolysis are also possible in alternate embodiments. Some of the acetate groups may further hydrolyze in use, so the final concentration of alcohol groups may be higher than the concentration at the time of packaging. The film may have a thickness of from about 25 to about 200 microns (μm), or from about 45 to about 100 μm, or from about 65 to about 90 μm in various embodiments. The film may include alternate materials in some embodiments, such as methyl hydroxy propyl cellulose and polyethylene oxide, but the film is water soluble in all embodiments. The unit dose pack may be formed from a container having a single section, but the unit dose pack may be formed from containers with two or more different sections in alternate embodiments. In embodiments with a container having two or more sections, the contents of the different sections may or may not be the same. In embodiments with two or more sections, at least one of the sections includes the wash composition. The other section may include the same or a different embodiment of the wash composition, but in alternate embodiments the other section includes a different composition, such as a fabric softening composition or other fabric treatment. In some embodiments, the unit dose pack is formulated and configured for cleaning laundry, but other cleaning purposes are also possible. The wash composition is positioned within the container, and the container is sealed to encapsulate and enclose the wash composition. The wash composition is typically in direct contact with the film of the container within the unit dose pack. The film of the container is sealable by heat, heat and water, ultrasonic methods, or other techniques, and one or more sealing techniques may be used to enclose the wash composition within the container. In an exemplary embodiment, the wash composition is liquid when encapsulated within the container. The liquid wash composition may have a viscosity of from about 100 to about 1,000 centipoise, or from about 100 to about 300 centipoise in different embodiments, where “viscosity,” as used herein, means the viscosity measured by a rotational viscometer at a temperature of 25 degrees Celsius (° C.) using an LV02 cylindrical spindle at about 20 revolutions per minute (RPM) with a Brookfield® DV2T viscometer. The liquid form facilitates rapid delivery and dispersion of the wash composition once the container ruptures, and this rapid dispersion can aid cleaning. In alternate embodiments, the wash composition is flowable, such as a gel, a liquid with suspended particulates, or other forms. In an exemplary embodiment, the unit dose pack is sized to provide a desired quantity of wash composition for one load of laundry or one batch of dishes in a dishwasher. The unit dose pack may also be sized for a fraction of a desired quantity, such as one half of a load of laundry, so a user can adjust the amount of detergent added without having to split a unit dose pack. In an exemplary embodiment, the unit dose pack has a weight of from about 5 to about 50 grams. In alternate embodiments, the unit dose pack is from about 10 to about 40 grams, or from about 15 to about 25 grams. A plurality of components are combined to form the wash composition, where the wash composition is typically prepared prior to encapsulation within the container. A total weight of the wash composition does not include the weight of the film or the container, where the total weight of the wash composition is generally referenced herein as the basis for the weight percent of components of the wash composition. Unless otherwise specified, the concentration of all components described herein, other than the film, is the weight percent of the named component based on the total weight of the wash composition. A solvent is a component that is utilized as a carrier in a formulation, where other components (solutes) are dissolved in the solvent. Solvents generally solvate solutes and act as bulk fillers for the formula when used below a certain use-level so as not to plasticize the film. Specific criteria that precisely and exactly define what is or is not a solvent are difficult to define, because some components may have more than one purpose. Generally, solvents for liquid formulations are liquids at standard conditions (i.e., 1 atmosphere pressure and 20 degrees Celsius (° C.)). Typically, ionic surfactants, non-ionic surfactants, optical brighteners, dyes or pigments, bleach activators or agents, enzymes, perfumes or other ingredients added for odor purposes, bittering agents, peroxy compounds, soil release agents, dye transfer inhibitors, foam inhibitors, chelators or other water softeners are not considered “solvents.” The wash composition includes water as one solvent, and the wash composition also includes a non-aqueous solvent. One solvent in the wash composition is water, as mentioned above. Water may be present in the wash composition at a concentration of from about 5 to about 45 weight percent, or present in an amount of from about 5 to about 35 weight percent, or present in an amount of from about 5 to about 28 weight percent, or present in an amount of from about 7 to about 25 weight percent in various embodiments, based on the total weight of the wash composition. The film is soluble in water, and non-aqueous solvents can help the film retain strength while encapsulating the wash composition with the water. The correct ratios of non-aqueous solvents and water allow for a stable unit dose pack with good film haptics. The wash composition also includes a non-aqueous solvent, and the non-aqueous solvent may include one or more components. The non-aqueous solvent may be present in the wash composition at from about 15 to about 60 weight percent, or at from about 25 to 40 weight percent in different embodiments, based on the total weight of the wash composition. As stated above, the definition of a solvent is not always clear, so in this description the following compounds are defined as “non-aqueous solvents:” glycerol; propylene glycol; ethylene glycol; ethanol; and 4C+ compounds. The term “4C+ compound” refers to one or more of: polypropylene glycol; polyethylene glycol; poloxamers; reverse poloxamers; polyethylene glycol esters such as polyethylene glycol stearate, propylene glycol laurate, and/or propylene glycol palmitate; methyl ester ethoxylate; diethylene glycol; dipropylene glycol; sorbitol; tetramethylene glycol; butylene glycol; pentanediol; hexylene glycol; heptylene glycol; octylene glycol; 2-methyl-1,3-propanediol; xylitol; mannitol; erythritol; dulcitol; inositol; adonitol; triethylene glycol; glycol ethers, such as ethylene glycol monobutyl ether, diethylene glycol monobutyl ether, triethylene glycol monobutyl ether, ethylene glycol monopropyl ether, diethylene glycol monoethyl ether, triethylene glycol monoethyl ether, diethylene glycol monomethyl ether, and triethylene glycol monomethyl ether; tris (2-hydroxyethyl)methyl ammonium methylsulfate; ethylene oxide/propylene oxide copolymers with a number average molecular weight of 3,500 Daltons or less; and ethoxylated fatty acids. The term “poloxamer,” as used herein, means a multi-block polymer with a central unit formed from hydrophobic poloxamer monomer component, where a polymer center of the poloxamer is relatively hydrophobic, as compared to the polymer ends. The “poloxamer” also has a plurality of polymer ends that are relatively hydrophilic, as compared to the polymer center. To be more specific, the solubility of the central unit is about 1 gram of polymer per 1,000 grams of water at 25° C., and the solubility of the polymer ends is about 100 grams of polymer per 1,000 grams of water, again at 25° C., where the solubility is determined based on a polymer comparable to just the polymer center or the polymer end. As such, the polymer center can be distinguished from the polymer ends by the water solubility of the components, where a polymer comparable to the polymer center has a water solubility of 1 gram polymer per 1,000 grams water or less, and a polymer comparable to the polymer ends has a water solubility of about 100 grams of polymer per 1,000 grams of water or more. The polymer ends are primarily formed from a hydrophilic poloxamer monomer component that is different from the hydrophobic poloxamer monomer component. The term “primarily,” as used herein, means at least 50% by weight, where it is possible for a limited number of hydrophilic poloxamer monomer components to be incorporated into the polymer ends or a limited number of hydrophobic poloxamer monomer components to be incorporated into the polymer center, not to the extent that the water solubility of the polymer center or the polymer ends falls outside of the range specified above. In a similar manner, the term “reverse poloxamer,” as used herein, means a multi-block polymer with a plurality of polymer ends and a polymer center, where the polymer ends are formed from a hydrophobic poloxamer monomer component and the polymer center is formed from a hydrophilic poloxamer monomer component. As such, the polymer center is relatively hydrophilic (compared to the polymer ends) and the polymer ends are relatively hydrophobic (compared to the polymer center.) More specifically, the polymer center has a water solubility of about 1 gram of polymer per 1,000 grams of water or less, at 25° C., and the polymer ends have a water solubility of about 100 grams of polymer per 1,000 grams of water or more, at 25° C. The polymer center is capped by the polymer ends, such that the poloxamer or reverse poloxamer center does not generally include a termination point for the polymer. In an exemplary embodiment, the reverse poloxamer has two poloxamer ends, so the reverse poloxamer is a tri-block polymer. The hydrophobic poloxamer monomer component is propylene oxide and the hydrophilic poloxamer monomer component is ethylene oxide in an exemplary embodiment, so the reverse poloxamer can be referred to as a PO-EO-PO reverse poloxamer (where PO stands for propylene oxide and EO stand for ethylene oxide.) An exemplary structure is illustrated below, where X may be from 1 to about 35, Y may be from 1 to about 35, and Z may be from about 1 to about 35. In the illustration above, the X and Z components represent portions of the reverse poloxamer formed from propylene oxide (PO), and the Y component represents a portion of the reverse poloxamer formed from ethylene oxide (EO). The ethylene oxide (EO) to propylene oxide (PO) ratio (the EO/PO ratio) of the reverse poloxamer may be from about 90:10 to about 10:90 in an exemplary embodiment, but in alternate embodiment the EO/PO ratio may be from about 50:50 to about 90:10, or from about 40:60 to about 60:40. The EO/PO ratio is based on the mass of the EO and PO monomer components present in the reverse poloxamer. In embodiments where the reverse poloxamer primarily or exclusively comprises EO and PO, the polymer center may include at least 2 PO monomers covalently bound together and no EO monomers covalently bound together, and each of the polymer ends may include at least 2 EO monomers covalently bound together and no PO monomers covalently bound together. In an exemplary embodiment, from about 90 to about 100 percent of the hydrophobic poloxamer monomer components are propylene oxide, from about 90 to about 100 percent of the hydrophilic poloxamer monomer components are ethylene oxide, and the reverse poloxamer includes from about 0 to about 20 percent hydrophilic and/or hydrophobic poloxamer monomer components that are not EO or PO, where the monomer percentages are weight/weight (i.e., weight of specific component compared to the weight of the named category, such as weight of propylene oxide compared to weight of the hydrophobic poloxamer monomer components.) In an alternate embodiment, about 100 percent of the hydrophobic poloxamer monomer component is propylene oxide, and about 100 percent of the hydrophilic poloxamer monomer component is ethylene oxide. It is hypothesized that the hydrophobic ends of the reverse poloxamer have little interaction with the water soluble film, and testing indicates use of the reverse poloxamer as a component of the non-aqueous solvents reduces efflorescence in the film, while maintaining good film haptics. However, there is no intention to be bound by theory in this disclosure. Testing results are detailed further below. The reverse poloxamer may be present in the non-aqueous solvent at from about 10 to about 80 weight percent, based on a total weight of the non-aqueous solvent, or from about 30 to about 80 weight percent in an alternate embodiment. In reference to the entire wash composition, the reverse poloxamer may be present at from about 3 to about 30 weight percent, based on the total weight of the wash composition. In an alternate embodiment, the reverse poloxamer may be present in the wash composition at from about 8 to about 30 weight percent, or from about 8 to about 20 weight percent, based on the total weight of the wash composition. The reverse poloxamer may have a number average molecular weight of from about 300 to about 5,000 in an exemplary embodiment, but other number average molecular weights are also possible. The non-aqueous solvent may include other components as well, such as polyethylene glycol is in some embodiments. The polyethylene glycol may be present at from about 0 to about 50 weight percent of the wash composition, or from about 0 to about 30 weight percent of the wash composition, or from about 5 to about 30 weight percent of the wash composition, based on the total weight of the wash composition. In some embodiments, the wash composition is free of polyethylene glycol. As used herein, “free of” means the named component is present in an amount of about 1 weight percent or less, based on a total weight of the named composition (such as the wash composition), unless otherwise specified. If a component is specified as being present at a concentration of less than about 1 weight percent, but more than 0 percent, the wash composition is not considered “free of” that component. The polyethylene glycol may have a number average molecular weight in the range from about 200 to about 1000 daltons in an exemplary embodiment, but other average molecular weights may be utilized in alternate embodiments. Glycerin may also be present in the non-aqueous solvents at from about 0 to about 70 weight percent, based on the total weight of the non-aqueous solvents, or from about 0 to about 30 weight percent based on the total weight of the wash composition. Other non-aqueous solvents as listed above may also be present in the wash composition, such as from about 0 to about 30 weight percent based on the total weight of the wash composition. The quantity and ratio of the non-aqueous solvents relative to the water should be adjusted to provide a film with suitable haptics, as discussed below, so the quantities of the components of the non-aqueous solvent are limited to internal ratios with limited efflorescence and satisfactory film haptics. The wash composition includes other components as well. For example, the wash composition may include one or more ionic surfactants, where the ionic surfactant is formulated for laundry in an exemplary embodiment. The ionic surfactant may include one or more surfactants, including cationic and/or anionic surfactants, in various embodiments. The ionic surfactant may be present in the wash composition at a concentration of from about 5 to about 55 weight percent in one embodiment, but the ionic surfactant may be present in the wash composition at a concentration of about 5 to about 45 weight percent, or from about 10 to about 40 weight percent, or from about 10 to about 35 weight percent, or from about 15 to about 30 weight percent in alternate embodiments, based on a total weight of the wash composition. Suitable ionic surfactants that are anionic include soaps which contain sulfate or sulfonate groups, including those with alkali metal ions as cations. In an exemplary embodiment, the wash composition includes an anionic sodium alcohol ethoxy sulfate surfactant at from about 3 to about 27 weight percent, or from about 6 to about 24 weight percent, or from about 6 to about 21 weight percent, or from about 9 to about 18 weight percent in alternate embodiments, based on a total weight of the wash composition. Usable soaps include alkali metal salts, amine salts, or other salts of saturated or unsaturated fatty acids with 12 to 18 carbon (C) atoms. Such fatty acids may also be used in incompletely neutralized form, such that some of the fatty acids are present in a salt form and other fatty acids are present in a free acid form where an acid group is protonated. Usable ionic surfactants of the sulfate type include sulfuric acid semi esters of fatty alcohols with 12 to 18 C atoms, and/or alcohol ethoxysulfates, where these compounds may be present in a salt form. Usable ionic surfactants of the sulfonate type include alkane sulfonates with 12 to 18 C atoms and olefin sulfonates with 12 to 18 C atoms, such as those that arise from the reaction of corresponding mono-olefins with sulfur trioxide. Another type of sulfonate surfactant includes alpha-sulfofatty acid esters such as those that arise from the sulfonation of fatty acid methyl or ethyl esters, and lauryl ether sulfates. In an exemplary embodiment, the wash composition includes linear alkyl benzene sulfonic acid surfactants as the ionic surfactant at a concentration of from about 1 to about 15 weight percent, or from about 2 to about 12 weight percent, or from about 4 to about 8 weight percent in different embodiments. In an exemplary embodiment, linear alkylbenzene sulfonates include 9 to 14 C atoms in the alkyl moiety. In alternate embodiments, the wash composition is free of linear alkyl benzene sulfonic acid surfactants. As used herein, “free of” means the named component is present in an amount of about 1 weight percent or less, based on a total weight of the named composition (such as the wash composition), unless otherwise specified. Suitable ionic surfactants that are cationic may include textile-softening substances of the general formula X, XI, or XII as illustrated below: in which each R1group is mutually independently selected from among C1-6alkyl, alkenyl or hydroxyalkyl groups; each R2group is mutually independently selected from among C8-28alkyl or alkenyl groups; R3═R1or (CH2)n-T-R2; R4═R1or R2or (CH2)n-T-R2; T=—CH2—, —O—CO—, or —CO—O—, and n is an integer from 0 to 5. The ionic surfactants that are cationic may include conventional anions of a nature and number required for charge balancing. Alternatively, the ionic surfactant may include anionic surfactants that may function to balance the charges with the cationic surfactants. In some embodiments, ionic surfactants that are cations may include hydroxyalkyltrialkylammonium compounds, such as C12-18alkyl(hydroxyethyl)dimethyl ammonium compounds, and may include the halides thereof, such as chlorides or other halides. The ionic surfactants that are cations may be especially useful for compositions intended for treating textiles. Non-ionic surfactants may optionally be present in the wash composition at a concentration of from about 0 to about 60 weight percent, or from about 5 to about 50 weight percent, or from about 10 to about 40 weight percent, or from about 15 to about 30 weight percent in various embodiments, based on the total weight of the wash composition. Suitable non-ionic surfactants include alkyl glycosides and ethoxylation and/or propoxylation products of alkyl glycosides or linear or branched alcohols in each case having 12 to 18 C atoms in the alkyl moiety and 3 to 20, or 4 to 10, alkyl ether groups. In an exemplary embodiment, the non-ionic surfactant is an alcohol ethoxylate surfactant, but other compounds are also possible. Corresponding ethoxylation and/or propoxylation products of N-alkylamines, vicinal diols, fatty acid esters and fatty acid amides, which correspond to the alkyl moiety in the stated long-chain alcohol derivatives, may furthermore be used. Alkylphenols having 5 to 12 C atoms may also be used in the alkyl moiety of the above described long-chain alcohol derivatives. Several other components may optionally be added to and included in the wash composition, including but not limited to water-binding saccharides, enzymes, peroxy compounds, bleach activators, anti-redeposition agents, pH adjusting agents, optical brighteners, foam inhibitors, bittering agents, dye transfer inhibitors, soil release agents, and other components. A partial, non-exclusive list of additional components that may be added to and included in the wash composition includes electrolytes, pH regulators, graying inhibitors, anti-crease components, processing aids, antimicrobial agents, and preservatives. Water binding saccharides are optionally included in the wash composition. In some embodiments, the saccharide is selected from the group of fructose, glucose, sucrose, xylitol, sorbitol, mannitol, erythritol, dulcitol, inositol, adonitol, tagatose, trehalose, galactose, rhamnose, cyclodextrin, maltodextrin, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, palatinose or isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, beet oligosaccharides, isomalto-oligosaccharides, xylo-oligosaccharides, gentio-oligoscaccharides, sorbose, nigero-oligosaccharides, palatinose oligosaccharides, fucose, fractooligosaccharides, maltotetraol, maltotriol, malto-oligosaccharides, lactulose, melibiose, raffinose, rhamnose, ribose, high fructose corn/starch syrup, coupling sugars, soybean oligosaccharides, or glucose syrup, and mixtures thereof. One example of a saccharide that may be utilized is high fructose corn syrup (HFCS.) HFCS typically refers to a blend of approximately 23% water and 77% saccharide. For example, HFCS 55 typically refers to a blend of water (about 23%), glucose (about 34%), and fructose (about 42%). However, in a dried form, HFCS 55 contains approximately 55% fructose by weight of dry HFCS, where the number after the abbreviation HFCS generally refers to the percentage of fructose in a dry state. Unless otherwise stated, HFCS used herein refers to a wet blend which contains water, as it is supplied from HFCS manufacturers. However, it should be understood that dry or essentially dry hybrids of monosaccharides (e.g. HFCS), wherein water has been removed partially or completely, can also be used. Other HFCS products may also be used, such as HFCS 42, HFCS 65, HFCS 90, and others. While pure fructose is very viscous and hard to handle, HFCS is more dilute and easier to handle. HFCS is also more cost-effective to manufacture. The United States Food and Drug Administration has even determined that HFCS is a safe ingredient for food and beverage manufacturing. It is certainly a safe and green ingredient for detergent products. Foam inhibitors may optionally be included in the wash composition. Suitable foam inhibitors include, but are not limited to, soaps of natural or synthetic origin, which include an elevated proportion of C18-C24fatty acids. Suitable non-surfactant foam inhibitors are, for example, organopolysiloxanes and mixtures thereof with microfine, optionally silanized silica as well as paraffins, waxes, microcrystalline waxes and mixtures thereof with silanized silica or bis-fatty acid alkylenediamides. Mixtures of different foam inhibitors may also be used, for example mixtures of silicones, paraffins or waxes. In an exemplary embodiment, coconut fatty acids are used as foam inhibitors, but other embodiments are possible, such as mixtures of paraffins and bistearylethylenediamide. The wash composition may include the foam inhibitor at an amount of from about 0 to about 15 weight percent, but in other embodiments the foam inhibitor may be present at an amount of from about 0.05 to about 10 weight percent, or an amount of from about 0.5 to about 8 weight percent, based on the total weight of the wash composition. PH adjusting agents may be added to and included in the wash composition. Exemplary pH adjusting agents include monoethanol amine, binary amines, buffers, triethanol amine, metal hydroxides, or other materials. Exemplary metal hydroxides are sodium hydroxide and/or potassium hydroxide, and other possible pH adjusting agents include compounds that adjust the pH of the wash composition. pH adjusting agents may be present in the wash composition at an amount of from about 0.1 to about 10 weight percent in some embodiments, based on the total weight of the wash composition, but in other embodiments the pH adjusting agent may be present in the wash composition at an amount of from about 0.5 to about 5 weight percent, or an amount of from about 1 to about 4 weight percent, based on the total weight of the wash composition. The pH adjusting agent may be utilized to adjust the pH of the wash composition to from about 6 to about 10, or from about 6.5 to about 9.5, or from about 7 to about 9 in various embodiments. The pH adjusting agent may form a cation that combines with an anionic surfactant and/or a coconut fatty acid or other foam inhibitor and/or another anionic component within the wash composition. In many cases, the pH adjusting agent forms a salt with an anionic component. As such, the anionic surfactant may be present in the wash composition as a surfactant salt, and the coconut fatty acid may be present in the wash composition as a coconut fatty acid salt. In some embodiments, the pH adjusting agent is included in a slight excess relative to the anionic surfactant or other acidic components to adjust the pH of the wash composition to within a desired range, such as the range(s) mentioned above. As used herein, the terms “anionic surfactant” and “coconut fatty acid” include the neutralization products thereof. Coconut fatty acids may optionally be utilized as a filler and as a stabilizing agent. Coconut fatty acids are relatively expensive, so non-aqueous solvent mixtures that allow for lower concentrations of coconut fatty acid while retaining a stable wash composition with good film haptics are desirable. The use of a reverse poloxamer allows for reduced concentrations of the coconut fatty acids in the wash composition. In an exemplary embodiment, the wash composition optionally includes coconut fatty acids at a concentration of from about 2 to about 15 weight percent, based on the total weight of the wash composition. However, in alternate embodiments, coconut fatty acids are present in the wash composition at from about 2 to about 10 weight percent, or from about 2 to about 7.5 weight percent, again based on the total weight of the wash composition. Possible enzymes that may be in the wash composition contemplated herein include one or more of a protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, (e.g., a laccase), and/or peroxidase, but others are also possible. In general, the properties of the selected enzyme(s) should be compatible with the selected wash composition, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.). The detergent enzyme(s) may be included in the wash composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all the enzymes that are added to the wash composition. The enzyme(s) should be present in the wash composition in effective amounts, such as from about 0 weight percent to about 5 weight percent of enzyme, or from about 0.001 to about 5 weight percent, or from about 0.001 to about 1 weight percent, or from about 0.2 to about 1 weight percent, or from about 0.5 to about 1 weight percent, based on the total weight of the wash composition, in various embodiments. In an exemplary embodiment, the wash composition includes three or more different enzymes. In one embodiment, the wash composition includes protease, mannanase, and amylase, but other embodiments are also possible. A peroxy compound may optionally be present in the wash composition. Exemplary peroxy compounds include organic peracids or peracidic salts of organic acids, such as phthalimidopercaproic acid, perbenzoic acid or salts of diperdodecanedioic acid, hydrogen peroxide and inorganic salts that release hydrogen peroxide under the washing conditions, such as perborate, percarbonate and/or persilicate. Hydrogen peroxide may also be produced with the assistance of an enzymatic system, i.e. an oxidase and its substrate. Other possible peroxy compounds include alkali metal percarbonates, alkali metal perborate monohydrates, alkali metal perborate tetrahydrates or hydrogen peroxide. Peroxy compounds may be present in the wash composition at an amount of from about 0 to about 15 weight percent, or an amount of from about 1 to about 10 weight percent, or an amount of from about 3 to about 5 weight percent, based on the total weight of the wash composition, in various embodiments. Bleach activators may optionally be added and included in the wash composition. Conventional bleach activators that form peroxycarboxylic acid or peroxyimidic acids under perhydrolysis conditions and/or conventional bleach-activating transition metal complexes may be used. The bleach activator optionally present may include, but is not limited to, one or more of: N- or O-acyl compounds, for example polyacylated alkylenediamines, such as tetraacetylethylenediamine; acylated glycolurils, such as tetraacetylglycoluril; N-acylated hydantoins; hydrazides; triazoles; urazoles; diketopiperazines; sulfurylamides and cyanurates; carboxylic anhydrides, such as phthalic anhydride; carboxylic acid esters, such as sodium isononanoylphenolsulfonate; acylated sugar derivatives, such as pentaacetyl glucose; and cationic nitrile derivatives such as trimethylammonium acetonitrile salts. To avoid interaction with peroxy compounds during storage, the bleach activators may be coated with shell substances or granulated prior to addition to the wash composition, in a known manner. As such, the bleach activator and/or other components may be present in a liquid wash composition as a free or floating particulate. Exemplary embodiments of the coating or shell substance include tetraacetylethylenediamine granulated with the assistance of carboxymethylcellulose and having an average grain size of 0.01 mm to 0.8 mm, granulated 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine, and/or trialkylammonium acetonitrile formulated in particulate form. In alternate embodiments, the peroxy compounds and bleach activators, if present, may be within separate chambers of the container to prevent premature interactions. In various embodiments, the bleach activators may be present in the wash composition in quantities of from about 0 to about 8 weight percent, or from about 0 to about 6 weight percent, or from about 0 to about 4 weight percent, in each case relative to the total weight of the wash composition. One or more anti-redeposition agents may also be optionally included in the wash composition. Anti-redeposition agents include polymers with a soil detachment capacity, which are also known as “soil repellents” due to their ability to provide a soil-repelling finish on the treated surface, such as a fiber. One example regarding polyesters includes copolyesters prepared from dicarboxylic acids, such as adipic acid, phthalic acid or terephthalic acid. In an exemplary embodiment, an anti-redeposition agents includes polyesters with a soil detachment capacity that include those compounds which, in formal terms, are obtainable by esterifying two monomer moieties, the first monomer being a dicarboxylic acid HOOC-Ph-COOH and the second monomer a diol HO—(CHR11—)aOH, which may also be present as a polymeric diol H—(O—(CHR11—)a)bOH. Ph here means an ortho-, meta- or para-phenylene residue that may bear 1 to 4 substituents selected from alkyl residues with 1 to 22 C atoms, sulfonic acid groups, carboxyl groups and mixtures thereof. R11means hydrogen or an alkyl residue with 1 to 22 C atoms and mixtures thereof. “a” means a number from 2 to 6 and “b” means a number from 1 to 300. The polyesters obtainable therefrom may contain not only monomer diol units —O—(CHR11—)aO— but also polymer diol units —(O—(CHR11—)a)bO—. The molar ratio of monomer diol units to polymer diol units may amount to from about 100:1 to about 1:100, or from about 10:1 to about 1:10 in another embodiment. In the polymer diol units, the degree of polymerization “b” may be in the range of from about 4 to about 200, or from about 12 to about 140 in an alternate embodiment. The number average molecular weight of the polyesters with a soil detachment capacity may be in the range of from about 250 to about 100,000, or from about 500 to about 50,000 in an alternate embodiment. The acid on which the residue Ph is based may be selected from terephthalic acid, isophthalic acid, phthalic acid, trimellitic acid, mellitic acid, the isomers of sulfophthalic acid, sulfoisophthalic acid and sulfoterephthalic acid and mixtures thereof. Where the acid groups thereof are not part of the ester bond in the polymer, they may be present in salt form, such as an alkali metal or ammonium salt. Exemplary embodiments include sodium and potassium salts. If desired, instead of the monomer HOOC-Ph-COOH, the polyester with a soil detachment capacity (the anti-redeposition agent) may include small proportions, such as no more than about 10 mole percent relative to the proportion of Ph with the above-stated meaning, of other acids that include at least two carboxyl groups. These include, for example, alkylene and alkenylene dicarboxylic acids such as malonic acid, succinic acid, fumaric acid, maleic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid and sebacic acid. Exemplary diols HO—(CHR11—)aOH include those in which R11is hydrogen and “a” is a number of from about 2 to about 6, and in another embodiment includes those in which “a” has the value of 2 and R11is selected from hydrogen and alkyl residues with 1 to 10 C atoms, or where R11is selected from hydrogen and alkyl residues with 1 to 3 C atoms in another embodiment. Examples of diol components are ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1,8-octanediol, 1,2-decanediol, 1,2-dodecanediol and neopentyl glycol. The polymeric diols include polyethylene glycol with a number average molar mass in the range from about 1000 to about 6000. If desired, these polyesters may also be end group-terminated, with end groups that may be alkyl groups with 1 to 22 C atoms or esters of monocarboxylic acids. The end groups attached via ester bonds may be based on alkyl, alkenyl and aryl monocarboxylic acids with 5 to 32 C atoms, or with 5 to 18 C atoms in another embodiment. These include valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, undecenoic acid, lauric acid, lauroleic acid, tridecanoic acid, myristic acid, myristoleic acid, pentadecanoic acid, palmitic acid, stearic acid, petroselinic acid, petroselaidic acid, oleic acid, linoleic acid, linolaidic acid, linolenic acid, eleostearic acid, arachidic acid, gadoleic acid, arachidonic acid, behenic acid, erucic acid, brassidic acid, clupanodonic acid, lignoceric acid, cerotic acid, melissic acid, and benzoic acid. These end groups may bear 1 to 5 substituents having a total of up to 25 C atoms, or 1 to 12 C atoms in another embodiment, for example tert-butylbenzoic acid. The end groups may also be based on hydroxymonocarboxylic acids with 5 to 22 C atoms, which for example include hydroxyvaleric acid, hydroxycaproic acid, ricinoleic acid, hydrogenation products thereof, hydroxystearic acid, and ortho-, meta- and para-hydroxybenzoic acid. The hydroxymonocarboxylic acids may in turn be joined to one another via their hydroxyl group and their carboxyl group and thus be repeatedly present in an end group. The number of hydroxymonocarboxylic acid units per end group, i.e. their degree of oligomerization, may be in the range of from 1 to 50, or in the range of from 1 to 10 in another embodiment. In an exemplary embodiment, polymers of ethylene terephthalate and polyethylene oxide terephthalate, in which the polyethylene glycol units have molar weights of from about 750 to about 5000 and the molar ratio of ethylene terephthalate to polyethylene oxide terephthalate of from about 50:50 to about 90:10, are used alone or in combination with cellulose derivatives. The anti-redeposition agent is present in the wash composition at an amount of from about 0 to about 5 weight percent, or an amount of from about 0 to about 4 weight percent, or an amount of from about 0 to about 3 weight percent, based on the total weight of the wash composition, in various embodiments. Optical brighteners may optionally be included in the wash composition. Optical brighteners adsorb ultraviolet and/or violet light and re-transmit it as visible light, typically a visible blue light. Optical brighteners include, but are not limited to, derivatives of diaminostilbene disulfonic acid or the alkali metal salts thereof. Suitable compounds are, for example, salts of 4,4′-bis(2-anilino-4-morpholino-1,3,5-triazinyl-6-amino)stilbene 2,2′-disulfonic acid or compounds of similar structure which, instead of the morpholino group, bear a diethanolamino group, a methylamino group, an anilino group or a 2-methoxyethylamino group. Optical brighteners of the substituted diphenylstyryl type may furthermore be present, such as the alkali metal salts of 4,4′-bis(2-sulfostyryl)diphenyl, 4,4′-bis(4-chloro-3-sulfostyryl)diphenyl, or 4-(4-chlorostyryl)-4′-(2-sulfostyryl)diphenyl. Mixtures of the above-stated optical brighteners may also be used. Optical brighteners may be present in the wash composition at an amount of from about 0 to about 5 weight percent in some embodiments, but in other embodiments optical brighteners are present in an amount of from about 0.005 to about 5 weight percent, or an amount of from about 0.01 to about 0.5 weight percent, or an amount of from about 0.05 to about 0.3 weight percent, based on the total weight of the wash composition. Bittering agents may optionally be added to hinder accidental ingestion of the unit dose pack or the wash composition. Bittering agents are compositions that taste bad, so children or others are discouraged from accidental ingestion. Exemplary bittering agents include denatonium benzoate, aloin, and others. Bittering agents may be present in the wash composition at an amount of from about 0 to about 1 weight percent, or an amount of from about 0.001 to about 0.5 weight percent, or an amount of from about 0.001 to about 0.25 weight percent in various embodiments, based on the total weight of the wash composition. The wash composition may optionally include sodium sulfite. Sodium sulfite is an oxygen scavenger, where sodium sulfite reacts with oxygen to form sodium sulfate. Free oxygen, such as oxygen dissolved in the wash composition, can react to produce metal oxides (rust) that reduce the life of the washing equipment. The metal oxides can also stain garments, dishes, or other items being washed. Dissolved oxygen can also react to produce other components, and some of those components may be colored bodies. Therefore, the sodium sulfite can help reduce the formation of colored bodies in the wash composition. However, sodium sulfite includes sodium, and sodium-containing compounds tend to produce efflorescent solids in the film. In various embodiments, the sodium sulfite is present in the wash composition at a concentration of from about 0.05 to about 4 weight percent, or from about 0.05 to about 3 weight percent, or from about 0.05 to about 2 weight percent, all based on the total weight of the wash composition. One or more chelating compounds may optionally be present in the wash composition at an amount of from about 0 to about 1.5 weight percent in an exemplary embodiment, but in alternate embodiments the chelating compound is present at an amount of from about 0 to about 1.25 weight percent, or an amount of from about 0 to about 1 weight percent, or an amount of from about 0 to about 0.5 weight percent, based on the total weight of the wash composition. Chelating compounds are sometimes referred to as water softeners. Many compounds can be used as chelating compounds, including but not limited to iminodisuccinate (IDS), ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid, diethylenetriaminepenta(methylenephosphonic acid), nitrilotris(methylenephosphonic acid), 1-hydroxyethane-1,1-diphosphonic acid, ethylenediamine-N,N′-disuccinic acid (EDDS), hydroxyethylenediaminetriacetic acid (HEDTA), or other chelating compounds. In some embodiments, the reverse poloxamer may interact with a chelating compound, and so the wash composition may be free of a chelating compound in some embodiments. One or more fragrances, or compounds that impart a desirable fragrance, may optionally be present in the wash composition in an amount of from about 0 to about 5 weight percent, based on the total weight of the wash composition. In alternate embodiments, the fragrance may be present in an amount of from about 0 to about 2 weight percent, or from about 0 to about 1 weight percent. The fragrance may be an encapsulated fragrance, or a combination of an encapsulated fragrance and fragrance that is not encapsulated, or just a fragrance that is not encapsulated. Encapsulation of fragrances prevents the fragrance from being released prematurely. The encapsulation may be ruptured at some time period after application to the garment, so fresh fragrance may be provided significantly after a garment is removed from a washing machine. The encapsulation may be ruptured by a wide variety of activities, such as physical contact from movement, melting, degradation from sunlight, degradation from oxidation, or other reasons. The encapsulation may be formed by aminoplast or cross-linked gelatin, polymeric materials, or other materials. The fragrance may be neat oil fragrance, an essential oil, botanical extracts, synthetic fragrance materials, or other compounds that provide a desirable odor. Many film manufacturers caution against the use of sodium-containing compounds in a wash composition because sodium can cause efflorescence solids to form in the film. Efflorescence results when a component is carried into the film, and that component or a portion thereof precipitates within the film. The solubility of the efflorescence component may be different in the film than in the wash composition, but other reasons for the efflorescence may also be possible. In any event, efflorescence is undesirable because it causes a “cloudy”, opaque, or otherwise unattractive appearance of the film, as well as gritty or rough feel as opposed to a smooth feel for film free of efflorescence. A chelating compound binds and removes various metals from water, such as calcium, magnesium, sodium, or other metals. The wash composition may be prepared by combining and mixing the components of the wash composition with a mixer. Once mixed, the wash composition is encapsulated in the container. The components of the wash composition may all be mixed at one time, or different components may be pre-mixed and then combined. A wide variety of mixers may be used in alternate embodiments, such as an agitator, an in-line mixer, a ribbon blender, an emulsifier, and others. The wash composition is placed in a container, and then the film of the container is sealed with a sealer, where the sealer may utilize heat, water, ultrasonic techniques, water and heat, pressure, or other techniques for sealing the container and forming the unit dose pack. Another exemplary embodiment contemplated herein is directed to the use of a unit dose pack as described above in a cleaning process, such as laundry and/or hard surface cleaning. In particular, an embodiment is directed to the use of a unit dose pack in laundering of textile and fabrics, such as house hold laundry washing and industrial laundry washing. A further exemplary embodiment is directed to the use of a unit dose pack in hard surface cleaning such as automated dish washing (ADW). The fabrics and/or garments subjected to a washing, cleaning or textile care process contemplated herein may be conventional washable laundry, such as household laundry. In some embodiments, the major part of the laundry is garments and fabrics, including but not limited to knits, woven fabrics, denims, non-woven fabrics, felts, yarns, and toweling. The fabrics may be cellulose based, such as natural cellulosics, including cotton, flax, linen, jute, ramie, sisal or coir or manmade cellulosics (e.g., originating from wood pulp) including viscose/rayon, ramie, cellulose acetate fibers (tricell), lyocell or blends thereof. The fabrics may also be non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit, and silk, or the fabric may be a synthetic polymer such as nylon, aramid, polyester, acrylic, polypropylene and spandex/elastin, or blends of any of the above-mentioned products. Examples of blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g., rayon/viscose, ramie, flax, linen, jute, cellulose acetate fibers, lyocell). In one embodiment, the fabrics and/or garments are added to a washing machine, and the unit dose pack is also added to the washing machine before wash water is added. In an alternate embodiment, the unit dose pack may be added to an automatic detergent addition system of a washing machine, where the contents of the unit dose pack are added to the wash water with the fabrics and/or garments after the washing process has begun. In yet another embodiment, the unit dose pack is manually added to the fabrics and/or garments with the wash water after the washing process has started. The film dissolves and releases the wash composition into the aqueous wash water. The film is dissolved and washes out of the washing machine with the excess wash water, so there is nothing to collect from the fabrics and/or garments after the wash cycle. The fabrics and/or garments are laundered with the wash water and the contents of the unit dose pack. The fabrics and/or garments may then be dried and processed as normal. In an alternate embodiment, the unit dose pack is added to a detergent charging system for an automatic dish washing machine. The detergent charging system opens and releases the unit dose pack to the wash water and a main compartment of the dish washing machine at a designated point in the wash cycle. Examples Samples of test wash compositions were prepared with the ingredients listed in Table 1 below. The non-aqueous solvent portions of the samples were varied while the remainder of the test wash compositions were held constant, where three non-aqueous solvents were evaluated: glycerin; polyethylene glycol with an average number molecular weight of about 400 (PEG 400); and a reverse poloxamer (Pluronic® 5R5, available from BASF). Three trials were run for each test. Test polyvinyl film strips were exposed to the test wash compositions, and the haptics of the test polyvinyl film strips were measured. TABLE 1SampleSampleSampleSampleSampleSampleSampleComponent1234567Polyethylene glycol32.320016.1616.16010.77Reverse poloxamer032.32016.16016.1610.77Glycerin0032.32016.1616.1610.77non-ionic alcohol23.07423.07423.07423.07423.07423.07423.074ethoxylate surfactantMonoethanol amine1.751.751.751.751.751.751.75Zeolite water (does7777777not include waterlisted as part ofother components)Linear alkyl benzene5555555sulfonic acidCoconut fatty acids4444444anionic sodium26262626262626alcohol ethoxysulfate surfactant(60% active in waterand ethanol)Bittering agent0.050.050.050.050.050.050.05(25% active inpropylene glycol)Optical brightener0.20.20.20.20.20.20.2Fragrance0.5850.5850.5850.5850.5850.5850.585Colorant0.0260.0260.0260.0260.0260.0260.0261. All compositions are listed as weight percent, based on a total weight of the wash composition.2. All compositions are at least 99% active, unless otherwise specified.3. Solvents in the compositions that are less than 100% active are not separately listed or totaled. The test results are provided in Table 2 below. The haptics testing included a % weight difference, where the weight of the test polyvinyl film strips was measure before and after a 24 hour exposure period. A spring constant was also measured for the test polyvinyl film strips after the 24 hour exposure, where the spring constant is the force needed to pull and stretch the test film across a 2 millimeter distance. The test polyvinyl strips were initially 2.5 inches (6.35 centimeters) by 1 inch (2.54 centimeters). A % weight gain of 10% or more was set as a failure, and a spring constant of 1.32 newtons or less was set as a failure. The results of the testing were charted in a triangular chart that indicates the ratios of the three non-aqueous solvents tested and the results of the haptics tests (i.e., the % weight gain and the spring constant.) The triangular chart is presented inFIG.1. Three trials were run for each of Samples 1-7, and the average results are listed in Table 2 below. TABLE 2TestSample 1Sample 2Sample 3Sample 4Sample 5Sample 6Sample 7Initial weight0.280.270.280.280.280.260.28Post weight0.290.260.430.280.350.330.33% Weight gain4−4572252317Spring constant4.444.090.963.931.631.352.041. Weights are in grams. Reference is made toFIG.1. The concentration of the reverse poloxamer is shown on the left side of the triangle, the concentration of glycerin is shown on the bottom side of the triangle, and the concentration of the polyethylene glycol is shown on the right hand side of the triangle. The spring constant failure area10is the smaller shaded area in the bottom left hand side of the chart. The spring constant failure area10shows the relative concentrations of the three tested non-aqueous solvents where the spring constant is greater than 1.32 newtons. The weight difference failure area20is the larger shaded area that covers most of the triangle and all of the bottom left hand side of the triangle. The weight difference failure area20overlies and encompasses the spring constant failure area10. The weight difference failure area shows where the film weight gain was greater than 10%. The acceptable haptics area30is the non-shaded area on the right hand side of the triangle. The non-aqueous solvent mixes illustrated in the acceptable haptics area30have a spring constant of higher than 1.32 and a weight difference of less than 10%. Efflorescence was tested for the reverse poloxamer solvent versus the polyethylene glycol solvent. The compositions of Samples 8, 9, 10, and 11 are listed in Table 3, below. The samples were then placed into monochamber unit dose packs at 24 grams of wash composition per unit dose pack. The film used was Aicello® GS-75 on both top and bottom. The samples were aged at 75° F., 105° F., 113° F., and 125° F. for 1 week. After aging 1 week, Samples 9 and 11 (with the reverse poloxamer) had no noticeable efflorescence on the film at any storage temperature. Samples 8 and 10 where not as transparent and felt rough, at all storage temperatures, due to effloresced salts on the film. Samples 9 and 11 were visually brighter than samples 8 and 10 at all storage temperatures as well. Therefore, the reverse poloxamer reduced efflorescence as compared to the polyethylene glycol. TABLE 3SampleSampleSampleSampleComponent891011Glycerin14.99214.99212.03812.0375Polyethylene glycol18.323014.710Reverse poloxamer018.323014.71Non-ionic alcohol ethoxylate23.07423.07423.07423.074surfactantMonoethanolamide1.751.751.751.75Water (does not include water6666as listed in other components.)Linear alkyl benzene sulfonic5555acidCoconut fatty acid4444Anionic sodium alcohol ethoxy26262626sulfate surfactant (60% activein water and ethanol)Bittering agent (25% active in0.050.050.050.05propylene glycol)Optical brightener0.20.20.30.3Fragrance A0.5850.58500Colorant0.0260.0260.0260.026Protease enzyme (8% active0022in water and propylene glycol)Mannanase enzyme (8%000.60.6active in water and propyleneglycol)Amylase enzyme (8% active in000.350.35water and propylene glycol)Anti-redeposition polymer001.61.6Sodium amino disuccinate000.90.9(34% active in water)Fragrance B001.61.61. All compositions are listed as weight percent, based on a total weight of the wash composition.2. All compositions are at least 99% active, unless otherwise specified.3. Solvents in the compositions that are less than 100% active are not separately listed or totaled.4. The non-ionic alcohol ethoxylate surfactant utilized had an average carbon chain length of 12 to 15 and 7 moles of ethoxylation.5. The anionic sodium alcohol ethoxy sulfate surfactant utilized had an average carbon chain length of 12 to 14 and 3 moles of ethoxylation. While at least one exemplary embodiment has been presented in the foregoing detailed description, it should be appreciated that a vast number of variations exist. It should also be appreciated that the exemplary embodiment or exemplary embodiments are only examples, and are not intended to limit the scope, applicability, or configuration of the subject matter in any way. Rather, the foregoing detailed description will provide those skilled in the art with a convenient road map for implementing an exemplary embodiment, it being understood that various changes may be made in the function and arrangement of elements described in an exemplary embodiment without departing from the scope as set forth in the appended claims and their legal equivalents. | 58,213 |
11859155 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention relates to detergent compositions that employ phosphinosuccinic acid and mono-, bis- and oligomeric phosphinosuccinic acid adducts with alkali metal hydroxides, alkali metal silicates, alkali metal metasilicates and combinations thereof. The detergent compositions may further include a compound selected from the group consisting of gluconic acid or salts thereof, a copolymer of acrylic and maleic acids or salts thereof, sodium hypochlorite, sodium dichloroisocyanurate and combinations thereof. The detergent compositions and methods of use thereof have many advantages over conventional alkaline detergents. For example, the detergent compositions minimize soil and hard water scale accumulation on hard surfaces under alkaline conditions from about 10 to about 13.5. The embodiments of this invention are not limited to particular alkaline detergent compositions, and methods of using the same, which can vary and are understood by skilled artisans. It is further to be understood that all terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the content clearly indicates otherwise. Further, all units, prefixes, and symbols may be denoted in its SI accepted form. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. So that the present invention may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present invention without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below. The term “about,” as used herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. The term “cleaning,” as used herein, refers to performing or aiding in any soil removal, bleaching, microbial population reduction, or combination thereof. The term “defoamer” or “defoaming agent,” as used herein, refers to a composition capable of reducing the stability of foam. Examples of defoaming agents include, but are not limited to: ethylene oxide/propylene block copolymers such as those available under the name Pluronic N-3; silicone compounds such as silica dispersed in polydimethylsiloxane, polydimethylsiloxane, and functionalized polydimethylsiloxane such as those available under the name Abil B9952; fatty amides, hydrocarbon waxes, fatty acids, fatty esters, fatty alcohols, fatty acid soaps, ethoxylates, mineral oils, polyethylene glycol esters, and alkyl phosphate esters such as monostearyl phosphate. A discussion of defoaming agents may be found, for example, in U.S. Pat. Nos. 3,048,548, 3,334,147, and 3,442,242, the disclosures of which are incorporated herein by reference. The terms “feed water,” “dilution water,” and “water” as used herein, refer to any source of water that can be used with the methods and compositions of the present invention. Water sources suitable for use in the present invention include a wide variety of both quality and pH, and include but are not limited to, city water, well water, water supplied by a municipal water system, water supplied by a private water system, and/or water directly from the system or well. Water can also include water from a used water reservoir, such as a recycle reservoir used for storage of recycled water, a storage tank, or any combination thereof. Water also includes food process or transport waters. It is to be understood that regardless of the source of incoming water for systems and methods of the invention, the water sources may be further treated within a manufacturing plant. For example, lime may be added for mineral precipitation, carbon filtration may remove odoriferous contaminants, additional chlorine or chlorine dioxide may be used for disinfection or water may be purified through reverse osmosis taking on properties similar to distilled water. As used herein, the term “microorganism” refers to any noncellular or unicellular (including colonial) organism. Microorganisms include all prokaryotes. Microorganisms include bacteria (including cyanobacteria), spores, lichens, fungi, protozoa, virinos, viroids, viruses, phages, and some algae. As used herein, the term “microbe” is synonymous with microorganism. For the purpose of this patent application, successful microbial reduction is achieved when the microbial populations are reduced by at least about 50%, or by significantly more than is achieved by a wash with water. Larger reductions in microbial population provide greater levels of protection. The term “substantially similar cleaning performance” refers generally to achievement by a substitute cleaning product or substitute cleaning system of generally the same degree (or at least not a significantly lesser degree) of cleanliness or with generally the same expenditure (or at least not a significantly lesser expenditure) of effort, or both. The term “weight percent,” “wt-%,” “percent by weight,” “% by weight,” and variations thereof, as used herein, refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, “percent,” “%,” and the like are intended to be synonymous with “weight percent,” “wt-%,” etc. The methods and compositions of the present invention may comprise, consist essentially of, or consist of the components and ingredients of the present invention as well as other ingredients described herein. As used herein, “consisting essentially of” means that the methods and compositions may include additional steps, components or ingredients, but only if the additional steps, components or ingredients do not materially alter the basic and novel characteristics of the claimed methods and compositions. Compositions According to an embodiment of the invention, alkaline detergents incorporate phosphinosuccinic acid (PSO) adducts. In an aspect, the alkaline detergents comprise, consist of and/or consist essentially of phosphinosuccinic acid (PSO) adducts and a source of alkalinity. In a further aspect, the alkaline detergents comprise, consist of and/or consist essentially of phosphinosuccinic acid (PSO) adducts, an alkali metal hydroxide, an alkali metal silicate and/or alkali metal metasilicate, and a polymer, such as polycarboxylic acids or hydrophobically modified polycarboxylic acids. The compositions may also include water, surfactants and/or other polymers, oxidizers, additional functional ingredients and any combination of the same. Additional detergent compositions may incorporate the PSO adducts according to the invention, including for example, those disclosed in U.S. Publication No. 2014/0073550, having beneficial solid, dimensional stability, which is herein incorporated by reference. An example of a suitable detergent composition for use according to the invention may comprise, consist and/or consist essentially of about 1-90 wt-% alkali metal hydroxide (or combinations of alkali metal hydroxide and alkali metal metasilicates and/or alkali metal silicates), from about 1-90 wt-% of the alkalinity source(s) from about 1-50 wt-% of the alkalinity source(s), and preferably about 1-40 wt-% alkali metal hydroxide, alkali metal metasilicates and/or alkali metal silicates; about 0.01-40 wt-% PSO adducts, preferably about 0.1-20 wt-% PSO adducts; about 0-45 wt-% polymers (e.g. polycarboxylic acids and/or hydrophobically modified polycarboxylic acids), preferably from about 0-25 wt-% polymers; and optionally other chelating agents, polymers and/or surfactants, oxidizers, and other functional ingredients, including for example preferably about 0-40 wt-% surfactant, and more preferably from about 0-25 wt-% surfactant. An example of a suitable detergent use solution composition for use according to the invention may comprise, consist and/or consist essentially of about from about 100-20,000 ppm of an alkalinity source, from about 1-2,000 ppm phosphinosuccinic acid adducts, and from about 1-1,000 ppm of a polymer having a use pH of between about 10 and about 13.5. Further description of suitable formulations is shown below: FormulationsWater0-90 wt-%20-90 wt-%40-80 wt-%Alkalinity source (e.g.1-90 wt-%1-50 wt-%1-40 wt-%sodium hydroxide (beads)and/or alkali metal silicatesand/or metasilicates)PSO adducts0.01-40 wt-%0.1-20 wt-%0.1-10 wt-%Optional Polymers (e.g.0-45 wt-%0-25 wt-%0-10 wt-%poly carboxylic acids)Optional Surfactant(s)0-40 wt-%0-25 wt-%0-10 wt-%Optional Additional Agents0-40 wt-%0-25 wt-%0-20 wt-% Use solutions of the detergent compositions have a pH greater than about 10. In further aspects, the pH of the detergent composition use solution is between about 10 and 13.5. Beneficially, the detergent compositions of the invention provide effective prevention of hardness scale accumulation on treated surfaces at such alkaline pH conditions. Without being limited to a particular theory of the invention, it is unexpected to have effective cleaning without the accumulation of hardness scaling at alkaline conditions above pH about 10 wherein alkalinity sources (e.g. sodium hydroxide, sodium metasilicate and/or sodium silicate) are employed. Beneficially, alkaline compositions according to the invention may be provided in various forms, including liquids, solids, powders, pastes and/or gels. Moreover, the alkaline compositions can be provided in use concentration and/or concentrates, such that use solutions may be obtained at a point of use or may be used without further dilution in the case of concentrate compositions. The alkaline compositions are suitable for dilution with a water source. Phosphinosuccinic Acid (PSO) Adducts The detergent compositions employ phosphinosuccinic acid (PSO) adducts providing water conditioning benefits including the reduction of hardness scale buildup. PSO adducts may also be described as phosphonic acid-based compositions. In an aspect of the invention, the PSO adducts are a combination of mono-, bis- and oligomeric phosphinosuccinic acid adducts and a phosphinosuccinic acid (PSA) adduct. The phosphinosuccinic acid (PSA) adducts have the formula (I) below: The mono-phosphinosuccinic acid adducts have the formula (II) below: The bis-phosphinosuccinic acid adducts have the formula (III) below: An exemplary structure for the oligomeric phosphinosuccinic acid adducts is shown in formula (IV) below: where M is H+, Na+, K+, NH4+, or mixtures thereof; and the sum of m plus n is greater than 2. In an aspect, the phosphinosuccinic acid adducts are a combination of various phosphinosuccinic acid adducts as shown in Formulas I-IV. In a preferred aspect, the phosphinosuccinic acid adduct of formula I constitutes between about 1-40 wt-% of the phosphinosuccinic acid adducts, the phosphinosuccinic acid adduct of formula II constitutes between about 1-25 wt-% of the phosphinosuccinic acid adducts, the phosphinosuccinic acid adduct of formula III constitutes between about 10-60 wt-% of the phosphinosuccinic acid adducts, the phosphinosuccinic acid adduct of formula IV constitutes between about 20-70 wt-% of the phosphinosuccinic acid adduct. Without being limited according to embodiments of the invention, all recited ranges for the phosphinosuccinic acid adducts are inclusive of the numbers defining the range and include each integer within the defined range. Additional oligomeric phosphinosuccinic acid adduct structures are set forth for example in U.S. Pat. Nos. 5,085,794, 5,023,000 and 5,018,577, each of which are incorporated herein by reference in their entirety. The oligomeric species may also contain esters of phosphinosuccinic acid, where the phosphonate group is esterified with a succinate-derived alkyl group. Furthermore, the oligomeric phosphinosuccinic acid adduct may comprise 1-20 wt % of additional monomers selected, including, but not limited to acrylic acid, methacrylic acid, itaconic acid, 2-acylamido-2-methylpropane sulfonic acid (AMPS), and acrylamide. The adducts of formula I, II, III and IV may be used in the acid or salt form. Further, in addition to the phosphinosuccinic acids and oligomeric species, the mixture may also contain some phosphinosuccinic acid adduct (I) from the oxidation of adduct II, as well as impurities such as various inorganic phosphorous byproducts of formula H2PO2—, HPO32−and PO43−. In an aspect, the mono-, bis- and oligomeric phosphinosuccinic acid adducts and the phosphinosuccinic acid (PSA) may be provided in the following mole and weight ratios as shown in Table 1. TABLE 1Species:MonoPSABisOligomerFormulaC4H7PO6C4H7PO7C8H11PO10C14.1H17.1PO16.1MW182198298475.5 (avg)Mole fraction0.2380.0270.4220.309(by NMR)Wt. fraction (as0.1350.0170.3910.457acid) Detergent compositions and methods of use may employ the phosphinosuccinic acid adducts and may include one or more of PSO adducts selected from mono-, bis- and oligomeric phosphinosuccinic acid and a phosphinosuccinic acid, wherein at least about 10 mol % of the adduct comprises a succinic acid:phosphorus ratio of about 1:1 to about 20:1. More preferably, the phosphinosuccinic acid adduct may include one or more of the PSO adducts selected from mono-, bis- and oligomeric phosphinosuccinic acid and optionally a phosphinosuccinic acid wherein at least about 10 mol % of the adduct comprises a succinic acid:phosphorus ratio of about 1:1 to about 15:1. Most preferably, the phosphinosuccinic acid adduct may include one or more adducts selected from mono-, bis- and oligomeric phosphinosuccinic acid and optionally a phosphinosuccinic acid wherein at least about 10 mol % of the adduct comprises a succinic acid:phosphorus ratio of about 1:1 to about 10:1. Additional description of suitable mono-, bis- and oligomeric phosphinosuccinic acid adducts for use as the PSO adducts of the present invention is provided in U.S. Pat. No. 6,572,789 which is incorporated herein by reference in its entirety. In aspects of the invention the detergent composition is nitrilotriacetic acid (NTA)-free to meet certain regulations. In additional aspects of the invention the detergent composition may be substantially phosphorous (and phosphate) free to meet certain regulations. The PSO adducts of the claimed invention may provide substantially phosphorous (and phosphate) free detergent compositions having less than about 0.5 wt-% of phosphorus (and phosphate). More preferably, the amount of phosphorus is a detergent composition may be less than about 0.1 wt-%. Accordingly, it is a benefit of the detergent compositions of the present invention to provide detergent compositions capable of controlling (i.e. preventing) hardness scale accumulation and soil redeposition on a substrate surface without the use of phosphates, such as tripolyphosphates including sodium tripolyphosphate, commonly used in detergents to prevent hardness scale and/or accumulation. Alkalinity Source According to an embodiment of the invention, the detergent compositions include an alkalinity source. Exemplary alkalinity sources include alkali metal hydroxides. In various aspects, a combination of both alkali metal hydroxides and alkali metal silicates and/or alkali metal metasilicates are employed as the alkalinity source. Alkali metal hydroxides used in the formulation of detergents are often referred to as caustic detergents. Examples of suitable alkali metal hydroxides include sodium hydroxide, potassium hydroxide, and lithium hydroxide. The alkali metal hydroxides may be added to the composition in any form known in the art, including as solid beads, dissolved in an aqueous solution, or a combination thereof. Alkali metal hydroxides are commercially available as a solid in the form of prilled solids or beads having a mix of particle sizes ranging from about 12-100 U.S. mesh, or as an aqueous solution, as for example, as a 45% and a 50% by weight solution. In addition to the first alkalinity source, i.e. the alkali metal hydroxide, the detergent composition may comprise a secondary alkalinity source. Examples of useful secondary alkaline sources include, but are not limited to: alkali metal silicates or metasilicates, such as sodium or potassium silicate or metasilicate; and ethanolamines and amines. Such alkalinity agents are commonly available in either aqueous or powdered form, either of which is useful in formulating the present detergent compositions. An effective amount of one or more alkalinity sources is provided in the detergent composition. An effective amount is referred to herein as an amount that provides a use composition having a pH of at least about 10, preferably at least about 10.5. When the use composition has a pH of about 10, it can be considered mildly alkaline, and when the pH is greater than about 12, the use composition can be considered caustic. In some circumstances, the detergent composition may provide a use composition that has a pH between about 10 and about 13.5. Additional Functional Ingredients The components of the detergent composition can be combined with various additional functional ingredients. In some embodiments, the detergent composition including the PSO adducts and alkalinity source(s) make up a large amount, or even substantially all of the total weight of the detergent composition, for example, in embodiments having few or no additional functional ingredients disposed therein. In these embodiments, the component concentrations ranges provided above for the detergent composition are representative of the ranges of those same components in the detergent composition. In other aspects, the detergent compositions include PSO adducts, alkali metal hydroxide and/or alkali metal silicate and/or metasilicate alkalinity source(s), threshold active polymer(s)/surfactant(s), and water, having few or no additional functional ingredients disposed therein. In still other aspects, the detergent compositions include PSO adducts, alkali metal hydroxide alkalinity source and/or alkali metal silicates and/or metasilicate, and a polycarboxylic acid polymer and/or hydrophobically modified polycarboxylic acid polymer, having few or no additional functional ingredients disposed therein. The functional ingredients provide desired properties and functionalities to the detergent composition. For the purpose of this application, the term “functional ingredients” includes an ingredient that when dispersed or dissolved in a use and/or concentrate, such as an aqueous solution, provides a beneficial property in a particular use. Some particular examples of functional ingredients are discussed in more detail below, although the particular materials discussed are given by way of example only, and that a broad variety of other functional ingredients may be used. For example, many of the functional ingredients discussed below relate to materials used in cleaning applications. However, other embodiments may include functional ingredients for use in other applications. Exemplary additional functional ingredients include for example: builders or water conditioners, including detergent builders; hardening agents; bleaching agents; fillers; defoaming agents; anti-redeposition agents; stabilizing agents; dispersants; oxidizers; chelants; fragrances and dyes; thickeners; etc. Further description of suitable additional functional ingredients is set forth in U.S. Patent Publication No. 2012/0165237, which is incorporated herein by reference in its entirety. Polymers In some embodiments, the compositions of the present invention include a water conditioning polymer. Water conditioning polymers suitable for use with the compositions of the present invention include, but are not limited to polycarboxylates or polycarboxylic acids. Exemplary polycarboxylates that can be used as builders and/or water conditioning polymers include, but are not limited to: those having pendant carboxylate (—CO2−) groups such as acrylic homopolymers, polyacrylic acid, maleic acid, maleic/olefin copolymer, sulfonated copolymer or terpolymer, acrylic/maleic copolymer, polymethacrylic acid, acrylic acid-methacrylic acid copolymers, hydrolyzed polyacrylamide, hydrolyzed polymethacrylamide, hydrolyzed polyamide-methacrylamide copolymers, hydrolyzed polyacrylonitrile, hydrolyzed polymethacrylonitrile, and hydrolyzed acrylonitrile-methacrylonitrile copolymers. In another aspect, the polycarboxylic acid polymer may be a non-phosphorus polymer. In a still further aspect, the polycarboxylic acid polymer may be hydrophobically modified. In a still further aspect, the polycarboxylic acid polymer may be a neutralized polycarboxylic acid polymer. An example of a suitable commercially-available polymer includes Acumer® 1000 (available from Dow Chemical). For a further discussion of water conditioning polymers, see Kirk-Othmer, Encyclopedia of Chemical Technology, Third Edition, volume 5, pages 339-366 and volume 23, pages 319-320, the disclosure of which is incorporated by reference herein. In an aspect where a water conditioning polymer is employed, it is preferred that between about 0-45 wt-% polymer are included in the composition, preferably from about 0-25 wt-% polymer, and more preferably from about 0-10 wt-% polymer. Surfactants In some embodiments, the compositions of the present invention include at least one surfactant. Surfactants suitable for use with the compositions of the present invention include, but are not limited to, anionic surfactants, nonionic surfactants, cationic surfactants, amphoteric surfactants and/or zwitterionic surfactants. In a preferred aspect, anionic surfactants are employed. In some embodiments, the compositions of the present invention include about 0-40 wt-% of a surfactant. In other embodiments the compositions of the present invention include about 0-25 wt-% of a surfactant. In certain embodiments of the invention the detergent composition does not require a surfactant and/or other polymer in addition to the PSO adducts. In alternative embodiments, the detergent compositions employ at least one anionic surfactant to provide improved detergency to the composition. In an embodiment, the detergent composition employs a sulfonate, sulphate or carboxylate anionic surfactant. In a further embodiment, the detergent compositions employ at least one nonionic surfactant and an anionic surfactant. Anionic Surfactants Also useful in the present invention are surface active substances which are categorized as anionics because the charge on the hydrophobe is negative; or surfactants in which the hydrophobic section of the molecule carries no charge unless the pH is elevated to neutrality or above (e.g. carboxylic acids). Carboxylate, sulfonate, sulfate and phosphate are the polar (hydrophilic) solubilizing groups found in anionic surfactants. Of the cations (counter ions) associated with these polar groups, sodium, lithium and potassium impart water solubility; ammonium and substituted ammonium ions provide both water and oil solubility; and, calcium, barium, and magnesium promote oil solubility. Generally, anionics have high foam profiles which may limit applications of use for cleaning systems such as CIP circuits that require strict foam control. However, other applications of use, including high foaming applications are suitable for using anionic surface active compounds to impart special chemical or physical properties. The majority of large volume commercial anionic surfactants can be subdivided into five major chemical classes and additional sub-groups known to those of skill in the art and described in “Surfactant Encyclopedia,” Cosmetics & Toiletries, Vol. 104 (2) 71-86 (1989). The first class includes acylamino acids (and salts), such as acylgluamates, acyl peptides, sarcosinates (e.g. N-acyl sarcosinates), taurates (e.g. N-acyl taurates and fatty acid amides of methyl tauride), and the like. The second class includes carboxylic acids (and salts), such as alkanoic acids (and alkanoates), ester carboxylic acids (e.g. alkyl succinates), ether carboxylic acids, and the like. The third class includes sulfonic acids (and salts), such as isethionates (e.g. acyl isethionates), alkylaryl sulfonates, alkyl sulfonates, sulfosuccinates (e.g. monoesters and diesters of sulfosuccinate), and the like. The fifth class includes sulfuric acid esters (and salts), such as alkyl ether sulfates, alkyl sulfates, and the like. Anionic sulfonate surfactants suitable for use in the present compositions include alkyl sulfonates, the linear and branched primary and secondary alkyl sulfonates, and the aromatic sulfonates with or without substituents. Anionic sulfate surfactants suitable for use in the present compositions include alkyl ether sulfates, alkyl sulfates, the linear and branched primary and secondary alkyl sulfates, alkyl ethoxysulfates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxide ether sulfates, the C5-C17acyl-N—(C1-C4alkyl) and —N—(C1-C2hydroxyalkyl) glucamine sulfates, and sulfates of alkylpolysaccharides such as the sulfates of alkylpolyglucoside, and the like. Also included are the alkyl sulfates, alkyl poly(ethyleneoxy) ether sulfates and aromatic poly(ethyleneoxy) sulfates such as the sulfates or condensation products of ethylene oxide and nonyl phenol (usually having 1 to 6 oxyethylene groups per molecule). Particularly suitable anionic sulfonates include alkyldiphenyloxide disulfonates, including for example C6 alkylated diphenyl oxide disulfonic acid, commercially-available under the tradename Dowfax. Anionic carboxylate surfactants suitable for use in the present compositions include carboxylic acids (and salts), such as alkanoic acids (and alkanoates), ester carboxylic acids (e.g. alkyl succinates), ether carboxylic acids, and the like. Such carboxylates include alkyl ethoxy carboxylates, alkyl aryl ethoxy carboxylates, alkyl polyethoxy polycarboxylate surfactants and soaps (e.g. alkyl carboxyls). Secondary carboxylates useful in the present compositions include those which contain a carboxyl unit connected to a secondary carbon. The secondary carbon can be in a ring structure, e.g. as in p-octyl benzoic acid, or as in alkyl-substituted cyclohexyl carboxylates. The secondary carboxylate surfactants typically contain no ether linkages, no ester linkages and no hydroxyl groups. Further, they typically lack nitrogen atoms in the head-group (amphiphilic portion). Suitable secondary soap surfactants typically contain 11-13 total carbon atoms, although more carbons atoms (e.g., up to 16) can be present. Suitable carboxylates also include acylamino acids (and salts), such as acylgluamates, acyl peptides, sarcosinates (e.g. N-acyl sarcosinates), taurates (e.g. N-acyl taurates and fatty acid amides of methyl tauride), and the like. Suitable anionic carboxylate surfactants may further include polycarboxylates or related copolymers. A variety of such polycarboxylate polymers and copolymers are known and described in patent and other literature, and are available commercially. Exemplary polycarboxylates that may be utilized according to the invention include for example: homopolymers and copolymers of polyacrylates; polymethacrylates; polymalates; materials such as acrylic, olefinic and/or maleic polymers and/or copolymers. Various examples of commercially-available agents, namely acrylic-maleic acid copolymers include, for example: Acusol 445N and Acusol 448 (available from Dow Chemical. Examples of suitable acrylic-maleic acid copolymers include, but are not limited to, acrylic-maleic acid copolymers having a molecular weight of between about 1,000 to about 100,000 g/mol, particularly between about 1,000 and about 75,000 g/mol and more particularly between about 1,000 and about 50,000 g/mol. Suitable anionic surfactants include alkyl or alkylaryl ethoxy carboxylates of the following formula: R—O—(CH2CH2O)n(CH2)m—CO2X (3) in which R is a C8to C22alkyl group or in which R1is a C4-C16alkyl group; n is an integer of 1-20; m is an integer of 1-3; and X is a counter ion, such as hydrogen, sodium, potassium, lithium, ammonium, or an amine salt such as monoethanolamine, diethanolamine or triethanolamine. In some embodiments, n is an integer of 4 to 10 and m is 1. In some embodiments, R is a C5-C16alkyl group. In some embodiments, R is a C12-C14alkyl group, n is 4, and m is 1. In other embodiments, R is and R1is a C6-C12alkyl group. In still yet other embodiments, R1is a C9alkyl group, n is 10 and m is 1. Such alkyl and alkylaryl ethoxy carboxylates are commercially available. These ethoxy carboxylates are typically available as the acid forms, which can be readily converted to the anionic or salt form. Commercially available carboxylates include, Neodox 23-4, a C12-13alkyl polyethoxy (4) carboxylic acid (Shell Chemical), and Emcol CNP-110, a C9alkylaryl polyethoxy (10) carboxylic acid (Witco Chemical). Carboxylates are also available from Clariant, e.g. the product Sandopan® DTC, a C13alkyl polyethoxy (7) carboxylic acid. Nonionic Surfactants Suitable nonionic surfactants suitable for use with the compositions of the present invention include alkoxylated surfactants. Suitable alkoxylated surfactants include EO/PO copolymers, capped EO/PO copolymers, alcohol alkoxylates, capped alcohol alkoxylates, mixtures thereof, or the like. Suitable alkoxylated surfactants for use as solvents include EO/PO block copolymers, such as the Pluronic® and reverse Pluronic® surfactants; alcohol alkoxylates; capped alcohol alkoxylates; mixtures thereof, or the like. Useful nonionic surfactants are generally characterized by the presence of an organic hydrophobic group and an organic hydrophilic group and are typically produced by the condensation of an organic aliphatic, alkyl aromatic or polyoxyalkylene hydrophobic compound with a hydrophilic alkaline oxide moiety which in common practice is ethylene oxide or a polyhydration product thereof, polyethylene glycol. Practically any hydrophobic compound having a hydroxyl, carboxyl, amino, or amido group with a reactive hydrogen atom can be condensed with ethylene oxide, or its polyhydration adducts, or its mixtures with alkoxylenes such as propylene oxide to form a nonionic surface-active agent. The length of the hydrophilic polyoxyalkylene moiety which is condensed with any particular hydrophobic compound can be readily adjusted to yield a water dispersible or water soluble compound having the desired degree of balance between hydrophilic and hydrophobic properties. Block polyoxypropylene-polyoxyethylene polymeric compounds based upon propylene glycol, ethylene glycol, glycerol, trimethylolpropane, and ethylenediamine as the initiator reactive hydrogen compound are suitable nonionic surfactants. Examples of polymeric compounds made from a sequential propoxylation and ethoxylation of initiator are commercially available under the trade names Pluronic® and Tetronic® manufactured by BASF Corp. Pluronic® compounds are difunctional (two reactive hydrogens) compounds formed by condensing ethylene oxide with a hydrophobic base formed by the addition of propylene oxide to the two hydroxyl groups of propylene glycol. This hydrophobic portion of the molecule weighs from about 1,000 to about 4,000. Ethylene oxide is then added to sandwich this hydrophobe between hydrophilic groups, controlled by length to constitute from about 10% by weight to about 80% by weight of the final molecule. Tetronic® compounds are tetra-functional block copolymers derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine. The molecular weight of the propylene oxide hydrotype ranges from about 500 to about 7,000; and, the hydrophile, ethylene oxide, is added to constitute from about 10% by weight to about 80% by weight of the molecule. Semi-Polar Nonionic Surfactants The semi-polar type of nonionic surface active agents are another class of nonionic surfactant useful in compositions of the present invention. Semi-polar nonionic surfactants include the amine oxides, phosphine oxides, sulfoxides and their alkoxylated derivatives. Amine oxides are tertiary amine oxides corresponding to the general formula: wherein the arrow is a conventional representation of a semi-polar bond; and, R1, R2, and R3may be aliphatic, aromatic, heterocyclic, alicyclic, or combinations thereof. Generally, for amine oxides of detergent interest, R1is an alkyl radical of from about 8 to about 24 carbon atoms; R2and R3are alkyl or hydroxyalkyl of 1-3 carbon atoms or a mixture thereof; R2and R3can be attached to each other, e.g. through an oxygen or nitrogen atom, to form a ring structure; R4is an alkylene or a hydroxyalkylene group containing 2 to 3 carbon atoms; and n ranges from 0 to about 20. An amine oxide can be generated from the corresponding amine and an oxidizing agent, such as hydrogen peroxide. Useful semi-polar nonionic surfactants also include the water soluble phosphine oxides having the following structure: wherein the arrow is a conventional representation of a semi-polar bond; and, R1is an alkyl, alkenyl or hydroxyalkyl moiety ranging from 10 to about 24 carbon atoms in chain length; and, R2and R3are each alkyl moieties separately selected from alkyl or hydroxyalkyl groups containing 1 to 3 carbon atoms. Examples of useful phosphine oxides include dimethyldecylphosphine oxide, dimethyltetradecylphosphine oxide, methylethyltetradecylphosphone oxide, dimethylhexadecylphosphine oxide, diethyl-2-hydroxyoctyldecylphosphine oxide, bis(2-hydroxyethyl)dodecylphosphine oxide, and bis(hydroxymethyl)tetradecylphosphine oxide. Useful water soluble amine oxide surfactants are selected from the octyl, decyl, dodecyl, isododecyl, coconut, or tallow alkyl di-(lower alkyl) amine oxides, specific examples of which are octyldimethylamine oxide, nonyldimethylamine oxide, decyldimethylamine oxide, undecyldimethylamine oxide, dodecyldimethylamine oxide, iso-dodecyldimethyl amine oxide, tridecyldimethylamine oxide, tetradecyldimethylamine oxide, pentadecyldimethylamine oxide, hexadecyldimethylamine oxide, heptadecyldimethylamine oxide, octadecyldimethylaine oxide, dodecyldipropylamine oxide, tetradecyldipropylamine oxide, hexadecyldipropylamine oxide, tetradecyldibutylamine oxide, octadecyldibutylamine oxide, bis(2-hydroxyethyl)dodecylamine oxide, bis(2-hydroxyethyl)-3-dodecoxy-1-hydroxypropylamine oxide, dimethyl-(2-hydroxydodecyl)amine oxide, 3,6,9-trioctadecyldimethylamine oxide and 3-dodecoxy-2-hydroxypropyldi-(2-hydroxyethyl)amine oxide. Semi-polar nonionic surfactants useful herein also include the water soluble sulfoxide compounds which have the structure: wherein the arrow is a conventional representation of a semi-polar bond; and, R1is an alkyl or hydroxyalkyl moiety of about 8 to about 28 carbon atoms, from 0 to about 5 ether linkages and from 0 to about 2 hydroxyl substituents; and R2is an alkyl moiety consisting of alkyl and hydroxyalkyl groups having 1 to 3 carbon atoms. Useful examples of these sulfoxides include dodecyl methyl sulfoxide; 3-hydroxy tridecyl methyl sulfoxide; 3-methoxy tridecyl methyl sulfoxide; and 3-hydroxy-4-dodecoxybutyl methyl sulfoxide. Preferred semi-polar nonionic surfactants for the compositions of the invention include dimethyl amine oxides, such as lauryl dimethyl amine oxide, myristyl dimethyl amine oxide, cetyl dimethyl amine oxide, combinations thereof, and the like. Alkoxylated amines or, most particularly, alcohol alkoxylated/aminated/alkoxylated surfactants are also suitable for use according to the invention. These non-ionic surfactants may be at least in part represented by the general formulae: R20—(PO)SN-(EO)tH, R20—(PO)SN-(EO)tH(EO)tH, and R20—N(EO)tH; in which R20is an alkyl, alkenyl or other aliphatic group, or an alkyl-aryl group of from 8 to 20, preferably 12 to 14 carbon atoms, EO is oxyethylene, PO is oxypropylene, s is 1 to 20, preferably 2-5, t is 1-10, preferably 2-5, and u is 1-10, preferably 2-5. Other variations on the scope of these compounds may be represented by the alternative formula: R20—(PO)V—N[(EO)wH][(EO)zMH] in which R20is as defined above, v is 1 to 20 (e.g., 1, 2, 3, or 4 (preferably 2)), and w and z are independently 1-10, preferably 2-5. These compounds are represented commercially by a line of products sold by Huntsman Chemicals as nonionic surfactants. Amphoteric Surfactants Amphoteric, or ampholytic, surfactants contain both a basic and an acidic hydrophilic group and an organic hydrophobic group. These ionic entities may be any of anionic or cationic groups described herein for other types of surfactants. A basic nitrogen and an acidic carboxylate group are the typical functional groups employed as the basic and acidic hydrophilic groups. In a few surfactants, sulfonate, sulfate, phosphonate or phosphate provide the negative charge. Amphoteric surfactants can be broadly described as derivatives of aliphatic secondary and tertiary amines, in which the aliphatic radical may be straight chain or branched and wherein one of the aliphatic substituents contains from about 8 to 18 carbon atoms and one contains an anionic water solubilizing group, e.g., carboxy, sulfo, sulfato, phosphato, or phosphino. Amphoteric surfactants are subdivided into two major classes known to those of skill in the art and described in “Surfactant Encyclopedia” Cosmetics & Toiletries, Vol. 104 (2) 69-71 (1989), which is herein incorporated by reference in its entirety. The first class includes acyl/dialkyl ethylenediamine derivatives (e.g. 2-alkyl hydroxyethyl imidazoline derivatives) and their salts. The second class includes N-alkylamino acids and their salts. Some amphoteric surfactants can be envisioned as fitting into both classes. Amphoteric surfactants can be synthesized by methods known to those of skill in the art. For example, 2-alkyl hydroxyethyl imidazoline is synthesized by condensation and ring closure of a long chain carboxylic acid (or a derivative) with dialkyl ethylenediamine. Commercial amphoteric surfactants are derivatized by subsequent hydrolysis and ring-opening of the imidazoline ring by alkylation—for example with chloroacetic acid or ethyl acetate. During alkylation, one or two carboxy-alkyl groups react to form a tertiary amine and an ether linkage with differing alkylating agents yielding different tertiary amines. Long chain imidazole derivatives having application in the present invention generally have the general formula: wherein R is an acyclic hydrophobic group containing from about 8 to 18 carbon atoms and M is a cation to neutralize the charge of the anion, generally sodium. Commercially prominent imidazoline-derived amphoterics that can be employed in the present compositions include for example: Cocoamphopropionate, Cocoamphocarboxy-propionate, Cocoamphoglycinate, Cocoamphocarboxy-glycinate, Cocoamphopropyl-sulfonate, and Cocoamphocarboxy-propionic acid. Amphocarboxylic acids can be produced from fatty imidazolines in which the dicarboxylic acid functionality of the amphodicarboxylic acid is diacetic acid and/or dipropionic acid. The carboxymethylated compounds (glycinates) described herein above frequently are called betaines. Betaines are a special class of amphoteric discussed herein below in the section entitled, Zwitterion Surfactants. Long chain N-alkylamino acids are readily prepared by reaction RNH2, in which R═C8-C18straight or branched chain alkyl, fatty amines with halogenated carboxylic acids. Alkylation of the primary amino groups of an amino acid leads to secondary and tertiary amines. Alkyl substituents may have additional amino groups that provide more than one reactive nitrogen center. Most commercial N-alkylamine acids are alkyl derivatives of beta-alanine or beta-N(2-carboxyethyl) alanine. Examples of commercial N-alkylamino acid ampholytes having application in this invention include alkyl beta-amino dipropionates, RN(C2H4COOM)2and RNHC2H4COOM. In an embodiment, R can be an acyclic hydrophobic group containing from about 8 to about 18 carbon atoms, and M is a cation to neutralize the charge of the anion. Suitable amphoteric surfactants include those derived from coconut products such as coconut oil or coconut fatty acid. Additional suitable coconut derived surfactants include as part of their structure an ethylenediamine moiety, an alkanolamide moiety, an amino acid moiety, e.g., glycine, or a combination thereof; and an aliphatic substituent of from about 8 to 18 (e.g., 12) carbon atoms. Such a surfactant can also be considered an alkyl amphodicarboxylic acid. These amphoteric surfactants can include chemical structures represented as: C12-alkyl-C(O)—NH—CH2—CH2—N+(CH2—CH2—CO2Na)2—CH2—CH2—OH or C12-alkyl-C(O)—N(H)—CH2—CH2—N+(CH2—CO2Na)2—CH2—CH2—OH. Disodium cocoampho dipropionate is one suitable amphoteric surfactant and is commercially available under the tradename Miranol™ FBS from Rhodia Inc., Cranbury, N.J. Another suitable coconut derived amphoteric surfactant with the chemical name disodium cocoampho diacetate is sold under the tradename Mirataine™ JCHA, also from Rhodia Inc., Cranbury, N.J. A typical listing of amphoteric classes, and species of these surfactants, is given in U.S. Pat. No. 3,929,678 issued to Laughlin and Heuring on Dec. 30, 1975. Further examples are given in “Surface Active Agents and Detergents” (Vol. I and II by Schwartz, Perry and Berch), which is herein incorporated by reference in its entirety. Cationic Surfactants Surface active substances are classified as cationic if the charge on the hydrotrope portion of the molecule is positive. Surfactants in which the hydrotrope carries no charge unless the pH is lowered close to neutrality or lower, but which are then cationic (e.g. alkyl amines), are also included in this group. In theory, cationic surfactants may be synthesized from any combination of elements containing an “onium” structure RnX+Y—and could include compounds other than nitrogen (ammonium) such as phosphorus (phosphonium) and sulfur (sulfonium). In practice, the cationic surfactant field is dominated by nitrogen containing compounds, probably because synthetic routes to nitrogenous cationics are simple and straightforward and give high yields of product, which can make them less expensive. Cationic surfactants preferably include, more preferably refer to, compounds containing at least one long carbon chain hydrophobic group and at least one positively charged nitrogen. The long carbon chain group may be attached directly to the nitrogen atom by simple substitution; or more preferably indirectly by a bridging functional group or groups in so-called interrupted alkylamines and amido amines. Such functional groups can make the molecule more hydrophilic and/or more water dispersible, more easily water solubilized by co-surfactant mixtures, and/or water soluble. For increased water solubility, additional primary, secondary or tertiary amino groups can be introduced or the amino nitrogen can be quaternized with low molecular weight alkyl groups. Further, the nitrogen can be a part of branched or straight chain moiety of varying degrees of unsaturation or of a saturated or unsaturated heterocyclic ring. In addition, cationic surfactants may contain complex linkages having more than one cationic nitrogen atom. The surfactant compounds classified as amine oxides, amphoterics and zwitterions are themselves typically cationic in near neutral to acidic pH solutions and can overlap surfactant classifications. Polyoxyethylated cationic surfactants generally behave like nonionic surfactants in alkaline solution and like cationic surfactants in acidic solution. The simplest cationic amines, amine salts and quaternary ammonium compounds can be schematically drawn thus: in which, R represents a long alkyl chain, R′, R″, and R′″ may be either long alkyl chains or smaller alkyl or aryl groups or hydrogen and X represents an anion. The amine salts and quaternary ammonium compounds are preferred for practical use in this invention due to their high degree of water solubility. The majority of large volume commercial cationic surfactants can be subdivided into four major classes and additional sub-groups known to those or skill in the art and described in “Surfactant Encyclopedia”, Cosmetics & Toiletries, Vol. 104 (2) 86-96 (1989), which is herein incorporated by reference in its entirety. The first class includes alkylamines and their salts. The second class includes alkyl imidazolines. The third class includes ethoxylated amines. The fourth class includes quaternaries, such as alkylbenzyldimethylammonium salts, alkyl benzene salts, heterocyclic ammonium salts, tetra alkylammonium salts, and the like. Cationic surfactants are known to have a variety of properties that can be beneficial in the present compositions. These desirable properties can include detergency in compositions of or below neutral pH, antimicrobial efficacy, thickening or gelling in cooperation with other agents, and the like. Cationic surfactants useful in the compositions of the present invention include those having the formula R1mR2×YLZ wherein each R1 is an organic group containing a straight or branched alkyl or alkenyl group optionally substituted with up to three phenyl or hydroxy groups and optionally interrupted by up to four of the following structures: or an isomer or mixture of these structures, and which contains from about 8 to 22 carbon atoms. The R1 groups can additionally contain up to 12 ethoxy groups. m is a number from 1 to 3. Preferably, no more than one R1 group in a molecule has 16 or more carbon atoms when m is 2 or more than 12 carbon atoms when m is 3. Each R2 is an alkyl or hydroxyalkyl group containing from 1 to 4 carbon atoms or a benzyl group with no more than one R2 in a molecule being benzyl, and x is a number from 0 to 11, preferably from 0 to 6. The remainder of any carbon atom positions on the Y group are filled by hydrogens. Y is can be a group including, but not limited to: or a mixture thereof. Preferably, L is 1 or 2, with the Y groups being separated by a moiety selected from R1 and R2 analogs (preferably alkylene or alkenylene) having from 1 to about 22 carbon atoms and two free carbon single bonds when L is 2. Z is a water soluble anion, such as a halide, sulfate, methylsulfate, hydroxide, or nitrate anion, particularly preferred being chloride, bromide, iodide, sulfate or methyl sulfate anions, in a number to give electrical neutrality of the cationic component. Zwitterionic Surfactants Zwitterionic surfactants can be thought of as a subset of the amphoteric surfactants and can include an anionic charge. Zwitterionic surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosphonium or tertiary sulfonium compounds. Typically, a zwitterionic surfactant includes a positive charged quaternary ammonium or, in some cases, a sulfonium or phosphonium ion; a negative charged carboxyl group; and an alkyl group. Zwitterionics generally contain cationic and anionic groups which ionize to a nearly equal degree in the isoelectric region of the molecule and which can develop strong “inner-salt” attraction between positive-negative charge centers. Examples of such zwitterionic synthetic surfactants include derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds, in which the aliphatic radicals can be straight chain or branched, and wherein one of the aliphatic substituents contains from 8 to 18 carbon atoms and one contains an anionic water solubilizing group, e.g., carboxy, sulfonate, sulfate, phosphate, or phosphonate. Betaine and sultaine surfactants are exemplary zwitterionic surfactants for use herein. A general formula for these compounds is: wherein R1contains an alkyl, alkenyl, or hydroxyalkyl radical of from 8 to 18 carbon atoms having from 0 to 10 ethylene oxide moieties and from 0 to 1 glyceryl moiety; Y is selected from the group consisting of nitrogen, phosphorus, and sulfur atoms; R2is an alkyl or monohydroxy alkyl group containing 1 to 3 carbon atoms; x is 1 when Y is a sulfur atom and 2 when Y is a nitrogen or phosphorus atom, R3is an alkylene or hydroxy alkylene or hydroxy alkylene of from 1 to 4 carbon atoms and Z is a radical selected from the group consisting of carboxylate, sulfonate, sulfate, phosphonate, and phosphate groups. Examples of zwitterionic surfactants having the structures listed above include: 4-[N,N-di(2-hydroxyethyl)-N-octadecylammonio]-butane-1-carboxylate; 5-[S-3-hydroxypropyl-S-hexadecylsulfonio]-3-hydroxypentane-1-sulfate; 3-[P,P-diethyl-P-3,6,9-trioxatetracosanephosphonio]-2-hydroxypropane-1-phosphate; 3-[N,N-dipropyl-N-3-dodecoxy-2-hydroxypropyl-ammonio]-propane-1-phosphonate; 3-(N,N-dimethyl-N-hexadecylammonio)-propane-1-sulfonate; 3-(N,N-dimethyl-N-hexadecylammonio)-2-hydroxy-propane-1-sulfonate; 4-[N,N-di(2(2-hydroxyethyl)-N(2-hydroxydodecyl)ammonio]-butane-1-carboxylate; 3-[S-ethyl-S-(3-dodecoxy-2-hydroxypropyl)sulfonio]-propane-1-phosphate; 3-[P,P-dimethyl-P-dodecylphosphonio]-propane-1-phosphonate; and S[N,N-di(3-hydroxypropyl)-N-hexadecylammonio]-2-hydroxy-pentane-1-sulfate. The alkyl groups contained in said detergent surfactants can be straight or branched and saturated or unsaturated. The zwitterionic surfactant suitable for use in the present compositions includes a betaine of the general structure: These surfactant betaines typically do not exhibit strong cationic or anionic characters at pH extremes nor do they show reduced water solubility in their isoelectric range. Unlike “external” quaternary ammonium salts, betaines are compatible with anionics. Examples of suitable betaines include coconut acylamidopropyldimethyl betaine; hexadecyl dimethyl betaine; C12-14acylamidopropylbetaine; C8-14acylamidohexyldiethyl betaine; 4-C14-16acylmethylamidodiethylammonio-1-carboxybutane; C16-18acylamidodimethylbetaine; C12-16 acylamidopentanediethylbetaine; and C12-16acylmethylamidodimethylbetaine. Sultaines useful in the present invention include those compounds having the formula (R(R1)2N+R2SO3−, in which R is a C6-C18hydrocarbyl group, each R1is typically independently C1-C3alkyl, e.g. methyl, and R2is a C1-C6hydrocarbyl group, e.g. a C1-C3alkylene or hydroxyalkylene group. A typical listing of zwitterionic classes, and species of these surfactants, is given in U.S. Pat. No. 3,929,678, which is herein incorporated by reference in its entirety. Further examples are given in “Surface Active Agents and Detergents” (Vol. I and II by Schwartz, Perry and Berch), which is herein incorporated by reference in its entirety. Detergent Builders The composition can include one or more building agents, also called chelating or sequestering agents (e.g., builders), including, but not limited to: condensed phosphates, alkali metal carbonates, phosphonates, aminocarboxylic acids, aminocarboxylates and their derivatives, ethylenediamine and ethylenetriamine derivatives, hydroxyacids, and mono-, di-, and tri-carboxylates and their corresponding acids, and/or polyacrylates. In general, a chelating agent is a molecule capable of coordinating (i.e., binding) the metal ions commonly found in natural water to prevent the metal ions from interfering with the action of the other detersive ingredients of a cleaning composition. In a preferred embodiment, the detergent composition does not comprise a phosphate builder. Other chelating agents include nitroloacetates and their derivatives, and mixtures thereof. Examples of aminocarboxylates include amino acetates and salts thereof. Suitable amino acetates include: N-hydroxyethylaminodiacetic acid; hydroxyethylenediaminetetraacetic acid; nitrilotriacetic acid (NTA); ethylenediaminetetraacetic acid (EDTA); N-hydroxyethyl-ethylenediaminetriacetic acid (HEDTA); tetrasodium ethylenediaminetetraacetic acid (EDTA); diethylenetriaminepentaacetic acid (DTPA); and alanine-N,N-diacetic acid; n-hydroxyethyliminodiacetic acid; and the like; their alkali metal salts; and mixtures thereof. Suitable aminophosphates include nitrilotrismethylene phosphates and other aminophosphates with alkyl or alkaline groups with less than 8 carbon atoms. Exemplary polycarboxylates iminodisuccinic acids (IDS), sodium polyacrylates, citric acid, gluconic acid, oxalic acid, salts thereof, mixtures thereof, and the like. Additional polycarboxylates include citric or citrate-type chelating agents, polymeric polycarboxylate, and acrylic or polyacrylic acid-type chelating agents. Additional chelating agents include polyaspartic acid or co-condensates of aspartic acid with other amino acids, C4-C25-mono-or-dicarboxylic acids and C4-C25-mono-or-diamines. Exemplary polymeric polycarboxylates include polyacrylic acid, maleic/olefin copolymer, acrylic/maleic copolymer, polymethacrylic acid, acrylic acid-methacrylic acid copolymers, hydrolyzed polyacrylamide, hydrolyzed polymethacrylamide, hydrolyzed poly amide-methacrylamide copolymers, hydrolyzed poly acrylonitrile, hydrolyzed polymethacrylonitrile, hydrolyzed acrylonitrile-methacrylonitrile copolymers, and the like. Useful aminocarboxylic acid materials containing little or no NTA include, but are not limited to: N-hydroxyethylaminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), hydroxyethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, N-hydroxyethyl-ethylenediaminetriacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), ethylenediaminesuccinic acid (EDDS), 2-hydroxyethyliminodiacetic acid (HEIDA), iminodisuccinic acid (IDS), 3-hydroxy-2-2′-iminodisuccinic acid (HIDS) and other similar acids or salts thereof having an amino group with a carboxylic acid substituent. In a preferred aspect, the chelant is gluconic acid, EDTA or an alkali metal salt thereof. Preferable levels of addition for builders that can also be chelating or sequestering agents are between about 0.001% to about 70% by weight, about 0.001% to about 60% by weight, or about 0.01% to about 50% by weight. If the composition is provided as a concentrate, the concentrate can include between approximately 0.001% to approximately 50% by weight, between approximately 0.001% to approximately 35% by weight, and between approximately 0.001% to approximately 30% by weight of the builders. Oxidizer An oxidizing agents for use in the detergent compositions may also be included, and may be referred to as a bleaching agent as it may provide lightening or whitening of a substrate. An oxidizer may include bleaching compounds capable of liberating an active halogen species, such as Cl2, Br2, —OCl and/or —OBr—, under conditions typically encountered during the cleansing process. Suitable bleaching agents for use in the present detergent compositions include, for example, chlorine-containing compounds such as a chlorine, a hypochlorite (e.g. sodium hypochlorite), and/or chloramine. Preferred halogen-releasing compounds include the alkali metal dichloroisocyanurates, such as sodium dichloroisocyanurate, chlorinated trisodium phosphate, the alkali metal hypochlorites, monochlorarrine and dichloramine, and the like. An oxidizer may also be a peroxygen or active oxygen source such as hydrogen peroxide, perborates, sodium carbonate peroxyhydrate, phosphate peroxyhydrates, potassium permonosulfate, and sodium perborate mono and tetrahydrate, with and without activators such as tetraacetylethylene diamine, and the like. A detergent composition may include a minor but effective amount of an oxidizer, preferably about 0.1-30 wt-%, and more preferably from about 1-15 wt-%. In a preferred aspect, the oxidizer is a alkali metal hypochlorite. Formulations The detergent compositions according to the invention may be formulated into solids, liquids, powders, pastes, gels, etc. Solid detergent compositions provide certain commercial advantages for use according to the invention. For example, use of concentrated solid detergent compositions decrease shipment costs as a result of the compact solid form, in comparison to bulkier liquid products. In certain embodiments of the invention, solid products may be provided in the form of a multiple-use solid, such as, a block or a plurality of pellets, and can be repeatedly used to generate aqueous use solutions of the detergent composition for multiple cycles or a predetermined number of dispensing cycles. In certain embodiments, the solid detergent compositions may have a mass greater than about 5 grams, such as for example from about 5 grams to 10 kilograms. In certain embodiments, a multiple-use form of the solid detergent composition has a mass of about 1 kilogram to about 10 kilogram or greater. Methods of Use The compositions of the invention are suitable for use in various applications and methods, including any application suitable for an alkali metal hydroxide, alkali metal metasilicate and/or alkali metal silicate detergent. In a particular aspect, the compositions of the invention are suitable for use in cleaning food, beverage and/or pharmaceutical equipment/processes as they beneficially reduce hard water scale within the cleaning applications. The methods of use may be desirable in additional applications where industrial standards are focused on the quality of the treated surface and/or the hard surfaces comprising the machinery or components wherein the surfaces are treated, such that the prevention of hard water scale build up provided by the detergent compositions of the invention are desirable. Preventing Hard Water Scale in Cleaning Applications The methods of the invention are particularly suited for methods employing alkaline detergents in need of preventing hard water scale accumulation on surfaces within food, beverage and/or pharmaceutical applications. In addition, the methods of the invention are well suited for controlling water hardness buildup on a plurality of surfaces. The methods of the invention prevent moderate to heavy accumulation hardness on treated substrate surfaces beneficially alleviating negative impacts of insufficient cleaning, decreasing product quality, reduced heat transfer and/or decreased water flow within a system. Moreover, the methods of the invention further improve the aesthetic appearance of the surface. In certain embodiments, surfaces in need of hard water scale accumulation prevention, include for example, plastics, metal and/or glass surfaces, namely those in food and beverage applications, such as clean-in-place systems. As used herein, clean-in-place (CIP) cleaning techniques refer a specific cleaning and/or disinfection regimen adapted for removing soils from the internal components of tanks, lines, pumps and other process equipment used for processing, often food and/or beverage processing. Typically the product streams are liquid such as beverages, milk, juices, etc. Clean-in-place cleaning involves passing cleaning solutions of the compositions according to the invention through the system without dismantling any system components. The methods for cleaning equipment using CIP cleaning procedures includes for example, such equipment as evaporators, heat exchangers (including tube-in-tube exchangers, direct steam injection, and plate-in-frame exchangers), heating coils (including steam, flame or heat transfer fluid heated) re-crystallizers, pan crystallizers, spray dryers, drum dryers, and tanks. The methods can be used in generally any applications where caked on soil or burned on soil, such as proteins or carbohydrates, needs to be removed; applications include the food and beverage industry (especially dairy), brewing, oil processing, industrial agriculture and ethanol processing. CIP processing is generally a well-known process, including applying a dilute solution (typically about 0.5-3%) onto the surface to be cleaned. The solution flows across the surface (typically about 3 to 6 feet/second), slowly removing the soil. Either new solution is re-applied to the surface, or the same solution is recirculated and re-applied to the surface. In a minimum aspect, the methods for a clean-in-place technique according to the invention involve passing a cleaning solution of the compositions of the invention through the equipment and then resuming normal processing. Beneficially, these clean-in-place cleaning techniques are adapted for removing soils from interior surfaces of a wide variety of parts of processing equipment, such as pipes, tubing, connections, tanks, storage reservoirs and the like. In further aspects, the methods remove a soil (including organic, inorganic or a mixture of the two components) can further include the steps of applying an acid solution wash and/or a fresh water rinse, in addition to the alkaline solution wash according to the compositions of the invention. Without being limited to a particular mechanism of action, the alkaline solution softens the soils and removes the organic alkaline soluble soils. The optional use of subsequent acid solution may be beneficial to remove mineral soils left behind by the alkaline cleaning step. The strength of the alkaline and acid solutions and the duration of the cleaning steps are typically dependent on the durability of the soil. The water rinse removes any residual solution and soils, and cleans the surface prior to the equipment being returned on-line. In an aspect of the invention, the CIP methods include an apparatus or system in need of cleaning, such as a tank. In an aspect, a feed line supplies the alkaline cleaning composition according to the invention to the tank, and a drain line removes the solution from tank. A system or apparatus may further have operably connected via appropriate pipes, valves, pumps, etc. equipment for the CIP process. A CIP process may further includes a tank for retaining the dilute CIP chemistry. A drain line from the tank is used to recirculate solution from tank back to CIP process and tank. The methods of the invention beneficially reduce the formation, precipitation and/or deposition of hard water scale, such as calcium carbonate, on hard surfaces contacted by the detergent compositions. In an embodiment, the detergent compositions are employed for the prevention of formation, precipitation and/or deposition of hard water scale on hard surfaces, such as those contacted in clean-in-place cleaning. The detergent compositions according to the invention beneficially provide such prevention of formation, precipitation and/or deposition of hard water scale despite the high alkalinity of the detergent composition use solutions (e.g. pH between about 10 and 13.5) in the presence of hard water. The compositions of the invention may be formulated prior to the point of use as a single or multiple component product. For example, the compositions of the invention may be formulated with both the alkali metal hydroxide and PSO adducts and may be used as a single cleaning composition between pH of about 10 and 13.5. The composition may comprise additional components such as for example, nonionic surfactants, anionic surfactants, polymers, oxidizers and corrosion inhibitors. The compositions of the invention may also be generated at the point of use. For example, the alkali metal hydroxide and PSO adducts may be added separately to the clean-in-place process. The PSO component may be added in acidic or neutralized form and combined with the alkali metal hydroxide to form a use solution between pH of about 10-13.5. Both the alkali metal hydroxide and PSO adduct solutions may comprise additional components such as for example, nonionic surfactants, anionic surfactants, polymers, oxidizers and corrosion inhibitors. Preventing Hard Water Scale in Foam Cleaning Applications The methods of the invention also suited for methods employing high foaming alkaline detergents in need of preventing hard water scale accumulation on treated surfaces. The methods of the invention prevent moderate to heavy accumulation hardness on treated substrate surfaces beneficially alleviating negative impacts of insufficient cleaning, providing improved aesthetic appearances, including on the visible, exterior surfaces of machinery and other hard surfaces. In certain embodiments, surfaces in need of hard water scale accumulation prevention, include for example, plastics, metal and/or glass surfaces, namely those in food and beverage applications, such as for example the exterior surfaces commonly found in food-and-beverage CIP systems. The methods for cleaning exterior portions/surfaces of equipment and hard surfaces in need of high foaming alkaline detergent compositions are particularly suitable for manual cleaning processes (as distinguished from the automated CIP cleaning procedures described above). Automated cleaning employing alkaline detergent compositions according to the invention can be done safely at a wide range of temperatures and a wide range of pressure applications (including under high pressure). In such aspects, cleaning solutions as well as rinse water is applied to a surface manually under a range of pressure to facilitate soil removal from the surfaces. Instead of the recirculation which may be employed in an automated systems (e.g. CIP), the mechanical solution flow can be used to remove soils according to manual methods. In an aspect of the invention employing manual cleaning operations, surfaces may include those in open, large facility environments. The alkaline detergent composition is applied to a surface in need of treatment through manual application. In such cleaning operations, residence time on a surface of the alkaline detergent composition (often in the form of foam or a gel, especially for vertical surfaces) provides cleaning efficacy without the accumulation of hardness scale. In other aspects, high temperature rinse water can be further employed to effectively clean a surface. In a minimum aspect, the methods for a manual cleaning technique according to the invention involve applying a cleaning solution of the compositions of the invention onto a hard surface and allowing residence time on the surface for the detergency effect. The methods further include the step of applying rinse water and/or other rinse aid to remove the alkaline detergent composition. In further aspects, the methods remove a soil (including organic, inorganic or a mixture of the two components) can further include the steps of applying an acid solution wash and/or a fresh water rinse, in addition to the alkaline solution wash according to the compositions of the invention. Without being limited to a particular mechanism of action, the alkaline solution softens the soils and removes the organic alkaline soluble soils. The optional use of subsequent acid solution may be beneficial to remove mineral soils left behind by the alkaline cleaning step. The strength of the alkaline and acid solutions and the duration of the cleaning steps are typically dependent on the durability of the soil. The water rinse removes any residual solution and soils, and cleans the surface prior to the equipment being returned on-line. The methods of the invention beneficially reduce the formation, precipitation and/or deposition of hard water scale, such as calcium carbonate, on hard surfaces contacted by the detergent compositions. In an embodiment, the detergent compositions are employed for the prevention of formation, precipitation and/or deposition of hard water scale on hard surfaces, such as external surfaces of machinery in food-and-beverage applications. The detergent compositions according to the invention beneficially provide such prevention of formation, precipitation and/or deposition of hard water scale despite the high alkalinity of the detergent composition use solutions (e.g. pH between about 10 and 13.5) in the presence of hard water. Preventing and or Minimizing Hardness Accumulation The methods of the invention are particularly suited for methods employing alkaline detergents in need of preventing hardness (e.g. calcium carbonate) accumulation on surfaces. Hardness accumulation is particularly detrimental to surfaces used in detergent cleaning applications for the interior surfaces, such as CIP applications, as it may result in the formation of build up or accumulation decreasing fluid transfer within the system, having distinct soiled appearance, in addition to the hardness scaling covering a surface. The methods of the invention are well suited for preventing hardness accumulation on a plurality of surfaces. The methods of the invention reduce and/or substantially prevent hardness accumulation on treated surfaces. In an aspect, the methods according to the invention provide reduction and/or prevention of hardness accumulation on treated surfaces over conventional phosphate-based alkaline detergents, such as those containing tripolyphosphates. In some aspects, the hardness accumulation is reduced by at least about 10% in comparison to conventional phosphate-based alkaline detergents, preferably at least about 20% in comparison to conventional phosphate-based alkaline detergents, or greater. In still a further aspect, the methods according to the invention provide at least substantially similar (e.g. meet performance) hardness accumulation prevention in comparison to phosphate-free alkaline detergents that do not contain the PSO adducts according to the invention. In an aspect, the methods of reducing hardness accumulation include contacting a hard surface with a detergent composition, wherein the detergent composition comprises, consists of and/or consists essentially of (a) an alkali metal hydroxide and/or alkali metal silicates and/or metasilicates, and (b) phosphinosuccinic acid adducts or adducts having at least one of the following formulas: where M is selected from the group consisting of H+, Na+, K+, NH4+, and mixtures thereof, wherein m plus n is greater than 2. The additional embodiments of the alkaline detergent composition are suitable for use according to the methods of the invention. Preferably, the contacting step with the detergent composition is during a washing step of a CIP cleaning cycle. The time for contacting the hard surface in need of treatment, namely within a CIP application, may vary depending on factors such as size, alkalinity of the detergent composition, amount of soil therein, etc. The detergent compositions are effective at preventing hard water scale accumulation in hard surface cleaning applications, including preferably CIP applications, using a variety of water sources, including hard water. The various methods of use according to the invention employ the use of the detergent composition, which may be formed prior to or at the point of use by combining the PSO adducts, alkalinity source and other desired components (e.g. optional polymers and/or surfactants) in the weight percentages disclosed herein. The detergent composition may be provided in various formulations. The methods of the invention may employ any of the formulations disclosed, including for example, liquids, semi-solids and/or other solids, powders, pastes and/or gel formulations. The methods of invention may also employ the detergent compositions which are provided (or sourced) in one or more parts. In an aspect, the detergent composition may be formed at a point of use such as where a two (or more) part composition is combined to form the detergent composition. In an exemplary aspect, the detergent composition comprising and/or consisting of the PSO derivations (and optionally polymers, surfactants, additional alkalinity sources and/or additional functional ingredients) may be combined with an alkali metal hydroxide alkalinity source (e.g. a commodity caustic source). The methods of the invention may also employ a concentrate and/or a use solution constituting an aqueous solution or dispersion of a concentrate. Such use solutions may be formed during the washing process. In aspects of the invention employing packaged solid detergent compositions, the products may first require removal from any applicable packaging (e.g. film). Thereafter, according to certain methods of use, the compositions can be inserted directly into a dispensing apparatus and/or provided to a water source for cleaning according to the invention. Examples of such dispensing systems include for example U.S. Pat. Nos. 4,826,661, 4,690,305, 4,687,121, 4,426,362 and U.S. Pat. Nos. RE 32,763 and 32,818, the disclosures of which are incorporated by reference herein in its entirety. Ideally, a solid detergent composition is configured or produced to closely fit the particular shape(s) of a dispensing system in order to prevent the introduction and dispensing of an incorrect solid product into the apparatus of the present invention. In certain embodiments, the detergent composition may be mixed with a water source prior to or at the point of use. In other embodiments, the detergent compositions do not require the formation of a use solution and/or further dilution and may be used without further dilution. In aspects of the invention employing solid detergent compositions, a water source contacts the detergent composition to convert solid detergent compositions, particularly powders, into use solutions. Additional dispensing systems may also be utilized which are more suited for converting alternative solid detergents compositions into use solutions. The methods of the present invention include use of a variety of solid detergent compositions, including, for example, extruded blocks or “capsule” types of package. In an aspect, a dispenser may be employed to spray water (e.g. in a spray pattern from a nozzle) to form a detergent use solution. For example, water may be sprayed toward an apparatus or other holding reservoir with the detergent composition, wherein the water reacts with the solid detergent composition to form the use solution. In certain embodiments of the methods of the invention, a use solution may be configured to drip downwardly due to gravity until the dissolved solution of the detergent composition is dispensed for use according to the invention. In an aspect, the use solution may be dispensed into a wash solution of a ware wash machine. All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated as incorporated by reference. EXAMPLES Embodiments of the present invention are further defined in the following non-limiting examples. It should be understood that these examples, while indicating certain embodiments of the invention, are given by way of illustration only. From the above discussion and the examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the invention to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Example 1 Hard water film accumulation testing was conducted using a light box evaluation of 100 cycle glasses. The 100 cycle experiment was performed using six 10 oz. Libby glasses on a Hobart AM-15 ware wash machine employing 17 grain water (hard water source). Initially the glasses were prepared using a cleaning cycle to completely remove all film and foreign material from the glass surface. The evaluated compositions are shown in Table 2. The experimental formulations shown in Table 3 provided 40% active salt and 31% active as an acid. A use concentration of 0.716 g/L was employed for the evaluated formulations. TABLE 2Raw materialEx 1Ex 2Ex 3Water14.314.314.3Sodium hydroxide69.869.869.8(beads)Pluronic N3: EP/PO0.90.90.9copolymersPSO adducts57.510Acusol 445N (45%):107.55polycarboxylic acid The ware wash machine controller was set to automatically dispense the indicated amount of detergent into the wash tank. Six clean glasses (G=glass tumblers) were placed in a Raburn rack. The ware wash machine automatically dispensed into the ware wash machine the detergent compositions to achieve the desired concentration and maintain the initial concentration. The glasses were dried overnight and then the film accumulation using a strong light source was evaluated. The light box test standardizes the evaluation of the glasses run in the 100 cycle test. The light box test is based on the use of an optical system including a photographic camera, a light box, a light source and a light meter. The system is controlled by a computer program (Spot Advance and Image Pro Plus). To evaluate the glasses after the 100 cycle test, each glass was placed on the light box resting on its side and the intensity of the light source was adjusted to a predetermined value using a light meter. The conditions of the 100 cycle test were entered into the computer. A picture of the glass was taken with the camera and saved on the computer for analysis by the program. The picture was analyzed using the upper half of the glass in order to avoid the gradient of darkness on the film from the top of the glass to the bottom of the glass, based on the shape of the glass. Generally, a lower light box rating indicates that more light was able to pass through the glass. Thus, the lower the light box rating, the more effective the composition was at preventing scaling on the surface of the glass. Light box evaluation of a clean, unused glass has a light box score of approximately 12,000 which corresponds to a score of 72,000 for the sum of 6 glasses. Table 2 shows the results of the light box test. Table 3 shows the results of the light box test. TABLE 3UseLight Box ScoresExampleConcentrationGlassesPlasticSumExample 1716 ppm20234633122235468Example 2716 ppm24685336741283594Example 3716 ppm17087037571208441 The results demonstrate that the PSO is suitable for combination with polymers according to an aspect of the invention. Examples 3-5 provided suitable performance for controlling hard water scale accumulation in an alkaline detergent applications. Example 2 A beaker test was employed to evaluate calcium carbonate inhibition for food and beverage applications. A hardness solution was prepared by dissolving 33.45 g of CaCl2)-2H2O and 23.24 g of MGCl2-6H2O in deionized water in a 1 L volumetric flask filled to volume. A sodium bicarbonate solution was prepared by dissolving NaHCO3-2H2O in DI water in a 1 L volumetric flask filled to volume. A beaker was placed on a heat plate/stirrer. To the beaker, 1000 ml deionized water and 5.00 ml of the sodium bicarbonate solution were added. The contents of the beaker were heated to 85° F. and then the hardness solution was added to provide a water harness of 17 grains. Then each component of the evaluated samples shown in Table 4 were added (4 ml, equivalent to 0.4% or 1 ounce/2 gallons) to the contents of the beaker in the identified concentrations. Exemplary samples 4 and 6 provide positive controls, providing a PBTC sodium salt instead of the PSO according to the invention. TABLE 4Raw materialEx 4Ex 5Ex 6Ex 7ControlSodium hydroxide4000 ppm4000 ppm4000 ppm4000 ppm4000 ppmBayhibit N (41%):400 ppm—400 ppm——PBTC Na saltPSO adducts, 40%—400 ppm—400 ppm—Acusol 1000 (48%):——476 ppm476 ppm—polyacrylic acidpH12.612.612.612.612.6 After the Sample was completely mixed into the beaker, an initial transmittance measurement at 560 nm was taken at 85° F., 140° F., and 160° F. The Sample was then allowed to cool to room temperature before a final measurement was taken. A “Clear” Sample as set forth in the tables below indicates that the beaker contents had a light transmission of at least about 95% when tested at 85° F., 140° F., 160° F. and room temperature, and was visibly clear without noticeable haziness, discoloration or precipitant formation. The fact that a particular sample was not indicated as being clear does not necessarily mean that the sample did not prevent scale. Rather, those sample that are indicated as being clear provide optimum scale protection under the conditions created in the experiment. The results are shown in Table 5. TABLE 585° F.140° F.160° F.averageaverageaverage85° F.140° F.160° F.(St Dev)(St Dev)(St Dev)Control96.268366.295.667.966.05Control9567.565.9(0.85)(0.57)(0.21)EXP 499.497.697.399.4596.7597EXP 499.595.996.7(0.07)(1.2)(0.42)EXP 595.594.393.895.8593.9593.75EXP 596.293.693.7(0.49)(0.49)(0.07)EXP 699.599.499.499.499.3599.35EXP 699.399.399.3(0.14)(0.07)(0.07)EXP 799.999.699.599.8599.599.45EXP 799.899.499.4(0.07)(0.14)(0.07) The results in Table 5 show the exemplary sample 5 according to an embodiment of the invention provided similar calcium carbonate inhibition as the positive control (sample 4 containing the PBTC sodium salt instead of the PSO according to the invention) at 85° F., 140° F., and 160° F. Additionally, exemplary sample 7 according to an embodiment of the invention provided similar calcium carbonate inhibition as the positive control (sample 6 containing the PBTC sodium salt and polyacrylate instead of the PSO/polyacrylate according to the invention) at 85° F., 140° F., and 160° F. All samples containing the polymer and/or phosphonate outperformed the Control (averaged results). Example 3 Hard water tolerance testing was conducted using formulations with the PSO adducts according to the invention in comparison to the formulations without the PSO adducts. The evaluated formulations are shown below in Table 6 wherein alkaline cleaning compositions including silicate and hydroxide alkalinity sources were combined with the PSO adducts and compared to the formulations without the PSO adducts (Control). TABLE 6EXP 8ControlDI water30-6030-60NaOH 50%10-2010-20Sodium Silicate Solution0.5-20.5-2PSO adducts, 40%1-50Sodium Hypochlorite, 10%20-4020-40Additional Functional Ingredients5-105-10100.00100 The formulations were combined with water sources having increasingly hard water (i.e. grains per gallon) as shown in Table 7. The hardness tolerance testing of the EXP 8 formulation and the control were conducted using 1% solutions in water with varying degrees of synthetic hardness created by adding various amounts of dissolved CaCl2) and MgCl2to a combination of deionized water and NaHCO3. Once the solutions reached 140° F. they were removed from the heat and let stand for 30 minutes. A failure was characterized by the presence of visible flocculent after the 30 minutes, whereas a passing evaluation was characterized by the absence of visible flocculent after the 30 minutes. The results are shown in Table 7. TABLE 7Grains perWater sourcegallonEXP 8Controlsynthetic hard water17PassPasssynthetic hard water18PassFailsynthetic hard water19PassFailReverse osmosis reject water (Eagan,22PassFailMN)Reverse osmosis reject water (Eagan,24PassFailMN)Reverse osmosis reject water (Eagan,26FailFailMN)Reverse osmosis reject water (Eagan,28FailFailMN) As shown in Table 7, the results indicate that the PSO-containing formulation of the alkaline detergent composition prevents hard water scale accumulation at hardness levels up to at least 24 grains, whereas the Control alkaline detergent formulation only prevented hard water scale accumulation at hardness levels up to 17 grains. Example 4 Testing to evaluate hard water tolerance of exemplary formulations of a high-foaming, higher alkaline chlorinated cleaner (with and without PSO) was conducted to determine the impact of the PSO on hard water tolerance. The evaluated formulations are shown below in Table 8 wherein alkaline cleaning compositions including hydroxide alkalinity sources were combined with the PSO adducts and compared to the formulations without the PSO adducts (Control). TABLE 8EXP 9ControlDI water25-5025-50NaOH 50%10-3010-30PSO adducts, 40%1-50Lauryl dimethylamine oxide 30%5-105-10Sodium Hypochlorite, 10%20-4020-40Additional Functional Ingredients5-105-10100.00100 The hardness tolerance testing of the EXP 9 formulation and the control were conducted using 1% solutions in water with varying degrees of synthetic hardness created by adding various amounts of dissolved CaCl2) and MgCl2to a combination of deionized water and NaHCO3. Once the solutions reached 140° F. they were removed from the heat and let stand for 30 minutes. A failure was characterized by the presence of visible flocculent after the 30 minutes, whereas a passing evaluation was characterized by the absence of visible flocculent after the 30 minutes. The results are shown in Table 9. TABLE 9Grains perWater sourcegallonEXP 9ControlSynthetic hard water16PassPassSynthetic hard water17PassPassSynthetic hard water18PassFailSynthetic hard water19PassFailSynthetic hard water20Fail—Synthetic hard water21Fail—Synthetic hard water22Fail—Synthetic hard water23Fail— As shown in Table 10, the exemplary high-foaming formulation (EXP 9) according to the invention containing the PSO adducts had increased hard water tolerance over cleaning compositions not containing the PSO adducts. The inventions being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the inventions and all such modifications are intended to be included within the scope of the following claims. | 87,424 |
11859156 | DETAILED DESCRIPTION OF THE INVENTION The present invention envisages an automatic dishwashing detergent composition. The composition comprises an alkoxylated polyalkyleneimine, bleach, it is free of bleach catalyst and bleach activator and comprises an enzymatic system. The composition provides improved removal of bleachable stains, in particular tea and coffee stains and enzymatic soils, including crème brule, starch, protein and complex mixtures of starch and proteins. There is also provided a method of automatic dishwashing using the composition of the invention and the use of the composition for the removal of bleachable stains (specially tea and coffee) and enzymatic soils from cookware and tableware. Alkoxylated Polyalkyleneimine The alkoxylated polyalkyleneimine preferably comprises polyethyleneimine and more preferably it is a polyethyleneimine. Preferably the composition of the invention comprises from 0.1% to about 5%, preferably from about 0.2% to about 3% by weight of the composition of the polyalkyleneimine. Preferably the method of the invention delivers from about 20 to about 100 ppm of the polyalkyleneimine. The alkoxylation of the polyalkyleneimine backbone comprises one or two alkoxylation modifications in a nitrogen atom, depending on whether the modification occurs at an internal nitrogen atom or at a terminal nitrogen atom in the polyalkyleneimine backbone, the alkoxylation modification involves the replacement of a hydrogen atom in a polyalkyleneimine by a monoalkoxylene or a polyalkoxylene chain preferably having an average of from about 1 to about 50 alkoxy units, wherein the terminal alkoxy unit of the polyalkoxylene chain is capped with hydrogen, C1-C4 alkyl or mixtures thereof. In addition, each nitrogen atom in the alkoxylated polyalkyleneimine may carry saturated or unsaturated, linear or branched alkyl, alkylaryl or aryl substituents, or combinations thereof, preferably benzyl substituents and/or C1-C12, preferably C1-C4 alkyl, aryl or alkylaryl substituents, resulting in neutral or cationic charge on each nitrogen atom depending on its total number of substituents. These modifications may result in permanent quaternization of polyalkyleneimine backbone nitrogen atoms. The degree of permanent quaternization is at least 5%, preferably at least 20%, more preferably from at least from 40% to 100% of the polyalkyleneimine backbone nitrogen atoms. Preferably, all the nitrogen atoms would comprise alkoxylation modification(s) although it might be possible to have polyalkyleneimines wherein only part of the nitrogen atoms have been alkoxylated. Examples of possible modifications are herein shown, the modifications correspond to terminal nitrogen atoms in the polyethyleneimine backbone where R represents an ethylene spacer and E represents a C1-C12alkyl unit and X−represents a suitable water soluble counterion, such as chlorine, bromine or iodine, sulphate (i.e. —O—SO3H or —O—SO3-), alkylsulfonate such as methylsulfonate, arylsulfonate such as tolylsulfonate, and alkyl sulphate, such as methosulphate (i.e. —O—SO2-OMe)). Examples of possible modifications are shown, the modifications correspond to internal nitrogen atoms in the polyethyleneimine backbone where R represents an ethylene spacer and E represents a C1-C12 alkyl unit and X— represents a suitable water soluble counterion. Also, for example, but not limited to, below is shown possible modifications to internal nitrogen atoms in the polyethyleneimine backbone where R represents an ethylene spacer and E represents a C1-C12 alkyl unit and X— represents a suitable water soluble counterion. The alkoxylation modification of the polyalkyleneimine backbone may comprise the replacement of a hydrogen atom by a polyalkoxylene chain having an average of about 1 to about 50 alkoxy units, preferably from about 2 to about 40 alkoxy units, more preferably from about 3 to about 30 units and especially from about 3 to about 20 alkoxy units. The alkoxy units are preferably selected from ethoxy (EO), 1,2-propoxy (1,2-PO), butoxy (BO), and combinations thereof. Preferably, the polyalkoxylene chain is selected from ethoxy units and a combination of ethoxy and propoxy units. More preferably, the polyalkoxylene chain comprises ethoxy units in an average degree of from about 1 to about 50, more preferably from about 2 to about 40 and especially from about 3 to 20. Polyalkyleneimines comprising this degree of ethoxy units have been found to provide best performance in terms of removal of bleachable stains, in particular tea and coffee stains. Also preferred in terms of bleachable stain removal are polyalkoxylene chains comprising a mixture of ethoxy and propoxy chains, preferably the polyalkoxylene chain comprises ethoxy units in an average of from about 1 to about 30 and more preferably propoxy units in an average degree of from about 0 to about 10, more preferably from about 2 to about 20 ethoxy units and from about 1 to about 10 propoxy units. An example of a preferred alkoxylated polyethyleneimine has the general structure of formula (I) or a quaternized version (II): wherein the polyethyleneimine backbone has a weight average molecular weight of from about 600 to about 5000 g/mole, n of formula (I) or (II) has an average of 3 to 20 and R of formula (I) is selected from hydrogen, a C1-C4 alkyl or benzyl, and mixtures thereof. The degree of quaternization of the polyalkyleneimine backbone of formula (II) may be at least 5%, more preferably at least 20% and especially 70% or higher of the polyalkyleneimine backbone nitrogen atoms. Another preferred polyethyleneimine has the general structure of formula (III), with the quaternized version shown as formula (IV): wherein the polyethyleneimine backbone has a weight average molecular weight of from about 600 to about 5000 g/mole, n of formulas (III) and (IV) has an average of 7, m of formulas (III) and (IV) have an average of 1 and R of formula (III) and (IV) is selected from hydrogen, a C1-C4 alkyl and mixtures thereof. The degree of permanent quaternization of formula (IV)) may be from 5% to 100%, preferably at least 10%, more preferably at least 20% of the polyethyleneimine backbone nitrogen atoms. Polyalkyleneimines suitable for the composition of the invention can be prepared, for example, by polymerizing ethyleneimine in the presence of a catalyst such as carbon dioxide, sodium bisulfite, sulfuric acid, hydrogen peroxide, hydrochloric acid, acetic acid, and the like. The alkoxylated polyalkylenimines may be prepared in a known manner by reaction of polyalkylene imines with alkoxy units, the process would herein be described for the ethoxylation of polyoxyethyleneimine. One preferred procedure consists in initially undertaking only an incipient ethoxylation of the polyalkylene imine in a first step. In this step, the polyalkylene imine is reacted only with a portion of the total amount of ethylene oxide used, which corresponds to about 1 mol of ethylene oxide per mole of NH unit. This reaction is undertaken generally in the absence of a catalyst in an aqueous solution at a reaction temperature from about 70 to about 200° C. and preferably from about 80 to about 160° C. This reaction may be affected at a pressure of up to about 10 bar, and in particular up to about 8 bar. In a second step, the further ethoxylation is then undertaken by subsequent reaction with the remaining amount of ethylene oxide. The further ethoxylation is undertaken typically in the presence of a basic catalyst. Examples of suitable catalysts are alkali metal and alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide and calcium hydroxide, alkali metal alkoxides, in particular sodium and potassium C1-C4-alkoxides, such as sodium methoxide, sodium ethoxide and potassium tert-butoxide, alkali metal and alkaline earth metal hydrides such as sodium hydride and calcium hydride, and alkali metal carbonates such as sodium carbonate and potassium carbonate. Preference is given to the alkali metal hydroxides and the alkali metal alkoxides, particular preference being given to potassium hydroxide and sodium hydroxide. Typical use amounts for the base are from 0.05 to 10% by weight, in particular from 0.5 to 2% by weight, based on the total amount of polyalkyleneimine and alkylene oxide. The further ethoxylation may be undertaken in substance (variant a)) or in an organic solvent (variant b)). In variant a), the aqueous solution of the incipiently ethoxylated polyalkylenimine obtained in the first step, after addition of the catalyst, is initially dewatered. This can be done in a simple manner by heating to from about 80 to about 150° C. and distilling off the water under a reduced pressure of from about 0.01 to about 0.5 bar. The subsequent reaction with the ethylene oxide is effected typically at a reaction temperature from about 70 to about 200° C. and preferably from about 100 to about 180° C. The subsequent reaction with the alkylene oxide is effected typically at a pressure of up to about 10 bar and in particular up to 8 bar. The reaction time of the subsequent reaction with the ethylene oxide is generally about 0.5 to about 4 hours. Suitable organic solvents for variant b) are in particular nonpolar and polar aprotic organic solvents. Examples of particularly suitable nonpolar aprotic solvents include aliphatic and aromatic hydrocarbons such as hexane, cyclohexane, toluene and xylene. Examples of particularly suitable polar aprotic solvents are ethers, in particular cyclic ethers such as tetrahydrofuran and dioxane, N,N-dialkylamides such as dimethylformamide and dimethylacetamide, and N-alkyllactams such as N-methylpyrrolidone. It is of course also possible to use mixtures of these organic solvents. Preferred organic solvents are xylene and toluene. In variant b), the solution obtained in the first step, after addition of catalyst and solvent, is initially dewatered, which is advantageously done by separating out the water at a temperature of from about 120 to about 180° C., preferably supported by a gentle nitrogen stream. The subsequent reaction with the alkylene oxide may be effected as in variant a). In variant a), the alkoxylated polyalkylenimine is obtained directly in substance and may be converted if desired to an aqueous solution. In variant b), the organic solvent is typically removed and replaced by water. The products may, of course, also be isolated in substance. The quaternization of alkoxylated polyethyleneimines is achieved preferably by introducing C1-C12 alkyl, aryl or alkylaryl groups and may be undertaken in a customary manner by reaction with corresponding alkyl-, alkylaryl-halides and dialkylsulfates, as described for example in WO2009060059. The quaternization of ethoxylated polyethyleneimines is achieved preferably by reacting the amines with at least one alkylating compound, which is selected from the compounds of the formula EX, wherein E is C1-C12 alkyl, aryl or alkyl and X is a leaving group, which is capable of being replaced by nitrogen (and C2-C6 alkylene oxide, especially ethylene oxide or propylene oxide). Suitable leaving groups X are halogen, especially chlorine, bromine or iodine, sulphate (i.e. —O SO3H or —O SO3-), alkylsulfonate such as methylsulfonate, arylsulfonate such as tolylsulfonate, and alkyl sulphate, such as methosulphate (i.e. —O SO2 OMe). Preferred alkylating agents EX are C1-C12 alkyl halides, bis (C1-C12-alkyl)sulfates, and benzyl halides. Examples of such alkylating agents are ethyl chloride, ethyl bromide, methyl chloride, methyl bromide, benzyl chloride, dimethyl sulphate, diethyl sulphate. The amount of alkylating agent determines the amount of quaternization of the amino groups in the polymer. The amount of the quaternization can be calculated from the difference of the amine number in the non-quaternized amine and the quaternized amine. The amine number can be determined according to the method described in DIN 16945. The reaction can be carried out without any solvent, however, a solvent or diluent like water, acetonitrile, dimethylsulfoxide, N-Methylpyrrolidone, etc. may be used. The reaction temperature is usually in the range from 10° C. to 150° C. and is preferably from 50° C. to 110° C. All molecular weights related to the alkoxylated polyalkyleneimine of the composition of the invention are weight-average molecular weights expressed as grams/mole, unless otherwise specified. The molecular weight can be measured using gel permeation chromatography. Molecular Weight Determination: Molecular weight is determined as weight-average molecular weight (Mw) by gel permeation chromatography (GPC) using a serial configuration of the GPC columns HEMA Bio linear, 40·8 mm 10 μm, HEMA Bio 100, 300·8 mm, 10 μm, HEMA Bio 1000, 300·8 mm, 10 μm and HEMA Bio 10000, 300·8 mm, 10 μm, (obtained from PSS Polymer Standards Service GmbH, Mainz, Germany). The eluent is 1.5% aqueous formic acid, flow is 1 ml/min, injected volume is 20 μl, sample concentration is 1%. The method is calibrated with a Pullulan standard (MW 342-1660000 g/mol, obtained from PSS Polymer Standards Service GmbH, Mainz, Germany). Preferably the polyalkyleneimine is preferably free of other alkyleneoxide units other than ethoxy and propoxy. SYNTHESIS EXAMPLES Example 1: Synthesis of PEI5000+7EO/NH, 50% Quaternized with Dimethyl Sulfate a) PEI5000+1EO/NH In a 3.5 l autoclave 2568.0 g of a polyethyleneimine 5000 (average molecular weight Mwof 5000, 50% solution in water) were heated to 80° C. and purged three times with nitrogen up to a pressure of 5 bar. After the temperature had been increased to 110° C., 1314.2 g ethylene oxide were added in portions up to 7 bar. To complete the reaction, the mixture was allowed to post-react for 2 h at 110° C. The reaction mixture was stripped with nitrogen and volatile compounds were removed in vacuum at 70° C. The temperature was increased to 90-110° C. and the mixture was dewatered for 2 hours in vacuum. 2580.0 g of polyethyleneimine 5000 with 1 mole of ethylene oxide per mole NH were obtained as a dark brown viscous oil (Amine value: 512 mg KOH/g). b) PEI5000+7EO/NH In a 5 l autoclave 997.6 g of the product obtained in Example 1 a) and 29.9 g of a 50% by weight aqueous solution of potassium hydroxide were heated to 80° C. and purged three times with nitrogen. The mixture was dewatered at 120° C. and a vacuum of 10 mbar for 2 h. After the vacuum had been removed with nitrogen, the temperature was increased to 140° C. and 3027.2 g ethylene oxide were added in portions up to 7 bar. To complete the reaction, the mixture was allowed to post-react for 2 h at 120° C. The reaction mixture was stripped with nitrogen and volatile compounds were removed in vacuum at 70° C. 4040.0 g of a polyethyleneimine 5000 with 7 mole of ethylene oxide per mole NH bond were obtained as a brown viscous liquid (Amine value: 137.4 mg KOH/g; pH of a 10% by weight aqueous solution: 11.7; viscosity (70° C.): 325 mPas). c) PEI5000+7EO/NH, 50% Quaternized with Dimethyl Sulfate In a 2 l reaction vessel 1500.0 g of the product from example 1 b) was heated to 70-75° C. under a constant stream of nitrogen. 232.0 g dimethyl sulfate was added within 2 h. The reaction mixture was stirred for additional 2 h at 75° C. 1720.0 g of light brown solid were obtained (Amine value: 63.3 mg KOH/g; pH of a 10% by weight aqueous solution: 7.8; Viscosity (70° C.): 838 mPas). Example 2: Synthesis of PEI600+10EO/NH, 75% Quaternized with Dimethyl Sulfate a) PEI600+1EO/NH In a 3.5 l autoclave 1328.5 g of a polyethyleneimine 600 (average molecular weight Mwof 600) and 66.4 g water were heated to 80° C. and purged three times with nitrogen up to a pressure of 5 bar. After the temperature had been increased to 120° C., 1359.4 g ethylene oxide were added in portions up to 7 bar. To complete the reaction, the mixture was allowed to post-react for 2 h at 120° C. The reaction mixture was stripped with nitrogen and volatile compounds were removed in vacuo at 70° C. The temperature was increased to 90-110° C. and the mixture was dewatered for 2 hours in vacuo. 2688.0 g of polyethyleneimine 600 with 1 mole of ethylene oxide per mole NH were obtained as a yellow viscous oil (Amine value: 549 mg KOH/g; pH of a 1% by weight aqueous solution: 11.06). b) PEI600+10 EO/NH In a 5 l autoclave 704.5 g of the product obtained in Example 1 a) and 21.1 g of a 50% by weight aqueous solution of potassium hydroxide were heated to 80° C. and purged three times with nitrogen. The mixture was dewatered at 120° C. and a vacuum of 10 mbar for 2 h. After the vacuum had been removed with nitrogen, the temperature was increased to 145° C. and 3206.7 g ethylene oxide were added in portions up to 7 bar. To complete the reaction, the mixture was allowed to post-react for 2 h at 120° C. The reaction mixture was stripped with nitrogen and volatile compounds were removed in vacuo at 70° C. 3968.0 g of a polyethyleneimine 600 with 10 mole of ethylene oxide per mole NH bond were obtained as a yellow-brown viscous liquid (Amine value: 101.5 mg KOH/g; pH of a 10% by weight aqueous solution: 11.6). c) PEI600+10 EO/NH, 75% Quatemized with Dimethyl Sulfate In a 0.5 l reaction vessel 120.0 g of the product from example 1 b) was heated to 70-75° C. under a constant stream of nitrogen. 20.5 g dimethyl sulfate was added within 15 min. The reaction mixture was stirred for additional 2 h at 75° C. For adjusting pH, 1.0 g NaOH (50% in water) was added. 110.0 g of light brown solid were obtained (Amine value: 23.5 mg KOH/g; pH of a 10% by weight aqueous solution: 9.3). Example 3: Synthesis of PEI600+7EO/NH, 75% Quaternized with Dimethyl Sulfate a) PEI600+7 EO/NH In a 2 l autoclave 261.0 g of the product obtained in Example 1 a) and 7.8 g of a 50% by weight aqueous solution of potassium hydroxide were heated to 80° C. and purged three times with nitrogen. The mixture was dewatered at 120° C. and a vacuum of 10 mbar for 2 h. After the vacuum had been removed with nitrogen, the temperature was increased to 145° C. and 792.0 g ethylene oxide were added in portions up to 7 bar. To complete the reaction, the mixture was allowed to post-react for 2 h at 120° C. The reaction mixture was stripped with nitrogen and volatile compounds were removed in vacuo at 70° C. 1056.0 g of a polyethyleneimine 600 with 7 mole of ethylene oxide per mole NH bond were obtained as a yellow-brown viscous liquid (Amine value: 147.8 mg KOH/g; pH of a 10% by weight aqueous solution: 11.6). b) PEI600+7 EO/NH, 75% Quatemized with Dimethyl Sulfate In a 0.5 l reaction vessel 250.0 g of the product from example 2 a) was heated to 70-75° C. under a constant stream of nitrogen. 58.4 g dimethyl sulfate was added within 15 min. The reaction mixture was stirred for additional 2 h at 75° C. 299.0 g of light brown solid were obtained (Amine value: 35.84 mg KOH/g; pH of a 10% by weight aqueous solution: 6.0; Iodine color number (10% in water): 4.0). Detergent Composition The detergent composition of the invention can be presented in any form. Preferably, the composition or part thereof is the form of loose powder and more preferable the composition is provided in unit-dose form, more preferably a unit dose form having a weight of from 10 to 20 grams. The composition of the invention is very well suited to be presented in the form of a multi-compartment pack, more in particular a multi-compartment pack comprising compartments with compositions in different physical forms, for example a compartment comprising a composition in the form of loose powder and another compartment comprising a composition in liquid form. The composition is preferably enveloped by a water-soluble film such as polyvinyl alcohol. The composition optionally but preferably comprises a complexing agent and/or a dispersant polymer. Preferably, the composition comprises the tri-sodium salt of MGDA, HEDP, dispersant polymer preferably a sulfonated polymer comprising 2-acrylamido-2-methylpropane sulfonic acid monomers, sodium carbonate, a bleach, preferably sodium percarbonate, protease and amylase enzymes and non-ionic surfactant and optionally crystalline silicaate. The composition is preferably free of citrate. The composition can further comprise a cationic polymer that provides anti-spotting benefits. The composition of the invention preferably has a pH as measured in 1% weight/volume aqueous solution in distilled water at 20° C. of from about 9 to about 12, more preferably from about 10 to less than about 11.5 and especially from about 10.5 to about 11.5. The composition of the invention preferably has a reserve alkalinity of from about 10 to about 20, more preferably from about 12 to about 18 at a pH of 9.5 as measured in NaOH with 100 mL of product at 20° C. Complexing Agent Complexing agents are materials capable of sequestering hardness ions, particularly calcium and/or magnesium. The composition of the invention comprises a high level of complexing agent, however the level should not be too high otherwise enzymes, in particular proteases can be negatively affected. Too high level of complexing agent can also negatively impact on glass care. The composition of the invention preferably comprises from 15% to 40%, preferably from 20% to 40%, more preferably from 20% to 35% by weight of the composition of a complexing agent selected from the group consisting of methylglycine-N,N-diacetic acid (MGDA), citric acid, glutamic acid-N,N-diacetic acid (GLDA) its salts and mixtures thereof. Especially preferred complexing agent for use herein is a salt of MGDA, in particular the trisodium salt of MGDA. Preferably, the composition of the invention comprises from 10% to 40% by weight of the composition of the trisodium salt of MGDA. Sodium Silicate The composition of the present invention may comprise silicate. If the composition comprises silicate, it preferably comprises from 2% to 8%, more preferably from 3% to 6% by weight of the composition of a crystalline sodium silicate. The crystalline sodium silicate, is preferably a layered silicate and preferably has the composition NaMSix O2x+1. y H2O, in which M denotes sodium or hydrogen, x is 1.9 to 4 and y is 0 to 20. The crystalline sodium silicates that can be optionally used in the composition of the invention can be layered in scanning electron microscope photographs. From the known compounds of the formula Na2SixO2x+1. y H2O, the corresponding compounds NaHSix O2x+1. y H2O can be prepared by treatment with acids and, in some cases, also with water. The water content given by the number y makes no differentiation between water of crystallization and adhering water. M preferably represents sodium. Preferred values of x are from 1.9 to 4. Compounds having the composition NaMSi 2 O5. y H2O are particularly preferred. Since the sodium silicates employed according to the invention are crystalline compounds, they can easily be characterized by their X-ray diffraction diagrams. Preferred layered crystalline silicates are those, in which x in the aforesaid general formula assumes the values 1.9 to 3.5. In particular, both delta- and beta-disodium disilicate (Na2Si2O5.yH2O) are preferred, with beta-disodium disilicate can be obtained, for example, by the process described in WO 91/08171 A1. Beta-disodium silicates with a molar ratio of SiO 2/Na 2 O between 1, 9 and 3.2 can be prepared according to Japanese Patent Application JP04/238809A or JP04/260610A. It can also be prepared from amorphous silicates, practically anhydrous crystalline alkali metal silicates of the abovementioned general formula (1), in which x is a number from 1, 9 to 2.1. In a further preferred embodiment of such agents, a crystalline sodium layer silicate with a molar ratio of SiO2/Na2O of 1.8 to 3 is used. In a preferred form, crystalline layered disodium disilicate builder is form from varying percentages of polymorphic phases alpha, beta and delta together. In commercially produced products, amorphous portions may also be present. The definitions of alpha, beta and delta disodium disilicate are known and can be found, for example, in EP0164514A1, as set forth below. The disodium state is preferably a layered crystalline disodium disilicate which consists of at least one of the polymorphic phases of the disodium disilicate and of sodium silicates of non-layered silicate nature. Particular preference is given to using crystalline sodium layer silicates having a content of from 80 to 100% by weight of delta-disodium disilicate. In a further preferred variant, it is also possible to use crystalline sodium layer silicates having a content of 70 to 100% by weight of beta disodium disilicate. Crystalline sodium layer silicates used with particular preference contain 1 to 40% by weight of alpha disodium disilicate, 0 to 50% by weight, in particular 0 to 45% by weight, of beta disodium disilicate, 50 to 98% by weight of delta disodium disilicate and 0 to 40% by weight of non-silicate sodium silicates (amorphous portions). Very particularly preferably used crystalline layered sodium silicates contain 7 to 21 wt % alpha disodium disilicate, 0 to 12 wt % beta disodium disilicate, 65 to 95 wt % delta disodium disilicate and 0 to 20 wt % amorphous shares. The abovementioned alpha-disodium disilicate corresponds to the Na-SK-S5 described in EP0164514 A1, characterized by those reproduced by X-ray diffraction data assigned to alpha-Na2Si2O5. The X-ray diffraction diagrams are available from the Joint Committee of Powder Diffraction Standards are registered under numbers 18-1241, 22-1397, 22-1397A, 19-1233, 19-1234 and 19-1237. The abovementioned beta-disodium disilicate corresponds to the Na-SKS-7 described in EP064514 A1, characterized by those reproduced there X-ray diffraction data assigned to beta-Na2Si2O5. The X-ray diffraction diagrams are available from the Joint Committee of Powder Diffraction Standards registered under the numbers 24-1123 and 29-1261. The abovementioned delta-disodium disilicate corresponds to that in EP0164514A described Na-SKS-6, characterized by the reproduced there X-ray diffraction data assigned to the delta-Na2Si2O5. The X-ray diffraction patterns are registered with the Joint Committee of Powder Diffraction Standards under the number 22-1396. The compositions according to the invention contain crystalline sodium layer silicate of the formula (1) in granulated form, and also cogranules containing crystalline sodium layer silicate and sparingly soluble metal carbonate, as described, for example, in WO2007/101622 A1. In a further preferred embodiment of the invention, the compositions of invention according to contain crystalline sodium disilicates Na2Si2O5.yH20 with y=0 to 2. In a preferred form, the crystalline layered sodium silicates additionally contain cationic and/or anionic constituents. The cationic constituents are preferably combinations of alkali metal and/or alkaline earth metal cations and/or Fe, W, Mo, Ta, Pb, A1, Zn, Ti, V, Cr, Mn, Co and/or Ni. The anionic constituents are preferably aluminates, sulfates, fluorides, chlorides, bromides, iodides, carbonates, bicarbonates, nitrates, oxide hydrates, phosphates and/or borates. In an alternative preferred form containing crystalline layered sodium silicates, based on the total content of SiO2, up to 10 mol % boron. In another alternative preferred form include the crystalline layered sodium silicates, based on the total content of SiO2, up to 20 mol % Phosphorus. Also, particularly preferred are sodium disilicates prepared hydrothermally of formula beta-Na are 2 Si2O5, as described in patent documents WO92/09526 A1, U.S. Pat. No. 5,417,951, DE 41 02 743 A1 and WO92/13935 A1, As sodium layer silicates, those according to WO00/09444 A1 are particularly preferred. Further preferred sodium layer silicates are those according to EP 0 550 048 A1 and EP 0 630 855 A1. The especially preferred silicate for use herein has the formula: Na2Si2O5. Carbonate The composition of the invention preferably comprise carbonate. It preferably comprises from 10% to 30%, preferably 5% to 25% by weight of the composition of sodium carbonate. Phosphonate Preferably the composition of the invention comprises phosphonate, preferably HEDP. It preferably comprise from 0.5% to 7%, preferably 1% to 6% by weight of the composition of HEDP. The composition is preferably free of phosphate, i.e., comprises less than 1%, more preferably less than 0.1% by weight of the composition of phosphate. Bleach Inorganic and organic bleaches are suitable for use herein. Inorganic bleaches include perhydrate salts such as perborate, percarbonate, perphosphate, persulfate and persilicate salts. The inorganic perhydrate salts are normally the alkali metal salts. The inorganic perhydrate salt may be included as the crystalline solid without additional protection. Alternatively, the salt can be coated. Alkali metal percarbonates, particularly sodium percarbonate is the preferred bleach for use herein. The percarbonate is most preferably incorporated into the products in a coated form which provides in-product stability. Potassium peroxymonopersulfate is another inorganic perhydrate salt of utility herein. Typical organic bleaches are organic peroxyacids, especially diperoxydodecanedioc acid, diperoxytetradecanedioc acid, and diperoxyhexadecanedioc acid. Mono- and diperazelaic acid, mono- and diperbrassylic acid are also suitable herein. Diacyl and Tetraacylperoxides, for instance dibenzoyl peroxide and dilauroyl peroxide, are other organic peroxides that can be used in the context of this invention. Further typical organic bleaches include the peroxyacids, particular examples being the alkylperoxy acids and the arylperoxy acids. Preferred representatives are (a) peroxybenzoic acid and its ring-substituted derivatives, such as alkylperoxybenzoic acids, but also peroxy-α-naphthoic acid and magnesium monoperphthalate, (b) the aliphatic or substituted aliphatic peroxy acids, such as peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid[phthaloiminoperoxyhexanoic acid (PAP)], o-carboxybenzamidoperoxycaproic acid, N-nonenylamidoperadipic acid and N-nonenylamidopersuccinates, and (c) aliphatic and araliphatic peroxydicarboxylic acids, such as 1,12-diperoxycarboxylic acid, 1,9-diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid, the diperoxyphthalic acids, 2-decyldiperoxybutane-1,4-dioic acid, N,N-terephthaloyldi(6-aminopercaproic acid). Preferably, the level of bleach in the composition of the invention is from about 1 to about 20%, more preferably from about 2 to about 25%, even more preferably from about 3 to about 20% by weight of the composition. Specially preferred are compositions comprising percarbonate. Dispersant Polymer The dispersant polymer is used in any suitable amount from about 1 to about 7%, preferably from 2 to about 6% by weight of the composition. The dispersant polymer is capable to suspend calcium or calcium carbonate in an automatic dishwashing process. Preferably, the dispersant polymers are sulfonated derivatives of polycarboxylic acids and may comprise two, three, four or more different monomer units. The preferred copolymers contain: At least one structural unit derived from a carboxylic acid monomer having the general formula (III): alkyl groups having from 2 to 12 carbon atoms, linear or branched mono or polyunsaturated alkenyl groups having from 2 to 12 carbon atoms, alkyl or alkenyl groups as aforementioned substituted with —NH2 or —OH, or —COOH, or COOR4, where R4 is selected from hydrogen, alkali metal, or a linear or branched, saturated or unsaturated alkyl or alkenyl group with 2 to 12 carbons; Preferred carboxylic acid monomers include one or more of the following: acrylic acid, maleic acid, maleic anhydride, itaconic acid, citraconic acid, 2-phenylacrylic acid, cinnamic acid, crotonic acid, fumaric acid, methacrylic acid, 2-ethylacrylic acid, methylenemalonic acid, or sorbic acid. Acrylic and methacrylic acids being more preferred. Optionally, one or more structural units derived from at least one nonionic monomer having the general formula (IV): Wherein R5 to R7 are independently selected from hydrogen, methyl, phenyl or hydroxyalkyl groups containing 1 to 6 carbon atoms, and can be part of a cyclic structure, X is an optionally present spacer group which is selected from —CH2-, —COO—, —CONH— or —CONR8-, and R8 is selected from linear or branched, saturated alkyl radicals having 1 to 22 carbon atoms or unsaturated, preferably aromatic, radicals having from 6 to 22 carbon atoms. Preferred non-ionic monomers include one or more of the following: butene, isobutene, pentene, 2-methylpent-1-ene, 3-methylpent-1-ene, 2,4,4-trimethylpent-1-ene, 2,4,4-trimethylpent-2-ene, cyclopentene, methylcyclopentene, 2-methyl-3-methyl-cyclopentene, hexene, 2,3-dimethylhex-1-ene, 2,4-dimethylhex-1-ene, 2,5-dimethylhex-1-ene, 3,5-dimethylhex-1-ene, 4,4-dimethylhex-1-ene, cyclohexene, methylcyclohexene, cycloheptene, alpha olefins having 10 or more carbon atoms such as, dec-1-ene, dodec-1-ene, hexadec-1-ene, octadec-1-ene and docos-1-ene, preferred aromatic monomers are styrene, alpha methylstyrene, 3-methylstyrene, 4-dodecylstyrene, 2-ethyl-4-bezylstyrene, 4-cyclohexylstyrene, 4-propylstyrol, 1-vinylnaphtalene, 2-vinylnaphtalene; preferred carboxylic ester monomers are methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, t-butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate and behenyl (meth)acrylate; preferred amides are N-methyl acrylamide, N-ethyl acrylamide, N-t-butyl acrylamide, N-2-ethylhexyl acrylamide, N-octyl acrylamide, N-lauryl acrylamide, N-stearyl acrylamide, N-behenyl acrylamide; and at least one structural unit derived from at least one sulfonic acid monomer having the general formula (V) and (VI): wherein R7 is a group comprising at least one sp2 bond, A is O, N, P, S, an amido or ester linkage, B is a mono- or polycyclic aromatic group or an aliphatic group, each t is independently 0 or 1, and M+ is a cation. In one aspect, R7 is a C2 to C6 alkene. In another aspect, R7 is ethene, butene or propene. Preferred sulfonated monomers include one or more of the following: 1-acrylamido-1-propanesulfonic acid, 2-acrylamido-2-propanesulfonic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-methacrylamido-2-methyl-1-propanesulfonic acid, 3-methacrylamido-2-hydroxy-propanesulfonic acid, allylsulfonic acid, methallylsulfonic acid, allyloxybenzenesulfonic acid, methallyloxybenzenesulfonic acid, 2-hydroxy-3-(2-propenyloxy) propanesulfonic acid, 2-methyl-2-propen-1-sulfonic acid, styrenesulfonic acid, vinylsulfonic acid, 3-sulfopropyl, 3-sulfo-propylmethacrylate, sulfomethacrylamide, sulfomethylmethacrylamide and mixtures of said acids or their water-soluble salts. Preferably, the polymer comprises the following levels of monomers: from about 40 to about 90%, preferably from about 60 to about 90% by weight of the polymer of one or more carboxylic acid monomer; from about 5 to about 50%, preferably from about 10 to about 40% by weight of the polymer of one or more sulfonic acid monomer; and optionally from about 1% to about 30%, preferably from about 2 to about 20% by weight of the polymer of one or more non-ionic monomer. An especially preferred polymer comprises about 70% to about 80% by weight of the polymer of at least one carboxylic acid monomer and from about 20% to about 30% by weight of the polymer of at least one sulfonic acid monomer. In the polymers, all or some of the carboxylic or sulfonic acid groups can be present in neutralized form, i.e. the acidic hydrogen atom of the carboxylic and/or sulfonic acid group in some or all acid groups can be replaced with metal ions, preferably alkali metal ions and in particular with sodium ions. The carboxylic acid is preferably (meth)acrylic acid. The sulfonic acid monomer is preferably 2-acrylamido-2-propanesulfonic acid (AMPS). Preferred commercial available polymers include: Alcosperse 240, Aquatreat AR 540 and Aquatreat MPS supplied by Alco Chemical; Acumer 3100, Acumer 2000, Acusol 587G and Acusol 588G supplied by Dow; Goodrich K-798, K-775 and K-797 supplied by BF Goodrich; and ACP 1042 supplied by ISP technologies Inc. Particularly preferred polymers are Acusol 587G and Acusol 588G supplied by Rohm & Haas. Suitable dispersant polymers include anionic carboxylic polymer of low molecular weight. They can be homopolymers or copolymers with a weight average molecular weight of less than or equal to about 200,000 g/mol, or less than or equal to about 75,000 g/mol, or less than or equal to about 50,000 g/mol, or from about 3,000 to about 50,000 g/mol, preferably from about 5,000 to about 45,000 g/mol. The dispersant polymer may be a low molecular weight homopolymer of polyacrylate, with an average molecular weight of from 1,000 to 20,000, particularly from 2,000 to 10,000, and particularly preferably from 3,000 to 5,000. The dispersant polymer may be a copolymer of acrylic with methacrylic acid, acrylic and/or methacrylic with maleic acid, and acrylic and/or methacrylic with fumaric acid, with a molecular weight of less than 70,000. Their molecular weight ranges from 2,000 to 80,000 and more preferably from 20,000 to 50,000 and in particular 30,000 to 40,000 g/mol, and a ratio of (meth)acrylate to maleate or fumarate segments of from 30:1 to 1:2. The dispersant polymer may be a copolymer of acrylamide and acrylate having a molecular weight of from 3,000 to 100,000, alternatively from 4,000 to 20,000, and an acrylamide content of less than 50%, alternatively less than 20%, by weight of the dispersant polymer can also be used. Alternatively, such dispersant polymer may have a molecular weight of from 4,000 to 20,000 and an acrylamide content of from 0% to 15%, by weight of the polymer. Dispersant polymers suitable herein also include itaconic acid homopolymers and copolymers. Alternatively, the dispersant polymer can be selected from the group consisting of alkoxylated polyalkyleneimines, alkoxylated polycarboxylates, polyethylene glycols, styrene co-polymers, cellulose sulfate esters, carboxylated polysaccharides, amphiphilic graft copolymers and mixtures thereof. Surfactant Surfactants suitable for use herein include non-ionic surfactants, preferably the compositions are free of any other surfactants. Traditionally, non-ionic surfactants have been used in automatic dishwashing for surface modification purposes in particular for sheeting to avoid filming and spotting and to improve shine. It has been found that non-ionic surfactants can also contribute to prevent redeposition of soils. Preferably the composition of the invention comprises a non-ionic surfactant or a non-ionic surfactant system, more preferably the non-ionic surfactant or a non-ionic surfactant system has a phase inversion temperature, as measured at a concentration of 1% in distilled water, between 40 and 70° C., preferably between 45 and 65° C. By a “non-ionic surfactant system” is meant herein a mixture of two or more non-ionic surfactants. Preferred for use herein are non-ionic surfactant systems. They seem to have improved cleaning and finishing properties and better stability in product than single non-ionic surfactants. Phase inversion temperature is the temperature below which a surfactant, or a mixture thereof, partitions preferentially into the water phase as oil-swollen micelles and above which it partitions preferentially into the oil phase as water swollen inverted micelles. Phase inversion temperature can be determined visually by identifying at which temperature cloudiness occurs. The phase inversion temperature of a non-ionic surfactant or system can be determined as follows: a solution containing 1% of the corresponding surfactant or mixture by weight of the solution in distilled water is prepared. The solution is stirred gently before phase inversion temperature analysis to ensure that the process occurs in chemical equilibrium. The phase inversion temperature is taken in a thermostable bath by immersing the solutions in 75 mm sealed glass test tube. To ensure the absence of leakage, the test tube is weighed before and after phase inversion temperature measurement. The temperature is gradually increased at a rate of less than 1° C. per minute, until the temperature reaches a few degrees below the pre-estimated phase inversion temperature. Phase inversion temperature is determined visually at the first sign of turbidity. Suitable nonionic surfactants include: i) ethoxylated non-ionic surfactants prepared by the reaction of a monohydroxy alkanol or alkyphenol with 6 to 20 carbon atoms with preferably at least 12 moles particularly preferred at least 16 moles, and still more preferred at least 20 moles of ethylene oxide per mole of alcohol or alkylphenol; ii) alcohol alkoxylated surfactants having a from 6 to 20 carbon atoms and at least one ethoxy and propoxy group. Preferred for use herein are mixtures of surfactants i) and ii). Another suitable non-ionic surfactants are epoxy-capped poly(oxyalkylated) alcohols represented by the formula: R1O[CH2CH(CH3)O]x[CH2CH2O]y[CH2CH(OH)R2] (I) wherein R1 is a linear or branched, aliphatic hydrocarbon radical having from 4 to 18 carbon atoms; R2 is a linear or branched aliphatic hydrocarbon radical having from 2 to 26 carbon atoms; x is an integer having an average value of from 0.5 to 1.5, more preferably about 1; and y is an integer having a value of at least 15, more preferably at least 20. Preferably, the surfactant of formula I, at least about 10 carbon atoms in the terminal epoxide unit [CH2CH(OH)R2]. Suitable surfactants of formula I, according to the present invention, are Olin Corporation's POLY-TERGENT® SLF-18B nonionic surfactants, as described, for example, in WO 94/22800, published Oct. 13, 1994 by Olin Corporation. Amine oxides surfactants useful herein include linear and branched compounds having the formula: wherein R3 is selected from an alkyl, hydroxyalkyl, acylamidopropoyl and alkyl phenyl group, or mixtures thereof, containing from 8 to 26 carbon atoms, preferably 8 to 18 carbon atoms; R4 is an alkylene or hydroxyalkylene group containing from 2 to 3 carbon atoms, preferably 2 carbon atoms, or mixtures thereof; x is from 0 to 5, preferably from 0 to 3; and each R5 is an alkyl or hydroxyalkyl group containing from 1 to 3, preferably from 1 to 2 carbon atoms, or a polyethylene oxide group containing from 1 to 3, preferable 1, ethylene oxide groups. The R5 groups can be attached to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure. These amine oxide surfactants in particular include C10-C18 alkyl dimethyl amine oxides and C8-C18 alkoxy ethyl dihydroxyethyl amine oxides. Examples of such materials include dimethyloctylamine oxide, diethyldecylamine oxide, bis-(2-hydroxyethyl)dodecylamine oxide, dimethyldodecylamine oxide, dipropyltetradecylamine oxide, methylethylhexadecylamine oxide, dodecylamidopropyl dimethylamine oxide, cetyl dimethylamine oxide, stearyl dimethylamine oxide, tallow dimethylamine oxide and dimethyl-2-hydroxyoctadecylamine oxide. Preferred are C10-C18 alkyl dimethylamine oxide, and C10-18 acylamido alkyl dimethylamine oxide. Surfactants may be present in amounts from 0 to 15% by weight, preferably from 0.1% to 10%, and most preferably from 0.25% to 8% by weight of the total composition. Enzymes In describing enzyme variants herein, the following nomenclature is used for ease of reference: Original amino acid(s):position(s):substituted amino acid(s). Standard enzyme IUPAC 1-letter codes for amino acids are used. Proteases The composition of the invention is beneficial in terms of removal of proteinaceous soils, in particular sugary burn soils such as crème brulee. The composition of the invention can comprise a protease. A mixture of two or more proteases can also contribute to an enhanced cleaning across a broader temperature, cycle duration, and/or substrate range, and provide superior shine benefits, especially when used in conjunction with an anti-redeposition agent and/or a sulfonated polymer. Suitable proteases include metalloproteases and serine proteases, including neutral or alkaline microbial serine proteases, such as subtilisins (EC 3.4.21.62). Suitable proteases include those of animal, vegetable or microbial origin. In one aspect, such suitable protease may be of microbial origin. The suitable proteases include chemically or genetically modified mutants of the aforementioned suitable proteases. In one aspect, the suitable protease may be a serine protease, such as an alkaline microbial protease or/and a trypsin-type protease. Examples of suitable neutral or alkaline proteases include: (a) subtilisins (EC 3.4.21.62), especially those derived fromBacillus, such asBacillussp.,B. lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, B. pumilus, B. gibsonii, andB. akibaiidescribed in WO2004067737, WO2015091989, WO2015091990, WO2015024739, WO2015143360, U.S. Pat. Nos. 6,312,936, 5,679,630, 4,760,025, DE102006022216A1, DE 102006022224A1, WO2015089447, WO2015089441, WO2016066756, WO2016066757, WO2016069557, WO2016069563, WO2016069569.(b) trypsin-type or chymotrypsin-type proteases, such as trypsin (e.g., of porcine or bovine origin), including theFusariumprotease described in WO 89/06270 and the chymotrypsin proteases derived from Cellumonas described in WO 05/052161 and WO 05/052146.(c) metalloproteases, especially those derived fromBacillus amyloliquefaciensdescribed in WO07/044993A2; fromBacillus, Brevibacillus, Thermoactinomyces, Geobacillus, Paenibacillus, Lysinibacillus orStreptomycesspp. described in WO2014194032, WO2014194054 and WO2014194117; fromKribella alluminosadescribed in WO2015193488; and fromStreptomycesand Lysobacter described in WO2016075078.(d) protease having at least 90% identity to the subtilase fromBacillussp. TY 145, NCIMB 40339, described in WO92/17577 (Novozymes A/S), including the variants of thisBacillussp TY145 subtilase described in WO2015024739, and WO2016066757.(e) protease having at least 90%, preferably at least 92% identity with the amino acid sequence of SEQ ID NO:85 from WO2016/205755 comprising at least one amino acid substitution (using the SEQ ID NO:85 numbering) selected from the group consisting of 1, 4, 9, 21, 24, 27, 36, 37, 39, 42, 43, 44, 47, 54, 55, 56, 74, 80, 85, 87, 99, 102, 114, 117, 119, 121, 126, 127, 128, 131, 143, 144, 158, 159, 160, 169, 182, 188, 190, 197, 198, 212, 224, 231, 232, 237, 242, 245, 246, 254, 255, 256, and 257, including the variants found in WO2016/205755 and WO2018/118950. Especially preferred proteases for the detergent of the invention are:(a) polypeptides demonstrating at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% and especially 100% identity with the wild-type enzyme fromBacillus lentus, comprising mutations in one or more, preferably two or more and more preferably three or more of the following positions, using the BPN′ numbering system and amino acid abbreviations as illustrated in WO00/37627, which is incorporated herein by reference:V68A, N76D, N87S, S99D, S99AD, S99A, S101G, S101M, S103A, V104N/I, G118V, G118R, 5128L, P129Q, 5130A, Y167A, R1705, A194P, V205I, Q206L/D/E, Y209W and/or M222S. and/or(b) protease having at least 95%, more preferably at least 98%, even more preferably at least 99% and especially 100% identity with the amino acid sequence of SEQ ID NO:85 from WO2016/205755 comprising at least one amino acid substitution (using the SEQ ID NO:85 numbering) selected from the group comprising:P54E/G/I/L/Q/S/TN; S99A/E/H/I/K/M/N/Q/R/TN;S126A/D/E/F/G/H/I/L/M/N/Q/R/TN/Y; D127A/E/F/G/H/I/L/MN/P/Q/S/TN/W/Y; F128A/C/D/E/G/H/I/K/L/M/N/P/Q/R/S/T/W, A37T, S39E, A47V, T56Y, 180V, N85S, E87D, T114Q, and N242D; Most preferably the additional protease is either selected from the group of proteases comprising the below mutations (BPN′ numbering system) versus either the PB92 wild-type (SEQ ID NO:2 in WO 08/010925) or the subtilisin 309 wild-type (sequence as per PB92 backbone, except comprising a natural variation of N87S).(i) G118V+S128L+P129Q+S130A(ii) S101M+G118V+S128L+P129Q+S130A(iii) N76D+N87R+G118R+S128L+P129Q+S130A+S188D+N248R(iv) N76D+N87R+G118R+S128L+P129Q+S130A+S188D+V244R(v) N76D+N87R+G118R+S128L+P129Q+S130A(vi) V68A+N87S+S101G+V104N(vii) S99AD or selected from the group of proteases comprising one or more, preferably two or more, preferably three or more, preferably four or more of the below mutations versus SEQ ID NO:1 from WO2018/118950:P54T, S99M, S126A/G, D127E, F128C/D/E/G, A37T, S39E, A47V, T56Y, 180V, N85S, E87D, T114Q, and N242D. Suitable commercially available additional protease enzymes include those sold under the trade names Alcalase®, Savinase®, Primase®, Durazym®, Polarzyme®, Kannase®, Liquanase®, Liquanase Ultra®, Savinase Ultra®, Savinase Evity®, Ovozyme®, Neutrase®, Everlase®, Coronase®, Blaze®, Blaze Ultra®, Blaze Evity® and Esperase® by Novozymes A/S (Denmark); those sold under the tradename Maxatase®, Maxacal®, Maxapem®, Properase®, Purafect®, Purafect Prime®, Purafect Ox®, FN3®, FN4®, Excellase®, Ultimase®, Extremase® and Purafect OXP® by Dupont; those sold under the tradename Opticlean® and Optimase® by Solvay Enzymes; and those available from Henkel/Kemira, namely BLAP (sequence shown in FIG. 29 of U.S. Pat. No. 5,352,604 with the following mutations S99D+S101 R+S103A+V104I+G159S, hereinafter referred to as BLAP), BLAP R (BLAP with S3T+V4I+V199M+V205I+L217D), BLAP X (BLAP with S3T+V4I+V205I) and BLAP F49 (BLAP with S3T+V4I+A194P+V199M+V205I+L217D); and KAP (Bacillus alkalophilussubtilisin with mutations A230V+S256G+S259N) from Kao. Especially preferred for use herein are commercial proteases selected from the group consisting of Properase®, Blaze®, Blaze Evity®, Savinase Evity®, Extremase®, Ultimase®, Everlase®, Savinase®, Excellase®, Blaze Ultra®, BLAP and BLAP variants. Preferred levels of protease in the product of the invention include from about 0.05 to about 20, more preferably from about 0.5 to about 10 and especially from about 1 to about 8 mg of active protease/g of composition. Amylases Preferably the composition of the invention may comprise an amylase. Suitable alpha-amylases include those of bacterial or fungal origin. Chemically or genetically modified mutants (variants) are included. A preferred alkaline alpha-amylase is derived from a strain ofBacillus, such asBacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus subtilis, or otherBacillussp., such asBacillussp. NCBI 12289, NCBI 12512, NCBI 12513, DSM 9375 (U.S. Pat. No. 7,153,818) DSM 12368, DSMZ no. 12649, KSM AP1378 (WO 97/00324), KSM K36 or KSM K38 (EP 1,022,334). Preferred amylases include:(a) variants described in WO 96/23873, WO00/60060, WO06/002643 and WO2017/192657, especially the variants with one or more substitutions in the following positions versus SEQ ID NO. 12 of WO06/002643:26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 202, 214, 231, 246, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 461, 471, 482, 484, preferably that also contain the deletions of D 183* and G184*.(b) variants exhibiting at least 90% identity with SEQ ID No. 4 in WO06/002643, the wild-type enzyme fromBacillusSP722, especially variants with deletions in the 183 and 184 positions and variants described in WO 00/60060, WO2011/100410 and WO2013/003659 which are incorporated herein by reference.(c) variants exhibiting at least 95% identity with the wild-type enzyme fromBacillussp. 707 (SEQ ID NO:7 in U.S. Pat. No. 6,093,562), especially those comprising one or more of mutations in the following positions M202, M208, S255, R172, and/or M261. Preferably said amylase comprises one or more of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N and/or R172Q. Particularly preferred are those comprising the M202L or M202T mutations.(d) variants described in WO 09/149130, preferably those exhibiting at least 90% identity with SEQ ID NO: 1 or SEQ ID NO: 2 in WO 09/149130, the wild-type enzyme fromGeobacillusStearophermophilus or a truncated version thereof.(e) variants exhibiting at least 89% identity with SEQ ID NO:1 in WO2016091688, especially those comprising deletions at positions H183+G184 and additionally one or more mutations at positions 405, 421, 422 and/or 428.(f) variants exhibiting at least 60% amino acid sequence identity with the “PcuAmyl a-amylase” fromPaenibacillus curdlanolyticusYK9 (SEQ ID NO:3 in WO2014099523).(g) variants exhibiting at least 60% amino acid sequence identity with the“CspAmy2 amylase” from Cytophaga sp. (SEQ ID NO:1 in WO2014164777).(h) variants exhibiting at least 85% identity with AmyE fromBacillus subtilis(SEQ ID NO:1 in WO2009149271).(i) variants exhibiting at least 90% identity with the wild-type amylase fromBacillussp. KSM-K38 with accession number AB051102.(j) variants exhibiting at least 80% identity with the mature amino acid sequence of AAI10 fromBacillussp (SEQ ID NO:7 in WO2016180748), preferably comprising a mutation in one or more of the following positions modification in one or more positions 1, 54, 56, 72, 109, 113, 116, 134, 140, 159, 167, 169, 172, 173, 174, 181, 182, 183, 184, 189, 194, 195, 206, 255, 260, 262, 265, 284, 289, 304, 305, 347, 391, 395, 439, 469, 444, 473, 476, or 477(k) variants exhibiting at least 80% identity with the mature amino acid sequence of the fusion peptide (SEQ ID NO:14 in US 2019/0169546), preferably comprising one or more of the mutations H1*, N54S+V56T, A60V, G109A, R116Q/H+W167F, L173V, A174S, Q172N, G182*, D183*, N195F, V206L/Y, V208L, K391A, K393A, I405L, A421H, A422P, A428T, G476K and/or G478K. Preferred amylases contain both the deletions G182* and G183* and optionally one or more of the following sets of mutations:1. H1*+G109A+N195F+V206Y+K391A;2. H1*+N54S+V56T+G109A+A1745+N195F+V206L+K391A+G476K)3. H1*+N54S+V56T+A60V+G109A+R116Q+W167F+Q172N+L173V+A1745+N195F+V206L+1405L+A421H+A422P+A428T4. H1*+N545+V56T+G109A+R116Q+A1745+N195F+V206L+1405L+A421H+A422P+A428T;5. H1*+N545+V56T+G109A+R116H+A1745+N195F+V208L+K393A+G478K;(l) variants exhibiting at least 80% identity with the mature amino acid sequence ofAlicyclobacillussp. amylase (SEQ ID NO:8 in WO2016180748) The amylase can be an engineered enzyme, wherein one or more of the amino acids prone to bleach oxidation have been substituted by an amino acid less prone to oxidation. In particular it is preferred that methionine residues are substituted with any other amino acid. In particular it is preferred that the methionine most prone to oxidation is substituted. Preferably the methionine in a position equivalent to 202 in SEQ ID NO:2 is substituted. Preferably, the methionine at this position is substituted with threonine or leucine, preferably leucine. Suitable commercially available alpha-amylases include DURAMYL®, LIQUEZYME®, TERMAMYL®, TERMAMYL ULTRA®, NATALASE®, SUPRAMYL®, STAINZYME®, STAINZYME PLUS®, FUNGAMYL®, ATLANTIC®, INTENSA® and BAN® (Novozymes A/S, Bagsvaerd, Denmark), KEMZYM® AT 9000 Biozym Biotech Trading GmbH Wehlistrasse 27b A-1200 Wien Austria, RAPIDASE®, PURASTAR®, ENZYSIZE®, OPTISIZE HT PLUS®, POWERASE®, PREFERENZ S® series (including PREFERENZ S1000® and PREFERENZ 52000® and PURASTAR OXAM® (DuPont, Palo Alto, Calif.) and KAM® (Kao, 14-10 Nihonbashi Kayabacho, 1-chome, Chuo-ku Tokyo 103-8210, Japan). In one aspect, suitable amylases include ATLANTIC®, STAINZYME®, POWERASE®, INTENSA® and STAINZYME PLUS®, ACHIEVE ALPHA® and mixtures thereof. Preferably, the product of the invention comprises at least 0.01 mg, preferably from about 0.05 to about 10, more preferably from about 0.1 to about 6, especially from about 0.2 to about 5 mg of active amylase/g of composition. Preferably, the protease and/or amylase of the composition of the invention are in the form of granulates, the granulates comprise more than 29% of sodium sulfate by weight of the granulate and/or the sodium sulfate and the active enzyme (protease and/or amylase) are in a weight ratio of between 3:1 and 100:1 or preferably between 4:1 and 30:1 or more preferably between 5:1 and 20:1. Metal Care Agents Metal care agents may prevent or reduce the tarnishing, corrosion or oxidation of metals, including aluminium, stainless steel and non-ferrous metals, such as silver and copper. Preferably the composition of the invention comprises from 0.1 to 5%, more preferably from 0.2 to 4% and especially from 0.3 to 3% by weight of the product of a metal care agent, preferably the metal care agent is benzo triazole (BTA). Glass Care Agents Glass care agents protect the appearance of glass items during the dishwashing process. Preferably the composition of the invention comprises from 0.1 to 5%, more preferably from 0.2 to 4% and specially from 0.3 to 3% by weight of the composition of a metal care agent, preferably the glass care agent is a zinc containing material, specially hydrozincite. Cationic Polymer The composition preferably comprises from 0.5 to 5%, preferably from 0.5 to 2% by weight of the composition of cationic polymer. The cationic polymer provides filming benefits. The cationic polymer comprises in copolymerized form from:i. 60% to 99% by weight of the cationic polymer of at least one monoethylenically unsaturated polyalkylene oxide monomer of the formula I (monomer (A)) in which the variables have the following meanings:X is —CH2- or —CO—, if Y is —O—;X is —CO—, if Y is —NH—;Y is —O— or —N14-;R1 is hydrogen or methyl;R2 are identical or different C2-C6-alkylene radicals;R3 is H or C1-C4 alkyl;n is an integer from 3 to 100, preferably from 15 to 60,ii. from 1 to 40% by weight of the cationic polymer of at least one quatemized nitrogen-containing monomer, selected from the group consisting of at least one of the monomers of the formula IIa to IId (monomer (B))i. )) in which the variables have the following meanings:R is C1-C4 alkyl or benzyl;R′ is hydrogen or methyl;Y is —O— or —NH—;A is C1-C6 alkylene;X— is halide, C1-C4-alkyl sulfate, C1-C4-alkylsulfonate and C1-C4-alkyl carbonate.iii. from 0 to 15% by weight of the cationic polymer of at least one anionic monoethylenically unsaturated monomer (monomer (C)), andiv. from 0 to 30% by weight of the cationic polymer of at least one other nonionic monoethylenically unsaturated monomer (monomer (D)), and the cationic polymer has a weight average molecular weight (Mw) from 2,000 to 500,000, preferably from 25,000 g/mol to 200,000 g/mol. In preferred cationic polymers the variables of monomer (A) have the following meanings:X is —CO—;Y is —O—;R1 is hydrogen or methyl;R2 is ethylene, linear or branched propylene or mixtures thereof;R3 is methyl;n is an integer from 15 to 60. Preferably, the cationic polymer comprises from 60 to 98% by weight of monomer (A) and from 1 to 39% by weight of monomer (B) and from 0.5 to 6% by weight of monomer (C). In preferred cationic polymers monomer (A) is methylpolyethylene glycol (meth)acrylate and wherein monomer (B) is a salt of 3-methyl-1-vinylimidazolium. Preferably, the cationic polymer comprises from 69 to 89% of monomer (A) and from 9 to 29% of monomer (B). In preferred cationic polymers, the weight ratio of monomer (A) to monomer (B) is ≥2:1 and for the case where the copolymer comprises a monomer (C), the weight ratio of monomer (B) to monomer (C) is also ≥2:1, more preferably is ≥2.5:1 and preferably monomer (A) comprises methylpolyethylene glycol (meth)acrylate and monomer (B) comprises a salt of 3-methyl-1-vinylimidazolium. A preferred composition according to the invention comprises:a) from 10% to 40% by weight of the composition of MGDA, preferably the trisodium salt of methylglycine-N,N-diacetic acid;b) optionally from 2% to 6% by weight of the composition of crystalline sodium silicate having a crystalline layered structure and the composition NaMSix O2x+1.y H2O, in which M denotes sodium or hydrogen, x is a number from 1.9 to 4 and y is a number from 0 to 20, preferably having the formula Na2Si2O5.c) from 10% to 30% by weight of the composition of carbonate;d) optionally from 1% to 6% by weight of the composition of HEDP;e) from 2% to 6% by weight of the composition of a dispersant polymer, preferably a sulfonate polymer;f) from 8% to 30% by weight of the composition of sodium percarbonate;g) non-ionic surfactant;h) amylase;i) protease; and optionallyj) glass and/or metal care agent. Method of Automatic Dishwashing The method of the invention comprises the step of subjecting tableware to the composition of the invention. The method provides very good cleaning of bleachable stains and enzymatic soils. EXAMPLES Example I Four automatic dishwashing Compositions (Compositions A to D) were made and tested as detailed below. I. Preparation of Test Compositions Tests were carried out using the following detergent compositions. Material additions are shown at total raw material level. Unless stated otherwise, the raw materials are 100% active. Composition AComposition BComposition CComposition D(Inventive)(Comparative)(Comparitive)(Inventive)g%g%g%g%Sodium Carbonate1.508.211.507.891.507.891.508.22Sodium 1-hydroxyethylidene-0.955.200.955.000.955.000.955.201,1-diphosphonate (84.2%active)Trilon ® M (78% active)6.7236.866.7235.386.7235.396.7236.86Tetraacetylethy lenediamine0.000.000.764.000.764.000.000.00(92% active)Acusol ™ 588GF (sulfonated0.784.270.784.100.784.100.784.27polymersupplied by DowChemical)(93% active)Amylase granule (4.2% active)0.291.570.291.500.291.500.291.57Protease granule (10% active)0.854.660.854.470.854.470.854.66Protease granule (8.1% active)0.231.260.231.210.231.210.231.26WeylClean ® MnTACN (98%0.0030.0170.0030.0160.000.000.000.00active)Sodium Percarbonate (13.4%3.4919.173.4918.403.4918.403.4919.17AvO)Plurafac ® SLF180 (non-ionic0.834.580.834.390.834.390.834.58surfactant supplied by BASF)Lutensol ® TO 7 (non-ionic0.894.900.894.700.894.700.894.90surfactant supplied by BASF)Benzotriazole0.0080.0430.0080.0420.0080.0420.0080.043PEI600EO7 75% Quat0.402.190.402.100.402.100.402.19Processing Aids, fillers,1.297.081.296.801.296.801.297.08minors & perfumeTotal (one dose)18.2310018.9910018.9910018.23100 II. Test Items The following test items were used: ItemDescriptionStainedFirma Schönwald white ceramic teacup, 98 L/0.19.TeacupsStained with tea, according to IKW method(Recommendations for the Quality Assessment of theCleaning Performance ofDishwasher Detergents (Part B, Update 2015)).MincedCeramic side plate, Arzberg form 2000, no. 10219,meat platesø 19 cmStained with minced meat, according to IKW method(Recommendations for the Quality Assessment of theCleaning Performance of Dishwasher Detergents(Part B, Update 2015)).CrèmeDessert plate, Arzberg, white, glazed porcelain,brûléeconforming with standard EN 50242, form 2000,platesno. 10219, ø 19 cm.Stained with crème brûlée according to the IKW method(Recommendations for the Quality Assessment of theCleaning Performance of Dishwasher Detergents(Part B, Update 2015)).CFT tilesCentre for Testmaterials BV melamine tiles stained withthe following:CFT Baked Light CheeseCFT Egg YolkCFT Rice StarchCFT Mixed StarchAdditionalPrepared according to the IKW methodBallast Soil(Recommendations for the Quality Assessment ofthe Cleaning Performance of DishwasherDetergents (Part B, Update 2015)). III. Test Wash Procedure Automatic Dishwasher:Miele, model GSL2Wash volume:5000mLWater temperature:45°C.Water hardness:20gpgDetergent addition:Added into the bottom of the automatic dish-washer after the initial pre-wash is complete.Positioning of test2× Ceramic Teacups on top rackitems:Cleaning Tiles placed on top rack2× 50 g pots of Additional ballast soil addedto top rack at the start of the wash cycle.5× Crème Brulee stained plate on bottomrack with unstained plate at front as ballast6× Ceramic Side-plate stained with 3 gminced meat mixture Dishwashers were loaded with the items as detailed above which were washed using one dose of Compositions A to D. Four external replicates were completed for each test product following Latin square rotation of machines and products. The stained tiles were graded using an Image Analysis System to measure Stain Removal Index (SRI), where higher SRI removal is desired. IV. Results Results-Melamine Cleaning TilesCFTBakedCFTLightCFT EggCFT RiceMixedCheeseYolkStarchStarchComposition A89.6BC98.1BCd82.9bC75.8BC(Inventive)Composition B73.59780.355.4(Comparative)Composition C73.996.979.561.8(Comparative)Composition D94.6ABC97.6bC83BC80.8BC(Inventive)Tukey's HSD3.820.612.6011.00UPPER CASE letters indicate significant difference between treatments at alpha = 0.05 using Tukey's HSD.Lower case letters indicate significant difference between treatments at alpha = 0.05 using Fisher's LSD UPPER CASE letters indicate significant difference between treatments at alpha=0.05 using Tukey's HSD. Lower case letters indicate significant difference between treatments at alpha=0.05 using Fisher's LSD As can be seen from the results above, compositions according to the invention provide higher levels of stain removal. Example II Two automatic dishwashing Compositions (Compositions E and F) were made and tested as detailed below. I. Preparation of Test Compositions Tests were carried out using the following detergent compositions. Material additions are shown at total raw material level. Unless stated otherwise, the raw materials are 100% active. Composition EComposition F(Comparative)(Inventive)g%g%Sodium Carbonate6.8236.026.8236.20Sodium 1-hydroxyethylidene-1,1-0.170.900.170.90diphosphonate (84.2% active)Sodium Sulfate2.8014.792.8014.86Trilon ® M (78% active)3.7319.703.7319.81Acusol ™ 588GF (sulfonated polymer1.296.811.296.85supplied by DowChemical) (93% active)Amylase granule (1.44% active)0.180.950.180.96Protease granule (8.1% active)0.120.650.120.66Cobalt Catalyst (PAAN) (2% active)0.1000.5280.0000.000(2 mg active)WeylClean ® MnTACN (98% active)0.0000.0000.00210.011(2 mg active)Sodium Percarbonate (13.4% AvO)1.638.581.638.63PEI600EO7 75% Quat0.251.320.251.33Plurafac ® SLF180 (non-ionic1.176.181.176.21surfactant supplied by BASF)Dipropylene Glycol0.442.320.442.34Amine Oxide (32% active)0.160.830.160.83Glycerine0.080.420.080.43Total18.9410018.84100 II. Test Items The following test items were used: ItemDescriptionStainedFirma Schönwald white ceramic teacup, 98 L/0.19.TeacupsStained with tea, according to IKW method(Recommendations for the Quality Assessment of theCleaning Performance ofDishwasher Detergents (Part B, Update 2015)).MincedCeramic side plate, Arzberg form 2000, no. 10219,meat platesø 19 cmStained with minced meat, according to IKW method(Recommendations for the QualityAssessment of the CleaningPerformance of Dishwasher Detergents(Part B, Update 2015)).CrèmeDessert plate, Arzberg, white, glazed porcelain,brûléeconforming with standard EN 50242, form 2000,platesno. 10219, ø 19 cm.Stained with crème brûlée according to the IKW method(Recommendations for the Quality Assessmentof the Cleaning Performance of DishwasherDetergents (Part B, Update 2015))AdditionalPrepared according to the IKW methodBallast Soil(Recommendations for the Quality Assessment of theCleaning Performance of DishwasherDetergents (Part B, Update 2015)). III. Test wash procedure AutomaticMiele, model GSL2Dishwasher:Wash volume:5000mlWater temperature:45°C.Water hardness:20gpgDetergent addition:Added into the bottom of the automatic dish-washer after the initial pre-wash is complete.Positioning of test2× Ceramic Teacups on top rackitems:5× Crème Brulee stained plate on bottomrack with unstained plate at front as ballast2× 50g pots of Additional ballast soil addedto top rack at the start of the wash cycle.6× Ceramic Side-plate stained with 3 gminced meat mixture Dishwashers were loaded with the items as detailed above which were washed using one dose of Composition E or F. Four external replicates were completed for each test product following Latin square rotation of machines and products. The teacups were visually graded according to the IKW method (Recommendations for the Quality Assessment of the Cleaning Performance of Dishwasher Detergents (Part B, Update 2015)), using a standard scale where higher soil removal is desired (maximum score is 10). IV. Results Teacup GradesStandard ErrorComposition E2.920.21(Comparitive)Composition F5.980.38(Inventive) As can be seen from the results above, the composition according to the invention provides a higher level of stain removal. The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm.” Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern. While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention. | 70,621 |
11859157 | Various embodiments of the exemplary cleaning compositions and methods of using the cleaning compositions are represented in the figures. Reference to various embodiments does not limit the scope of the invention. Figures represented herein are not limitations to the various embodiments according to the invention and are presented for exemplary illustration of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present disclosure relates to compositions comprising an enzyme composition and a nonionic surfactant having an HLB value between 10 and 22. These compositions are useful for cleaning of bacterial cellulose deposits and such methods are described herein. The compositions have many advantages over existing bacterial cellulose treatment compositions. For example, an advantage of the compositions is that they provide improved removal of bacterial cellulose deposits. It is a further advantage that the compositions do not require PPE. Yet another advantage of the compositions is that they have a synergistic reaction between the enzyme composition and surfactants that provides surprising efficacy against bacterial cellulose deposits. Still a further advantage is that the compositions to preferably comprise less than about 0.5 wt. % active protein concentration, more preferably less than about 0.1 wt. % active protein concentration, while maintaining cleaning efficacy. The embodiments described herein are not limited to particular bacterial cellulose deposits, which can vary in makeup and in location. It is further to be understood that all terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the content clearly indicates otherwise. Further, all units, prefixes, and symbols may be denoted in its SI accepted form. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of the compositions and methods are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 1½, and 4¾. This applies regardless of the breadth of the range. References to elements herein are intended to encompass any or all of their oxidative states and isotopes. Definitions So that the present disclosure may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the disclosure pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present disclosure without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present disclosure, the following terminology will be used in accordance with the definitions set out below. The term “about,” as used herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, wave length, frequency, voltage, current, and electromagnetic field. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. The term “about” also encompasses these variations. Whether or not modified by the term “about,” the claims include equivalents to the quantities. As used herein, the term “oligomer” refers to a molecular complex comprised of between one and ten monomeric units. For example, dimers, trimers, and tetramers, are considered oligomers. Furthermore, unless otherwise specifically limited, the term “oligomer” shall include all possible isomeric configurations of the molecule, including, but are not limited to isotactic, syndiotactic and random symmetries, and combinations thereof. Furthermore, unless otherwise specifically limited, the term “oligomer” shall include all possible geometrical configurations of the molecule. As used herein the term “polymer” refers to a molecular complex comprised of more than ten monomeric units and generally includes, but is not limited to, homopolymers, copolymers, such as for example, block, graft, random and alternating copolymers, terpolymers, and higher “x”mers, further including their analogs, derivatives, combinations, and blends thereof. Furthermore, unless otherwise specifically limited, the term “polymer” shall include all possible isomeric configurations of the molecule, including, but are not limited to isotactic, syndiotactic and random symmetries, and combinations thereof. Furthermore, unless otherwise specifically limited, the term “polymer” shall include all possible geometrical configurations of the molecule. The methods and compositions of the present disclosure may comprise, consist essentially of, or consist of the components and ingredients of the present disclosure as well as other ingredients described herein. As used herein, “consisting essentially of” means that the methods, systems, apparatuses and compositions may include additional steps, components or ingredients, but only if the additional steps, components or ingredients do not materially alter the basic and novel characteristics of the claimed methods, systems, apparatuses, and compositions. The term “actives” or “percent actives” or “percent by weight actives” or “actives concentration” are used interchangeably herein and refers to the concentration of those ingredients involved in cleaning expressed as a percentage minus inert ingredients such as water or salts. It is also sometimes indicated by a percentage in parentheses, for example, “chemical (10%).” As used herein, the term “alkyl” or “alkyl groups” refers to saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), cyclic alkyl groups (or “cycloalkyl” or “alicyclic” or “carbocyclic” groups) (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.), branched-chain alkyl groups (e.g., isopropyl, tert-butyl, sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl groups (e.g., alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups). Unless otherwise specified, the term “alkyl” includes both “unsubstituted alkyls” and “substituted alkyls.” As used herein, the term “substituted alkyls” refers to alkyl groups having substituents replacing one or more hydrogens on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, alkenyl, alkynyl, halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonates, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclic, alkylaryl, or aromatic (including heteroaromatic) groups. In some embodiments, substituted alkyls can include a heterocyclic group. As used herein, the term “heterocyclic group” includes closed ring structures analogous to carbocyclic groups in which one or more of the carbon atoms in the ring is an element other than carbon, for example, nitrogen, sulfur or oxygen. Heterocyclic groups may be saturated or unsaturated. Exemplary heterocyclic groups include, but are not limited to, aziridine, ethylene oxide (epoxides, oxiranes), thiirane (episulfides), dioxirane, azetidine, oxetane, thietane, dioxetane, dithietane, dithiete, azolidine, pyrrolidine, pyrroline, oxolane, dihydrofuran, and furan. The term “weight percent,” “.%,” “wt. %,” “percent by weight,” “% by weight,” and variations thereof, as used herein, refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. As used herein, the term “cleaning” refers to a method used to facilitate or aid in soil removal, bleaching, microbial population reduction, and any combination thereof. As used herein, the term “microorganism” refers to any noncellular or unicellular (including colonial) organism. Microorganisms include all prokaryotes. Microorganisms include bacteria (including cyanobacteria), spores, lichens, fungi, protozoa, virinos, viroids, viruses, phages, and some algae. As used herein, the term “microbe” is synonymous with microorganism. As used herein, the term “disinfectant” refers to an agent that kills all vegetative cells including most recognized pathogenic microorganisms, using the procedure described inA.O.A.C. Use Dilution Methods, Official Methods of Analysis of the Association of Official Analytical Chemists, paragraph 955.14 and applicable sections, 15th Edition, 1990 (EPA Guideline 91-2). As used herein, the term “high level disinfection” or “high level disinfectant” refers to a compound or composition that kills substantially all organisms, except high levels of bacterial spores, and is affected with a chemical germicide cleared for marketing as a sterilant by the Food and Drug Administration. As used herein, the term “intermediate-level disinfection” or “intermediate level disinfectant” refers to a compound or composition that kills mycobacteria, most viruses, and bacteria with a chemical germicide registered as a tuberculocide by the Environmental Protection Agency (EPA). As used herein, the term “low-level disinfection” or “low level disinfectant” refers to a compound or composition that kills some viruses and bacteria with a chemical germicide registered as a hospital disinfectant by the EPA. As used herein, the term “malodor,” is synonymous with phrases like “objectionable odor” and “offensive odor,” which refer to a sharp, pungent, or acrid odor or atmospheric environment from which a typical person withdraws if they are able to. Hedonic tone provides a measure of the degree to which an odor is pleasant or unpleasant. A “malodor” has a hedonic tone rating it as unpleasant as or more unpleasant than a solution of 5 wt. % acetic acid, propionic acid, butyric acid, or mixtures thereof. For the purpose of this patent application, successful microbial reduction is achieved when the microbial populations are reduced by at least about 50%, or by significantly more than is achieved by a wash with water. Larger reductions in microbial population provide greater levels of protection. Differentiation of antimicrobial “-cidal” or “-static” activity, the definitions which describe the degree of efficacy, and the official laboratory protocols for measuring this efficacy are considerations for understanding the relevance of antimicrobial agents and compositions. Antimicrobial compositions can affect two kinds of microbial cell damage. The first is a lethal, irreversible action resulting in complete microbial cell destruction or incapacitation. The second type of cell damage is reversible, such that if the organism is rendered free of the agent, it can again multiply. The former is termed microbiocidal and the later, microbistatic. A sanitizer and a disinfectant are, by definition, agents which provide antimicrobial or microbiocidal activity. In contrast, a preservative is generally described as an inhibitor or microbistatic composition As used herein, the term “substantially free” refers to compositions completely lacking the component or having such a small amount of the component that the component does not affect the performance of the composition. The component may be present as an impurity or as a contaminant and shall be less than 0.5 wt. %. In another embodiment, the amount of the component is less than 0.1 wt. % and in yet another embodiment, the amount of component is less than 0.01 wt. %. The terms “water soluble” and “water dispersible” as used herein, means that the polymer is soluble or dispersible in water in the inventive compositions. In general, the polymer should be soluble or dispersible at 25° C. at a concentration of 0.0001% by weight of the water solution and/or water carrier, preferably at 0.001%, more preferably at 0.01% and most preferably at 0.1%. The term “weight percent,” “.%,” “percent by weight,” “% by weight,” and variations thereof, as used herein, refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, “percent,” “%,” and the like are intended to be synonymous with “weight percent,” “wt. %,” etc. The methods, systems, apparatuses, and compositions of the present disclosure may comprise, consist essentially of, or consist of the components and ingredients of the present disclosure as well as other ingredients described herein. As used herein, “consisting essentially of” means that the methods, systems, apparatuses and compositions may include additional steps, components or ingredients, but only if the additional steps, components or ingredients do not materially alter the basic and novel characteristics of the claimed methods, systems, apparatuses, and compositions. It should also be noted that, as used in this specification and the appended claims, the term “configured” describes a system, apparatus, or other structure that is constructed or configured to perform a particular task or adopt a particular configuration. The term “configured” can be used interchangeably with other similar phrases such as arranged and configured, constructed and arranged, adapted and configured, adapted, constructed, manufactured and arranged, and the like. Compositions Preferably, the compositions comprise an enzyme composition, a nonionic surfactant having an HLB value between 10 and 22, and water. In a preferred embodiment, the compositions further comprise a pH modifier and/or stabilizing agent. In a more preferred embodiment, the compositions comprise an enzyme composition, a nonionic surfactant having an HLB value between about 13 and about 18, a pH modifier and/or stabilizing agent, and water. Preferably the compositions have a pH between about 2 and about 5. The compositions can be in concentrated form or a diluted ready to use form. The compositions can be a premixed composition or a multi-part system mixed prior to use or at the time of use. For example, a multi-part system, can be prepared with two, three, four, or more parts each having different components, that are combined and mixed prior to or at the time of use. The premixed compositions and multi-part systems are preferably concentrated compositions, which are diluted; however, in some embodiments they may be use concentrations. The concentrated compositions can be in solid, liquid, or gel form. The ready to use forms can be in liquid or gel form. In a preferred embodiment, the concentrated and ready-to-use compositions are liquid. In a preferred embodiment, the composition can be a dissolvable solid. Preferably the dissolvable solid can be added to a drain such that when fluid goes down the drain the solid is partially dissolved forming a use solution that contacts the drain. In a preferred embodiment, the concentrated compositions are prepared at a concentration that is 10, 9, 8, 7, 6, 5, 4, 3, 2 times the concentration of the desired use solution. In an embodiment, the concentrated composition is diluted at a ratio of between about 1:1 and 1:10. Preferably, the concentrated compositions are diluted at a ratio of about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1. Preferred embodiments of the compositions are described in Table 1 below. TABLE 1CompositionPreferredMore PreferredMost PreferredEnzyme0.01-20.1-1.50.2-1Composition (wt. %)Nonionic Surfactant0.001-70.01-50.1-4.5(wt. %)Water (wt. %)55-9975-9780-95Additional0-350.1-300.5-25Ingredients (wt. %) Enzyme Composition The compositions contain an enzyme composition. The enzyme composition may comprise one or more (e.g., several) enzymes comprising, consisting essentially of, or consisting of a cellulase, an AA9 polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, a swollenin, or a combination or mixture thereof. Preferably, the enzyme composition comprises a cellulase. The cellulase is preferably one or more (e.g., several) enzymes comprising, consisting essentially of, or consisting of an endoglucanase, a cellobiohydrolase, a beta-glucosidase, or a combination or mixture thereof. In another aspect, the hemicellulase is preferably one or more (e.g., several) enzymes comprising, consisting essentially of, or consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, a xylosidase, or a combination or mixture thereof. In an embodiment, the enzyme composition comprises a cellulolytic enzyme composition comprising one or more (e.g., several) enzymes comprising, consisting essentially of, or consisting of a cellobiohydrolase, an endoglucanase, a beta glucosidase an AA9 polypeptide having cellulolytic enhancing activity, or a combination or mixture thereof. In a further embodiment, the enzyme composition comprises one or more cellulases and one or more hemicellulases. One or more (e.g., several) of the enzymes may be wild-type proteins, recombinant proteins, or a combination of wild-type proteins and recombinant proteins. For example, one or more (e.g., several) enzymes may be native proteins of a cell, which is used as a host cell to express recombinantly the enzyme composition. The enzyme composition may also be a fermentation broth formulation or a cell composition. The host cell may be any filamentous fungal cell useful in the recombinant production of an enzyme or protein. In an embodiment the enzyme composition is derived from a fungal host cell. In an embodiment the fungal host cell isTrichoderma reesei. In one embodiment the enzyme composition is or comprises an expression product ofTrichoderma reesei. In one embodiment the enzyme composition is or comprises a cellulolytic enzyme composition derived fromTrichoderma reeseicomprised ofTrichoderma reeseienzymes having cellulase activity and effective to degrade cellulose to, at least glucose. In one embodiment the enzyme composition has an endoglucanase, and a cellobiohydrolase. In another embodiment the enzyme composition has an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. In a further embodiment the enzyme composition further comprisesA. nigerbeta-glucosidase. In a still further embodiment the enzyme composition has an endoglucanase, a cellobiohydrolase, a beta-glucosidase and an AA9 polypeptide having cellulolytic enhancing activity. In another embodiment the enzyme composition is a cellulolytic enzyme composition comprising an AA9, a beta-glucosidase, a CBHI, and a CBHII. In a further embodiment the cellulolytic enzyme composition further comprises a xylanase and/or a xylosidase. In a further embodiment, the cellulolytic enzyme composition is a cellulolytic enzyme composition derived fromTrichoderma reeseifurther comprising aPenicilliumsp. (emersonii) AA9 (GH61) polypeptide having cellulolytic enhancing activity, anAspergillus fumigatusbeta-glucosidase variant, anAspergillus fumigatuscellobiohydrolase I, and anAspergillus fumigatuscellobiohydrolase II. In a still further embodiment the cellulolytic enzyme composition further comprises anAspergillus fumigatusxylanase, and anAspergillus fumigatusbeta-xylosidase. For example, the enzyme composition is a composition described in WO 2013/028928. In an embodiment the enzyme composition is or comprises a commercial enzyme preparation. Examples of commercial enzyme preparations suitable for use in the compositions include, but are not limited to, ACCELLERASE® (Danisco US Inc.), ACCELLERASE® XY (Danisco US Inc.), ACCELLERASE® XC (Danisco US Inc.), ACCELLERASE® TRIO (Danisco US Inc.), ALTERNA FUEL 100P (Dyadic), ALTERNA FUEL 200P (Dyadic), CELLIC® CTec (Novozymes A/S), CELLIC® CTec2 (Novozymes A/S), CELLIC® Ctec3 (Novozymes A/S), CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC® HTec3 (Novozymes A/S), CELLUCLAST® (Novozymes A/S), CELLUCLAST CONCENTRATED BG® (Novozymes A/S), CELLUCLEAN CLASSIC 700T® (Novozymes A/S), CELLUZYIVIIE™ (Novozymes A/S), CEREFLO® (Novo Nordisk A/S), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), DEPOL™ 762P (Biocatalysts Limit, Wales, UK), DRAIN EASE FLOW™ (Novozymes A/S), ECOPULP® TX-200A (Roal Oy LLC), FIBREZYME® LBR (Dyadic International, Inc.), FIBREZYME® LDI (Dyadic International, Inc.), LAM IN EX® (Danisco US Inc.), HSP 6000 Xylanase (DSM), MULTI FECT® Xylanase (Danisco US Inc.), PULPZYME® HC (Novozymes A/S), ROHAMENT® 7069 W (AB Enzymes), 5HEARZYIVffi™ (Novozymes A/S), SPEZYME® CP (Danisco US Inc.), ULTRAFLO® (Novozymes A/S), VISCOSTAR™ 150L (Dyadic International, Inc.), or VISCOZYME® (Novozymes A/S). Preferably, the compositions include from about 0.01 wt. % to about 2 wt. % enzyme composition, more preferably from about 0.1 wt. % to about 1.5 wt. % enzyme composition, and most preferably from about 0.2 wt. % to about 1 wt. % enzyme composition. Nonionic Surfactant The compositions contain a nonionic surfactant having an HLB value between 10 and 22. Preferably, the HLB value is between about 11 and about 20, more preferably between about 12 and about 19, most preferably between about 13 and about 18. Preferably, the surfactant is an alkoxylated surfactant. Suitable alkoxylated surfactants include EO/PO copolymers, capped EO/PO copolymers, alcohol alkoxylates, capped alcohol alkoxylates, mixtures thereof, or the like. Preferred surfactants, including, but are not limited to, alcohol ethoxylates, polyethylene glycol sorbitan ester, polyethylene glycol ether, polyoxyethylene ether, a poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol), poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), or mixture thereof so long as the surfactant selected has an HLB value between 10 and 22. Suitable alkoxylated surfactants for use as solvents include EO/PO block copolymers, such as the Pluronic® and reverse Pluronic® surfactants; alcohol alkoxylates, such as Dehypon® LS-54 (R-(EO)5(PO)4) and Dehypon® LS-36 (R-(EO)3(PO)6); and capped alcohol alkoxylates, such as Plurafac® LF221 and Tegoten® EC11; mixtures thereof, or the like. Preferred surfactants include, but are not limited to, polyethylene glycol sorbitan monolaurate (commercially available as Tween® 20 from Sigma-Aldrich), polyethylene glycol sorbitan monooleate (commercially available as Tween® 80 from Sigma-Aldrich), polyethylene glycol tert-octylphenyl ether (commercially available as Triton™ X-100 from Sigma-Aldrich), polyethylene glycol trimethylnonyl ether (commercially available as Tergitol™ TMN-6 from Sigma-Aldrich), poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) (commercially available as Pluronic® 10R5 from Sigma-Aldrich, preferably having an average molecular weight of 1950), polyoxyethylene (23) lauryl ether (commercially available as Brij® L23 from Sigma-Aldrich), and mixtures thereof. Preferably, the compositions include from about 0.001 wt. % to about 7 wt. % nonionic surfactant, more preferably from about 0.01 wt. % to about 5 wt. % nonionic surfactant, still more preferably from about 0.1 wt. % to about 4.5 wt. %, and most preferably from about 1 wt. % to about 4 wt. % nonionic surfactant. pH In an acidic embodiment, the compositions preferably have a pH equal to or less than about 5, more preferably, between about 2 and about 4.75, most preferably between about 3 and about 4.5. It has been found that the compositions lose stability at a pH of above 5 with most buffers and stabilizers. However, using the buffer CAPS it was found that the compositions perform well at a pH of between about 8 and about 11, more preferably between about 9 and about 10.5, most preferably at a pH of about 10. In an embodiment having a multi-part system combined prior to or at the time of use, the compositions can be prepared with a pH between about 2 and about 11. However, if the pH is between about 4.5 and about 8, the stability of the compositions may require use fairly quickly after combining the different parts. Thus, in a preferred embodiment having a pH between 4.5 and 8, the compositions are used no more than 2 hours, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute, after combination. Water The compositions contain water. In a preferred embodiment comprising water as a carrier, the water is deionized water or softened water. The water typically makes up the remaining volume after the addition of all other ingredients. Preferably, the compositions include from about 55 wt. % to about 99 wt. % water, more preferably from about 75 wt. % to about 97 wt. % water, and most preferably from about 80 wt. % to about 95 wt. % water. Additional Optional Ingredients The compositions can include a number of optional ingredients in various embodiments. Many additional optional ingredients can be added to provide desired properties to the compositions. Optional ingredients can include, but are not limited to, a buffering agent, a colorant, an additional enzyme, a fragrance, a pH modifier, a stabilizing agent, an additional surfactant, a thickening agent, and mixtures thereof. Buffering Agent The compositions can optionally include a buffering agent. As used herein the term “buffer” and “buffering agent” are synonymous. Preferred buffering agents include, but are not limited to, N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-(2-hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES), pH modifiers (discussed below), tris(hydroxymethyl)aminomethane (Tris), and mixtures thereof. Most preferred buffers, include CAPS, CHES, citric acid and its salts (including in particular sodium citrate), and mixtures thereof. Specifically, CAPS and CHES are most preferred for alkaline pH and citric acid, citrate, acetic acid, and acetate are most preferred for acidic pH. Preferably, the compositions include from about 0.1 wt. % to about 5 wt. % buffering agent, more preferably from about 0.5 wt. % to about 3 wt. % buffering agent, and most preferably from about 1 wt. % to about 2 wt. % buffering agent. Colorant The compositions can optionally comprise a colorant. The colorant can be a dye or an additive that provides a visible color or color change. Preferred colorants, including, but are not limited to, copper(II) salts, Direct Blue 86, available from Mac Dye-Chem Industries, Ahmedabad, India; Fastusol Blue, available from Mobay Chemical Corporation, Pittsburgh, Pa.; Acid Orange 7, available from American Cyanamid Company, Wayne, N.J.; Basic Violet 10 and Sandolan Blue/Acid Blue 182, available from Sandoz, Princeton, N.J.; Acid Yellow 23, available from Chemos GmbH, Regenstauf, Germany; Acid Yellow 17, available from Sigma Chemical, St. Louis, Mo.; Sap Green and Metanil Yellow, available from Keyston Analine and Chemical, Chicago, Ill.; Acid Blue 9, available from Emerald Hilton Davis, LLC, Cincinnati, Ohio; Hisol Fast Red and Fluorescein, available from Capitol Color and Chemical Company, Newark, N.J.; and Acid Green 25, Ciba Specialty Chemicals Corporation, Greenboro, N.C. Preferably the colorant can be in a concentration between about 0 wt. % and about 2 wt. %, more preferably between about 0.001 wt. % and about 1 wt. %. Additional Enzyme The compositions can optionally include an additional enzyme. Suitable additional enzymes, include, but are not limited to, a protease, a xylanase, a nuclease, and mixtures thereof. If the compositions contain an additional enzyme, it is preferably in a concentration from about 0.01 wt. % to about 2 wt. %, more preferably from about 0.1 wt. % to about 1.5 wt. %, and most preferably from about 0.5 wt. % to about 1 wt. %. Fragrance The compositions can optionally comprise a fragrance. Preferred fragrances include, but are not limited to, terpenoids such as citronellol, aldehydes such as amyl cinnamaldehyde, a jasmine such as C1S-jasmine or jasmal, vanillin, and the like. Preferably the fragrance can be in a concentration between about 0 wt. % and about 1 wt. %, more preferably between about 0.01 wt. % and about 1 wt. %. pH Modifier The compositions can include a pH modifier to adjust the pH or act as a buffer. Suitable pH modifiers can include water soluble acids. Preferred acids can be organic and/or inorganic acids and their salts that are water soluble. Preferred inorganic acids include, but are not limited to, boric acid, hydrobromic acid, hydrochloric acid, hydrofluoric acid, hydroiodic acid, hypophosphorous acid, phosphoric acid, phosphorous acid, polyphosphoric acid, sulfamic acid, sulfuric acid, sulfurous acid, sodium bisulfate, sodium bisulfite, their salts and mixtures thereof. Preferred organic acids include, but are not limited to, acetic acid, acrylic acids, adipic acid, benzoic acid, butyric acid, caproic acid, citric acid, formic acid, fumaric acid, gluconic acid or its precursor glucono-6-lactone, glutaric acid, hydroxy acetic acid, isophthalic acid, lactic acid, lauric acid, maleic acid, malic acid, malonic acid, palmitic acid, pimelic acid, polymaleic-acrylic acids, polyacrylic acids, propionic acid, sebacic acid, stearic acid, suberic acid, succinic acid, tartaric acid, terephthalic acid, uric acid, valeric acid, their salts and mixtures thereof. Preferred acid salts include, but are not limited to, acetic acid salts, citric acid salts, formic acid salts, and mixtures thereof. Preferably, the compositions include from about 0.1 wt. % to about 5 wt. % pH modifier, more preferably from about 0.5 wt. % to about 3 wt. % pH modifier, and most preferably from about 1 wt. % to about 2 wt. % pH modifier. Stabilizing Agent The compositions can optionally comprise a stabilizing agent. Preferred stabilizing agents include, but are not limited to, borate, calcium/magnesium ions, glycerol, polyethylene glycol 200, polyethylene glycol 400, propylene glycol, sucrose, and mixtures thereof. When the compositions include a stabilizing agent, it can be included in an amount that provides the desired level of stability to the composition. Preferably, the compositions include from about 0.01 wt. % to about 30 wt. % stabilizing agent, more preferably from about 0.5 wt. % to about 25 wt. % stabilizing agent, and most preferably from about 1 wt. % to about 25 wt. % stabilizing agent. Additional Surfactant In some embodiments, the compositions include an additional surfactant besides the nonionic surfactant having an HLB between 10 and 22. Additional surfactants suitable for use in the compositions include, but are not limited to, anionic surfactants, cationic surfactants, nonionic surfactants, and zwitterionic surfactants. Preferred additional surfactants, include, but are not limited to, nonionic seed oil surfactants, such as the alcohol ethoxylate Ecosurf™ SA-9 (commercially available from DOW Chemical), cocamidopropyl betaine (commercially available as Amphosol® CG from Stepan), alkyl polyglucosides, including, for example decyl glucoside (commercially available as APG® 325N from BASF), cocoamine oxide (commercially available as Barlox™ 12 from Lonza), sodium xylene sulfonate, ethylene oxide/propylene oxide block copolymers, such as the Pluronic® surfactant line available from BASF (such as Pluronic® 25R and Pluronic® 10R5), cocamidopropyl hydroxysultaine (commercially available as Mackam® 50-SB from Solvay), and mixtures thereof. When the compositions include an additional surfactant, preferably it is in a concentration from about 0.01 wt. % to about 5 wt. %. Thickening Agent The compositions can optionally include a thickening agent. A wide variety of thickening agents can be included. Preferred thickening agents can be organic or inorganic. When a thickening agent is included, it is preferably in an amount between about 0.01 wt. % and about 5 wt. %. Preferred organic thickening agents include, but are not limited to, acrylic copolymers, carboxyvinyl polymers, corn starch, crosslinked polyacrylic acid-type thickening agents, fatty acid thixotropic thickeners, guar gum, guar hydroxy propyltrimonium chloride, polyacrylate polymers, poly(methylvinylether/maleic) anhydride polymers, and mixtures thereof. As used herein, “polyacrylic acid-type” is intended to refer to water soluble homopolymers of acrylic acid or methacrylic acid or water-dispersible or water-soluble salts, esters and amides thereof, or water-soluble copolymers of these acids or their salts, esters or amides with each other or with one or more ethylenically unsaturated monomers, such as styrene, maleic acid, maleic anhydride, 2-hydroxyethylacrylate, acrylonitrile, vinyl acetate, ethylene, propylene, or the like. Preferably, the polyacrylic thickening agent is one of the crosslinked polyacrylic acid-type thickening agents commercially available as CARBOPOL™. The CARBOPOL™ resins, also known as carbomer resins, are hydrophilic, high molecular weight, crosslinked acrylic acid polymers. The CARBOPOL′ resins are crosslinked with a polyalkenyl polyether, such as a polyalkyl ether of sucrose having an average of 5.8 alkyl groups per molecule of sucrose. Other suitable carbomer thickening agents include the PNC carbomers. Suitable fatty acid thixotropic thickeners, include, but are not limited to, higher aliphatic fatty monocarboxylic acids having from about 8 to about 22 carbon atoms, inclusive of the carbon atom of the carboxyl group of the fatty acid. The aliphatic radicals are saturated and can be straight or branched. Mixtures of fatty acids may be used, such as those derived from natural sources, such as tallow fatty acid, coco fatty acid, soya fatty acid, etc., or from synthetic sources available from industrial manufacturing processes. Examples of the fatty acids which can be used as thickeners include, for example, decanoic acid, lauric acid, dodecanoic acid, palmitic acid, myristic acid, stearic acid, oleic acid, eicosanoic acid, tallow fatty acid, coco fatty acid, soya fatty acid and mixtures of these acids. The metal salts of the above fatty acids can also be used in as thixotropic thickener agents, such as salts of the monovalent and polyvalent metals such as sodium, potassium, magnesium, calcium, aluminum and zinc. Suitable metal salts, include, but are not limited to, aluminum salts in triacid form, e.g., aluminum tristearate, Al(OCOC17H35)3, monoacid salts, e.g., aluminum monostearate, Al(OH)2(OCOC17H35) and diacid salts, e.g. aluminum distearate, Al(OH)(OCOC17H35)2, and mixtures of two or three of the mono-, di- and triacid salts can be used for those metals, e.g., Al, with valences of +3, and mixtures of the mono- and diacid salts can be used for those metals, e.g., Zn, with valences of +2. The thickening agent used can also be any one of a number of natural or synthetic inorganic materials, such as clays, silicas, aluminas, titanium dioxide (pyrogenic) and calcium and/or magnesium oxides. All of these materials are readily available from commercial sources. Various types of clays which are useful include kaolins such as kaolinite, dicktite, nacrite, halloysite and endillite; serpentine clays such as chrysotile and amesite; smectites such as montmorillonite (derived from bentonite rock), beidellite, nontronite, hectorite, saponite and sauconite; illites or micas; glauconite; chlorites and vermiculites; attapulgite and sepiolite. Mixed layer clays exhibiting intercalation of mineral sandwiches with one another may be used, such as, for example, mixed-layer clay mineral sheets of illite interspersed randomly or regularly with montmorillonite, or chlorite with one of the other types of clay, such as vermiculite. Other useful clays include amorphous clays, such as allophane and imogolite, and high-alumina clay minerals such as diaspore, boehmite, bibbsite and cliachite. Various types of silicas which are useful include diatomite, precipitated silica and fumed silica. Various types of aluminas may be used, as well as various types of calcium and magnesium oxides. Methods of Preparing the Compositions The compositions can be prepared by adding and mixing the desired ingredients. Preferably the ingredients are mixed until they are homogeneous or substantially homogenous. The compositions can be prepared manually or by a system that adds the components in desired quantities to achieve a particular concentration of ingredients. In a preferred embodiment, the compositions are prepared as a concentrated composition and diluted on site prior or during use. In a preferred embodiment, the ingredients are mixed at the time of use prior to contacting a surface or at the time of contacting a surface to be cleaned. The compositions can be prepared as a multi Methods of Using the Compositions The compositions can be used by contacting a hard surface, preferably a drain, with the composition. Typically, the hard surface has a bacterial cellulose deposit or may be susceptible to the development of a bacterial cellulose deposit. Such hard surfaces, including, but are not limited to, drains, floors, sinks, beverage tower fluid lines, or combination thereof. In an aspect of the method of use, the composition can be allowed to contact the hard surface for a sufficient time to at least partially degrade the bacterial cellulose deposit, whereby the at least partially degraded material is removed from the hard surface. In another aspect of the method of use, the composition is allowed to coat the hard surface to prevent or at least reduce the development of a bacterial cellulose deposit. In one embodiment of the present method used to remove or prevent bacterial cellulose deposits is added directly to a hard surface, preferably a drain system through an opening in the system, such as a floor drain or any other opening that will allow access to the drain interior. Preferably the composition is in contact with the hard surface for a time prior to use or rinsing of at least about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 11 seconds, 12 seconds, 13 seconds 14 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 90 seconds, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3, hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 48 hours, 60 hours, 72 hours. Optionally, the hard surface can be rinsed after allowing the composition to contact the hard surface for sufficient time. In a preferred embodiment, the hard surface is not rinsed after contact with the composition. In another preferred embodiment, the hard surface is rinsed with water. The water can have a temperature between 10° C. and about 100° C., preferably between about 25° C. and about 90° C., more preferably between about 35° C. and about 80° C. The method of use requires no particular mode of contacting the composition to the bacterial cellulose deposit to be removed, provided the contact takes place for a time sufficient to allow at least partial degradation of the bacterial cellulose deposit. Optionally, the bacterial cellulose can be removed with minimal mechanical or manual effort, such as by flushing or rinsing, by gentle mechanical agitation, or by continued use of the compositions described herein. Preferably, the composition is permitted to contact the deposits for at least two to three hours. The drain cleaners, compositions, and methods can be applied to effect both prevention and removal of bacterial cellulose deposits. When used to clean drain pipes, such as soft drink and alcoholic beverage station drain pipes, the condition of the drain must be ascertained, i.e., whether the drain is fully or partially clogged. If fully clogged, the drain can be partially unblocked, typically by mechanical means such as snaking, rotor rooting, water jetting, etc., to allow the composition to contact as much of the deposited bacterial cellulose as possible. However, it is also possible to apply the compositions to a fully clogged drain in small amounts repeatedly as it degrades the bacterial cellulose deposit. In a preferred embodiment, the compositions provide a synergistic degradation of bacterial cellulose deposits. Further, they can provide removal of malodor and have a cidal effect on insects, particularly flies, that tend to feed off of bacterial cellulose deposits. All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated as incorporated by reference. EXAMPLES Embodiments of the present disclosure are further defined in the following non-limiting Examples. It should be understood that these Examples, while indicating certain embodiments, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of the compositions and methods, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt it to various usages and conditions. Thus, various modifications of the embodiments, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The materials used in the following Examples are provided herein: Amphosol® CG: an amphoteric surfactant, cocamidopropyl betaine, available from Stepan. APG® 325N: a nonionic surfactant, alkyl polyglucoside (decyl glucoside) having an HLB of about 13, available from BASF Corp. Barlox™ 12: a zwitterionic surfactant, cocoamine oxide, available from Lonza. Bioterge® AS-40K: an anionic surfactant, sodium C14-16 alpha olefin sulfonate, available from Stepan. Brij® L23: a nonionic surfactant, polyoxyethylene (23) lauryl ether having an HLB of 17, available from Sigma-Aldrich. Biological Formula 2-24 InstantDrosophilaMedium: a culture medium available from Carolina. Cellulase C: an exemplary enzyme composition comprising a cellulase enzyme obtained from Novozymes. Cellulase C comprises between 70 wt. % and about 75 wt. % water, between 10 wt. % and about 20 wt. % cellulase, between about 5 wt. % and about 15 wt. % sorbitol, and less than 1 wt. % proxel. CELLUCLAST CONCENTRATED BG®: an exemplary enzyme composition available from Novozymes. CELLUCLEAN CLASSIC 700T®: an exemplary enzyme composition available from Novozymes. DRAIN EASE FLOW™: an exemplary enzyme composition comprising a cellulase enzyme obtained from Novozymes. DRAIN EASE FLOW comprises water, a polysaccharide, a cellulase, sodium benzoate, and potassium sorbate. The water comprises between about 40 wt. % and about 50 wt. % of the enzyme composition. The polysaccharide comprises between about 25 wt. % and about 35 wt. % of the enzyme composition. The polysaccharide comprises sucrose, glucose, or a mixture thereof. The cellulase comprises between about 20 wt. % and about 25 wt. % of the enzyme composition. Ecosurf™ SA-9: a nonionic alcohol ethoxylate seed oil surfactant having an HLB of 11-13, available from DOW Chemical. Mackam 50-SB®: a zwitterionic surfactant, cocamidopropyl hydroxysultaine, available from Solvay. PEG 200®: a polyethylene glycol available from a number of commercial sources including, Sigma-Aldrich. Pluronic F108®: a nonionic surfactant, ethylene oxide and propylene oxide block copolymer having an HLB greater than 24, available from BASF. Pluronic L31®: a nonionic surfactant, ethylene oxide and propylene oxide block copolymer having an HLB of 1-7, available from BASF. Pluronic 10R5®: a nonionic surfactant, poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) having an HLB of 12-18, available from Sigma-Aldrich. Pluronic 25R®: a nonionic surfactant, ethylene oxide and propylene oxide block copolymer having an HLB of 7-12, available from BASF. Sodium xylene sulfonate (SXS), an anionic surfactant available from multiple commercial sources. Tergitol™ NP-12: a nonionic nonylphenol ethoxylate having an HLB of 13.8, available from DOW Chemical. Tergitol™ TMN-6: a nonionic surfactant, polyethylene glycol trimethylnonyl ether having an HLB of 13.1, available from DOW Chemical. Triton™ X-100: a nonionic surfactant, polyethylene glycol tert-octylphenyl ether having an HLB of 13.5, available from Sigma-Aldrich. Tween® 20: a nonionic surfactant, polyethylene glycol sorbitan monolaurate having an HLB of 16.7, available from Sigma-Aldrich. Tween® 80: a nonionic surfactant, polyethylene glycol sorbitan monooleate having an HLB of 15, available from Sigma-Aldrich. Additional ingredients available from a number of sources include, citric acid, glycerol, sodium citrate, and water (5 grain). Concentration percentages of ingredients provided in the Examples below are in weight percent unless indicated as percent active. Example 1 In order to test interactions between enzyme compositions comprising cellulase(s) and detergent components the “Cellulase catalyzed hydrolysis of bacterial cellulose” method was used. For this method, the sugar snake is weighed before and after treatment with the listed chemistry. The percent sugar snake degraded after two hours was calculated and recorded. The general formulation is listed in Table 2 below. TABLE 2ConcentrationComponentSpecies(wt. %)BufferCitric Acid (50%)1.12%BufferTrisodium Citrate0.6%SurfactantSee Table 30-4%EnzymeDRAIN EASE FLOW0.5%Water5 grainQ.S. Aside from enzyme RM, all percentages are percent active concentrations. The type of surfactant was varied and compared to the control formulation which did not contain surfactant. If the amount of sugar snake degraded after a set time was statistically greater than the control reaction across replicate tests it was deemed synergistic. If the amount of degradation was comparable the surfactant interaction was deemed compatible, and if the amount of degradation was less than the control the surfactant interaction was deemed less favorable. The results are provided below in Table 3 showing the percent degradation of the sugar snake an assessment of the enzyme compatibility. TABLE 3DegradationCompatibilitySurfactantHLBIncrease (%)RatingPluronic L311-70.22CompatiblePluronic 10R512-1820SynergisticTergitol TMN-613.120.6SynergisticTriton X-10013.530.1SynergisticTween 801521.9SynergisticTween 2016.720.4SynergisticBrij L231710.6SynergisticPluronic F108>24−0.9*Compatible*Pluronic F108 was not statistically different than the no-surfactant control The results from Table 3 are also shown inFIG.1. As can be seen in Table 3 and inFIG.1, nonionic surfactants with HLB values ranging from about 10-22 showed enhanced performance, with unexpected synergy in the range of about 13 HLB to about 18 HLB. The data also shows that Pluronic® L31 (HLB 1-7) and Pluronic® F108 (>24) were not statistically different from the no-surfactant control. Example 2 Another wider test was completed using a method in which a piece of sugar snake (weight previously recorded) was incubated in chemistry containing surfactant. After a set time, the remaining sugar snake was removed and quantified by water displacement in a graduated cylinder. After treatment, sugar snake degradation was determined by subtracting the volume (mL) of water displacement from the starting weight (grams). Each variation was compared to a control where the surfactant was omitted. The same three terms as above (synergistic, compatible, and less favorable) were used to identify the enzyme compatibility of each surfactant. Results are shown in Table 4. TABLE 4Concen-IncubationPercentCompat-trationTimeDegra-ibilitySurfactant(wt. %)pH(hr)dationRatingTergitol442461LessNP-12FavorableEcosurf SA-944885CompatibleAmphosol CG44854CompatibleMackam 50-SB44851CompatibleBioterge44827LessAS-40KFavorableAPG 325N44785CompatibleAPG 325N410743LessFavorableBarlox 1244785CompatibleBarlox 12410716LessFavorableSXS44785CompatibleSXS24711CompatiblePluronic 25R44858Compatible Example 3 Multiple enzyme compositions comprising a cellulase were evaluated to determine sugar snake degradation performance as a baseline without potential surfactant synergy to assess the role of the enzyme composition versus improvement based on surfactant synergy. Compositions were prepared with 0.5 wt. % enzyme composition, 1.7 wt. % sodium citrate buffer, and water at a pH of 4.25. The four enzyme compositions tested were obtained from Novozymes and included: DRAIN EASE FLOW™, CELLUCLEAN CLASSIC 700T®, CELLUCLAST CONCENTRATED BG®, and Cellulase C. A sugar snake of equal mass was measured and the cleaning compositions were applied to it. The percent degradation of the sugar snake (based on mass) was evaluated after 2 hours of contact and after 24 hours of contact. The percent degradation is shown inFIG.2where 100 indicates 100% degradation. As can be seen inFIG.2, CELLUCLAST CONCENTRATED BG® provided the best sugar snake degradation at both the 2-hour time and 24-hour time. DRAIN EASE FLOW™ and Cellulase C performed substantially similar and CELLUCLEAN CLASSIC 700T® did not appear to degrade the sugar snake at all. Example 4 The three enzyme compositions that had a good baseline were tested in an exemplary cleaning composition containing Tween® 20 to assess which would exhibit synergistic performance with a surfactant. Compositions were prepared with 0.5 wt. % enzyme composition, 1.7 wt. % sodium citrate buffer, 2 wt. % Tween® 20, and water at a pH of 4.25. The three enzyme compositions tested were DRAIN EASE FLOW™ CELLUCLAST CONCENTRATED BG®, and Cellulase C, all from Novozymes. Control compositions were also prepared without the surfactant (Tween® 20) having 0.5 wt. % enzyme composition, 1.7 wt. % sodium citrate buffer, and water at a pH of 4.25. A sugar snake of equal mass was measured and the cleaning compositions were applied to it. The percent degradation of the sugar snake (based on mass) was evaluated after 2 hours of contact and after 18 hours of contact. The percent degradation is shown inFIGS.3A-3Cwhere 100 indicates 100% degradation. As can be seen inFIGS.3A-3C, all three of the enzyme compositions demonstrated synergistic improvement with the surfactant added. DRAIN EASE FLOW™ and CELLUCLAST CONCENTRATED BG® both degraded the sugar snake entirely after 18 hours. The observed synergy became more apparent with increased incubation time. T-tests were done on all the data to assess statistical improvement. The results of the t-test are summarized in Table 5. TABLE 5Difference95% ConfidenceCellulase RM(+/−2% Tween 20)p valueIntervalDrain Ease Flow−27.30%0.032−48.8-5.9Cellulase C−42.70%0.02−69.3, 16.0Celluclast Conc−25.00%0.039−47.1, −3.0BG The T-test analysis shows that each sample had a statistical difference between +/−2% Tween® 20 at 18 hours incubation time. Example 5 Enzyme stability was tested in cleaning compositions prepared with differing stabilizers. All test compositions were prepared containing 0.5% DRAIN EASE FLOW™ 2% Tween® 20, and 1.8% sodium citrate buffer in water prepared at a pH of about 4.5 Enzyme stability was assessed by an activity assay. The results are provided inFIG.4. Formulations containing 20% PEG 400® and 20% glycerol showed 84% and 83% retention of DRAIN EASE FLOW™ activity after 8 weeks at 37° C. Formulations containing 20% propylene glycol showed 100% retention of DRAIN EASE FLOW™ activity under the same conditions. Indicating the stabilizers did provide enzyme stability and retention. Example 6 DRAIN EASE FLOW™ performance was also assessed using the “Cellulase catalyzed hydrolysis of bacterial cellulose” test at increasing stabilizer concentrations. The initial screening was done using about 1 gram of sugar snake dosed at 20 mL/gram chemistry. The relative enzyme performance was determined at each concentration by comparing the performance to control formulations lacking stabilizer. The percent degradation is shown inFIG.5where 100 indicates 100% degradation and N/A indicates immediate precipitation upon the addition of the enzyme. Of all the potential stabilizers tested, glucose showed the highest inhibition whereas PEG 200® had the lowest impact on performance. After incubating sugar snake for 24 hours in exemplary cleaning compositions, the samples were visually assessed for amount of sugar snake remaining. Formulations containing 0.5% DRAIN EASE FLOW™, 2% Tween® 20, and 1.8% sodium citrate buffer in water prepared at a pH of about 4.5 were found to fully degrade the sugar snake. Formulations containing 20% PEG 200®, 20% PEG 400®, and 20% sucrose, respectively, were also each found to fully degraded the sugar snake. Formulations containing other stabilizers did not result in complete degradation of sugar snake even after 24 hours despite fairly high performance after 2 hours. The inventions being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the inventions and all such modifications are intended to be included within the scope of the following claims. The above specification provides a description of the manufacture and use of the disclosed compositions and methods. Since many embodiments can be made without departing from the spirit and scope of the invention, the invention resides in the claims. | 56,474 |
11859158 | DETAILED DESCRIPTION As defined herein, “essentially free of” a component means that no amount of that component is deliberately incorporated into the respective premix, or composition. Preferably, “essentially free of” a component means that no amount of that component is present in the respective premix, or composition, but may be present as trace impurities. As used herein, “isotropic” means a clear mixture, having little or no visible haziness, phase separation and/or dispersed particles, and having a uniform transparent appearance. As defined herein, “stable” means that no visible phase separation is observed for a premix kept at 25° C. for a period of at least two weeks, or at least four weeks, or greater than a month or greater than four months, as measured using the Floc Formation Test, described in USPA 2008/0263780 A1. By “Low volatile organic compound hard surface cleaning composition”, it is meant herein a finished product having low volatile organic compound (“VOC”) content like, for example, a maximum of 0.5% by weight of the composition of VOCs, however, it is noted that fragrance is exempted from this value up to 2% by the weight of the finished product. All percentages, ratios and proportions used herein are by weight percent of the premix, unless otherwise specified. All average values are calculated “by weight” of the premix, unless otherwise expressly indicated. All measurements are performed at 25° C. unless otherwise specified. Unless otherwise noted, all component or composition levels are in reference to the active portion of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources of such components or compositions. Liquid Hard Surface Cleaning Compositions By “liquid hard surface cleaning composition”, it is meant herein a liquid composition for cleaning hard surfaces found in households, especially domestic households. Surfaces to be cleaned include kitchens and bathrooms, e.g., floors, walls, tiles, windows, cupboards, sinks, showers, shower plastified curtains, wash basins, WCs, fixtures and fittings and the like made of different materials like ceramic, vinyl, no-wax vinyl, linoleum, melamine, glass, steel, kitchen work surfaces, any plastics, wood, plastified wood, metal or any painted or varnished or sealed surface and the like. Household hard surfaces also include household appliances including, but not limited to refrigerators, freezers, washing machines, automatic dryers, ovens, microwave ovens, dishwashers and so on. Such hard surfaces may be found both in private households as well as in commercial, institutional and industrial environments. In a preferred embodiment, the liquid compositions herein are aqueous compositions. Therefore, they may comprise from 92% to 99.7% by weight of the total composition of water, preferably at least about 93 wt %, more preferably at least about 95 wt %, more preferably at least about 97 wt %, most preferably at least about 98 wt % water. The compositions of the present disclosure preferably have a viscosity from lcps to 650 cps, more preferably of from 100 cps to 550 cps, more preferably from 150 cps to 450 cps, most preferably from 250 cps to 350 cps when measured at 20° C. with a AD1000 Advanced Rheometer from Atlas® shear rate 10 s−1 with a coned spindle of 40 mm with a cone angle 2° and a truncation of ±60 μm. The pH is preferably from 3.5 to 9.5, more preferably from 5 to 8 and most preferably from 6 to 7. It will be understood that the compositions herein may further comprise an acid or base to adjust pH as appropriate. A suitable acid for use herein is an organic and/or an inorganic acid. A preferred organic acid for use herein has a pKa of less than 7. A suitable organic acid is selected from the group consisting of: citric acid, lactic acid, glycolic acid, maleic acid, malic acid, succinic acid, glutaric acid and adipic acid and mixtures thereof. A suitable inorganic acid can be selected from the group consisting of: hydrochloric acid, sulphuric acid, phosphoric acid and mixtures thereof. A typical level of such acids, when present, is from 0.001% to 1.0% by weight of the total composition, preferably from 0.005% to 0.5% and more preferably from 0.01% to 0.05%. A suitable base to be used herein is an organic and/or inorganic base. Suitable bases for use herein are the caustic alkalis, such as sodium hydroxide, potassium hydroxide and/or lithium hydroxide, and/or the alkali metal oxides such, as sodium and/or potassium oxide or mixtures thereof. A preferred base is a caustic alkali, more preferably sodium hydroxide and/or potassium hydroxide. Other suitable bases include ammonia, ammonium carbonate, K2CO3, Na2CO3and alkanolamines (such as monoethanolamine, triethanolamine, aminomethylpropanol, and mixtures thereof). Typical levels of such bases, when present, are from 0.001% to 1.0% by weight of the total composition, preferably from 0.005% to 0.5% and more preferably from 0.01% to 0.05%. Solvent The liquid hard surface cleaning compositions preferably comprises a propylene phenyl glycol esthers solvent. The solvent comprises a propylene glycol phenyl ether or dipropylene glycol phenyl ethers solvent and a combination thereof with an HLB value of 5.5 to 6.5. It was found that a composition having a propylene phenyl glycol esthers solvent with an HLB value of 5.5 to 6.5 delivered good cleaning with an unexpectedly low streaks and water marks as compared to compositions with traditional solvent systems. Without being bound by theory, a composition having a solvent or solvents with the preferred HLB value for example, propylene glycol phenyl ether or dipropylene glycol phenyl ethers solvent, provides sufficient water solubility for solution stability and enough oil compatibility for oil or lipid solubility. Propylene glycol phenyl ether and dipropylene glycol phenyl ethers solvents or solvents deliver optimum oil solubility for cleaning, soil dissolution, and absorption while maintaining a low level of streaks and water marks coming from their ability to dewett during the drying process. A phenyl glycol ether having an HLB between 5.5 and 6.5 may be selected from the group consisting of: propylene glycol phenyl ether and dipropylene glycol phenyl ether, and combinations thereof. Exemplary glycol ethers having an HLB between 5.5 and 6.5 are DOWANOL™ PPH, DOWANOL™ DiPPH Glycol Ether, and DOWANOL™ CNTR from the Dow Chemical Company. The phenyl glycol ether solvent may be present at a level of 0.05 wt. % to 3.50 wt. %, more preferably 0.1 wt. % to 1.5 wt. %, most preferably 0.3 wt. % to 0.9 wt. %, by weight of the overall composition. The composition may comprise less than 0.5 wt. % ethanol, more preferably less than 0.4 wt. % ethanol, and most preferably less than 0.3 wt. % ethanol. Without wishing to be bound by theory, it is believed that higher levels of ethanol negatively impacting the hysteresis of the composition and increase the streaking of the composition on the hard surface. During the cleaning process, surfaces may undergo four transformations, cycles or stages: application of the solution to wet the surface, wetting and spreading of the solution on the surface, optional removal of the solution from the surface that can include absorption into a cleaning substrate, and drying of any residual solution which on horizontals surfaces, like floors, occurs mainly by evaporation. However, during the drying cycle the wetting and spreading characteristics of the solution, which are controlled by the interaction between fluid and the surface interface, are important for the final appearance of the cleaning surface. The effect of the wetting behavior of the solution during the drying cycle is a phenomenal that is not well understood. During the drying cycle the solution evaporates forming beads and depending on their chemical composition these beads would evaporate by pining or dewetting. As shown inFIG.4, these two phenomena, pinning or dewetting, can significantly impact spotting and/or streaking of the solution left behind. When a solution evaporates through a pinning process, the area and the radius of the bead contacting the surface remains constant while the volume and the contact angle between the fluid and the surface decreases. This results in streaks and/or water marks of the size of the area or radius of the original bead which has a negative impact in cleaning appearance and shine. On the other hand, when a solution evaporates through a dewetting process, the area, the radius of the bead contacting the surface, and its volume decreases while the contact angle between the fluid and the surface remains contact. This results in the reduction of the area and radius of the original bead which reduces its foot print on the surface as the beads evaporate resulting in smaller and less visible streaks and water marks. One parameter that is important for the dewetting behaviour of the solution is its degree of hysterisis, which is defined as the substraction of its advancing contact minus its receiding contact angle. The hysterisis of the solution controls how the solution wets or spread and dewetts or recede on the surface. This hysterisis is a dynamic process with its advancing component controlling wetting, for example during the wetting cycle; and its receding component controlling dewetting during the drying process. High hysteresis solutions, or solutions with higher advancing contact angle component, have a higher tendency to spread and are less prone to dewett. While low hysteresis solutions, or solutions with lower advancing contact angle or higher receding contact angle component, have a higher tendency to recede and are more prone to deweet. It has been found that solutions containing high hydrophobic solvents with a Hydrophile-Lipophile Balance between 5.5 to 6.5, such as phenyl glycol ethers, more specifically propylene phenyl glycol ethers results in low hysteresis less than 30°, more preferably less than 20°, and most preferably less than 10° offer dewetting benefits. Without being bound by theory, it is believed that optimum dewetting and shine results are found with a solutions preferably having a hysteresis than lower than 30°, more preferably lower than 20°, and most preferably lower than 10°. This benefit is of particular importance in cleaning application without a rinse or that are not completely absorbed during the cleaning process. The benefit of these solvents can also show benefits when the composition comprises an antibacterial (as discussed further herein), such as a quaternary ammonium. Specifically, antibacterial compositions, such as those with quaternary ammonium are known to be poor dewetters and tend to cause stickiness problems if used at high concentration or if left behind on the cleaning surface. The benefit with antibacterial formulations can be further improved when the pad or wipe contains airlaid cellulose because, without being bound by theory, the negative charge sites in the cellulose bind to the quaternary ammonium and reduce their free or expressed concentration that can reach the cleaning surface reducing their negative impact in cleaning while still delivering high micro efficacy. Non-Ionic Surfactant The liquid hard surface cleaning composition of the present disclosure may include an amine oxide surfactant a non-ionic surfactant. The non-ionic surfactant may be present at a level of 0.005 wt. % to 0.5 wt. %, more preferably 0.01 wt. % to 0.1 wt. %, most preferably 0.04 wt. % to 0.06 wt. %, by weight of the overall composition. Suitable non-ionic surfactants include amine oxide surfactants which include: R1R2R3NO wherein each of R1, R2and R3is independently a saturated or unsaturated, substituted or unsubstituted, linear or branched hydrocarbon chain having from 10 to 30 carbon atoms. Preferred amine oxide surfactants are amine oxides having the following formula: R1R2R3NO wherein R1is an hydrocarbon chain comprising from 1 to 30 carbon atoms, preferably from 6 to 20, more preferably from 8 to 16 and wherein R2and R3are independently saturated or unsaturated, substituted or unsubstituted, linear or branched hydrocarbon chains comprising from 1 to 4 carbon atoms, preferably from 1 to 3 carbon atoms, and more preferably are methyl groups. R1may be a saturated or unsaturated, substituted or unsubstituted linear or branched hydrocarbon chain. A highly preferred amine oxide is C12-C14dimethyl amine oxide, commercially available from Albright & Wilson, C12-C14amine oxides commercially available under the trade name Genaminox® LA from Clariant or AROMOX® DMC from AKZO Nobel. Another suitable non-ionic surfactants are ethoxylated alkoxylated nonionic surfactant. The ethoxylated alkoxylated nonionic surfactant is preferably selected from the group consisting of: esterified alkyl alkoxylated surfactant; alkyl ethoxy alkoxy alcohol, wherein the alkoxy part of the molecule is preferably propoxy, or butoxy, or propoxy-butoxy; polyoxyalkylene block copolymers, and mixtures thereof. The preferred ethoxylated alkoxylated nonionic surfactant is an esterified alkyl alkoxylated surfactant of general formula (I): whereR is a branched or unbranched alkyl radical having 8 to 16 carbon atoms, preferably from 10 to 16 and more preferably from 12 to 15;R3, R1 independently of one another, are hydrogen or a branched or unbranched alkyl radical having 1 to 5 carbon atoms; preferably R3 and R1 are hydrogenR2 is an unbranched alkyl radical having 5 to 17 carbon atoms; preferably from 6 to 14 carbon atomsl, n independently of one another, are a number from 1 to 5 andm is a number from 8 to 50; and Preferably, the weight average molecular weight of the ethoxylated alkoxylated nonionic surfactant of formula (I) is from 950 to 2300 g/mol, more preferably from 1200 to 1900 g/mol. R is preferably from 12 to 15, preferably 13 carbon atoms. R3 and R1 are preferably hydrogen. Component l is preferably 5. n is preferably 1. m is preferably from 13 to 35, more preferably 15 to 25, most preferably 22. R2 is preferably from 6 to 14 carbon atoms. The hard surface cleaning composition of the invention provides especially high shine when the esterified alkyl akoxylated surfactant is as follows: R has from 12 to 15, preferably 13 carbon atoms, R3 is hydrogen, R1 is hydrogen, component l is 5, n is 1, m is from 15 to 25, preferably 22 and R2 has from 6 to 14 carbon atoms and the alcohol ethoxylated has an aliphatic alcohol chain containing from 10 to 14, more preferably 13 carbon atoms and from 5 to 8, more preferably 7 molecules of ethylene oxide. Preferably, the ethoxylated alkoxylated nonionic surfactant can be a polyoxyalkylene copolymer. The polyoxyalkylene copolymer can be a block-heteric ethoxylated alkoxylated nonionic surfactant, though block-block surfactants are preferred. Suitable polyoxyalkylene block copolymers include ethylene oxide/propylene oxide block polymers, of formula (II): (EO)x(PO)y(EO)x, or (II) (PO)x(EO)y(PO)x (II) wherein EO represents an ethylene oxide unit, PO represents a propylene oxide unit, and x and y are numbers detailing the average number of moles ethylene oxide and propylene oxide in each mole of product. Such materials tend to have higher molecular weights than most non-ionic surfactants, and as such can range between 1000 and 30000 g/mol, although the molecular weight should be above 2200 and preferably below 13000. A preferred range for the molecular weight of the polymeric non-ionic surfactant is from 2400 to 11500 Daltons. BASF (Mount Olive, N.J.) manufactures a suitable set of derivatives and markets them under the Pluronic trademarks. Examples of these are Pluronic (trademark) F77, L62 and F88 which have the molecular weight of 6600, 2450 and 11400 g/mol respectively. An especially preferred example of a useful polymeric non-ionic surfactant is Pluronic (trademark) F77. Other suitable ethoxylated alkoxylated nonionic surfactants are described in Chapter 7 of Surfactant Science and Technology, Third Edition, Wiley Press, ISBN 978-0-471-68024-6. The ethoxylated alkoxylated nonionic surfactant preferably provides a wetting effect of from 15 to 350 s, more preferably from 60 to 200 s, even more preferably from 75 to 150 s. The wetting effect is measured according to EN 1772, using 1 g/l of the ethoxylated alkoxylated nonionic surfactant in distilled water, at 23° C., with 2 g soda/l. The ethoxylated alkoxylated nonionic surfactants preferably are low foaming non-ionic surfactants that are alkoxylated and include unbranched fatty alcohols that may contain high amounts of alkene oxide and ethylene oxide. For example, preferred ethoxylated alkoxylated nonionic surfactants may include those sold by BASF under the “Plurafac” trademark, especially Plurafac LF 131 (wetting effect of 25 s), LF 132 (wetting effect of 70 s), LF 231 (wetting effect of 40 s), LF 431 (wetting effect of 30 s), LF 1530 (wetting effect>300 s), LF 731 (wetting effect of 100 s), LF 1430 (wetting effect>300 s) and LF 7319 (wetting effect of 100 s). The ethoxylated alkoxylated nonionic surfactants preferably are not hydrogenated and, therefore, the fatty alcohol chains do not terminate in a hydrogen group. Examples of such hydrogenated non-ionic surfactants include Plurafac 305 and Plurafac 204. Another suitable non-ionic surfactants are alkoxylated nonionic surfactants, alkyl polyglycosides, and mixture thereof. Suitable alkoxylated nonionic surfactants include primary C6-C16alcohol polyglycol ether i.e. ethoxylated alcohols having 6 to 16 carbon atoms in the alkyl moiety and 4 to 30 ethylene oxide (EO) units. When referred to for example C9-14it is meant average carbons and alternative reference to for example EO8 is meant average ethylene oxide units. Suitable alkoxylated nonionic surfactants are according to the formula RO-(A)nH, wherein: R is a C6to C18, preferably a C8to C16, more preferably a C8to C12alkyl chain, or a C6to C28alkyl benzene chain; A is an ethoxy or propoxy or butoxy unit, and wherein n is from 1 to 30, preferably from 1 to 15 and, more preferably from 4 to 12 even more preferably from 5 to 10. Preferred R chains for use herein are the C8to C22alkyl chains. Even more preferred R chains for use herein are the C9to C12alkyl chains. R can be linear or branched alkyl chain. Suitable ethoxylated nonionic surfactants for use herein are Dobanol® 91-2.5 (HLB=8.1; R is a mixture of C9and C11alkyl chains, n is 2.5), Dobanol® 91-10 (HLB=14.2; R is a mixture of C9to C11alkyl chains, n is 10), Dobanol® 91-12 (HLB=14.5 ; R is a mixture of C9to C11alkyl chains, n is 12), Greenbentine DE80 (HLB=13.8, 98 wt % C10 linear alkyl chain, n is 8), Marlipal 10-8 (HLB=13.8, R is a C10 linear alkyl chain, n is 8), Lialethl® 11-5 (R is a C11alkyl chain, n is 5), Isalchem® 11-5 (R is a mixture of linear and branched C11 alkyl chain, n is 5), Lialethl® 11-21 (R is a mixture of linear and branched C11alkyl chain, n is 21), Isalchem® 11-21 (R is a C11branched alkyl chain, n is 21), Empilan® KBE21 (R is a mixture of C12and C14alkyl chains, n is 21) or mixtures thereof. Preferred herein are Dobanol® 91-5, Neodol® 11-5, Lialethl® 11-21 Lialethl® 11-5 Isalchem® 11-5 Isalchem® 11-21 Dobanol® 91-8, or Dobanol® 91-10, or Dobanol® 91-12, or mixtures thereof. These Dobanol®/Neodol® surfactants are commercially available from SHELL. These Lutensol® surfactants are commercially available from BASF and these Tergitol® surfactants are commercially available from Dow Chemicals. Suitable chemical processes for preparing the alkoxylated nonionic surfactants for use herein include condensation of corresponding alcohols with alkylene oxide, in the desired proportions. Such processes are well known to the person skilled in the art and have been extensively described in the art, including the OXO process and various derivatives thereof. Suitable alkoxylated fatty alcohol nonionic surfactants, produced using the OXO process, have been marketed under the tradename NEODOL® by the Shell Chemical Company. Alternatively, suitable alkoxylated nonionic surfactants can be prepared by other processes such as the Ziegler process, in addition to derivatives of the OXO or Ziegler processes. Preferably, said alkoxylated nonionic surfactant is a C9-11EO5 alkylethoxylate, C12-14EO5 alkylethoxylate, a C11EO5 alkylethoxylate, C12-14EO21 alkylethoxylate, or a C9-11EO8 alkylethoxylate or a mixture thereof. Most preferably, said alkoxylated nonionic surfactant is a C11EO5 alkylethoxylate or a C9-11EO8 alkylethoxylate or a mixture thereof. Another suitable non-ionic surfactants are Alkyl polyglycosides, which are biodegradable nonionic surfactants which are well known in the art. Suitable alkyl polyglycosides can have the general formula CnH2n+1O(C6H10O5)xH wherein n is preferably from 9 to 16, more preferably 11 to 14, and x is preferably from 1 to 2, more preferably 1.3 to 1.6. Such alkyl polyglycosides provide a good balance between anti-foam activity and detergency. Alkyl polyglycoside surfactants are commercially available in a large variety. An example of a very suitable alkyl poly glycoside product is Planteren APG 600, which is essentially an aqueous dispersion of alkyl polyglycosides wherein n is about 13 and x is about 1.4. The additional nonionic surfactant is preferably a low molecular weight nonionic surfactant, having a molecular weight of less than 950 g/mol, more preferably less than 500 g/mol. Another suitable non-ionic surfactants are suitable zwitterionic surfactants. Zwitterionic surfactants typically contain both cationic and anionic groups in substantially equivalent proportions so as to be electrically neutral at the pH of use. The typical cationic group is a quaternary ammonium group, other positively charged groups like phosphonium, imidazolium and sulfonium groups can be used. The typical anionic hydrophilic groups are carboxylates and sulfonates, although other groups like sulfates, phosphonates, and the like can be used. Some common examples of zwitterionic surfactants (such as betaine/sulphobetaine surfacants) are described in U.S. Pat. Nos. 2,082,275, 2,702,279 and 2,255,082. For example Coconut dimethyl betaine is commercially available from Seppic under the trade name of Amonyl 265®. Lauryl betaine is commercially available from Albright & Wilson under the trade name Empigen BB/L®. A further example of betaine is Lauryl-imminodipropionate commercially available from Rhodia under the trade name Mirataine H2C-HA®. Sulfobetaine surfactants are particularly preferred, since they can improve soap scum cleaning. Examples of suitable sulfobetaine surfactants include tallow bis(hydroxyethyl) sulphobetaine, cocoamido propyl hydroxy sulphobetaines which are commercially available from Rhodia and Witco, under the trade name of Mirataine CBS® and ReWoteric AM CAS 15® respectively. Another suitable non-ionic surfactants are amphoteric surfactants. Amphoteric surfactants can be either cationic or anionic depending upon the pH of the composition. Suitable amphoteric surfactants include dodecylbeta-alanine, N-alkyltaurines such as the one prepared by reacting dodecylamine with sodium isethionate, as taught in U.S. Pat. No. 2,658,072, N-higher alkylaspartic acids such as those taught in U.S. Pat. No. 2,438,091, and the products sold under the trade name “Miranol”, as described in U.S. Pat. No. 2,528,378. Other suitable additional surfactants can be found in McCutcheon's Detergents and Emulsifers, North American Ed. 1980. Copolymer: The cleaning composition may comprise from 0.005% to 1.5%, more preferably from 0.01% to 1%, yet more preferably from 0.01% to 5%, most preferably from 0.01 to 0.06% by weight of the cleaning composition, of a copolymer that comprises monomers selected from the group comprising monomers of formula (III) (Monomer A) and monomers of formula (IVa-IVd) (Monomer B) (hereinafter referred to as “the copolymer”). The copolymer comprises from 60 to 99%, preferably from 70 to 95% and especially from 80 to 90% by weight of at least one monoethylenically unsaturated polyalkylene oxide monomer of the formula (III) (monomer A) wherein Y of formula (III) is selected from —O— and —NH—; if Y of formula (III) is —O—, X of formula (III) is selected from —CH2— or —CO—, if Y of formula (III) is —NH—, X of formula (III) is —CO—; R1of formula (III) is selected from hydrogen, methyl, and mixtures thereof; R2of formula (III) is independently selected from linear or branched C2-C6-alkylene radicals, which may be arranged blockwise or randomly; R3of formula (III) is selected from hydrogen, C1-C4-alkyl, and mixtures thereof; n of formula (III) is an integer from 5 to 100, preferably from 10 to 70 and more preferably from 20 to 50. The copolymer comprises from 1 to 40%, preferably from 2 to 30% and especially from 5 to 20% by weight of at least one quaternized nitrogen-containing monoethylenically unsaturated monomer of formula (IVa-IVd) (monomer B). The monomers are selected such that the copolymer has a weight average molecular weight (Mw) of from 5,000 to 500,000 g/mol, preferably from greater than 7,000 to 150,000 g/mol and especially from 10,000 to 80,000 g/mol. The copolymer preferably has a net positive charge at a pH of 3 or above. The copolymer for use in the present disclosure may further comprise monomers C and/or D. Monomer C may comprise from 0% to 15%, preferably from 0 to 10% and especially from 1 to 7% by weight of the copolymer of an anionic monoethylenically unsaturated monomer. Monomer D may comprise from 0% to 40%, preferably from 1 to 30% and especially from 5 to 20% by weight of the copolymer of other non-ionic monoethylenically unsaturated monomers. Preferred copolymers according to the present disclosure comprise, as copolymerized Monomer A, monoethylenically unsaturated polyalkylene oxide monomers of formula (III) in which Y of formula (III) is —O—; X of formula (III) is —CO—; R1of formula (III) is hydrogen or methyl; R2of formula (III) is independently selected from linear or branched C2-C4-alkylene radicals arranged blockwise or randomly, preferably ethylene, 1,2- or 1,3-propylene or mixtures thereof, particularly preferably ethylene; R3of formula (III) is methyl; and n is an integer from 20 to 50. Monomer A A monomer A for use in the copolymer of the present disclosure may be, for example:(a) reaction products of (meth)acrylic acid with polyalkylene glycols which are not terminally capped, terminally capped at one end by alkyl radicals; and(b) alkenyl ethers of polyalkylene glycols which are not terminally capped or terminally capped at one end by alkyl radicals. Preferred monomer A is the (meth)acrylates and the allyl ethers, where the acrylates and primarily the methacrylates are particularly preferred. Particularly suitable examples of the monomer A are:(a) methylpolyethylene glycol (meth)acrylate and (meth)acrylamide, methylpolypropylene glycol (meth)acrylate and (meth)acrylamide, methylpolybutylene glycol (meth)acrylate and (meth)acrylamide, methylpoly(propylene oxide-co-ethylene oxide) (meth)acrylate and (meth)acrylamide, ethylpolyethylene glycol (meth)acrylate and (meth)acrylamide, ethylpolypropylene glycol (meth)acrylate and (meth)acrylamide, ethylpolybutylene glycol (meth)acrylate and (meth)acrylamide and ethylpoly(propylene oxide-co-ethylene oxide) (meth)acrylate and (meth)acrylamide, each with 5 to 100, preferably 10 to 70 and particularly preferably 20 to 50, alkylene oxide units, where methylpolyethylene glycol acrylate is preferred and methylpolyethylene glycol methacrylate is particularly preferred;(b) ethylene glycol allyl ethers and methylethylene glycol allyl ethers, propylene glycol allyl ethers and methylpropylene glycol allyl ethers each with 5 to 100, preferably 10 to 70 and particularly preferably 20 to 50, alkylene oxide units. The proportion of Monomer A in the copolymer according to the present disclosure is 60% to 99% by weight, preferably 70% to 95%, more preferably from 80% to 90% by weight of the copolymer. Monomer B A monomer B that is particularly suitable for the copolymer of the present disclosure includes the quaternization products of 1-vinylimidazoles, of vinylpyridines, of (meth)acrylic esters with amino alcohols, in particular N,N-di-C1-C4-alkylamino-C2-C6-alcohols, of amino-containing (meth)acrylamides, in particular N,N-di-C1-C4-alkyl-amino-C2-C6-alkylamides of (meth)acrylic acid, and of diallylalkylamines, in particular diallyl-C1-C4-alkylamines. Suitable monomers B have the formula IVa to IVd: wherein R of formula IVa to IVd is selected from C1-C4-alkyl or benzyl, preferably methyl, ethyl or benzyl; R′ of formula IVc is selected from hydrogen or methyl; Y of formula IVc is selected from —O— or —NH—; A of formula IVc is selected from C1-C6-alkylene, preferably straight-chain or branched C2-C4-alkylene, in particular 1,2-ethylene, 1,3- and 1,2-propylene or 1,4-butylene; X− of formula IVa to IVd is selected from halide, such as iodide and preferably chloride or bromide, C1-C4-alkyl sulfate, preferably methyl sulfate or ethyl sulfate, C1-C4-alkylsulfonate, preferably methylsulfonate or ethylsulfonate, C1-C4-alkyl carbonate; and mixtures thereof. Specific examples of preferred monomer B that may be utilized in the present disclosure are:(a) 3-methyl-1-vinylimidazolium chloride, 3-methyl-1-vinylimidazolium methyl sulfate, 3-ethyl-1-vinylimidazolium ethyl sulfate, 3-ethyl-1-vinylimidazolium chloride and 3-benzyl-1-vinylimidazolium chloride;(b) 1-methyl-4-vinylpyridinium chloride, 1-methyl-4-vinylpyridinium methyl sulfate and 1-benzyl-4-vinylpyridinium chloride;(c) 3-methacrylamido-N,N,N-trimethylpropan-1-aminium chloride, 3-acryl-N,N,N-trimethylpropan-1-aminium chloride, 3-acryl -N,N,N-trimethylpropan-1-aminium methylsulfate, 3-methacryl-N,N,N-trimethylpropan-1-aminium chloride, 3-methacryl-N,N,N-trimethylpropan-1-aminium methylsulfate, 2-acrylamido-N,N,N-trimethylethan-1-aminium chloride, 2-acryl-N,N,N-trimethylethan-1-aminium chloride, 2-acryl-N,N,N-trimethylethan-1-aminium methyl sulfate, 2-methacryl-N,N,N-trimethylethan-1-aminium chloride, 2-methacryl-N,N,N-trimethylethan-1-aminium methyl sulfate, 2-acryl-N,N-dimethyl-N-ethylethan-1-aminium ethylsulfate, 2-methacryl-N,N-dimethyl-N-ethylethan-1-aminium ethylsulfate, and(d) dimethyldiallylammonium chloride and diethyldiallylammonium chloride. A preferred monomer B is selected from 3-methyl-1-vinylimidazolium chloride, 3-methyl-1-vinylimidazolium methyl sulfate, 3-methacryl-N,N,N-trimethylpropan-1-aminium chloride, 2-methacryl-N,N,N-trimethylethan-1-aminium chloride, 2-methacryl-N,N-dimethyl-N-ethylethan-1-aminium ethylsulfate, and dimethyldiallylammonium chloride. The copolymer according to the present disclosure comprises 1% to 40% by weight, preferably 2% to 30%, and especially preferable from 5 to 20% by weight of the copolymer, of Monomer B. The weight ratio of Monomer A to Monomer B is preferably equal to or greater than 2:1, preferably 3:1 to 5:1. Monomer C As optional components of the copolymer of the present disclosure, monomers C and D may also be utilized. Monomer C is selected from anionic monoethylenically unsaturated monomers. Suitable monomer C may be selected from:(a) α,β-unsaturated monocarboxylic acids which preferably have 3 to 6 carbon atoms, such as acrylic acid, methacrylic acid, 2-methylenebutanoic acid, crotonic acid and vinylacetic acid, preference being given to acrylic acid and methacrylic acid;(b) unsaturated dicarboxylic acids, which preferably have 4 to 6 carbon atoms, such as itaconic acid and maleic acid, anhydrides thereof, such as maleic anhydride;(c) ethylenically unsaturated sulfonic acids, such as vinylsulfonic acid, acrylamido-propanesulfonic acid, methallylsulfonic acid, methacrylsulfonic acid, m- and p-styrenesulfonic acid, (meth)acrylamidomethanesulfonic acid, (meth)acrylamidoethanesulfonic acid, (meth)acrylamidopropanesulfonic acid, 2-(meth)acrylamido-2-methylpropanesulfonic acid, 2-acrylamido-2-butanesulfonic acid, 3-methacrylamido-2-hydroxypropanesulfonic acid, methanesulfonic acid acrylate, ethanesulfonic acid acrylate, propanesulfonic acid acrylate, allyloxybenzenesulfonic acid, methallyloxybenzenesulfonic acid and 1-allyloxy-2-hydroxypropanesulfonic acid; and(d) ethylenically unsaturated phosphonic acids, such as vinylphosphonic acid and m- and p-styrenephosphonic acid. The anionic Monomer C can be present in the form of water soluble free acids or in water-soluble salt form, especially in the form of alkali metal and ammonium, in particular alkylammonium, salts, and preferred salts being the sodium salts. A preferred Monomer C may be selected from acrylic acid, methacrylic acid, maleic acid, vinylsulfonic acid, 2-(meth) acrylamido-2-methylpropanesulfonic acid and vinylphosphonic acid, particular preference being given to acrylic acid, methacrylic acid and 2-acrylamido-2-methylpropanesulfonic acid. The proportion of monomer C in the copolymer of the present disclosure can be up to 15% by weight, preferably from 1% to 5% by weight of the copolymer. If monomer C is present in the copolymer of the present disclosure, then, the molar ratio of monomer B to monomer C is greater than 1. The weight ratio of Monomer A to monomer C is preferably equal to or greater than 4:1, more preferably equal to or greater than 5:1. Additionally, the weight ratio of monomer B to monomer C is equal or greater than 2:1, and even more preferable from 2.5:1 Monomer D As an optional component of the copolymer of the present disclosure, monomer D may also be utilized. Monomer D is selected from nonionic monoethylenically unsaturated monomers selected from:(a) esters of monoethylenically unsaturated C3-C6-carboxylic acids, especially acrylic acid and methacrylic acid, with monohydric C1-C22-alcohols, in particular C1-C16-alcohols; and hydroxyalkyl esters of monoethylenically unsaturated C3-C6-carboyxlic acids, especially acrylic acid and methacrylic acid, with divalent C2-C4-alcohols, such as methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, sec-butyl (meth)acrylate, tert-butyl (meth)acrylate, ethylhexyl (meth)acrylate, decyl (meth)acrylate, lauryl (meth)acrylate, isobornyl (meth)acrylate, cetyl (meth)acrylate, palmityl (meth)acrylate and stearyl (meth)acrylate, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate and hydroxybutyl (meth)acrylate;(b) amides of monoethylenically unsaturated C3-C6-carboxylic acids, especially acrylic acid and methacrylic acid, with C1-C12-alkylamines and di(C1-C4-alkyl)amines, such as N-methyl(meth)acrylamide, N,N-dimethyl(meth)acrylamide, N-ethyl(meth)acrylamide, N-propyl(meth)acrylamide, N-tert-butyl(meth)acrylamide, N-tert-octyl(meth)acrylamide and N-undecyl(meth)acrylamide, and (meth)acrylamide;(c) vinyl esters of saturated C2-C30-carboxylic acids, in particular C2-C14-carboxylic acids, such as vinyl acetate, vinyl propionate, vinyl butyrate, vinyl 2-ethylhexanoate and vinyl laurate;(d) vinyl C1-C30-alkyl ethers, in particular vinyl C1-C18-alkyl ethers, such as vinyl methyl ether, vinyl ethyl ether, vinyl n-propyl ether, vinyl isopropyl ether, vinyl n-butyl ether, vinyl isobutyl ether, vinyl 2-ethylhexyl ether and vinyl octadecyl ether;(e) N-vinylamides and N-vinyllactams, such as N-vinylformamide, N-vinyl-N-methyl-formamide, N-vinylacetamide, N-vinyl-N-methylacetamide, N-vinylimidazol, N-vinylpyrrolidone, N-vinylpiperidone and N-vinylcaprolactam;(f) aliphatic and aromatic olefins, such as ethylene, propylene, C4-C24-α-olefins, in particular C4-C16-α-olefins, e.g. butylene, isobutylene, diisobutene, styrene and α-methylstyrene, and also diolefins with an active double bond, e.g. butadiene;(g) unsaturated nitriles, such as acrylonitrile and methacrylonitrile. A preferred monomer D is selected from methyl (meth)acrylate, ethyl (meth)acrylate, (meth)acrylamide, vinyl acetate, vinyl propionate, vinyl methyl ether, N-vinylformamide, N-vinylpyrrolidone, N-vinylimidazole and N-vinylcaprolactam. N-vinylimidazol is particularly preferred. If the monomer D is present in the copolymer of the present disclosure, then the proportion of monomer D may be up to 40%, preferably from 1% to 30%, more preferably from 5% to 20% by weight of the copolymer. Preferred copolymers of the present disclosure include: wherein indices y and z are such that the monomer ratio (z:y) is from 3:1 to 20:1 and the indices x and z are such that the monomer ratio (z:x) is from 1.5:1 to 20:1, and the polymer has a weight average molecular weight of from 20,000 to 500,000 g/mol, preferably from greater than 25,000 to 150,000 g/mol and especially from 30,000 to 80,000 g/mol. The copolymers according to the present disclosure can be prepared by free-radical polymerization of the Monomers A and B and if desired C and/or D. The free-radical polymerization of the monomers can be carried out in accordance with all known methods, preference being given to the processes of solution polymerization and of emulsion polymerization. Suitable polymerization initiators are compounds which decompose thermally or photochemically (photoinitiators) to form free radicals, such as benzophenone, acetophenone, benzoin ether, benzyl dialkyl ketones and derivatives thereof. The polymerization initiators are used according to the requirements of the material to be polymerized, usually in amounts of from 0.01% to 15%, preferably 0.5% to 5% by weight based on the monomers to be polymerized, and can be used individually or in combination with one another. Instead of a quaternized Monomer B, it is also possible to use the corresponding tertiary amines. In this case, the quaternization is carried out after the polymerization by reacting the resulting copolymer with alkylating agents, such as alkyl halides, dialkyl sulfates and dialkyl carbonates, or benzyl halides, such as benzyl chloride. Examples of suitable alkylating agents which may be mentioned are, methyl chloride, bromide and iodide, ethyl chloride and bromide, dimethyl sulfate, diethyl sulfate, dimethyl carbonate and diethyl carbonate. The anionic monomer C can be used in the polymerization either in the form of the free acids or in a form partially or completely neutralized with bases. Specific examples that may be listed are: sodium hydroxide solution, potassium hydroxide solution, sodium carbonate, sodium hydrogen carbonate, ethanolamine, diethanolamine and triethanolamine. To limit the molar masses of the copolymers according to the present disclosure, customary regulators can be added during the polymerization, e.g. mercapto compounds, such as mercaptoethanol, thioglycolic acid and sodium disulfite. Suitable amounts of regulator are 0.1% to 5% by weight based on the monomers to be polymerized. Quaternary Compound The liquid hard surface cleaning composition may comprise a quaternary compound. Preferably, the liquid hard surface cleaning composition comprises the quaternary compound at a level of from 0.001 to 2% wt %, more preferably from 0.002 to 0.5 wt %, most preferably from 0.005 wt % to 01 wt % of the composition. Traditionally, compositions comprising quaternary compounds tend to leave unsightly filming and/or streaking on the treated surfaces. However, compositions as presently disclosed surprisingly provide improved shine and reduced streaking. Without being bound to theory this improved shine benefit is a result of the dewetting property of the solvent. Furthermore, the compositions disclosed provide high antibacterial benefit delivering 5 logs micro efficacy while still delivering high shine. In the case of premoisten wipes, without being bound to theory, the cleaning and shine benefit are improved when the pad contains cellulose, preferentially between 50 to 200 gsm, more preferentially between 80 to 150 gsm, so that the negative sites of the cellulose bind some of the quaternary compounds reducing the expressed or release level of the quaternary compounds and thereby reducing their negative impacts on cleaning, shine and haze while maintaining its micro bacterial elimination efficacy. Quaternary compounds useful herein are preferably selected from the group consisting of C6-C18 alkyltrimethylammonium chlorides, C6-C18dialkyldimethylammonium chlorides, and mixtures thereof. Preferably, the quaternary compound is selected from the group consisting of a C8-C12 alkyltrimethylammonium chloride, a C8-C12 dialkyldimethylammonium chloride, and mixtures thereof. Most preferably, the quaternary compound is C10 dialkyldimethylammonium chloride. Non-limiting examples of useful quaternary compounds include: (1) Maquat® (available from Mason), and Hyamine® (available from Lonza); (2) di(C6-C14)alkyl di short chain (C1-4 alkyl and/or hydroxyalkl) quaternary such as Uniquat® and Bardac® products of Lonza, (3) N-(3-chloroallyl) hexaminium chlorides such as Dowicil® and Dowicil® available from Dow; and (4) di(C8-C12)dialkyl dimethyl ammonium chloride, such as didecyldimethylammonium chloride (Bardac 22, Uniquat 2250 or Bardac 2250), and dioctyldimethylammonium chloride (Bardac 2050). The quaternary compounds preferably are not benzyl quats. An example of such benzyl quat includes alkyl dimethyl benzyl ammonium chloride (Uniquat QAC). Nitrogen-Containing Polymer The liquid hard surface cleaning composition may comprise a nitrogen-containing polymer. Nitrogen-containing polymers useful herein include polymers that contain amines (primary, secondary, and tertiary), amine-N-oxide, amides, urethanes, and/or quaternary ammonium groups. When present, it is important that the polymers herein contain nitrogen-containing groups that tend to strongly interact with the surface being treated in order to displace any present cationic quaternary compound from the surface. Preferably, the polymers herein contain basic nitrogen groups. Basic nitrogen groups include primary, secondary, and tertiary amines capable of acting as proton acceptors. Thus, the preferred polymers herein can be nonionic or cationic, depending upon the pH of the solution. Polymers useful herein can include other functional groups, in addition to nitrogen groups. The preferred polymers herein are also essentially free of, or free of, quaternary ammonium groups. Preferably, the polymers herein are branched polymers, especially highly branched polymers including comb, graft, starburst, and dendritic structures. Preferably, the polymers herein are not linear polymers. The nitrogen-containing polymers herein can be an unmodified or modified polyamine, especially an unmodified or modified polyalkyleneimine. Preferably, the nitrogen containing polymers herein are modified polyamines. Poly (C2-C12alkyleneimines) include simple polyethyleneimines and polypropyleneimines as well as more complex polymers containing these polyamines. Polyethyleneimines are common commercial materials produced by polymerization of aziridine or reaction of (di)amines with alkylenedichlorides. Polypropyleneimines are also included herein. Although modified polyamines are preferred, linear or branched polyalkyleneimines, especially polyethyleneimines or polypropyleneimines, can be suitable in the present compositions to mitigate filming and/or streaking resulting from such compositions containing quaternary compounds. Branched polyalkyleneimines are preferred to linear polyalkyleneimines. Suitable polyalkyleneimines typically have a molecular weight of from about 1,000 to about 30,000 Daltons, and preferably from about 4,000 to about 25,000 Daltons. Such polyalkyleneimines are free of any ethoxylated and/or propoxylated groups, as it has been found that ethoxylation or propoxylation of polyalkyleneimines reduces or eliminates their ability to mitigate the filming and/or streaking problems caused by compositions containing quaternary compounds. In preferred low-surfactant compositions for use in no-rinse cleaning methods, such compositions typically comprise nitrogen-containing polymer at a level of from about 0.005% to about 1%, preferably from about 0.005% to about 0.3%, and more preferably from about 0.005% to about 0.1%, by weight of the composition. Examples of preferred modified polyamines useful as nitrogen-containing polymers herein are branched polyethyleneimines with a molecular weight of about 25,000 Daltons, and Lupasol® SK and Lupasol® SK(A) available from BASF. Additional Polymers The liquid hard surface cleaning composition may comprise an additional polymer. It has been found that the presence of a specific polymer as described herein, when present, allows further improving the grease removal performance of the liquid composition due to the specific sudsing/foaming characteristics they provide to the composition. Suitable polymers for use herein are disclosed in co-pending EP patent application EP2272942 (09164872.5) and granted European patent EP2025743 (07113156.9). The polymer can be selected from the group consisting of: a vinylpyrrolidone homopolymer (PVP); a polyethyleneglycol dimethylether (DM-PEG); a vinylpyrrolidone/dialkylaminoalkyl acrylate or methacrylate copolymers; a polystyrenesulphonate polymer (PSS); a poly vinyl pyridine-N-oxide (PVNO); a polyvinylpyrrolidone/vinylimidazole copolymer (PVP-VI); a polyvinylpyrrolidone/polyacrylic acid copolymer (PVP-AA); a polyvinylpyrrolidone/vinylacetate copolymer (PVP-VA); a polyacrylic polymer or polyacrylicmaleic copolymer; and a polyacrylic or polyacrylic maleic phosphono end group copolymer; and mixtures thereof. Typically, the liquid hard surface cleaning composition may comprise from 0.001% to 1.0% by weight of the total composition of said polymer, preferably from 0.005% to 0.5%, more preferably from 0.01% to 0.05% and most preferably from 0.01% to 0.03%. Fatty Acid The liquid hard surface cleaning composition may comprise a fatty acid as a highly preferred optional ingredient, particularly as suds supressors. Fatty acids are desired herein as they reduce the sudsing of the liquid composition when the composition is rinsed off the surface to which it has been applied. Suitable fatty acids include the alkali salts of a C8-C24fatty acid. Such alkali salts include the metal fully saturated salts like sodium, potassium and/or lithium salts as well as the ammonium and/or alkylammonium salts of fatty acids, preferably the sodium salt. Preferred fatty acids for use herein contain from 8 to 22, preferably from 8 to 20 and more preferably from 8 to 18 carbon atoms. Suitable fatty acids may be selected from caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and mixtures of fatty acids suitably hardened, derived from natural sources such as plant or animal esters (e.g., palm oil, olive oil, coconut oil, soybean oil, castor oil, tallow, ground oil, whale and fish oils and/or babassu oil. For example coconut fatty acid is commercially available from KLK OLEA under the name PALMERAB1211. Typically, the liquid hard surface cleaning composition may comprise up to 0.5% by weight of the total composition of said fatty acid, preferably from 0.05% to 0.3%, more preferably from 0.05% to 0.2% and most preferably from 0.07% to 0.1% by weight of the total composition of said fatty acid. Branched Fatty Alcohol The liquid hard surface cleaning composition may comprise a branched fatty alcohol, particularly as suds suppressors. Suitable branched fatty alcohols include the 2-alkyl alkanols having an alkyl chain comprising from 6 to 16, preferably from 7 to 13, more preferably from 8 to 12, most preferably from 8 to 10 carbon atoms and a terminal hydroxy group, said alkyl chain being substituted in the α position (i.e., position number 2) by an alkyl chain comprising from 1 to 10, preferably from 2 to 8 and more preferably 4 to 6 carbon atoms. Such suitable compounds are commercially available, for instance, as the Isofol® series such as Isofol® 12 (2-butyl octanol) or Isofol® 16 (2-hexyl decanol) commercially available from Sasol Typically, the liquid hard surface cleaning composition may comprise up to 2.0% by weight of the total composition of said branched fatty alcohol, preferably from 0.10% to 1.0%, more preferably from 0.1% to 0.8% and most preferably from 0.1% to 0.5%. Perfumes The liquid hard surface cleaning compositions preferably comprise a perfume. Suitable perfumes provide an olfactory aesthetic benefit and/or mask any “chemical” odor that the product may have. Other Optional Ingredients The liquid hard surface cleaning compositions may comprise a variety of other optional ingredients depending on the technical benefit aimed for and the surface treated. Suitable optional ingredients for use herein include builders, other polymers, buffers, bactericides, hydrotropes, colorants, stabilizers, radical scavengers, abrasives, soil suspenders, brighteners, anti-dusting agents, dispersants, dye transfer inhibitors, pigments, silicones and/or dyes. Cleaning Pad The liquid hard surface cleaning composition may be used in combination with a cleaning pad of the present disclosure. The cleaning pad may be dry and may contact a surface wetted with a cleaning composition, or the cleaning pad may be pre-moistened. The cleaning pad may comprise one or more layers. Referring toFIGS.1A,1B and2, the cleaning pad10may comprise plural layers, to provide for absorption and storage of cleaning fluid and other liquids deposited on the target surface. The target surface will be described herein as a floor, although one of skill will recognize the invention is not so limited. The target surface can be any hard surface, such as a table or countertop, from which it is desired to absorb and retain liquids such as spill, cleaning solutions, etc. The cleaning pad10may comprise a liquid pervious floor sheet14which contacts the floor during cleaning and preferably provides a desired coefficient of friction during cleaning. An absorbent core16, preferably comprising an absorbent gelling material (“AGM”)16A is disposed on, and optionally joined to an inwardly facing surface of the floor sheet14. The floor sheet may have an absorbency of at least 30%, more preferably at least 35%. It is to be appreciated that if the cleaning pad is to be used to clean a surface other than a floor, the floor sheet may be the sheet that contacts the surface to be cleaned. The floor sheet of the cleaning pad may have a thickness from about 1 mm to about 5 mm, more preferably about 1.5 mm to about 3.0 mm and most preferably about 1.2 mm. A smoothing strip12may be disposed on the outwardly facing surface of the floor sheet14. Optionally, a back sheet18may be joined to the core16opposite the floor sheet14, to provide for attachment of the cleaning pad10to an implement30. The back sheet18may have an outwardly facing surface with one or more attachment strips20to particularly facilitate attachment to an implement30. The cleaning pad10may be generally planar and define an XY plane and associated X, Y axes. The Z axis is perpendicular thereto and generally vertical when the cleaning pad10is in use on a floor. If desired, the core16may comprise AGM16A to increase the absorbent capacity of the cleaning pad10. The AGM16A may be in the form of particles may be distributed within the cleaning pad10in such a manner to avoid rapid absorbency and absorb fluids slowly, to provide for the most effective use of the cleaning pad10. The AGM16A also entraps dirty liquid absorbed from the floor, preventing redeposition. If desired foam absorbent material or fibrous material may be incorporated into the core16. Examining the cleaning pad10in more detail, the cleaning pad10may comprise plural layers disposed in a laminate. The lowest, or downwardly facing outer layer, may comprise apertures to allow for transmission of liquid therethrough and to promote the scrubbing of the target surface. One, two or more cores16layers may provide for storage of the liquids and may comprise the absorbent gelling materials. The cleaning pad10may have an absorbent capacity of at least 10, 15, or 20 grams of cleaning solution per gram of dry cleaning pad10, as set forth in commonly assigned U.S. Pat. Nos. 6,003,191 and 6,601,261. The optional top, or upwardly facing layer, is a back sheet18, and may be liquid impervious in order to minimize loss of absorbed fluids and to protect the user's hand if the cleaning pad10is used without an implement30. The top layer may further provide for releasable attachment of the cleaning pad10to a cleaning implement30. The top layer may be made of a polyolefinic film, such as LDPE. A suitable back sheet18comprises a PE/PP film having a basis weight of 10 to 30 gsm. The optional top, or upwardly facing layer, is a back sheet18, and may be liquid impervious in order to minimize loss of absorbed fluids and to protect the user's hand if the cleaning pad10is used without an implement30. This top layer may also be pervious to liquid and made of a polyolefinic nonwoven to provide a soft feel for the user. The top layer may further provide for releasable attachment of the cleaning pad10to a cleaning implement30. The impervious top layer may be made of a polyolefinic film, such as LDPE. A suitable back sheet18comprises a PE/PP film having a basis weight of 10 to 30 gsm. For a liquid pervious top layer, it may comprise a polyolefinic nonwoven, such as a PP nonwoven. Attached to the back sheet18may be one or more optional attachment strips20. The attachment strips20may comprise adhesive, preferably pressure sensitive adhesive, or may comprise loops for removable attachment to complementary hooks on an implement30. Suitable loop attachment strips20may comprise a laminate of PE film and Nylon loops. The back sheet18and floor sheet14may be peripherally joined, as is known in the art. This arrangement creates a pocket for securely holding the core16. The core16may be juxtaposed with, and optionally joined to the respective inwardly facing surfaces of the floor sheet14and back sheet18. The core16may comprise a single layer or two or more layers. If plural layers are selected for the core16, the width of the layers may decrease as the floor sheet14is approached, as shown. The core16may comprise airlaid cellulose and optionally polymer fiber, as available from Glatfelter of York, PA. If two airlaid cellulose core16layers are selected, each layer of the core16may have a basis weight of at least about 75, 100, 125, 150, 175, 200, or 225 gsm and less than about 300 gsm. Preferably each layer of the core16comprises AGM16A. The AGM16A may absorb at least 10, 15 or 20 times its own weight. The AGM16A may be blown into the airlaid core16layer during manufacture as is known in the art. Suitable AGM16A is available as Z3070G from Evonik of Essen, Germany. Arlaid material containing a gradient AGM16A distribution is available from Glatfelter of York, PA. The gradient distribution AGM16A may be achieved by using more than one forming head. For example, an airfelt/AGM16A line may have three forming heads. The first head may distribute a relatively large amount of AGM16A relative to the cellulose distributed from that head. The second forming head may distribute a less amount of AGM16A relative to the cellulose base, with this mixture being laid onto top of the first AGM16A/cellulose base. This pattern may be repeated using as many forming heads as desired. If desired the final forming head may distribute pure cellulose and no AGM16A. Generally, the layer from each forming head does not intermix with adjacent layers. Adhesive bonding and/or thermal bonding may hold superposed layers in place and provide structural rigidity. Suitable core16layers and a suitable apparatus and process for making one or more layers of a core16having a gradient AGM16A distribution are found in U.S. Pat. No. 8,603,622 issued Dec. 10, 2013. The teachings of U.S. Pat. No. 8,603,622 are incorporated herein by reference at column 5, lines 8-14 for the teaching of a suitable core16layer and atFIGS.3-4, with the accompanying discussion at column 16, line 41 to column 17, line 59 for the teaching of production devices suitable to make a core16layer for the present invention. If two airlaid cellulose core16layers are selected, the lower core layer16L, juxtaposed with the floor sheet14, may comprise about 10 to 20 weight percent AGM16A, with about 15 percent being found suitable. The upper core layer16U, juxtaposed with the optional back sheet18, if any, may comprise about 20 to about 30 weight percent AGM16A, with about 25 percent being found suitable. The total core16, with all layers thereof considered, may comprise 5 to 50 w %, or 10 to 45 w % AGM16A, the amount and gradient distribution of AGM16A being found helpful for the present invention. The percentage of AGM16A, as described and claimed herein refers to the weight percentage of AGM16A in that particular core16layer (16U or16L), without regard to the floor sheet14, back sheet18, smoothing strip12or attachment strips20. Each core layer16L,16U and particularly the upper core layer16U may be further stratified to provide greater absorbency benefit. The upper core layer16U may have three strata, as formed. The strata may comprise 0, 25, and 50 weight percent, monotonically increasing as the back sheet18, if any, is approached, to provide a gradient distribution. Generally, it is desired that the upper core layer16U comprise more AGM16A, on both an absolute basis and a weight percentage basis than the lower core layer16L. The arrangement provides the benefit that gel blocking in the lower core layer16L does not prevent full absorption of liquid from the target surface and that liquids are transported upwardly and away from the floor sheet14. Any arrangement that provides more AGM16A, preferably on an absolute basis or optionally on a weight percentage basis is suitable. Alternatively, either core16layer or a single core16layer may have increasing AGM16A concentration in the Z direction. Any such process, as is known in the art, or arrangement, which provides for increasing AGM16A in the Z direction as the back sheet18is approached is herein considered an AGM16A gradient. It is to be recognized that the AGM16A gradient may be smooth, comprise one or more stepwise increments or any combination thereof. The floor sheet14may comprise a discrete apertured nonwoven having a basis weight of about 20 to about 80 gsm and particularly about 28 to 60 gsm. The floor sheet14may be hydrophobic and made of synthetic fibers. A suitable floor sheet14is a 60 gsm PE/PP discrete apertured spunbond nonwoven available as SofSpan from Fitsea of Simpsonville, SC. The floor sheet14may have a contact angle of 101 to 180 degrees with water. The floor sheet14may comprise a smoothing strip12. The smoothing strip12may have a width less than the floor sheet14and may comprise at least about 10, 20, 30, 40, 50, 60 or 70% of the floor sheet14width. The smoothing strip12may have a width of at least 10, 20, 30, 40, 50, 100, 150, 200, 250, mm and less than 70, 80, 100, 200 or 300 mm, with a width of 24 to 44 mm being suitable and a width of 34 mm being preferred. The smoothing strip12may be hydrophilic. As used herein hydrophilic means having a contact angle of 0 to 100 degrees, as measured by the test method set forth herein. The smoothing strip12may particularly have a contact angle of 30 to 100 degrees and more particularly 55 to 90 degrees. The smoothing strip12may comprise at least 50% cellulosic content to be hydrophilic. More particularly, a suitable smoothing strip12may comprise a laminate of cellulose fibers and synthetic fibers. Such a laminate is believed to be helpful in attaining the performance of the cleaning pads10described herein. The cellulose fiber lamina may be outwardly facing, to provide friction and absorbency on the floor. The synthetic fiber layer may be positioned on contacting relationship with the floor sheet14to provide integrity during use. A 23 gsm tissue and 17 gsm polypropylene spunbond hydroentagled, sold as 40 gsm Genesis tissue by Suominen of Helsinki, Finland has been found to be a suitable smoothing strip12. Another suitable smoothing strip12may comprise 28 gsm tissue and 17 gsm polypropylene spunbond hydroentagled, sold as 45 gsm Hydratexture tissue by Suominen. The smoothing strip12may have a surface texture less than 0.5 mm, 0.4 mm or less than 0.3 mm and even be essentially 0 mm. Surface texture is measured as the peak to valley distance, independent of the smoothing strip12thickness. A surface texture of less than 0.5 mm is believed to minimize streaking during cleaning, particularly when the floor dries and more particularly when a dark floor dries. Wipe The cleaning pad may be in the form of a cleaning wipe. The cleaning wipe may be used as a pre-moistened cleaning wipe or a dry wipe for use with a cleaning composition. If the cleaning wipe is pre-moistened, it is pre-moistened with a cleaning composition, as described in further detail above, which provides for cleaning of the target surface, such as a floor, but yet does not require a post-cleaning rinsing operation. The cleaning wipe used in conjunction with this cleaning composition may comprise natural or synthetic fibers. The fibers may be hydrophilic, hydrophobic or a combination thereof, provided that the cleaning wipe is generally absorbent to hold, and express upon demand, the above described cleaning composition. In one embodiment, the cleaning wipe may comprise at least 50 weight percent or at least 70 weight percent cellulose fibers, such as air laid SSK fibers. If desired, the cleaning wipe may comprise plural layers to provide for scrubbing, liquid storage, and other particularized tasks for the cleaning operation. If one or multiple airlaid cellulose core layers are selected, each layer of the core16may have a basis weight of at least about 50, 100, 125, 150, 175, 200, or 225 gsm and less than about 300 gsm. The level of airlaid cellulose may be important with respect to the retention and control release of the cleaning solution increasing its mileage with respect to cleaning performance in pre-moistened wipes and reducing flooding at the beginning of the cleaning process. In addition, it may be important formulations that include antibacterials due to their controlled release of the quaternary active ingredient. A cleaning wipe may have a thickness from about 1 mm to about 5 mm, more preferably about 1.5 mm to about 3.0 mm and most preferably about 1.2 mm. Optionally, the cleaning wipe may further comprise a scrubbing strip. A scrubbing strip is a portion of the cleaning wipe which provides for more aggressive cleaning of the target surface. A suitable scrubbing strip may comprise a polyolefinic film, such as LDPE, and have outwardly extending perforations, etc. The scrubbing strip may be made and used according to commonly assigned U.S. Pat. Nos. 8,250,700; 8,407,848; D551,409 S and/or D614,408 S. A suitable pre-moistened cleaning wipe maybe made according to the teachings of commonly assigned U.S. Pat. Nos. 6,716,805; D614,408; D629,211 and/or D652,633. Cleaning Implement The cleaning pad10and cleaning composition may be used by hand or with a cleaning implement30. Referring toFIG.3, the cleaning implement30may comprise a plastic head32for holding the cleaning pad10and an elongate handle34connected thereto. The handle34may comprise a metal or plastic tube or solid rod. The head32may have a downwardly facing surface, to which the cleaning pad10may be attached. The downwardly facing surface may be generally flat, or slightly convex. The head32may further have an upwardly facing surface. The upwardly facing surface may have a universal joint to facilitate connection of the elongate handle34to the head32. A hook and loop system may be used to attach the cleaning pad10directly to the bottom of the head. Alternatively, the upwardly facing surface may further comprise a mechanism, such as resilient grippers, for removably attaching the cleaning pad10to the implement30. If grippers are used with the cleaning implement30, the grippers may be made according to commonly assigned U.S. Pat. Nos. 6,305,046; 6,484,346; 6,651,290 and/or D487,173. The cleaning implement may further comprise a reservoir for storage of the cleaning composition, a described in further detail above. The reservoir may be replaced when the cleaning composition is depleted and/or refilled as desired. The reservoir may be disposed on the head or the handle of the cleaning implement of the reservoir may be separate from the cleaning implement. The neck of the reservoir may be offset per commonly assigned U.S. Pat. No. 6,390,335. The reservoir may be in the form of a spray bottle. The cleaning implement30may further comprise a pump for dispensing cleaning solution from the reservoir onto the target surface, such as a floor. The pump may be battery powered or operated by line voltage. Alternatively, the cleaning solution may be dispensed by gravity flow. The cleaning solution may be sprayed through one or more nozzles to provide for distribution of the cleaning solution onto the target surface in an efficacious pattern. If a replaceable reservoir is utilized, the replaceable reservoir may be inverted to provide for gravity flow of the cleaning solution. Or the cleaning solution may be pumped to the dispensing nozzles. The reservoir may be a bottle, and may be made of plastic, such as a polyolefin. The cleaning implement30may have a sleeve (36), which removably receives the bottle, or other reservoir. The cleaning implement30may have a needle, optionally disposed in the sleeve (36) to receive the cleaning solution from the bottle. The bottle may have a needle pierceable membrane, complementary to the needle, and which is resealed to prevent undesired dripping of the cleaning solution during insertion and removal of the replaceable reservoir. Alternatively or additionally, If desired, the implement30may also provide for steam to be delivered to the cleaning pad10and/or to the floor or other target surface. A suitable reservoir of cleaning solution and fitment therefore may be made according to the teachings of commonly assigned U.S. Pat. Nos. 6,386,392, 7,172,099; D388,705; D484,804; D485,178. A suitable cleaning implement30may be made according to the teachings of commonly assigned U.S. Pat. Nos. 5,888,006; 5,960,508; 5,988,920; 6,045,622; 6,101,661; 6,142,750; 6,579,023; 6,601,261; 6,722,806; 6,766,552; D477,701 and/or D487,174. A steam implement30may be made according to the teachings of jointly assigned 2013/0319463. Method of Cleaning a Surface Cleaning pads, cleaning wipes, and cleaning implements using cleaning pads and cleaning wipes may be used along with a liquid hard surface cleaning composition having an advancing contact higher than 30° for cleaning hard surfaces. Preferably cleaning pads, cleaning wipes, and cleaning implements using cleaning pads and cleaning wipes may be used along with a liquid hard surface cleaning composition having from about 0.001 wt % to about 0.015 wt % of an ethoxylated alkoxylated nonionic surface or a copolymer of the present disclosure and at least about 93 wt % water are suitable for cleaning household surfaces. More preferably, the liquid hard surface cleaning composition is used with a cleaning pad having a floor sheet with a thickness of less than 1.2 mm or a cleaning wipe having a thickness of less than 1.2 mm. Such combination of cleaning composition and cleaning pad or cleaning wipe provide improved shine, increased absorbency and faster drying. For general cleaning, especially of floors, a preferred method of cleaning comprises the steps of: wetting a hard surface with a cleaning composition and removing the cleaning composition from the hard surface by wiping the hard surface with a cleaning pad or cleaning wipe of the present disclosure. The step of wetting the hard surface may involve spraying the hard surface with a liquid hard surface cleaning composition or contacting the hard surface with a pre-moistened wipe or cleaning pad to wet the hard surface. A cleaning implement comprising a pre-moistened or dry cleaning pad or cleaning wipe may also be used to wet and/or remove the cleaning composition from the hard surface. Test Methods: A) Shine Test for Floor Cleaning: The shine test is done with soil mixture which consists of a mixture of consumer relevant soils such as oil, particulates, pet hair, sugar etc. The dark colored engineered hardwood flooring is soiled with the soil mixture and cleaned with the liquid hard surface cleaning composition(s) and a cleaning pad is wiped up and down for a total of six (6) times to cover the entire flooring, after letting them dry, results are analyzed by using grading scale described below. and PSU Scale Versus a ReferenceGrading in absolute scale:(average of 3 graders):0 = as new/no streaks and/or film0 = I see no difference1 = very slight streaks and/or film1 = I think there is difference2 = slight streaks and/or film2 = I am sure there is a slight difference3 = slight to moderate streaks and/or film3 = I am sure there is a difference4 = moderate streaks and/or film4 = I am sure there is a big difference5 = moderate/heavy streaks and/or film6 = heavy streaks and/or film B) Fluid Hysteresis—Advancing minus Receding Contact Angle A contact angle goniometer is used to measure the hysteresis of the fluid. The method described herein below is derived from ASTM D5946-09. The apparatus for measuring hysteresis and advancing and receding contact angle has: (1) a liquid dispenser capable of suspending a sessile drop, as specified, from the tip of the dispenser, (2) a sample holder that allows a sample to lay flat without unintended wrinkles or distortions, and hold the sample so that the surface being measured is horizontal, (3) provision for bringing the sample and suspended droplet towards each other in a controlled manner to accomplish droplet transfer onto the test surface, and (4) means for capturing a profile image of the drop with minimal distortion. A 5-degree lookdown angle is used, so that the line of sight is raised 5 degrees from the horizontal and the baseline of the drop is clearly visible when in contact with the sample. The apparatus has means for direct angle measurements, such as image analysis of the drop dimensions and position on the sample. A FTÅ200 dynamic contact angle video system analyzer manufactured by First Ten Angstroms, Portsmouth, VA has been found suitable. FTÅ software supplied by First Ten Angstroms (Build 362, Version 2.1) has been found suitable. Lighting is adjusted so a clear image is resolvable by the software, to extract the baseline and droplet contour without user input. The test liquid shall be kept in clean containers. The substrate used for this testing is an engineered, interlocking tongue and groove planked, hardwood floor with aluminum oxide polyurethane coating. The floor has a contact angle measured with deionized water of 100 degrees+/−15 degrees and has a 60 degrees gloss reading of 85+/−5 Gloss Units. A Home Legend Santos Mahogany Engineered Hardwood floor, UPC 664646301473, has been found suitable. The area of test sample (i.e., floor sheet or smoothing strip) is sufficient to prevent spreading of the test drop to the edge of the sample being tested or drops from contacting each other. The test surface is not directly touched during preparation or testing, to avoid finger contamination. The glossy surface of the floor material is carefully cleaned using an 80/20 deionized water/isopropyl alcohol solution prior to use in any test. The temperature and humidity of the lab must be controlled to 25° C.±2° C. temperature and 40±5% humidity. Temperature and humidity is recorded during the measurement process. The wooden flooring substrate is placed onto the specimen holder of the instrument ensuring that the substrate is lying flat and its glossy surface is facing upwards toward the test fluid droplet. A single droplet of 6.5+/−1.5 μL of the test fluid is suspended at the end of a 27 gauge syringe needle. The mounted substrate sample is brought upward until it touches the pendant drop. Droplets should not be dropped or squirted onto the substrate surface. The needle is lowered into the drop until it is at least 0.5 mm from surface. Images of the profile of the drop are collected at a rate of at least 20 images/s. The test fluid is slowly pumped at a rate of 1 μL/s until 10 μL has been added to the drop. This is the advancing contact angle portion of the test. After waiting 15 seconds, the direction of fluid flow in the syringe is reversed in order to slowly remove test fluid from the droplet on the surface of the sample at −1 μL/s until 10 μL has been removed. This is the receding contact angle portion of the measurement. The flooring substrate is moved, in order to place the next droplet of the test fluid onto a clean, undisturbed area of the substrate, preferably at least 25 mm away from any previous measurements. A total of five contact angle measurements from the advancing and receding portion of the test are taken on the substrate sample using the same test fluid. The advancing contact angle is extracted from the video immediately after the diameter of the drop expands as test fluid is pumped to the surface by addition through the needle. The drop may glide across the surface. Averaging values during this gliding portion would constitute an advancing contact angle as long as the diameter of the drop is expanding. Test fluid must be added to the drop at 1 μL/s until the diameter increases. Immediately after the expansion in diameter, the contact angle is obtained as an advancing contact angle. The advancing contact angle of the test fluid is reported as the average advancing contact angle of the five measurements. The receding contact angle is extracted from the video immediately after the diameter of the drop contracts as test fluid is removed from the surface through the needle. Averaging values during this gliding portion would constitute a receding contact angle as long as the diameter of the drop is contracting. Test fluid must be removed from the drop at 1 μL/s until the diameter decreases. Immediately after the contraction in diameter, the contact angle is obtained as a receding contact angle. The hysteresis in calculated from the advancing contact angle minus the receding contact angle and it is reported as the average hysteresis of the five measurements. Hysteresis that is less than at least 30, more preferably less than at least 20, and most preferably less than 10 is consumer acceptable. Without being bound to theory, the hysteresis of the fluid corelates to the size or appearance of streaks or water marks of the left behind fluid on the cleaned surface. Hysteresis below 10 are hardly noticeable by the consumer's naked eyes and are deemed as having high level of shine and cleanness. C) pH Measurement: The pH is measured on the neat composition, at 25° C., using a Sartarius PT-10P pH meter with gel-filled probe (such as the Toledo probe, part number 52 000 100), calibrated according to the instructions manual. D) Measurement of Quaternary Compound Express Level: The quantification of express level of the Quaternary Compound is done by High performance liquid chromatography-Charged Aerosol Detector (HPLC-CAD) using an external calibration curve determined at known concentration of quaternary compound. Premoistened pads aged for two weeks are expressed and their solutions analysed by HPLC-CAD. The signal from the HPLC-CAD is compare with the calibration curve to determine the expressed or free unbound concentration of the Quaternary Compound released from the pre-moistened wipe. EXAMPLES TABLE 1Ex 4Ex 5Wt %Wt %DiethyleneEthyleneEx 1Ex 2Ex 3glycolglycolEx 6Ex 7Ex 8Ex 9Wt %Wt %Wt %monohexylmonohexylWt %Wt %Wt %Wt %TripropylenePropyleneDipropyleneetheretherEthylenePropyleneDipropyleneTripropyleneglycolGlycolglycol(Hexyl(HexylglycolglycolglycolglycolmethylN-butylN-butylCARBITCELLOSphenylphenylphenylphenyletheretherEtherOL ™OLVE ™etheretheretheretherSolvent(TPM)(PnB)(DPnB)Solvent)Solvent)(EPH)(PPH)DiPPh(TPPH)Surface3027.528.429.227.74238.137.739Tension(dyne/cm)HLB8.16.96.86.76.46.45.95.85.5pH6.56.56.56.56.56.56.56.56.5Minorsto 100%to 100%to 100%to 100%to 100%to 100%to 100%to 100%to 100%andWaterShine32221.510.50.53.5Result(absolute)Shine−1.50−0.50−0.50−0.50Reference0.501.001.00−2.00Result(PSU)CA35252530302081030Hysteresis(Adv-Rec) As shown in Table 1, compositions having 0.05 wt. of amine oxide, 0.01 wt. of polymer Mirapol 300, 0.02 wt. % of Uniquat 2250, 0.5 wt. % of a Propylene glycol n-butyl ether, 0.03% of perfume, 98.5 wt. % water, by weight of the overall composition with 0.4% of the preferred HLB solvents, provide consumer acceptable hysteresis, streaks and shine as compared to composition using solvents outside of the preferred HLB range. TABLE 2Ex 10Ex 11Ex 12Ex 13Ex 14Ex 15Ex 16Ex 17Wt %Wt %Wt %Wt %Wt %Wt %Wt %Wt %Solvent PPH0.90.90.90.90.90.90.90.9Surfactant0.050.050.050.050.050.050.050.05Amine OxideMEA0.00.030.0150.010.0050.00.00.0Citric Acid0.00.00.00.00.00.030.090.15pH6.5109.59.08.04.54.03.5Minorsto 100%to 100%to 100%to 100%to 100%to 100%to 100%to 100%andWaterShine0.52.51.01.00.50.50.50.5Result(absolute)ShineReference−2.00−0.50−0.500.00.00.00.0Result(PSU)Contact83015108558AngleHysteresis(Adv-Rec) As shown in Table 2, compositions with 0.9 of the preferred solvent Dipropylene glycol phenyl ether having 0.05 wt. of amine oxide, 0.01 wt. of polymer Mirapol 300, 0.02 wt. % of Uniquat 2250, 0.5 wt. % of a Propylene glycol n-butyl ether, 0.03% of perfume, 98.5 wt. % water, by weight of the overall composition, provide consumer acceptable hysteresis, streaks and shine when used in a pH range of 3.5 to 9.5 as compared to composition at pH higher than 10. TABLE 3Ex 18Ex 19Ex 20Ex 21Ex 22Ex 23Ex 24Ex 25Ex 26Wt %Wt %Wt %Wt %Wt %Wt %Wt %Wt %Wt %Solvent PPH0.40.40.40.40.40.40.40.40.4SurfactantAmine Oxide0.050.050.050.050.050.050.050.050.05Uniquat 22500.020.0700.0650.0730.1000.1250.1400.2200.280gsm of Pulp1480.080111111136148148148in padpH5.05.05.05.05.05.05.05.05.0Minorsto 100%to 100%to 100%to 100%to 100%to 100%to 100%to 100%to 100%andWaterUniquat 22505670200220300320300470650Express Level(PPM)MicroFailPassPassPassPassPassPassPassPassEfficacyShine0.52.51.00.51.00.50.51.02.5Result(absolute)ShineReference−2.00−0.500.0−0.500.00.0−0.50−2.00Result(PSU)CA84012812581530Hysteresis(Adv-Rec) As shown in Table 3, pre-moistened wipes with 0.4 of the preferred solvent Dipropylene glycol phenyl ether and Uniquat 2250 between 700 to 2200 ppm, having 0.05 wt. of amine oxide, 0.02 wt. of polymer Mirapol 300, 0.5 wt. % of a Propylene glycol n-butyl ether, and 0.03% of perfume, by weight of the overall composition, provide consumer acceptable hysteresis, resulting in improved streaks and shine in addition to micro efficacy benefits when used in a premoistened pad containing cellulose between 80 to 148 gsm over a pure synthetic pad or non-cellulose containing pad when the expressed or release level of Uniquat is between 200 and 600 ppm. The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm”. Every document cited herein, including any cross referenced or related patent or application and any patent application or patent to which this application claims priority or benefit thereof, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern. While particular embodiments of the present disclosure have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention. | 81,115 |
11859159 | DETAILED DESCRIPTION OF THE INVENTION The compositions of the present invention may comprise: polyethylene glycol; free perfume and/or perfume microcapsules; and optionally a dye. In one embodiment, the composition is essentially free of detergent surfactants and/or fabric softening actives. Polyethylene Glycol (PEG) Polyethylene glycol (PEG) has a relatively low cost, may be formed into many different shapes and sizes, minimizes free perfume diffusion, and dissolves well in water. PEG comes in various molecular weights. A suitable molecular weight range of PEG for the purposes of freshening laundry includes from about 3,000 to about 13,000, from about 4,000 to about 12,000, alternatively from about 5,000 to about 11,000, alternatively from about 6,000 to about 10,000, alternatively from about 6,000 to about 10,000, alternatively from about 7,000 to about 9,000, alternatively combinations thereof. PEG is available from BASF, for example PLURIOL E 8000. The compositions of the present invention may comprise from about 65% to about 99% by weight of the composition of PEG. Alternatively, the composition can comprise from about % to about 91%, alternatively from about 85% to about 91%, more than about 75%, alternatively from about 70% to about 98%, alternatively from about 80% to about 95%, alternatively combinations thereof, of PEG by weight of the composition. Free Perfume The compositions of the present invention may comprise a free perfume and/or a perfume microcapsule. Perfumes are generally described in U.S. Pat. No. 7,186,680 at column 10, line 56, to column 25, line 22. In one embodiment, the composition comprises free perfume and is essentially free of perfume carriers, such as a perfume microcapsule. In yet another embodiment, the composition comprises perfume carrier materials (and perfume contained therein). Examples of perfume carrier materials are described in U.S. Pat. No. 7,186,680, column 25, line 23, to column 31, line 7. Specific examples of perfume carrier materials may include cyclodextrin and zeolites. In one embodiment, the composition comprises free (neat) perfume but is free or essentially free of a perfume carrier. In such an embodiment, the composition may comprise less than about 20%, alternatively less than about 25%, alternatively from about 9% to about 20%, alternatively from about 10% to about 18%, alternatively from about 11% to about 13%, alternatively combinations thereof, of free perfume by weight of the composition. In one embodiment, the composition consists essentially of: (a) from about 80% to about 91% by weight of the composition of polyethylene glycol, wherein the polyethylene glycol has a molecular weight from about 5,000 to about 11,000; and (b) from about 9% to about 20% by weight of the composition free perfume; wherein the composition is essentially free of a perfume carrier; and wherein the composition is shaped in a pastille having a mass from about 0.95 mg to about 2 g. In an alternative embodiment, the composition consists essentially of: (a) more than about 75% by weight of the composition of polyethylene glycol, wherein the polyethylene glycol has a molecular weight from about 5,000 to about 11,000; and (b) less than about 25% by weight of the composition free perfume; wherein the composition is essentially free of a perfume carrier; and wherein the composition is shaped in a pastille having a mass from about 0.95 mg to about 2 g. In another embodiment, the composition comprises free perfume and perfume microcapsules. In this embodiment, the composition may comprise from about 2% to about 12%, alternatively from about 1% to about 10%, alternatively from about 3% to about 8%, alternatively from about 4% to about 7%, alternatively from about 5% to about 7%, alternatively combinations thereof, of the free perfume by weight of the composition. In yet another embodiment, the composition comprises free (neat) perfume and a perfume microcapsule but is free or essentially free of other perfume carriers. Perfume Microcapsules The compositions of the present invention can comprise perfume oil encapsulated in a perfume microcapsule (PMC). The PMC can be a friable PMC. The term “PMC” and “perfume microcapsule” are used interchangeably and refers to a plurality of perfume microcapsules. Suitable perfume microcapsules and perfume nanocapsules can include: U.S. Patent Publication Nos. 2003215417 A1; 2003216488 A1; 2003158344 A1; 2003165692 A1; 2004071742 A1; 2004071746 A1; 2004072719 A1; 2004072720 A1; 2003203829 A1; 2003195133 A1; 2004087477 A1; and 20040106536 A1; U.S. Pat. Nos. 6,645,479; 6,200,949; 4,882,220; 4,917,920; 4,514,461; and 4,234,627; and U.S. Re. 32,713, and European Patent Publication EP 1393706 A1. For purposes of the present invention, the term “perfume microcapsules” or “PMC” describes both perfume microcapsules and perfume nanocapsules. The PMCs can be friable (verses, for example, moisture activated PMCs). The PMCs can be moisture activated. In one embodiment, the PMC comprises a melamine/formaldehyde shell. Encapsulated perfume and/or PMC may be obtained from Appleton, Quest International, or International Flavor & Fragrances, or other suitable source. In one embodiment, the PMC shell is coated with polymer to enhance the ability of the PMCs to adhere to fabric, as describe in U.S. Pat. Nos. 7,125,835; 7,196,049; and 7,119,057. In one embodiment, the composition comprises a PMC but is free or essentially free or free of (neat) perfume. In such an embodiment, the composition may comprise less than about %, alternatively less than about 25%, alternatively from about 9% to about 20%, alternatively from about 9% to about 15%, alternatively from about 10% to about 14%, alternatively from about 11% to about 13%, alternatively combinations thereof, of PMC (including the encapsulated perfume) by weight of the composition. In such an embodiment, the perfume encapsulated by the PMC may comprise from about 0.6% to about 4% of perfume by weight of the composition. In one embodiment, the composition consists essentially of: (a) from about 80% to about 91% by weight of the composition of polyethylene glycol, wherein the polyethylene glycol has a molecular weight from about 5,000 to about 11,000; and (b) from about 9% to about 20% by weight of the composition of a friable perfume microcapsule, wherein the perfume microcapsule comprises encapsulated perfume; wherein the composition is essentially free of free perfume; and wherein the composition is shaped in a pastille having a mass from about 0.95 mg to about 2 g. In such an embodiment, the perfume encapsulated by the PMC may comprise from about 0.6% to about 4% of perfume by weight of the composition. In another embodiment, the composition comprises PMC and free perfume. In such an embodiment, the composition may comprise from about 1% to about 10%, alternatively from about 2% to about 12%, alternatively from about 2% to about 8%, alternatively from about 3% to about 8%, alternatively from about 4% to about 7%, alternatively from about 5% to about 7%, alternatively combinations thereof, of PMC (including the encapsulated perfume) by weight of the composition. In this embodiment, the perfume encapsulated by the PMC may comprise from about 0.6% to about 4% of perfume by weight of the composition. In one embodiment, the composition may consist essentially of: (a) from about 80% to about 91% by weight of the composition of polyethylene glycol, wherein the polyethylene glycol has a molecular weight from about 5,000 to about 11,000; (b) from about 2% to about 12% by weight of the composition free perfume; and (c) from about 2% to about 12% by weight of the composition of friable perfume microcapsule, wherein the perfume microcapsule comprises encapsulated perfume; wherein the composition is shaped in a pastille having a mass from about 0.95 mg to about 2 g. In this embodiment, the perfume encapsulated by the PMC may comprise from about 0.6% to about 4% of perfume by weight of the composition. In one embodiment, the composition comprises (a) from about 80% to about 91% by weight of the composition of polyethylene glycol, wherein the polyethylene glycol has a molecular weight from about 5,000 to about 11,000; (b) from about 2% to about 12% by weight of the composition free perfume; and (c) from about 2% to about 12% by weight of the composition of a friable perfume microcapsule, wherein the perfume microcapsule comprises encapsulated perfume; wherein the composition is shaped in a pastille, each of the pastilles has a mass from about 0.95 mg to about 2 g. Such a formulation is thought to provide for a balanced scent experience to the user of the composition. With the level of polyethylene glycol between about 80% and about 91% by weight of the composition, the about 2% to about 12% by weight of the composition of free perfume can provide for a pleasant scent experience to the user upon opening of the package containing the composition and as the user pours the composition into a dosing device and transfers the composition to her washing machine. That is the user can experience the scent at an appreciably detectable level but is not overwhelmed by the scent. Similarly, the about 2% to about 12% by weight of the composition of friable perfume microcapsule can provide physical and/or chemical stability of the pastille and for a sufficient quantity of friable perfume microcapsule to deposit on a user's clothing during washing when the pastilles are applied in the wash in a unit dose. Further, it can be beneficial for the composition to consist essentially of the above ingredients at the prescribed levels as additional components might interfere with the physical and/or chemical stability of the pastilles and recognizing that other components, such as surfactants, fabric softeners, or other such ingredients, might be delivered by other mechanisms, such as the detergent or dryer added product, and there would be the potential that the user might over apply such ingredients during washing and/or drying. In yet another embodiment, the composition can comprise perfume microcapsule but is free or essentially free of other perfume carriers and/or free (neat) perfume. In yet still another embodiment, the composition may comprise a formaldehyde scavenger. In yet still another embodiment, the scent of the present composition is coordinated with scent(s) of other fabric care products (e.g., laundry detergent, fabric softener). This way, consumers who like APRIL FRESH scent, may use a pastille having an APRIL FRESH scent, thereby coordinating the scent experience of washing their laundry with their scent experience from using APRIL FRESH. The pastilles of the present invention may be sold as a product array (with laundry detergent and/or fabric softener) having coordinated scents. Dye The composition may comprise dye. The dye may include those that are typically used in laundry detergent or fabric softeners. The composition may comprises from about 0.001% to about 0.1%, alternatively from about 0.01% to about 0.02%, alternatively combinations thereof, of dye by weight of the composition. An example of a dye includes LIQUITINT BLUE BL from Millikin Chemical Free of Laundry Actives and Softeners The composition may be free of laundry active and/or fabric softener actives. To reduce costs and avoid formulation capability issues, one aspect of the invention may include compositions that are free or essentially free of laundry actives and/or fabric softener actives. In one embodiment, the composition comprises less than about 3%, alternatively less than about 2% by weight of the composition, alternatively less than about 1% by weight of the composition, alternatively less than about 0.1% by weight of the composition, alternatively are about free, of laundry actives and/or fabric softener actives (or combinations thereof). A laundry active includes: detergent surfactants, detergent builders, bleaching agents, enzymes, mixtures thereof, and the like. It is appreciated that a non-detersive level of surfactant may be used to help solubilize perfume contained in the composition. Pastilles The composition of the present invention may be formed into pastilles by those methods known in the art, including methods disclosed in U.S. Pat. Nos. 5,013,498 and 5,770,235. The composition of the present invention may be prepared in either batch or continuous mode. In batch mode, molten PEG is loaded into a mixing vessel having temperature control. PMC is then added and mixed with PEG until homogeneous. Perfume is then added to the vessel and the components are further mixed for a period of time until the entire mixture is homogeneous. In continuous mode, molten PEG is mixed with perfume and PMC in an in-line mixer such as a static mixer or a high shear mixer and the resulting homogeneous mixture is then used for pastillation. PMC and perfume can be added to PEG in any order or simultaneously and dye can be added at a step prior to pastillation. The pastilles may be formed into different shapes include tablets, pills, spheres, and the like. A pastille can have a shape selected from the group consisting of spherical, hemispherical, compressed hemispherical, lentil shaped, and oblong. Lentil shaped refers to the shape of a lentil bean. Compressed hemispherical refers to a shape corresponding to a hemisphere that is at least partially flattened such that the curvature of the curved surface is less, on average, than the curvature of a hemisphere having the same radius. A compressed hemispherical pastille can have a ratio of height to diameter of from about 0.01 to about 0.4, alternatively from about 0.1 to about alternatively from about 0.2 to about 0.3. Oblong shaped refers to a shape having a maximum dimension and a maximum secondary dimension orthogonal to the maximum dimension, wherein the ratio of maximum dimension to the maximum secondary dimension is greater than about 1.2. An oblong shape can have a ratio of maximum dimension to maximum secondary dimension greater than about 1.5. An oblong shape can have a ratio of maximum dimension to maximum secondary dimension greater than about 2. Oblong shaped particles can have a maximum dimension from about 2 mm to about 6 mm, a maximum secondary dimension of from about 2 mm to about 4 mm. In alternative embodiments of any of the formulations disclosed herein, each individual pastille can have a mass from about 0.95 mg to about 2 g, alternatively from about 10 mg to about 1 g, alternatively from about 10 mg to about 500 mg, alternatively from about 10 mg to about 250 mg, alternatively from about 0.95 mg to about 125 mg, alternatively combinations thereof. In a plurality of pastilles, individual pastilles can have a shape selected from the group consisting of spherical, hemispherical, compressed hemispherical, lentil shaped, and oblong. An individual pastille may have a volume from about 0.003 cm 3 to about 0.15 cm 3. A plurality of pastilles may collectively comprise a unit dose for dosing to a laundry washing machine or laundry was basin. A single unit dose of the pastilles may comprise from about 13 g to about 27 g, alternatively from about 14 g to about 20 g, alternatively from about 15 g to about 19 g, alternatively from about 16 g to about 18 g, alternatively combinations thereof. The individual pastilles forming the plurality of pastilles that make up the unit dose can each have a mass from about 0.95 mg to about 2 g. The plurality of pastilles can be made up of pastilles of different size, shape, and/or mass. The pastilles in a unit dose can have a maximum dimension less than about 1 centimeter. The composition may be manufactured by a pastillation process. A schematic of a pastillation apparatus100is illustrated inFIG.1. The steps of manufacturing according to such process can comprise providing the desired formulation as a viscous material50. The viscous material50can comprise or consists of any of the possible formulations disclosed herein. In one embodiment, the viscous material50comprises: (a) from about 80% to about 91% by weight of the composition of polyethylene glycol, wherein the polyethylene glycol has a molecular weight from about 5,000 to about 11,000; (b) from about 2% to about 12% by weight of the composition free perfume; and (c) from about 2% to about 12% by weight of the composition of friable perfume microcapsule, wherein the perfume microcapsule comprises encapsulated perfume. The viscous material50can be provided at a processing temperature less than about 20 degrees Celsius above the onset of solidification temperature as determined by differential scanning calorimetry. In one embodiment, the PMC can be added as a slurry to the polyethylene glycol and free perfume to form the viscous material50. The PMC can be added as a powder to the polyethylene glycol and free perfume to form the viscous material50. The viscous material50is passed through small openings10and onto a moving conveyor surface20upon which the viscous material50is cooled below the glass transition temperature to form a plurality of pastilles30. As illustrated inFIG.1, the small openings10can be on a rotatable pastillation roll Viscous material50can be distributed to the small openings10by a viscous material distributor40. Pastilles can be formed on a ROTOFORMER, available from Sandvik Materials Technology. Package A unit dose or a plurality of unit doses may be contained in a package. The package may be a bottle, bag, or other container. In one embodiment, the package is a bottle, preferably a PET bottle comprising a translucent portion to showcase the pastilles to a viewing consumer. In one embodiment, the package comprises a single unit dose (e.g., trial size sachet); or multiple unit doses (e.g., from about 15 unit doses to about 30 unit doses). Dosing The aforementioned package may comprise a dosing means for dispensing the pastilles from the package to a laundry washing machine (or laundry wash basin in hand washing applications). The user may use the dosing means to meter the recommended unit dose amount or simply use the dosing means to meter the pastilles according to the user's own scent preference. Examples of a dosing means may be a dispensing cap, dome, or the like, that is functionally attached to the package. The dosing means can be releasably detachable from the package and re-attachable to the package, such as for example, a cup mountable on the package. The dosing means may be tethered (e.g., by hinge or string) to the rest of the package (or alternatively un-tethered). The dosing means may have one or more demarcations (e.g., fill-line) to indicate a recommend unit dose amount. The packaging may include instructions instructing the user to open the removable opening of the package, and dispense (e.g., pour) the pastilles contained in the package into the dosing means. Thereafter, the user may be instructed to dose the pastilles contained in the dosing means to a laundry washing machine or laundry wash basin. The pastille of the present invention may be used to add freshness to laundry. The package including the dosing means may be made of plastic. One embodiment can be a unit dose of a fabric treatment composition comprising a plurality of pastilles, wherein each pastille comprises: (a) from about 80% to about 91% by weight of the composition of polyethylene glycol, wherein the polyethylene glycol has a molecular weight from about 5,000 to about 11,000; (b) from about 2% to about 12% by weight of the composition free perfume; and (c) from about 2% to about 12% by weight of the composition of friable perfume microcapsule, wherein the perfume microcapsule comprises encapsulated perfume; wherein each pastille has a mass from about 0.95 mg to about 2 g; and wherein the plurality of pastilles has a mass from about 13 g to about 27 g to comprise the unit dose. In one embodiment, the pastilles of the present invention can be administered to a laundry machine as used during the “wash cycle” of the washing machine (but a “rinse cycle” may also be used). In another embodiment, the pastilles of the present invention are administered in a laundry wash basin—during washing and/or rinsing laundry. In a laundry hand rinsing application, the pastille may further comprise an “antifoam agent” such as those available from Wacker. Antifoam agents (suds suppressing systems) are described in U.S. Patent Publication No. 20030060389 at 65-77. EXAMPLEGrams in a% WeightIngredient:17 g unit Doseof CompositionPEG 80001588.24%Free (neat) Perfume15.88%Perfume Microcapsule115.88%(Encapsulated(0.32)(1.88%)perfume)2Dye0.00250.015%1PMC is a friable PMC with a urea-formaldehyde shell from Appleton. About 50% water by weight of the PMC (including encapsulated perfume) is assumed.2Encapsulated perfume (within PMC) assumes about 32% active. The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm.” Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern. While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention. | 22,438 |
11859160 | DETAILED DESCRIPTION Overview This disclosure is directed to oval-shaped metal tanks that create a torus shaped vortex of fermenting wine that are relatively lightweight, easily produced, less labor intensive to clean than compared to egg-shaped concrete tanks and are less costly than egg-shaped concrete tanks. Moreover, these oval-shaped metal tanks are not susceptible to discoloring an appearance or “pinking” (e.g., causing a blush color, a red blush color, etc.) of a white wine that is made in a tank subsequent to producing a red wine in the same tank. In an embodiment, the tanks may include a cone-shaped wall formed of a steel attached to a first dome-shaped surface formed of the steel and a second dome-shaped surface formed of the steel. The cone-shaped wall attached to the first dome-shaped surface and the second dome-shaped surface define an oval-shape that is void of any angled corners on the inside surface of the tank. Because the oval-shape on the inside surface of the tank is void of any angled corners, this provides the necessary smooth arcuate egg-shaped inside surface to displace a product (e.g., wine, red wine, white wine, etc.) in a torus shaped vortex, which produces a continuous and gentle mix of the product void of any dead circulation areas during a fermentation of the product. The cone-shaped wall (e.g., tapered wall) may be narrower at the top of the cone-shaped wall relative to the bottom of the cone-shaped wall, which provides for compressing a cap (e.g., grape solids, skins, seeds, stems, etc.) throughout the fermentation of the product. Because the cone-shaped wall compresses the cap, this provides for a majority of the cap to remain submerged and in constant contact with the product. Mixing the product in the torus shaped vortex eliminates the need for any intervention by a user (e.g., winemaker, worker, etc.) to produce a complete and complex product. For example, a user may simply initiate the fermentation process, and the smooth arcuate egg-shaped inside surface causes the product to be displaced in the torus shaped vortex, but without user intervention to mix the product with lees, mix the product with yeast, etc. Stated otherwise, the torus shaped vortex may be started by a worker initiating the fermentation process in the tank that gently mixes the product such that the product is in constant gentle contact with the lees and the yeast, without intervention by a worker. The tanks may include a cooling jacket attached to the cone-shaped wall. The cooling jacket may be attached to a top half of the cone-shaped wall. The cooling jacket may be thermostatically controlled. The cooling jacket may cool the product when the product encounters an inside top half surface of the cone-shaped wall. As the cooling jacket cools the product, the product is displaced down (e.g., sinks) toward the second domed shape surface (e.g., metal bottom dome, bottom head, etc.). Because the oval-shape on the inside surface of the tank is void of any angled corners, this provides the smooth arcuate egg-shaped inside surface for the cooled product to be displaced down to the second domed shape surface without any dead circulation areas, which provides a homogeneous mixture of the product. Moreover, the exothermic reaction of the fermentation process provides for the product located at the center of the tank to remain warmer than the cooled product located at the inside surface of the tank, which provides for displacing the product back up towards the first domed shape surface (e.g., metal top dome, top head, etc.) at which point the product is again cooled by the cooling jacket to displace the product back down toward the second domed shape surface. During the fermentation of the product, the heating and cooling of the product displaces the product in the tank in the torus shaped vortex, which homogeneously mixes the product continuously and gently without any dead circulation areas to produce a complete and complex product. The tanks may further include an oxygenation system (e.g., a micro-oxygenation system, micro-ox system, a macro-oxygenation system, etc.). The oxygenation system may provide for oxygenation of the product contained in the tank. For example, the oxygenation system may be inserted in the tank which has an oxygenation stone (e.g., stainless steel O2 stone, stainless diffusion stone, micro diffusion stone, oxygen aeration stone, oxygen stone, etc.) disposed proximate to the second domed shape surface. The oxygenation system may provide for piping controlled quantities of pure oxygen (O2) into the product contained in the tank. The tanks may be formed of stainless steel. For example, one or more of the first domed shape surface, the second domed shape surface, and/or the cone-shaped wall may be formed of stainless steel. The use of stainless steel may reduce the weight of the tank so as to weigh about 80% less than similarly sized or capacity egg-shaped concrete fermentation tank. Therefore, the tank according to the instant disclosure may be more easily transported, set, and removed without specialized moving equipment required by the heavier egg-shaped concrete fermentation tanks. For example, a 1,600 liters (420 gallons) egg-shaped concrete fermentation tank weighs about 2 tons (˜1800 kilograms), whereas a 1,600 liters metal tank weighs about 800 pounds (˜360 kilograms). The elimination of the need for specialized moving equipment may significantly reduce the higher costs associated with concrete fermentation tanks. Moreover, because the tanks may be formed of stainless steel, the tanks may be easily produced in larger sizes than egg-shaped concrete fermentation tanks. For example, because of the mass, weight, and/or casting limitations of concrete, the maximum size an egg-shaped concrete fermentation tank that has been produced using existing techniques is about 3,400 liters (900 gallons). In contrast, a size of an tank may be produced greater than about 38,000 liters (˜10,000 gallons). Because the tanks may be produced in larger sizes than egg-shaped concrete fermentation tanks, the tanks provide for greater economies of scale for a user (e.g., wine maker) as compared to the egg-shaped concrete fermentation tanks. For example, the tanks provide for maximizing a yield of floor space by about 11 times more than the egg-shaped concrete fermentation tanks. Thus, a user may produce more volume of product in the same or smaller area of floor space with the tanks than a volume of product produced in the egg-shaped concrete fermentation tanks. Further, because the tanks may be formed of stainless steel, the tanks may be more easily cleaned as compared to the egg-shaped concrete fermentation tanks. For example, the tanks are easily cleaned using typical cleaning protocols involving scrubbers, metal, hot water, ozone, chlorine, strong acids, and bases, whereas the egg-shaped concrete fermentation tanks are porous, which allows microbes and bacteria to lodge into these pores, and they are susceptible to being damaged by the scrubbers, metal, hot water, ozone, chlorine, strong acids, and bases. Illustrative Oval-Shaped Metal Tank FIG.1illustrates a front, top, right-side perspective view100of an example tank102. The tank102may be a fermentation tank such as a red wine fermenter for holding a juice, for example. The tank102may be an oval-shaped metal tank. In an embodiment, the tank102may include a cone-shaped wall104formed of a steel sheet attached between a first dome-shaped surface106formed of steel and a second dome-shaped surface108formed of steel. In one possible implementation, a size of the cone-shaped wall may have a height of about 66 inches. The first dome-shaped surface106may be a metal top dome (e.g., a top head) of the tank102. The second dome-shaped surface108may be a metal bottom dome (e.g., a bottom head) of the tank102. The cone-shaped wall104may include a first wall portion110formed of the steel and attached to a second wall portion112formed of the steel. For example, the first wall portion110may be welded (e.g., seam welded) to the second wall portion112. The first wall portion110attached to the second wall portion112may define a seam114having an elliptical shape. The elliptical shape of the seam114may circumnavigate the cone-shaped wall104convolutely (e.g., twisted, coiled, etc.) along a longitudinal length of the cone-shaped wall104. Additionally, the first wall portion110may be attached to the second wall portion112such that the seam114is void of angled corners, steps, and/or flats on the inside surface of the tank102. The first wall portion110has a top perimeter116and the second wall portion has a bottom perimeter118opposite the top perimeter116. The top perimeter116of the first wall portion110may be attached to a perimeter of the first dome-shaped surface106. For example, the top perimeter116of the first wall portion110may be welded (e.g., seam welded) to the perimeter of the first dome-shaped surface106. The top perimeter116of the first wall portion110may be attached to the perimeter of the first dome-shaped surface106such that the attachment is void of angled corners, steps, and/or flats on the inside surface of the tank102. The bottom perimeter118of the second wall portion112may be attached to a perimeter of the second dome-shaped surface108. For example, the bottom perimeter118of the second wall portion112may be welded (e.g., seam welded) to the perimeter of the second dome-shaped surface108. The bottom perimeter118of the second wall portion112may be attached to the perimeter of the second dome-shaped surface108such that the attachment is void of angled corners, steps, and/or flats on the inside surface of the tank102. In this way, an inside surface of the tank102has an oval-shape (e.g., egg shape) void of angled corners on the inside surface of tank102such that when a product contained in the tank102is displaced, the product is displaced in a torus shaped vortex between the first dome-shaped surface106and the second dome-shaped surface108. In an embodiment, the tank102may include a cooling jacket120attached to the cone-shaped wall104. For example, the cooling jacket120may be attached to a top half of the cone-shaped wall104. In another example, the cooling jacket120may be attached to the first wall portion110of the cone-shaped wall104. The tank102may further include fitting(s)122. One or more of the fittings122may be an oxygenation port. The oxygenation port may receive at least a portion of an oxygenation system (e.g., a micro-oxygenation system, micro-ox system, a macro-oxygenation system, etc.) (not shown). For example, an oxygenation system may be inserted into the tank102via the fitting122such that an oxygenation stone (e.g., stainless steel O2 stone, stainless diffusion stone, micro diffusion stone, oxygen aeration stone, oxygen stone, etc.) (not shown) may be disposed proximate to the second domed-shaped surface108. In one example, the fitting122may be disposed in the first dome-shaped surface106(not depicted). In another example, the fitting122may be disposed in a manway assembly124attached to the first dome-shaped surface106(depicted inFIG.1). The tank102may include a manway assembly126attached to the second dome-shaped surface108. FIG.2illustrates a back, top, left-side perspective view200of the tank102shown inFIG.1.FIG.2illustrates the cone-shaped wall104of the tank102may include a seam202. For example, a first vertical edge of the cone-shaped wall104may be attached to a second vertical edge of the cone-shaped wall104. For example, the first vertical edge of the cone-shaped wall104may be welded (e.g., seam welded) to the second vertical edge of the cone-shaped wall104. The seam202of the cone-shaped wall104may extend rectilinearly along a longitudinal length of the cone-shaped wall104. The cooling jacket120may have the same cone shape as the cone-shaped wall104to provide for interfacing with the outside surface of the cone-shaped wall104. For example, the top perimeter116of the cone-shaped wall104may be narrower relative to the bottom perimeter118of the cone-shaped wall104, and the cooling jacket120may have a cone shape (e.g., tapered shape) having a narrower top perimeter relative to a bottom perimeter that are equal to the top perimeter116and bottom perimeter118of the cone-shaped wall104to fit on the cone shape of the exterior surface of the cone-shaped wall104. The cooling jacket120may include one or more ports204(only two are depicted). The one or more ports204may provide for a coolant (e.g., glycol coolant) to be pumped through the cooling jacket120. One of the one or more ports204may be an “in” port and one of the one or more ports204may be an “out” port located in the cooling jacket120to maximize a flow rate of the coolant through the cooling jacket120. The flow rate be about 5 gallons per minute (gpm) at about 50 pounds per square inch (psi). The cooling jacket120may be a resistance spot-welded dimpled jacket attached to the outside surface of the cone-shaped wall104. The cooling jacket120may have a gap of about 0.08 inches between the outside surface of the cone-shaped wall104and the inside surface of the cooling jacket120facing the outside surface of the cone-shaped wall104. For example, the cooling jacket120may be pillowed (e.g., inflated) to provide a gap of about 0.08 inches between the outside surface of the cone-shaped wall104and the inside surface of the cooling jacket120facing the outside surface of the cone-shaped wall104. The coolant may be pumped through the cooling jacket120(e.g., through the gap between the outside surface of the cone-shaped wall104and the inside surface of the cooling jacket120) via a refrigeration system. For example, the coolant may be pumped through the cooling jacket120via a central refrigeration system of a winery. The temperature of the cooling jacket120may be controlled via a tank monitoring system. The temperature of the cooling jacket120may be determined by a winemaker, which may be dependent upon a type of grape, a type of yeast, and/or a type of wine being produced. In one implementation, a size of the tank102may have a minimum outside diameter of about 48 inches and a maximum diameter of about 64 inches. For example, in an implementation, the top perimeter116of tank102may have a minimum outside diameter of about 48 inches and the bottom perimeter118of the tank102may have a maximum diameter of about 64 inches. A tank having components with the dimensions described herein may have a volume of about 950 gallons. While the specification describes a tank having a minimum outside diameter of about 48 inches, a maximum diameter of about 64 inches, and a volume of about 950 gallons, it is contemplated that the tank may be of any size and or shape. In an alternative implementation, a size of the tank102may have a minimum diameter smaller than 48 inches, a maximum diameter smaller than 64 inches, and a volume less than 950 gallons. In this example, where the tank102has a minimum diameter smaller than 48 inches, a maximum diameter smaller than 64 inches, and a volume less than 950 gallons, the cone-shaped wall104may not include both of the first wall portion110and the second wall portion112. That is, in view of capabilities and/or limitations of standard manufactured sizes of stainless steel sheets, the cone-shaped wall104may with only a first wall portion110to form a tank having the volume less than 950 gallons. In contrast, as indicated above, in an example where the minimum diameter is larger than 48 inches, the maximum diameter larger is than 64 inches, and the volume desired is greater than 950 gallons, the cone-shaped wall104may include one or more additional wall portions attached to the first wall portion110and/or the second wall portion112. For example, because the tank102has a minimum diameter larger than 48 inches, a maximum diameter larger than 64 inches, and a volume greater than 950 gallons, the cone-shaped wall104may require one or more additional wall portions welded (e.g., seam welded) to the first wall portion110and/or the second wall portion112to form a tank having the volume greater than 950 gallons. The minimum diameter and the maximum diameter of the tank may depend on a desired volume of the tank102, and the quantity of wall portions may depend on a desired volume of the tank. The tank102having the volume of about 950 gallons may have a height of about 94 inches from the top outside surface of the first dome-shaped surface106to a bottom outside surface of the second dome-shaped surface108. Notably, the tank102may have any height. The tank102may include support legs206. For example, the tank102may include legs and/or bracing welded to the tank102. A height of the tank102may be adjusted via the legs206. In an implementation, a size of the tank102having the volume of about 950 gallons may have an overall height of about 142 inches. FIG.3illustrates a transparent view300of the tank102shown inFIG.1to depict a product302being displaced in a torus shaped vortex304within the tank102.FIG.3illustrates the first dome-shaped portion106, the cone-shaped wall104, and the cooling jacket120as being transparent to show displacement of the product302. As the cooling jacket120attached to the cone-shaped wall104cools the product302encountering an inside top half surface306of the cone-shaped wall104, the product302is displaced in a direction308down toward the second dome-shaped surface108. As the exothermic reaction of the fermentation process of the product302releases heat, the product302located at a center310of the tank102is displaced in a direction312back up towards the first domed-shape surface106. Thus, the exothermic reaction heating the product302and the cooling jacket120cooling the product302displaces the product302in the tank102in the torus shaped vortex304, and homogeneously mixes the product302continuously and gently without any dead circulation areas to produce a complete and complex product302. Because the first dome-shaped portion106of the cone-shaped wall104is narrower than the second dome-shaped portion108of the cone-shaped wall104, a cap314of grape solids, skins, seeds, stems, etc. may be compressed and remain submerged in the product302, such that the cap314is in constant contact with the product302throughout the fermentation of the product302. FIG.4illustrates a planar view400of an example first wall portion402of the tank102shown inFIG.1. The first wall portion402may be the same as the first wall portion110shown inFIGS.1and2. The first wall portion402may be cut from a coil stock. For example, the first wall portion402may be cut from a coil stock of 12 gauge (GA), steel (e.g., A240-T304 stainless steel (SS), #4 finish)). The coil stock may have a width of about 60 inches. For example, the standard coil stock width (e.g., largest width) available for purchase (e.g., off-the-shelf) may be 60 inches. The first wall portion402may have a bottom length404of about 194 inches. The first wall portion402may have a top length406of about 147 inches (i.e., straight line distance between point A and point B). The first wall portion402may have a width408of about 59 inches. The first wall portion402may have a grain direction410that is parallel to the bottom length404and/or the top length406. The grain direction410of the first wall portion402may be the same as a grain direction of the 12 GA coil stock, where the grain direction of the 12 GA coil stock is in a direction of a roll of the 12 GA coil stock perpendicular to the width of the 12 GA coil stock. FIG.5illustrates a planar view500of an example second wall portion502of the tank102shown inFIG.1. The second wall portion502may be the same as the second wall portion112shown inFIGS.1and2. The second wall portion502may be cut from a coil stock. For example, the second wall portion502may be cut from a coil stock of 12 gauge (GA), steel (e.g., A240-T304 stainless steel (SS), #4 finish). The second wall portion502may have a bottom length504of about 196 inches (i.e., straight line distance between point C and point D). The second wall portion502may have a top length506of about 194 inches. The second wall portion502may have a width508of about 22 inches. The second wall portion502may have a grain direction510that is parallel to the bottom length504and/or the top length506. The grain direction510of the second wall portion502may be the same as the grain direction410of the first wall portion402. FIG.6illustrates a planar view600of an example cooling jacket602of the tank102shown inFIG.1. The cooling jacket602may be the same as the cooling jacket120shown inFIGS.1and2. The cooling jacket602may be cut from a coil stock. For example, the cooling jacket602may be cut from a coil stock of 20 gauge (GA), steel (e.g., A240-T304 stainless steel (SS), #4 finish). The cooling jacket602may have a bottom length604of about 170 inches. The cooling jacket602may have a width606of about 46 inches. The cooling jacket602may have a grain direction608that is parallel to the bottom length604. The grain direction608of the cooling jacket602may be the same as a grain direction of the 20 GA coil stock, where the grain direction of the 20 GA coil stock is in a direction of a roll of the 20 GA coil stock perpendicular to the width of the 20 GA coil stock. The cooling jacket602may have a width610of about 33 inches. The cooling jacket602may have a radius of about 469 inches. FIG.7illustrates a top view700of an example cone-shaped wall assembly702including the first wall portion402, the second wall portion502, and the cooling jacket602shown inFIGS.4,5, and6. The cone-shaped wall assembly702may be the same as the cone-shaped wall104shown inFIGS.1,2, and3. The bottom length404of the first wall portion402may be attached to the top length506of the second wall portion502. For example, the bottom length404of the first wall portion402may be seam welded to the top length506of the second wall portion502. The cooling jacket602may be attached to a top surface704of the first wall portion402. For example, the cooling jacket602may be resistance spot welded to the top surface704of the first wall portion402. FIG.8illustrates a side view800of an example first dome-shaped surface802shown inFIG.1. The first dome-shaped surface802may be the same as the first dome-shaped surface106shown inFIGS.1,2, and3. In an implementation, a possible size of the first dome-shaped surface802may have an outside diameter804of about 48 inches. The first dome-shaped surface802may have a height806of about 12 inches. The first dome-shaped surface802may have knuckle radius808of about 8 inches. The first dome-shaped surface802may have crown radius810of about 42 inches. The first dome-shaped surface802may be formed of a steel (e.g., A240-T304 stainless steel (SS)). FIG.9illustrates a side view900of an example second dome-shaped surface shown inFIG.1. The second dome-shaped surface902may be the same as the second dome-shaped surface108shown inFIGS.1,2, and3. The second dome-shaped surface902may have an outside diameter904of about 64 inches. The second dome-shaped surface902may have a height906of about 17 inches. The second dome-shaped surface902may have knuckle radius908of about 12 inches. The second dome-shaped surface902may have crown radius910of about 56 inches. The second dome-shaped surface902may be formed of a steel (e.g., A240-T304 stainless steel (SS)). Example Method of Making an Oval-Shaped Metal Tank FIGS.10A-Bcollectively illustrate an example method1000of making an example oval-shaped metal (e.g., tank102) that, when completed, has an oval shape on an inside surface of the tank that is void of any angled corners. The oval-shaped inside surface provides the smooth arcuate egg-shaped inside surface to displace a product (e.g., product302) in a torus shaped vortex (e.g., torus shaped vortex304) that continuously and gently mixes the product void of any dead circulation areas during a fermentation of the product. Method1000may include an operation1002, which represents measuring a circumference of a first dome-shaped surface (e.g., first dome-shaped surface106or802) formed of steel and measuring a circumference of a second dome-shaped surface (e.g., second dome-shaped surface108or902) formed of steel. For example, operation1002may include measuring a circumference of a first dome-shaped surface and a circumference of a second dome-shaped surface that may have been provided by a manufacture (e.g., third party manufacture) of heads for tanks. Method1000may proceed to operation1004, which represents cutting coil stock to produce a first wall portion (e.g., first wall portion110or402) and a second wall portion (e.g., second wall portion112or502) of the tank. For example, the first wall portion and/or the second wall portion may be laser cut, water jet cut, etc. from a 12-gauge (GA) coil stock of steel (e.g., A240-T304 stainless steel (SS), #4 finish). The first wall portion may be cut from the coil stock based at least in part on the measurement of the circumference of the first dome-shaped surface. For example, a top length (e.g., top length406) of the first wall portion may be cut from the coil stock to match the circumference of the first dome-shaped surface. The second wall portion may be cut from the coil stock based at least in part on the measurement of the circumference of the second dome-shaped surface. For example, a bottom length (e.g., bottom length504) of the second wall portion may be cut from the coil stock to match the circumference of the second dome-shaped surface. Operation1004may include cutting a bottom length (e.g., bottom length404) of the first wall portion and cutting a top length (e.g., top length506) of the second wall portion from the coil stock such that the bottom length of the first wall portion matches the top length of the second wall portion. Method1000may include operation1006, which represents attaching the first wall portion to the second wall portion. For example, the bottom length of the first wall portion may be attached to the top length of the second wall portion. For example, the bottom length of the first wall portion may be seam welded to the top length of the second wall portion. Method1000may include operation1008, which represents cutting coil stock to produce a cooling jacket (e.g., cooling jacket120, cooling jacket602). For example, the cooling jacket may be laser cut, water jet cut, etc. from a 12-gauge (GA) coil stock of steel (e.g., A240-T304 stainless steel (SS), #4 finish). The cooling jacket may be cut from the coil stock based at least in part on a cone shape (e.g., tapered shape) of the cone-shaped wall (e.g., cone-shaped wall104). For example, the cooling jacket may be cut from the coil stock to have a narrower top perimeter relative to a bottom perimeter that are equal to a top perimeter (e.g., top perimeter116) and bottom perimeter (e.g., bottom perimeter118) of the cone-shaped wall to fit on the cone shape of the exterior surface of the cone-shaped wall. Method1000may include operation1010, which represents attaching the cooling jacket to the first wall portion. For example, the cooling jacket may be resistance spot welded to the first wall portion. The first wall portion attached to the second wall portion, and the cooling jacket attached to the first wall portion defining a cone-shaped wall assembly (e.g., cone-shaped wall assembly702). Method1000may be include operation1012, which represents rolling the cone-shaped wall assembly into a cone shape. For example, the cone-shaped wall assembly may have a substantially planar cross-sectional profile subsequent to the assembly of the cone-shaped assembly, and the planar cone-shaped assembly may be rolled via a needle roller bearing to impart a desired cone shape to the cone-shaped assembly. FIG.2Bcontinues the illustration of the method1000, which may include operation1014, which represent attaching a first vertical edge of the cone-shaped wall to a second vertical edge of the cone-shaped wall. For example, the first vertical edge of the cone-shaped wall may be welded (e.g., tack welded) to the second vertical edge of the cone-shaped wall. The first vertical edge attached to the second vertical edge defining a seam (e.g., seam202) of the cone-shaped wall. Method1000may include operation1016, which represents attaching the first dome-shaped surface and the second dome-shaped surface to the cone-shaped wall. For example, a perimeter (e.g., circumference) of the first dome-shaped surface may be fitted and tack welded to a top perimeter (e.g., top perimeter116) of the first wall portion of the cone-shaped wall, and a perimeter (e.g., circumference) of the second dome-shaped surface may be fitted and tack welded to a bottom perimeter (e.g., bottom perimeter118) of the second wall portion of the cone-shaped wall. Method1000may include operation1018, which represents finish welding the attachments, interfaces, seams, etc. between the first dome-shaped surface, the cone-shaped wall, and the second dome-shaped surface. For example, the first vertical edge of the cone shaped wall may be finished welded to the second vertical edge of the cone-shaped wall, the perimeter of the first dome-shaped surface may be finished welded to the top perimeter of the first wall portion, and the perimeter of the second dome-shape surface may be finished welded to the bottom perimeter of the second wall portion. Operation1018may also represent finish welding the cooling jacket to the first wall portion. Method1000may include operation1020, which represents pillowing the cooling jacket. For example, the cooling jacket that is finish welded to the first wall portion may be inflated to provide about a 0.08-inch gap between the outside surface of the first wall portion and the inside surface of the cooling jacket120facing the outside of the first wall portion. Method1000may include operation1022, which represents attach fittings (e.g., fitting(s)122), manways (e.g., manway assemblies124and126), legs and/or bracing to the tank. For example, fittings, manways, legs, and/or bracing may be welded to the tank. Method1000may be complete at operation1024, which represents testing the tank. For example, the tank may be leak tested, pressure tested, corrosion tested, etc. CONCLUSION Although the invention has been described in language specific to structural features and/or methodological acts, it is to be understood that the invention is not necessarily limited to the specific features or acts described. Rather, the specific features and acts are disclosed as illustrative forms of implementing the invention. For example, while embodiments are described having certain shapes, sizes, and configurations, these shapes, sizes, and configurations are merely illustrative. | 30,937 |
11859161 | DESCRIPTION Certain details of scale-up processes and the complications experienced by workers in this field are described in, for example,Physiological Similarity and Bioreactor Scale-up, Votruba et al., Folia Microbiology, vol 37 (5), p. 331, 1992, Shake Flask, to Fermentor: What Have We Learned, Humphrey, Biotechnology Progress, vol. 14, p. 3, 1998, and Living With Heterogeneities in Bioreactors, Lara et al., Molecular Biotechnology, vol. 24, p. 355, 2006, each incorporated herein by reference in its entirety. It has been suggested by many authors and summarized by Ju (Improved scale-up strategies of bioreactors, Bioprocess Engineering vol. 8, p. 49, 1992 all information incorporated herein) that in order to effectively scale-up a bioprocess the following process characteristics would be maintained as constant:1. Reactor geometry;2. The volumetric oxygen transfer coefficient kLa (m/s m2/m3);3. Maximum shear experienced by cells (kg/m2s);4. Power input per unit volume of liquid P(kW)/V (m3);5. Volumetric gas flow rate per unit volume of liquid Q (m3/s)/V(m3)6. Superficial gas velocity v (m/s)7. Mixing time (s)8. Impeller Reynolds Number Rei=fluid density×impeller speed×(density Impeller tip speed)2/kinematic viscosity (dimensionless quantity)9. Momentum factor (cm3/s) Item 1 refers to the actual shape and aspect ratio (height vs. width) of the bioreactor. This has a large effect on how the bioreactor functions and the extent to which it can be only partially filled (e.g.: the bioreactor's operational range in fill volume or turn-down ratio). Additional parameters related to the bioreactor shape and size are the impeller designs and ratio of their diameters to the diameter of the bioreactor. Item 2 refers to the rate at which oxygen can be transferred to the media—typically from sparged oxygen or air. In general, there is a tradeoff between making the bubbles smaller (increased surface area to volume ratio with decreased size due to their spherical shape) and foaming that occurs from the surface tension of the high number density of breaking bubbles. Item 3 and Item 4 are related to the shear force that will actually damage a cell. Mammalian cells are far more susceptible to this type of damage than bacterial cells. Shear forces are induced by many things including the surface tension of a bubble bursting or from a vortex formed at the tip of the impeller (Animal Cell Culture in Stirred Bioreactors: Observations on Scale-up, Y. Christi, Process Biochemistry, vol. 28, 0. 511, 1992). Items 5 and 6, like item 2 are related to the ability to transfer gas into or out of the media. Item 7, mixing time, refers to the time it takes a bioreactor to reach a set degree of uniformity in concentration of one or more monitored analytes or cell density. The set degree can differ in the literature, but a typical definition is that the concentration differs by less than 10% at any point in the bioreactor. The mixing time can also be defined as the time it takes for a parameter (or tracer) to be homogeneously distributed within the bioreactor to the level required for a successful process. (Modeling of mixing times for stirred bioreactors.3. Mixing time for aerated broths, D. Cascaval et al, Chem. Ind. Vol. 56 (12), p 506, 2002) incorporated herein by reference in its entirety. Unless otherwise stated herein, this last definition will be used herein. Item 8, the impeller Reynolds number is inversely related to the mixing time of the bioreactor. Generally speaking, the higher Reynolds number, the higher the turbulence but the shorter the mixing time. The cost for shorter mixing times is higher shear; this is another tradeoff that must be balanced for the exact system under study. Item 9, apparently less frequently used in the literature, is the momentum factor and is related to both shear and mass transfer in the bioreactor. The number of process parameters and combination of process parameters from the above list that are desired to be held constant during scaling changes based on a large number of considerations. These considerations include but are likely not limited to: the cell line chosen, the type or product being produced, the yield required from the product, the type of bioreactor used, the details of the process implemented, and the degree of scale-up desired. It can be gleaned from the brief review of the aforementioned literature that scale-up is not generally a simple process and has occupied the time and efforts of many skilled workers in the field of bioproces sing for many years. For the process to maintain a consistent yield and be clinically and economically viable, the cells/process must minimize, or at worst maintain, bioreactor heterogeneity of the above parameters (and sometimes related parameters) during the scale-up process. As mentioned above, this is to date a significant challenge even for those skilled in this art. This often results in lower titer or cell density for the end of a seed train than for the small scale bioreactors in the laboratory. Ideally then, a system by which the volume can be scaled up without changing the bioreactor scale would yield optimal results and allow straightforward changes from R&D through production. This is in contrast with scale-up and scale-down today where most of the optimization work is performed to match the titer and productivity of small glass vessels (<15 L) at the production volumes envisaged (biocompare.com/Editorial-Articles/165203-Scaling-Up-Your-Cell-cultures-to-Bioreactors/), and where scalability inconsistencies are still being observed in both titer and product quality as a result of the different bioreactor volumes [J. Li, G. Zhang, et al. “Challenges of scale down model for disposable bioreactors: case studies on growth & product quality impacts”, Single-Use Technologies, Leesburg, VA, Oct. 18-21, 2015 (incorporated herein by reference in its entirety) or infors-ht.com/index.php/en/applications/scale-up]. Similarly, the performance of new large-scale bioreactors is always compared to running the same process in a glass vessel (Pall Application Note: USD2926, “Cultivation of CHO Cells in Allegro™ STR 200 Single-Use Stirred Tank Bioreactor System,” incorporated herein by reference in its entirety or biopharminternational.com/evaluation-single-use-bioreactor-fed-batch-production-monoclonal-antibody). The embodiments described here address many of these challenges. Current conventional (e.g., stirred tank) bioreactor design at this time can practically speaking allow scalability/turn-down ratio of up to a factor of 5 [GE Healthcare Life Sciences Data file 29-0929-25 AA “Xcellerex™ XDR cell culture bioreactor systems” (incorporated herein by reference in its entirety) or emdmillipore.com/US/en/mobius-single-use-manufacturing/mobius-single-use-bioreactors/mobius-bioreactors/fCyb.qB.1TkAAAFEF9sMfopc,nav or bioprocessintl.com/upstream-processing/fermentation/single-use-processing-for-microbial-fermentations/1. As mentioned above, the terminology often used in the bioprocessing industry is “turn-down ratio” or the ratio from the maximum working volume to the minimum working volume. For example, then, a single-use bioreactor properly designed could scale from 130 L to 650 L with a 5:1 turndown ratio. The goal for design of the turn-down ratio is that within this scaling, many of the 9 criteria mentioned above can be considered “conserved” or similar enough that the cell densities, cell viabilities, and titers are will be the same; specifically, for a bioreactor with a 5:1 turn-down ratio at any volume from 20% to 100% of the working volume (maximum usable volume) one can consider the properties of the bioreactor very similar. However, if it is desired to scale-up past the maximum volume, to date, a new larger bioreactor would be required to increase the total number of cells or the titer. As discussed earlier, a larger bioreactor cannot generally be treated as having the same 9 aforementioned quantities, thus creating the problem discussed above. For example, mixing time is not preserved in a 2000 L bioreactor between full volume and half-volume operation (Thermo Fischer 2000 L SUB Scale-up Summary or Sub Validation guide page 41/42, incorporated herein by reference). Note that when micro-carriers or other suspension mechanisms are introduced into a bioreactor, the scale-up problem is significantly more difficult (bioprocessintl.com/manufacturing/antibody-non-antibody/considerations-in-scale-up-of-viral-vaccine-production-320990/). Furthermore, with certain types of shear-sensitive cells such as stem cells, scale-up is paramount to producing the cellular product. One method to circumvent this issue is to simultaneously run multiple, or N, bioreactors of the same size to scale-up the volume. This is, however, often considered an unsatisfying or even unacceptable way to do scale-up as each bioreactor is independent and can have slightly different conditions and must be set up independently. This means that each bioreactor is controlled separately (e.g., each has its own set of PID loop controllers), and therefore the batches from each bioreactor are considered to be independent, namely, different from a yield, traceability, quality, and general behavior perspective. Furthermore, in order to eventually utilize the N bioreactors' products as one batch, the contents must be combined or “pooled” before being processed in the downstream (e.g.: filtration, chromatography, virus inactivation). Quality groups and/or regulatory bodies often object to pooling as this creates a lack of traceability to the source of any issues (e.g.: contamination, lower yield or efficacy etc.). Given these facts, the concept of running N independent (smaller) bioreactor vessels is often eschewed for scale-up in favor of one much larger bioreactor. Certain embodiments of this disclosure provide a way to circumvent, or at least significantly mitigate the aforementioned problems of scale-up by introducing coupling between the N bioreactors; here coupling means the physical transfer of the contents (cells and supernatant) between the bioreactors. This can be viewed as an extension to a very well-known set of problems in mathematics called “mixing problems”. If one couples a set of N bioreactors together by exchanging fluid contents between them such as to allow the contents of all of the N bioreactors to be considered homogenous (and therefore identical), we have for all intents and purposes, one batch. This allows multiple bioreactors to be utilized to create one batch without pooling while bypassing the scale-up issues and without the need for scrutiny from a quality or regulatory group Going back to the issue of coupling the bioreactors, we will review the mathematical machinery of mixing between tanks. As an introduction to the concept of mixing in tanks we will start with a simple and common problem—single analyte (e.g., brine or salt) mixing. The most basic problem usually has one tank partially filled with an analyte (e.g., salt) dissolved in water. Typical problems require one to work out the solution to an ordinary differential equation (ODE) and calculate how long will it take for the system to reach equilibrium. The typical problem also requires one to calculate the eventual equilibrium concentration of the analyte for a given influx at a stated rate and concentration of analyte in water with given initial concentrations and volume. FIG.2shows a mixing tank with a situation similar to that described above. Assume:S(t) is the amount of salt at time t;V(t) is the solution volume in the tank at time t;FInis the flow rate at which the solution flows into the tank;FOutis the flow rate at which the mixture flows out of the tank;Cinis the concentration of salt in the solution flowing into the tank;Coutis the concentration of salt in the solution flowing out of the tank;Rinis the rate at which salt is poured into the tank Rin=FInRIn×CinEquation 1Routis the rate at which the salt is poured out of the tank Rout=FoutRout×CoutEquation 2 Given these assumptions, we can define: Cout(t)=S(t)/V(t) Equation 3 dS(t)/dt=the rate of change of salt in the tank asRin−RoutEquation 4 This can be re-stated as: dS(t)/dt=FIn×Cin−Fout×S(t)/V(t) Equation 5 This is a standard form of ordinary differential equation (ODE) of type dS/dt+pS=qEquation 7 The solution to this ODE is: S(t)=∫µ(t)q(t)dt+Cµ(t)whereµ(t)=e∫p(t)dtEquation8 Therefore, we can solve for the concentration of salt, S(t), in the tank at any time, t, given the initial conditions and flow rates. The point of the above example is simply give insight into how one utilizes standard differential equations to solve this type of problem. Now the discussion will turn to the system and mathematics that describe in detail certain embodiments described here. The term “vessel” will be used as a generic term describing a liquid container which can mean either a tank or a bioreactor. Tanks will be passive or mixing containers while bioreactors will be vessels with the potential to actively control parameters of interest for bio-processing. Consider the arrangement shown inFIG.3taken from Mixing Problems with Many Tanks, A. Slavik, Mathematical Association of America, p.806, 2013 (incorporated herein by reference in its entirety). This figure shows an arrangement of tanks in what is referred to as a “star configuration,” which has a central vessel and three or more other vessels directly fluidically connected to the central vessel. The star configuration is one possible configuration, but in no way the only configuration that allows for mixing between vessels. InFIG.3, the tanks are labeled as T1. . . T8. This coupled system is one arrangement that more realistically depicts how tanks or bioreactors can be networked for scalability. If one solves rigorously and unambiguously for flows of mixtures of a substance (e.g., salt) flowing between coupled tanks, one can also solve rigorously for the mixtures of cells and/or media (general supernatant) moving in between bioreactors. This means that one can also create models to predict what flow rates from initial conditions will result in a stationary and homogenous solution. Many of the cases of interest have been summarized in the aforementioned paper. The paper provides a general set of equations which describe the problem of mixing in multiple coupled tanks and allow for solutions. These equations are the general extension of the single tank mixing problem we introduced above. Salt is specifically used in Slavik's paper, but any uniformly mixed substance can be substituted for salt. Equations 9 and 10 below from Slavik use slightly different notation from our simplified example: dx1(t)dt=-(n-1)fx1(t)V+∑i=2nfxi(t)VEquation9dxi(t)dt=fx1(t)V-fxi(t)Vfor2≤i≤nEquation10V is the volumeT1the central tank is labeled as tank 1T2. . . Tnare the (n−1) tanks connected to tank 1xi(t) is the amount of salt in tank I, (Ti), at any given time t Equation 9 describes the change in amount of the additive (salt) as a function of time in the central tank (tank 1), while equation 10 describes the change in salt concentration as a function of time in any tank Tithat is not the central tank. For a given set of initial conditions, the amount of salt in each tank at “time 0” can be solved and the details are worked out Slavik's paper. As mentioned previously, since the mathematical solutions for any mixture can be solved, this solution will also accurately describe the contents of a bioreactor. Considering the star configuration inFIG.3further, it can be seen that N vessels (e.g., eight or even more) can be coupled, where N is only limited by practical considerations (geometrical and space issue, distances, cost). AsFIG.3is a mathematical idealization of the physical problem, many considerations for applying the aforementioned mathematical machinery to bioreactors has not been specifically addressed. FIG.4shows two more mathematical representations of tank arrangements that can used for mixing. Reference numeral4-100is a linear chain with fluid moving bi-directionally in-between each tank, while4-101is a ring configuration that also employs bidirectional mixing. Note that for well characterized vessels that have been designed with appropriate scale-up/scale-down consistency in their operating parameters, any of these configurations could also contain vessels of different sizes. Bidirectional flow between vessels should increase the speed with which the vessels reach equilibrium, but if real mixing can be ensured this directionality is not strictly necessary. This has also been mathematically modelled and two such unidirectional configurations are shown inFIG.5. Reference numeral5-100is the unidirectional analog of the ring4-100shown inFIG.4, while5-101is the unidirectional analog of the chain4-101. Reference numeral5-102is the inlet and5-103is the outlet which will need to be tied together in a fluid conservative system. FIG.6presents a further example. As shown there, the central bioreactor6-100acts as the “master” or controlling bioreactor, while expansion or satellite bioreactors6-106and6-108are the “slave” bioreactors. As shown, a central bioreactor6-100is outfitted with sensors used for control of various analytes or physical levels including but not limited to sensors for: dissolved oxygen, pH, temperature, CO2, cell density, conductivity, and cell viability. The master bioreactor is also equipped with a mixing system and impeller6-102, and spargers6-107to control the dissolved oxygen level or strip CO2. These features are typically used with a control loop (e.g., PID loop) to maintain optimal levels of any of the aforementioned quantities. The control inFIG.6occurs though measurements in the master bioreactor, while a slave system is used for monitoring and feedback to both the master loops and the pumps6-103and6-104. In certain embodiments such as those depicted inFIG.4, the capacity of the bioreactor can be expanded or scaled by attaching other bioreactors such as bioreactors6-106and6-108to the central bioreactor6-100. The bioreactors, all typically pre-sterilized, are connected using single-use aseptic connectors and associated tubing sets or other fluidic connectors 6-109 and 6-110 on the master and slave bioreactors respectively. The connectors are shown on both the master and slave but only one set is strictly necessary. These single-use aseptic connectors are now common in the single-use bioprocessing arena and are made by companies such as Pall, GE, and Colder (e.g., pall.com/main/biopharmaceuticals/product.page?id=34125, gelifesciences.com/web app/wcs/stores/servlet/productById/en/GELifeSciences-us/28936612). Also note that the slave bioreactors may be passive vessels or vessels having structure (e.g., plate or micro-carrier aggregate) for adherent cells where an agitation mechanism is not possible. The ability to transfer the contents of the master bioreactor to the slave bioreactor is enabled by pumps6-103which are shown removing the liquid from the bottom of the bioreactor for hydrostatic pressure considerations. The pumps6-104are shown bringing the contents of the slave bioreactor from the bottoms of these vessels back to the master bioreactor. The pumps can be peristaltic pumps or any other known mechanism for moving fluid from one location to another through an enclosed path. Minimal damage to the cells is currently expected when the pumps utilized are low shear centrifugal pumps (e.g., Levitronix pumps, levitronix.com) but this also not strictly necessary and will depend on the speed of transfer required and the robustness of the cell line in use. In some embodiments, the bioreactors are disposed on scales or load cells6-111,6-112,6-113in order to maintain equal mass/volumes in each vessel. If the total volume is known in advance it is possible to have only a subset of the bioreactors on scales by using a simple algorithm incorporating the requisite addition or subtraction of the volumes using mass balance. It should be noted that by connecting bioreactors of known scalability (parameters uniform enough for successful scale-up) the issue of scale-up as discussed earlier has been circumvented. In various implementations, the pumps force exchange of contents between the bioreactors quickly enough that for all intents and purposes the system can be considered one bioreactor. Quickly enough means that all of the bioreactors are uniform in terms of certain parameters of groups of parameters and that a sample from one of the bioreactors cannot be distinguished from a sample taken from another at the same time. Any of the parameters described above can provide the measure of uniformity; such parameters generally relate to fluid dynamic parameters (e.g., shear experienced by cells, Reynolds number), composition parameters (e.g., concentrations of dissolved components and/or pH), and mass transfer parameters (e.g., oxygen transfer coefficient). Though each cell line and process is different, if the exchange rate between the vessels exceeds the rate at which such parameters change (and, e.g., cell growth (doubling) rates) the requisite uniformity is preserved. This can be proven experimentally for each cell line and process and controlled rigorously after this by adjusting the pump speeds accordingly and/or by monitoring the key process sensors. The sensors can be utilized as a feedback mechanism for the pump speed to maintain homogeneity within the typical error (or dead-band) of the associated control loop. It will be helpful to review here the cell growth process in terms of the disclosed embodiments. Specifically, how do cell populations grow and change as a function of time? A typical cell growth curve is shown inFIG.7. As shown in the figure, there is a lag phase immediately after seeding of the vessel where the cells are said to be recovering from the stress of being immersed in new media. Next is the log or exponential phase where the growth rates accelerate until the media is exhausted or other detrimental effect occurs. This is followed by the stationary phase and the decline phase. One measure of the cell growth rate is the “doubling time” or the time it takes for the cell population to double in population (—see, ATCC Animal Cell Guide) during the “log” phase of the growth cycle. The doubling time for the cell lines under the growth conditions is typically well characterized. For example, a typical CHO (Chinese hamster ovary) cell line has a doubling time of >15 hours, while human cardiac cells and mesenchymal stem cells from mice have median doubling times of approximately 29 and 22 hours respectively. Microbial cell doubling times are much faster;Escherichia colihave a doubling time on the order of 15-30 minutes. The doubling time can provide a reasonable measure of the rate of change of the growth process and there serve as a benchmark for the rate at which material needs to be exchanged between vessels in order to maintain homogeneity of the cell population. In the lag, stationary, and decline phases, the rate of exchange required for homogeneity will be reduced from the exponential growth phase where the cell doubling rate is the fastest. During the lag phase where the cells are doubling very slowly, the exchange rate can be commensurately slow. If a slave vessel is added during the log phase, a starting point for achieving homogeneity can use the volumetric flow/exchange rate (pump rates in and out of the vessels) benchmarked against the cell doubling time as a metric. The question of the required rate of volumetric exchange can be reduced to the question of how many times does the volume of the vessel need to be exchanged compared to the doubling time. If the vessel contents are exchanged at least an order of magnitude faster than the doubling time, the multiple vessel contents will almost certainly be homogeneous. How much slower can the rate of exchange of vessel contents be before homogeneity is lost will depend on the specifics of the cell line and the process? As an example, if a CHO cell growth process were started in a 100 L working volume bioreactor with a 4:1 turndown ratio completely filled with media and cells and the process was in the lag phase, the group running the process might require that a second identical and perhaps third identical bioreactor are attached as slave vessels in order to meet their product and/or titer goals. In this scenario to set up the system, the outgoing pumps from the master would turn on such that the second and third bioreactors are filled with >25 L with the volumes being determined by change in measured values on the load cells. As long as the cells and supernatant are transferred in a time period such that there is little change in the cell population (e.g.: the cell population can still be considered to be firmly in the lag phase), the master and slaves can still be treated as homogenous or uniform. The return pumps (from slave vessels to the master vessel) would remain off until the desired volume was reached in the slaves and then active bi-directional exchange between the vessels would commence. Additional media can be slowly added to the master or to multiple vessels again based on the total required volume and/or yield for the process; the master and slaves can all eventually be filled to the working volume. The rate at which the media is introduced into the system might have to correspond to the rate of change of cell density so that the cells are not substantially perturbed. During the lag phase, as mentioned before, the cells are not growing rapidly and therefore the exchange rate volumetric flow requirements are low. In the log phase, if the doubling time is 15 hours, a starting point based on the above discussion is to exchange the vessel volume every 1.5 hours. This means that 100 L/90 minutes or about 1.1 SLPM for the volumetric transfer rate. Based on measurements of relevant cell growth parameters including but not limited to the bioreactor pH, metabolite concentration, cell density, cell viability, etc. the volumetric exchange rates can be reduced. In certain embodiments, different processes use different algorithms to control the volumetric exchange rates and therefore maintain homogeneity. In certain embodiments, simply maintaining a very high volumetric exchange rate (compared to the fastest doubling time) throughout the entire growth process would simplify the system and uncertainty about homogeneity. As mentioned, in certain embodiments, the master vessel will generally control the important quantities such as dissolved oxygen, pH, and temperature. But in certain embodiments there can also be actuators in the slave vessels as shown inFIG.6, reference numerals6-106and6-108. Some control architectures depend on the physical embodiment of the system. For example, if the exchange rate between the vessels is fast compared to a thermal time scale of any one vessel, in some implementations, there is not only no need to control temperature in each individual vessel there is also no need for an actuator (e.g., heating blanket) that is set at a nominal value. Having a single master control loop avoids “pulling” or communication between control loops and simplifies the control scheme. Having actuators and sensors in the slave bioreactors as well allows the control to be performed through the master and allows the speed of the pumps to equilibrate the master and slave bioreactors; the sensors in the slave bioreactors compared to the sensors in the master allow a feedback path for the control of the pumps if desired. For example, having impellers, sensors, and spargers in the slave bioreactors controlled, but having the sparging and mixing in the slave vessels controlled by the master is a suitable control scheme. Passive control by design is also possible, where the speed of the pumps allows the slave vessels to be essentially passive elements or tanks. FIG.8extends this concept of scale-up by multiple connected vessels and may fully utilize the mathematics described in equations 9 and 10. InFIG.8, reference numeral8-100shows a simplified version of the master bioreactor where the spargers, sensors, impellers etc. are not shown but can be considered as included. The pumps (bidirectional) are shown as a unit8-109, and for the sake of clarity inFIG.8the bioreactors8-101through8-107are shown in simplified form and without showing the aseptic connectors, while a bioreactor8-108block illustrates the extension to any number of vessels. This limit is set by physical factors, cost, and practical considerations—not by theoretical extension or mathematics. If scaling the system (pumps, connectors, etc.) becomes cost prohibitive, this scaling by discrete addition of tanks can be used in conjunction with a conventional scale-up process to limit the number of tanks required. For example, if a bioreactor with a 5:1 turndown ratio is used, one can start the process in say a 30 L bioreactor turned down to 6 L, and then fill it to 30 L. After this a second or third bioreactor can be added which takes the total working volume to 100 L. The production can then move to the 650 L vessel which can be turned down to approximately 115 L, yet is expandable with two additional vessels to 1950 L. The largest single-use bioreactor in commercial production today is 2000 L, and a large number of bioreactor volumes in between are required to scale to this volume which means more parts, more scaling, more effort, and more cost. The example just reviewed allows scaling from 6 L to almost 2000 L with only one scale-up step in terms of bioreactor maximum working volume. Another application for this type of scalable system is in the personalized medicine arena. This area of application includes but is not limited to stem cell therapies, chimeric antigen receptor T Cells (CAR-T) therapies, and tumor infiltrating lymphocyte (TIL) based treatments where small but potentially expandable volumes are required. Currently for CAR-T type therapies a typical batch size is on the order of 1 L, and the US Food and Drug Administration (FDA) requires approximately one-half of this quality and safety testing. In such immune-oncology applications, the starting volume of cells collected and selected from a patient sample is very small (30 mL) compared to the required batch size (3 L) and represents a 100× amplification at high cell density in a single-patient dose scenario. The amplification challenge is even greater for heterologous treatments where a batch size of 300 L is targeted (10,000 amplification), if the treatments costs are to be affordable and quality assurance is to be amortized over a few thousand doses. The disclosed embodiments provide a useful mode of meeting such challenges. In personalized medicine applications, if the batch is lost a patient can lose an opportunity for live saving treatment at the last stages of his or her illness. Batches often fail owing to contamination of the process. Therefore, the ability to expand a 30 mL sample in a linear progression of effectively identical bioreactors is very appealing as the process can start in one 300 mL vessel, and be scaled to two and eventually three or more vessels simultaneously as the cells grow. Once the process is scaled into three or more vessels, the contents of one vessel can be removed for storage in case of a contamination of the whole batch, analyzed for quality/or stored as a treatment dose in cold storage. The two vessels can then be scaled back into the empty vessel and a second vessel volume removed at that point. This process can be repeated as necessary with a batch collected every week, for example, without compromising the viability of the cell culture as a vessel can be drained and refilled with media or detached and a new vessel aseptically attached (or all vessels in the train can be attached at the start with some vessels being empty and a sterile valve being used to determine when and if they are added to the process chain). This can then be viewed as a continuous fed-batch process which supports discrete volume changes. Batches can also be pooled into a single dose as they can be shown to be identical material. In the case of very shear sensitive cells, the satellite vessel concept in this disclosure can be applied with a set of master vessels having impellers and full sensor analytics being utilized to control the oxygen, pH and metabolite concentrations of the media, and the satellite vessel(s) being passive with either a packed bed of micro-carriers, hollow fibers or adhesion plates. With the pumps, such as inFIG.9, connecting the passive vessels to the master vessels, any viruses or bacteria can be isolated to a single vessel by the addition of appropriate filters8-119(typically <0.22 μm for viruses) in the lines between the vessels. The volume of the original cell sample would be divided among the satellites thereby de-risking the entire sample from failure to expand. FIG.9shows a system of three networked vessels, though it is expandable to N vessels as well. A main bioreactor9-100networked to satellite/slave vessels9-108and9-109with aseptic connectors9-118, bi-directional pumps9-107, in line virus filtration9-119, along with aseptic connectorized tubing sets9-115which are both called out specifically only on one vessel,9-109, but shown on all three vessels. The main bioreactor, or master,9-100is shown with an impeller9-102and sparger9-104. These features are not explicitly shown on the slave vessels9-108and9-109as they may not be required. In this embodiment, if the cells are sensitive to shear and/or for lower cost, the vessels can have the mixing, oxygenation and pH adjust all take place in the master tank. Given this possibility, the master tank here is shown as larger in size and volume than either of the slave tanks so that it can also act as both ballast and a reservoir for the oxygenated, pH adjusted media. Sensors9-103are shown in all three vessels so that there is feedback for monitoring and control purposes. Specifically, either changing the pumps speeds or changes to the analyte values in the master bioreactor. Additionally, inFIG.9, the slave bioreactors are shown being utilized in perfusion mode or a cell separation mode depending on the application. A tube9-113which allows the contents of the bioreactor to be brought up to the cell retention/separation device (acoustic separator, filter etc. (e.g.: applikon-bio.com/en/news2/itemlist/category/52-biosep, spectrumlabs.com/filtration/KR2System.html),9-114is shown with a return loop9-115to the bioreactor with a pump,9-116, or other device to move the fluid in the required path. The vessels can therefore be used to implement a perfusion process with actual growth process occurring in the slave vessels, and oxygenation and control residing in the master vessel, or in a cell expansion mode with a similar division of tasks. The cell expansion mode is of interest as T-Cell, stem cell expansion, or adherent cell growth can occur in the slaves without a need for an impeller and without the accompanying shear. The vessels are again shown on scales or load cells9-111,9-112,9-113for mass/volume balancing between the vessels. Finally, the entire system can be enclosed in an incubator or incubator shaker9-101for temperature control, as well as mixing if none of the vessels were equipped with impellers (e.g., shake flasks or bioreactors specifically designed to work without an impeller kuhner.com/en/product/shakers/single-use/sb200-x.html). This can further reduce the per-use cost of the vessels. As indicated, a multi-vessel bioreactor system includes two, three, or more fluidically coupled vessels configured to collectively carry out a bioreaction. They do so by maintaining nominally uniform or consistent conditions between the vessels during the bioreaction. As a consequence, the vessels share reactants and products between the vessels during the bioreaction. This may be accomplished by including fluidic paths (sometimes implemented herein as fluidic connectors, e.g., tubes) coupling the vessels to one another during the bioreaction and fluid transfer devices (pumps) in at least some of the one or more fluidic paths or connectors. Typically, the bioreactor system also includes a control system configured to control reaction conditions in the vessels to carry out the bioreaction. Among other responsibilities, the control system may be tasked with maintaining uniform process conditions between the vessels during the bioreaction. To this end, the control system may be configured to (i) read or receive values of at least one parameter characterizing a culture medium or other reaction fluid in one or more of the vessels during the bioreaction, (ii) use the values to determine an adjusted flow rate in at least one of the fluidic connectors to maintain substantially uniform values of the parameter in the reaction fluid from vessel-to-vessel among the vessels, and (iii) control at least one of the fluid transfer devices to adjust the flow rate determined in (ii). By controlling the flow of reaction fluid in this manner, the temperature, pH, selected analyte concentrations (e.g., dissolved oxygen concentration, glucose concentration), hydrodynamic conditions, etc. can be maintained substantially consistent or uniform across the vessels. Substantially consistent or uniform is determined from perspectives relative to the bioreaction, so, for example, the cell viability, cell productivity, titer, etc. are consistent from vessel-to-vessel. Magnitude differences within which a process condition is still consistent are determined in various ways, as appropriate, for performance of the bioreaction. For example, it may be appropriate for the mass concentration of a selected analyte or product to deviate by no more than about 5% from one vessel to another, or for pH to vary in magnitude by no more than about 0.2 pH units from one vessel to another, for temperature to vary by no more than about 0.5 degrees C. from one vessel to another. Again, the main point is that the variability is within bounds that produce consistent bioreaction results from vessel-to-vessel. These results may be product titer and/or concentration, cell viability, etc. In certain embodiments, the control system is configured to (i) read or receive parameter values characterizing the culture medium or other reaction fluid in one or more of the vessels during the bioreaction, and (ii) control the one or more fluid transfer devices to transfer the culture medium between the two or more vessels such that the time required to exchange the culture medium in the vessels is within an order of magnitude of the time required for cells in the vessels to double under conditions in the vessels. For example, the time required to exchange the culture medium in the vessels may be at most about one-half the time required for cells in the vessels to double under conditions in the vessels. Under more stringent operating conditions, the control system will direct transfer for the culture medium faster, such that the time required to exchange the culture medium in the vessels is at most about one-third, or one-fifth, or one-tenth (or even one-twentieth) the time required for cells in the vessels to double under conditions in the vessels. The actual rate will depend in part on the current conditions in the bioreactor system and degree of uniformity needed (e.g., the pH should not vary by more than about 0.1 pH unit from vessel-to-vessel). Note that the time required to exchange culture medium in a vessel or vessels is based on the amount of culture medium currently in the vessel(s), which is not necessarily the working volume of the vessel(s). (In practice, of course, the system would not remove all the culture medium from a vessel during the exchange; the system would flow in new medium and at the same time push out a corresponding amount of old medium.) Further, the cell doubling time varies depending up a number of factors including the cells' current growth phase, and where in the phase most of the cells currently reside. For example, the doubling time is dramatically different for cells in the lag phase and cells in the log/exponential phase. See the discussion ofFIG.7. In certain embodiments, the control system drives flow rates of the culture medium into each of the vessels such that the time require to exchange culture medium in any of the vessels is shorter than the mixing time of the culture medium for the respective vessels. In certain embodiments, the exchange time is within an order of magnitude of the mixing time. For example, the exchange time may be no greater than about one-half the mixing time, or no greater than about one-third, or one-fifth, or one-tenth of the mixing time. In certain embodiments, the exchange time is no greater than about one-tenth the mixing time. One way to consider this is that the time required fully exchange culture medium present in a vessel using the operating flow rate into the vessel is faster than the time required to mix the components of the vessel using the intrinsic mixing drivers of a vessel (e.g., convection, diffusion, etc.). As explained, mixing time may be defined in various ways. For purposes of this embodiment, is assumed that the mixing rate is defined by the time it takes for a recently introduced tracer to be homogeneously distributed within the bioreactor to the level required for a successful bioprocess. This assumes that culture medium is not entering or leaving the vessel during the mixing; i.e., it assumes that the vessel where the mixing occurs is a closed system. In certain embodiments, homogeneous distribution is defined such that the concentration of tracer varies by no more than about 10% between any two points in the vessel. Vessel As should be apparent, there are many benefits in using relatively small vessels. Examples of such benefits include avoiding certain scale up challenges (e.g., less experimentation and uncertainty), reducing intra-vessel variation in process conditions, etc. Therefore, the sizes of the individual vessels in the multi-vessel bioreactor system are relatively small. As an example, each vessel has a working volume of not greater than about 700 liters, or not greater than about 500 liters, or not greater than about 100 liters, or not greater than about 50 liters. It should be understood that the terms “total volume” and “working volume” are sometimes used in the industry, and consistent with that use, the term total volume refers to a vessel's total capacity, regardless of limits of fluid volume, while the term working volume refers to the maximum volume of fluid that can be filled in a vessel for undergoing a bioreaction. As indicated, a bioreactor system may include two, three, or more vessels. The numbers of vessels influences, but does not completely dictate, the ratio of total working volume of the bioreactor system (e.g., the sum of the working volumes of the two or more vessels making up bioreactor system) to the working volume of largest of the two or more vessels. In certain embodiments, that ratio is at least about 2 or at least about 3. Ratios in the range of about 2-4 are often appropriate for relatively large total volume bioreactors systems; e.g., systems having a total volume of about 1000 liters or greater, or about 1500 liters or greater, or about 2000 liters or greater. For some applications, such as smaller scale applications (e.g., about 500 liters total working volume or less) or personalized medicine applications, the ratio of total working volume of the bioreactor system to the working volume of largest of the vessels may be a larger, e.g., at least about 6. The total number of vessels employed in the bioreactor at any given time during the bioreaction may be at least about 5, at least about 6, at least about 10, or at least about 12. The number is chosen based on a number of factors including the bioreactor product or application, the total working volume of the bioreactor system, the need to replace, remove, or add vessels over the course of a bioreaction, etc. As an example of the power of this approach when coupled with typical vessel turn down ratios, consider a 4:1 or 5:1 turn down ratio in a 500 liter vessel, the working volume of the coupled multi-vessel system can scale from 100-125 L all the way to 1500 L to 2000 L without changing vessel sizes. This means a manufacturer of bioreactor vessels can have a focused product offering in which only a few vessel sizes are needed to provide an extremely wide range of effective bioreactor volumes. For example, the train of four or more bioreactor sizes inFIG.1can be replaced with two or three total bioreactors for the entire scale up process. In some implementations, the individual vessels of the bioreactor system are similar to one another in working volume, geometry, materials of construction, and/or agitation/mixing system. For example, the vessels may have similar turndown ratios; e.g., no two vessels have turndown ratios varying from one another by more within about 20%. In some embodiments, all vessels are made from the same material; e.g., all vessels have polymeric vessel walls or vessels have stainless steel walls. In certain embodiments, as explained, one of the multiple vessels is a master vessel and the others are satellite or slave vessels. Frequently, though not necessarily, the master vessel has a larger working volume than any of the satellite vessels. Typically, the master vessel is used to provide some measure of control over the conditions in the satellite vessels. For example, the control system may monitor and adjust conditions first in the master vessel and then use inter-vessel fluid transfer have conditions in the satellite vessels follow those in the master. In certain embodiments, the master vessel includes one or more sensors for the pH, temperature, a cell metabolite concentration, and dissolved oxygen in the culture medium. In certain embodiments, the control system receives values of such parameters from sensors on the master and/or satellite vessels and uses these values to first adjust conditions in the master vessel. In certain embodiments, the control system includes a single control loop configured to control the master vessel, and in some cases at least a mixing system configured to agitate the culture medium in the master vessel. In certain embodiments, the satellite vessels contain spargers set at a substantially constant rate. In such cases, sparging air or oxygen can occur in the satellite vessels, while the primary control point employs sensors in the master vessel. In some cases, the master vessel may include mixing system configured to agitate the culture medium in the master. In some of these cases, none of the satellite vessels includes a mixing system. Examples of suitable mixing systems, whether implemented in a master or satellite vessel, include impellers, orbital shakers, wave rockers, and plungers. As explained, the arrangement of vessels, as dictated by direct connections between individual vessels in the bioreactor system, may have many configurations. Examples include a star configuration, a linear configuration, closed loop configuration, and a combination of any two more of these. For example, the arrangement may be a spoke a wheel configuration, or a lasso configuration having a closed loop with a tail extending from one of the loop vessels, or a fused ring configuration. The arrangement can be bidirectional fluidic connections or simply a closed loop fluidic connection. As illustrated, a closed loop configuration forms a closed loop for fluid flow. In certain embodiments, the bioreactor systems includes an additional vessel initially unconnected to the two or more fluidically connected vessels, but it includes a supplemental fluidic connection for connecting to the bioreactor system after the bioreactor system has been operating. This allows the systems to scale up during the course of the bioreaction, as may be appropriate when culturing a patient's own cells (or a modified variant thereof) to be used in a subsequent treatment administered to the patient. In some cases the control system is configured to at least partially fill the additional vessel with culture medium from one or more other vessels during the bioreaction. Alternatively or in addition, one or more of the vessels of the system is configured to be removed, before the bioreaction completes, along with their culture medium and cells grown in the medium. Control System As mentioned, the control system may be tasked with maintaining uniform process conditions between the vessels during the bioreaction. This may require that, for example, cell growth rates and/or product titers do not differ by more than a few percent (e.g., no more than about 10%) from vessel-to-vessel. In various embodiments, the control system is configured to maintain substantially uniform values of pH from vessel-to-vessel. For example, the control system may be configured to adjust conditions in the two or more vessels to ensure that the mean pH of the culture medium in the two or more vessels does not vary from one vessel to another by more than about 0.2 pH units (or by no more than about 0.1 pH units or by no more than about 0.05 pH units). This is particularly useful when the individual vessels are small enough and/or well-mixed enough that the internal variation in conditions is minimal. For example, the two or more vessels and the control system may be designed or configured such that pH of the culture medium within any of the vessels has variance of at most about 0.1 pH units. In certain embodiments, the control system is further configured to adjust conditions in the two or more vessels to ensure that the mean culture medium temperature is held from vessel-to-vessel to within about 0.5 degree C. (or within about 0.1 degree C.). In certain embodiments, the control system is further configured to adjust conditions in the two or more vessels to ensure that shear forces experienced by cells in the two or more vessels are substantially equal from one vessel to another during the bioreaction. For example, the number of cells dying due to shear may be within 5% between any two vessels. Ideally, during the course of a bioreaction, few cells in any vessel or die due to shear forces or at least no more than about 10% die due to shear forces.” In certain embodiments, the control system is further configured to adjust conditions in the two or more vessels to ensure that culture medium gas transfer rates in the two or more vessels are substantially equal from one vessel to another during the bioreaction (e.g., the mean values of any one or more of the above-listed transfer parameters do not vary by more than about 5% from vessel-to-vessel). Of course, the dissolved oxygen level should not be below a level at which cells begin dying from lack of oxygen. In certain embodiments, the one or more of the vessels in the bioreactor system are provided on scales and/or load cells to monitor their masses during the bioreaction. The control system may be configured to monitor outputs from the scales and/or load cells and adjust flow rates between vessels to ensure the volume/mass in the individual vessels remains within specification. Broadly speaking, the controller may be electronics having various integrated circuits, logic, memory, and/or software that receive instructions, issue instructions, control operation, enable cleaning operations, enable measurements, and the like. The controller hardware may contain one or more processors, memory devices, and interfaces for communicating with sensors, pumps, spargers, mixers, and the like. The processors may include chips in the form of firmware that store program instructions, digital signal processors (DSPs), chips defined as application specific integrated circuits (ASICs), and/or one or more microprocessors, or microcontrollers that execute program instructions (e.g., software). Program instructions may be instructions communicated to the controller in the form of various individual settings (or program files), defining operational parameters for carrying out a particular process on a bioreactor. The controller may be integrated with electronics for controlling operation before, during, and after execution of a bioprocess. The controller, depending on the processing requirements and/or the type of system, may be programmed to control any of the processes disclosed herein, including pumping media between vessels, adjusting temperature, controlling media conditions such as pH, nutrient concentration, etc., and removing products of bioreactions. The controller, in some implementations, may be a part of or coupled to a computer that is integrated with, coupled to the system, otherwise networked to the system, or a combination thereof. For example, the controller may be in the “cloud” or all or a part of a facilities host computer system, which can allow for remote access of the bioprocessing. The computer may enable remote access to the system to monitor current progress of bioprocess operations, examine a history of past bioprocessing operations, examine trends or performance metrics from a plurality of bioprocessing operations, to change parameters of current processing, to set processing steps to follow a current processing, or to start a new process. In some examples, a remote computer (e.g. a server) can provide process operating instructions to a system over a network, which may include a local network or the Internet. The remote computer may include a user interface that enables entry or programming of parameters and/or settings, which are then communicated to the system from the remote computer. In some examples, the controller receives instructions in the form of data, which specify parameters for each of the processing steps to be performed during one or more operations. Thus as described above, the controller may be distributed, such as by comprising one or more discrete controllers that are networked together and working towards a common purpose, such as the processes and controls described herein. An example of a distributed controller for such purposes would be one or more integrated circuits on a reactor in communication with one or more integrated circuits located remotely (such as at the platform level or as part of a remote computer) that combine to control a process on the reactor. Each algorithm or other computational element of a controller may be implemented as an organized collection of computer data and instructions. In certain embodiments, a module for controlling flow between vessels, a module for adjusting temperature of one or more vessels, a module for adjusting introduction and/or removal of material from one or more vessels can each be viewed as a form of application software that interfaces with a user and with system software. System software typically interfaces with computer hardware, typically implemented as one or more processors (e.g., CPUs or ASICs as described) and associated memory. In certain embodiments, the system software includes operating system software and/or firmware, as well as any middleware and drivers installed in the system. The system software provides basic non-task-specific functions of the computer. In contrast, the modules and other application software are used to accomplish specific tasks. Each native instruction for a module is stored in a memory device and is represented by a numeric value. At one level a computational element is implemented as a set of commands prepared by the programmer/developer. However, the module software that can be executed by the computer hardware is executable code committed to memory using “machine codes” selected from the specific machine language instruction set, or “native instructions,” designed into the hardware processor. The machine language instruction set, or native instruction set, is known to, and essentially built into, the hardware processor(s). This is the “language” by which the system and application software communicates with the hardware processors. Each native instruction is a discrete code that is recognized by the processing architecture and that can specify particular registers for arithmetic, addressing, or control functions; particular memory locations or offsets; and particular addressing modes used to interpret operands. More complex operations are built up by combining these simple native instructions, which are executed sequentially, or as otherwise directed by control flow instructions. The inter-relationship between the executable software instructions and the hardware processor is structural. In other words, the instructions per se are a series of symbols or numeric values. They do not intrinsically convey any information. It is the processor, which by design was preconfigured to interpret the symbols/numeric values, which imparts meaning to the instructions. Fluidic Connections and Fluid Transfer Devices As mentioned, one or more fluidic connectors, or more generally fluidic paths, connect the vessels. In general, the connectors are configured to provide flow of culture medium between the two or more vessels during the bioreaction. They may be pipes, tubes, weirs, and the like. They have a fluid travel length, cross-sectional diameter, surface roughness condition, rigidity/flexibility, etc. as necessary to support the flow rates and flow conditions (e.g., laminar, turbulent, transitional) for transferring culture medium or other reaction fluid to maintain the required level(s) of uniformity from vessel-to-vessel. In certain embodiments, the fluidic paths are made from material that is USP Class VI/IS010993, animal component derived free, latex free, phthalate free, and/or gamma/beta radiation stable. In some implementations each of the fluidic connectors is configured to permit bidirectional flow of the culture medium between two of the vessels. In such cases, two or more flow paths may be provided between vessels or a two-way pump may be provided in a fluidic connector. Various physical structures of the fluidic connectors may be employed. One example includes tubing and aseptic connectors attached to the two or more vessels. In some implementations, at least one fluid path of the fluidic connectors attach at or near to bottoms of the two or more vessels. This allows the fluid flow to take advantage of hydrostatic pressure head, particularly in large vessels. Of course, other vessel designs may suggest the locations of the inlet and outlet fluid paths. For example, in the case of a two way fluidic connector and a vessel having an impeller directing fluid downward, the outlet of fluidic connector may attach below the bottom level of the impeller, and the inlet fluidic connector may attach at or above the top level of the impeller. Various types of fluid transfer devices may be employed. For example, at least one of the fluid transfer devices may be a pump. A pump is broadly defined to include all types used to move liquid by mechanical action. Examples include rotodynamic pumps (e.g., centrifugal or axial) and positive displacement pumps (e.g., a syringe type, a gear type, diaphragm type, a piston type, a plunger type, a screw type, or vane type). Perfusion As explained, one or more vessels of the bioreactor system may be configured to operate in a perfusion mode, whereby culture medium is distributed (flows) over the cells constrained to the vessel. Typically, in perfusion mode, conditions are maintained such that cells are retained in a vessel while fresh media is brought in. The rate that the media is brought in depends on the cell line and the phase of growth. A goal is to maintain a substantially optimal growth environment (in terms of nutrient concentrations, product titer, temperature, hydrodynamic conditions, etc.), and, as a consequence, the cells in a vessel maintain a relatively consistent biological state in terms of production rate, viability, etc. for a longer period of time compared to batch mode or fed batch modes of growth. In certain embodiments, a bioreactor system configured to operate in perfusion mode includes two or more fluidically coupled vessels configured to collectively carry out a bioreaction, where at least one of the vessels is a perfusion vessel having a fluidic inlet, a fluidic outlet, and a filter or other mechanism configured to prevent biological cells from leaving the perfusion vessel during the bioreaction. Additionally, as with some other systems described herein, a perfusion mode bioreactor system may include one or more fluidic connectors (or more generally fluidic paths) coupling the two or more vessels to one another and, during the bioreaction. The system typically includes one or more fluid transfer devices (e.g., pumps) in at least one of the one or more fluidic connectors. Collectively, the fluidic connectors and the fluid transfer devices are configured to provide flow of culture medium between the two or more vessels. Still further a perfusion mode bioreactor system may contain a control system configured to (i) read or receive values of two or more parameters characterizing the culture medium in one or more of the vessels during the bioreaction, (ii) use the values to determine process conditions to maintain substantially uniform values of the two or parameters in the culture medium from vessel-to-vessel among the two or more vessels, and (iii) introduce the culture media to the perfusion vessel through the fluidic inlet, flow the culture media over the biological cells while they are retained in the perfusion vessel, and flow the culture media out the fluidic outlet, to thereby operate in a perfusion mode. Stated another way, at least one of the fluidically coupled vessels includes a fluidic inlet and a fluidic outlet and the bioreactor system is configured to introduce the fresh culture media to the at least one vessel through the fluidic inlet, allow the flow of the fresh culture media into the bioreactor containing biological cells while they are retained in that vessel, and flow the exhausted culture media out the fluidic outlet, to thereby operate the at least one vessel in a perfusion mode. Various mechanisms may be employed to ensure that the cells remain in the vessel. For example, a fluidic outlet may include a filter or trap configured to prevent the biological cells in the vessel from leaving the at least one vessel or its fluidic outlet. In some implementations, a vessel configured to operate in perfusion mode includes micro-carriers, hollow fiber filter, a cell settler, and/or an acoustic separator, any of which may be employed to retain cells within a perfusion mode vessel. Any individual one or more of the vessels in the multi-vessel bioreactor system may be configured to operate in perfusion mode. In some implementations, at least two of the fluidically coupled vessels each include a fluidic inlet and a fluidic outlet and the bioreactor system is configured to introduce the fresh culture medium to the at least two vessels through the fluidic inlets, flow the fresh medium into the vessels while the cells are retained in the at least two vessels, and flow the exhausted culture media out the fluidic outlets, to thereby operate the at least two vessels in a perfusion mode. In some instances, one vessel simply supplies the nutrient and oxygen laden media to the perfusion system, thereby acting as a reservoir for new media and to oxygenate and stabilize the fluid returning from the vessel where the perfusion growth process is occurring. In some implementations, one of the vessels is a master vessel configured to provide culture media to the at least one other vessel through the fluidic inlet to provide perfusion. Further, the control system and master vessel may be together configured to maintain values of dissolved oxygen concentration, temperature, and pH in the culture medium in the master vessel and in the at least one vessel. In certain perfusion-configured bioreactor systems, the master vessel includes a mixing system, while the at least one vessel does not include any mixing system. Methods of Scaling a Bioprocess As explained, the disclosed concepts facilitate scaling a bioprocess working volume from that of a small-scale bioreactor to that of a large-scale bioreactor. In certain embodiments, a scaling process initially involves determining appropriate process conditions for performing the bioprocess in a test vessel having a relative small working volume (e.g., no greater than about 700 liters). Examples of process conditions that may be designed for the test vessel include values of two or more parameters characterizing culture medium in the vessel during the bioprocess. Relevant parameters discussed above including temperature, pH, dissolved oxygen concentration, etc. may be employed. After determining process conditions for the test vessel, the scaling process involves designing a bioreactor system having a control system and two or more fluidically connected production vessels. Each of production vessels has a working volume that is similar to that of the test vessel (e.g., within the range of the turndown ratio for the test vessel. In some embodiments, each production vessel has a working volume that is between about 0.7 and 1.5 times the working volume of the test vessel. In certain embodiments, the sum of the working volumes of the two or more production vessels is at least about 2 times larger than the working volume of the test vessel. In certain embodiments, the design process involves designing the control system to maintain substantially uniform values of one or more parameters, from production vessel to production vessel, during the intended to bioreaction. As discussed above, a suitable control system may (i) read or receive production values of the two or more parameters characterizing the culture medium in one or more of the two or more vessels during the bioprocess, (ii) use the read or received production values to determine an adjusted flow rate of the culture medium between the two or more production vessels to maintain substantially uniform production values of the two or parameters in the culture medium from vessel-to-vessel among the two or more production vessels during the bioprocess, and (iii) control one of the fluid transfer devices disposed between the two or more production vessels to adjust the flow rate as determined in (ii). It should be noted that such control loop keeps conditions substantially the same from vessel-to-vessel in the bioreactor system, as opposed to a different control loop, that may be implemented in a master vessel that keeps the parameter values within the ranges required (absolute ranges). Often the process of designing the control system for large-scale bioreactor system involves designing the control system to maintain, during the bioprocess, the production values of the two or more parameters to be substantially equal to the test values of the two more parameters. After designing the production vessels and the control system, the process involves actually constructing and/or arranging the two or more production vessels and the control system to as specified to produce the large-scale bioreactor. Finally, the bioprocess may be performed in the large-scale bioreactor. In some implementations, the large-scale bioreactor system is configured to permit bidirectional flow of the culture medium between two of the production vessels, and/or the two or more production vessels are arranged in closed loop. During the bioprocess, the total amount of culture medium in the bioreactor system may increase. For example, the bioprocess may be conducted at a first total working volume of culture medium in the bioreactor, and then the bioprocess may be conducted at a second total working volume of culture medium that is greater than the first total working volume but still uses only the two or more production vessels of the large-scale bioreactor. This may be accomplished by adding culture medium to the bioreactor system before the bioprocess completes. In another approach, at least a portion of the bioprocess may be conducted in the bioreactor, and then, one more additional production vessels are added to or activated in the bioreactor system. Thereafter, the bioreactor system may be operated to at least partially fill the one or more additional production vessels with culture medium from one or more other production vessels of the bioreactor system. Yet another mode of operating involves (i) conducting the bioprocess in the bioreactor system, and then (ii) removing one or more of the two or more production vessels containing culture medium and cells grown in the culture medium. This approach may be appropriate when the cells in a vessel are cultivated for treating a patient. In certain embodiments, the large-scale bioreactor system includes more than two production vessels—e.g., as explained above, three, four, five, six, or more production vessels—configured to collectively carry out the bioprocess. In certain embodiments, each production vessel has a working volume of no greater than about 500 liters. In some implementations, the ratio of total working volume of the two or more production vessels to the working volume of largest of the two or more production vessels is at least about 3. In certain embodiments, the two or more production vessels are connected in a star configuration or in a closed loop configuration. In general, the large-scale bioreactor system may have any one or more of the features described elsewhere herein for such systems. For example, each of the two or more production vessels and the test vessel may have a turndown ratio, and the turndown ratios of any one the two or more production vessels and the test vessel may be limited vary by no more than about 20%. Additionally, the control system may be configured to adjust conditions in the two or more production vessels to ensure that the mean pH of the culture medium in the two or more production vessels does not vary from one vessel to another by more than about 0.1 pH units. In many embodiments, one of the two or more production vessels is a master vessel and the others are satellite vessels, where the master vessel includes a mixing system configured to agitate the culture medium. Any one of the satellite vessels may or may not include a mixing system. In some such embodiments, the master vessel has a larger working volume than any of the satellite vessels. In some implementations, the master vessel includes one or more sensors for the pH, temperature, a cell metabolite concentration, and dissolved oxygen in the culture medium. | 71,208 |
11859162 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Further, the singular forms and the articles “a”, “an” and “the” are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and/or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and/or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present. It will be understood that although terms such as “first” and “second” are used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Thus, an element discussed below could be termed a second element, and similarly, a second element may be termed a first element without departing from the teachings of the present invention. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The invention generally relates to approaches for transferring one or more material(s) or agent(s), referred to herein as “cargo” or “payload”, into or out of cells. In many cases, the cells are eukaryotic cells, typically having a diameter within the range of from about 10 to about 100 microns (μm). Cargo also can be transferred to or from other membrane bound structures, such as, for instance, liposomes, exosomes, micelles, etc. Examples of cargo materials include but are not limited to small molecules, chromosomes, DNA, RNA, (e.g., mRNA, siRNA, gRNA, ssRNA), other genetic materials, oligomers, biomarkers, proteins, transposons, biomolecule complexes, small molecules, therapeutic agents, and so forth. Often, the cargo is an agent useful in the treatment, prophylaxis or diagnosis of a condition in a human or animal subject. In many of its aspects, the invention relates to a system that supports a hands-free, continuous flow transfer of cargo to cells or other substrates. Processes conducted in the system can be partially or completely automated. Shown inFIG.1, for example, is system10which includes several components or modules: incubator12, (first) buffer exchanger14, electroporation assembly16, (second) buffer exchanger18and (second) incubator20. The system can further include one or more reservoirs such as reservoirs22,24and26, for example and pumps30,32,34and36. Voltages required for electroporation are provided by electrical function generator40and acoustic energy is provided via acoustic function generator42. The system is controlled by controller44. Often the controller is a microprocessor in a computer system such as a single board computer system. In other cases, the controller is a microcontroller with integrated memory and analog to digital converters and digital to analog converters. Either or both incubators12and20(disposed, respectively, upstream of and downstream of electroporation assembly16) can be benchtop incubation chambers configured for housing cells before and/or after electroporation and can have an internal volume of about 0.3 to about 50 liters (L). In many cases one or both incubators have a miniaturized design. Typically, incubator12and/or20is/are provided with means for transferring cells to and/or from the incubators. For example, incubator12can be equipped with a receptacle for a cell container or reservoir (a flask, conical tube, etc.). The container can be a sealed, sterile container such as a blood bag, for instance. In illustrative examples, it provides cells at a concentration of 105to 5×108cell/mL) suspended in a high-conductivity (e.g., about 1 to 2 S/m) culture buffer, such as TexMACS or RPMI (Roswell Park Memorial Institute) medium for T cells. A closed loop or another suitable arrangement can be included to control the cell culture conditions, e.g., the incubator temperature (often maintained at 37° C.), the incubator gas composition (CO2and/or humidity levels, for instance), sensors for metabolic or general processing readouts (pH, O2, etc.) and so forth. Examples of cells that can be housed in incubator12include suspension cells such as primary T cells, NK cells, hematopoietic stem cells, or adherent cells such as MSCs, CHO cells, and many others. In some implementations, incubators12and/or20are designed or adapted to support membrane-bound structures such as liposomes, exosomes, micelles and so forth. Suitable buffers and conditions for keeping these targets stable can be selected as known in the art and/or determined experimentally. With cells (or other membrane-bound structures) that may not be neutrally buoyant in the culture medium, the incubator can be fitted with an agitation mechanism for generating a (gentle) movement in the container that houses the cells, reducing, minimizing or preventing settling or sinking. This helps to ensure that the concentration of cells that is delivered into downstream operations (media exchange and electroporation) is controlled and consistent. One illustrative design is described below with reference toFIG.2. In some cases, rather than utilizing separate incubator chambers for the start and end of the process (see, e.g., elements12and20inFIG.1), the entire system is housed in an incubator chamber. In many of the embodiments described herein, electroporation assembly16supports electroporation processes conducted in continuous fashion, using a sheath flow configuration, in a microfluidic channel, for example. One specific implementation brings cells and cargo into contact in a central flow or stream, that typically utilizes a low conductivity fluid, also referred to herein as an electroporation fluid. The central stream flows between two sheath (also referred to as “side” or “lateral”) streams that typically employ high conductivity fluids. The difference in conductivity between the center and side fluids leads to a concentration of the electric field (supplied by voltage generator40) in the central (low-conductivity) region of the flow, allowing an effective amplification of the electric field strength and preventing cells in the central stream from coming into physical contact with the electrodes. Considering that preferred media for cell cultures typically have high electrical conductivity and the sheath flow arrangement described above preferably places the cells in a low conductivity medium during electroporation, system10uses buffer exchange arrangement14for transferring cells from the cell culture medium to an electroporation buffer medium. In many embodiments, the rapid buffer exchange represented by component14inFIG.1involves driving cells from one flow stream into another acoustically, with acoustic frequencies being generated through component42. After electroporation, cells can be transferred from the electroporation medium into a cell medium. One approach illustrated inFIG.1employs buffer exchange module18which can further include a cell concentration function. Traditionally, cell concentration is typically accomplished in batch processes using centrifugation, but could be accomplished in flow configurations using acoustophoresis, dielectrophoresis, electrophoresis, inertial effects, or integrated porous membranes or sieves. In one embodiment, at least one of buffer exchange devices14and18is a rapid buffer exchange device and cell concentrator. Another embodiment utilizes a design in which one or both buffer exchange (switching) modules and the electroporation device16(which can employ the sheath flow configuration described above) are integrated into a single apparatus. In a further embodiment, at least one buffer exchange is conducted in a device that is separate from the electroporation device. In one example of this approach, buffer exchanger14, flow electroporation assembly16and buffer exchanger18are connected to one another by conduits, e.g., suitable tubing, that can provide fluid communication between these components. Some aspects of the invention employ acoustically-driven rapid buffer switching in both devices14and18. In other aspects, non-acoustically-driven buffer switching is utilized in at least one of the buffer exchange devices. One implementation utilizes an acoustically-driven buffer exchange device14and a non-acoustically-driven buffer exchange device18. Construction and operational details for acoustically-driven buffer exchanges are provided in U.S. patent application Ser. No. 16/359,626, with the title Acoustically-Driven Buffer Switching for Microparticles, filed on Mar. 20, 2019, which is incorporated herein by this reference. Techniques that can be employed to obtain non-acoustically driven buffer switching include but are not limited to inertial techniques, microchannels with integrated porous membranes or sieves, or diffusion-b based techniques. Output cells are collected in incubator20. In one example, these cells are primary human T cells that contain mRNA. Such cells can be used in gene editing applications, or as transient therapeutic systems (mRNA CAR-T). In other examples, the output cells are used for protein or extracellular vesicle production (e.g., modified CHO cells or MSCs). Throughout the system, flow is driven (actuated) by a pump system, including pumps that are commercial, off-the shelf pumps often of peristaltic design. Other suitable pump types can be employed. In general, one pump is used to actuate flow of the cell suspension out of the first incubation chamber and through the entire system with a flow rate that ranges, e.g., from 100 μL/min to 2 mL/min. One additional pump is needed for each buffer exchange that occurs in the system. Nominally, at least two pumps are needed: one involved in moving cells into the electroporation buffer, and one that later returns cells to a culture buffer. In order to protect against flow rate differentials between devices, fluidic capacitors or reservoirs can be placed between devices that act as ballast. In this case, each microfluidic device has its own set of pumps to control flow rate. In system10, cells to be electroporated are withdrawn from incubator12using pump26, which can be a syringe pump capable of controlling fluid flow. System10also includes reservoir22and pump32, e.g., a syringe pump, for supplying electroporation buffer to electroporation arrangement16. High conductivity fluid for the sheath flow can be added from reservoir24by means of pump34, e.g., a syringe pump. Cell medium is supplied to buffer exchange module18from reservoir26, using pump36, e.g., a syringe pump. System10provides various options regarding the reservoir that houses the cargo to be incorporated into the cells (or into other types of membrane bound structures. In one example that uses primary human T cells, mRNA in electroporation buffer can be introduced from reservoir22into media exchange device14, which transfers cells into the mRNA-laden electroporation media before it flows into electroporation device16. Other arrangements supply a cargo such as plasmid DNA, single-stranded linear DNA, double-stranded linear DNA, linearized plasmid DNA, single-stranded donor oligonucleotides, ribonucleoproteins (e.g., Cas9 protein complexed with guide RNA), proteins, or small molecules from reservoir22. Cells can also be manually suspended in electroporation media laden with cargo, introduced into incubator12, and the flowed directly into flow electroporation device16. Silastic or other suitable tubing can be employed for some or all connections providing fluid communication between the various modules (components). Controller44can include one or more computers, hardware, software, sensors, interfaces, etc. for controlling the operation of system10or components thereof, to reach partial or complete automation. In many embodiments, controller44controls the electrical parameters applied for electroporation and/or the acoustic frequencies employed in buffer exchange device14and, optionally, in buffer exchange device18. Controller44can monitor or control incubator parameters, the operation of one or more of pumps30,32,34and/or36, the flow and parameters of central and/or sheath streams described above and so forth. Various embodiments that can be included in system10and/or its operation are further described below. For instance, as noted above, system10can utilize one or more agitators for preventing cells maintained in an incubator from settling. An example is presented inFIGS.2A and2B. Shown in these drawings is front end agitator apparatus11, which can be housed in incubator12(FIG.1). Agitator apparatus11includes a cell reservoir13for supplying cells in a suitable cell buffer to buffer exchanger14(FIG.1) and impeller15, for stirring the cells and keeping them from settling. In specific embodiments, cell reservoir13includes a cylindrical (tubular) upper section17and a conical lower section19. Outlet21provides fluidic communication between the conical lower section19of cell reservoir13to pump30inFIG.1. A lid23caps the top opening of section17. Cell reservoir13can have a volume within the range of from about 15 mL to about 1 L. In many cases, selecting a suitable impeller design takes into consideration the need to protect the cells from excessive shear forces in the cell reservoir while promoting sufficient agitation to reduce, minimize or entirely prevent their sinking to the bottom of the cell reservoir. In the specific embodiment ofFIGS.2A and2B, impeller15has a profile that is similar to that of the cell reservoir13and includes an upper section25and a tapered lower section27, as illustrated inFIG.2C. In the embodiment ofFIG.2C, the impeller is a thin, flat structure configured to fit inside cell reservoir13. The degree of taper, the relative length of sections25and27as well as other parameters can be optimized experimentally. Furthermore, other types of impellers can be employed. Impeller15is connected to motor29via magnetic coupling31. The motor, magnetic coupling and the cell reservoir can be installed on a platform33, which, in some implementations, is fabricated from aluminum breadboard. Cell reservoir13is stabilized by stabilization plate35, while holder plate37holds the cell reservoir13at lid23. Holder plate37can have a U-shape opening, e.g., for loading and unloading the cell reservoir and keeping it stable during agitation. Stabilization plate35and holder plate37can be mounted on platform33using pillar posts or other suitable means. In the embodiment ofFIGS.2A and2B, apparatus11includes 3 male female pillar posts39, which can be 6 inches long. Motor subassembly41, which can include 3 screws43, is mounted on holder plate37using, for example, male female pillar posts45, which can be 3 inches long. The motor spins a magnet that magnetically couples to the impeller, causing it to spin in the cell reservoir without direct contact. Parameters for operating impeller15take into consideration the need to protect cells from excessive shear, while keeping the cells from sinking and settling at the bottom of the cell reservoir. In specific implementations, impeller15is operated at160rotations per minute, and imparts a maximum shear stress of about 2 dyne/cm2to the cells. It is important that shear stress remain below about 10 dyne/cm2. If a peristaltic pump is used to drive fluid out from outlet21, a compliant structure can be placed in line to dampen flow oscillations created by the pump, providing a smooth flow to upstream devices. The compliant structure can be, for example, a fluidic capacitor, i.e. a microchannel with a floor and/or ceiling fabricated from a compliant material such as a polymer membrane that deforms in response to fluid pressure. An example is shown inFIGS.2D(an exploded view of the fluidic capacitor61) and2E (a view of the assembled fluidic capacitor). As seen in these drawings, the fluidic capacitor61includes top (ceiling) plate63, middle plate65and bottom (floor) plate67, separated by two polymer membranes, namely sheets69, which can be fabricated, for example, from high purity, high temperature silicone rubber or another suitable material. Top plate63, first sheet69, middle plate65, second sheet69and bottom plate67are held together by screws71that can be threaded into threaded holes73. Other arrangements for clamping together the plates and sheets can be employed. Ports75provide fluidic communication between, to and from other system components. In one example, outlet21(FIGS.2A and2B) is connected to a first (inlet) port75, while the second (outlet port75is connected to pump30(FIG.1). FIGS.2F,2G and2Hshow, respectively, plates63,65and67. In more detail, a microchannel77is cut through the middle plate65. Through holes79accommodate screws71while through holes81are configured to fit inlet/outlet ports75. During operation, fluid flowing through microchannel77is sandwiched between sheets69, which are pliant and can accommodate volume fluctuations to smooth out the flow. FIG.3is a top view of an exemplary electroporation assembly16that can support a sheath flow configuration such as described above. The arrangement includes microfluidic center channel46having trifurcating inlets (elements46a,46band46c) and trifurcating outlets (elements46a′,46b′ and46c′) inFIG.3. Microfluidic channel46can be fabricated in a substrate52such as hard plastic (which, for many materials, renders the device disposable). Examples include but are not limited to cyclic olefin copolymer (COC) thermoplastic, a polyimide film, such as Kapton®, polystyrene, PEI (polyetherimide), e.g., Ultem®, or a combination of various polymers. Other materials such as glass, quartz, silicon, suitable ceramics, and so forth also can be employed. The channel dimensions can range from 500 micrometer (μm) to 3 millimeter (mm) in width, 1 centimeter (cm) to 5 cm in length, and 125 μm to 500 μm in height. A pair of coplanar rhomboid-shaped electrodes (48aand48b) are patterned onto the polymer layer beneath the floor of the microchannel with square wire bond or solder pad areas defined by cutouts in the polymer layers that expose the electrodes for external access. A masking layer is placed between the electrode layer and the microfluidic channel46, with cutouts that define the portion of electrode that is exposed to fluid in the microchannel. Typically, the electrodes are formed from an electrochemically stable material, such as platinum metal (Pt). The portion of the electrodes that are exposed to the fluid in the channel have dimensions of 100-250 μm in width and 8-45 mm in length and interface to the electrical function generator40via connection to the square soldering pads (elements50aand50binFIG.3). In an arrangement such as that ofFIG.3, the relative flow rates of the center vs. side streams can be tuned. In one example, the relative flow is adjusted so that the electrodes only make contact with the side streams. The total flow rate can range from 375 μL/min to 6 ml/min. In specific examples, the flow ratio for the side streams vs. the center stream is typically in the range of 1:0.5 to 1:1 (single side:center). When the conductivity of the solution comprising the center stream is much lower than the conductivities of the solutions comprising the side streams (e.g., 10× or more), the center stream dominates the electrical resistance of the circuit, such that, when voltage is applied to the electrodes, most of the voltage is dropped across the center stream. The voltage (from the electrical function generator40inFIG.1) is applied across the square soldering pads50aand50band may take the form of sinusoids with periods ranging from 10 nanoseconds (ns) to 10 milliseconds (ms), or pulse trains with pulse widths ranging from 10 ns to 10 ms. The magnitude of the applied voltage can vary, so as to generate an electric field across the center stream that ranges from about 2-1000 kV/m, and pulse widths ranging from 10 ns to 10 ms. The frequency of the pulse train can be varied as well, and ranges, for example, from one pulse per cell residence time, to 10 pulses per residence time or more. An arrangement such as that inFIG.3can support the use of different buffers. In specific embodiments, the sheath side streams are characterized by high electrical conductivity (σ), e.g., in the range of from about 1 to about 2 Siemens per meter (S/m), while the central sheath stream has a low σ, e.g., within a range of from 10 to 1000 micro Siemens per centimeter (μS/cm). This approach is compatible with the use of buffers suitable for cell culture and/or buffers suitable for electroporation. Thus, in one implementation, the cells, in their preferred buffer, are provided via the center sheath stream46c. The two side streams46aand46bare supplied from the high conductivity media reservoir24by pump34inFIG.1. It is common for such a cell preferred buffer to have a high σ, e.g., in the range of from about 10,000 to about 20,000 (μS/cm). Low σ electroporation buffer flows in central stream46cand is supplied from electroporation reservoir22by pump32inFIG.1. Prior to entering the electroporation module16(FIG.3), cells coming from incubator12are flowed into the side stream port110of an acoustic media exchange module ofFIG.4Awhere they are driven or pushed, e.g., acoustically, from the high conductivity side sheath streams to the center stream, which contains a low σ electroporation buffer and cargo. As a result, the cells become suspended in the central, electroporation buffer (which is then delivered to the center stream port46cof module16). The acoustic energy to drive the cells from one buffer to another is supplied from the acoustic function generator42to an acoustic transducer154attached to the channel substrate152. After the electroporation operation (conducted in electroporation assembly16inFIGS.1and3), cargo-containing product cells can remain suspended in the central stream and can be collected from outlet46c′. Fluid obtained from outlets46a′ and46b′ is handled as waste or recycled. In other embodiments, a second buffer exchange (see buffer exchanger18inFIG.1) can be performed to move the cargo-containing product cells from the low σ electroporation buffer in the central stream to the high σ fluid in the side streams. In this configuration, cargo-containing product cells can be collected from outlets46a′ and46b′. Fluid from outlet46c′ is handled as waste or directed to a collection arrangement for reuse. Schematically shown inFIG.4Ais one arrangement of the (first) buffer exchanger14for moving cells from sheath streams containing high conductivity cell buffer, to a low conductivity electroporation buffer flowing in the central stream. Cells in their preferred medium, i.e., cell buffer, are introduced through the cell inlet110. The stream is supplied from the incubator12by the pump30. The medium input into the inlet is bifurcated into two cell subchannels110A,110B. The cell subchannels110A,110B diverge from each other in the y-axis direction and then converge as they progress in the positive x-axis direction. The cells are acoustically driven from the sheath cell buffer streams into the central electroporation buffer stream in the acoustic focusing region119. The electroporation buffer is supplied from the electroporation buffer reservoir22by pump32. The acoustic energy is supplied from the acoustic function generator42to an acoustic transducer154attached to the channel substrate152. Output cells are collected from the central outlet121, while the cell buffer is collected at outlets123A and123B as waste or directed to a collection arrangement for reuse. As discussed previously, in one configuration, the output cells from outlet121are supplied to center sheath stream46cof the electroporation module16ofFIG.3. FIG.4Bshows a two-stage, acoustically-driven, rapid buffer exchange system100used for electroporation of cells, which performs the functions of the first buffer exchanger14, electroporation assembly16, and the second buffer exchanger18described inFIG.1. This example comprises two or more connected microchannels102,104. Typically, the channels are fabricated from a hard polymer substrate or substrates108, such as polystyrene, or other hard substrates such as silicon, glass, and quartz. The prototypical two-channel system is described here, but additional channels can be added in kind. In the illustrated example, each of the microchannels102,104is fabricated in a separate substrate108-1,108-2. Each microchannel102,104supports a sheath or co-flow, with a center stream124and streams126on either side. In specific examples, the center stream124has a composition that is different from the composition of the side streams126. Flow is maintained in the laminar regime, so mixing between the streams is minimal. In order to maintain this laminar flow, the fluid velocities of the center stream124and the sheath or side streams126are such that the Reynolds number, Re, in the system is small (Re<<˜2000) in the region of the trifurcated inlets in convergence region127. Preferably, Re is less than 500 and is preferably less than 10. The microchannels102,104may be rectangular in cross section with width and height dimensions that range from 100 micrometers (μm) to 1000 μm. The length of each of the microchannels102,104ranges from 5 millimeters (mm) to 30 mm. Other embodiments are possible, however. Another example has a concentric flow geometry wherein the sheath stream126surrounds the center stream124on all sides. Particles, such as cells10, initially introduced into the one or more sheath streams126, e.g., at an input region such as inlet port110, can range in diameter from 100 nanometers (nm) to 25 μm. For example, T-cells, a typical particle for electroporation transfection, range in size from about 6 μm to 12 μm. In one application, the system100is used to rapidly move cells into and out of a specialized electroporation buffer, each microchannel has two inlets110,112and a trifurcating outlet114. Electroporation buffer is introduced directly into the first microchannel102through inlet112and forms or comprises the center stream. The electroporation buffer is supplied from the electroporation buffer reservoir22by pump32inFIG.1. Cells in their preferred media, i.e., cell buffer, are introduced through the cell inlet110. The cells are supplied from the incubator12by the pump30. The media input into the inlet is bifurcated into two cell subchannels110A,110B. The cell sub channels110A,110B diverge from each other in the y-axis direction and then converge as they progress in the positive x-axis direction. The cell buffer and the electroporation buffer generally differ from each other in terms of how long the cells can survive in the respective buffers. An example of a cell buffer for T cells would be TexMACS (sold by Miltenyi Biotec Inc.) or RPMI (sold by Thermo Fisher Scientific Inc.). Such cell buffer typically contains physiological salt concentrations that match cell osmolarity and nutrients. On the other hand, an example of an electroporation buffer would be BTX low-conductivity buffer (sold by BTX). Such electroporation buffer typically has lower salt concentration to reduce the conductivity but has added sugars to reduce osmotic shock to the cells. The cell sub channels110A,110B converge toward each other, on either side of an electroporation buffer flowing from subchannel112A to create a trifurcated inlet in the convergence region127. In this way, all three subchannels110A,110B,112A deliver their flow into a switching channel125. The flow of streams126, containing the cells, converges around the center stream124as two side sheath streams of the flow. At the other distal end of the first microchannel102, the switching channel125delivers flow to two side outlet subchannels130A,130B in divergence region129. Here, the subchannels130A,130B diverge from each other in the y-axis direction as they progress in the x-axis direction and also diverge from a center outlet subchannel132. The two side outlet subchannels130A,130B carry flow largely from the original sheath input streams126and exit as waste or are collected for reuse. The center outlet subchannel132carries flow from the center stream of the switching channel. It contains the cells10in the electroporation buffer. Here, also laminar flow is preferably maintained. The fluid velocities within a divergence region129are such that the Reynolds number, Re, in the system is small (Re<<˜2000) in the region of the trifurcated outlets in divergence region129. Preferably, Re is less than 500 and is preferably less than 10. The center outlet subchannel132of the first microchannel102directs flow to inlet134of the second microchannel104, preferably fabricated in a separate substrate108-2. The second microchannel inlet134bifurcates into two cell subchannels134A,134B. The cell sub channels134A,134B diverge from each other in the y-axis direction and then reconverge as they progress in the positive x-axis direction. The cells' preferred media or a secondary media (possibly containing a different biomarker or cargo to be transfected) is introduced into the other inlet116of the second microchannel104. This media is supplied from the cell media reservoir26by the pump36inFIG.1. The electroporated cell sub channels134A,134B converge toward each other, on either side of a cell buffer subchannel116A, which carries the flowing media in the positive x-axis direction. The electroporated cell sub channels134A,134B and the cell buffer subchannel116A deliver their flow into a second switching channel140. Here, the cells10are directed, from the side streams162, to the center stream160of the second switching channel140of the second microchannel104. As before, the Reynolds number, Re, is small (Re<<˜2000) in the region of the trifurcated inlets in convergence region127. Preferably, Re is less than 500 and is preferably less than 10. Cells can be collected from the center outlet subchannel142of the second microchannel104. The collected cells from the microchannel142are transferred to the incubator20shown inFIG.1. Two lateral outlet subchannels144A,144B, at the end of the switching channel140and on either side of the center outlet subchannel142, carry fluid to waste or a collection arrangement for reuse. Alternatively, cells can be directed toward an additional microchannel and so forth, depending on the number of buffer exchanges that are desired. Typical input flow rates range from 1 microliter per minute (μl/min) to 1 milliliter per minute (ml/min). In one example, the substrates108-1,108-2are bonded to separate lead zirconate titanate piezoelectric transducers118-1,118-2using cyanoacrylate adhesive, and it is shorter than the microchannel. The transducers118-1,118-2are connected to and driven by separate drivers150-1,150-2of the acoustic function generator42shown inFIG.1, each of which includes a radio frequency amplifier which is driven by a function generator that creates the sinusoidal signal which excites the respective channel102,104. This device configuration has been shown to support an acoustic resonance frequency between 900 to 990 kHz, where a stable standing pressure wave is generated across the width of each of the switching channels125,140. The transducers and microchannel substrates are also preferably mounted to aluminum plates which acts as a heat sink. A thermoelectric cooler (TEC) element and base plate sit beneath the aluminum plate. A thermistor is connected on top of the transducer near the microchannel, which is connected to a TEC controller along with the TEC, to make a closed-loop temperature control system. The temperature is preferably held at approximately 26° C. An acoustic isolator146prevents acoustic energy from each of the acoustic transducers118-1,118-2from affecting the other microchannel. This prevents cross-talk between the two microchannels and allows them to be separately driven and tuned. In the present example, isolation is achieved by fabricating the microchannels102,104in separate substrates108-1,108-2and then connecting the substrates with flexible tubing to avoid acoustic crosstalk. These transducers118-1,118-2are actuated by separate drivers150-1,150-2of the acoustic function generator42ofFIG.1. Each of these drivers applies a separately tunable sinusoidally varying voltage, for example. The frequency is chosen such that a stable standing pressure wave is generated across the width of each switching channel125,140of the respective microchannel102,104(transverse to the fluid flow direction). In this way, the transducers118-1,118-2drive the operation of the (first) buffer exchanger14(FIG.1) and second buffer exchanger18(FIG.1), respectively, in the two-stage, acoustically-driven, rapid buffer exchange system100ofFIG.4B. For the fundamental focusing mode there is a single pressure node in the fluid. The acoustic radiation pressure exerts a force on the cells in the direction of the pressure node. This results in the migration of cells out of the side streams and into the center stream, toward the centerline of the cross-section of the channel. In the first microchannel102, this action results in cells moving out of their preferred buffer and into the electroporation buffer, where they are electroporated. In the second microchannel104, this action results in cells moving out of the electroporation buffer and back into their preferred or a new buffer. This results in a residence time of cells in the electroporation buffer of seconds or less. A pair of electroporation electrodes120drive the operation of the electroporation assembly16(FIG.1) in the two-stage, acoustically-driven, rapid buffer exchange system100ofFIG.4B. The electroporation electrodes120can be positioned in the region between the trifurcated inlet and trifurcating outlet of the first microchannel102; for example, halfway between the trifurcation inlet and trifurcation outlet. If multiple stages of electroporation, with multiple sequential payloads being required, electrodes may also be fabricated in the 2nd and any additional microchannels, so long as the final microchannel returns cells to their preferred buffer. The electrodes120are placed such that cells pass through the electroporation field after being focused into the electroporation buffer in the center stream124of in the switching channel125. The electrodes are driven by the electrical function generator40shown inFIG.1. The electrodes120may be patterned using photolithographic processes onto the floor and ceiling of the switching channel125, or onto the sidewalls of the channel125. Electrode area (especially the dimension along the flow axis (x-axis direction inFIG.4B) of the channel) and flow rate determine the residence time of cells in the electric field. Chosen residence times can vary from 100 microseconds (is) to about a second. Alternatively, “remote electrodes” can be used, comprising fluidic connections from open ports to the main channel, and wire electrodes placed in the ports (such a configuration requires Faradaic current to pass through the electrodes). An AC (for example, sinusoids or pulse trains with periods/pulse widths ranging from 10 ns to 100 s of microseconds) or a DC electric field is established and remains active while cells flow through the device. The magnitude of the field is tuned for the specific cell type to a value sufficient to achieve permeabilization, and is typically in the range of 2-200 kV/m. Cargo can be mixed into either the electroporation buffer introduced into the first microchannel102at inlet112, or with the cells in the preferred cell buffer that is introduced into the cell inlet110. The former enables tuning of the cells' exposure times to the cargo by adjusting the timing of transit into the second microchannel. In some embodiments, the individual microchannels are fabricated separately, connected fluidically by polymer tubing, and are acoustically-actuated independently. The individual microchannels might even be fabricated on the same substrate and actuated together using a single piezoelectric transducer. In some embodiments the “waste” streams in the two side outlet subchannels130A,130B from the first microchannel102, containing the cells' preferred media, are directed and coupled into the center stream via inlet116of the second microchannel104(instead of a second pump delivering media directly into the center stream of the second microchannel). In some embodiments, multiple sequential microchannel setups are laid out in parallel with manifolds for introducing cells and buffer, increasing throughput. FIGS.4C and4Dshow two alternate embodiments of the rapid buffer exchange system100. Here, in a compound microchannel106, the cells are focused into the center stream for electroporation, and then focused back into the outer stream (called “de-focused” here) for collection. In more detail, electroporation buffer (supplied from electroporation buffer reservoir22by pump32inFIG.1) is introduced directly into the compound microchannel106through inlet112and comprises the center stream of the sheath flow, as shown inFIG.4C. Cells in their preferred media (supplied from incubator20by pump30inFIG.1) are introduced through the cell inlet110. The media input into the inlet is bifurcated into two cell subchannels110A,110B. The cell sub channels110A,110B diverge from each other in the y-axis direction and then converge as they progress in the positive x-axis direction. The cell sub channels110A,110B converge toward each other, on either side of an electroporation buffer subchannel112A to create a trifurcated inlet. In this way, the subchannels110A,110B,112A deliver their flow into a compound switching channel125. The flow126, containing the cells, converges around the center stream124as two side sheath streams of the flow as in previous embodiments. At the other distal end of the first microchannel102, the switching channel125delivers flow to two side outlet subchannels130A,130B, which diverge from each other in the y-axis direction as they progress in the x-axis direction. The two sided outlet subchannels130A,130B carry flow largely from the original sheath streams126, but in this example, the cells have been moved into the sheath streams126upstream of the side outlet subchannels130A,130B. The long compound switching channel125is divided into two regions by a set of electroporation electrodes120on either lateral side of the channel at a distance L1, measured along the x-axis, from the trifurcating inlet, which distance, for example, can range from 20 to 40 mm. The channel has a width of w, which can range from 420 to 740 μm. Acoustic actuation at frequency, f1, which typically ranges from 400 to 1000 kHz, is provided by acoustic function generator42inFIG.1, and applied by the first driver150-1to the first acoustic wave transducers118-1and is used to drive cells to the center, low-conductivity stream upstream of the electroporation electrodes120. In the region downstream of the electrodes120, a different frequency, f2, typically greater than f1, by a factor of 1.5 to 2.5, for example (provided by a second acoustic function generator, e.g., similar to acoustic function generator42inFIG.1) is applied by the second driver150-2to the second acoustic wave transducers118-2, which is used to drive the cells out of the center stream. The center outlet subchannel132at the distal end of the microchannel102carries flow from the center stream of the switching channel, e.g., to waste or recycling. InFIG.4C, a single acoustic driving frequency is used, but the channel downstream of the electrodes120is wider, having a width w2that is greater than w1(the width upstream of electrodes120) by a factor of 1.5 to 2.5. This alters the nodal structure of the soundwaves in the channel and achieves the similar forcing of the cells to the side streams. In both the embodiments ofFIGS.4C and4D, the width t of the substrate108with respect to the width of the fluid channel w, w1, w2is an important parameter. It ranges from 550 to 1050 μm. In still a further embodiment, the microchannel(s) continue to sit atop a piezoelectric transducer (or surface acoustic wave transducer) for generating the acoustic standing mode which acts on the microparticles (or cells) in the microchannel(s). However, this embodiment does not employ a set of electroporation electrodes for generating electric fields in the fluid. Such a configuration, i.e., without electroporation electrodes, is useful for “washing” cells or for transferring them from one media to another, for example. It can represent a good alternative to the conventional method that involves spinning down the microparticles (cells) in a centrifuge, removing the supernatant, adding the second buffer, and resuspending the microparticles. Shown inFIGS.5A and5Bare microscopy images demonstrating acoustically-driven rapid buffer exchange. When the acoustics are off (FIG.5A), cells in the side streams pass through without being deflected and remain in their buffer. When the acoustics are activated (FIG.5B), cells are deflected from the side streams into the center stream, which can include a different buffer or contain different reagents. In some embodiments of the invention, the system illustrated inFIG.1employs an electroporation assembly16that consists essentially of or comprises at least one microchannel arrangement (device) such as that shown inFIG.3. An electroporation assembly also can include two or more (i.e., multiple or a plurality of) electroporation arrangements in a parallel configuration as further described below. Such an assembly can be used not only in the high throughput, high efficiency continuous flow systems illustrated inFIG.1, but also in other systems or independently, as flow electroporation devices for transfection of cells (or other targets such as exosomes, etc.) that have been loaded into an appropriate, low-conductivity, electroporation buffer using conventional means (e.g., centrifugation and resuspension). In this configuration, transfected cells are collected at the output and returned to incubation or storage conditions manually. Further implementations utilize the electroporation assembly with parallel channels in systems that do not employ acoustically-driven buffer exchanges. FIG.6is an exploded view of assembly200which includes several layers or plates (namely layers210,230,250,270and290) further described inFIGS.8-12. The layers can be laminated in a stacked configuration as shown inFIGS.7(top view) and8(bottom view). In one implementation, the laminate is prepared by curing a suitable adhesive film disposed between adjacent plates. Turning to the individual layers, shown inFIG.9is viewing layer210which includes openings212that provide access for solder bonding to the electrodes (48aand48b) and can have a thickness of 10 millimeters (mm or mil). In one example, layer210is made of Ultem® material. FIG.10shows fluidic channel layer230including a plurality (four being shown in the figure) of fluidic microchannels, each having the trifurcating inlets and outlets for a central and side streams and the microfluidic center channel46, essentially as described with reference toFIG.2B. The specific embodiment described above has four parallel fluidic microchannels. In a preferred arrangement, these would be used in parallel to process cells from a single common incubator12. The use of the parallel microchannels allows for higher throughput, such as higher than 4 million cells per minute. In general, micro channels cannot be simply made wider since the electric fields are optimally placed across a few or even single cells. Thus, in further implementations, higher numbers of parallel fluidic microchannels are fabricated in a single assembly200. One embodiment includes at least 10, 20, 30, 40, 50 or more fluidic microchannels that are operated in parallel between an input common incubator12to output modified cells to a common output incubator20. In more detail, each fluidic microchannel has trifurcating inlets232a,232band232cand trifurcating outlets232a′,232b′ and232c′. Hypodermic stainless steel tubing (e.g., nominally 25 Gauge tubing, 0.4 inches long) inserted into these inlets/outlets and sealed with epoxy can serve as an interface to the device for introducing cells and fluids. Plastic tubing (e.g., 0.38 millimeter inner diameter, vinyl) can be press fit onto the stainless steel tubing. As seen in the figure, the fluidic microchannels are arranged in a parallel configuration. Fluidic channel plate230can have a thickness of about 10 mil and can be fabricated from Ultem®, for example. Preferably, the channels, and especially the microfluidic center channel46are fabricated in the fluidic channel layer230by forming slots all the way through the layer. Different technologies can be used upon the slots. In the current implementation, the slots are formed with laser machining. Other options are mechanical milling and photolithographic processes, to list a few examples. The layer shown inFIG.11is electrode frame250, provided with electrode slots (or trenches)254aand254b. These slots are formed all the way through the electrode frame250using a fabrication technique as described earlier. The slots enable fluid communication between the proximal (long) edges of each of the rhomboid electrodes and the fluid in microfluidic center channel46. The electrode frame can be made from Ultem® or Kapton®, with a thickness of about 1 mil, for instance. Holes256are provided for lamination alignment dowel pins. FIG.12shows electrode layer270including four pairs of rhomboid metalizations272aand272bthat form the pairs of coplanar rhomboid electrodes (48aand48b). The metalizations can be fabricated by the deposition (e.g., sputtering through a shadow mask) of an electrochemically stable material, platinum metal, for example, on an Ultem® film of 5 mil, for example. Fiducial markers274are provided for alignment of the deposition mask used in the fabrication of the electrode layer. Shown inFIG.13is port plate290, made, for example, from a 3/32″ thick Ultem® material and provided with openings292a,292band292c, for access to the trifurcating inlets46a,46band46c(FIGS.2B and9), and openings292a′,292b′ and292c′ for access to trifurcated outlets46a′,46b′ and46c′ (FIGS.2B and10). These openings are sized to accommodate tubes300shown inFIG.5. As described above, hypodermic stainless steel tubing (nominally 25 Gauge tubing, 0.4 inches long) can be inserted through these openings, connected to the inlets/outlets of the microchannels and sealed with epoxy to form an interface to the device for introducing cells and fluids. A suitable gauge plastic tubing can be press fit onto the stainless steel tubing. For proper orientation during assembly and fabrication, plates210,230,250,270and290can be marked by chamber297. The plates are preferably adhered to each other. In one example, an adhesive film such as R/flex 1000 sheets is attached to some plates prior to laser machining. The adhesive sheets are then cured with high temperature and pressure, for example. A system such as system10(FIG.1), optionally including one or more of the subassemblies described above (e.g., with reference toFIGS.2-4D and6-13), can be operated as follows. A cell container, such as a blood bag, is introduced into the first incubator12and attached to an agitation mechanism that prevents the cells from settling due to sedimentation. Silastic tubing makes a fluidic connection between the blood bag and a first rapid buffer exchange module and a peristaltic pump30drives a flow of cells into the buffer exchange device. At the entrance to the buffer exchange device, the flow bifurcates to form the outer streams of a sheath flow. A second pump32(e.g., a syringe pump) drives the flow of electroporation buffer containing cargo from the reservoir22to be delivered to the cells (mRNA, pDNA, RNP, etc.) into the center stream of the sheath flow in the buffer exchange device. An acoustic field driven by a piezoelectric actuator driven by the acoustic function generator42attached to the device drives cells from the outer streams into the center stream, into the cargo-containing, low-conductivity electroporation buffer. The entire sheath flow then passes into a flow electroporation module (which can be directly integrated into the buffer exchange device), where electrodes in contact with the outer streams are energized to expose the cells to electric fields. In other arrangements, it is just the center stream that passes into the electroporation device, with fresh high-conductivity buffer being introduced into the side streams. The outer streams then flow to waste, while the center stream is introduced into a second buffer exchange device. At the entrance to the second buffer exchange device, the flow coming from the electroporation module bifurcates to form the outer streams of a sheath flow. A pump36(e.g., a syringe pump) pumps cell culture or recovery buffer from the cell media reservoir26into the second rapid buffer exchange device to form the center of a sheath flow. An acoustic field driven by a piezoelectric actuator driven by the acoustic function generator42then drives the cells from the outer streams into the center stream, returning them to a culture buffer which can be the same or different from the initial buffer in the blood bag in the first incubator. The cells then flow into a collection receptacle in a second incubation chamber20. To facilitate or enhance the transfer of the payload into permeabilized cells, applying acoustic energy (using a sonicator, for example) can increase collisions between cells and the cargo material, thereby increasing the probability of bringing the cell and cargo material in close proximity and loading the cargo into the cells. An increase in the collision rate between cells and cargo also can be obtained using mechanical agitation or other suitable means. This happens naturally in the acoustic buffer exchange modules, but additional acoustic agitation can be added (e.g., using a piezoelectric transducer bonded to the outside of the device) to the flow electroporation device shown inFIG.3. Some embodiments provide for multiple buffer exchanges and/or multiple delivery events (multiple electroporation operations) to take place before cells are returned to the culture buffer in an incubator. This may require additional buffer exchange modules, flow electroporation modules, and pumps. At least some of the various transfers of cells into the electroporation buffer(s), electroporation, and transfer of cells out of electroporation buffer(s) back into a suitable cell culture buffer can be integrated into a single module. In many embodiments, processes carried out in system10are conducted in continuous fashion. Since the system can be modular, it can be customized based on the application. For example, some processes may require only a single buffer exchange, while others may require several sequential buffer exchanges and several electroporation events for delivery of multiple payloads in sequence. In yet other embodiments, the cells and/or reagents are recirculated for multiple passes. Principles described herein also can be employed to remove some or all the contents held in cells or other membrane bound structures; that is, opening pores and allowing the internal contents to diffuse out either passively or via an active force. As with conventional bulk electroporation techniques, the flow arrangement described herein remains compatible with the electroporation of cells in small batches. However, this is time and touch-labor intensive and will be intractable for large-scale processing. Equipment and techniques described herein can increase throughput and/or improve the efficiency with which cargo is transferred to cells or other membrane bound structures. As described, various measures are taken to protect cells before, during and after the electroporation process, improving cell viability. Thus, in some embodiments, cell electroporation can be conducted with a throughput of at least 4 million cells processed per minute, e.g., a throughput within the range of from about 4 million to about 50 million cells per minute. Transfection efficiencies for a genetic cargo such as mRNA to primary human T cells can be as high as 90% (with less than a 5% reduction in cell viability), within the range of from about 65% to about 90%, as indicated by expression of a fluorescent reporter protein measured by flow cytometry. In some cases, the efficiency of transferring cargo to cells can be increased by raising the cargo concentration, by promoting collisions between cells and cargo, increasing the electric field dosage (potentially at the cost of viability) and/or optimizing the electroporation buffer. Cell viability, measured, for example, by flow cytometry, can be as high as 95% of the initial cell viability, within the range of from about 80% to about 95%, for instance. Features described herein are consistent with scale up and commercial manufacturing goals and thus, embodiments described herein can find many applications. Examples include but are not limited to the production of Autologous or Allogeneic CAR-T, Allogeneic or Autologous TCR, TRnC cells, modified TILs, CAR-NKTs, CAR-NKs, CAR-Macs, CAR-CIK or modified gamma delta cells. The features described herein can also be used to engineer cargo-loaded exosomes, or produce gene modified stem or suspension cells to treat genetic diseases or disorders. One illustrative application relates to cellular therapy manufacturing. Recent developments in adoptive cell transfer based immunotherapies have increased the demand for improved cell bioprocessing and gene delivery technologies. For instance, the FDA has granted approval for the use of T cells modified to express chimeric antigen receptor (CAR) genes for treatment of certain hematological cancers. However, the manufacturing chain for CAR T cell based therapeutics currently involves lentiviral-based transduction for gene delivery. These vectors are complex and expensive to manufacture and have limited payload capacity. Since they integrate genetic information into the genome in an uncontrolled way this approach also presents safety concerns. To address some of these problems, the non-limiting example below was conducted to investigate the feasibility of using embodiments described herein in the electrotransfection of primary human T cells for cellular therapy manufacturing. EXAMPLE Experiments were conducted using an assembly such as described above with reference toFIGS.6-13. In more detail, the assembly was constructed by laminating a stack of machined thin polymer layers together. Sheets of polyetherimide (PEI) were then machined either on a conventional CNC milling machine or an ultraviolet laser cutter. Each layer is backed with a layer of adhesive. To demonstrate proof of concept, the arrangement used was simplified and did not include a buffer exchange function. Rather, a microfluidic hydrodynamic sheath flow configuration was established by directing a low-conductivity electroporation buffer containing primary human T cells and mCherry-encoding mRNA (CleanCap mCherry mRNA, TriLink Biotechnologies, San Diego, CA) through the central inlet (inlet46cinFIG.3). High-conductivity culture buffer was supplied through the two side inlets (46a,46binFIG.3). The cells and media entered and exited the microchannel through ports at the upstream and downstream ends, respectively. Stainless steel (SS) tubing was inserted and epoxied in place at the fluid inlets and outlets. Each inlet and outlet had a trifurcation design which allowed for the generation of a stable sheath flow configuration wherein cells entered the center inlet in low-conductivity media and high-conductivity buffer was run in both side inlets, surrounding the center flow. Metal electrodes that were sputter-deposited on the floor of the channel were connected to external control circuitry by soldered wire leads. The patterned electrodes were rectangular in geometry and were positioned to make contact only with the sheath fluid. The arrangement facilitated a concentration of the electric field across the width of the low-conductivity media (negligible voltage drop across the high-conductivity buffer) and prevented the cells from making physical contact with the electrodes and the sidewalls of the channel. This was believed to promote cell recovery and viability. Experiments were conducted to investigate how electric field pulse magnitude, pulse duration, and the number of pulses applied affect transfection efficiency of mRNA into primary human T cells and the concomitant changes in cell viability and overall cell recovery. Higher electric field pulse magnitudes, and with longer exposure times, were found to increase transfection efficiency. According to one set of data, no transfection could be observed for field magnitudes of 67 kV/m and below. Transfection efficiency increased with increasing field magnitude in all cases starting at a magnitude of 102 kV/m. This suggested a critical electric field magnitude between 67 kV/m and 102 kV/m for transfection of mRNA into primary human T cells in this particular device. Generally, the data showed that it was possible to electroporate the cells in a continuous flow arrangement. A throughput of up to 8×106primary cells could be processed per minute, while achieving 72% electroporation efficiency as measured by the expression of a fluorescent reporter (mCherry) 24 hours after mRNA delivery. The cell viability was observed to be reduced by only 9% by the electroporation process and the total system recovery of cells was 61%. In specific examples, primary human T cells were electroporated using a commercial bulk electroporation system after being held in BTX electroporation media for 0, 15, or 60 minutes. Data is presented inFIGS.14A and14B, which shows average of replicates from 3 independent, healthy donors. Error bars represent the standard error of the mean. As seen inFIG.14A, transfection efficiency, as measured by flow cytometry 24 hours after electroporation, is reduced when T cells are held in BTXpress media with mRNA for 15 min, and is reduced further if the hold time is increased to 60 min. Post-transfection viability (FIG.14B), as measured by flow cytometry 24 hours after electroporation, decreased with increasing hold time, but the decrease is not statistically significant. FIG.15shows the viability and transfection efficiency (as percent of cells) for cells obtained from two different donors similarly electroporated in continuous flow according to aspects of the invention. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. | 60,604 |
11859163 | It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements can be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. DETAILED DESCRIPTION In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the bioreactors described herein, and use thereof. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the bioreactors described herein and uses thereof. The present application discloses a cell culturing processing and manipulating system including bioreactors and bioreactor systems designed for culturing of cells and microorganisms in changing densities and adaptive culture volumes starting from isolation to final formulation. The bioreactors disclosed herein are configured to continuously allow all the necessary steps of selecting, culturing, modifying, activating, expanding, washing, concentrating and formulating in one single unit. According to some embodiments, the bioreactors can be used in a batch mode, fed batch mode and perfusion mode and can be fully controlled in a closed, aseptic environment and can be implemented for a single use (to be disposed after one culturing cycle) as well as for multiple cycle uses. Before explaining the various embodiments of the bioreactors and systems thereof as disclosed herein in detail, it is noted that the bioreactors and systems thereof disclosed, are not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth in the following description and/or illustrated in the drawings and/or the Examples. The bioreactors and systems thereof disclosed herein can encompass other embodiments or of being practiced or carried out in various ways. The present application in some embodiments thereof, discloses a flow or a stream of a “medium”, “liquid”, “gas”, “wash buffer”, “solution” or “fluid”. A skilled artisan would appreciate that these terms are alternatively used and having a characteristic of a substance that continually deforms (flows) under an applied pressure and/or an applied shear stress. The present application in some embodiments thereof, discloses bioreactors for growing living cells or microorganisms, and methods thereof for growing cells or microorganisms in these bioreactors including all culturing steps from isolation to final formulation. A skilled artisan would appreciate that the terms “cell” and “cells” may encompass any living cells. In some embodiments, cells that may be grown in a bioreactor disclosed herein comprise any prokaryotic or eukaryotic cell. In some embodiments, cells that may be grown in a bioreactor disclosed herein comprise unicellular and multicellular microorganisms, for example bacteria, archaebacteria, viruses, yeast cells, plant cells, or insect cells. In some embodiments, eukaryotic cells comprise plant cells, insect cells, animal cells, or fungi. In some embodiments, cells comprise tissue culture cells, primary cells, or reproductive cells. In some embodiments, tissue culture cells or primary cells comprise stem cells, adult cells, transdifferentiated cells, dedifferentiated cells, or differentiated cells. In some embodiments, animal cells comprise mammalian cells. For example, mammalian cells may comprise cells originating from a baboon, buffalo, cat, chicken, cow, dog, goat, guinea pig, hamster, horse, human, monkey, mouse, pig, quail, or rabbit. In some embodiments, mammalian cells comprise primary cells comprising stem cells, embryonic cells, adult cells, transdifferentiated cells, dedifferentiated cells, or differentiated cells. In some embodiments, mammalian cells comprise tissue culture cells comprising stem cells, embryonic cells, adult cells, transdifferentiated cells, dedifferentiated cells, or differentiated cells. In some embodiments, the cell types compatible with growth in a bioreactor disclosed herein include stem cells, Acinar cells, Adipocytes, Alveolar cells, Ameloblasts, Annulus Fibrosus Cells, Arachnoidal cells, Astrocytes, Blastoderms, Calvarial Cells, Cancerous cells (Adenocarcinomas, Fibrosarcomas, Glioblastomas, Hepatomas, Melanomas, Myeloid Leukemias, Neuroblastomas, Osteosarcomas, Sarcomas) Cardiomyocytes, Chondrocytes, Chordoma Cells, Chromaffin Cells, Cumulus Cells, Endothelial cells, Endothelial-like cells, Ensheathing cells, Epithelial cells, Fibroblasts, Fibroblast-like cells, Germ cells, Hepatocytes, Hybridomas, Insulin producing cells, Intersticial Cells, Islets, Keatinocytes, Lymphocytic cells, Macrophages, Mast cells, Melanocytes, Meniscus Cells, Mesangial cells, Mesenchymal Precursor Cells, Monocytes, Mononuclear Cells, Myeloblasts, Myoblasts, Myofibroblasts, Neuronal cells, Nucleus cells, Odontoblasts, Oocytes, Osteoblasts, Osteoblast-like cells, Osteoclasts, Osteoclast precursor cells. Oval Cells, Papilla cells. Parenchymal cells, Pericytes, Peridontal Ligament Cells, Periosteal cells, Platelets, Pneumocytes, Preadipocytes, Proepicardium cells, Renal cells, Salisphere cells, Schwann cells, Secretory cells, Smooth Muscle cells, Sperm cells, Stellate Cells. Stem Cells, Stem Cell-like cells, Stertoli Cells, Stromal cells, Synovial cells, Synoviocytes, T Cells, Tenocytes, T-lymphoblasts, Trophoblasts, Natural killer cells, dendritic cells, Urothelial cells, Vitreous cells, and the like; the cells originating from, for example and without limitation, any of the following tissues: Adipose Tissue, Adrenal gland, Amniotic fluid, Amniotic sac, Aorta, Artery (Carotid, Coronary, Pulmonary), Bile Duct, Bladder, Blood, Bone, Bone Marrow, Brain (including Cerebral Cortex), Breast, Bronchi, Cartilage, Cervix, Chorionic Villi, Colon, Conjunctiva, Connective Tissue, Cornea, Dental Pulp, Duodenum, Dura Mater, Ear, Endometriotic cyst, Endometrium, Esophagus, Eye, Foreskin, Gallbladder, Ganglia, Gingiva, Head/Neck, Heart, Heart Valve, Hippocampus, Iliac, Intervertebral Disc, Joint, Jugular vein, Kidney, Knee, Lacrimal Gland, Ligament, Liver, Lung, Lymph node, Mammary gland, Mandible, Meninges, Mesoderm, Microvasculature, Mucosa, Muscle-derived (MD), Myeloid Lukemia, Myeloma, Nasal, Nasopharyngeal, Nerve, Nucleus Pulposus, Oral Mucosa, Ovary, Pancreas, Parotid Gland, Penis, Placenta, Prostate, Renal, Respiratory Tract, Retina, Salivary Gland, Saphenous Vein, Sciatic Nerve, Skeletal Muscle, Skin, Small Intestine, Sphincter, Spine, Spleen, Stomach, Synovium, Teeth, Tendon, Testes, Thyroid, Tonsil, Trachea, Umbilical Artery, Umbilical Cord, Umbilical Cord Blood, Umbilical Cord Vein, Umbilical Cord (Wartons Jelly), Urinary tract, Uterus, Vasculature, Ventricle, Vocal folds and cells, or any combination thereof. In some embodiments, the cells grown in a bioreactor disclosed herein may comprise a combination of different cell types. As used herein, in some embodiments the terms “cells” and “microorganisms” may be used interchangeably having all the same meanings and qualities. In some embodiments, the products of the cells or microorganisms grown in a bioreactor disclosed herein are collected, for example proteins, peptides, antibiotics or amino acids. In some embodiments, any product of a cell or microorganism grown in a large-scale manner in a bioreactor disclosed herein and synthesized by the cell or microorganism, can be collected. The bioreactors disclosed in the present application, non-limiting of which are presented inFIG.1(10),FIG.2(110),FIG.3(210),FIG.4A(300),FIG.4B(310),FIG.4C(320),FIG.4D(330),FIG.4E(340),FIG.4F(350),FIG.4G(360),FIG.4H(370),FIG.4I(380),FIGS.6A and6B(510),FIGS.6C and6D(550).FIG.7(610),FIG.8(710),FIG.9(810),FIGS.10A and10B(910),FIGS.11A and11B(1010),FIGS.12A and12B(1110), andFIG.14A, can be shaped like a hollow vessel including a perforated barrier that divides the internal volume or space within the vessel into a first (lower) chamber and a second (upper) chamber disposed above the first chamber. According to some embodiments, a bioreactor described herein for growing cells or microorganisms therein, the bioreactor comprising:a closed vessel enclosing a space therein;a barrier having a plurality of perforations therein, the barrier is sealingly disposed within the space configured to divide the space into a first chamber and a second chamber, wherein the second chamber is configured to accommodate the growing cells or microorganisms therein, and wherein a diameter of the perforations is configured to allow a fluid flow solely between the first chamber and the second chamber and vice versa,one or more fluid inlet ports for introducing the fluid into the first chamber; andone or more fluid outlet ports for allowing the fluid to exit from the second chamber. According to some embodiments, the bioreactor vessel can be constructed of at least two parts. And according to some embodiments, the barrier can be attached between the two parts. According to some embodiments, more perforated barriers can be provided, in some cases between the different parts of the vessel. According to some embodiments, the barrier is disposed in contact with walls of the vessel (as demonstrated inFIGS.1-4,6-13,15-16). According to some embodiments, the first chamber is a lower chamber and the second chamber is an upper chamber and wherein the fluid flow is an upstream flow from the lower chamber towards the upper chamber (against gravity direction). Without being limiting, in some embodiments, a bioreactor comprises a chamber comprising a widening shape, for example a conical frustum shape, or a portion thereof, which is configured to lead to reduction of velocity of a fluid. In some embodiments, a bioreactor comprises a chamber of two parts divided by a perforated barrier, wherein the barrier allows a constant fluid flow, for example but not limited to a fluid growth media, and wherein the cells are retained in the second (upper) chamber. In some embodiments, a bioreactor comprises reduced velocity of flow of a fluid in the second (upper) chamber and a uniform and gentle flow of a fluid throughout the vessel. In some embodiments, the gentle and uniform flow combined with the reduced velocity in the second (upper) chamber results in a balance between the mass of cells (cell mass) and the velocity of the fluid resulting in a steady mass of cells known as a “floating cake”. In some embodiments, a floating cake of cells localized to the lower portion of the second (upper) chamber. In some embodiments, use of a bioreactor described herein results in a constant fluid flow. In some embodiments, use of a bioreactor results in a constant flow of growth media and cell feeding during the culturing process. In some embodiments, a fluid, for example a growth media, can be exchanged during culturing, wherein very small volumes and/or very large volumes provide the for adaptive and optimal cell feeding. In some embodiments, use of a bioreactor described herein comprises cell washing and harvesting to a selected media in a very gentle and efficient manner without the need to open the bioreactor chamber. In some embodiments, use of a bioreactor described herein provides for optimal and adaptive culturing, wherein manipulation of cells or microorganisms is performed in a closed system, wherein the manipulation can be automated, and wherein cells experience minimal sheer force. In some embodiments, use of a bioreactor described herein supports high density growth of cells or microorganisms. In some embodiments, the density achieved, by the bioreactors disclosed herein, can be greater than 10-fold that observed using standard culturing conditions. A skilled artisan would appreciate that the term “perforated barrier” may be used interchangeable with the term “filter” or “membrane” or “perforated plate” having all the same qualities and meanings. In some embodiments, the perforated barrier comprises a plurality of perforations therein that is configured to allow bidirectional flow of a liquid, for example a growth media through the perforations of the perforated barrier such that liquid can flow from the first chamber to the second chamber and also from the second chamber to the first chamber. A skilled artisan would appreciate that the term “first chamber” as used herein, may in some embodiments be used interchangeable with the term “lower chamber” having all the same meanings and qualities thereof. A skilled artisan would appreciate that the term “second chamber” as used herein, may in some embodiments be used interchangeable with the term “upper chamber” having all the same meanings and qualities thereof. In some embodiments, cells are cultured in the second chamber of bioreactor vessel. In some embodiments, the perforated barrier is configured to allow bidirectional flow of liquid including additional factors through the perforations of the perforated barrier such that liquid and additional factor or factors can flow from the first chamber to the second chamber and from the second chamber to the first chamber. In some embodiments, the perforation diameter is configured to allow liquid flow solely from the first chamber to the second chamber and from the second chamber to the first chamber. In some embodiments, the perforation diameter is configured to allow liquid including a factor or factors to flow solely from the first chamber to the second chamber and from the second chamber to the first chamber. In some embodiments, the factor or factors does not include cells or microorganisms. In some embodiments, the perforated barrier comprising a plurality of perforations, which do not allow cells or microorganisms grown in the vessel of the bioreactor to pass through the perforated barrier. A skilled artisan would appreciate that flow may encompass flow of a liquid fluid comprising a growth media, a washing solution, a nutrient solution, a selection solution, an enzyme mixture solution, a collection solution, a final formulation solution, a storage solution, or any combination thereof. In some embodiments, a liquid comprises a growth media, a washing solution, a nutrient solution, a collection solution, a harvesting solution, a storage solution, or any combination thereof. In some embodiments, a liquid comprises additional factors, wherein non-limiting examples of factors that may be added include nutrients, gasses, activation factors, induction factors, antibiotics, antifungal agents, and salts. In some embodiments, any factor beneficial for the growth and collection of cells or microorganisms in bioreactor systems described herein may be added to a liquid. In some embodiments, a factor dissolves within the liquid, wherein the liquid represents a solvent and the factor a solute to form a solution. In some embodiments, a factor remains as a particulate within the liquid. A skilled artisan would appreciate that the term “plurality” may encompass the number of perforations (pores) in a perforated barrier. In some embodiments, the plurality of perforations is determined based on a needed rate of exchange of media or other liquid flowing from a first chamber to a second chamber, or from a second chamber to a first chamber. In some embodiments, the plurality of perforations is determined based on the flow rate of media or other liquid flowing from a first chamber to a second chamber, or from a second chamber to a first chamber. In some embodiments, the plurality of perforations is determined based on the pattern of flow of media or other liquid flowing from a first chamber to a second chamber, or from a second chamber to a first chamber. In some embodiments, the arrangement of perforations within a perforated barrier is configured to affect the pattern of flow of a media or other liquid flowing from a first chamber to a second chamber, or from a second chamber to a first chamber. In some embodiments, a perforated barrier comprises an evenly spaced plurality of perforations. In some embodiments, a perforated barrier comprises an uneven spacing of a plurality of perforations. In some embodiments, the mean perforation diameter or effective mean diameter of the perforations in the perforated barrier is selected such that it does not allow cells or microorganisms grown in the bioreactor to pass through the perforated barrier. For example, in some embodiments, determining of the size of a perforation diameter comprises measuring a cell or microorganism size and determining a cell or microorganism shape, choosing a perforation diameter (perforation pore size) that would prevent the cell or microorganism from passing through a perforated barrier having the chosen pore size. According to some related embodiments, the mean perforation diameter or effective mean diameter of the perforations in the perforated barrier is selected to be smaller than: 120 micrometer, or 100 micrometer or, 75 micrometer, or 50 micrometer, or 25 micrometer, or 15 micrometer. According to some related embodiments, the mean perforation diameter or effective mean diameter of the perforations in the perforated barrier is selected to be larger than: 0.1 micrometer, or 0.2 micrometer, or 0.3 micro meter or, 0.45 micrometer, or 0.75 micrometer or, 1.0 micrometer. According to some related embodiments, the mean perforation diameter or effective mean diameter of the perforations in the perforated barrier is selected between 0.1 micrometer and 120 micrometer. According to some related embodiments, the mean perforation diameter or the effective mean diameter of the perforations in the perforated barrier does not allow cells or microorganisms to pass from one chamber to a second chamber. For example, the mean perforation diameter or the effective mean diameter of the perforations in the perforated barrier is selected so that cells or microorganisms grown in an upper chamber may not pass into the lower chamber. In some embodiments, the cell or microorganism have a spherical shape, accordingly the diameter of the cell or microorganism is used in determining perforation size. In some embodiments, the cell or microorganism may not have a spherical shape. In some embodiments, a cell or a microorganism may comprise a non-symmetrical shape, for example but in no way limiting a rod shape. Wherein a cell or a microorganism has a non-symmetrical shape, measurement for determining pore size would be based on the smallest diameter presented by a cell. In some embodiments, a cell may have the capacity to change shapes. Wherein a cell or a microorganism has the capacity to change shape, measurement for determining pore size would be based on the smallest diameter presented by the cell or microorganism that would allow passage of a cell or microorganism through a pore. In some embodiments, a cell or a microorganism may be deformable. Wherein a cell or a microorganism is deformable, cell size determination takes into account the diameter of the deformed cell or microorganism. In some embodiments, a plurality of perforations comprises perforations of all the same size. In some embodiments, a plurality of perforation comprises perforations that am not all the same size. In some embodiments, perforations of different sizes comprise a random distribution. In some embodiments, the distribution of perforations of different sizes is determined based on fluid flow patterns from the flow of a liquid from a first chamber to the second chamber and from the second chamber to the first chamber. In some embodiments, the shape of the perforations is symmetrical. In some embodiments, the shape of the perforations is non-symmetrical. In some embodiments, the shape of the perforation comprises a circular shape, an irregular in shape, an elliptical shape, or a polygonal. In some embodiment, a plurality of perforations comprises perforations all of the same shape. In some embodiment, a plurality of perforations comprises perforations of different shapes. In some embodiments, the mean perforation diameter or effective mean diameter of the perforations in the perforated barrier is determined by selecting a diameter configured to allow the flow of a liquid from a first chamber to the second chamber and also from the second chamber to the first chamber, and does not allow cells or microorganisms grown in the bioreactor to pass through the perforated barrier. In some embodiments, the mean perforation diameter or effective mean diameter of the perforations in the perforated barrier is determined by selecting a diameter that allows for the flow of a liquid comprising additional factors from a first chamber to the second chamber and also from the second chamber to the first chamber, and does not allow cells or microorganisms grown in the bioreactor to pass through the perforated barrier. In some embodiments, the mean perforation diameter or effective mean diameter of the perforations in the perforated barrier is determined by selecting a diameter that allows for the flow of a liquid comprising additional factors and products produced from the cells or microorganisms from a first chamber to the second chamber and also from the second chamber to the first chamber, and does not allow cells or microorganisms grown in the bioreactor to pass through the perforated barrier. In some embodiments, the perforation diameter (pore size) or effective mean diameter comprises about 0.1 to 40 micrometer. In some embodiments, the perforation diameter (pore size) or effective mean diameter comprises about 0.2 to 10 micrometer. In some embodiments, the perforation diameter (pore size) or effective mean diameter comprises about 10 to 40 micrometer. In some embodiments, the perforation diameter (pore size) or effective mean diameter is larger than 40 micrometers. In some embodiments, the perforation diameter (pore size) or effective mean diameter comprises about 40 to 60 micrometer. In some embodiments, the perforation diameter (pore size) or effective mean diameter comprises about 60 to 100 micrometer. In some embodiments, the perforation diameter (pore size) or effective mean diameter is configured to prevent cells or microorganisms, to flow through the pore. In some embodiments, the perforation diameter or effective mean diameter is configured to prevent cells or microorganisms bound to beads to flow through the pore. In some embodiments, the pore diameter, of the perforations of a perforated barrier having a plurality of perforations therein, is configured to allow solely liquid flow from the first chamber to the second chamber and from the second chamber to the first chamber. In some embodiments the liquid can comprise solutes and/or added factors. In some embodiments, the pore diameter of the perforations of a perforated barrier having a plurality of perforations therein, is configured to allow solely liquid flow from the first chamber to the second chamber and from the second chamber to the first chamber, wherein the pore diameter is configure to not allow the passage of cells or microorganisms from the first chamber to the second chamber and from the second chamber to the first chamber. In some embodiments, the perforated barrier is configured and useful, for example, in confining the grown cells to the second chamber within the reactor and in harvesting the cells. According to some embodiments, the present application also discloses bioreactor systems including the bioreactors and methods for growing cells or microorganisms in the bioreactors and bioreactor systems from isolation to final formulation. In some embodiments, a bioreactor comprises an additional lower perforated barrier12D below the perforated barrier12(which is present at the bottom of the upper chamber); see for exampleFIG.1(12) wherein the perforated barrier12comprises the bottom of the upper chamber. In some embodiments, the additional lower perforated barrier12D is located between the bottom surface of the vessel (at the lower chamber) and the perforated barrier12(which is forming the bottom surface of the upper chamber); for example between10B and12ofFIG.1. In some embodiments, the upstream flow of liquid from a lower chamber to an upper chamber passes through the two perforated barriers12and12D. The additional lower perforated barrier12D is configured to assist in aligning the flow of a liquid (straitening, providing linearity and uniform flow thereto) before it reaches the perforated barrier12that comprises the bottom of the upper chamber. This arrangement is configured to improve the linearity (and uniformity) of a liquid's flow. According to some embodiments, aligning the stream comprises providing an approximately even longitudinal flow rate along different radial locations of the perforated barrier [v(r1)≈v r2)], or in other words the flow rate is substantially equal at every distance of the geometrical center of the perforated barrier. According to some embodiments the lower perforated barrier is sealingly attached to the walls of the lower chamber, and wherein its pores size is configured the prevent passage of cells or microorganism. According to some embodiments, both the perforated barrier12and the lower perforated barrier12D are configured to align the liquid flow rate. According to some related embodiments, the mean perforation diameter or effective mean diameter of the perforations in the lower perforated barrier12D is selected between 0.1 micrometer and 1 millimeter. According to some embodiments, the lower perforated barrier12D is configured to control the fluid velocity. A non limiting example for such a velocity controlling barrier1600is detailed inFIG.16. As demonstrated inFIG.16, the pores1601of a velocity controlling perforated barrier1600can comprise conical shapes; conical shape of the pores can be similar or different between the different pores, some pores can be similar and some can be different. According to some embodiments, the wider base of the conical pores is located at the bottom side of the barrier; such a configuration can provide the flow with an increasing flow rate towards the upper side of the barrier. According to some embodiments, pore/s1602closer to the center of the barrier can have a wider cone, or a wider opening at the upper side of the barrier, than of the peripheral pores1601; such a configuration can provide an approximately even longitudinal flow rate along the different radial locations [v(r1)≈v(r2)] of the perforated barrier1600. According to such embodiments, a fluid impeller may not be required. In some embodiments, the presences of the additional lower perforated barrier12D is configured to trap air bubbles, air clusters, and debris which would otherwise clog and block flow through perforations of the upper perforated barrier12and interfere with the linearity and uniformity of flow. In some embodiments, a bioreactor comprises an additional screening perforated barrier1502above the perforated barrier (first perforated barrier)1512(which is present at the bottom of the upper chamber), the screening perforated barrier is disposed sealingly to the walls of the upper chamber.FIG.15Ademonstrates the first perforated barrier1512and the additional screening perforated barrier1502, which is positioned above the level of the cells mass3. According to some embodiments, the additional screening perforated barrier1502is configured to prevent cells or microorganism passage for example to prevent the cells from leaving the bioreactor. In some embodiments, the bioreactor vessel is in an inverted position (See also Example 3 below) the flow of liquid is downstream1520(approximately with gravity direction) from an upper (the second1540) chamber to a lower (the first1550) chamber. This configuration is configured in some embodiments to be used during washing of cells or exchange of media or liquid solutions allowing wider surface area barrier, which enables to reduce a clogging of the barrier by the cell mass. According to related embodiments, the bioreactor comprises, three perforated barriers:a primary perforated barrier1512(FIG.15A), configured to separate between the upper and the lower chambers (1540,1550) of the bioreactor's vessel and to prevent passage of cells and microorganism there between;an upper perforated barrier1502(FIG.15A), located in the upper chamber1540above cell mass3configured to prevent passage of cells and microorganism; therefore cell mass is kept between the primary and the upper perforated barriers (1512,1502) and;a lower perforated barrier12D (FIG.1), located in the first chamber14A below primary perforated barrier12, configured to align and/or control the fluid flow before reaching to the primary perforated barrier12. According to some related embodiments, the primary and the upper perforated barriers (1512,1502,FIG.15A) comprise similar pores size configured to prevent passage of cells or microorganisms. According to some embodiments, the size of the pores of the lower perforated barrier (12D,FIG.1) can be similar to—or can be different than—the size of the pores of the primary and the upper barriers (1512,1502,FIG.15A). One skilled in the art would appreciate that the range, shape, and distribution of pores may be similar or different between the different perforated barriers. In some embodiments, the diameter or effective diameter of the perforations (pores) of an additional perforated barrier comprise different sizes of pores than is present in the perforated barrier that separates the first and second chambers. In some embodiments, the diameter or effective diameter of the perforations (pores) of an additional perforated barrier comprise similar sizes of pores than perforated barrier that separates the first and second chambers. In some embodiments, the shape of the perforations (pores) of an additional perforated barrier comprises different shapes of pores than is present in the perforated barrier that separates the first and second chambers. In some embodiments, the shape of the perforations (pores) of an additional perforated barrier comprises similar shapes of pores than the perforated barrier that separates the first and second chambers. In some embodiments, the distribution of the perforations (pores) of an additional perforated barrier comprises different distribution of pores than is present in the perforated barrier that separates the first and second chambers. In some embodiments, the distribution of the perforations (pores) of an additional perforated barrier comprises similar distribution of pores than the perforated barrier that separates the first and second chambers. In some embodiments, a bioreactor comprises an additional barrier with the second chamber above the cells and an additional barrier within the first chamber below the barrier that separates the first and second chambers. One skilled in the art would appreciate that the surface area of an additional perforated barrier can be greater than or less than the surface area of the barrier that separates the first chamber from the second chamber. In some embodiments, an additional perforated barrier has a larger surface area than the surface area of the barrier that separates the first chamber from the second chamber. In some embodiments, an additional perforated barrier has a smaller surface area than the surface area of the barrier that separates the first chamber from the second chamber. The disclosed bioreactors and bioreactor systems allows growing, processing and formulating the cells or other microorganisms in one closed single or multiple use system minimizing the risk of contamination and allowing efficient processing. According to some embodiments, bioreactors disclosed herein are configured to allow growing cells or other microorganisms to a desired concentration. In one embodiment, bioreactors disclosed herein provide a sterile environment. In one embodiment, bioreactor systems disclosed herein provide a sterile environment. Furthermore, as the cells or microorganisms are cultured and propagated they require more media and nutrients and larger culturing volumes. Some embodiments of the bioreactors described hereinafter include adaptive controlled volume changes (variable bioreactor volume) and media refreshment without the need to transfer the cells or microorganisms to a larger container. In some embodiments, the bioreactors of the present application are configured to be used for growing non-adherent cells, which are suspended in the growth medium. In some embodiments, the bioreactors disclosed herein are configured to be used for growing adherent cells by including or adding a suitable cell supporting matrix into the second chamber of the bioreactor. The cell supporting matrix can be any type of cell supporting matrix known in the art to which the cells can adhere. If such a cell supporting matrix is being used in the bioreactor, it may be necessary to detach the cells from the cell supporting matrix by using detachment methods known in the art. As used herein, in some embodiments, the terms “cell supporting matrix” and “cell carrier matrix” and conjugates thereof may be used interchangeably having all the same meanings and qualities. The bioreactors of the present application are configured to have a fixed volume or a variable volume. A skilled artisan would appreciate that in some embodiments, the terms “bioreactor” and “vessel” may be used interchangeable having all the same meanings and qualities. In embodiments wherein the bioreactor comprises a fixed volume, the rate of flow of a liquid, for example a growth medium can be controlled but the level and volume of the liquid, for example a growth medium in the bioreactor is substantially fixed. In embodiments wherein the bioreactor comprises a variable volume, the rate of flow of the liquid, for example a growth medium can be controlled and the level and volume of growth medium in the bioreactor can be variable. In some embodiments, variable the liquid levels, for example growth medium levels can be achieved by using multiple fluid outlet ports opening into the second chamber of the bioreactor at various different heights along the length of the walls of the bioreactor. A non-limiting example of this is presented inFIG.2. In some embodiments, the working volume of media is low, wherein cells are grown to high density cultures. In some embodiments, wherein the working volume is low, the rate of flow is also low or no flow at all. In some embodiments, the flow rate is low. In some embodiments, there is no flow from a first chamber to the second or from the second chamber to the first. In some embodiments, there is no flow from a first chamber to the second and from the second chamber to the first. In some embodiments, wherein the working volume is low, the medium is optimized for high density growth of cells. In some embodiments, wherein the working volume is low, cell growth is optimized for higher yields and lower media needs than are achievable in other bioreactors. In some embodiments, when a culture comprises a small number of cells, for example less than the maximal number of cells that can be cultured in a bioreactor described herein, the cells are cultured in a low volume of growth media, as cells proliferate and the number of cells increases the volume within the chamber comprising the cells can be increased. At a point a flow cycle can be implemented, wherein the flow of liquid, for example growth media, increases as the quantity of cells increases. In some embodiments, nutrients can be added to the liquid, e.g., a growth media based on cell growth needs. In some embodiments, culturing cells in a bioreactor described herein maintains cells within a cell density range by adjusting the volume of liquid, e.g., growth media, within the bioreactor. In some embodiments, use of a flow cycle as described herein results in lower growth media needs for culturing an equivalent number of cells. In some embodiments, a flow cycle is used in a bioreactor described herein, wherein the supply of a growth media is regulated based on cells' needs. In other words, cells are fed only as needed. In some embodiments, the flow cycle controls the proliferation rate of cells. According to some embodiments, each of the multiple outlet ports are configured to have a valve therein and configured be connected and disconnected fluidically to a common manifold feeding a pump. The level of a liquid, e.g., a growth medium in the bioreactor of such embodiments can be varied by suitably opening the valve of a selected fluid outlet port and closing all the valves of the remaining fluid outlet ports. According to some embodiments, controlling the volume of a liquid, e.g., a growth medium in the bioreactor advantageously allows expanding the culture as the cells continue to proliferate without opening the bioreactor and without the need of using methods used in other bioreactor systems, such as, for example cell passaging and dish/container replacement. In some embodiments, the bioreactors are configured to include a fluid impeller or fluid disperser disposed in the first (lower) chamber of the bioreactor's vessel. In some embodiments, the bioreactor is configured to include an oxygenating system for oxygenating the growth medium. Bubbles may in certain embodiments be created by the oxygenating system. Bubbles in a lower chamber may in some embodiments, have a negative impact on a bioreactor, as the bubbles may stick to a perforated barrier and interfere with the flow of liquid from one chamber to the next chamber. Additionally, nano bubbles that pass through the perforations of the barrier tend to lift cells up, which may interfere with the high density growth of a floating cell cake. According to some embodiments, the lower perforated12D (FIG.1) is configured to prevent passage of bubbles created or formed in the lower chamber from reaching and blocking the perforated barrier12; bubbles created for example by the oxygenating system. According to some embodiments, bubbles with an approximate diameter of several nanometers do pass the lower perforated barrier12D and the perforated barrier12and assist in lifting the cells or microorganism up the liquid's flow. According to some embodiments, the bioreactors disclosed herein are configured to have various different shapes and at least the portions of the walls of the bioreactors, which define the second chamber is configured to be straight (vertical) or configured to be slanted at an angle to the vertical (or slanted with respect to a longitudinal axis of the bioreactor). In some embodiments, some of the walls surrounding the second chambers are configured to be vertical and some of the walls are configured to be slanted. Non-limiting examples of shapes of the bioreactor vessel are presented inFIG.4A(304A and304B),FIG.4B(314A and314B),FIG.4C(324A and324B),FIG.4D(334A and334B),FIG.4E(344A and344B),FIG.4F(354A and354B),FIG.4G(364A and364B),FIG.4H(374A and374B),FIG.4I(384A and30B). The upward increasing transversal cross-sectional area of the second chamber in such embodiments is configured to allow a fluid velocity gradient to be established along the vertical direction (along the longitudinal axis of the bioreactor), such that the growth medium flow velocity decreases with increasing transversal cross-sectional area. According to some embodiments, this flow velocity gradient combined with the gravitational force acting on the cells suspended in the growth medium assists in suspending the cells at some desired region within the volume of growth medium contained in the second chamber. In some embodiments, regulation of flow rates of medium maintains cells in a desired position within a bioreactor. In some embodiments, regulation of flow rates of medium maintains cells in a desired position within a bioreactor. In some embodiments, regulation of flow rates in relation to the radius of the bioreactor, or chamber thereof, of medium maintains cells in a desired position within a bioreactor. In some embodiments, the desired position is lower than the exit port. For example seeFIG.1, if cells suspended within a liquid rise within the upper chamber at a flow rate of 1 mm per min (middle set of arrows37B), in the lower part there can be for example a flow rate of 3 mm per min (arrows at the level of the barrier37A), in the middle a flow rate of 1 mm per min (37B), and a few cm above were the media exits the chamber via a port/valve the flow rate can be 0.2 mm per min (would be above upper set of arrows37C and above the level of the exit port26). In some embodiments, the position of the cells is determined by the flow rate. In some embodiments, the position of the cells is lower than the exit port. A position for cells lower than the exit port can be desired when washing cells, when removing sub-populations of cells, when exchanging a liquid, when adding factors, or any combination thereof. A skilled artisan would appreciate that a cell population may comprise cells of different sizes, charge, and mass. In some embodiments, cells can be separated within different positions within a bioreactor disclosed herein, based on cell characteristics including size, charge, and mass. In some embodiments, cells are maintained within different positions within a bioreactor disclosed herein based on cell characteristics including size, charge, and mass. A skilled artisan would appreciate that cell size varies based on the type of cell. For example a red blood cell is about 6-8 mm in diameter, a T-lymphocyte is about 9-12 mm in diameter, a mesenchymal stem cell (MSC) is about 15-21 mm in diameter, and a macrophage is about 50 mm in diameter. The volume between cells can be dramatically different as well. In some embodiments, a bioreactor system disclosed herein is configured to be used to separate blood cells by regulating the flow rate. In some embodiments, the flow rate comprises a range of about 0.01 mm per minute to 50 mm per minute. In some embodiment, the flow rate comprises a range of about 0.01 mm/min to 0.1 mm/min. In some embodiment, the flow rate comprises a range of about 0.1 mm/min to 1.0 mm/min. In some embodiment, the flow rate comprises a range of about 1.0 mm/min to 2.0 mm/min. In some embodiment, the flow rate comprises a range of about 2.0 mm/min to 3.0 mm/min. In some embodiment, the flow rate comprises a range of about 3.0 mm/min to 4.0 mm/min. In some embodiment, the flow rate comprises a range of about 4.0 mm/min to 5.0 mm/min. In some embodiment, the flow rate comprises a range of about 5.0 mm/min to 10.0 mm/min. In some embodiment, the flow rate comprises a range of about 10 mm/min to 15 mm/min. In some embodiment, the flow rate comprises a range of about 15 mm/min to 20 mm/min. In some embodiment, in the flow rate comprises a range of about 20 mm/min to 25 mm/min. In some embodiment, the flow rate comprises a range of about 25 mm/min to 30 mm/min. In some embodiment, the flow rate comprises a range of about 30 mm/min to 35 mm/min. In some embodiment, the flow rate comprises a range of about 35 mm/min to 40 mm/min. In some embodiment, the flow rate comprises a range of about 40 mm/min to 45 mm/min. In some embodiment, the flow rate comprises a range of about 45 mm/min to 50 mm/min. In some embodiments, the flow rate within a bioreactor is different in different positions within the bioreactor (See for exampleFIG.1and the accompanying explanation thereof below, and the representative flow rate arrows37A.37B, and37C, orFIG.13and representative flow rate arrows37A and37C). In some embodiments, the size, charge, and/or mass of a population of cells can be artificially changed. For example, in some embodiments, cells can be cultured with beads, wherein the cells bind to the beads resulting in cell-bead complexes having a higher mass and different shape then the cells not attached to beads. In some embodiments, 100% of cells are bound to a bead. In some embodiments, a sub-set of cells are bound to a bead. In some embodiments, at least 90% of cells, 80% of cells, 70% of cells, 60% of cells, 50% of cells, 40% of cells, 30% of cells, 20% of cells, or 10% of cells are bound to a bead. In some embodiments, less than 10% of cells are bound to a bead. In some embodiments, cells bound to beads are excluded from collection of the final cell population. In some embodiments, cells bound to beads are the cells desired to be collected as the final cell population. For example, in one embodiment, following addition of beads, wherein a subpopulation of cells binds to the beads in a specific fashion, increasing the flow rate will result in the cells not bound to beads rising at an increased rate compared with the cells bound to the beads, so these non-bound cells can exit the vessel chamber from an exit port wherein the bound cells remain in a position lower than the exit port. In some embodiments, the non-bound cells are collected upon exiting the bioreactor chamber. In some embodiments, the non-bound cells are disposed of upon exiting the bioreactor chamber and the bound cells are harvested. In some embodiments, the surface of beads can comprise an antibody, a receptor ligand, a carbohydrate binding molecule, a lectin, or a component of a binding pair for example biotin. In some embodiments, the surface of beads comprises a positive surface charge. In some embodiments, binding between beads and cells or a subpopulation thereof is reversible. In some embodiments, binding between beads and cells or a subpopulation thereof is irreversible. In some embodiments, bioreactors are configured to include one or more harvesting ports that are configured to open into the second chamber at the vicinity of the perforated barrier, or, alternatively, are configured to open at the upper surface of the perforated barrier. Non-limiting examples of harvesting ports that are configured to open into the second chamber or at the upper surface of the perforated barrier are presented inFIG.1(21),FIG.2(127),FIG.6AandFIG.6B(521),FIGS.6C and6D(531),FIGS.7(627),FIG.8(727),FIG.9(827),FIGS.10A and10B(927),FIGS.11A and11B(927), andFIGS.12A and12B(1127). In accordance with some embodiments, the entire reactor or perforated barrier are configured to be tiltable at an angle to the vertical to assist the harvesting of the cells. In some embodiments, harvesting of the cells, microorganisms, or products thereof, grown in a bioreactor disclosed herein comprises sterile harvesting of the cells, microorganisms, or products thereof. Non-limiting examples of perforated barriers are presented inFIG.1(12),FIG.7(612),FIG.8(712),FIG.9(812).FIGS.10A and10B(912),FIGS.11A and11B(1012), andFIGS.12A and12B(1112). In accordance with some embodiments of the bioreactor, the perforated barrier is configured to be a fixed (non-movable) barrier. In some embodiments, a fixed perforated barrier is sealingly attached to the vessel walls. In accordance with some other embodiments, the perforated barrier is configured to be a movable and/or tiltable perforated barrier. In accordance with some embodiments of the bioreactor fixed perforated barriers is configured to be a flat perforated barrier, a flat perforated barrier inclined at an angle to a longitudinal axis of the bioreactor, a concave perforated barrier with a concave upper surface facing the top of the bioreactor, a tapering perforated barrier, or a conical perforated barrier, or any combination thereof. In accordance with some embodiments of the bioreactor, the movable perforated barriers are configured to be a movable perforated barrier sealingly attached to the vessel walls of the bioreactor by a flexible and/or stretchable member. The flexible and/or stretchable member is scalingly attached to a perimeter of the perforated barrier and sealingly attached to the vessel wall. In accordance with some embodiments of the bioreactor, the movable perforated barrier is configured to be a deformable and/or flexible perforated barrier, or a convex buckling perforated barrier with a convex upper surface facing the top of the bioreactor. A skilled artisan would appreciate that the term “sealingly” and different grammatical forms thereof, refers to an attachment between the barrier and the vessel wall wherein there is no flow through the barrier of any kind of material unless through perforations. In some embodiments, bioreactor systems including the bioreactors of the present application are configured to also include temperature control systems, pumps for circulating the growth medium, one or more fluid reservoirs connectable to the bioreactor for introducing volumes of growth medium and/or additives and/or substances required for maintaining the level of nutrients and/or any other materials necessary for cell growth. Other substances required for any steps of growing and/or maintaining, washing, and/or proliferating and/or differentiating and/or activating and/or detaching the cells for harvesting can also be added through such fluid reservoirs, including various enzymes, growth factors, activating factors, differentiating factors, washing buffers, pH adjustments, dissolved Oxygen adjustments, Nutrients or any other necessary substances or compounds. In some embodiments, living cells can also be added for co-culturing with or activating the cells within the bioreactor. In some embodiments, other substances required for inducing and/or maintaining induction of a cell product or microorganism product can also be added to medium within the bioreactor. In some embodiments, bioreactor systems disclosed herein are configured to also include a controller for controlling the operation of the bioreactor, for opening and/or closing various different valves of the bioreactor, for controlling the flow of growth medium or other fluids through the bioreactor by controlling the pump and/or various different valves. As used herein, one skilled in the art would appreciate that the term “flow velocity” may be used interchangeable with “flow rate” having all the same meanings and qualities. As used herein, one skilled in the art would appreciate that the term “perforations” may be used interchangeable with “pores” having all the same meanings and qualities. In some embodiments, the flow rate directly or indirectly influences the density of cells cultured in a bioreactor disclosed herein. In some embodiments, a low flow rate is used to culture very high density cell cultures. In some embodiments, bioreactor systems and bioreactors disclosed herein are configured to also include one or more sensors suitably connected to the controller for monitoring and/or regulating various physical and/or chemical parameters within the growth medium (such as, for example, temperature, pH, glucose concentration, dissolved oxygen concentration the concentration of dissolved carbon dioxide or of HCO3ions, the concentration of lactate, and ionic strength) in the growth medium, all can be sensed monitored and controlled in the bioreactor and/or bioreactor headspace and/or in a fluid reservoir connectable to the bioreactor and/or at the various inlets or outlet ports. In some embodiments, sensors are configured to detect a product synthesized by a cell or microorganism grown in the bioreactor. In some embodiments, control of some of the features above may require mixing of the growth medium, the mixing can be provided at the fluid reservoir. Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the bioreactors and systems thereof pertains.FIGS.1-16and the accompanying description thereof, provide numerous embodiments of bioreactors and systems thereof. A skilled artisan would recognize that other methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments disclosed herein. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. Implementation of the method and/or system of embodiments of the bioreactor and systems thereof disclosed herein can involve performing or completing selected tasks manually, automatically, or a combination thereof. Moreover, according to actual instrumentation and equipment of embodiments of the method and/or system disclosed herein, several selected tasks could be implemented by hardware, by software or by firmware or by a combination thereof using an operating system. For example, hardware for performing selected tasks according to some embodiments could be implemented as a chip or a circuit. As software, selected tasks according to some embodiments could be implemented as a plurality of software instructions being executed by a computer using any suitable operating system. In one embodiment, one or more tasks according to the methods and/or systems as described herein, can be performed by a data processor, such as a computing platform for executing a plurality of instructions. Optionally, the data processor includes a volatile memory for storing instructions and/or data and/or a non-volatile storage, for example, a magnetic hard-disk and/or removable media, for storing instructions and/or data. Optionally, a network connection is provided as well. A display and/or a user input device such as a keyboard or mouse are optionally provided as well. Reference is now made toFIG.1, which is a schematic part cross-sectional diagram illustrating a bioreactor system including a bioreactor having a perforated barrier, in accordance with some embodiments of the bioreactors of the present application. According to some embodiments, the bioreactor system50includes a bioreactor10, a pump4, a controller30and a growth medium reservoir20. The pump4can be any type of fluid pump known in the art and capable of receiving a fluid such as a growth medium received at the pump's inlet port and pumping it through an outlet port thereof at a controllable pumping rate without compromising the sterility of the growth medium. For example, the pump4can be a variable flow rate peristaltic pump, such as, for example, a model530process pump commercially available from Watson-Marlow fluid technology group (UK) or any other suitable type of pump known in the art. The bioreactor10has a bioreactor wall10A having a bottom part10B and a top part10C. In the embodiment of the bioreactor presented inFIG.1, the bioreactor10comprises a top part10C that has a threaded opening10E into which a threaded cover10D is sealingly threaded. The cover10D is configured to also (optionally) have one or more openings therein such as for example, the opening10F into, which a sensor unit22is configured to be sealingly inserted into the volume enclosed within the walls10A of the bioreactor10. According to some embodiments, the threaded opening10G is configured to be sealed by a threaded sealing cap10H when not in use. The bioreactor cover10D is configured to (optionally) include several additional sealable openings (not shown inFIG.1), which are configured to be used for inserting therein additional sensors (not shown inFIG.1), or other needed devices such as, for example, a heating unit (not shown) an oxygenating unit (not shown), a thermometer (not shown) or any other device needed for operating the bioreactor10and/or monitoring the contents of the bioreactor10and/or ports allowing sampling and introduction of materials to the content of bioreactor10. According to some embodiments, the bioreactor10can be made from any suitable biocompatible material known in the art, such as a suitably biocompatible plastic or polymer based material. In some embodiments, the reactor10is made from a transparent material to enable an operator to see the contents of the bioreactor10. In some embodiments, non-limiting examples of materials that can be used in the construction of the bioreactor10include but are not limited to, polystyrene, stainless steel, polyetheretherketone (PEEK), polysulfone, and various types of polytetrafluoroethylene (PTFE) plastics, for example Rulon®. In some embodiments, materials for use in the construction of a bioreactor described herein are selected based on their low coefficient of friction, excellent abrasion resistance, Gamma radiation sterilization, wide range of operating temperatures, or chemical inertness, or any combination thereof. The bioreactor10further comprises a perforated barrier12sealingly attached to the walls10A of the bioreactor10. The perforated barrier12divides the volume enclosed within the bioreactor10into a first (lower) chamber14A and a second (upper) chamber14B. The perforated barrier12is made from a material which has multiple perforations therein. The average diameter of the perforations formed in the perforated barrier12is selected such that the cells3(or microorganisms) suspended in a growth medium2cannot penetrate into the perforations of the perforated barrier12, while the growth medium2can flow into and through the perforations. The perforated barrier12operates as a cell (or microorganism) barrier while allowing the growth medium2to flow and pass there through. According to some embodiments the construction of the perforated barrier12is also configured to align a medium flow. According to some embodiments, the alignment comprises improving the linearity and uniformity of a medium flow towards the cell mass3and throughout the upper chamber. The perforated barrier12can be made from any suitable perforated biocompatible material, such as, for example, a suitable biocompatible plastic or polymer based material having a selected perforation average perforation (or pore) diameter. The thickness and strength of the perforated barrier12and the type of perforated material selected for the perforated barrier12can depend, for example, on the average size of the cells or microorganisms to be grown in the bioreactor12, the desired rate of flow of the growth medium2through the bioreactor, the maximal allowable level of pressure of the growth medium within the first chamber14A, or the method of harvesting cells or microorganisms as implemented in the design of the bioreactor, or any combination thereof. For example, if the perforated barrier needs to be flexible as explained in detail hereinafter (See for exampleFIGS.7-8), a thinner perforated barrier can be selected for use. In some embodiments, the types of materials from which the perforated barriers can be made can include but are not limited to cellulose nitrate, cellulose acetate, polytetrafluorethylene (FTFE), hydrophobic FIFE, hydrophilic FTFE, aliphatic or semi-aromaticpolyamides—for example Nylon®, polycarbonate, polysulfone, polyethylene, polyethersoulfone, polyvinylidene, stainless steel, and regenerated cellulose. In some embodiments, the thickness of the perforated barrier12can be in the range of 0.5-5.0 millimeter. In other embodiments, thinner perforated barriers can be used depending on the application, the mechanical properties of the material from which the perforated barrier is made, total surface area and shape of the perforated barrier and other considerations. In other embodiments, thicker perforated barriers can be used depending on the application, the mechanical properties of the material from which the perforated barrier is made, total surface area and shape of the perforated barrier and other considerations. The bioreactor10has a fluid inlet port16through which growth medium2can be pumped into the first chamber14A. The fluid inlet port16is configured to receive the growth medium2under pressure from the pump4of the bioreactor system50. The growth medium entering the fluid inlet port16can pass into a fluid impeller18disposed within the first chamber14A. The (optional) fluid impeller18is configured to be a hollow disc-like perforated member having multiple passages18P therein. The fluid impeller18is configured to receive growth medium2from the inlet port16and disperse the growth medium2through the multiple perforations18P in multiple jets19of growth medium to enhance the mixing of the growth medium2entering the inlet port16with the growth medium2disposed within the chamber14A. It is noted to that the specific structure of fluid impeller18illustrated inFIG.1is one embodiment of a fluid impeller and not obligatory. Many other different types of fluid impellers/dispersers having various different shapes, structures, dimensions and using passages and/or nozzles can be used, as is well known in the art including impeller types such as a pinch blade or marine type. According to some embodiments, in operation of the system50, cells (or microorganisms) are suspended in a growth medium and placed within the second (upper) chamber14B of the bioreactor10by inserting the suspended cells through the opening10E or through the opening10G of the cover10D (which can then be sealed with the cap10H). Alternatively, the cell suspension can be inserted into the second (upper) chamber14B through any other suitable port, such as for example, a harvesting port21opening into the second chamber14B just above the surface12A of the perforated barrier12. The growth medium2injected into the chamber14B by the fluid impeller18increases the pressure of the growth medium2in the first chamber14A and causes the growth medium2to flow through the perforations of the perforated member12into the second chamber14B effectively perfusing the cells mass3suspended in the growth medium2held within the second chamber14B. The growth medium2rises within the second chamber14B and reaches the level of a fluid outlet26, where it is drained out of the bioreactor10and carried by a conduit28to the pump4where it is recirculated into the bioreactor12through the inlet port16. In some embodiments, the bioreactor10has a generally frustoconical shape. The diameter of the bottom part10B is smaller than the diameter of the top part10C and the walls10A are sloped. Due to the frustoconical shape of the bioreactor, the diameter of the bioreactor increases as the growth medium moves upwards (towards the top part10D) within the bioreactor. As the pump4pushes the growth medium into the inlet port16at a constant flow rate, the flow velocity (fluid velocity) of the growth medium2adjacent the surface12A of the perforated barrier12is higher than the flow speed of the growth medium near the top part10D, effectively resulting in establishing a fluid flow velocity gradient along the longitudinal axis35of the bioreactor10. The flow velocity gradient is schematically indicated by the length and thickness of the solid arrows37A,37B and37C. The flow velocity represented by the arrow37A is greater than the flow velocity represented by the arrow37B and the flow velocity represented by the arrow37B is greater than the flow velocity represented by the arrow37C. The suspended cells3are carried upwards by the upward moving flow of the growth medium2, which counteracts the tendency of the cells3(which have a higher specific gravity than the specific gravity of the growth medium2) to move downwards and to settle on the surface12A due to the force of gravity acting on the cells3. The flow rate of the growth medium can therefore be controlled and adjusted to result in an adequate suspension of the cells within the volume of the growth medium2contained in the second chamber14B avoiding the settling of the cells3on the surface12A of the perforated barrier12, while leaving most of the cells3suspended in the growth medium2at a region within the chamber14B, which is adequately lower than the upper surface2A of the growth medium2so as to minimize or adequately reduce the number of cells entering the fluid outlet port26(which greatly reduces loss of cells3). According to some embodiments the outlet port26comprises a perforated barrier or filter (not shown), configured to prevent the cells or microorganisms from leaving the bioreactor. In some embodiments, the flow rate of the growth medium2through the second chamber14B is low enough to avoid substantial shear forces which can be detrimental to the cells3. When the proper flow rate of the growth medium2through the bioreactor10is established, the pump4circulates the growth medium2through the volume of the bioreactor10by pumping any growth medium2exiting the fluid outlet port26back into the bioreactor through the fluid inlet port16in a closed loop. During the cell growth, when there arises a need to add new nutrients to the growth medium2(to compensate for depletion thereof by absorption into cells) or to add activating substances or any other additive or substance into the growth medium2, this can be done by flowing some fresh growth medium2from the medium reservoir20of the system50by way of a media tube (38). The medium reservoir20is configured to be connected to an inlet port4A of the pump4by a suitable hollow conduit38. A suitable controllable valve (or stopcock)39is configured to be attached between the conduit38and the pump inlet4A, such that the flow of growth medium from the fluid reservoir20into the pump inlet port4A can be controlled. The valve39is configured to be controllably closed to stop feeding fluid from fluid reservoir20into the pump inlet port4A or is configured to be opened to enable feeding fluid from fluid reservoir20into the pump inlet port4A allowing media refreshment and high density cell culturing. In some embodiments, regulation of flow rates correlates with the density of cells being grown and propagated. In some embodiments, very low flow rates provide for high density culturing of cells in the bioreactors disclosed herein. In some embodiments, the working volume of media in which the cells are grown is low, as is the flow rate allowing for the maintenance of high density culturing of cells. This low working volume and low flow rate, can in certain embodiments, lead to higher yields and lower media needs. In some embodiments, the bioreactors disclosed herein and methods of use thereof, are advantageous compared with other bioreactors known in the art due to their ability achieve and maintain high density cultures of cells or organisms, which results in higher yield and lower media needs. In some embodiments, a bioreactor disclosed herein comprises a smaller physical footprint minimizing the bioreactor size, and thereby reducing media use. According to some embodiments, the bioreactor10is configured to also (optionally) include an additional outlet port27opening at the bottom part10B of the bioreactor. The outlet port27includes a valve (or stopcock)25that is configured to allow draining an amount of the growth medium2from the first chamber14A of the bioreactor10if necessary. For example, if an amount of new growth medium2is added to the bioreactor10from the fluid reservoir20, a similar amount of growth medium can be bled out of the bioreactor10to restore the level of growth medium2within the second chamber14B. According to some embodiments, growth medium2can also be bled out of the bioreactor10through the outlet port25when it is desired to reduce the total volume of the growth medium2within the second chamber14B in order to concentrate the cells3for cell harvesting. When such a cell concentrating is performed, the smaller volume of the growth medium2remaining in the lower part of the second chamber14B has a higher cell court (in cells/ml of growth medium) since the cells3cannot pass the perforated barrier12and are therefore concentrating. The concentrated suspension of cells3remaining in the chamber14B can then be harvested through the harvesting port21which is configured to include a valve (or stopcock)23as illustrated inFIG.1. In some embodiments, in order to prevent clogging the perforated barrier12most the growth medium can be drained via at least one of the outlet ports126A-126D (detailed in the following), and only a minimal volume of the growth medium may be drained via outlet port25. It is noted that while any desired additives and/or substances can be introduced into the bioreactor10by introducing such substances and/or additives into the growth medium2held within the fluid reservoir20and allowing a volume of the growth medium2including such substances and/or additives to flow into the chamber14A, as disclosed hereinabove, it can also be possible to directly introduce such substances and/or additives into the bioreactor by introducing a relatively small volume of fluid or growth medium including a suitably high concentration of the substances and/or additives into the bioreactor10through any suitable opening or inlet port of the bioreactor10and allowing the added small volume to mix with the volume of growth medium2circulating within the reactor to reach the desired concentration. For example, such small volumes of fluid or growth medium including additives and/or substances can be introduced though the opening10G by temporarily removing the cap10H and resealing the opening10G. In some other embodiments, the cap10H is configured to include a penetrable sealing diaphragm (not shown in detail inFIG.1) made from rubber, latex or any other suitable sealing material as is known in the art and commonly used in bottles containing injectable liquid formulations and the small volume of fluid with substances and/or additives can be loaded within a sterile syringe having a sterilized needle and where the needle is configured to be pushed into the sealing diaphragm of the cap until it penetrates the sealing diaphragm, the contents of the syringe can then be injected into the growth medium2within the second chamber14B, and the needle of the injector can be withdrawn from the sealing membrane as is known in the art. This method can advantageously reduce the risk of contamination of the growth medium by any undesirable microorganisms. Additionally, cap10H is configured to have a deep tube touching the growth medium2with a one way seal allowing media sampling in a sterile way. In some other embodiments, the cap10H is configured to include a filter (not shown inFIG.1). The cap's filter is configured to allow a flow of air to the headspace (space between the bioreactor top10C and the media's surface) or for reduction pressure from the headspace. According to some embodiments, the bioreactor system50is configured to use the controller30and the sensor unit22for monitoring the operation of the system. The sensor unit22is configured to include a sensor or multiple sensors (the individual sensors are not shown in detail inFIG.1for the sake of clarity of illustration), which can be disposed in several locations for example: via the end part22A of the sensor unit22that is immersed in the growth medium2, or via at least one of the outlet ports (126A-126D), or via harvesting port (21), or via inlet port (116), or via outlet port (27) or via side wall (10A) or any combination thereof. The sensor(s) of the sensor unit22can be used to determine the concentration of several chemical species within the growth medium2, such as, for example, the concentration of H+ions (to determine the pH of the growth medium2), the concentration of dissolved oxygen in the growth medium2, the concentration of dissolved carbon dioxide in the growth medium2or of HCO3−ions in the growth medium2, the concentration of glucose, the concentration of lactate, and ionic strength. Such sensor or sensors can be single use sensors using optic sensing without the need to penetrate the wall or can be located on10A touching the liquid. According to some embodiments, the sensors of the sensor unit22are configured to also be sensors for sensing physical parameters of the growth medium2, such as but not limited to, the temperature and/or the turbidity and/or the optical density of the growth medium2, and/or any other desired physical parameter of the growth medium2such as, conductivity, capacitance, pressure, flow rates, viscosity, turbidity and others. According to some embodiments, the signal(s) from the sensor unit22representing any of the chemical and/or physical parameters sensed by the sensors can be fed into the controller30by suitable electrical conductors (or conductor pairs)22B. The controller30is configured to process such sensor signals to determine of the values of the sensed parameters as is well known in the art. According to some embodiments, the controller30is configured to be or configured to include one or more processing devices such as, for example, a microprocessor or a microcontroller or a digital signal processor, a personal computer or any other suitable means for processing received signals and any type of memory device known in the art for storing any computed data therein for the purpose of off-line or on-line presentation of all determined sensor data and the history of operation of the bioreactor (including, but not limited to, the rate of flow of growth medium2through the bioreactor10, the time of introducing and the volume of growth medium from the fluid reservoir20, the time of introducing and the volume and concentration of any other added substance or additive during the operation of the system50). According to some embodiments, the controller30is configured to also include any display device known in the art for displaying processed results and the values of any sensed parameters to an operator or user of the system50. The controller30is configured to also include one or more user interface device (such as, but not limited to a mouse, a light pen, a pointing device, a keyboard, a touch sensitive screen, or any other input device known in the art) which is configured to be used by the user or operator of the system50for inputting data and/or suitable commands into the controller30. For example, the user can control the rate of flow of the growth medium2through the bioreactor10by entering suitable commands into the controller30resulting in suitable control signals being sent by the controller30to the pump4through a communication line29connecting the controller and the pump4. In some embodiments of the systems of the present application, the valves23,24,25, and39of the system50are configured to be manual valves or stopcocks, which can be manually closed or opened. In some other embodiments, one or more of the valves23,24,25, and39are configured to be electrically operated valves that can be operated by receiving appropriate command signals from the controller30. For example, any of the valves23,24,25, and39can be electrically operable solenoid based valves which can be opened and/or closed controllably and/or automatically by applying suitable voltage or current signals to the solenoids by the controller30. It is noted that for the sake of clarity of illustration any electrical wires connected between the controller30and any of the valves23,24,25, and39are not shown inFIG.1. However, such optional connections are shown in the schematic diagram ofFIG.5. It is noted that while in the bioreactor system50the level of the upper surface2A of growth medium2in the second chamber14B is fixed, this is not obligatory and in some embodiments of the bioreactor systems, the level (height) of the growth medium in the bioreactor can be controllably changed. Reference is now made toFIG.2, which is a schematic part cross-sectional diagram illustrating a bioreactor system having a bioreactor with multiple fluid outlet ports for controllably adjusting the level of the growth medium in the bioreactor, in accordance with some embodiments of the bioreactors of the present application. According to some embodiments, the bioreactor system150includes a bioreactor110, the controller30as disclosed in detail hereinabove, the pump4as disclosed in detail hereinabove and the fluid reservoir20as disclosed in detail hereinabove. The bioreactor system150is configured to also include an oxygenating system160. The bioreactor110can be made from any of the materials disclosed in detail hereinabove for the bioreactor110. The bioreactor110has a bioreactor wall110A, a bottom part110B and a bioreactor top part110C. According to some embodiments, the top part110C is configured to have a threaded opening110F therein for sealingly inserting there through a threaded sensor unit122. A top opening in the top of the bioreactor110D can be effectively closed using a cap110E, wherein the seal of the opening in the head plate of the bioreactor is represented by110G. According to some embodiments, the sensor unit122is configured to include any number of sensors (not shown individually inFIG.2for the sake of clarity of illustration) attached to or included in the end122A of the sensor unit122for sensing any desired chemical or physical property of the growth medium2within, which the end122A of the sensor unit122can be immersed. It is noted that the position of the end122A can be changed by threading the sensor unit122up or down within the threaded opening110F such that the end122A can be immersed in the growth medium2at any level of the growth medium2within the bioreactor110. A perforated barrier112is sealingly attached to the wall110A of the bioreactor110such that the perforated barrier112divides the internal volume of the bioreactor110into a first (lower) chamber114A and a second (upper) chamber114B, as disclosed in detail hereinabove for the bioreactor10and the perforated barrier12of the bioreactor system150. According to some embodiments, the perforated barrier112can be made from similar material(s) and can have similar perforation mean sizes as disclosed in detail hereinabove for the perforated barrier12. However, according to some embodiments, while the bioreactor10(ofFIG.1) has a single fluid outlet port26in the second chamber14B, the bioreactor110has plurality of different fluid outlet ports at different heights and corresponding valves, for example four different fluid outlet ports126A,126B,126C and126D in the second chamber114B. The outlet ports126A,126B,126C and126D are disposed along the length of the second chamber114B at different positions and each of the fluid outlet ports outlet ports126A,126B,126C and126D has a corresponding valve124A,124B,124C and124D (respectively) attached thereto. The valves124A,124B,124C and124D are fluidically connected to a common fluid manifold128which is fluidically connected to the pump4. The arrangement of the four valves124A,124B,124C and124D at different positions allows the level of the growth medium2to be selected from four different levels schematically represented inFIG.2by the dashed lines A, B, and C and the line D. In some embodiments, if the valve124D is opened and the valves124A,124B,124C are closed (as illustrated inFIG.2), the growth medium2reaches the level represented by the solid line D and the growth medium2leaving the second chamber114B through the fluid outlet port126D enters the manifold128and is re-circulated into the bioreactor110by the pump4pumping the growth medium2through the pump outlet4B into the fluid inlet port116and through the perforations19the fluid impeller18. In some embodiments, if it is desired to increase the level of growth medium2in the second chamber114B, the valves126A,126B and126D can be closed and the valve126C can be opened while the valve39can be opened for a period of time allowing an amount of growth medium2from the reservoir20to be pumped by the pump4into the first chamber114A until the level of the growth medium2to reach the level represented by the dashed line C at which time the valve39can be closed and the growth medium2leaves the second chamber through the fluid outlet port126C. Similarly, in some embodiments if it is desired to further increase the level of growth medium2in the second chamber114B, the valves126A.126C and126D can be closed and the valve126B can be opened while the valve39can be opened for a period of time allowing an additional amount of growth medium2from the reservoir20to be pumped by the pump4into the first chamber114A until the level of the growth medium2to reach the level represented by the dashed line B at which time the valve39can be closed and the growth medium2leaves the second chamber through the fluid outlet port126B. Furthermore, if it is desired to even further increase the level of growth medium in the second chamber114B, the valves,126B,126C and126D, according to some embodiments, can be closed and the valve126A opened while the valve39can be opened for a period of time allowing an additional amount of growth medium2from the reservoir20to be pumped by the pump4into the first chamber114A until the level of the growth medium2reaches the level represented by the dashed line A, at which time the valve39can be closed and the growth medium2leaves the second chamber through the fluid outlet port126A. It will be appreciated by those skilled in the art that while the bioreactor110includes four fluid outlet ports126A,126B,126C and126D levels allowing four different levels, this is not obligatory of the growth medium2to be achieved during closed loop perfusion (recirculation) of the growth medium2, this is by no means intended to be obligatory. Rather, in some embodiments of the bioreactors of the present applications, the number of the outlet ports (and the corresponding valves attached thereto) opening into the second chamber of the bioreactor can be varied as desired and can be smaller or larger than four (with suitable modification of the manifold128to accommodate the required number of valves), in such a way as to allow any desired practical number of growth medium2levels to be achieved in the second chamber of the bioreactor by suitable opening and closing of the valves as disclosed in detail hereinabove. An advantage of being able to set different levels of growth medium2within the second chamber of the bioreactor is that it can allow the increasing or decreasing of the total volume of growth medium2in the second chamber114B in order to increase (or decrease, respectively) the number of cells (or microorganisms) which can be grown within the bioreactor, if necessary. This mechanism allows culturing of cells in high density and adapting the refreshment of media and nutrients as the cell proliferate reducing or eliminating the need for passaging and dish/container replacement. According to some embodiments, at least some of the plurality of different fluid outlet ports at the different heights and together with their corresponding valves are configured also as fluid inlets ports. In some embodiments, the plurality of different fluid outlet/inlet ports is configured to circulate out of the bioreactor a portion of the cells or microorganisms. In some embodiments, cells or microorganisms may be circulated out of the upper chamber of the bioreactor in order to process cells wherein the processed cells are then circulated back into the bioreactor (not shown). In some embodiments, cells may for example be selected by depleting or enriching of a specific cell type or genetically modified, for example but not limited to, to express a polypeptide or fragment thereof not previously expressed, or to increase or decrease expression of a polypeptide or fragment thereof. In some embodiments, processing comprising inducing cells to increase or decrease expression of a specific gene or gene variant. Methods of genetic modification and control of gene expression are well known in the art. In some embodiments, cells may be transformed (genetically modified) using any method known in the art. In some embodiments, cells may be processed wherein polypeptide expression is modified using any method known in the art. In a related embodiment, the outlet/inlet fluid ports and their corresponding valves are selected to circulate the cell mass, according to the cells mass current level (height). It is noted that according to some embodiments, the frustoconical shape of the bioreactor110allows the establishment of a fluid velocity gradient along the length of the bioreactor110in order to gently float the cells mass3and keep most of the cells mass3suspended within a defined region of the growth medium2contained in the second chamber114B to avoid cell accumulation on (and/or adhering) to the upper surface112A of the perforated barrier112as well as to reduce cell loss by exiting through a fluid outlet port being used for recirculation of the growth medium2. According to some embodiments, the provided bioreactor comprises a vessel or at least an upper chamber with an inverted frustoconical shape configured to allow the cell (or microorganism) growing mass to float and elevate to a larger surface, due to the medium's upstream flow (against gravity direction) and the pressure equilibrium (mass gravity vs. upstream liquid's flow). Further, due to constant volumetric-flow, a slower flow of the medium runs through the cell (or microorganism) mass at the upper and larger areas of the inverted frustoconical shape, which assist in concentrating the cells mass, and reduces shear forces applied by the medium's flow. It is noted that like in the bioreactor10ofFIG.1, the vessel walls110A are slanted at an angle with respect to a longitudinal axis135of the bioreactor110as can be seen in the part longitudinal cross section view ofFIG.2. According to some embodiments, the angle at which the vessel walls110A are configured to be slanted with respect to the longitudinal axis135can be in the range of 0 to 175 degrees. However, higher or lower slant angles can also be used, depending, inter alia, on the particular application. It is noted that not all the walls of the bioreactors of the present application need be slanted and only some of the walls are configured to be slanted depending on the specific shape of the bioreactor (for example, see the bioreactor ofFIG.4I, herein after). Thus, the area of a transversal cross section of the bioreactor taken at a level represented by the dashed line A is larger than the area of a transversal cross section taken at a level D. According to some embodiments, the transversal cross sectional area of the bioreactor110becomes larger as one moves upwards along the longitudinal axis135within the second chamber114B results in the establishing of a fluid velocity gradient in the growth medium2such that the fluid velocity of the growth medium2gradually decreases as one moves upwards in the direction from the surface112A towards the top part110C. This fluid velocity gradient assists in suspending most of the cells or microorganisms in a zone or region within the growth medium2of the second chamber114B in which the force of gravity acting downwards on the cells3(or microorganisms) balances out the mean upward directed force exerted on the cells by the upward flowing growth medium2as is disclosed in detail hereinabove for the bioreactor10. Thus, in the bioreactor110, the controlling of the level (or height) of the growth medium2within the second chamber114B together with controlling of the flow rate of the growth medium2(by controlling the pump flow rate) can advantageously allow finer control of the zone or region within which most of the cells are suspended within the second chamber2. Additionally, the flow rate control allows minimizing the sheer forces introduced to the cells and maintains the ability to optimize and refresh media in correlation to the cells proliferation and density which could result in high cell density culturing. According to some embodiments, the perforated barrier112of the bioreactor110is a flat (planar) barrier. According to some embodiments, a harvesting port127is configured to be used for harvesting cells from the bioreactor110. According to some embodiments, the harvesting port127is shaped as a hollow member or tube that includes a first hollow part127A and a second hollow pat127B. The part127A is sealingly attached to the perforated barrier112(in some embodiments at the center of the perforated barrier112) and has an opening127C which opens into the second chamber114B at the upper surface112A of the perforated barrier. The second hollow part127B is contiguous with the first hollow member127A and bent at an angle thereto such that it passes through the vessel wall110A of the first chamber114A and is sealingly attached to the vessel walls110A. The second part127B exits the vessel walls110A and extends outside the bioreactor110. The second part127B includes a valve (or a stopcock)123which is disposed within the portion of the second part127B that extends outside of the bioreactor110. When it is desired to harvest cells3from the bioreactor, this can be performed by concentrating the cells by reducing the level of the growth medium2within the second chamber114B. For example, the level of the growth medium2can be brought to the level represented by the line D, or, alternatively, to a level lower than the level D by draining additional growth medium from the first chamber through a suitable outlet port (not shown inFIG.2) disposed in the bottom pat110B of the bioreactor110(such as, for example, an outlet port similar to the outlet port27or ports126A-126D illustrated inFIG.1). After the cells3are concentrated, the suspension of cells3in the growth medium2can be harvested through the harvesting port127by opening the valve123and receiving the cell suspension in an appropriate collecting vessel (not shown). According to some embodiments, the valves126A,126B,126C,126D,39and123can be manual valves (or stopcocks), but may, in accordance with some embodiments of the bioreactor110, controllably and/or automatically operable as disclosed in detail hereinabove with respect to the valves24,23,25and39ofFIG.1. For example, any of the valves126A,126B,126C,126D,39and123are configured to be electrically operable solenoid valves which can be controlled to open and closed by the controller30of the bioreactor system150(it is noted that any lines connecting any of the valves126A,126B,126C,126D,39and123to the controller30if the valves are indeed implemented as solenoid based valves, are not shown inFIG.2for the sake of clarity of illustration. However, such schematic lines are shown in more detail inFIG.5hereinafter). According to some embodiments, the controller20is configured to be suitably connected through connecting wires22B to a sensor unit122which is configured to include any number of sensors for sensing any chemical and/or physical properties of the growth medium2as disclosed in detail hereinabove for the sensor unit22ofFIG.1. It is noted that while the position of the end22A of the sensor unit22can be fixed (since the level of the growth medium2in the second chamber14B of the bioreactor10does not change significantly during perfusion, the sensor unit122is configured to be substantially longer than the sensor unit22and is configured to be implemented in such a way that the position of the end122A of the sensor unit122can be changed, if necessary to accommodate any changes in the level of the surface of the growth medium2within the second chamber114B. For example, a substantial part of the length of the sensor unit122can be threaded and the opening110F, into which the sensor unit122fits, can also be internally threaded to allow changing the position of the end122A within the second chamber by suitably screwing the sensor122in or out as necessary. Alternatively, the surface of the sensor unit122can be smooth and the position of the end122A of the sensor122can be varied by suitably sealingly pushing or pulling the sensor unit122within a suitable gasket (not shown inFIG.2) sealingly disposed between the opening110F and the sensor unit122. According to some embodiments, the oxygenating system160of the system150is configured to include an oxygen source160A for supplying oxygen gas to the bioreactor110, and a gas dispersing head160(optionally) disposed within the first chamber114A. According to some embodiments, the oxygen source160A is configured to be connected through a gas valve160D to the gas dispersing head by a suitable hollow member160C sealingly passing through the wall110A of the bioreactor110such as, for example Suitable hollow flexible tubing. Alternatively, according to some embodiments, the oxygen source160A is configured to be suitably connected through a suitable gas valve160D to a fixed inlet formed as an integral part of the wall110A to which the gas dispersing head can be suitably attached. According to some embodiments, the gas valve160D is configured to be a manually operated valve manually opened or closed by an operator. However, in some embodiments, the gas valve160D may is configured to be an actuator controlled valve that can be suitably opened or closed by receiving suitable electrical command signals from the controller30(it is noted that any command lines connecting the controller30with the gas valve160D are not shown inFIG.2for the sake of clarity of illustration. According to some embodiments, the oxygen source160A can be a compressed oxygen tank as is known in the art, but can alternatively be any type of oxygen generator known in the art, such as but not limited to an electrolytic oxygen generator or any other source of gaseous oxygen known in the art. Alternatively, the oxygen source can be a source of any mixture of gases which contains a substantial amount of oxygen (such as, for example, air, a mixture of oxygen and nitrogen, a mixture of oxygen, nitrogen and carbon dioxide, or any other suitable mixture of gases suitable for the purpose of oxygenation of a growth medium as is known in the art.). According to some other embodiments, the oxygenation of the liquid medium is provided at the liquid's reservoir20. When the gas valve160D is open, oxygen gas from the oxygen source160A passes through the gas dispersing head160B and is dispersed in the form of small oxygen containing bubbles that rise up within the first chamber114A. The gas dispersing head160B can be any type of head including perforations therein and capable of dispersing a gas passing there through a liquid (such as, for example the growth medium2) in the form of small bubbles. For example, the gas dispersing head160B can be a block of perforated ceramic material, a block of perforated stainless steel, a block of perforated titanium, or any other type of sterilizable dispersing head known in the art (such a gas dispersing head can be similar in construction and operation to the gas dispersing heads used to oxygenate the water in fish aquaria, as is well known in the art). It is noted that while the oxygenating system160illustrated inFIG.2directly provides oxygen to the growth medium within the first (lower) chamber114A of the bioreactor110, this is in no way obligatory for practicing the bioreactor or bioreactor systems disclosed herein. For example, the oxygenating system160can provide oxygen to other different parts of the bioreactor system150, such as, for example to the second chamber114B or to the manifold128, or to the fluid reservoir20, or can provide oxygen to more than one part of the bioreactor system150(such as, for example, both to the first chamber114A and to the fluid reservoir20). Alternatively, the oxygen level in the medium can be controlled by controlling the oxygen levels in the headspace between the bioreactor top110C and the media D surface allowing oxidation by diffusion. This can be implemented by placing the oxygen dispersing head160B in the desired part of the system or by providing several oxygen dispersing heads all suitable connected to the oxygen source160A and disposed in any selected parts of the bioreactor system150for oxygenating any growth medium disposed in such parts. All such alternative oxygen supply methods are contemplated for use in some of the embodiments of the bioreactors and/or bioreactor systems as disclosed herein. It is further noted that, since the sensors, for example the dissolved oxygen sensor, can be placed in the various inlets and outlets of the bioreactor (as mentioned above), the monitoring of the dissolved oxygen concentration within the growth medium is enabled at any time or process stage (either continuously, or at preset and/or programmable and/or predetermined time intervals). Accordingly, it enables to automate the oxygenation of the growth medium2in the bioreactor110by automatically regulating the rate of gas flow of oxygen (or oxygen containing gas mixture) through the dispersing head160B (or heads if there is more than one such head in the system150) to maintain a desired level of dissolved oxygen in the growth medium. According to some embodiments, the increasing of the medium's oxygen level, at the bioreactor vessel, can be provided by increasing the medium's oxygen level at the reservoir, and by increasing perfusion rate of the medium at the first chamber. It is noted that the shape of the bioreactors of the present application are not limited to the frustoconical shape as illustrated inFIGS.1-2. For example, the bioreactors are configured to have, inter alia, conical shape, a frustoconical shape, a tapering shape, a cylindrical shape, a polygonal prism shape, a tapering shape having an ellipsoidal transversal cross section, a tapering shape having a polygonal transversal cross section, a shape having a cylindrical part and a tapering part, and a shape having a conical or tapered part and a hemispherical part. However, other different bioreactor shapes can also be implemented in accordance with some embodiments of the bioreactor, depending, inter alia, on the specific application and on manufacturing considerations. Several possible exemplary shapes of the bioreactors are schematically illustrated inFIG.3andFIGS.4A-4I. Reference is now made toFIG.3, which is a schematic part cross-sectional diagram illustrating a bioreactor system including a bioreactor having a cylindrical shape including a perforated barrier, in accordance with another embodiment of the bioreactors of the present application. According to some embodiments, the bioreactor system250includes a bioreactor210, the controller30as disclosed in detail hereinabove, the pump4as disclosed in detail hereinabove and the fluid reservoir20as disclosed in detail hereinabove. According to some embodiments, the bioreactor system250also includes the oxygenating system160as disclosed in detail hereinabove. The bioreactor210can be made from any of the materials disclosed in detail hereinabove for the bioreactors10and110. The bioreactor210has vessel walls210A, a bottom part210B and a bioreactor top part210C. The top part210C may have an opening210G therein and a self-sealing gasket211can be disposed within the opening for sealing the opening. The self-sealing gasket211can be sealably penetrated by a needle (not shown inFIG.3) for introducing a suspension of cells or microorganisms in a growth medium, or any other fluid or solution containing any substance or additive into the bioreactor210, as disclosed in detail hereinabove. It is noted that the cells or microorganisms can also be introduced into the second chamber of the bioreactor through any suitable one way valve (not shown inFIG.3) disposed in the walls or top of the bioreactor such that the one way valve allows the injecting of a cell suspension or a microorganism suspension there through and into the second chamber of the bioreactor without compromising the sterility of the bioreactor. In accordance with one embodiment of the bioreactors, the one way valve can be a luer-lock like valve which can be shaped to accept the end of a standard syringe containing the cell or microorganism suspension. The use of such a one way valve can be advantageous because the orifice of the valve can be made sufficiently large to reduce the shearing forces affecting the cells when the suspension is injected into the bioreactor. It is noted that any of the bioreactors of the present application are configured to have any combination of such opening(s), self-sealing gasket(s) and one way valve(s). According to some embodiments, the vessel walls210A are configured to have an opening210F for sealingly inserting there through a threaded sensor unit222. The sensor unit222is configured to include any number of sensors (not shown individually inFIG.3for the sake of clarity of illustration) attached to or included in sensor unit222for sensing any desired chemical or physical property of the growth medium2as disclosed in detail hereinabove with respect to the sensor unit122inFIG.2. According to some embodiments, a perforated barrier212is sealingly attached to the vessel wall210A of the bioreactor210such that the perforated barrier212divides the internal volume of the bioreactor210into a first (lower) chamber214A and a second (upper) chamber214B, as disclosed in detail hereinabove for the bioreactor10and the perforated barrier12of the bioreactor system50ofFIG.1. The perforated barrier212can be made from similar material(s) and can have similar perforation mean sizes as disclosed in detail hereinabove for the perforated barriers, for example 12 ofFIG.1. However, while the bioreactor10(ofFIG.1) has a single fluid outlet port26in the second chamber14B, the bioreactor210can comprise several different fluid outlet ports (not shown) in the second chamber214B, wherein the outlet ports comprise an individual outlet and valve (not shown). According to some embodiments, the valves are fluidically connected to a common fluid manifold280A which is fluidically connected to the pump4. The arrangement of the four valves at different positions, as illustrated inFIG.2, allows the level of the growth medium2to be selected from four different levels. According to some embodiments, the number of the outlet ports (and the corresponding valves attached thereto) opening into the second chamber of the bioreactor can be varied (the number of outlet ports can be smaller or larger than 4, with suitable modification of the manifold280to accommodate the required number of valves) in such a way as to allow any desired practical number of growth medium2levels to be achieved in the second chamber of the bioreactor by suitable opening and closing of the valves as disclosed in detail hereinabove. According to some embodiments, the oxygenating system160of the system250includes an oxygen source160A for supplying oxygen gas to the bioreactor110, and a gas dispersing head160(optionally) disposed within the first chamber214A. The oxygen source160A is configured to be connected through a gas valve160D to the gas dispersing head by a suitable hollow member160C sealingly passing through the wall210A of the bioreactor110such as, for example Suitable hollow flexible tubing. Alternatively, the oxygen source160A is configured to be suitably connected through a suitable gas valve160D to a fixed inlet formed as an integral part of the wall210A to which the gas dispersing head can be suitably attached. Additionally, the concentration of oxygen can also be controlled by controlling the oxygen concentration in the headspace between the top part210C and liquid level D allowing oxygenation of the growth medium2via diffusion. In some embodiments, the pH may be adjusted. For example but not limited to controlling CO2concentration, the pH can be controlled by controlling the CO2concentration in the headspace via diffusion. According to some embodiments, the gas valve160D is configured to be a manually operated valve manually opened or closed by an operator. However, in some embodiments, the gas valve160D is configured to be an actuator controlled valve that can be suitably opened or closed by receiving suitable electrical command signals from the controller30(it is noted that any command lines connecting the controller30with the gas valve160D are not shown inFIG.3for the sake of clarity of illustration. According to some embodiments, the oxygen source160A can be a compressed oxygen tank, as is known in the art, but can alternatively be any type of oxygen generator known in the art, such as but not limited to an electrolytic oxygen generator or any other source of gaseous oxygen known in the art. Alternatively, the oxygen source can be a source of any mixture of gases which contains a substantial amount of oxygen (such as, for example, air, a mixture of oxygen and nitrogen, a mixture of oxygen, nitrogen and carbon dioxide, or any other suitable mixture of gases suitable for the purpose of oxygenation of a growth medium as is known in the art.) When the gas valve160D is open, oxygen gas from the oxygen source160A passes through the gas dispersing head160B and is dispersed in the form of small oxygen containing bubbles that rise up within the first chamber214A. The gas dispersing head160B can be any type of head including perforations therein and capable of dispersing a gas passing through a liquid (such as, for example the growth medium2) in the form of small bubbles. For example, the gas dispersing head160B can be a block of perforated ceramic material, a block of perforated stainless steel, a block of perforated titanium, or any other type of sterilizable dispersing head known in the art (such a gas dispersing head can be similar in construction and operation to the gas dispersing heads used to oxygenate the water in fish aquaria, as is well known in the art). Reference is now made toFIGS.4A-4Iwhich are schematic cross-sectional diagrams illustrating several exemplary shapes of bioreactors including a perforated barrier in accordance with several embodiments of the bioreactors of the present application. It is noted that, for the sake of clarity of illustration, the schematic drawings of Mgs,4A-4I illustrate only the general shape of the walls of the bioreactors and the perforated barrier included therein and do not show any details of any additional components of the bioreactors or bioreactor systems (such as, for example, various openings in the walls of the bioreactors, sensor units, fluid inlet ports, fluid outlet ports, draining ports, harvesting ports, heating units, cooling/heating units, fluid impellers, gas dispersing heads, valves, pumps, controllers, self-sealable gaskets, fluid manifolds or any other components) which are not important to understanding the shape of the bioreactors. It will be appreciated by those skilled in the art that any such components not shown inFIGS.4A-4Imay be included in any non mutually exclusive combinations and/or permutations in any of the bioreactors schematically illustrated inFIGS.4A-4I, as is disclosed herein in detail herein and illustrated in the drawing figures. It is further noted that while the perforated barriers illustrated inFIGS.4A-4Iare illustrated as a flat fixed perforated barriers, this is shown by way of example only and it is contemplated that any of the bioreactors having shapes as disclosed inFIGS.4A-4Imay also be implemented as any of the types of perforated barriers disclosed in the present application (including any of the flat or non-flat, fixed and movable perforated barriers, buckling perforated barriers and all other perforated barrier forms disclosed in the present application). Turning toFIG.4A, the bioreactor300includes the perforated barrier12as disclosed hereinabove which divides the bioreactor300into a first chamber304A shaped as a cylindrical part of the bioreactor300and a second chamber304B shaped as a frustoconical part of the bioreactor300. Thus the bioreactor300has a shape that has a cylindrical part and a frustoconical part. Turning toFIG.4B, the bioreactor310includes the perforated barrier12as disclosed hereinabove which divides the bioreactor310into a first chamber314A shaped as a cylindrical part of the bioreactor300and a second chamber314B shaped as a tapering part of the bioreactor300. Thus, the bioreactor300has a shape that has a cylindrical part and a tapering part. The tapering walls308of the second chamber314B have a convex outer surface308A. Turning toFIG.4C, the bioreactor320includes the perforated barrier12as disclosed hereinabove which divides the bioreactor320into a first chamber324A shaped as a cylindrical part of the bioreactor320and a second chamber324B shaped as a tapering part of the bioreactor320. The bioreactor320has a shape that has a cylindrical part and a tapering part. The tapering walls328of the second chamber324B have a concave outer surface328A. Turning toFIG.4D, the bioreactor330includes the perforated barrier12as disclosed hereinabove which divides the bioreactor330into a first chamber334A shaped as a tapering part of the bioreactor330and a second chamber334B shaped as a tapering part of the bioreactor330. The bioreactor330has a tapering shape. The tapering walls338of the bioreactor330have a convex outer surface338A. Turning toFIG.4E, the bioreactor340includes the perforated barrier12as disclosed hereinabove which divides the bioreactor340into a first chamber344A shaped as a tapering part of the bioreactor340and a second chamber344B shaped as a tapering part of the bioreactor300. The bioreactor340has a tapering shape. The tapering walls348of the bioreactor340have a convex outer surface348A. Turning toFIG.4F, the bioreactor350includes the perforated barrier12as disclosed hereinabove which divides the bioreactor350into a first chamber354A shaped as a conical of part of the bioreactor350and a second chamber354B shaped as a frustoconical part of the bioreactor300. The bioreactor350has a conical shape. Turning toFIG.4G, the bioreactor360includes the perforated barrier12as disclosed hereinabove which divides the bioreactor360into a first chamber364A shaped as a cylindrical part of the bioreactor360and a second chamber364B shaped as a cylindrical part of the bioreactor360. The bioreactor360has a cylindrical shape. Turning toFIG.4H, the bioreactor370includes the perforated barrier12as disclosed hereinabove which divides the bioreactor370into a first chamber374A shaped as a hemispherical part of the bioreactor370and a second chamber374B shaped as a frustoconical part of the bioreactor370. The bioreactor370has a shape similar to a chalice. Turning toFIG.4I, the bioreactor380includes the perforated barrier12as disclosed hereinabove which divides the bioreactor380into a first chamber384A and a second chamber384B. The bioreactor380includes a vertical wall portion380H that is orthogonal to the bottom part380B of the bioreactor380(the wall portion380H forms an angle of 90 degrees with the bottom part380B) and a slanted wall portion380E that is slanted at an angle α1 relative to the wall portion380H (the dashed line385is parallel to the vertical wall portion380H). Typically the angle α1<90° and in some embodiments but not obligatorily α1<45°. Reference is now made toFIG.4, which is a top view of the bioreactor380ofFIG.4I. The top part380C of the bioreactor380is shaped such that it has a semi-circular portion380E, two straight portions380F and380G and a straight portion380H. The bottom part380B of the bioreactor380(schematically illustrated by the dashed line380B inFIG.4J) can have a shape or contour similar to the shape or contour of the top part but has a smaller cross-sectional area than the cross-sectional area of the top part380C due to the slanting of the wall portion380E. It is noted that while the shape of the top part38C of the bioreactor380is as disclosed hereinabove with respect toFIG.4Q, this is not obligatory and other different shapes of the top part380C and the bottom part380B can be used in some embodiment of the bioreactors having a slanted wall portion or part. In some embodiments of the bioreactors having a slanted wall portion and a non-slanted wall portion, the top and/or bottom parts of the bioreactor can have any other desired shape including but not limited to, a semi-elliptical shape, a semi-circular shape, a rectangular shape, a square shape, a trapazoidal shape, a polygonal shape, or any other suitable regular or irregular shape. It is noted that while in several of the embodiments of the bioreactors disclosed hereinabove transversal cross sections of the bioreactor can be circular, in other embodiment of the bioreactors of the present application, transversal cross sections of the bioreactor can have other shapes, including, but not limited to an elliptical shape, a polygonal shape, a regular polygonal shape, or any other suitable shape. It is further noted that in some of the bioreactors disclosed herein different transversal cross sections taken at different positions along a longitudinal axis of the bioreactor can have different shapes. For example, returning toFIG.4C, while the transversal cross section taken along the lines I-I and II-II (which are both orthogonal to the longitudinal axis335) can both be circular in shape, in accordance with another embodiment of the bioreactor, the transversal cross section taken along the line I-I can be circular in shape, and the transversal cross section taken along the line II-II can be elliptical in shape. Furthermore, in accordance with some embodiments of the bioreactor, the shape of the bioreactor can be a conical shape, a frustoconical shape, a tapering shape, a cylindrical shape, a polygonal prism shape, a tapering shape having an ellipsoidal transversal cross section, a tapering shape having a polygonal transversal cross section, a shape having a cylindrical part and a tapering part, and a shape having a conical or tapered part and a hemispherical part. Reference is now made toFIG.5which is a schematic block diagram illustrating the components of a bioreactor system, in accordance with some embodiments of the bioreactor systems of the present application. The bioreactor system400includes a bioreactor410, a pump404, the bioreactor system can also include N+1 controllable valves424A-424N (wherein N is an integer number) and another controllable valves439. The bioreactor system can also include an (optional) controller430, an (optional) fluid reservoir420, an (optional) fluid impeller418an (optional) oxygenating system460and an (optional) heater/cooler unit470. In some embodiments, a bioreactor system disclosed herein further comprises a controller. In some embodiments, a bioreactor system further comprises a fluid reserve. In some embodiments, a bioreactor system further comprises a fluid impeller. In some embodiments, a bioreactor system further comprises an oxygenating system. In some embodiments, a bioreactor system further comprises a heater unit. In some embodiments, heating on the liquid medium can be provided via a heating jacket or any provided bioreactor surrounding environment (not shown). In some embodiments, a bioreactor system further comprises a cooler unit. In some embodiments, a bioreactor system further comprises a heater unit and a cooler unit. According to some embodiments the liquid's temperature can be controlled (heated/cooled to a desired temperature) at the liquid's reservoir. In some embodiments, a bioreactor system comprises a control signal to an outlet valve (426). In some embodiments, a bioreactor system comprises a control signal (439A) for a pump. According to some embodiments, the bioreactor410can be any of the bioreactors that have multiple fluid outlet ports (as disclosed in the present application and illustrated in the drawing figures) which include a first (lower) chamber and a second (upper) chamber (the first and second chambers are not shown in detail in the schematic block diagram ofFIG.5, but can be seen as illustrated, for example, inFIG.2). Each of the multiple fluid outlet ports opening into the second chamber (not shown in the schematic diagram ofFIG.5for the sake of clarity) is fluidically connectable to a fluid manifold428through one of the respective N valves424A-424N. According to some embodiments, the fluid manifold428is configured to feed the growth medium collected from the second chamber of the bioreactor410to the pump404which is configured to pump the growth medium back into the first chamber of the bioreactor410through the fluid inlet port448which opens into the first chamber of the bioreactor410. The fluid input port448is configured to (optionally) feed the growth medium to the (optional) fluid impeller418as disclosed in detail hereinabove with respect toFIG.2. The sensor unit422can be implemented as disclosed hereinabove with respect to any of the sensor units22,122and222(ofFIGS.1,2and3, respectively). According to some embodiments, the fluid reservoir420can be a fluid reservoir external to the bioreactor410, as disclosed hereinabove, and is configured to be fluidically and controllably coupled to the pump404through the valve439. Each of the N valves404A-404N is suitably connected to the controller430by a respective communication lines429A-429N to receive control signals from the controller for opening or closing any of the valves424A-424N. The valve439is connected to the controller430by a suitable communication line for receiving control signals there from to open or close the valve439for allowing growth medium to flow from the reservoir420into the pump404and there from into the bioreactor410as disclosed in detail hereinabove for the valve39(ofFIG.1). According to some embodiments, the pump404is configured to be suitably connected to the controller430by a suitable communication line for controlling the operation of the pump404. For example, such control signals can turn the pump on or off and can also control the rate of flow of growth medium through the pump404(or the rate of pumping of the growth medium by the pump404. According to some embodiments, the (optional) heater/cooler470is configured to be disposed in the bioreactor410(in some embodiments within the first chamber thereof) to heat or cool the growth medium within the bioreactor410to maintain a desired temperature of the growth medium. Optionally, a water jacket (not shown) or blanket (not shown) or any other controlled temperature environment can be used for temperature control of the bioreactor. According to some embodiments, if the sensor unit422includes a temperature sensor, signals representing the sensed temperature can be sent from the temperature sensor to the controller430through a communication line(s)422A. The controller430is configured to process such signals and send appropriate signals to the heater/cooler470for maintaining a desired temperature, or a set temperature or a preset temperature within the bioreactor as is well known in the art of temperature control. Any other sensors included within the sensor unit422are configured to (optionally) send through the communication line(s)422A sensor signals representing any sensed physical or chemical parameter of the growth medium in the bioreactor410, as disclosed in detail hereinabove. According to some embodiments, the controller430is configured to process any such sensor signals to determine the status of the growth medium and can also use the processed either display status data or about any monitored or sensed physical or chemical parameters to an operator or user of the bioreactor system400by an (optional) display unit (not shown in detail inFIG.5) included in an (optional) user interface431included in the controller430, as is disclosed hereinabove in detail. For example, in a case in which the sensor unit includes a dissolved oxygen sensor for sensing the amount of oxygen dissolved in the growth medium within the bioreactor430, the sensor signals can be processed by the controller430and if the concentration of dissolved oxygen is different than a desired set, preset, or predetermined) value, the controller430is configured to send control signals to the oxygenating system460for stopping or starting the introducing of oxygen containing gas into the growth medium within the bioreactor430(or within the fluid reservoir420, depending on the specific implementation of the bioreactor system400to suitably adjust the dissolved oxygen level to the desired level. It is noted that as disclosed in detail hereinabove with respect to the controller30(ofFIG.1), the controller unit430is configured to include any type of suitable processor (digital and/or analog) which can be operated by suitable software to automatically or semi-automatically control the operation of the bioreactor430or at least some of the operational functions thereof. For example, while the determining of the growth medium level and rate of flow within the second chamber of the bioreactor410can be set manually by an operator by using the user interface431, the regulation of the bioreactor's temperature and/or dissolved oxygen concentration within the growth medium can be automatically controlled by suitable software operating on the controller430. Similarly, the addition of amounts of fresh growth medium from the reservoir420can be fully automated by periodically draining an amount of the growth medium from the first chamber through a draining port427by turning the404off and opening a draining valve425, and then closing the draining valve425, opening the valve439and turning the pump404on to allow an amount of fresh growth medium to be pumped into the first chamber and then closing the valve439to restart the recirculation of the growth medium through the bioreactor410. A similar method can be used in the reservoir430resulting in media refreshment. When the cells or microorganisms grown within the bioreactor need to be harvested, the harvesting can be performed is several different ways in accordance with the specific structure of the bioreactor. In some embodiments of the bioreactor (such as, for example in the bioreactor ofFIG.1), the perforated barrier is fixed and immovably attached to the walls of the bioreactor and the harvesting. The harvesting of cells in such a bioreactor, can be performed by using one or more harvesting ports disposed in the vessel walls of the bioreactor and opening into the second chamber in the vicinity of the upper surface of the perforated barrier (such as, for example, the single harvesting port21of the bioreactor10which opens into the second chamber14B in the vicinity of the surface12A of the perforated barrier12ofFIG.1. However, since the flat surface12A of the bioreactor10is horizontal during harvesting, the harvesting may be somewhat hampered as some of the cells3may not reach the opening of the harvesting port21. Reference is now made toFIGS.6A-6Bwhich are schematic part cross-sectional diagrams illustrating two possible positional states of a tiltable bioreactor, in accordance with some embodiments of the bioreactors of the present application. It is noted that the bioreactor510ofFIGS.6A-6Bis only schematically illustrated in outline and only the components necessary for understanding the harvesting operation thereof are shown in detail. Other components of the bioreactor510not necessary for understanding of the tilting action and the cell harvesting are not shown inFIGS.6A-6Bfor the sake of clarity of illustration and can be implemented as disclosed in detail for the bioreactors ofFIG.1-5or any other bioreactors disclosed herein. In the tiltable bioreactor510ofFIG.6A, the bioreactor includes vessel walls510A, top part510C and bottom part510B. The space within the bioreactor510is divided into a first chamber514A and a second chamber514B by a perforated barrier512. Any other components of the bioreactor510not shown in detail inFIGS.6A-Dcan be as disclosed in detail hereinabove with respect to the bioreactor10ofFIG.1. InFIG.6A, the bioreactor510is in a vertical state in which the longitudinal axis535of the bioreactor510is vertical (inFIG.6Athis is represented by the longitudinal axis535being aligned along the vertical axis V). The bioreactor510includes a harvesting port521and a valve523. InFIG.6A, the valve523is shown in the closed state and the bioreactor510is shown to contain a small amount of growth medium2in which the cells3to be harvested are suspended after most (but not all) of the growth medium2has been drained from the bioreactor510through an outlet port527opening into the first chamber514A by opening the valve525. During draining, according to some embodiments, some of the growth medium2held in the second chamber514B passes downstream through the perforations of the perforated barrier and into the first chamber514A and exits from the outlet port527but the cells3are retained in the second chamber514B as they cannot pass through the perforations of the perforated barrier. According to some embodiments, the draining can also be provided via a deep tube (not shown) that can be inserted to the upper chamber via for example one of the outlet ports126A-126D (shown inFIG.2), as long as the deep tube is positioned above cell mass concentration. According to some embodiments, the draining can also be provided by opening the valve of one of the outlet ports126A-126D (shown inFIG.2), as long as the outlet port is located above cell mass concentration. This results in concentrating the cells in the second chamber514B due to the reduction of the amount of growth medium2remaining in the second chamber. When the level of the growth medium2in the second chamber514B has been sufficiently reduced, the valve525can be closed. According to some embodiments, in order to perform the cell harvesting, the bioreactor510is now tilted as illustrated inFIG.6B, which illustrates the bioreactor510in a tilted state. In the tilted state, the longitudinal axis535of the bioreactor510is tilted at an angle α to the vertical direction (represented inFIG.6Bby the vertical dashed line V). The angle α can be any convenient angle in the range 0<α<90 degrees. After the bioreactor510is tilted (for example at an angle α=45 degrees), the suspended cells3can be harvested into a suitable collecting vessel such as a test tube511by opening the valve523as illustrated inFIG.6B. The advantage of such tiltable bioreactors is that during harvesting, the yield of collected cells can be higher as compared to the yield of harvesting performed in non-tiltable bioreactors such as the bioreactor10ofFIG.1. AsFIGS.6B,6C, and6Dare embodiments of the bioreactor510ofFIG.6A, the elements inFIGS.6B,6C, and6Dthat are identified above forFIG.6Ahave the same meaning and qualities as these elements inFIG.6A. According to some embodiments, the tilting action of the bioreactor510(or of any other type of tiltable bioreactor implemented as disclosed in the present application) can be performed by any mechanical means known in the art, such as, but not limited to, by tilting the bioreactor within any mechanical support structure (not shown) holding the bioreactor510. Additionally, in accordance with some additional embodiments of the bioreactor, the bioreactor510is configured to be tiltably supported within a fork-like gantry (not shown) having two opposing arms tiltably holding a bracket within which the bioreactor510can be supported. Such mechanical structures for tiltably holding a vessel such that it can be vertically aligned or tilted at any desired angle to the vertical are well known in the art, and are therefore not described in detail hereinafter. Reference is now made toFIGS.6C and6Dwhich are schematic part cross-sectional views illustrating a bioreactor having a fixed slanted perforated barrier, in accordance with some embodiments of the bioreactors of the present application; It is noted that the bioreactor550ofFIGS.6C-6Dis only schematically illustrated in outline and only the components necessary for understanding the harvesting operation thereof are shown in detail. Other components of the bioreactor550that are not necessary for understanding of the cell harvesting method are not shown inFIGS.6C-6Dfor the sake of clarity of illustration and can be implemented as disclosed in detail for the bioreactors ofFIGS.1-2and5or any other bioreactors disclosed herein. The bioreactor550ofFIG.6Cincludes vessel walls550A, a top part550C and a bottom part550B. The space within the bioreactor550is divided into a first chamber520A and a second chamber520B by a perforated barrier512. Any other components of the bioreactor550not shown in detail inFIGS.6C and6Dare disclosed in detail hereinabove with respect to the bioreactor10ofFIG.1. The perforated barrier522is sealingly and fixedly attached to the vessel walls550A and is slanted at an angle β relative to the horizontal plane H of the bioreactor550(the horizontal plane is schematically represented by the dashed line H inFIGS.6C and6D). The angle Q can be any angle in the range 0.2<β<45 degrees, but other angles smaller or larger than this range can be used, depending, inter alia, upon the application. In typical applications the angle Q can be in the range of 0.2<β<15 degrees. The bioreactor550includes a harvesting port531having a valve533. The valve533of the harvesting port531is illustrated inFIG.6Cin a closed state and the bioreactor550is shown to contain an amount of growth medium2including the cells3suspended in the growth medium2. Turning now toFIG.6D, when the cells3need to be harvested, most (but not all) of the growth medium2is drained from the bioreactor550through an outlet port527opening into the first chamber520A by opening the valve525of the outlet port527. During draining, most of the growth medium2(or a washing buffer used to wash the cells3) flows into the first chamber520A by passing through the perforations in the perforated barrier522and exits from the outlet port527but the cells3are retained in the second chamber520B as they cannot pass through the perforations in the perforated barrier. This results in concentrating the cells3in the second chamber520B due to the reduction of the amount of growth medium2remaining in the second chamber520B. When the level of the growth medium2in the second chamber520B has been sufficiently reduced, the valve525can be closed. Turning toFIG.6D, the bioreactor550is illustrated with the second chamber520B containing the cells3concentrated in the small amount of the growth medium2remaining within the second chamber520B after most of the growth medium2was drained from the second chamber520B; for example, by opening the valve525of the outlet port until the desired amount of growth medium is drained from the bioreactor550and then closing the valve525, and/or via the deep tube (as mentioned above) and/or one of the second chamber's outlet ports (as mentioned above). The harvesting of the cells can be performed by opening the valve533of the harvesting port531and connecting a collecting vessel511to the end of the harvesting port531. Reference is now made toFIGS.7-9, which are schematic, part cross-sectional diagrams illustrating three different embodiments of bioreactors including three different types of non-planar (not flat) perforated barriers, in accordance with some embodiments of the bioreactors of the present application. It is noted that for the sake of clarity of illustration, the schematic drawings ofFIGS.7-9illustrate only the general shape of the walls of the bioreactors and the shape of the perforated barrier included therein and of the harvesting port associated with the perforated barrier and do not show any details of any additional components of the bioreactors or bioreactor systems (such as, for example, various openings in the walls of the bioreactors, sensor units, fluid inlet ports, fluid outlet ports, draining ports, harvesting ports, heating units, cooling units, fluid impellers, gas dispersing heads, valves, pumps, controllers, self-sealable gaskets, fluid manifolds or any other components) which are not important to understanding the shape of the perforated barriers shown of the bioreactors. It will be appreciated by those skilled in the art that any such components which are not shown inFIGS.7-9, can be included in any non-mutually exclusive combinations and/or permutations in any of the bioreactors schematically illustrated inFIGS.7-9, as is disclosed herein in detail herein and as illustrated in the drawing figures. Turning toFIG.7, the bioreactor610has vessel walls610A, a curved perforated barrier612is fixedly (non-movably) and sealingly attached to the vessel walls610A, dividing the space within the bioreactor610into a first chamber614A and a second chamber614B. The bioreactor610further comprises a harvesting port627which is a hollow member that includes a valve623. The harvesting port627is similar in structure to the harvesting port127ofFIG.2. The harvesting port627is sealingly attached to the curved perforated barrier612and opens at the surface612A into the second chamber614B. As disclosed in detail hereinabove for the harvesting port127(ofFIG.2), the harvesting port627scalingly passes through the vessel walls610A to exit the bioreactor610. The upper surface612A of the curved perforated barrier612facing the top part610C of the bioreactor610is concave, which can advantageously increase the yield of harvested cells as compared to the yield of harvested cells in a bioreactor having a fixed (non-movable) flat (planar) perforated barrier (such as, for example, the bioreactor110ofFIG.2). Turning toFIG.8, the bioreactor710has vessel walls710A, a conical perforated barrier712is fixedly (non-movably) and sealingly attached to the vessel walls710A, dividing the space within the bioreactor710into a first chamber714A and a second chamber714B. The bioreactor710further comprises a harvesting port727which is a hollow member that includes a valve723. The harvesting port727is similar in structure to the harvesting port127ofFIG.2. H represents the horizontal plane H of the bioreactor (710). According to some embodiments, the harvesting port727is sealingly attached to the conical perforated barrier712and opens at the surface712A into the second chamber714B. As disclosed in detail hereinabove for the harvesting port127(ofFIG.2), the harvesting port727sealingly passes through the vessel walls710A to exit the bioreactor710. The upper surface712A of the conical perforated barrier712facing the top part710C of the bioreactor710is a conical surface, which can advantageously increase the yield of harvested cells as compared to the yield of harvested cells in a bioreactor having a fixed (non-movable) flat (planar) perforated barrier (such as, for example, the bioreactor110ofFIG.2). Turning toFIG.9, the bioreactor810has vessel walls810A, a tapering perforated barrier812is fixedly (non-movably) and sealingly attached to the vessel walls810A, dividing the space within the bioreactor810into a first chamber814A and a second chamber814B. The bioreactor810further comprises a harvesting port827which is a hollow member that includes a valve823. The harvesting port827is similar in structure to the harvesting port127ofFIG.2. The harvesting port827is sealingly attached to the tapering perforated barrier812and opens at the surface812A into the second chamber814B. As disclosed in detail hereinabove for the harvesting port127(ofFIG.2), the harvesting port827scalingly passes through the vessel walls810A to exit the bioreactor810. The upper surface812A of the tapering perforated barrier812facing the top part810C of the bioreactor810is a tapering surface, which can advantageously increase the yield of harvested cells as compared to the yield of harvested cells in a bioreactor having a fixed (non-movable) flat (planar) perforated barrier (such as, for example, the bioreactor110ofFIG.2). It is noted that while all the bioreactors disclosed hereinabove and illustrated inFIGS.1-3,4A-4I,6A-6B and7-9include fixed non-movable perforated barriers, this is not obligatory to practicing the using the bioreactors or systems thereof disclosed herein, and in accordance with some embodiments, the bioreactors are configured to include movable (non-fixed) perforated barriers or tiltable perforated barriers. Reference is now made toFIGS.1A-10B,11A-11B and12A-12B, which illustrated some embodiments of reactors having movable and/or tiltable perforated barriers.FIGS.1A-10Bare schematic part cross-sectional diagrams illustrating two different states of a bioreactor including a deformable perforated barrier, in accordance with some embodiments of the bioreactors of the present application. FIGS.11A-11Bare schematic part cross-sectional diagrams illustrating two different states of a bioreactor including a buckling perforated barrier, in accordance with some embodiments of the bioreactors of the present application, andFIGS.12A-12Bare schematic part cross-sectional diagrams illustrating two different states of a bioreactor including a tiltable perforated barrier, in accordance with some embodiments of the bioreactors of the present application. It is noted that, for the sake of clarity of illustration, the schematic drawings ofFIGS.10A-10B,11A-11B and12A-12B, illustrate only the general shape of the walls of the bioreactors and the shape and arrangement of the movable or deformable or tiltable or buckling perforated barrier included therein and of the harvesting port associated with the perforated barrier and do not show any details of any additional components of the bioreactors or bioreactor systems (such as, for example, various openings in the walls of the bioreactors, sensor units, fluid inlet ports, fluid outlet ports, draining ports, harvesting ports, heating units, cooling units, fluid impellers, gas dispersing heads, valves, pumps, controllers, self-sealable gaskets, fluid manifolds or any other components) which are not important to understanding the shape of the perforated barriers shown of the bioreactors. It will be appreciated by those skilled in the art that any in such components which not shown inFIGS.10A-10B,11A-11B, and12A-12Bcan be included in any non mutually exclusive combinations and/or permutations in any of the bioreactors schematically illustrated inFIGS.0A-10B,11A-11B,12A-12B, and13as is disclosed in detail herein and as illustrated in the drawing figures. Turning now toFIGS.1A-10B, the bioreactor910has vessel walls910A, a deformable perforated barrier912is fixedly and scalingly attached to the vessel walls910A, dividing the space within the bioreactor910into a first chamber914A and a second chamber914B. The deformable perforated barrier912includes multiple perforations as disclosed in detail hereinabove and allows the growth medium2to bidirectionally pass there through (from the first chamber914A to the second chamber914B, and vice versa) but blocks the passage of cells or organisms there through as is disclosed in detail hereinabove. According to some embodiments, the perforated barrier912can be made from a material that is biocompatible for the growing of cells or microorganisms and is also flexible or deformable such that a force applied to the perforated barrier912can deform its shape. The bioreactor910further comprises a harvesting port927which is a hollow member that includes a valve923. The harvesting port927is sealingly attached to the deformable perforated barrier912and opens at the surface912A into the second chamber914B. The harvesting port927sealingly passes through the vessel walls910A to exit the bioreactor910. The harvesting port927is a hollow member that has a first rigid (non movable) part (or portion)927A disposed within the first chamber914A. The first rigid part927A sealingly passes through the vessel walls910A and exits outside the bioreactor910. The first rigid part927A has a valve923therein for opening or closing the harvesting port927. According to some embodiments, the harvesting port927further comprises a second flexible and/or compressible part (or portion)927B which is sealingly attached to the first part927A at one end thereof. The flexible and/or compressible part927B and the rigid part927A are connected together to form the hollow member opening to the second chamber914B at the end of the flexible part927B which is sealingly attached to the deformable perforated barrier912and open at the surface912A thereof. It is noted that while the harvesting ports disclosed in some embodiments of the present application are open at the upper surface of the perforated barrier, alternative embodiments can include harvesting ports which are closed or scaled at their end connected to the perforated barrier by a thin sealing membrane (not shown). In such embodiments, when the harvesting port needs to be used for harvesting cells from the second chamber of the bioreactor, the sealing membrane is configured to burst open by either inserting a sharp sterile wire-like instrument through the harvesting port and bursting the sealing membrane, or by inserting a sharp sterile instrument through any of the openings in the top part of the bioreactor into the second chamber and bursting the scaling membrane. Any other mechanical or magnetic mechanisms can also be used for bursting the sealing membrane of such sealed harvesting ports as is known in the art. According to some embodiments, the bioreactor910includes a magnetic member915attached to the second compressible (or flexible part)927B, as illustrated inFIGS.10A-10B. Alternatively, in accordance with yet another embodiment of the bioreactor910, the magnetic member915is configured to be attached to the deformable perforated barrier912, in some embodiments near the central part of the perforated barrier912(not shown inFIGS.1A-10B). The magnetic member915is configured to be (optionally) shaped like an annular member made from a permanently magnetized material. For example, the magnetic member915can be made from a FeNdB (Iron Neodymium Boron) permanent magnet, a samarium-cobalt permanent magnet or any other magnetic or paramagnetic material known in the art such as, for example, Iron. If necessary, the magnetic member915can be coated with, or embedded in a biocompatible material such as, for example, a biocompatible plastic or any suitable biocompatible polymer based material, a biocompatible ceramic layer or any other suitable biocompatible and (in some embodiments) sterilizable material. Turning now toFIG.10B, when the cells3need to be harvested from the bioreactor910, an amount of growth medium2can be drained from the first chamber914A of the bioreactor910through a suitable outlet port (not shown inFIGS.10A-10B, for the sake of clarity of illustration, but similar to the outlet port27ofFIG.1or to the outlet port227ofFIG.3) as disclosed hereinabove for concentrating the cells3in the remaining growth medium2. A strong magnet M can then be suitably placed near the bioreactor910as illustrated inFIG.10B. The magnet M can be any suitable permanent magnet or an electromagnet known in the art. The placement of the magnet M near the bioreactor910exerts a magnetic force represented by the arrows F which is directed towards the magnet M. The force pulls the second part927B downwards causing the deformable perforated barrier912attached to the second compressible part927to be also pulled downwards and to deform. When the magnetic force is acting on the second compressible (or flexible or shortenable) part927B, the second compressible part927is compressed such that it's length shortens, allowing the part of the perforated barrier912attached to the second part927B to move downwards, causing the shape of the perforated barrier to deform into a deformed state (as illustrated inFIG.10B). The deformation of the deformable perforated barrier912, results in the perforated barrier912assuming a slightly curved shape, such that the upper surface912A of the perforated barrier912in the deformed state can nearly resemble a parabolloidal surface. Returning toFIG.10A, the bioreactor910is shown with the deformable perforated barrier912in a flat non-deformed state. In this non-deformed state, the upper surface912A of the perforated barrier912is substantially planar (flat). In this state the cells3can be grown in the second chamber914B as is described in detail for other bioreactor embodiments disclosed hereinabove. Returning now toFIG.10B, the bioreactor910is illustrated with the deformable perforated barrier912in a deformed state. In this deformed state, the upper surface912A of the perforated barrier912is a curved surface. In this deformed state, the concentrated cells3suspended in the growth medium2can be harvested by opening the valve923of the harvesting port927and collecting the cell3suspended in the growth medium2into a collection vessel511as disclosed hereinabove. The concave surface912A of the curved shape of the deformed perforated barrier912can advantageously increase the yield of harvested cells as compared to the yield of harvested cells in a bioreactor having a fixed (non-movable) flat (planar) perforated barrier (such as, for example, the bioreactor110ofFIG.2). Turning now toFIGS.11A-11B, the bioreactor1010has vessel walls1010A. A buckling perforated barrier1012is fixedly and sealingly attached to the vessel walls1010A, dividing the space within the bioreactor1010into a first chamber1014A and a second chamber1014B. The buckling perforated barrier1012includes multiple perforations as disclosed in detail hereinabove and allows the growth medium2to bidirectionally pass there through (from the first chamber1014A to the second chamber1014B, and vice versa) but blocks the passage of cells or microorganisms there through as is disclosed in detail hereinabove. According to some embodiments, the buckling perforated barrier1012can be made from a stiff but flexible material which is biocompatible for the growing of cells or microorganisms. According to some embodiments, the perimeter of the buckling perforated barrier1012is sealingly attached to the vessel walls1010A such that in a first stable state of the buckling perforated barrier (illustrated inFIG.11A), the perforated barrier1012is convex in shape and the upper surface1012A of the perforated barrier1012which faces the top part1010C of the bioreactor1010is a convex surface. According to some embodiments, if a force of sufficient magnitude is applied to the buckling perforated barrier1012, the buckling perforated barrier1012will flip into a second stable state (illustrated inFIG.11B). As compared with the barrier (1012) inFIG.11A, the barrier (1012) inFIG.11Bis tilted a bit towards the bottom of the bioreactor vessel. In the second state of the perforated barrier1012, the perforated harrier1012is concave in shape and the upper surface1012A of the perforated barrier1012which faces the top part1010C of the bioreactor1010is a concave surface. According to some embodiments, the buckling perforated barrier1012is configured such that it is in a bi-stable configuration in which a transition between the two stable states of the buckling perforated barrier requires the application of sufficient force to the perforated barrier1012. According to some embodiments, the bioreactor910further comprises the harvesting port927which is a hollow member that includes a valve923. The harvesting port927is sealingly attached to the buckling perforated barrier1012and opens at the upper surface1012A into the second chamber1014B. The harvesting port927sealingly passes through the vessel walls1010A to exit the bioreactor1010. The harvesting port927is a hollow member that has a first rigid (non-movable) part (or portion)927A disposed within the first chamber1014A. According to some embodiments, the first rigid part927A sealingly passes through the vessel walls1010A and exits outside the bioreactor1010. The first rigid part927A has a valve923therein for opening or closing the harvesting port927. According to some embodiments, the harvesting port927further comprises a second flexible and/or compressible part (or portion)927B which is sealingly attached to the first part927A at an end thereof. According to some embodiments, the flexible and/or compressible part927B and the rigid part927A are connected together to form the hollow member opening to the second chamber1014B at the end of the flexible part927B which is sealingly attached to the buckling perforated barrier1012and open at the surface1012A thereof. According to some embodiments, the bioreactor1010includes a magnetic member1015. The magnetic member1015is configured to (optionally) have an annular shaped magnetic member attached to the deformable perforated barrier1012, as illustrated inFIGS.11A-11B. Alternatively, in accordance with yet another embodiment of the bioreactor1010, the magnetic member1015is configured to be attached to the second compressible (or flexible) part927B of the harvesting port927(this embodiment is not shown inFIGS.10A-10B). However, the magnetic member1015can have any other shape suitable for applying an appropriately downward directed force to the buckling perforated barrier or to the second compressible (or flexible) part927B of the harvesting port927(depending on the part to which the magnetic member1015is attached in the above disclosed different alternative embodiments). According to some embodiments, the magnetic member1015can be made from a permanently magnetized material or from a paramagnetic material or from any other magnetizable material as disclosed hereinabove in detail with respect to the magnetic member1015. If necessary, the magnetic member1015can be coated with or embedded in a biocompatible material such as a biocompatible plastic or any suitable biocompatible polymer based material, a biocompatible ceramic layer or any other suitable biocompatible and (in some embodiments) sterilizable material, as disclosed hereinabove with respect to the magnetic member915. Turning now toFIG.11B, when cells (not shown) need to be harvested from the bioreactor1010, an amount of growth medium (not shown) can be drained from the first chamber1014A of the bioreactor1010through a suitable outlet port (not shown inFIGS.11A-11B, for the sake of clarity of illustration, but similar to the outlet port27ofFIG.1or to the outlet port227ofFIG.3) as disclosed hereinabove for concentrating the cells in the remaining growth medium. According to some embodiments, a magnet M is configured to then be suitably placed near the bioreactor1010as illustrated inFIG.11B. The magnet M can be any suitable permanent magnet or an electromagnet known in the art, as disclosed in detail with respect toFIG.10Bhereinabove. The placement of the magnet M near the bioreactor1010exerts a magnetic force on the magnetic member1015represented by the arrows F which is directed towards the magnet M. The force pulls the buckling perforated barrier1012downward in the direction represented by the arrows F. According to some embodiments, the magnetic force is of a magnitude that is more than sufficient to cause the buckling perforated barrier1012to flip from the first stable (convex) state to the second stable (concave) state (as is illustrated inFIGS.11A-11B). According to some embodiments, when the perforated barrier1012flips from the first state to the second state, the central part of the buckling perforated barrier1012moves downwards and causes the second compressible part927B to be compressed such that the length of the part927B shortens, allowing the part of the buckling perforated barrier1012attached to the second part927B to move downwards. According to some embodiments, the flipping of the buckling perforated barrier1012from the first state to the second state can also be achieved mechanically using a weal (not shown) or a vertical rod-like pushing/pulling member (not shown) which is configured to be attached at one end thereof to the buckling perforated barrier1012while the second end thereof sealingly and slidably passes through a suitable sealing gasket (not shown) disposed in an opening (not Shown) in the top part1010C of the bioreactor1010. According to some embodiments, when the buckling perforated barrier1012is in the first state, pushing such a pushing/puling member downwards is configured to flip the buckling perforated barrier1012from the first state to the second state. However, it will be appreciated by those skilled in the art that any other mechanical or magnetic or electromagnetic mechanism or combinations of such mechanisms can be used to flip the buckling perforated barrier from the first state into the second state and all such mechanisms or combinations of mechanisms are deemed to be included within the scope of the embodiments of the present application. InFIG.11Bthe bioreactor1010is illustrated with the buckling perforated barrier1012in the second stable state. In this second state, the upper surface1012A of the buckling perforated barrier1012is a concavely curved surface. In this state, the concentrated cells (not shown) suspended in the growth medium (not shown) within the second chamber1014B can be harvested by opening the valve923of the harvesting port927and collecting the cell suspension into a collection vessel (not shown) as disclosed hereinabove. According to some embodiments, the concave surface1012A of the buckling perforated barrier1012in the second stable state can advantageously increase the yield of harvested cells as compared to the yield of harvested cells in a bioreactor having a fixed (non-movable) flat (planar) perforated barrier (such as, for example, the bioreactor110ofFIG.2). Turning now toFIGS.12A-12B, the bioreactor1110has vessel walls1110A. A tiltable perforated barrier1112is sealingly attached to the vessel walls1110A, dividing the space within the bioreactor1110into a first chamber1114A and a second chamber1114B. The perimeter of the tiltable perforated barrier1112is sealingly attached to a flexible and/or deformable and/or stretchable annular member1113. Typically, the annular sheet1113does not have any perforations therein. The annular member1113can be made from a flexible or pliable and/or stretchable material, such as, for example, rubber or latex or a flexible polysilane based thin material and is also sealably attached to the vessel walls1110A of the bioreactor1110. In some embodiments, the annular member can be non-permeable to either the cells3and to the growth medium2. The tiltable perforated barrier1112has multiple perforations therein as disclosed in detail hereinabove and allows the growth medium2to bi-directionally pass there through (from the first chamber1114A to the second chamber1114B and vice versa) but blocks the passage of cells or microorganisms there through as is disclosed in detail hereinabove. According to some embodiments, the perforated barrier1112can be (optionally) made from a stiff or rigid material which is biocompatible for the growing of cells or microorganisms. According to some embodiments, the bioreactor1110further comprises the harvesting port1127which is a hollow member that includes a valve1123. A first end1127A of the harvesting port1127is disposed within the first chamber1114A and is sealingly attached to the annular member1113such that the end1127A opens into the second chamber1114B through an opening1113B on the upper surface1113A of the annular member1113. The harvesting port1127sealingly passes through the vessel walls1110A to exit the bioreactor1110. The harvesting port1127is a hollow member. A second end1127B of the harvesting port1127is disposed outside the bioreactor1110and includes a valve1123therein for opening or closing the harvesting port1127. According to some embodiments, the bioreactor1110also includes a magnetic member1115. The magnetic member1115is configured to (optionally) be a bar shaped magnetic member attached to the perforated barrier1112near the perimeter of the perforated barrier1112, as illustrated inFIGS.12A-12B. However, the magnetic member1115can have any other shape suitable for applying an appropriately downward directed force to the tiltable perforated barrier1112. When no force is applied to the tiltable perforated barrier1112, the perforated barrier1112is horizontal or nearly horizontal as illustrated inFIG.12A. According to some embodiments, the magnetic member1115can be made from a permanently magnetized material or from a paramagnetic material or a ferromagnetic material or from any other magnetizable material and can (optionally) be coated with or embedded in a biocompatible material, as disclosed hereinabove in detail with respect to the magnetic member915. Turning toFIG.12B, when the cells3need to be harvested from the bioreactor1110, an amount of growth medium (not shown) can be drained from the first chamber1114A of the bioreactor1110through a suitable outlet port (not shown inFIGS.12A-12B, for the sake of clarity of illustration, but similar to the outlet port27ofFIG.1or to the outlet port227ofFIG.3) as disclosed hereinabove for concentrating the cells in the remaining growth medium2. A magnet M can be suitably placed near the bioreactor1110as illustrated inFIG.12B. The magnet M can be any suitable permanent magnet or an electromagnet known in the art, as disclosed in detail with respect toFIG.10Bhereinabove. According to some embodiments, the placement of the magnet M near the bioreactor1110exerts a magnetic forte on the magnetic member1115represented by the arrow F which is directed towards the magnet M. The magnetic force pulls the side1112B of the perforated barrier1112to which the magnetic member is attached downwards in the direction represented by the arrows F. As a result of the applied magnetic force F, the perforated barrier1112is tilted such that the side1112B of the perforated barrier1112is to lower than the side1112A of the perforated member1112. InFIG.12B, the bioreactor1110is illustrated with the perforated barrier1112in a tilted state after a magnetic force has been applied by the magnet M to the magnetic member1115. In this tilted state, the concentrated cells3suspended in the growth medium2within the second chamber1114B can be harvested by opening the valve1123of the harvesting port1127and collecting the cell suspension into a collection vessel (not shown) as disclosed hereinabove. The tilt (relative to the horizon) of the tiltable perforated barrier1112can advantageously increase the yield of harvested cells as compared to the yield of harvested cells in a bioreactor having a fixed (non-movable) flat (planar) perforated barrier (such as, for example, the bioreactor110ofFIG.2). It is noted that during operating the bioreactors and bioreactor systems of the present application, a liquid, e.g., a growth medium can be supplied by perfusion (constant replacement of media by recirculation as disclosed in detail), or by fed batch (addition of specific nutrients to the growth medium2) or by hatch (replacement of the growth medium or part of the growth medium periodically if needed). According to some embodiments, during harvesting of the cells/microorganisms grown in the bioreactors of the present application, a need may arise to further concentrate the cells being harvested. Such concentrating can be achieved without needing to perform additional actions outside the bioreactor (such as, for example, centrifugation in a centrifuge) which can adversely increase the probability of contaminating the harvested cells by using an inline concentrating filter connected to the harvesting port. According to some embodiments, washing of the cells in the bioreactors can be done performed by replacing the growth medium2with a wash buffer as is known in the art. The replacement of the growth medium2can be performed by draining the growth medium2from the bioreactor and filling the bioreactor with new wash buffer several times. According to some embodiments, the draining can be performed by using any of the draining ports included in the first (lower) chamber of any of the bioreactors (such as, for example, the outlet port27of the bioreactor10ofFIG.1, or the outlet port227of the bioreactor210ofFIG.3) or by using the output ports opening into the second (upper) chamber included in bioreactor embodiments that allow controlling of the level of growth medium in the second chamber of the bioreactors (such as, for example, the outlet port126D of the bioreactor110ofFIG.2). According to some embodiments, the bioreactors of the present application are configured to allow cell separation and/or cell selection. Cell separation such as magnetic bead binding or antibody binding can be performed inside the second chamber of some embodiments of the bioreactors by using magnetic bead methods as is well known in the at According to some embodiments, magnetic beads (such as, for example magnetic cell specific antibody-coated beads can be inserted into the second chamber through any of the closable openings at the top part of the bioreactors (such as, for example through the opening110E of the bioreactor110ofFIG.2). According to some embodiments, once the cells are bonded to the beads, the beads can be collected by using a magnet as is well known in the art, or by using a large filter that is adapted for selecting between the bead size and cells. Such filters can be positive or negative selectors based on the filter's pore size. For Example, cells attached to beads will not pass the filter whereas native cells not attached to beads will pass through the pores in the filter. Optionally, according to some embodiments the filter is configured to have an affinity to the beads and can retain the beads and the cells attached to the beads on the filter, while allowing unattached cells to pass through the filter. Alternatively, it is possible to use a “tea hag” shaped enclosure enclosing heads coated with a cell specific antibody that allows free passage of unbound cells through the pores in the “tea bag” but retains any antibody coated beads and the cells that are bonded to the beads within the “tea bag”. According to some embodiments, cells can pass through the “tea bag” membrane but the beads are bigger and stay in the bag. According to some embodiments, cells that are attached to the beads can be retained in the “tea bag” and taken out of the bioreactor or can be retained depending on the intended use and application. According to some embodiments, the bioreactor can further comprise a 3D hollow container (for example but not limited to a column-like container560) in its upper chamber (demonstrated inFIG.6A), configured to be used for cell sorting; for a non-limiting example, precipitating CAR-T cells with magnetic beads. In some embodiments, the upper chamber (second chamber) is configured to comprise an immobilized matrix and or beads in order to select cells or microorganisms having a particular binding activity. In some embodiment, the cells or microorganisms comprised in the fluid, for example but not limited to a growth media or wash media, can be circulated through an inner 3D container comprising the immobilized matrix or beads. In some embodiments, the container walls permit cell and media flow in and out of the container but beads and cells bound to beads or the immobilized matrix are not permitted egress from the container. In some embodiments, the container comprises an immobilized matrix. In some embodiments, beads comprise an affinity molecule on their surface. In some embodiments, an affinity molecule comprises a polypeptide, or portion thereof or a peptide or a carbohydrate binding molecule. In some embodiments, an affinity molecule comprises an antibody, biotin, avidin, a receptor or part thereof, an agglutinin, a lectin, or any other molecule known in the art to which a cell or microorganism can bind. In some embodiments, the beads comprise magnetic beads. In the case of a magnet, magnetic beads can be retained in the container by positioning a magnet near the container and retaining the positive cells attached to the magnetic heads in the container while circulating back the negative cells. In some embodiments, an immobilized matrix comprises an affinity molecule on its surface. In some embodiments, an affinity molecule comprises a polypeptide, or portion thereof or a peptide or a carbohydrate binding molecule. In some embodiments, an affinity molecule comprises an antibody, biotin, avidin, a receptor or part thereof, an agglutinin, a lectin, or any other molecule known in the art to which a cell or microorganism can bind. In some embodiments, cells pass through the container, wherein if the cells or microorganism possess a binding partner to the surface marker present on the beads or immobilized matrix, the cells can bind to the surface of the beads or immobilized matrix and be retained within the container. In some embodiments, the container comprises a “tea bag” like structure, wherein the sides are configured to be flexible. According to some embodiments, a material such as Retro-Nectin can be added to the barrier or to the affinity matrix in order to enhance infection rate of viruses, such as retor or lenti virus, as commonly used for CAR T. According to some embodiments, the barrier and/or the affinity matrix can be coated with relevant antibodies. Activation of cells such as, for example, T cells can be achieved by adding cytokines and activation signals to the growth medium2or by co-culturing the T-cells with cytokine secreting cells that can be adhered to the perforated barrier or to any other type of suitable carrier, or adhered to a “tea bag” or floating in a “tea bag” or on magnetic beads, as disclosed hereinabove. Additionally, the activation of T-cells can be performed by co-culturing T-cells with Antigen presenting cells, as is known in the art. It is noted that co-culturing of different types of cells is not limited to cell activation only. For a non-limiting example, anti CD3/CD28 conjugated beads can also be used to activate T cells. In another non-limiting example, Anti CD3 and Anti CD28 antibodies can also be used for activating T cells. According to some embodiments, the bioreactors of the present application are configured to also be used for co-culturing other types of cells for achieving other results. For example, when culturing embryonic stem cells, the bioreactors of the present application are configured to also be used to co-culture the embryonic stem cells with feeder cells (such as, for example, fibroblasts) which can release into the growth medium substances and/or factors necessary for maintaining growth and proliferation of the stem cells and/or for inducing differentiation of the stem cells. It is noted that for increasing harvesting efficiency the entire second (upper) chamber of the bioreactors disclosed hereinabove or the upper surface of the perforated barriers included within such bioreactors can be washed by growth medium can be perfused or added to the second chamber of the bioreactors from the top or bottom of the second chamber (such as, for example by adding growth medium through the opening110E of the bioreactor110, or trough the opening10G at the top part10C of the bioreactor10ofFIG.1, or by injecting growth medium through the self-sealing gasket211of the bioreactor210ofFIG.3by using a syringe filled with sterile growth medium2). Such washing of the walls of the second chamber and/or of the perforated barriers can result in pushing the cells towards the opening of any harvesting port opening into the second (upper) chamber of the bioreactor as disclosed hereinabove. According to some embodiments, cells that are grown within the bioreactors disclosed in the present application can be counted on line and concentrated by using a circulation loop with a conic shaped concentrating filter to allow volume reduction. The cell counting can be performed by indirect measurements such as by using capacitance measurements, optical density measurements, and/or other optical sensors as is well known in the art. According to some embodiments, the bioreactors of the present application are configured to allow culturing of adherent cells on an attachment surface such as a carrier packed bed or even plenary surfaces above the perforated barrier. Detachment of the cells adhering to the perforated barrier can be performed enzymatically, as is well known in the art. Such enzymatic treatment can also be combined with flushing the attachment surface with growth medium or a wash buffer and/or with applying vibrations to the attachment surface. Reference is now made toFIG.13which is a schematic part cross-sectional diagram illustrating a bioreactor system including a bioreactor having a perforated barrier and a cell carrier matrix, in accordance with an embodiment of the bioreactor of the present application. Descriptions of elements presented inFIG.13not specifically detailed herein below, are presented in the description ofFIG.1above. The bioreactor system1250is similar to the bioreactor system50ofFIG.1except that the bioreactor10of the bioreactor system1250further comprises a supporting matrix1260which is disposed within the second chamber14B. While the supporting matrix1260of the system1250occupies only a portion of the volume immersed within the growth medium2, in other embodiments of the bioreactor systems, the supporting matrix is configured to extend up to the surface2A of the growth medium2and can also extend downwards towards the upper surface12A of the perforated barrier12. The volume occupied by the support matrix1260can depend, inter alia, upon the specific application, the resistance of the cell supporting matrix1260to the flow of the growth medium2, the final amount of required cells or microorganisms and other consideration. According to some embodiments, the bioreactor system1250of the present application is configured to allow culturing of adherent cells on an attachment surface such as, for example, a cell carrier matrix packed bed or even plenary surfaces above the perforated barrier. According to some embodiments, the packed bed of the cell supporting matrix1260is configured to be positioned above the perforated barrier12of the bioreactor10allowing grow medium (or other solutions) to circulate through the immobile (or less mobile) cell supporting matrix1260for feeding the cells attached to the surface(s) of the cell supporting matrix1260. This arrangement enables constant feeding of the cells attached to the cell supporting matrix1260, allowing high density cell culturing with a high surface to volume ratio and very low sheer forces while constantly feeding the cells3. Such cell supporting matrix1260can comprise, inter alia, woven and none woven fibers, electrospin-meshes, plastic beads, plastic surfaces, biodegradable materials such as, for example alginate or any other suitable matrices or carriers having two dimensional and/or three dimensional surface(s), as is well known in the art. According to some embodiments, once there is a need to harvest the cells attached to the cell supporting matrix1260, the cells3can be enzymatically detached from packed the surface(s) of the cell supporting matrix1260as is well known in the art. The enzymatic treatment can be combined together with flushing the attachment surface with growth medium or a wash buffer and/or with vibrating of the surface to facilitate detachment of the adhered cells. According to some embodiments, Enzymatic detachment of adhered cells can be performed by adding one or more enzymes to the growth medium2and incubation of the adherent cells in the enzyme containing growth medium for a prescribed time period. Enzymes useful for performing cell detachment can include but are not limited to a protease (such as, for example, trypsin, pepsin or papain) or a suitable collagenase, or any combinations of a collagenase and a protease. Once the cells are harvested from the attachment surface, washing and processing of the cells can be done as described carrier. Furthermore, in accordance with some embodiments of the bioreactors of the present application, the second (upper) chamber of any of the bioreactors disclosed herein is configured to also include a cell supporting matrix similar to the above disclosed cell supporting matrix1260which is configured to be introduced into the second chamber through any of the openings available in the top part of the bioreactors (such as, for example, through the closable opening110E of the bioreactor110ofFIG.2). While growing non-adherent cells in the bioreactors disclosed herein in which the cells are suspended in the growth medium and do not typically adhere to a surface, the bioreactors disclosed herein are configured to also be used for growing adherent cells that require some surface or substrate to adhere to. While such adherent cells can adhere to the perforated barrier of the bioreactor, it can be desirable to increase the surface area available for such adherent cells in order to increase cell yield. Therefore, in accordance with some embodiments of the bioreactors of the present application, any of the bioreactors disclosed herein are configured to include a suitable cell supporting matrix disposed within the second chamber of the bioreactor. According to some embodiments, the cell supporting matrix can be any type of cell supporting matrix known in the art to which the cells can adhere. For example, the cell supporting matrix can include a collagen based matrix, woven and none woven fibers, electro-spin meshes, plastic (polymer based) beads, plastic (polymer based) particles surfaces, biodegradable materials such as, for example alginate, any type of collagen or any other suitable matrices or cell carriers having two dimensional and/or three dimensional surface(s) with a high surface to volume ratio, as is well known in the art. It is noted that the bioreactors and bioreactor systems disclosed in the present application are configured to be used for many different applications including, inter alia, the growing of microorganisms like bacteria or any other single cell or multicellular microorganisms, isolated living cells of any type, including but not limited to, living cells from insects, living cells of invertebrates, living cells of vertebrates, living mammalian cells, and various different types of human cells. The total volume, shape and other components and/or characteristics of the various embodiments of the bioreactors and bioreactor systems disclosed hereinabove are configured to be scaled and adapted to each specific application. According to some embodiments, the bioreactor1250is configured to be used to co-culture together adherent and non-adherent suspended cells that need co-culturing were the adherent cells are attached to the cell supporting matrix1260and the suspended non-adhering cells are suspended in the medium above the perforated barrier12and below the cell supporting matrix1260. For example the bioreactor1250or any other of the bioreactors containing a cell supporting matrix are configured to be used for culturing of embryonic stem cells which are suspended non-adherent cells with feeder cells such as adherent fibroblasts. One example application of the bioreactors and bioreactor systems is the growing of cells for cell therapy. Cell therapy is an evolving industry where cells are used as therapeutic agents. The cells can be obtained from an autologous source (from the patient) or an allogeneic source (different individual donor). In cases of use of autologous cells, such as immune-cell therapy (using T cells, and/or B cells and/or dendritic cells, and/or natural killer cells) and/or mesenchymal stem cells. The therapeutic dosages can range from several million cells to several billion typically cultured in volumes of a few litters (1-20 L). In allogeneic therapies the bio-manufacturing of therapeutic agents can reach volumes of up to thousands of litters per bioreactor. In some of the embodiments of the bioreactors of the present application, providing for adaptive culturing (using variable medium levels) which allow incremental volume changes, media perfusion and refreshments and high density culturing (such as, but not limited to, in the bioreactor20ofFIG.2) the working volume and bioreactor size can be advantageously reduced dramatically by about 2-100 fold as compared to prior art bioreactors. For example, a typical bioreactor having a total volume in the range of 1-2 litter can be used for culturing the cells required for autologous therapy. Such relatively small bioreactor volumes can allow the growing of a few billion cells. According to some embodiments, the ability to use the relatively small bioreactors of the present application can advantageously save space and reduce operating costs significantly in the facility by allowing the use of many small bioreactors in the same workspace, allowing many small bioreactors to share common services (such as, for example, by sharing a central oxygenating supply space, sharing other facilities, such as computers, controllers and/or workspace temperature controlling devices and air conditioning devices and other shareable devices and systems. It is noted that similar workspace reductions and cost savings can also be obtained in larger bioreactors adapted for use in allogeneic culturing in which larger bioreactor volumes are required. Such allogeneic cell culturing can require using embodiments of the bioreactors disclosed in the present application having bioreactor volumes in the range of 10-1000 liter (with a typical exemplary, but not obligatory, bioreactor volume of about 100 liter). It is noted that all the above disclosed bioreactor volume ranges in both applications of growing allogeneic cells and/or autologous cells are given by way of example only and are not obligatory. Thus, bioreactors having volumes that are cither larger or smaller than the above ranges can also be used in certain applications and are included within the scope of the volumes of the bioreactors of the present application. For example, in some applications such as, for example, growing algae, bacteria or other microorganisms for obtaining biofuels or other products, the volume of any of the bioreactors of the present application are configured to be scaled up to volumes much higher than 1000 liter. According to some embodiments, the above mentioned washing methods using the above mentioned bioreactors can be applied to any provided cell mass, even if originally incubated in a different bioreactor. According to some embodiments, the bioreactors' designs as mentioned above, are configured to allow cell washing and formulating in a very gentle and efficient manner without the need of opening the bioreactor chamber or interfering thereto. According to some embodiments, the bioreactors' designs as mentioned above, are configured to allow continuous, optimal and adaptive cell culturing at changing volumes, feeding schemes, activating, manipulating, washing and formulating, all in a closed and automated bioreactor with minimal sheer force applied onto the cell mass. It is appreciated that certain features of the bioreactors and systems thereof disclosed herein, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the bioreactors and systems thereof disclosed herein, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the bioreactors and systems thereof disclosed herein. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Although the bioreactors and systems thereof disclosed herein have been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present bioreactors and systems thereof disclosed herein. To the extent that section headings are used, they should not be construed as necessarily limiting. As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof. Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. A skilled artisan would appreciate that the term “medium” may encompass in some embodiments any type of growth medium suitable for growing cells (either eukaryotic or prokaryotic) or any other type of unicellular or multi-cellular microorganisms. In some embodiments, the term “medium” comprises any type of solution used for cell or microorganism processing including but not limited to wash buffers, nutrient buffers, enzyme mixtures, selection solutions, and final formulation solutions. As used herein, in one embodiment the term “about” refers to ±10%. In another embodiment, the term “about” refers to ±9%. In another embodiment, the term “about” refers to ±9%. In another embodiment, the term “about” refers to ±8%. In another embodiment, the term “about” refers to ±7%. In another embodiment, the term “about” refers to ±6%. In another embodiment, the term “about” refers to ±5%. In another embodiment, the term “about” refers to ±4%. In another embodiment, the term “about” refers to ±3%. In another embodiment, the term “about” refers to ±2%. In another embodiment, the term “about” refers to ±1%. As used herein, the term “optionally” encompasses the meaning that some element “is provided in some embodiments and not provided in other embodiments.” Any particular embodiment disclosed herein may include a plurality of “optional” features unless such features conflict. Additional objects, advantages, and novel features disclosed herein will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, various embodiments and aspects disclosed herein as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples. EXAMPLES The bioreactor system used in the following examples included a bioreactor schematically presented inFIG.14A, which comprises a bioreactor similar to that shown inFIG.1. The perforated barrier was circular in shape with a 50 cm2diameter and 1 micrometer thickness. The upper chamber had a conical shape and a 120 cm2top. The total volume of the growth chamber (upper chamber) was 250 ml. The term “footprint” used herein refers to the lower perforated barrier surface area and total chamber area. Cells, used to exemplify bioreactor use and effectiveness, were T-lymphocytes, but this in no way should be considered limiting. The flow rate used in the Examples was about 2-3 mm per mins. This is a representative embodiment of the flow rate for the cells used, wherein the skilled artisan would appreciate that flow rate may change depending on cells used. Thus, the flow rate used in the Examples should in no way be considered limiting. For example, a skilled artisan would appreciate that when culturing larger cells, such as mesenchymal stem cells (MSC), the flow rate may reach 10 mm per minute, and for even larger cells, such as macrophages, flow rate may reach 20 mm (data not shown). Example 1: Growth of High Density Cell Cultures Objective: High density culturing of cells. Methods: Cells (T cell lymphocytes) were grown on a 50 square cm perforated barrier system with 150 ml media for 7 days, starting at the maximum known cell density for these cells of about 4 million cells per ml. Based on knowledge in the art, this is the density at which these cells would normally be passaged and then maintained at 1 million cells per ml. The media was perfused so the total media used was increased but the volume of media in the chamber remained at 150 ml. Results: TABLE 1DaysCM2Cells(E6/ml)Total CellsCells/cm20503.580667,000,00013,340,0002505.230784,500,00015,690,0004509.2671,390,050,00027,801,00075024.553,683,632,50073,672,650 The data shows that using a bioreactor disclosed herein, the cells were grown at a density (cells/ml) that is more than 24-fold of the normally expected density for these cells (1×106/ml). Similarly, the data shows that growing cells in a bioreactor system having a footprint of 50 cm2, that starting at 13.3 million per cm2(as opposed to the maximum reported of 10×106/cm2), use of a bioreactor described herein resulted in having 73.6×106per cm2. Conclusion: Cells can be grown at high density using a bioreactor comprising a very small footprint (50 cm2) of the culturing system. Thus, the bioreactor provides for a system that allowed optimal and adaptive cell culturing at changing volumes and feeding schemes, allowed for activating, manipulating, feeding, washing, and formulating cells in a closed automated manner with minimal sheer force (See, Examples 2-3 as well). Additional cell incubators or centrifuges are not required for culturing and collection of cells, respectively. Example 2: Comparison Cell Cultures: Bioreactors vs. Tissue Culture Flasks Objective: Compare culturing cells in a bioreactor comprising a 50 cm2 perforated barrier with culturing cells in tissue culture flasks. Methods: Cells (T cell lymphocytes) were cultured for 14 days in the same dishes as follows: in either a 50 cm2perforated barrier bioreactor system with perfusion, or a T75 flask without media change, or T75 flask with media exchange every 4 days. Results: FIGS.14B-14Cpresent growth curves from two representative culturing experiments, showing that cells could be continuously grown in a bioreactor system having a 50 cm2perforated barrier without the need to replace media (a pour out/pour in complete exchange), passage or change the container. Further, that cells grown in the closed continuous bioreactor system (yellow) continued to proliferate for at least 14 days, and achieved a total cell number of 1,633,996,000 cells compared with only about 4,3200,000 cells in the T75 flask without media change (grey), and only about 300,000,000 cells in the T75 flask with media change (blue). The 14-day time frame was used based on the fact that growth of cells in the bioreactor surpassed that in the flasks after a week. Cells can be cultured for more than two weeks in the bioreactor (data not shown). Conclusion: Culturing of cells in a bioreactor system described herein is more effective than culturing of cells in flasks even with media exchange. Example 3: Processing of Cells Grown in a Bioreactor Objective: Processing of cells (or microorganisms) includes washing the cells, media replacement, and concentrating the cells. These steps are normally accomplished in the prior art by repeated centrifugation and pelleting of the cells. There are two additional technologies known in the art for replacing media which are a TFF (tangential force filtration) centrifugation and a counter flow centrifuge. The objective of this example was to examine cell recovery from a bioreactor as disclosed herein, including the viability of the cells recovered. Methods: In the bioreactor system used (demonstrated inFIG.15A), in order to wash the cells and replace the growth media, the wash buffer was perfused upstream1510from the bottom of the bioreactor vessel (lower chamber1550), wherein the wash buffer flowed through a first perforated barrier1512into the upper chamber1540and was extracted from the highest valve1530. This perfusion flow diluted the media until growth media had been replaced by the wash solution. In some embodiments, the valve1530can comprise a perforated barrier or a filter (not shown), configured to prevent the cells from leaving the bioreactor (during the liquids change). At this point, the final formulation media may be perfused through the system, replacing the wash buffer. In addition, in some embodiments, some of the growth media could be drawn-off from the upper chamber (optionally via a second screening perforated barrier (FIG.15A1502) configured to prevent the cells from leaving the bioreactor) until a level where the cells are located, thereby reducing the volume and concentrating the cells, before the final formulation media is perfused (FIG.15A). As demonstrated inFIG.15A, the provided bioreactor with an inverted frustoconical shape allows the cells (or microorganisms) growing mass to float and to elevate to a larger surface, due to the wash solution upstream flow (against gravity direction) and the pressure equilibrium (mass gravity vs. upstream liquid's flow). Further, due to constant volumetric-flow, a slower flow of the wash solution runs through the cells (or microorganisms) mass3at the upper and larger areas of the inverted frustoconical shape, which assist in concentrating the cells mass, and reduces shear forces applied by the wash solution flow. In another embodiment, larger volumes of wash solution can be exchanged with growth media by using a bioreactor with an additional barrier located above the level of the cells (when looking at Hg.15A) and inverting the bioreactor (as shown inFIG.15B). The bioreactor vessel is configured to be flipped such that the upper chamber (or what is now the lower chamber1540) will have perforated barriers both below1502and above1512the mass of cells. This practically allows more media or wash solution to be downstream perfused due to the larger surface area of the second barrier (barrier2inFIG.15B). A skilled artisan would recognize that more volume on wider surface area results in the same velocity (flow rate) so the cells stay near the second barrier (barrier2inFIG.15B) and larger volumes of cells mass can be washed. FIGS.15C and15Ddemonstrate a bioreactor1590comprising a vessel constructed of two frusto-conical parts having same diameter for their wider base, yet their narrower base can comprise a different diameter. The two parts are sited one on top of the other coaxially joined together at their wider (similar) base. The vessel is divided into three chambers by two perforated harriers; a first perforated barrier1505and a second (screening) perforated barrier1506, which are sealingly disposed at the walls of the bioreactors vessel, according to some embodiments.FIG.15Cdemonstrates the bioreactor during cell growth stage, where the first lower chamber1591(having the narrowest base as its bottom) is configured to be introduced (not shown here) with the growth medium, which flows upstream via the first perforated barrier1505, and into the second middle chamber1592(which was created by the two perforated barrier); the middle chamber is configured to be introduced with (not shown here) and to accommodate the cells. As shown, the second middle chamber1592comprises the area with the largest/widest cross-section surface1595, therefore with the slowest medium's flow rate. According to some embodiments, the aim is not to have the cells pass this largest/widest area, during the growing stage; this could be achieved for example by controlling the medium's flow velocity. Above the widest area a second perforated barrier1506is shown, which serves as the bottom of upper third chamber1593, which is configured to be introduced with a washing medium (not shown here). FIG.15Ddemonstrates the bioreactor1590at its flipped or inverted position during a washing stage. During the washing stage, the washing media is introduced to downstream via the third chamber1593(not shown) and then down via the cells mass accommodated in the middle chamber1952and then drained out via the third chamber1593. The second perforated barrier1506is configured to prevent cells passage; therefore washed cells are retained in the second middle chamber. According to some embodiments, a bioreactor configuration such as demonstrated inFIGS.15C and15D, where one base of the vessel is wider than the other, can serve for growing cells in two steps. In the first step, the growing can start where the smaller base is facing down, as demonstrated inFIG.15C, with very low amounts of cells, allowed to grow to higher surface areas. In the second step when the cell mass is grown, instead of moving to the cells into a larger chamber of another bioreactor, the bioreactor1590can be flipped or inverted to have now the wider base facing down, as shown inFIG.15D, allowing the cell mass larger surface area and lower medium's flow rates. The downstream washing/collecting process was tested in an embodiment of a bioreactor with a single perforated harder, wherein three different surface velocities were examined near the perforated barrier: 3.6 mm/min, 1.8 mm/min, and 1.2 mm/min. Following removal of media with a deep tube (FIG.15A) 15 ml of cells in growth media remained. The total wash volume used was 600 ml, wherein the final volume of liquid comprising the cells was again reduced to 15 ml. Media replacement was performed for 40 cycles (Forty (40)×15 ml washes=600 ml total wash volume). There is not a limit to the volume of media that can be replaced. Results: In order to examine the effect of flow rate during exchange of a liquid solution, the volume of liquid used in the downstream washing/collection was maintained but the rate at which the liquid flowed was differed. Thus, an exchange in a shorter time period was a result of a higher flow, and a longer time period was the result of a lower flow rate. After 30 mins of media exchange at 3.6 mm/min, 60.3% of the cells recovered having viability of 87.8%. After 60 mins of media exchange at 1.8 mm/min, 100% of the cells were recovered having 91% viability. After 90 minutes of media exchange at 1.2 m/min, 100% of the cells were recovered with 92.1% viability. Conclusion: Media replacement was comparable to other methods known in the art, such as TFF, which replaces/dilutes 5 volumes. Significantly, using the method described here to wash and collect cells avoids the high flow rate and shear of the continues flow centrifuge (1-2 liters per minute), as the low flow rates used were 1,000 to 10,000 fold lower with much less shear. While certain features of the bioreactors and systems thereof disclosed herein have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the bioreactors and systems thereof disclosed herein. | 196,774 |
11859164 | DETAILED DESCRIPTION The invention involves novel, safe, scalable, biomaterial-based bioreactors and method of using the same. In one embodiment, the bioreactor is made using perforated plates stacked such that the assembled bioreactor has the necessary manifolds and chambers to transport gas and liquids to the biomaterial and to remove the reaction products. In one embodiment, the bioreactor may be used to contact gases and liquid medium with a reactor chamber containing, biomaterial. As used herein, the term “biomaterial” is intended, at a minimum, to cover all types of biomaterial that can be used to convert CO2to bio-based products, including but not limited to, bacteria and algae. The term “biomaterial” may also include biomaterial that does not use and/or require CO2. In one embodiment, the biomaterial comprises a living organism capable of using hydrogen gas as a source of energy. In one embodiment, the biomaterial comprises bacteria capable of using hydrogen gas as a source of energy. In one embodiment, the biomaterial, is an autotroph. In one embodiment, the biomaterial comprisesRalstonia eutropha, e.g., H16 orCupriavidus. In another embodiment, the reactor may be used to grow anaerobic organisms (using oxygen nonpermeable membranes about the biomaterial) and aerobic organisms that use alternative carbon sources. In one embodiment, the biomaterial reactor chamber may contain a natural or artificial biofilm grown on a membrane or other fibrous support structure, or the biomaterial can be sandwiched in a chamber cavity between two membranes. On one side of the biomaterial reactor chamber containing the biomaterial, a gas, such as hydrogen, may be introduced. On the opposite side of the chamber, a liquid medium, providing nutrients, oxygen, and carbon dioxide for the biomaterial may be circulated. Thus, in at least one embodiment, when a first element is said to be “in fluid communication with” a second element (e.g., a gas reactor chamber “in fluid communication with” a first side of the biomaterial reactor chamber; or a medium reactor chamber “in fluid communication with a second side of the biomaterial reactor chamber”) in fact, the two elements may be in fluid communication through the membrane(s). The liquid medium may comprise any suitable liquid medium that may supply nutrients and oxygen, and, in some embodiments, carbon dioxide, such as are well known in the art. For example, the liquid medium may comprise Repaske's medium or a modified version thereof. The biomaterial converts the carbon dioxide to bio-based products or another product(s), which can be outletted or otherwise removed on either the gas, side or the liquid medium side. In various embodiments, the bioreactor may be used to make bio-based products, including, but not limited to, lubricants and greases, lubricant additives, biofuels bio-based chemicals, oil remediation dispersants and sorbents, health supplements, nutraceutical, cosmeceufical, and pharmaceutical product ingredients, horticultural and aquacultural feed or supplements, and intermediates to the foregoing. As used herein, the term “biofuels” broadly refers to bio-based products suitable for use as a fuel or a combustion source, including fuels suitable for transportation and power generation. Biofuels include, but are not limited to, biogasoline, biodiesel, jet fuels, ethanol, methanol, butanol, and the like. Bio-based materials and chemicals include, but are not limited to: Polyhydmxyalkanoates, lactic acid, acetic acid, succinic acid, malic acid, 1-butanol, isobutanol, 2-butanol, other alcohols, amino acids, 1,3-propanediol, ethylene, glycerine, β-lactam antibiotics, cephalosporin, alkanes, terpenes, and the like. In one embodiment, the bioreactor may be made up of a stack of plates. Each plate may be stamped with a perforation pattern such that when the plates are stacked together, a bioreactor is created, with gas and medium reactor chambers on each side of a biomaterial reactor chamber containing the biomaterial. Gas may be supplied on one side of the biomaterial reactor chamber through gas manifolds, and medium may be supplied on the other side of the biomaterial reactor chamber through medium manifolds. In some embodiments, costs for preparation of the bioreactor may be minimized because the plates can be mass produced using a stamping process. In addition, in some embodiments, reactor capacity may be readily scalable by increasing the number of plates in the stack and/or the dimensions of the membranes. The reactor may also provide increased safety, as the small size of each individual cell will limit the magnitude of potentially destructive energy release. If desirable or appropriate, flame arrestors may be added in the gas manifolds, as well as between the plates, to prevent flame propagation if hydrogen and oxygen were to react explosively. In some, embodiments, the reactor may be configured so that different reactions take place in adjacent cells. In one embodiment, the stacked reactors may be configured so that products produced in one reactor become feedstocks for another reactor, and so forth, like a daisy chain to the final product. For example: Stack Reactor 1=Product 1 (feedstock for Reactor 2)→Stack Reactor 2=Product 2 (feedstock for Reactor 3)→→→final product. For example, hydrogen can be produced by one type of biomaterial in one cell, and this hydrogen can be transported to an adjacent cell, where it can be used as a feedstock to produce a secondary product. In another example, other intermediates (e.g., metabolites, peptides, building blocks) may be prepared and/or supplied for more complex products. Indeed, using such a design may provide for practically limitless bioconversions (e.g., chiral specific conversions of pharmaceuticals and their precursors, as well as oxygenation and/or dehydrogenation and/or methylation and/or acetylation, of numerous compounds). The possibilities are vast and, in view of disclosure of the present application, achievable. In one embodiment, CO2may be supplied in the form of CO2emitted from a commercial process. In one embodiment, CO2, H2, and hydrocarbon or hydrocarbon-like (hydrogen and carbon-rich) gas may be supplied in the form of a product from pyrolysis of a biomass. FIGS.1-2illustrate one embodiment of a bioreactor100. More specifically,FIGS.1A-Eillustrate a series of plates that can be used to form one embodiment of bioreactor100.FIG.1Aillustrates manifold plate110.FIG.1Bshows a gas delivery plate115.FIG.1Cshows a biomaterial plate120.FIG.1Dshows a medium delivery plate125. AndFIG.1Eshows an end plate130. The plates can be made of any suitable material, such as metal, plastic, ceramic, acrylic, polycarbonate, polypropylene, Delrin® manufactured by Dupont, polyetheretherketone, polyvinyl chloride (PVC), stainless steel, and the like. The plates can be any suitable shape, such as square, rectangular, circular, and the like. The bioreactor may be secured together in any suitable manner, as would be understood by those of skill in the art. For example, bolts, screws, or clamps may be used. In some embodiments, adjacent plates could have interlocking parts to secure the adjacent plates together. The interlocking parts may be releasable or permanent. For instance, permanent interlocks may be used if the bioreactor was to be disposable and not intended to be disassembled. In one embodiment, each of the plates has bolt holes135on the corners for bolting the plates together to assemble the bioreactor. As shown, manifold plate110has a gas inlet140and a gas outlet145on two opposing sides, and a medium inlet150and medium outlet155on the other two opposing sides of manifold plate110. As shown, gas delivery plate115has a gas reactor chamber160in the middle, biomaterial plate120has a biomaterial reactor chamber161in the middle, and medium delivery plate125has a medium reactor chamber162in the middle. Gas delivery plate115has a gas inlet manifold165and a gas outlet manifold170on opposite sides of the plate outside of gas reactor chamber160, and a medium inlet manifold175and a medium outlet manifold180outside of gas reactor chamber160on the other two opposing sides. Biomaterial plate120has a gas inlet manifold165and a gas outlet manifold170on opposite sides of the plate outside of biomaterial reactor chamber161, and a medium inlet manifold175and a medium outlet manifold180outside of biomaterial reactor chamber161on the other two opposing sides. Medium delivery plate125has a gas inlet manifold165and a gas: outlet manifold170on opposite sides of the plate outside of medium reactor chamber162, and a medium inlet manifold175and a medium outlet manifold180outside of medium reactor chamber162on the other two opposing sides. As shown, gas delivery plate115has gas inlet channels185between gas inlet manifold165and gas reactor chamber160, and gas outlet channels190between gas reactor chamber160and gas outlet manifold170. Biomaterial plate120may have a membrane195in biomaterial reactor chamber161. In one embodiment, membrane195may contain an artificial or natural biofilm which is used to immobilize biomaterial. Membrane195may be fabricated using composite materials and may serve to provide one or more of the following attributes: (a) structural support or scaffolding to the biofilm; (b) a seal between the gas and the medium phases; and (c) pathways for the gases, nutrients, and products to and from the biomaterial immobilized in the biofilm. Alternatively, biomaterial reactor chamber161may contain free-floating biomaterial. As shown, medium delivery plate125has medium inlet channels1100between medium inlet manifold175and medium reactor chamber162, and medium outlet channels1105between medium reactor chamber162and medium outlet manifold180. In one embodiment, end plate130may be solid except for bolt holes135. In one embodiment, plates110,115,120,125, and130are bolted together by placing bolts1110in bolt holes135in the corners of the plates, as shown inFIG.2. There may additionally be gaskets1115between plates110,115,120,125, and130to seal bioreactor100. In one embodiment, H2gas may enter bioreactor100through gas inlet140in manifold plate110, and may flow through gas inlet channels185in gas delivery plate115into gas reactor chamber160, where the gas is available for use by the biomaterial on membrane195in biomaterial reactor chamber161. The gas flows out, through gas outlet channels190to gas outlet manifold170, and exits bioreactor100through gas outlet145in manifold plate110. In one embodiment, medium may enter bioreactor100though medium inlet150in manifold plate110. In one embodiment, O2and CO2are mixed in the medium. Medium flows through medium inlet manifold175in gas delivery plate115, biomaterial plate120, and medium delivery plate125. The medium flows through medium inlet channels1100to medium reactor chamber162in medium delivery plate125, where the medium is available for use by the biomaterial in biomaterial reactor chamber161of biomaterial plate120. The medium flows out through medium outlet channels1105to medium outlet manifold180in medium delivery plate125, biomaterial plate120, and gas delivery plate115. The medium exits bioreactor100through medium outlet155in manifold plate110. Alternatively, CO2may be supplied with the H2gas; or CO2may be supplied with both the H2gas and the medium. Alternatively, in one embodiment, a different gas may be introduced from each side (such as H2/O2) of the biomaterial reactor chamber, and the medium may be flowed slowly through the biomaterial reactor chamber to provide mixing within the biomaterial reactor chamber. In such an embodiment, of course, the innermost membranes about the biomaterial may be hydrophobic. In one embodiment, membrane195in biomaterial plate120mitigates or governs the concentrations of reactants in the interaction between the H2gas in gas reactor chamber160in gas delivery plate115and the CO2and the O2in the medium in medium reactor chamber162in medium delivery plate125. In one embodiment, the biomaterial on membrane195may consume the H2from gas reactor chamber160in gas delivery plate115and the CO2and O2in the medium in medium reactor chamber162in medium delivery plate125and metabolizes them to the bio-based product. In one embodiment, the bio-based product may flow out with the medium and can be separated in a distillation process. The process is easily scalable by adding plates to the reactor. For example, instead of end plate130after medium delivery plate125, there may be another biomaterial plate120, and gas delivery plate115. The addition may be extended to tens or hundreds of plates. If there is a puncture in membrane195and O2leaks into the H2, there is only a small volume of H2present in gas reactor chamber160in gas delivery plate115, so any energy discharge if the O2and H2ignite would be small. Flame arrestors may also be included to quench any flame front generated to further reduce the danger of explosion. The flame arrestors may be located in one or more of the gas manifolds, the gas reactor chamber, the medium manifolds, or the medium reactor chamber. Suitable flame arrestors include, but are not limited to, wire mesh, or metal, plastic, or ceramic foam materials. | 13,293 |
11859165 | It is not intended that the figures be limiting. The open-top cavity/chamber can have various geometries other than the one depicted above: e.g. oval, rectangular slot, ellipse. Structural anchors can have various geometries other than the one depicted above. For example, they can have different ‘head’ geometries and sizes. Alternatively, the gel can be maintained with a mesh wall or micro-pillar array.FIG.20A-Bshows a top view and elevated side view of one embodiment of a micro-pillar array. FIG.21A-Bshows a top view and elevated side view of one embodiment of a mesh wall or insert. The bottom-layer microfluidics can have various channel geometries other than the one depicted above, i.e. the channel height, channel width, and channel path geometry can be changed.FIG.22shows a different design for the microfluidic channels. FIGS.23-26show various embodiments for microfluidic devices as contemplated herein that are configured for electrophysiological measurements (e.g., for example, patch clamp measurements using transepithelial electric resistance (TEER). FIG.27illustrates one embodiment of a top view of an assembled open-top chip microfluidic device of the device depicted inFIG.13. FIG.28illustrates one embodiment of an array of open chambers in an open-top chip device as contemplated herein. FIG.29A-Billustrates one embodiment of a stretchable open top chip device. FIG.29A: A bottom structure with a spiral microchannel with an inlet well and and outlet well. FIG.29B: A top view of a spiral microchannel configured with a circular vacuum chamber. FIG.30illustrates an exploded view of one embodiment of a stretchable open top chip device demonstrating the layering of a fluidic top, top structure and bottom structure. FIG.31A-Billustrates a cut-away view of one embodiment of a stretchable open top chip device showing the regional placement of assay cells (e.g., epithelial cells, dermal cells and/or vascular cells). FIG.32illustrates a fully assembled view of one embodiment of a stretchable open top chip device. FIGS.33A and33Billustrate exploded views of two embodiments of a stretchable open top chip device. FIGS.34A and34Billustrate assembled views of a stretchable open top chip device as depicted inFIGS.33A and33B. FIGS.35A and35Brespectively illustrate an assembled isometric view and an exploded view of a tall channel stretchable open top chip device. FIG.36presents a top assembled view of one embodiment of a stretchable open-top microfluidic chip comprising a fluidic cover and a single channel. FIG.37A-Bpresents a crossectional view of a first embodiment of a stretchable open top microfluidic chip along plane A ofFIG.36. FIG.37A: Illustrates a fluidic cover in a closed position. FIG.37B: Illustrates a fluidic cover in an open position. FIG.38A-Bpresents a crossection view of a second embodiment of a stretchable open top microfluidic chip along plane A ofFIG.36. FIG.38A: Illustrates a fluidic cover in a closed position. FIG.38B: Illustrates a fluidic cover in an open position. FIG.39presents an exploded view of the array device depicted inFIG.28. DESCRIPTION OF THE INVENTION A microfluidic device is contemplated comprising an open-top cavity with structural anchors on the vertical wall surfaces that serve to prevent gel shrinkage-induced delamination, a porous membrane (optionally stretchable) positioned in the middle over a microfluidic channel(s). The device can be used in many ways with many types of tissues and cells. For example, the organ mimic device described herein can be used for the identification of markers of disease; assessing efficacy of anti-cancer therapeutics; testing gene therapy vectors; drug development; screening; and for studies of particular cells (and arrangements of cells). In one embodiment, the device serves as a skin model. In this embodiment, the open-top device provides an uncovered chamber comprising a skin-like, human or animal tissue that can be tested with drugs, including topicals and aerosols. A. Gel-Containing Skin Model In one embodiment, the present invention contemplates a construct comprising a “dermis” with fibroblasts embedded in a matrix having a thickness between 0.2 and 6.0 mm, e.g. a collagen I gel matrix, and an “epidermis”, which is comprised of keratinocytes, e.g. stratified, differentiated keratinocytes. A matrix such as a collagen gel provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. In one embodiment, the construct further comprises a functional basement membrane, which separates the epidermis from the dermis. In one embodiment, the present invention contemplates a layered structure comprising i) fluidic channels covered by ii) a porous membrane, said membrane comprising iii) a layer of endothelial cells and said membrane position below iv) a gel matrix comprising fibroblasts and keratinocytes. In a preferred embodiment, the fibroblasts are within the gel matrix and the keratinocytes are on top of the gel matrix. In a preferred embodiment, the keratinocytes comprise more than one layer on top of the gel matrix. In a preferred embodiment, the layer of endothelial cells is positioned on the bottom of the membrane and is in contact with the fluidic channels. In a preferred embodiment, the fluidic channels provide shear to said endothelial cells. It is not intended that the present invention be limited to the thickness of the gel matrix. However, a preferred range of thickness is between 0.2 and 6 mm, and more preferably between 0.5 mm and 3.5 mm, and still more preferably approximately 1-2 mm. In a preferred embodiment, the gel matrix is stretchable. In a preferred embodiment, the gel matrix is stretched in a manner such that the entire gel matrix expands, not just a portion of the gel matrix (such as only the bottom or top of the matrix). In a preferred embodiment, the gel matrix is stretched by vacuum channels that are designed to provide pneumatic stretching that is uniform across the thickness of the gel. In a preferred embodiment, the layered structure is positioned in an open-top microfluidic device (i.e. a device lacking a top covering), wherein the gel matrix is secured in a chamber of the device by anchors. In a preferred embodiment, the surfaces of the device that contact the gel matrix have been treated to enhance attachment of the gel matrix. In a preferred embodiment, the surfaces have been plasma treated, i.e. the surface is activated with ionized gas. It has been found that the surface treatment, in combination with the anchors, prevent delamination of the gel from the walls of the chamber. In one embodiment, the fluidic channels bring one or more compounds that will induce the endothelial cells to differentiate. In one embodiment, the fluidic channels comprise a solution comprising a vascular endothelial growth factor (VEGF). The open-top device provides an uncovered chamber comprising a skin-like tissue that can be tested with topicals and aerosols. In one embodiment, drugs are applied topically or transdermally to the keratinocyte layer(s). As used herein, the term “topical” refers to administration of an agent or agents (e.g. cosmetic, medication, vitamin, etc.) on the skin. “Transdermal” refers to the delivery of an agent (e.g. cosmetic, medication, vitamin, etc.) through the skin (e.g. so that at least some portion of the population of molecules reaches underlying layers of the skin). In one embodiment, a candidate cosmetic is applied to the keratinocyte layer(s). As used herein, a “cosmetic” refers to a substance that aids in the enhancement or protection of the appearance (e.g. color, texture, look, feel, etc.) or odor of a subject's skin. A cosmetic may or may not change the underlying structure of the skin. In this skin model, a layer of endothelial cells (ECs) is positioned on the underside of the membrane facing the fluidic channels. Endothelial cells and endothelial stem cells will, under appropriate conditions, migrate and differentiate. In terms of migration, while not limited to any particular mechanism, it is believed that this motile process is directionally regulated by chemotactic, haptotactic, and mechanotactic stimuli and (where applicable) may require degradation of the extracellular matrix to enable progression of the migrating cells. It is believed to involve the activation of several signaling pathways that converge on cytoskeletal remodeling. Generally, it is been observed that the endothelial cells extend, contract, and progress forward. In a preferred embodiment, ECs are grown on a membrane with a porosity sufficient to allow for this cell migration, i.e. through the membrane. In some embodiments, growth factors or compounds that enhance the production of the desired cell type(s) can be added to the perfusion fluid in the fluidic channels. By way of non-limiting example, erythropoietin stimulates the production of red blood cells, VEGF stimulates angiogenesis, and thrombopoietin stimulates the production of megakaryocytes and platelets. “Vascular growth” is defined here as at least one of vasculogenesis and angiogenesis and includes formation of one or more of the following: capillaries, arteries, veins or lymphatic vessels. Blood vessel formation de novo (vasculogenesis) and from existing vessels (angiogenesis) results in blood vessels lined by endothelial cells (ECs). Vascular endothelial growth factor (VEGF) is an interesting inducer of angiogenesis and lymphangiogenesis because it is highly specific endothelial cells. The VEGF family currently comprises seven members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PlGF. All members have a common VEGF homology domain. Signal transduction involves binding to tyrosine kinase receptors and results in endothelial cell proliferation, migration, and new vessel formation. In a preferred embodiment, VEGF (and/or other known angiogenic or arteriogenic growth factors) is used to induce EC differentiation, proliferation, infiltration, angiogenesis, vascularization, etc., or any combination thereof. It is not intended that the present invention be limited to only one source or type of endothelial cell (EC). In one embodiment, primary ECs are used in the open-top device. In one embodiment, freshly isolated small vessel human dermal microvascular endothelial cells are employed on the open-top-chip. In one embodiment, an endothelial cell line is employed. In yet another embodiment, human umbilical vein endothelial cells (HUVECs) are used. In still another embodiment, bone marrow-derived endothelial progenitor cells are seeded in the chip. In still another embodiment, stem cells that can differentiate into ECs are used. It is not intended that the present invention be limited to only one place for seeding the open-top-chip with ECs. While placement of ECs on the underside of the membrane (in contact with the fluidics) is preferred, placement on the topside of the membrane and placement within the gel matrix itself are alternative embodiments. With regard to the latter, in one embodiment, microfluidic pathways in the gel itself are created that are thereafter seeded with the endothelial cells. For example, in one embodiment, microfluidic vessel networks are engineered by seeding human endothelial cells [e.g. umbilical vein endothelial cells (HUVECs)] into microfluidic circuits formed via soft lithography in a type I collagen gel. Native, type I collagen at 6-10 mg/mL is of an appropriate stiffness to allow high reproducibility of vessel microstructure and also enables remodeling through degradation and deposition of extracellular matrix. The lithographic process enables the formation of endothelium along the microfluidic channels and the incorporation of living cells within the bulk collagen gel matrix within the open-top-chip. In one embodiment, endothelial cells are seeded into the gel containing (or onto confluent lawns of) human fibroblasts and cultured in the presence of high levels of ascorbate 2-phosphate to create a tissue-like structure in which endothelial cells organize into tube-like structures. It is not intended that the skin model be limited to just one type of keratinocyte. Indeed, the model can be used with many types of cells of the integumentary system including but not limited to Keratinizing epithelial cells, Epidermal keratinocyte (differentiating epidermal cell), Epidermal basal cell (stem cell), Keratinocyte of fingernails and toenails, Nail bed basal cell (stem cell), Medullary hair shaft cell, Cortical hair shaft cell, Cuticular hair shaft cell, Cuticular hair root sheath cell, Hair root sheath cell of Huxley's layer, Hair root sheath cell of Henle's layer, External hair root sheath cell, and Hair matrix cells (stem cell). In one embodiment, human foreskin keratinocytes are employed. B. Other Cells and Tissues A variety of different cells and tissue types can be modeled and tested with the open-top spacer chip described herein. Indeed, the system can virtually be adapted to all epithelial tissues. In addition to skin, preferred models include (but are not limited) to Lung, the Small Airway, the gut, muscle (including skeletal, cardiac and or smooth muscle, and the Blood Brain Barrier (BBB). Both human and animal cells are contemplated. Cell types which can be used in the open-top devices include, but are not limited to Wet stratified barrier epithelial cells, such as Surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, Urinary epithelium cell (lining urinary bladder and urinary ducts); Exocrine secretory epithelial cells, such as Salivary gland mucous cell (polysaccharide-rich secretion), Salivary gland serous cell (glycoprotein enzyme-rich secretion), Von Ebner's gland cell in tongue (washes taste buds), Mammary gland cell (milk secretion), Lacrimal gland cell (tear secretion), Ceruminous gland cell in ear (wax secretion), Eccrine sweat gland dark cell (glycoprotein secretion), Eccrine sweat gland clear cell (small molecule secretion), Apocrine sweat gland cell (odoriferous secretion, sex-hormone sensitive), Gland of Moll cell in eyelid (specialized sweat gland), Sebaceous gland cell (lipid-rich sebum secretion), Bowman's gland cell in nose (washes olfactory epithelium), Brunner's gland cell in duodenum (enzymes and alkaline mucus), Seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm), Prostate gland cell (secretes seminal fluid components), Bulbourethral gland cell (mucus secretion), Bartholin's gland cell (vaginal lubricant secretion), Gland of Littre cell (mucus secretion), Uterus endometrium cell (carbohydrate secretion), Isolated goblet cell of respiratory and digestive tracts (mucus secretion), Stomach lining mucous cell (mucus secretion), Gastric gland zymogenic cell (pepsinogen secretion), Gastric gland oxyntic cell (hydrochloric acid secretion), Pancreatic acinar cell (bicarbonate and digestive enzyme secretion), pancreatic endocrine cells, Paneth cell of small intestine (lysozyme secretion), intestinal epithelial cells, Types I and II pneumocytes of lung (surfactant secretion), and/or Clara cell of lung. One can also coat the membrane with Hormone secreting cells, such as endocrine cells of the islet of Langerhands of the pancreas, Anterior pituitary cells, Somatotropes, Lactotropes, Thyrotropes, Gonadotropes, Corticotropes, Intermediate pituitary cell, secreting melanocyte-stimulating hormone; and Magnocellular neurosecretory cells secreting oxytocin or vasopressin; Gut and respiratory tract cells secreting serotonin, endorphin, somatostatin, gastrin, secretin, cholecystokinin, insulin, glucagon, bombesin; Thyroid gland cells such as thyroid epithelial cell, parafollicular cell, Parathyroid gland cells, Parathyroid chief cell, Oxyphil cell, Adrenal gland cells, chromaffin cells secreting steroid hormones (mineralcorticoids and gluco corticoids), Leydig cell of testes secreting testosterone,Theca internacell of ovarian follicle secreting estrogen, Corpusluteumcell of ruptured ovarian follicle secreting progesterone, Granulosa lutein cells,Thecalutein cells, Juxtaglomerular cell (renin secretion),Macula densacell of kidney, Peripolar cell of kidney, and/or Mesangial cell of kidney. Additionally or alternatively, one can treat at least one side of the membrane with Metabolism and storage cells such as Hepatocyte (liver cell), White fat cell, Brown fat cell, Liver lipocyte. One can also use Barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract) or Kidney cells such as Kidney glomerulus parietal cell, Kidney glomerulus podocyte, Kidney proximal tubule brush border cell, Loop of Henle thin segment cell, Kidney distal tubule cell, and/or Kidney collecting duct cell. Different geometries can be employed with dimensions related to the different tissue types. For example, in one embodiment, relatively tall spacer open top chip dimensions are contemplated for the skin model, bronchial model, Kidney model and Gut model, i.e. chamber height between 500 microns to 5 mm, chamber width 1 mm, chamber length 1.6 mm. In another embodiment, relatively short spacer open top chip dimensions are employed: chamber height between 100 to 500 microns, chamber width 1 mm, chamber length 1.6 mm. These dimensions are better suited to the Brain barrier and Lung models. An example of the importance of its application is the small airway model: Small Airway cells feel the paracrine stimulation of neighbor cells, which stimulate their fully differentiation. In the normal chip design cytokines are continuously flushed away from the epithelial compartment by the constant flow, and this reduces or impedes epithelial cell differentiation. The presence of this porous matrix efficiently buffers the effect of flow reducing or annul the effect of flow under cells. The physical properties of the gels and fluids can vary (in addition to the different geometries and dimensions for each of the different tissue types). For example, for the Skin model and bronchial model, a relatively high concentration collagen (8-11 mg/ml) is used. For the Kidney model and Gut model, a 1:1 mixture of high concentration collagen:Matrigel is employed. For the Brain barrier and lung models, a 1:1 low concentration (e.g. 3 mg/ml) of collagen/matrigel and/or fibronectin is employed. All in all, concentrations above 0.3 mg/ml are required to form gels. Preferred concentrations range between 3 mg/ml and 10 mg/ml. However, concentrations above 5 mg/ml are particularly suitable for use in the open top chip. Not all of the organ models require a gel. Indeed, some organ chips are ideally used without a gel (e.g. lung). When gels are used, more than one gel layer can be employed. For example, hepatocytes can have a gel on both sides of the cells (e.g. a matrigel layer on top and a collagen layer on the bottom. Importantly, the gel can have a variety of thicknesses, including a thin (molecular) coating. In one embodiment, the coating is made with by ink jet printing. Some cells do very well on patterned gels. For example, muscle cells do well when they can deform to the surface. Indeed, in one embodiment, the present invention contemplates a gel pattern such that the sarcomeres align. Importantly, the present invention contemplates electrophysiological measurements in more than the blood brain barrier (BBB) model. The present invention contemplates such measurements for muscle (whether skeletal, cardiac or smooth muscle) cells. DETAILED DESCRIPTION OF THE INVENTION While this invention is susceptible of embodiment in many different forms, there is shown in the drawings and will herein be described in detail preferred embodiments of the invention with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and is not intended to limit the broad embodiment of the invention to the embodiment illustrated. For purposes of the present detailed description, the singular includes the plural and vice versa (unless specifically disclaimed); the word “or” shall be both conjunctive and disjunctive; the word “all” means “any and all”; the word “any” means “any and all”; and the word “including” means “including without limitation.” Those of ordinary skill in the art will realize that the following description is illustrative only and is not intended to be in any way limiting. Other embodiments will readily suggest themselves to such skilled persons having the benefit of this disclosure. Reference will now be made in detail to implementations of the example embodiments as illustrated in the accompanying drawings. The same or similar reference indicators will be used throughout the drawings and the following description to refer to the same or like items. It is understood that the phrase “an embodiment” encompasses more than one embodiment and is thus not limited to only one embodiment. As used herein, the term “rigid” refers to a material that is stiff and does not stretch easily, or maintains very close to its original form after a force or pressure has been applied to it. The term “elastomeric” as used herein refers to a material or a composite material that is not rigid as defined herein. An elastomeric material can be generally moldable, extrudable, cuttable, machinable, castable, and/or curable, and can have an elastic property that enables the material to deform (e.g., stretching, expanding, contracting, retracting, compressing, twisting, and/or bending) when subjected to a mechanical force or pressure and partially or completely resume its original form or position in the absence of the mechanical force or pressure. In some embodiments, the term “elastomeric” can also refer to a material that is flexible/stretchable but it does not resume its original form or position after pressure has been applied to it and removed thereafter. The terms “elastomeric” and “flexible” are used interchangeably herein. The functionality of cells, tissue types, organs, or organ-components can be implemented in one or more microfluidic devices or “chips” that enable researchers to study these cells, tissue types, organs, or organ-components outside of the body while mimicking much of the stimuli and environment that the tissue is exposed to in-vivo. In some embodiments, it is desirable to implement these microfluidic devices into interconnected components that can simulate the function of groups of organs, organ-components, or tissue systems. In some cases it is desirable to configure the microfluidic devices so that they can be easily inserted and removed from an underlying fluidic system that connects to these devices in order to vary the simulated in-vivo conditions and organ systems (e.g., in situ conditions). Many of the problems associated with earlier systems can be solved by providing an open-top style microfluidic device that allows topical access to one or more parts of the device or cells that it comprises. For example, the microfluidic device can include a removable cover, that when removed, provides access to the cells of interest in the microfluidic device. In some embodiments, the microfluidic devices include systems that constrain fluids, cells, or biological components to desired area(s). The improved systems provide for more versatile experimentation when using microfluidic devices, including improved application of treatments being tested, improved seeding of additional cells, and/or improved aerosol delivery for select tissue types. In a preferred embodiment, the open-top microfluidic device comprises a gel matrix. The present disclosure additionally relates to organ-on-chips (“OOCs”), such as fluidic devices comprising one or more cells types for the simulation one or more of the function of organs or organ-components. Accordingly, the present disclosure additionally describes open-top organ-on-chips that solve problems associated with earlier fluidic systems. Without limitation, specific examples include models of skin, bronchial, and gut. It is also desirable in some embodiments to provide access to regions of a cell-culture device. For example, it can be desirable to provide topical access to cells to (i) apply topical treatments with liquid, gaseous, solid, semi-solid, or aerosolized reagents, (ii) obtain samples and biopsies, or (iii) add additional cells or biological/chemical components. The present disclosure relates to fluidic systems that include a fluidic device, such as a microfluidic device with an opening that provides direct access to device regions or components (e.g. access to the gel region, access to one or more cellular components, etc.). Although the present disclosure provides an embodiment wherein the opening is at the top of the device (referred to herein with the term “open top”), the present invention contemplates other embodiments where the opening is in another position on the device. For example, in one embodiment, the opening is on the bottom of the device. In another embodiment, the opening is on one or more of the sides of the device. In another embodiment, there is a combination of openings (e.g. top and sides, top and bottom, bottom and side, etc.). While detailed discussion of the “open top” embodiment is provided herein, those of ordinary skill in the art will appreciate that many embodiments of the “open top” embodiment apply similarly to open bottom embodiments, as well as open side embodiments or embodiments with openings in any other regions or directions, or combinations thereof. Similarly, the device need not remain “open” throughout its use; rather, as several embodiments described herein illustrate, the device may further comprise a cover or seal, which may be affixed reversibly or irreversibly. For example, removal of a removable cover creates an opening, while placement of the cover back on the device closes the device. The opening, and in particular the opening at the top, provides a number of advantages, for example, allowing (i) the creation of one or more gel layers for simulating the application of topical treatments on the cells, tissues, or organs, or (ii) the addition of chemical or biological components such as the seeding of additional cell types for simulating the function of tissue and organ systems. The present disclosure further relates to improvement in fluidic system(s) that improve the delivery of aerosols to simulate the function of tissue and organ systems, such as simulated function of lung tissues. Furthermore, the present disclosure contemplates improvements to fluidic systems that include a fluidic device, such as a microfluidic device with an open-top region that reduces the impact of stress that can cause the delamination of tissue or related component(s) (e.g., such as a gel layer). Improvements to microfluidic devices for simulating the function of a tissue are contemplated by the present disclosure that include one or more of an open-top microfluidic device with two or more chambers (e.g., microchannels) separated by a membrane. In some embodiments, one or more of the devices further comprises a gel in a chamber (e.g., microchannel or cavity) accessible through an opening, including but not limited to an open-top structure, of the microfluidic device. In some embodiments, the device further comprises a removable or permanent cover for the microfluidic device where the cover optionally has a fluidic chamber or microchannel therein. Other desirable improvements that are contemplated include a patterned gel in a microfluidic device. The present disclosure further describes a method for culturing cells in open-top devices. In some embodiments, the method comprises placing a gel into an open-top structure. In some embodiments, the method further comprises patterning the gel using a shaping device, such as a patterned plunger stamp, a shaping stamp, or similar devices. In some embodiments, the method comprises permanently or reversibly applying a cover or other shaping device to the open-top. The present disclosure further relates to the use of fluidic systems that include a fluidic device, such as a microfluidic device with an open-top, to construct a model simulating the structure and/or one or more functions of, for example, skin, bronchial, or gut. In some embodiments, these models benefit from the presence of gels, which for example, can provide a mechanical, biochemical environment for one or more cells types, augment the mass-transport characteristics, or provide an additional compartment that may be used, for example, to house an additional cell type (e.g. fibroblasts). A system that provides for the use of a gel can be particularly desirable for a skin model. For example, the current state-of-the-art skin model, the living skin equivalent (LSE), is a 3D gel, 2 mm to 3 mm thick, that is embedded with fibroblasts with differentiated keratinocytes on top of the gel. The actual thickness of the gel can range from 0.1 mm to 5 mm. It is known that a 3D gel is preferred to properly culture the fibroblasts that, in turn, enables keratinocytes to fully differentiate. An open-top architecture as described by some embodiments herein is desirable because it enables LSE-like and similar cultures of fibroblasts and keratinocytes, while further allowing the introduction of an endothelial layer, the application of shear forces, and the application of stretching to create a more physiologically relevant model. Each of these optional features, individually and collectively, provides desirable improvements over current state-of-the-art LSE-like skin models. Referring now toFIGS.1and2, one type of a microfluidic device referred to as an organ-on-chip (“OOC”) device100is illustrated that may be modified to include open-top embodiments that are described in more detail later in this disclosure (see, e.g.,FIGS.3-5A-F and8A-C-12). The OOC device100includes a body112that typically comprises an upper body segment101and a lower body segment103. The upper body segment101and the lower body segment103are typically made of a polymeric material, including, but not limited to, PDMS (poly-dimethylsiloxane), polycarbonate, polyethylene terephthalate, polystyrene, polypropylene, cyclo-olefin polymers, polyurethanes, fluoropolymers, styrene derivatives like styrene ethylene butylene styrene (SEBS), or other polymer materials. The upper body segment101, while illustrated with a first fluid inlet117and a second fluid inlet118, can be modified to include an open region104(not shown) to optionally allow the application of a gel layer150(not shown) to a membrane140and optionally modified to exclude the illustrated first fluid inlet117and/or second fluid inlet118. A first fluid path for a first fluid includes the first fluid inlet117, a first seeding channel127, an upper microchannel134, an exit channel131, and then the first fluid outlet124. A second fluid path for a second fluid includes the second fluid inlet118, a second seeding channel128, a lower microchannel136, an outlet channel133, and then the second fluid outlet126. Referring toFIG.2, a membrane240extends between the upper body segment201and the lower body segment203. The membrane240is preferably an inert, polymeric, micro-molded membrane having uniformly distributed pores with sizes normally in the range of about 0.1 μm to 20 μm, though other pore sizes are also contemplated. In some embodiments, the pore size is in the range of about 0.1 μm to 20 μm. The overall dimensions of the membrane240include any size that is compatible with or otherwise based on the dimensions of upper body segment201and lower body segment103, such as about 0.05-100 mm (channel width) by about 0.5-300 mm (channel length), though other overall dimensions are also contemplated. In some embodiments, the overall dimensions of the membrane240are about 1-100 mm (channel width) by about 1-100 mm (channel length). In one embodiment, the thickness of the membrane240is generally in the range of about 5 μm to about 500 μm, and in some embodiments, the thickness is about 20-50 μm. In some embodiments, the thickness can be less than 1 μm or greater than 500 μm. It is contemplated that the membrane240can be made of materials including, but not limited to poly-dimethylsiloxane (PDMS), polycarbonate, polyethylene terephthalate, styrene derivatives (e.g, styrene ethylene butylene styrene, SEBS), fluoropolymers, and/or other elastomeric or rigid materials. Additionally, the membrane240can be made of biological materials including, but not limited to, polylactic acid, collagen, gelatin, cellulose and its derivatives, poly(lactic-co-glycolic acid), and/or comprise such materials in addition to one or more polymeric materials. The membrane240separates an upper microchannel234from the lower microchannel236in an active region237, which includes a bilayer of cells in the illustrated embodiment. In some embodiments, a first cell layer242is adhered to a first side of the membrane240, and in some embodiments a second cell layer244is adhered to a second side of the membrane240. The first cell layer242may include the same type of cells as the second cell layer244. Or, the first cell layer242may include a different type of cell than the second cell layer244. And, while a single layer of cells is shown for the first cell layer242and the second cell layer244, either the first cell layer242, the second cell layer244, or both may include multiple cell layers or cells in a non-layer structure. Further, while the illustrated embodiment includes a bilayer of cells on the membrane240, the membrane240may include only cells disposed on one of its sides. Furthermore, while the illustrated embodiment includes cells adherent to the membrane, cells on one or both sides may instead be not be adherent to the membrane as drawn; rather, cells may be adherent on the opposing chamber surface or embedded in a substrate. In some embodiments, the said substrate may be a gel. The OOC device100is configured to simulate a biological function that typically includes cellular communication between the first cell layer242and the second cell layer244, as would be experienced in-vivo within organs, tissues, cells, etc. Depending on the application, the membrane240is designed to have a porosity to permit the migration of cells, particulates, media, proteins, and/or chemicals between an upper microchannel234and a lower microchannel36. The working fluids within microchannels234and236may be the same fluid or different fluids. As one example, as OOC device100simulating a lung may have air as a fluid in one channel and a fluid simulating blood in the other channel. As another example, when developing the cell layers242and244on the membrane240, the working fluids may be a tissue-culturing fluid. Although it is not necessary to understand the mechanism of an invention, it is believed that an organ-on-chip device offers utility even in the absence of cells on one side of the membrane, as the independent perfusion on either side of the membrane can serve to better simulate the functions of mass-transport, shear forces, and other embodiments of the biological environment. In one embodiment, the active region237defined by an upper microchannel234and a lower microchannel236having lengths of about 0.1-10 cm, and widths of about 10-2000 μm. The OOC device100preferably includes an optical window that permits viewing of the fluids, media, particulates, etc. as they move across the first cell layer242and the second cell layer244. Various image-gathering techniques, such as spectroscopy and microscopy, can be used to quantify and evaluate the effects of the fluid flow in an upper microchannel234and a lower microchannel236, as well as cellular behavior and cellular communication through the membrane240. More details on OOC devices can be found in, for example, U.S. Pat. No. 8,647,861, and is incorporated by reference in its entirety. Consistent with the disclosure in U.S. Pat. No. 8,647,861, in one preferred embodiment, the membrane240is capable of stretching and expanding in one or more planes to simulate functions of the physiological effects of expansion and contraction forces that are commonly experienced by cells. Micro- and mesofluidic devices and membranes can be fabricated from or coated with or otherwise produced from a variety of materials, including, but not limited to, plastics, glass, silicones, biological materials (e.g., gelatin, collagen, fibronectin, laminin, Matrigel®, chitosan, and others). Turning now toFIGS.3through12various exemplary open-top microfluidic devices (e.g., open-top OOC devices) and components are illustrated that can be used for creating gel layers, such as for an open-top skin-on-a-chip device or for creating gel layers for an open-top OOC device for simulating other biological functions. FIG.3illustrates an exploded perspective view of a cross-section through an exemplary open-top microfluidic device300(e.g., an open-top OOC device). Open-top microfluidic devices, such as an open-top OOC device, that allow access to the top of a chip offer several benefits. Topical treatment, such as for a skin-on-a-chip, can be applied directly through the open top to the tissue of interest. Topical treatments can include, for example, liquid, gas, gel, semi-solid, solid, particulate or aerosol. Furthermore, additional chemical or biological components can be added by means of the open top; as a particular example, additional cell types can be seeded within the open top of the device. Aerosol delivery, such as for a lung-tissue chip, is also contemplated and can be completed through the open top, as well. The microfluidic device300can optionally include a base305, such as a glass slide, polymeric or metal support or a similar structure, optionally providing an optical window. The base305can support a bottom structure325of the microfluidic device300. The bottom structure325defines a bottom chamber336connected to a bottom fluidic channel409in the microfluidic device300. Above the bottom structure325is a membrane340having a membrane top side348and a membrane bottom side349. The membrane bottom side349is disposed on the top surface of bottom structure325such that membrane bottom side349rests above the bottom chamber306. A top structure320is disposed on the membrane top side348of membrane340and defines an open region304for the open-top microfluidic device300(e.g., the open-top chip). When the top structure320is disposed on the membrane340, it may be desirable that all or substantially all of the open region304is bounded on the bottom by the membrane top side348of the membrane340. In some embodiments, the chamber of the top structure320can further include a top microfluidic cover fluidic channel308(not shown) such as a top microfluidic cover fluidic channel508(e.g., as illustrated inFIG.5A). InFIG.5A-F, such a top microfluidic cover fluidic channel508may permit perfusion of a top chamber507, particularly while top chamber507is covered by an optional fluidic cover510(FIG.5B). In some embodiments, the present invention contemplates that embodiments one or both of a bottom structure fluidic channel509and a top microfluidic cover fluidic channel508are microchannels. In some embodiments, the present invention further contemplates that embodiments, an optional fluidic covers, such as fluidic cover410or fluidic cover510(see,FIGS.4and5A-F, respectively) are disposed above a top structure520and may further be in fluid communication with, and define a top chamber507and an open region504. Although it is not necessary to understand the mechanism of an invention, it is believed that a fluidic cover, such as fluidic cover410, may be designed for a one-time application (e.g. by means of bonding it in place) or for subsequent removal. An open region304in the open-top structure320may have any shape, but is preferably a notch. In one embodiment, the purpose of open region304is believed to allow direct access to the membrane340or any matter disposed above it, before, during, and/or after experimentation; such access is not available in earlier closed microfluidic devices for simulating tissues. While previous microfluidic devices, such as OOC, may have allowed for low viscosity fluids to be directed through limited-access channels to a membrane, such as illustrated inFIGS.1and2, the open region304in top structure320additionally allows for the placement of high viscosity gels, high viscosity fluids, solids, aerosols, and powders on an area of interest of membrane340(e.g., on the membrane inclusive of a predetermined tissue culture). Turning now toFIG.4, an exploded perspective view of an exemplary open-top microfluidic device400includes a fluidic cover410. The microfluidic device400includes an optional base405that supports a bottom structure425. The bottom structure425defines a bottom chamber406. Above the bottom structure425and the bottom chamber406is an interface region488that comprises a membrane440. The membrane440is disposed on the bottom structure425and above the bottom chamber406. A top structure420is disposed above the membrane440and includes a top chamber407with an open region404. When the top structure420is disposed on the membrane440during assembly of the device400, it may be desirable that all or substantially all of the open region404is bounded along the bottom by the membrane440. The fluidic cover410may be designed to permit the perfusion of the open region404while the fluidic cover410is present. In some embodiments, the present invention contemplates that this configuration provides an advantage over previous similar devices that allows the perfusion of the open region404by way of a top fluidic cover fluidic channel408in the top structure420. One of the benefits of including a top fluidic cover fluidic channel408in the fluidic cover410instead of the top structure420, is that cells, gel or other materials disposed in the open region404are not allowed to leak or spread into the top fluidic cover fluidic channel408, where they may be undesirable. For example, cells in the top fluidic cover fluidic channel408will not be allowed to lie away from the active region437of membrane440. In contrast, by disposing a top fluidic cover fluidic channel408in the fluidic cover410, a benefit is provided of top fluidic cover fluidic channels408being absent when a fluidic cover410is removed, which disallows top fluidic cover fluidic channels408from being similarly filled with cells during seeding, as would happen with channels being directly disposed in the top structure420. To minimize “leakage” of a substance of interest placed into an open region404into areas where the substance is not desired, different configurations of the open-top microfluidic device are contemplated. For example, a fluidic cover410can include a top chamber407(which may be a channel or part thereof) that substantially aligns with all or a portion of the open region404cover disposed in top structure420. The top chamber407may optionally be hydraulically connected to one or more fluidic cover inlet ports414and/or fluidic cover outlet ports416(see also, fluidic cover inlet port514,FIG.5D), which in some embodiments may be similar to the ports described for upper body segment101inFIGS.1and2. The presence of the top chamber407is especially significant where the open region404is filled with a gel or other substance that impedes fluid flow. In such a case, the top chamber407may be filled or perfused, enabling its contents to fluidically interact with the substance in the open region404. For example, if the open region404holds a gel containing cells, flowing tissue-culture media through the top chamber407(or even incubating this media without flow) would allow nutrients and reagents to be delivered to the cells, as well as for waste products to be removed. Through the use of a clamping device the fluidic cover410can be mechanically secured to the top structure420(e.g., seeFIG.5E) to prevent or minimize leakage of any fluidic substance of interest from the open region404of the open-top microfluidic device400. For example, a spring-loaded clamp can be used to provide compression to a biocompatible polymer that uniformly seals the open region without adhesives. Such sealing can be further improved by including an elastomeric, pliable or soft material in at least one of the fluidic cover410or top structure420; one with ordinary skill in the art will appreciate that many forms of gasketing and sealing may be applied here. An advantage of some embodiments that employ clamping is that they facilitate the application, removal and potentially the reapplication of a lid or cover, which may desirably allow access to the open region404after it was covered. Allowing access to an open region404of a microfluidic device during experimentation can be useful, for example, in (i) the application of topical treatment, aerosol, additional cells or other biological reagents, (ii) change of fluidic (e.g. tissue-culture media), (iii) sampling of fluidic or solid matter, or (iv) imaging using optical or other techniques. The option to reposition the cover or apply a different cover further permits the continued use of the device (e.g. in a biological experiment). Alternatively, the lid or cover may be removed at the end of the device's use to permit sampling that may be destructive, such as taking biopsies or otherwise removing samples, staining, fixing, or imaging. In some embodiments, the present invention contemplates that a fluidic cover410can also, or alternatively, be bonded or otherwise disposed onto the top structure420. For example, for fluidic or gas sealing, an adhesive membrane, laminate, film, or sheet can be used to temporarily or permanently seal the open region at the interface between the top structure that defines the open region and a removable cover. It is also contemplated that biocompatible polymer plugs or pistons can be used to seal off the open region. It is further contemplated that an open region404of an open-top microfluidic device400can be simply covered (e.g., similar to cell culture plates) with a cover or plate that limits evaporation and improves sterile handling. In embodiments one embodiment, the present invention contemplates that a top structure420can be used with an open region404, similar to a well, or with a removable fluidic cover410that may be akin to a flat layer that seals the top structure420. An optional configuration inFIG.4includes a top chamber407with a fluidic cover fluidic channel408that can also introduce fluids into the microfluidic device such as for perfusion or the introduction of other liquids into the system. As discussed above, open-top microfluidic devices described herein offer a number of advantages. For example, these devices allow the topical application of compounds to a membrane, including compounds in the form or a gel or powder. The open-top design also allows for aerosol delivery to effect a simulated function of a tissue directly from the top of the microfluidic device. Furthermore, the open-top configuration allows access to apply simulated effects of wounding to a tissue (e.g., simulate effects of a burn or scratch on the skin or intestine) during the course of testing and the application of a treatment of interest all within the same microfluidic device and as part of the same experimentation cycle. Furthermore, the open-top configurations described herein also allow direct access to the epithelium, and thus, allow the ability to biopsy a sample during testing. An open-top configuration also allows microscopy to be applied during use of a chip, such as the application of electron microscopy, high-magnification imaging methods, and laser-based imaging methods by removing the top cover of the microfluidic device, while optionally maintaining the integrity of the experiment. In some embodiments, it is desirable to simulate one or more functions of lung, as such function simulations may be beneficial, for example, in testing compound transport and absorption through the lung, the effect of aerosolized or inhaled compounds, model lung disease, or otherwise observe lung response. In vitro models are known in the art, including for example a lung-on-a-chip microdevice disclosures in U.S. Pat. No. 8,647,861, entitled, “Organ Mimic Device with Microchannels and Methods of Use and Manufacturing Thereof,” and the small-airway on-a-chip microdevice disclosures in International Publication No. WO 2015/0138034, entitled, “Low Shear Microfluidic Devices and Methods of Use and Manufacturing Thereof,” both of which are hereby incorporated by reference herein in their entireties. A lung model that combines several of desired features in the same model would be beneficial. Desired features include recapitulation of various elements of lung structure and morphology, and the ability to satisfactorily introduce compounds or materials as aerosols, fluidic access (e.g. to emulate blood or air flow), or mechanical forces. For example, a lung model is desirable that minimizes loss of aerosol that can occur in delivery tubing and channels and variation in the aerosol delivery along the length of the channel. According to some embodiments of the present disclosure, a lung model that includes one or more of such desired features can be constructed. For example, in one embodiment, a lung module is constructed using an open-top device, such as that illustrated inFIG.4(whether employing a fluidic cover410, the optional cover ofFIG.3, or no cover). Accordingly, lung epithelial cells (e.g. alveolar epithelial cells) can be included or deposited within the open region404. Optionally, the bottom structure425may include endothelial cells, motivated by the presence of similar cells in the vasculature (e.g. capillary bed) of an in vivo lung. It is also contemplated that using the various embodiments of open-top devices described herein, a lung model may be biologically cultured or operated statically (i.e., for example, without continuous flow or with discrete exchanges of some portion of the liquid in the device) or under flow in either fluidic channels disposed in, for example, the bottom structure425, top structure420, or cover410, as well as any combination of these modalities, which may optionally be varied during operation (e.g. begin with discrete fluid exchanges, then introduce flow). In addition, the open region404or cell layers within it may be cultured dry, under an air-liquid interface, or submerged, with this mode of culture optionally varied during use. For example, following the example of the lung-on-a-chip and small-airway-on-a-chip devices, it may be desirable to begin lung culture under submerged conditions and transition to an air-liquid interface culture after some maturation period (e.g. ranging without limitation from 1 hour to 7 days, or from 1 day to 14 days). A particular advantage of the various open-top embodiments of the present disclosure is that aerosol may be delivered to the lung cells in the open region, such as open region404. In one exemplary embodiment, while operating the device without the optional cover (or by removing the cover), aerosol can be delivered directly into the open region404from above (or substantially above). The aerosol may be generated using any of a variety of aerosol-generation techniques known in the art. Alternatively, an aerosol generation means may be included in a cover that can be placed on top of the open region404. A cover may be optionally removed or exchanged during use; for example, an aerosol-generating cover may be applied when aerosol is desired and replaced with a fluidic cover410when fluidic perfusion is desired. In some embodiments, non-aerosol materials or samples can be applied to cells present in an open region, such as open region404. This may include, but are not limited to, materials or samples that are difficult to apply fluidically due to their properties, such as slurries, pastes, solids, or viscous fluids. Referring now toFIGS.5A-5F, multiple perspective views, including additional cross-sectional details through an exemplary open-top microfluidic device, are illustrated. The microfluidic device500includes a membrane540disposed between a bottom structure525and a top structure520. The bottom structure defines a bottom chamber506, ‘and the top structure520includes a top chamber506that defines an open region504, of the microfluidic device500. In some embodiments, it is desirable that the open region504, includes a gel layer550, comprising a porous volume, or another material for testing (e.g., an extracellular matrix or cells embedded in an extracellular matrix). For example, a gel layer550can include gels used in an organ-on-chip model of the skin to house fibroblasts and to support a layer or keratinocytes. InFIG.5B, a gel layer550is introduced into the open region504(seeFIG.5A) where the gel layer550is bounded on the bottom by membrane540. In some embodiments, a gel layer550, or porous volume, is formed by injecting one or more suitable precursors through one or more fluidic channels included in the top structure520(such optional channels are depicted inFIGS.5A-5C). The one or more precursors can then be treated as desired to form the gel or porous volume (e.g. UV light, chemical treatment, temperature treatment and/or incubation/waiting). Alternatively, the one or more precursors are in a final or near-final form, where no additional active process is applied in order to generate the gel or porous volume. While the approach of injecting the one or more precursors through one or more fluidic channels included in the top structure520can be adapted to permit consistent filling with gel or other porous volume, it typically results in the gel or porous volume filling at least part of the said fluidic channels. This may be undesirable in some situations; for example, when dealing with a gel containing cells, it is desirable to limit the cells to the active region, least they may not receive sufficient nutrient or biochemical cues through the membrane. Alternatively, the one of more precursors can be placed into the top of the open-top microfluidic device via the open region504. Such an approach permits alternative embodiments that eliminate or limit spaces into which the precursors may spread (e.g. one may avoid fluidic channels included in the top structure520that are in fluidic communication with the open region504). In other embodiments, the one or more precursors may be injected into the open region504, by means of a fluidic cover510that includes one or more fluidic cover fluidic channels508(an example is illustrated inFIG.5D). Although such embodiments may also result in a gel layer550formed in the fluidic cover fluidic channels508, the fluidic cover510can be removed and optionally replaced, removing at least part of the undesired material. In some embodiments, it is desirable to limit or shape the gel volume or porous volume. For example, in an organ-on-chip model of the skin, it is may be desirable to limit the thickness of a gel layer housing fibroblasts and supporting keratinocytes to a selected thickness. Without limitation, such thickness may be chosen from one or more of the ranges of 10 um to 200 um, 100 um to 1 mm, 0.5 mm to 5 mm, or 1 mm to 10 mm. According to some embodiments, the extent of a gel layer550, or porous volume, may be limited by a shaping device559(e.g., a shaping cover, a plunger560with a patterned base) that is present during the introduction or formation of the gel or porous volume. This shaping device559may be removed and optionally replaced with a cover (e.g., a fluidic cover510) once a gel layer550, or porous volume, has formed. The shaping device559may optionally include a chamber into which the gel or porous volume can conform, at least in part. Alternatively, a shaping device559may include one or more features that protrude into the open region504.FIG.5Cillustrates one type of a shaping device with features that protrude into the open region504, which takes the form of a plunger stamp560. In some embodiments, shaping devices are applied before the introduction of one or more precursors for a gel or porous volume; for example, it could be introduced through fluidic channels present in the top structure520, a fluidic cover510or even in the shaping device itself. In other embodiments, the one or more precursors are introduced before the application of the shaping device, whether through fluidic channels in the top structure520or fluidic cover510, or introduced directly into the open region504(e.g. using a syringe, pipette or printing process). In such cases, the shaping device may optionally include features (e.g. holes, fluidic channels, cavities) designed to allow the capture of excess precursor. In some embodiments, the shaping device comprises a plurality of layers. For example, the shaping device may include a spacer layer used to define gel height and a flat cover to prevent the gel from passing the spacer's height. All or only a subset of these layers may be removed once the gel or porous volume is defined, with the remaining layers (e.g. spacer layer) potentially remaining during device use or experimentation. In some embodiments, the top structure520may be removed after gel or porous volume formation, and can be optionally replaced with a different structure or cover, that may or may not include an open region. In one embodiment, the present invention contemplates a gel layer2050comprising a plurality of gel micropillars2053.FIG.20A. For example, such gel micropillars2053may be arranged in symmetrical rows along the surface of the gel layer2050.FIG.20B. In one embodiment, the present invention contemplates a gel layer formed as a gel mesh2054.FIG.20A. For example, such a gel mesh2054may be formed as an insert within a top chamber2006or bottom chamber2007.FIG.20B. In one embodiment, the present invention contemplates a shaping device comprising a plunger stamp560having a patterned surface665that creates a pattern in the gel or porous volume at a patterning interface555. Depending on the properties of the precursor materials (e.g. viscosity of the precursor and its change through curing), the shaping device may be removed before the gel or porous volume have fully formed. FIG.5Dnext illustrates a perspective view of the exemplary open-top microfluidic device ofFIG.5Cafter a plunger stamp560has been removed, including a patterned top surface557in the gel layer550. The patterning includes depressions558in the patterned top surface557of the gel layer550. The removable fluidic cover510can then be placed onto microfluidic device500such that top chamber507aligns with bottom chamber506. An exemplary fluidic cover510can optionally include fluidic channels. In the example illustrated, one of the fluidic channels508extends from inlet hole514to the top chamber507. An outlet fluidic chamber515ends at outlet hole516wherein the outlet fluidic chamber515extends downwardly through the fluidic cover510, and connects through an opening in the membrane540, such that it is fluidically connected with chamber506. The fluidic cover510may be removable, and once removed it may be optionally reapplied or optionally replaced with a different cover. FIG.5Eillustrates the exemplary open-top microfluidic device disposed within an exemplary clamping device570. A clamping device570can be desirable because no glue or bonding is needed to hold the various layers of the microfluidic device together. The clamping device applied to an open-top microfluidic device optionally allows efficient removal of the removable cover during an experiment. The clamping device570for the microfluidic device500can include an optional platform585for engaging a first side (e.g., the bottom side) of the microfluidic device500. In some embodiments, a plurality of elongated posts590can extend upwardly from the platform585. A compression plate580, which may flat or may in some embodiments be uneven, is movably coupled to the plurality of elongated posts590such that the compression plate580is vertically slidable along the posts590. In some embodiments, the compression plate580engages a second side (e.g., the top side) of the microfluidic device500; in other embodiments, the compression plate580retains a cover to the microfluidic device500. A compression device580provides compressive forces (e.g., see arrows598) generally in a direction along the elongated posts590. The compression device (e.g., springs595, elastomers, flextures, etc.) is operatively connected to the compression plate580such that the compressive forces (e.g., see arrows598) create a substantially uniform pressure on the second side (e.g., the top side) of the microfluidic device500. Clamping device components can be made from different types of materials, including, but not limited to, PMMA (e.g., acrylic), thermoplastics, thermoset polymers, other polymer materials, metals, wood, glass, or ceramics. In alternate embodiments, the compressive plate580may be held in place using a retention mechanism including, but not limited to, one or more of screws, clips, tacky/sticky materials, other retention mechanisms known in the art, or the combination of any of these mechanisms and/or the aforementioned compression device. In some embodiments, a retention mechanism retains a compressive plate580with respect to or against a platform585. In alternate embodiments, a retention mechanism retains a compression plate580with respect to or against a microfluidic device500. For example, screws can be used to fasten a compression plate580against a microfluidic device500with a corresponding threaded holes included in a microfluidic device500. As another example, a compression plate580can include a clip feature (as a retention mechanism) that clips into a suitable receiving feature of a microfluidic device. In some embodiments, the compression plate580comprises a cover for an open area included in a microfluidic device500. In other embodiments, a compression plate580retains an additional substrate that comprises a cover for an open area included in a microfluidic device500. In some embodiments, a compression plate580may include at least one access hole581that substantially aligns with a corresponding fluid port (e.g., inlet hole514or outlet hole516) on a microfluidic device500or an optional cover. In some embodiments, an access hole581securely holds or comprises a fluid connector. Such a fluidic connector may be beneficial in fluidically interfacing with a microfluidic device500or optional cover without necessitating that a connector be included in a microfluidic device500or optional cover. A bottom surface area of the compression plate580may be greater or smaller than a top surface area of the microfluidic device500. In some embodiments, the platform585can have a width such that the compression plate width is greater than the base width. The compression plate580can further include finger nubs or tabs (not shown) protruding from a central portion of the compression plate and extending beyond the base such that a compression plate width with the finger nubs is greater than the base width. In embodiments that include elongated posts590, it is contemplated that the plurality of elongated posts590are substantially parallel and the compression plate580includes a plurality of apertures operative to allow an elongated post to pass through a respective aperture. The plurality of elongated posts590supports the compression device (e.g., springs595). The compression device can include at least one spring595extending around an outer boundary of at least one of the plurality of elongated posts590. In some embodiments, a compression plate580comprises two springs595that provide a substantial uniform or equalized pressure to a compression plate where a compression plate is a mobile part of the clamping device570that moves easily up and down (or along other axes) to allow for easy manipulation of the clamped system. For example, the use of springs in a clamping device can be desirable because springs constants can provide for a wide range of translation distances and forces and are versatile for situations where a clamping device may be positioned upside down for extended periods of time. A compression plate580can be modified in area, shape, thickness, or material. Although it is not necessary to understand the mechanism of an invention, it is believed that a maximum compressive force provided to a microfluidic device by a clamping device is determined based on the force required to create a fluidic seal between a compression plate580or optional cover and a microfluidic device500(if such a seal is desired), and a propensity for the collapse of microfluidic channels or chambers within the microfluidic device500or optional cover. In some embodiments, compressive forces provided can range from approximately 50 Pa (approximately 0.007 psi) to approximately 400 kPa (approximately 58 psi). In some embodiments, compressive forces provided can range from approximately 5 kPa (0.7 psi) to approximately 200 kPa (29 psi). In some embodiments, it is desirable that the amount of force or pressure applied by a compression plate580to a microfluidic device500keep a microfluidic device sealed or properly sandwiched between the compression plate580and a platform585while not being so extreme as to cause the collapse of the microfluidic channels or to prevent desired gas exchange. A glass slide or other transparent window (e.g. made of PMMA, polycarbonate, sapphire, etc.) can be integrated into a clamping device570to provide a rigid support for the microfluidic device which improves pressure distribution for flexible devices (such as those made from PDMS silicone) while enabling good optical access for macroscopic, visual, or microscopic imaging that may be desirable through viewing portions of the clamp system. In one embodiment, the present invention contemplates that the described clamping device can facilitate the use or positioning of the device in an upside down position. This can be a particularly desirable feature during cell seeding of the underside of a chip membrane, commonly done during OOC co-culture. A compression device for the clamping device570can include alternatives to springs or other aforementioned compression devices or retention mechanisms. For example, hydraulic or pneumatic compression systems are contemplated. It is also contemplated that for rigid microfluidic devices compliant gaskets can be used. For example, the clamping device570can be fitted with a compliant gasket that has a level of springiness to it rather than a spring itself. The compliant gasket materials would create an interface between the compression plate580and the microfluidic device500or between an optional cover and the microfluidic device500. It is also contemplated that in some embodiments a compression device can utilize geometric shapes, such as cantilevered beams, as part of the device design to provide compressive force resulting from the case material flexure or compression. In some embodiments, the compressive force can also be provided with magnetic or electromagnetic systems. FIG.5Fillustrates a perspective view of an alternative exemplary cross-section through an open-top microfluidic device, similar to device500, with a bottom chamber506and open region504that are generally circular from a top or bottom view perspective. Other embodiments can include an oval or football shape. Another exemplary feature includes a membrane540disposed between the bottom structure525and the top structure520, where the bottom structure defines the bottom chamber506and the top structure defines the open region504. The illustrated membrane540limits passage between the channels (e.g., the open region504and the bottom chamber506) to a plurality of holes541that in some embodiments comprise less than the entire surface area of the membrane540within the open region504and bottom chamber506. The plurality of holes541may include laser cut holes for passage of a gel, a porous volume, or another material (e.g., an extracellular matrix or cells embedded in an extracellular matrix) that has been disposed in the open region for testing. In some embodiments, an open-top microfluidic device allows for the direct deposition of a matrix, for example a gel or a porous volume or a biodegradable polyester such as polycaprolactone, into the open region or open portion of an open-top microfluidic device. For example, a gel-forming solution or precursor can be placed in a mold that is separate from the microfluidic device. The mold can approximate the shape of the chamber or open region into which the gel volume will be disposed for a desired experiment. Similar to setting a gel layer550directly into the microfluidic device500(seeFIGS.5C-5D), a plunger stamp560is placed into the gel solution in the mold such that a bottom surface of the plunger stamp is in contact with the gel solution in the mold. The bottom surface of the plunger stamp includes the pattern of features555for imprinting into the gel solution. After the gel solution has at least partially solidified, the plunger stamp is then removed from the gel solution, thereby creating a patterned gel surface557to simulate the functions of a tissue microstructure. Once the gel has solidified to the point where the gel will not break apart or otherwise separate, the patterned gel can be removed from the mold and be inserted into the similarly shaped open region of the actual microfluidic device to be used for experimentation. Alternatively, or in combination, a suitably shaped volume or gel or porous volume can be cut to size, 3D printed or aggregated from smaller volumes, then disposed into the open region. Further, a gel or porous volume can be 3D printed directly into the open region. In another related embodiment, a matrix (e.g., gel or porous volume) such as one formed as described forFIGS.5C-5D, can also be easily extracted (whether whole or in part) from the top structure of an open-top microfluidic device, which provides benefits by overcoming the problem of staining and high-resolution imaging without having to stain an entire chip or having to reconstruct cell-monolayers. The removal or insertion of a gel, porous material and/or biological sample (e.g. biopsy, blood) to or from the open region of an open-top microfluidic device is also desirable because it can allow access for testing of the subject tissue sample in the microfluidic device and/or then the subsequent removal of the sample from an OOC device, which can then be used for other applications (e.g., for implantation into a patient; additional analysis in another device). In an alternative embodiment, the gel or gel containing cells or tissue can be patterned following culture of cells in the gel material. In some embodiments of a microfluidic device, it is desirable to include a cover that comprises sensors or actuators. For example, a cover can comprise one or more electrodes that can be used for measurement of electrical excitation. In some embodiments, such as where the device comprises a membrane (e.g., membrane540), the one or more electrodes can be used to perform a measurement of trans-epithelial electrical resistance (TEER) for the membrane. It may also be desirable to include one or more electrodes on the opposite side of the membrane540. In some embodiments, the electrodes can be included in a bottom structure (e.g., bottom structure525). In some embodiments, the bottom structure can be an open bottom with bottom electrodes included on a bottom cover that can be brought into contact with the bottom structure. The bottom cover may support any of the features or variations discussed herein in the context of a top cover, including, for example, removability, fluidic channels, multiple layers, clamping features, etc. In some embodiments, it is desirable to simulate one or more functions of skin, for example, in testing compound transport and absorption through the skin, the effect of topical treatments on skin aging or healing, modeling skin disease, or observing skin response such as damage or sensitization. While in vitro skin models are known, such as living skin equivalent (LSE), a skin model that combines several features in the same model would is desirable. For example, desirable features can include recapitulation of various elements of skin structure and morphology, topical access, fluidic access (e.g. to emulate blood flow), or mechanical forces. According to some embodiments of the present invention, a skin model that includes one or more of such desired features can be constructed. In one exemplary embodiment, the skin model is constructed using the open-top device illustrated inFIG.5D. Accordingly, a gel layer550, which may be considered to correspond to the skin's dermal layer, is present in or introduced into (e.g. using any of the aforementioned methods) the open region504. Optionally, the gel layer550(or other matrix) may include embedded fibroblasts or related cells, motivated by the presence of similar cells in the dermal layer of in vivo skin. Furthermore, the gel layer550is topped by keratinocytes, which are a primary cell type of the skin. The keratinocytes may, for example, be deposited on top of the gel layer550(which can be done, for example, directly through the open top or introduced fluidically through channels present in the top structure520or cover510) or present in the gel or other device component and allowed to biologically mature or develop into a cell layer at the top of the gel layer550. Optionally, the bottom structure525includes endothelial cells, motivated by the presence of similar cells in the vasculature (e.g. capillary bed) of in vivo skin. Using various embodiments of the open-top device described herein, the resulting skin model may be biologically cultured or operated statically (i.e., for example, without continuous flow or with discrete exchanges of some portion of the liquid in the device) or under flow in either fluidic channels disposed in the bottom structure525top structure520or cover510, as well as any combination of these modalities, which may optionally be varied during operation (e.g. begin with discrete fluid exchanges, then introduce flow). In addition, the open region504or cell layers within the open-top microfluidic device may be cultured dry, under an air-liquid interface, or submerged, with this mode of culture optionally varied during use. For example, following the example of prior skin models such as the LSE, it may be desirable to begin keratinocyte culture under submerged conditions and transition to an air-liquid interface culture after some maturation period (e.g. ranging without limitation from 1 hour to 3 days, or from 1 day to 14 days). The gel layer550may comprise a biological or synthetic gel or other porous volume, including for example, collagen I, collagen IV, fibronectin, elastin, laminin, gelatin, polyacrylamide, alginate, or Matrigel®. Collagen I in particular has been used by prior skin models, whereas it is known that elastin is present in in vivo skin, motivating its use in the disclosed in vitro model. In some embodiments, it can be similarly desirable to simulate one or more functions of the intestine, for example, in testing compound transport and absorption through the intestine or its parts, the effect of treatments on intestine health or healing, modeling intestinal disease, or observing intestinal response such as damage or sensitization. In vitro intestinal models are known in the art, including for example transwell-based systems or the gut-on-a-chip microdevice disclosures in U.S. Patent Publication No. 2014/0038279, entitled “Cell Culture System,” which is incorporated by reference herein in its entirety. In some embodiments, it is desirable construct an intestinal model that combines several of the desired features in the same model, including recapitulation of various elements of intestinal structure and morphology, fluidic access (e.g. to emulate luminal transport or blood flow), or mechanical forces. According to some embodiments of the present disclosure, an intestine model that includes one or more of such desired features can be constructed. In one exemplary embodiment, the intestine model is constructed using the open-top device illustrated inFIG.5D. Accordingly, a gel layer550, is present in or introduced into (e.g. using any of the aforementioned methods) the open region504. Furthermore, the gel layer550is topped by intestinal epithelial cells. The intestinal epithelial cells may, for example, be deposited on top of the gel layer550(which can be done, for example, directly through the open top or introduced fluidically through channels present in the top structure520or cover510) or be present in the gel or other device component and allowed to biologically mature or develop into a cell layer at the top of the gel layer550. Optionally, the bottom structure525includes endothelial cells, motivated by the presence of similar cells in the vasculature (e.g. capillary bed) of in vivo intestines. Optionally, the gel layer550includes cells, for example, smooth muscle cells, neuronal cells, lymphatic cells or other cells types, cultures within the gel layer550. Using various embodiments of the open-top device described herein, the resulting model may be biologically cultured or operated statically (i.e., for example, without continuous flow or with discrete exchanges of some portion of the liquid in the device) or under flow in either fluidic channels disposed in the bottom structure525top structure520, or cover510, as well as any combination of these modalities, which may optionally be varied during operation (e.g. begin with discrete fluid exchanges, then introduce flow). Although cells of the intestine are typically cultured submerged, the open-top device also permits the open region504or cell layers within it to be cultured dry or under an air-liquid interface, to simulate intestinal gas or various pathologies (e.g. swallowed air or gas presence with irritable bowel syndrome or lactose intolerance), or cultured with highly viscous or solid particulate material (e.g., food, fecal matter, etc.) with the mode of culture optionally varied during use. The gel layer550may comprise a biological or synthetic gel or porous volume, including for example, collagen I, collagen IV, fibronectin, elastin, laminin, gelatin, polyacrylamide, alginate, or Matrigel®. It some embodiments, it can be similarly desirable to simulate one or more functions of the small airway, for example, in testing compound transport and absorption through the airway or its parts, the effect of treatments on airway health or healing, modeling airway disease, or observing airway response such as damage or sensitization. In vitro small airway models are known in the art, including for example the small-airway on-a-chip microdevice disclosures in International Publication No. WO 2015/0138034, entitled, “Low Shear Microfluidic Devices and Methods of Use and Manufacturing Thereof,” which is hereby incorporated by reference herein in its entirety. According to some embodiments of the present disclosure, a small-airway model can be constructed to include one or more desired features, including for example fluidic access to airway and vasculature, several of the differentiated cell types found in the in vivo airway (e.g. ciliated cells, mucus-producing cells), and immune response. In one exemplary embodiment, the small-airway model is constructed using the open-top device illustrated inFIG.5D. Accordingly, a gel layer550, is present in or introduced into (e.g. using any of the aforementioned methods) the open region504. Furthermore, the gel layer550is topped by small-airway epithelial cells. The small-airway epithelial cells may, for example, be deposited on top of the gel layer550(which can be done, for example, directly through the open top or introduced fluidically through channels present in the top structure520or cover510). Optionally, the bottom structure525includes endothelial cells, motivated by the presence of similar cells in the vasculature (e.g. capillary bed) of in vivo airway. Using various embodiments of the open-top device described herein, the resulting model may be biologically cultured or operated statically (without continuous flow or with discrete exchanges of some portion of the liquid in the device) or under flow in either fluidic channels disposed in the bottom structure525, top structure520, or cover510, as well as any combination of these modalities, which may optionally be varied during operation (e.g. begin with discrete fluid exchanges, then introduce flow). In addition, the open region504or cell layers within it may be cultured dry, under an air-liquid interface, or submerged, with this mode of culture optionally varied during use. The gel layer550may comprise a biological or synthetic gel or porous volume, including for example, collagen I, collagen IV, fibronectin, elastin, laminin, gelatin, polyacrylamide, alginate, or Matrigel®. In some embodiments, it is desirable to provide mechanical strain or force to at least a portion of the fluidic device. In particular, it may be desirable to apply mechanical force to at least some cells present within the fluidic device. According to some embodiments, a mechanical force is applied to at least one portion of an open-top device by incorporating an actuation mechanism. In some embodiments, this actuation mechanism can include one or more operational channels, similar to ones described by U.S. Pat. No. 8,647,861, which is hereby incorporated by reference herein in its entirety. Such operational channels can be evacuated or pressurized to cause the application of force to a portion of the device, for example, a membrane separating a top and bottom fluidic channels. In this example, any cells present on top or below the membrane may experience the mechanical force, leading to a potential biological effect. In some embodiments, an open-top device is included in a system that additionally includes an actuation mechanism. In some embodiments, this actuation mechanism comprises a system for mechanically engaging the open-top device and a system for applying a stretch or compression force. A number of examples of actuation systems included in a fluidic device or in systems that include a fluidic device are described by International Application No. PCT/US2014/071570, filed Dec. 19, 2014, entitled “Organomimetic Devices and Methods of Use and Manufacturing Thereof”, which is hereby incorporated by reference herein in its entirety. In one exemplary embodiment, a system comprises an open-top device, a mechanical engaging device including one or more clamps or pins, and a mechanical actuation device including one or more electrical motors or pneumatic cylinders. According to one method to employ such a system, the open-top device is engaged with the mechanical engaging mechanism (e.g. by slipping the one or more pins into corresponding holes included in the open-top device), and actuating said one or more electrical motors or pneumatic cylinders to apply a cyclical mechanical force on at least part of the open-top device. Turning now toFIG.6, another exemplary shaping device560(in this case a plunger stamp660) with a textured bottom surface666is illustrated for simulating biological conditions in an open-top microfluidic device (e.g., an open-top OOC device). The plunger stamp660can be used in a similar manner as illustrated inFIGS.5C and5D. Plunger stamps can also be used to create gel layers of a defined thickness in the open region of an open-top microfluidic device. This can be particularly beneficial where a separate section or layer may be needed to introduce a dermal equivalent layer, such as a collagen plus a fibroblast. A plunger stamp can also be beneficial for skin development in, for example, an open-top OOC device, by allowing the creation of a thick gel layer (e.g., about 50 micrometers to about 10 millimeter thick, about 100 micrometers to about 1 millimeter thick), such as for an in vivo skin section. The plunger stamp can also be used in applications where cells are embedded into a system, such as an ECM with the introduction of cells into the matrix. Application of a plunger stamp to a gel in an open region of an open-top microfluidic device also allows for the embedding of fibroblasts into the gel layer. Patterned surfaces created with a shaping device (e.g. plunger stamp) can provide for more accurate simulation of tissue or organ characteristics, such as for skin tissue, small-airway tissue and intestine. For example, a gel layer for a skin model can be formed to be undulating, with the undulations mimicking features of in vivo papillae or rete peg structures. Such structures are hallmarks of in vivo skin and can vary with skin health and age. Accordingly, the ability to form and control structures in the open-top chip that mimic in vivo structures is a beneficial embodiment of the disclosed open-top microfluidic systems. As a further example, patterning using a shaping device (e.g. plunger stamp660) can be used to recreate structure in an intestinal model that mimic intestinal villi. Villi are understood to be a predominant cellular structure of the in vivo intestine, as amongst other things, they are believed to correspond to a villus-crypt axis of cell differentiation. An ability to controllably form structures that mimic villi in an intestinal model is another beneficial embodiment of the disclosed open-top microfluidic systems. A type of pattern formed on a gel or porous volume may also determine if desired cell types will form in, or on, the said gel or porous volume. For example, adult keratinocyte cells may not differentiate and may die if the geometry of the gel does not sufficiently simulate the cells' native environment. Using a patterned shaping device (e.g. patterned plunger stamp660) that allows the imprinting of specific and sophisticated patterns (e.g., patterning and/or geometries simulating the native environment for cells being cultured) into the gel or porous volume surface, a desirable micro-environment can be created that may allow for cell survival and cell differentiation. Turning now toFIG.7, an exemplary pattern for a plunger stamp760is illustrated. The plunger stamp760includes a patterned bottom surface with a plurality of papillae structures767that simulate the papillae structure of the dermis, which when imprinted into the surface of a gel layer can be useful for differentiation of an adult skin equivalent. In some embodiments, a gel layer is first placed into an open region of a top structure of a microfluidic device or placed into a mold (e.g., simulating an open region) followed by stamping of a gel surface with a plunger stamp. In other embodiments, a plunger stamp is first inserted into an open region to a predetermined desired based on a desired gel layer thickness and a pre-polymerized gel with a lower-viscosity than in its final cured form is placed or allowed to flow into an open region confined by a plunger stamp, a membrane, and the sides of the open region. A plunger stamp is dimensioned such that there are sufficient tolerances (e.g., gaps) between the side of a plunger stamp and the side walls of an open region (e.g., channel) so that a gel does not ooze or leak up the side of an open region when a pre-polymerized gel is imprinted with a patterned surface of a plunger stamp. Referring now toFIGS.8A-C-10, an exemplary embodiment of an open-top device800including round open regions804a,804b,804cis illustrated. The round open regions804a,804b,804coffer advantages in the use of the device. For example, the device is amenable to biopsy with round biopsy punches typical for in vivo work, there is broad area available for topical treatments or experimental procedures, and they may provide a more isotropic biological environment than, for example, elongated sections. A more isotropic environment can be especially beneficial when present cells affect contractile or expansive forces, as is often the case with fibroblasts such as those present in the dermal-like layer of skin models. Although the depicted embodiments inFIG.8A-Care round, some of the aforementioned advantages also apply to other shapes, including for example ovals, shapes that inscribe round sections, or other broad shapes. FIGS.8A-C-10specifically illustrate stretchable embodiments of an open-top microfluidic device800. A stretchable open-top microfluidic device, such as the one illustrated inFIGS.8A-C-10can include open regions shaped in various ways including linear sections, although circular, elliptical (e.g., from circular to a 1:2 ratio), or ovoid top region seem to reduce the impact of tissue-induced stress that can lead to delamination of the tissue culture of interest (e.g., skin tissues). A stretchable device may allow for flow in a bottom fluidic layer that is separated from a top fluidic layer by a permeable membrane (not shown), similar to the open-top microfluidic devices described forFIGS.3-5A-F. While the open-top microfluidic device800is described as a stretchable device, it can be used with membranes other than stretchable membranes (e.g., PDMS membranes) for applications where membrane stretch is not desired. Turning toFIG.8A, a top view of the exemplary assembled stretchable open-top microfluidic device800is illustrated. The device800includes a top structure820that has three apertures therethrough which define a plurality of open-top open regions804a,804b,804cthat may include a gel or porous volume. The open-top open regions may extend through the entire thickness of the top structure820. As mentioned, mechanical actuation can be effected in a variety of ways; in the illustrated example, mechanical stretch is attained using one or more operating channels that on the perimeter of the open region. The top structure820further includes a plurality of vacuum port pairs: i)830a,832a; ii)830b,832b; and iii)830c,832c, that are in communication with the one or more vacuum chambers837a,838a,837b,838band837c,838c. The vacuum port pairs can be connected to a vacuum device that is used to generate pressure differences that cause, for example, a membrane (not shown) to stretch (e.g., radially). Each open-top open region (e.g.,804a) is illustrated as having two opposing vacuum ports (e.g.,830a,832a), thereby forming a vacuum port pair. The illustrated configuration permits a mechanical stretch generated by the opposing vacuum chambers837a-cand838a-cto apply a biaxial force on the device's membrane active regions. Combined with the circular shape of the open regions, the device approximates isotropic stretch, which may be desirable in the recapitulation of the biological mechanical environment of some organs, including the skin. In alternative embodiments, the shape of the open regions and vacuum chambers can be modified to augment the directionality and non-isotropicity of the stretch. Moreover, devices that include a plurality of vacuum chambers corresponding to one or more of the open regions allow the application of different pressures (including vacuum levels) permitting the selection of stretch directionality during use. The top structure820further includes a plurality of bottom fluidic layer inlet ports819a,819b,819cand outlet ports822a,822b,822cthat allow for the introduction and extraction of fluids (e.g., for perfusion) from the open-top microfluidic device800.FIG.8Billustrates a perspective view of the top structure of the exemplary stretchable open-top microfluidic device ofFIG.8A, and in particular shows how the open-top open regions, vacuum ports, vacuum chambers and bottom fluidic layer extend through the entire top structure820. More or fewer (e.g., one, two, four, five or more) open-top open regions and related support features are contemplated. Turning now toFIG.8C, a perspective view of the bottom structure825of the exemplary stretchable open-top microfluidic device800is illustrated. Similar to the previously described embodiments of an open-top microfluidic device, a permeable membrane (not shown) is disposed along the interface between the top structure820and the bottom structure825. The bottom structure includes feeding channels839a-ccomprising a plurality of feeding channel wells (e.g., first feeding channel well835a, second feeding channel well835b, third feeding channel well835c) that align with open-top inlet and outlet ports (e.g.,514and516), respectively. A membrane (not shown) separates the open-top open region (e.g.,804a) from the feeding channels839a-cand feeding channel wells (e.g.,835a-c). It is contemplated that a gel layer in the device800can be formed on top of the membrane in the open-top openings similar to what is described elsewhere herein (see, e.g.,FIGS.5C-5D). FIGS.9and10illustrate exemplary perspective views of cross-sections9-9and10-10through the stretchable open-top microfluidic device. ofFIG.8A. With the top and bottom structure assembled, the bottom fluidic layer inlet (e.g.,819b) and outlet ports (e.g.,822b) each extend through the membrane (not shown) such that the ports are each hydraulically connected to feeding channels839a,839b,839c(e.g., illustrated as long narrow channels) in the bottom structure825to allow for the circulation or introduction of fluids into the open-top microfluidic device.FIG.8A-C. Similarly, inFIG.9the vacuum port pairs (e.g.,930a,932a;930b,932b;930c,932c) in the top structure920each extend to vacuum chamber pairs: i)937a,938a; ii)937b,938b; and iii)937c,938cformed by the interfacing of the top structure920and bottom structure925. The vacuum chambers are at least partially defined by a stretchable or deformable surface pairs such as1045band1046bthat introduces pressure changes to actuate the membranes (not shown) at the interface of each of the open-top openings (e.g.,1004b) and with the bottom wells (e.g.,1006b.FIG.10. Although it is not necessary to understand the mechanism of an invention, it is believed that the presently disclosed vacuum chambers function to provide a pneumatic stretching of a membrane. For example, when placed under a vacuum, a first deformable surface1645and second deformable surface1646deflect towards each other as depicted by a deflection line1647.FIG.16. It is further believed that since the top portion of the deformable surfaces are deflected at a greater angle than the bottom portion of the deformable surfaces, the induced stress is transferred to the underlying membrane, thereby causing the membrane to stretch. A more detailed depiction of deformable surfaces1945,1946induce a deflection1947that causes bending around the corner of the vacuum chamber wall, as shown by the change in position of the inner and outer dotted lines.FIG.22. FIGS.11and12illustrate exemplary views of different bottom fluidic channel configurations. In the embodiment illustrated inFIG.11, a lower microchannel1136is split into a number of constituent channels1129. Although it is not necessary to understand the mechanism of an invention, it is believed that the smaller diameter of these constituent channels1129, as compared to the diameter of a lower microchannel1136, may offer an advantage in terms of bubble/debris clearance and flow uniformity compared to the single wider channel. Alternatively, as illustrated inFIG.12, the lower microchannel1236can be take a spiral form1251, or a serpentine or meandering form2252as illustrated inFIG.22. Although it is not necessary to understand the mechanism of an invention, it is believed that the configuration ofFIG.12can provide increased robustness in the face of bubbles and debris that may be present, and can provide a more even flow rate than the lower microchannel1136design illustrated inFIG.11. However, the resulting channel length of the lower microchannel1236configuration inFIG.12is typically longer than in the lower microchannel1136designs similar toFIG.11, with a shorter microchannel length being advantageous in some applications. For example, the spiral lower microchannel1251design illustrated inFIG.12first winds inwardly towards the center of the active region and the winds outwardly. An alternative design avoids the outward winding by flowing downward, either to a fluidic port or to an additional fluidic channel that may run underneath the spiral channel. In one embodiment, the present invention contemplates an open-top microfluidic device1300comprising at least two open regions1304. Each open region11304may be configured with an inlet port1314, an outlet port1316and a vacuum port pair (1330,1332). In one embodiment, the present invention contemplates an open top microfluidic device1400comprising a top chamber1407or a bottom chamber1406, said chambers having side walls1443where a plurality of projections1413protrude into a chamber lumen1421.FIG.14AandFIG.14B. In one embodiment, the present invention contemplates an open-top chip device1500comprising at least two spiral lower microchannels1551, wherein each of the microchannels are in fluidic communication with an inlet port1519and an outlet port1522.FIG.15. In one embodiment, the present invention contemplates an open-top chip device1700comprising: i) a first chamber1763and a second chamber1764, wherein each chamber is surrounded by a deformable surface1745; and ii) at least two spiral microchannels1751located on the bottom surface of the chambers, wherein each of the microchannels are in fluidic communication with an inlet port1719and an outlet port1722and are respectively configured with a first vacuum port1730or a second vacuum port1732, such that each vacuum port is respectively connected to a first vacuum chamber1737or a second vacuum chamber1738.FIG.17. An exploded view of the embodiment depictedFIG.17shows an open-top chip device1800, wherein a membrane1840resides between the bottom surface of the first chamber1863and the second chamber1864and the at least two spiral microchannels1851.FIG.18. In some embodiments, the present invention contemplates an open-top chip device2700comprising at least two spiral lower microchannels2751, wherein the microchannels are in fluidic communication with an inlet port2719and an outlet port2722.FIG.27. The spiral lower microchannel2751is also flanked by a vacuum port2730configured with a vacuum chamber2737. A deformable surface2745is configured on the inside surface of the vacuum chamber2737, In some embodiments, the present invention contemplates an array device2811comprising a plurality of open top chip devices2800. Each of the open top chip devices2800is configured with at least an open region2804and flanked by an inlet port2814and an outlet port2816, or alternatively, a first vacuum port2730and a second vacuum port2732.FIG.28. An exploded view of an array device3911is provided showing the open top chip devices3900in top structure3920and a bottom chamber3906in bottom structure3925with a membrane3940layered between the top structure3920and bottom structure3925.FIG.39. Although it is not necessary to understand the mechanism of an invention, it is believed that an array device comprising open top chips represents a fundamental shift in architecture as compared to conventional “tissue-on-a-chip” designs. It is further believed that this array design facilitates multiplexing of 3D scaffold models for scaffold optimization. Furthermore, the array test platforms are designed to be compatible with existing 3D scaffold models in transwell. For example, array devices as contemplated herein are useful for 3D scaffold models, ECM/gel optimization and tissue chips including, but not limited to, skin, lung and intestine (e.g., gut). In one embodiment, an array device2811may have the following specifications: Body MaterialPDMS Sylgard 184Membrane MaterialPDMS Sylgard 184DimensionsWidth51.8mmLength50.8mmHeight8.0mmOpen-Top Chamber DimensionsTop Chamber Diameter6.3mmTop Chamber Height6mmTop Channel Volume193.02mm3Top Culture Area32.17mm2Bottom Chamber DimensionsBottom Chamber Diameter5.4mmBottom Channel Height1mmBottom Channel Volume22.90mm3Bottom Culture Area22.90mm2Membrane DimensionsPore Diameter7μmPore Spacing40 μm (hexagonallypacked)Thickness50μmCo-culture Area22.9mm2Minimum Imaging2mmDistance (top of membrane) Additional exemplary embodiments of open-top microfluidic devices, such as the devices discussed above inFIGS.1-12, are now described further. In some embodiments, the dimensions of the top area of the open region in a top structure for a chip can range from about 0.1 to about 17 millimeters (or 1 to about 7 millimeters) along in the narrowest dimension. In some embodiments, the dimensions range from about 0.5 to about 200 or more millimeters (or about 0.5 to about 20 millimeters). The lower end of the range of the narrowest dimension of the open region is also desirably sized to allow accessibility to the region for pipettes or syringes that are used to place, for example cell cultures or gel materials. The open region can be sized to limit any capillary action, which may be undesirable in some applications (capillary action may nevertheless be desirable in other applications). It is further desirable in some applications for the upper range of the open region dimensions to be sized to maintain accuracy in the flow distribution for the bottom channel across the cell culture area. In some embodiments, the depth of the open region (e.g., measuring vertically upward in the open region from the interface of the top structure with the membrane) can vary from about 0.1 to about 20 millimeters (or about 1 to about 5 millimeters). In some embodiments, an additional well or spacer may be added to increase the well volume of the open region, such as where the full depth of the open region is completely filled. It is contemplated that aspect ratios of the dimensions for the top area to the depth of the open region in some applications should range from about 1 to above 100, or in some applications from about less than 0.01 to 2. In some embodiments, it is desirable to have different geometries for the open region based on the type of tissue that is subject to experimentation. For example, certain types of tissue, such as skin, are highly contractile during culturing. When placed into high-aspect ratio (e.g., 16 millimeters by 1 millimeter) channels, delamination of the tissue can occur along the narrow dimension. However, an open region that has a circular (e.g., open region804a) provides radial symmetry that can allow tissue to shrink uniformly and not move out of plane. A wider channel geometry that minimizes edge effects can also be beneficial for other organ systems that may require multiple layers, such as the blood-brain barrier, airways, or digestive tract, because the layers can be more easily formed by the sequential deposition of thin gel or cellular tissue layers, which is difficult to do in closed channels or chambers. In some embodiments, the geometry of the open region is something different than the rectangles or circles illustrated in the exemplary embodiments ofFIGS.5A-Fand8A-C. For example, a triangular or star geometry can be used to look at the effects of cell crowding or diffusion of signaling molecules as affected by geometry. In another example, a “FIG.8A-C” shape can be beneficial for analyzing the interaction between two three-dimensional cultures For fluidic channel(s) disposed in the top structure of an open-top device that might be used for skin, bronchial, or gut tissue simulations, the geometry and dimensions for the open region of a chamber can include a channel-type geometry with a channel height ranging, for example, from about 0.02 millimeters to about 10 millimeters, a channel width of about 0.05 millimeter to 20 millimeter, and a channel length of about 0.5 millimeters to about 300 millimeters. In some embodiments, the geometry and dimensions for the open region of a top chamber can include a channel-type shape with a height ranging, for example, from about 0.02 millimeters to about 10 millimeter and a fluidic cover fluidic channel width of about 0.05 millimeter to 20 millimeter. The base or bottom chamber can also have a channel-type shape with a height ranging, for example, from about 0.02 millimeters to about 10 millimeter. For an optional top structure420that might be used for brain-barrier and lung tissue simulations, the geometry and dimensions for the top structure, for example, include a height of about 0.05 millimeters to about 5 millimeter. A taller top structure spacer in an open-top microfluidic device is often used for simulations where three-dimensionality is desirable, such as where fibroblast or other cells are embedded in the gel layer for the formation of, for example, a dermal layer. A shorter top structure spacer in an open-top microfluidic device can be used, for example, for simulations where two- or three-dimensionality is desired, such as for small airway simulations where small airway cells feel the paracrine stimulation of neighbor cells, which stimulates their full differentiation. Various tissue types are contemplated for testing in an open-top microfluidic device (e.g., an open-top OOC device), such as skin, small-airway, and alveolar tissues. However, open-top microfluidic devices can also accommodate other types of tissues, as well, including other epithelial tissues. The properties of gels or porous volumes that can be used for an open-top microfluidic device can vary and the properties will often depend on the different tissue type that is being tested. For example, different tissue types or specific models may employ different extracellular matrix proteins (ECMs) and ECM mixtures (for example, collagen I, collagen IV, Matrigel®, laminin, fibronectin, gelatin, elastin, etc., and combinations thereof). Additionally, some embodiments may employ synthetic polymer gels (e.g. polyacrylamide, polyvinyl alcohol, etc.) or various other gels known in the art (e.g. agarose, alginate, etc.) alone, in mixture, or in combinations with ECMs. Similarly, porous volumes used for an open-top microfluidic device may include a variety of open-cell foams, for example, expanded polyurethane, expanded polystyrene, expanded cellulose, expanded polylactic acid, etc. Without being bound by example, for the simulation of a skin or bronchial tissue, the gel can have a higher concentration of collagen, roughly at about 1 to about 11 milligrams per milliliter of gel. For the simulation of gut tissue function, one exemplary embodiment contemplates a gel with a 1:1 ratio of a high concentration collagen to an ECM such as the Corning® Matrigel® matrix available from Corning Life Sciences, is desirable. For the simulation of alveolar tissue function, one exemplary embodiment contemplates a gel with a 1:1 ratio of a low concentration of collagen (e.g., about 3 milligrams per milliliter of gel) to ECM, such as the Corning® Matrigel® matrix or fibronectin, is desirable. It is contemplated in one embodiment that extracellular matrices or other gel precursors that form gels with concentrations of above 5 milligrams per milliliter of gel, or ranging from about 3 to about 15 milligrams per milliliter of gel, or ranging from about 0.2 to 4 milligrams, can be used in the open-top microfluidic devices described herein. Moreover, cross-linking agents such as, but not limited to, transglutaminase, glutaraldehyde, bis(sulfosuccinimidyl)suberate, and many other cross-linkers known in the art, can be used to increase gel stiffness and optionally lower gel concentration. With the use of cross-linkers, it is contemplated that extracellular matrices or other gel precursors that form gels with concentrations ranging from about 0.05 to 5 milligrams per milliliter of gel, or ranging from about 1 to 10 milligrams per milliliter of gel, can be used in the open-top microfluidic devices described herein. While the described open-top microfluidic devices, including open-top OOC devices, are compatible with standard microfluidic fluids having relatively low viscosities (e.g., about 1 to about 10 centipoise or less), the open-top devices are well-suited for high viscosity solutions and gels having a viscosity equal to or greater than 10 centipoise along with being well-suited for the polymerization of gels in situ for later removal from the microfluidic device and other manipulation of the gel. For example, collagen gels with a high protein content (e.g., 3 milligrams per milliliter) can be directly pipetted into the open tops and gelled in place without shearing cells or requiring high pressure actuation. For drug testing applications, creams and similar high-viscosity materials can be spread directly on the tissue using the open tops to test compounds in the final formulations rather than dissolved drugs alone. Thick gels layers can also be easily generated for three-dimensional culture applications with the potential for providing mechanical stretch. Other desirable embodiments of open-top microfluidic devices include the open tops are readily compatible with aerosol and other particulate (e.g., liquid or solid) delivery while minimizing loss, which allows for enabling high dosing accuracy. Because the particles can be delivered directly to the tissue, there is minimal loss due to adsorption to other surfaces, such as tubing and microchannels. In some embodiments, the gel layer described in the above embodiments does not need to be patterned. It is also contemplated that a gel or other material suitable for growing tissues can be patterned externally, shaped to fit the open region of the channel or chamber of the top structure, and subsequently inserted into the open-top microfluidic device for cell culture. The gel or other material could also be a large sheet that is compressed using the spring loaded clamps with the two chambers or channels on either side of the gel or other material, where the gel or other material acts as a membrane in the open-top microfluidic device. The externally-prepared material can include biological tissue such as a biopsy from a patient or small piece of artificial tissue prior to implantation, and thus allow the performance of assays on tissue to determine drug response, tissue quality, and other factors. It is further contemplated that the gel or a similar material from the open-top microfluidic device can be extracted via the open top and used for in vivo applications. For example, the microfluidic device could be used to pattern and mature the tissue prior to implantation. Numerous skin substitutes are commercially available, such as epidermal substitutes, dermal substitutes, and bilayer substitutes. These can be employed together with the devices, layered structures and methods described above. PREFERRED EMBODIMENTS A. Blood Brain Barrier Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. The blood-brain barrier is of major clinical relevance because dysfunction of the blood-brain barrier leads to degeneration of the neurovascular unit, and also because drugs that are supposed to treat neurological disorders often fail to permeate the blood-brain barrier. Due to its importance in disease and medical treatment, it would be highly advantageous to have a predictive model of the human blood-brain barrier that recapitulates aspects of the cerebral endothelial microenvironment in a controlled way. In one embodiment, the present invention contemplates a layered structure comprising i) fluidic channels covered by ii) a porous membrane, said membrane comprising iii) a layer of brain microvascular endothelial cells and said membrane positioned below iv) a gel matrix (or other porous volume). The present invention contemplates, in one embodiment, living neuronal cells (e.g. neurons, astrocytes, pericytes, etc.) on, in or under the gel matrix. It is preferred that some portion of the device can be opened for access to these cells. In one embodiment, the device comprises a removable top. The gel can be patterned to control the positioning and/or orientation of the cells or portions thereof. For example, the pattern on the gel matrix can direct neurite growth for neurons seeded on the patterned surface. B. Transepithelial Electric Resistance There are many ways to evaluate the integrity and physiology of an in vitro system that mimics the blood brain barrier. Two of the most common methods are Transepithelial Electric Resistance (TEER) and Lucifer Yellow (LY) rejection. Lucifer Yellow (LY) travels across cell monolayers only through passive paracellular diffusion (through spaces between cells) and has low permeability. Therefore it is considerably impeded in passing across cell monolayers with tight junctions. Permeability (Papp) for LY of ≤5 to 12 nm/s has been reported to be indicative of well-established monolayers. One of skill in the art would understand that manipulations should be performed using aseptic techniques in order for the cells to remain in culture without contamination. TEER measures the resistance to pass current across one or more cell layers on a membrane. Specifically, this electrical resistance is a direct measurement of the resistance of the cell monolayer to the transport of ions. The measurement may be affected by the pore size and density of the membrane, but it aims to ascertain cell and/or tissue properties. The TEER value is considered a good measure of the integrity of the cell monolayer. For TEER measurements, an embodiment is contemplated wherein a layered structure or microfluidic device2300has a top electrode2371and a bottom electrode2372configured for measuring the electrophysiology of said brain microvascular endothelial cells.FIG.23In one embodiment, the top electrode2371is a chromium/gold (Cr—Au) electrode. In one embodiment, the bottom electrode2372is a chromium/gold (Cr—Au) electrode. However, it is not intended that the present invention be limited to only TEER measurements. In one embodiment, the present invention contemplates a method of testing, comprising 1) providing a layered TEER microfluidic device2300comprising i) a bottom structure2325comprising at least one upper microfluidic channel2334covered by ii) a porous membrane2340, said membrane comprising iii) a layer of brain microvascular endothelial cells in contact with said at least one upper microfluidic channel, said membrane position below iv) a gel matrix (or other porous volume), said gel matrix (preferably) under a removable cover; and 2) measuring the electrophysiology of said brain microvascular endothelial cells. In one embodiment, the porous membrane2340is covered by a top structure2320. In one embodiment, the layered TEER microfluidic device2300further comprises a top clamp2379and a bottom clamp2384, wherein said top clamp2379has at least one access hole2381. In one embodiment, the at least one access hole2381is configured to align with a port adapter2383. In some embodiments, a glass slide2382is placed between the bottom electrode2372and the bottom clamp2384. In one embodiment, the top clamp2379comprises a lasercut acrylic material. In one embodiment, the port adapter2383comprises a cast PDMS material. In one embodiment, the top electrode2371comprises a lasercut PET material. In one embodiment, the bottom electrode2372comprises a lasercut PET material. In one embodiment, the top structure2320comprises an open-top channel gasket having a cast PDMS material. In one embodiment, the bottom structure2325comprises an open-bottom channel gasket having a spincoated and lasercut PDMS material. In one embodiment, the bottom clamp2384comprises a 3D printed ABS plastic material. Although not limiting, the top clamp2379and bottom clamp2384may be attached with M4 screws2386and M4 nuts2387. Although it is not necessary to understand the mechanism of an invention, it is believed that a TEER microfluidic device is clamped because the various layered components described above would be difficult to glue (e.g., bonding). It is further believed that a clamp facilitates an ability to open the device and have direct access to cells for patch-clamp measurements. Alternatively, if this openable feature is not desired, the device layers can be bonded together. A fully assembled layered TEER chip2400between a top clamp479and bottom clamp2384is presented inFIG.24. A variety of techniques are contemplated including but not limited to using a multi-electrode array or patch clamping. In one embodiment, the present invention contemplates an “open top” design that allows for patch clamping through the opening. For example, an open-top patch clamp layered TEER microfluidic device2500may comprise an optional top microfluidic cover2510comprising an open region2504, an optional top microfluidic cover fluidic channel2508and inlet port2514, wherein the open region2504provides access to an open-top channel gasket2573. In one embodiment, the TEER microfluidic subassembly device2500comprises an open-top channel gasket2573having at least one upper microchannel2534in fluid communication with at least one upper microchannel well2523. A porous membrane2540is placed between the open-top channel gasket2573and an open-bottom channel gasket2574, wherein the open-bottom channel gasket2574comprises at least one lower microchannel2536. A bottom electrode2572is placed underneath the open-top channel gasket/porous membrane/open-bottom channel gasket layered stack. In one embodiment, the bottom electrode2572is a chromium/gold electrode.FIG.25 An open-top TEER microfluidic subassembly patch clamp device2600may be exposed to allow access with a micro-manipulator2661.FIG.26. For example, a micromanipulator arm2661may be placed directly within an upper microchannel2634. Although it is not necessary to understand the mechanism of an invention, it is believed that the micromanipulator arm2661may, for example, add reagents, remove a fluid sample, add cells and/or remove cells. This allows the configuration of the patch clamp device2600to interchangeably go between a flow configuration (e.g., where the upper microchannel2634is not exposed) and an open configuration (e.g., where the upper microchannel2634is exposed). C. Stretchable Open Top Chips In one embodiment, the present invention contemplates a stretchable open top chip device2900comprising at least one spiral microchannel2951configured with at least one fluid inlet2917and at least one fluid outlet2924.FIG.29A. In one embodiment, the microfluidic chip device2900further comprises a upper microchannel with a circular chamber2956configured with a first fluid or gas port pair2975and second fluid or gas port pair2976, a first vacuum port2930connected to a first vacuum chamber2937and a second vacuum port2932connected to a second vacuum chamber2938, wherein the vacuum chambers are proximally configured around the spiral microchannel. In one embodiment, the upper microchannel with a circular chamber2956is positioned above the spiral microchannel2951.FIG.29B. Although it is not necessary to understand the mechanism of an invention it is believed that the stretchable open top chip design represents a fundamental shift in architecture as compared to conventional “tissue-on-a-chip” designs. It is further believed that the open top design is compatible with 3D scaffold models. For example, an open top chip design may include, but is not limited to, three layers exemplified by a bottom channel, a middle chamber and a top channel. In one embodiment, the bottom channel layout may be spiral in shape in order to fit within the circular shape of the chamber. In another embodiment, the top channel allows for the ability to run media solutions or humidity-controlled gases (e.g., for example, air and/or oxygen-carbon dioxide mixtures such as 95% O2/5% CO2) to prevent gel evaporation. In another embodiment, the membrane is porous to facilitate cell-to-cell communication. Other embodiments provide a vacuum channel design that provides a mechanical stretch to the entire 3D scaffold thickness. Furthermore, the open top stretchable chips as contemplated herein are useful for biological interfaces, co-cultures, multiple cell type cultures, tissue stretching, 3D scaffold models, micro-patterning and tissue chips including, but not limited to, skin, lung and intestine (e.g., gut). In one embodiment, an open top stretchable device may have the following specifications: Body MaterialPDMS Sylgard 184Membrane MaterialPDMS Sylgard 184DimensionsWidth15.87mmLength35.87mmHeight6.0mmTop Channel DimensionsTop Channel Height200μmTop Chamber Diameter5.70mmTop Chamber DimensionsTop Chamber Diameter5.70mmTop Chamber Height4.00mmTop Channel Volume102.07mmTop Culture Area25.52mm2Bottom Channel DimensionsBottom Channel Width600μmBottom Channel Height400μmBottom Channel Volume5.446mm3Bottom Culture Area13.6mm2Membrane DimensionsPore Diameter7.0μmPore Spacing40 μm (hexagonallypacked)Thickness50μmMinimum Imaging850mmDistance (top of membrane) In one embodiment, the present invention contemplates a stretchable open top chip device3000comprising: i) a fluidic cover3010comprising an upper microchannel with a circular chamber3056configured with a first fluid or gas port pair3075and second fluid or gas port pair3076; a fluid inlet port3014, a fluid outlet port3016, a first vacuum port3030and a second vacuum port3032; ii) a top structure3020comprising a chamber3063, a first vacuum chamber3037connected to the first vacuum port3030, and a second vacuum chamber3038, connected to the second vacuum port3032, wherein the upper microchannel with a circular chamber3056overlays the top surface of the chamber3063; and iii) a bottom structure3025comprising a spiral microchannel3051comprising an inlet well3068connected to the fluid inlet port3014and an outlet well3069connected to the fluid outlet port3016, wherein a membrane3040is layered between the top structure3020and bottom structure3025.FIG.30. In one embodiment, the present invention contemplates a stretchable open top chip device3100comprising a chamber3163comprising an epithelial region3177and a dermal region3178. In one embodiment, the epithelial region comprises an epithelial cell layer. In one embodiment, the dermal region comprises a dermal cell layer, wherein said epithelial cell layer adheres to the surface of the dermal cell layer. In one embodiment, the device further comprises a spiral microchannel3151in fluid communication with a fluid inlet port3114, wherein the microchannel comprises a plurality of vascular cells. In one embodiment, a membrane3140is placed between the chamber dermal cell layer and the microchannel plurality of vascular cells. In one embodiment, the device further comprises an upper microchannel with a circular chamber3156connected to a fluid or gas port pair3175. In one embodiment, the device further comprises a first vacuum port3130connected to a first vacuum chamber3137and a second vacuum port3132connected to a second vacuum chamber3138. In one embodiment, the membrane3140comprises a PDMS membrane comprising a plurality of pores3141, wherein said pores3141are approximately 50 μm thick, approximately 7 μm in diameter, packed as 40 μm hexagons, wherein each pore has a surface area of approximately 0.32 cm2. Although it is not necessary to understand the mechanism of an invention, it is believed that the pore surface area contacts a gel layer (if present).FIGS.31A and31B. In one embodiment, the present invention contemplates a stretchable open top chip device3200comprising: i) a fluidic cover3210comprising an upper microchannel with a circular chamber3256configured with a first fluid or gas port pair3275and second fluid or gas port pair3276; a fluid inlet port3214, a fluid outlet port3216, a first vacuum port3230and a second vacuum port3232; ii) a top structure3220comprising a chamber3263, a first vacuum chamber3237connected to the first vacuum port3230, and a second vacuum chamber3238, connected to the second vacuum port3232, wherein the upper microchannel with a circular chamber3256seals with the top surface of the chamber3263; and iii) a bottom structure3225layered underneath said top structure3220.FIG.32. FIGS.33A and33Billustrate exploded views of two embodiments of a stretchable open top chip device comprising: i) a fluidic cover3310comprising an upper microchannel with a circular chamber3356configured with a first fluid or gas port pair3375and second fluid or gas port pair3376; a fluid inlet port3314, a fluid outlet port3316, a first vacuum port3330and a second vacuum port3332; ii) a top structure3320comprising a chamber3363, a first vacuum chamber3337connected to the first vacuum port3330, and a second vacuum chamber3338, connected to the second vacuum port3332, wherein the upper microchannel with a circular chamber3356overlays the top surface of the chamber3363and a first membrane3340layered between the fluidic cover3310and the top structure3320; and iii) a bottom structure3325layered underneath said top structure3220, wherein a second membrane3340is layered between the bottom structure3325and the top structure3320.FIG.33A-B. FIG.34Aillustrates an assembled top view of a stretchable open top chip device as shown inFIG.33A.FIG.34Billustrates a cutaway assembled side view of a stretchable open top chip device as shown inFIG.33A. In one embodiment, the present invention contemplates a tall channel stretchable open top chip device3500comprising: i) a fluidic cover3510comprising an open region3504; ii) a top structure3520comprising an upper microchannel3534attached to the fluidic cover3510; iii) a bottom structure3525comprising a lower microchannel3536attached to the top structure3520; and iv) a membrane3540layer between the bottom structure3525and the top structure3520. In one embodiment, the open region3504, upper microchannel3534and lower microchannel3536are configured to at least partially overlay each other.FIG.35AandFIG.35B. Although not intended to be limiting, the tall channel stretchable open top chip device3500may also comprise a vacuum port pair and/or inlet/outlet ports as shown and described above. Although it is not necessary to understand the mechanism of an invention it is believed that a tall channel stretchable open top chip design represents a fundamental shift in architecture as compared to conventional “tissue-on-a-chip” designs. It is further believed that this tall channel open top design incorporates an openable lid for direct access to the top channel that allows for the ability to load thick gel matrices as well as micro-patterning of the gel. Furthermore, the open top stretchable test platforms as contemplated herein are useful for biological interfaces, co-cultures, multiple cell type cultures, tissue stretching, 3D scaffold models, micro-patterning and tissue chips including, but not limited to, skin, lung and intestine (e.g., gut). In one embodiment, a tall channel open top stretchable device may have the following specifications: Body MaterialPDMS Sylgard 184Membrane MaterialPDMS Sylgard 184DimensionsWidth15.87mmLength35.87mmHeight5.85mmTop Channel DimensionsTop Channel Width1000μmTop Channel Height (closed)1000μmTop Channel Height (open)2000μmTop Channel Volume28.041mm3Top Culture Area28.0mm2Bottom Channel DimensionsBottom Channel Width1000μmBottom Channel Height200μmBottom Channel Volume5.584mm3Bottom Culture Area24.5mm2Membrane DimensionsPore Diameter7.0μmPore Spacing40 μm (hexagonallypacked)Thickness50μmCo-culture Area17.1mm2Minimum Imaging850mmDistance (top of membrane) In one embodiment, the present invention contemplates a fully assembled stretchable open top microfluidic device3600comprising a fluidic cover3610comprising microfluidic channel3608, a first vacuum port3630and a second vacuum port3632, wherein the microfluidic channel3608terminates at either end an an inlet port3614and an outlet port3616, respectively.FIG.36. A first cross-sectional view across plane A ofFIG.36presents an open top microfluidic device3700in an assembled configuration comprising a fluidic cover3710attached to a membrane3740, wherein the membrane3740overlays an open region3704(shown as hidden open region3604inFIG.36) within a top structure3720that is attached to a bottom structure3725.FIG.37A. A second cross-section view across plane A ofFIG.36presents an open top microfluidic device3700in a separated configuration where a fluidic top3710comprising a membrane3740is removed from top structure3720thereby providing access to an open region3704, wherein a microfluidic channel3608is configured within the fluidic cover3710.FIG.37B. A third cross-sectional view across plane A ofFIG.36presents an open top microfluidic device3800in an assembled configuration comprising a fluidic cover3810attached to a membrane3840, wherein the membrane3840overlays an open region3804(shown as hidden open region3604inFIG.36) within a top structure3820that is attached to a bottom structure3825.FIG.38A. A fourth cross-section view across plane A ofFIG.36presents an open top microfluidic device3800in a separated configuration where a fluidic top3810comprising a membrane3840is removed from top structure3820thereby providing access to an open region3804, wherein a microfluidic channel3608is configured to traverse between fluidic cover3810and top structure3820.FIG.38B. EXPERIMENTAL Example 1—Keratinocyte and Fibroblast Cell Culture This example describes the preparation of keratinocytes, and in particular human foreskin keratinocytes (HFKs). An aliquot of Lonza Gold KGM media (Lonza 192060) is placed in a 50 ml tube (i.e. with 1 cryovial of HFK cells, one needs 12 ml for the flask, 10 ml for the washing step and 1 to 5 ml to break the pellet for a total of about 25 ml). The medium is warmed by putting it into the water bath for 5-10 min and then transferred inside the sterile hood. The needed number of 15 and 50 ml conical tubes are prepared, along with the needed number of flasks. These are filled with the appropriate amount of Lonza medium. To thaw the HFKs, a cryovial is removed from the liquid nitrogen container and transferred into the basket containing dry ice. The cryovial is placed into the water bath until the freezing medium inside it is completely melted. The cryovial is sprayed with ethanol and brought to the sterile hood. The cryovial is opened in the hood and the contents are collected from the cryovial (freezing medium+cells) using a 1000 μl pipette. The contents are transferred into the 15 ml conical tube containing Lonza Gold KGM medium previously warmed. This conical tube is closed and then tilted to mix. Thereafter, it is centrifuged at 1000 rpm for 5 minutes. The conical tube is sprayed with ethanol and returned to the sterile hood. It is opened and the supernatant is withdrawn, leaving the cell pellet. The pellet is re-suspended using fresh pre-warmed Lonza Gold KGM and the mixture is transferred to a flask (or flasks), which were previously filled with Lonza Gold KGM medium. The flasks are gently agitated to make sure that the medium covers the entire bottom surface. The flasks are then transferred to the incubator. The keratinocytes are fed with new media approximately every other day (about every 36 hours). To thaw the fibroblasts, a cryovial is removed from the liquid nitrogen tank and transferred into the basket containing dry ice. The cryovial is placed into the water bath until the freezing medium inside it is completely melted. The cryovial is sprayed with ethanol and brought to the sterile hood. The cryovial is opened in the hood and the contents are collected from the cryovial (freezing medium+cells) using a 1000 μl pipette. Tee contents are transferred into the 15 ml conical tube containing Lonza FGM-2 medium previously warmed. This conical tube is closed and then tilted to mix. Thereafter, it is centrifuged at 1200 rpm for 5 minutes. The conical tube is sprayed with ethanol and returned to the sterile hood. It is opened and the supernatant is withdrawn, leaving the cell pellet. The pellet is re-suspended using fresh pre-warmed Lonza FGM-2 and the mixture is transferred to a flask (or flasks) which were previously filled with Lonza FGM-2 medium. The flasks are gently agitated to make sure that the medium covers the entire bottom surface. The flasks are then transferred to the incubator. The fibroblasts are fed with new media approximately every other day (about every 36 hours). For detaching the HFKs by trypsinization, the protocol is as follows. First, an aliquot Lonza Gold KGM (Lonza 192060), Lonza reagent subculture reagent CC-5034 and E-medium (or variants) 10% FBS medium is placed in 15 ml and 50 ml tubes. It is convenient to us 4 mls of Lonza reagent subculture reagent CC-5034 per T75 flask and to add 8 mls of 10% FBS medium to the flask (which corresponds to 2 ml for each ml of reagent Lonza reagent subculture reagent CC-5034). The media and enzymes are warmed by putting it into the water bath for 5-10 min. The flask containing HFK (typically when the cells are between 50 and 70% confluence) is removed from the incubator, sterilized on the outside with ethanol, and transferred into the hood. The flask is opened and the the Lonza Gold KGM medium is aspirated, being careful to not scratch the bottom flask surface where the cells are attached. Fresh pre-warmed Lonza Gold KGM medium (e.g. 5 mls) is then added to wash the cells. This media is also aspirated carefully. Then, 4 ml of 0.05% trypsin/EDTA (Corning 25-052 CL) is added to the flask and the flask is returned to the incubator. The detaching cells can be monitored using the microscope if desired. As a rule of thumb, keratinocytes should detach in about 2-3 minutes. Longer exposure to Lonza subculture reagent CC-5034 (or 0.05 EDTA trypsin Invitrogen 25200-056) could damage keratinocytes irreversibly. When the cells detach completely, the outside of the flask is sterilized and brought to the hood. The flask is opened and 8 ml of 10% FBS E-medium (or variants) is added to the flask (2 ml for each ml of 0.05 EDTA trypsin Corning 25-052-CL). Thereafter, the contents of the flask are conveniently transferred to a 15 ml conical tube. The tube is closed and centrifuged at 1000 rpm for 5 min. The tube is then sterilized with ethanol, returned to the hood and opened. The supernatant is gently aspirated, being careful not to disturb the cell pellet. After the supernatant is removed, the pellet is re-suspended using fresh pre-warmed Lonza Gold KGM medium. The mixture is then transferred to the flask/flasks, which were previously filled with Lonza Gold KGM medium. The flasks are gently agitated to make sure that the medium covers the entire bottom surface, and they are returned to the incubator. Feeding is as stated above. For detaching the fibroblasts by trypsinization, the protocol is as follows. An aliquot of Lonza FGM-2 medium (Lonza CC-3132), Lonza reagent subculture reagent CC-5034 and 10% FBS medium is added in 15 ml and 50 ml tubes. It is convenient to use 4 ml Lonza reagent subculture reagent CC-5034 per T75 flask and 8 ml of 10% FBS medium to the flask (which corresponds 2 ml for each ml of reagent Lonza reagent subculture reagent CC-5034). The media and enzymes are warmed by putting them into the water bath for 5-10 min. The flask containing fibroblasts (typically when the cells are between 50 and 70% confluence) is removed from the incubator, sterilized on the outside with ethanol, and transferred into the hood. The flask is opened and the media is aspirated gently, being careful to not scratch the bottom flask surface containing the cell layer. 5 ml of fresh PBS is added to wash the cells (this can be done twice). The PBS is aspirated carefully, and 4 ml of 0.05% trypsin/EDTA (Lonza CC-5012) is added and the flask is returned to the incubator. The detaching cells can be monitored using the microscope if desired. As a rule of thumb, fibroblasts should detach in about 2-3 minutes. Longer exposure could damage the cells irreversibly. When the cells detach completely, the outside of the flask is sterilized and brought to the hood. The flask is opened and 8 ml of Trypsin Neutralizing Solution(CC-5002) [2 ml for each ml of 0.05% trypsin/EDTA (Lonza CC-5002)] is added. The flask contents are transferred to a 15 ml conical tube and this tube is centrifuged at 1000 rpm for 5 min. The tube is sterilized with ethanol and returned to the hood. The supernatant is aspirated, being careful not to disturb the cell pellet. Then, the pellet is re-suspended using fresh pre-warmed Lonza FGM-2 medium and the contents are transferred to the flask/flasks, which were previously filled with Lonza FGM-2 medium. The flasks are gently agitated to make sure that the medium covers the entire bottom surface and then returned to the incubator. Feeding is as indicated above. Example 2—Embedding Cells in the Dermal Layer For embedding fibroblasts into the dermal layer (e.g. gel matrix), the protocol is as follows. First, the fibroblasts are detached using the trypsinization protocol described above. However, the pellet is re-suspended in complete E-medium low calcium (0.6 mM Ca++), supplemented with 0.5% (V/V) FBS (Invitrogen 16140071) and 2% penicillin/streptomycin (invitrogen 15140-122) and then added back to the flasks, where they are allowed to reach 50-60% confluence. Once again, the fibroblasts are detached according to the protocol described above. Once re-suspended, they are embedded into the dermal layer. From Day 0 to Day 1-2, the cells in the dermal layer are fed using complete E-medium low calcium (0.6 mM Ca++), supplemented with 0.5% (V/V) FBS (Invitrogen 16140071) and 100 μm ascorbic acid, RM/TI transglutaminase 50 μg/ml. From Day 1-2 to Day 3-4, the cells in the dermal layer are fed using complete E-medium low calcium (1.2 mM Ca++), supplemented with 0.5% (V/V) FBS (Invitrogen 16140071) and 100 μm ascorbic acid and RM/TI transglutaminase 50 μg/ml. From Day 14-18 on, the cells in the dermal layer are fed using complete cornification medium (1.8 mM Ca++), supplemented with 5% (V/V) FBS (Invitrogen 16140071) and 100 μm ascorbic acid and RM/TI transglutaminase 50 μg/ml. Example 3—Preparing the Dermal Layer First, pipette tips are cooled by putting into refrigerator for 15-30 min (Pipettes need to be cold when working with rat-tail type I collagen in order to avoid coagulation). Both the pipette tips and the ECM matrix should stay in the ice box during the procedure. In order to calculate the final volume of rat-tail type I collagen mixture needed, one calculates the number of dermal equivalent cultures that are needed. This calculation is based on 12 well+3 extra (those are needed to compensate for the ECM matrix that adheres to the surface of pipette). Where 2×104neonatal or adult Human Foreskin Fibroblast per raft are employed and 12+3 rafts are prepared, one needs 15×2×104=30×104fibroblasts (or 300,000 fibroblasts).To impede fibroblasts proliferation, one can irradiate the fibroblast with 70Gy. Now, to make 150 μl/raft×(12+3) rafts=2.25 ml. 10% 10×DMEM or variants*=0.225 ml or 225 μl. 10% reconstruction buffer+=0.225 ml or 225 μl. 80% ECM matrix=1.8 ml or 1800 μl. (1.8 ml ECM matrix×2.4×10 1N NaOH (1M))=43.2 μl 1M NaOH (1M) (NaOH makes ECM matrix to coagulate). This is put into INCUBATOR 37° C. for 2-4 Hours. One can trypsinize the fibroblasts using 0.05% trypsin/EDTA (Corning 25-052 CL) according to protocol described above. One can then re-suspend the fibroblast pellet in the predetermined amount of 10×DMEM or variants. This is mixed with the necessary amount of reconstitution buffer. (Note: best results are obtained when fibroblasts are collected in active growth phase, which occurs when fibroblast are between 50 and 70% confluence). 100 μl ECM+fibroblast are added to each well and this is incubated (37° C. for 2 Hours). Thereafter, 100 μl of E medium is added to the top of each collagen gel. 100 μl of E medium+RM TG* is then added to the bottom of each collagen gel. This is incubated (37° C. for 12-16 Hours). A variety of collagen containing matrices are contemplated for making an artificial derma and ECM to embed fibroblasts: Tropoelastin:Collagen I:Collagen III:Dermatan sulfate (1 mg:3 mg:3 mg:0.5 mg) Col I (3 mg/ml)/Elastin (3 mg/ml) Col I (3 mg/ml)/Elastin (1 mg/ml) Col I (10 mg/ml)/MaxGEL Col I (3 mg/ml)/Elastin (3 mg/ml) 1:1 MaxGel Col I (3 mg/ml)/Elastin (3 mg/ml)/Col III (3 mg/ml) 1:1:1 MaxGel Col I (10 mg/ml)/Elastin (10 mg/ml) Additional embodiments are contemplated: 1. A device comprising i) a chamber, said chamber comprising a lumen, said lumen positioned under ii) a removable top and above iii) a porous membrane, said membrane positioned above one or more iv) fluidic channels. 2. The device of Claim 1, further comprising a gel matrix. 3. The device of Claim 2, further comprising parenchymal cells on or in the gel matrix, or both. 4. The device of Claim 3, wherein said parenchymal cells are selected from the group consisting of epithelial cells of the lung and epithelial cells of the skin. 5. The device of Claim 4, wherein said epithelial cells of the lung are selected from the group consisting of alveolar epithelial cells and airway epithelial cells. 6. The device of Claim 4, wherein said epithelial cells of the skin comprise keratinocytes. 7. The device of Claim 1, further comprising positioned on the bottom of the membrane so as to be in contact with the fluidic channels. 8. The device of Claim 7, wherein the endothelial cells are primary cells. 9. The device of Claim 8, wherein said primary cells are small vessel human dermal microvascular endothelial cells. 10. The device of Claim 8, wherein said primary cells are human umbilical vein endothelial cells. 11. The device of Claim 8, wherein said primary cells are bone marrow-derived endothelial progenitor cells. 12. The device of Claim 6, wherein said keratinocytes are epidermal keratinocytes. 13. The device of Claim 6, wherein said keratinocytes are human foreskin keratinocytes. 14. The device of Claim 1, wherein said device is a microfluidic device and said fluidic channels are microfluidic channels. 15. A device comprising i) a chamber, said chamber comprising a lumen, said lumen comprising ii) a gel matrix, said gel matrix comprising parenchymal cells, said gel matrix positioned above iii) a porous membrane, said membrane comprising endothelial cells in contact with iv) fluidic channels. 16. The device of Claim 15, wherein said parenchymal cells are selected from the group consisting of epithelial cells of the lung and epithelial cells of the skin. 17. The device of Claim 16, wherein said epithelial cells of the lung are selected from the group consisting of alveolar epithelial cells and airway epithelial cells. 18. The device of Claim 16, wherein said epithelial cells of the skin comprise keratinocytes. 19. The device of Claim 18, further comprising fibroblasts within the gel matrix, wherein the keratinocytes are on top of the gel matrix. 20. The device of Claim 19, wherein the keratinocytes comprise more than one layer on top of the gel matrix. 21. The device of Claim 15, wherein the endothelial cells are primary cells. 22. The device of Claim 21, wherein said primary cells are small vessel human dermal microvascular endothelial cells. 23. The device of Claim 21, wherein said primary cells are human umbilical vein endothelial cells. 24. The device of Claim 21, wherein said primary cells are bone marrow-derived endothelial progenitor cells. 25. The device of Claim 18, wherein said keratinocytes are epidermal keratinocytes. 26. The device of Claim 18, wherein said keratinocytes are human foreskin keratinocytes. 27. The device of Claim 15, further comprising an open region in contact with at least one of said gel, said membrane, said parenchymal cells or said endothelial cells. 28. A method of testing a drug, comprising 1) providing a) a candidate drug and b) device comprising i) a chamber, said chamber comprising a lumen, said lumen positioned above ii) a porous membrane, said membrane comprising parenchymal cells and positioned above one or more iii) fluidic channels; and 2) contacting said parenchymal cells with said candidate drug. 29. The method of Claim 28, wherein said parenchymal cells are selected from the group consisting of epithelial cells of the lung and epithelial cells of the skin. 30. The method of Claim 29, wherein said epithelial cells of the lung are selected from the group consisting of alveolar epithelial cells and airway epithelial cells. 31. The method of Claim 29, wherein said epithelial cells of the skin comprise keratinocytes. 32. The method of Claim 31, further comprising fibroblasts within the gel matrix, wherein the keratinocytes are on top of the gel matrix. 33. The method of Claim 28, wherein said chamber lacks a covering and said candidate drug is introduced into said lumen under conditions such that said parenchymal cells are contacted. 34. The method of Claim 28, wherein said candidate drug is in an aerosol. 35. The method of Claim 28, wherein said candidate drug is in a paste. 36. The method of Claim 28, wherein said device further comprises a removable top and said method further comprises, prior to step 2), removing said removable top. 37. A method of testing an agent comprising 1) providing a) an agent and b) microfluidic device comprising i) a chamber, said chamber comprising a lumen, said lumen comprising ii) a gel matrix comprising cells in, on or under said gel matrix, said gel matrix positioned above iii) a porous membrane and under iv) a removable cover, said membrane positioned above one or more v) fluidic channels; 2) removing said removable cover; and 3) contacting said cells in, on or under said gel matrix with said agent. 38. The method of Claim 37, wherein said agent is in an aerosol. 39. The method of Claim 37, wherein said agent is in a paste. 40. The method of Claim 37, wherein said agent is in a liquid, gas, gel, semi-solid, solid, or particulate form. 41. A device comprising i) a chamber, said chamber comprising a lumen and projections into the lumen, said lumen comprising ii) a gel matrix anchored by said projections, said gel matrix positioned above iii) a porous membrane, said membrane positioned above one or more iv) fluidic channels. 42. The device of Claim 41, wherein fibroblasts are within the gel matrix and keratinocytes are on top of the gel matrix. 43. The device of Claim 42, wherein the keratinocytes comprise more than one layer on top of the gel matrix. 44. The device of Claim 41, wherein a layer of endothelial cells is positioned on the bottom of the membrane so as to be in contact with the fluidic channels. 45. The device of Claim 44, wherein the endothelial cells are primary cells. 46. The device of Claim 45, wherein said primary cells are small vessel human dermal microvascular endothelial cells. 47. The device of Claim 45, wherein said primary cells are human umbilical vein endothelial cells. 48. The device of Claim 45, wherein said primary cells are bone marrow-derived endothelial progenitor cells. 49. The device of Claim 42, wherein said keratinocytes are epidermal keratinocytes. 50. The device of Claim 42, wherein said keratinocytes are human foreskin keratinocytes. 51. The device of Claim 41, further comprising a removable cover. 52. The device of Claim 41, wherein said device is a microfluidic device and said fluidic channels are microfluidic channels. 53. A microfluidic device comprising i) a chamber, said chamber comprising a lumen and projections into the lumen, said lumen comprising ii) a gel matrix anchored by said projections, said gel matrix comprising fibroblasts and keratinocytes, said gel matrix positioned above iii) a porous membrane, said membrane comprising endothelial cells in contact with iv) microfluidic channels. 54. The device of Claim 53, wherein the membrane is above said fluidic channels and wherein the layer of endothelial cells is positioned on the bottom of the membrane so as to be in contact with the fluidic channels. 55. The device of Claim 53, wherein the fibroblasts are within the gel matrix and the keratinocytes are on top of the gel matrix. 56. The device of Claim 55, wherein the keratinocytes comprise more than one layer on top of the gel matrix. 57. The device of Claim 53, wherein the endothelial cells are primary cells. 58. The device of Claim 57, wherein said primary cells are small vessel human dermal microvascular endothelial cells. 59. The device of Claim 57, wherein said primary cells are human umbilical vein endothelial cells. 60. The device of Claim 57, wherein said primary cells are bone marrow-derived endothelial progenitor cells. 61. The device of Claim 53, wherein said keratinocytes are epidermal keratinocytes. 62. The device of Claim 53, wherein said keratinocytes are human foreskin keratinocytes. 63. The device of Claim 53, wherein said matrix comprises collagen. 64. The device of Claim 53, wherein said collagen matrix is between 0.2 and 6 mm in thickness. 65. A method of testing a drug on keratinocytes, comprising 1) providing a) a candidate drug and b) microfluidic device comprising i) a chamber, said chamber comprising a lumen and projections into the lumen, said lumen comprising ii) a gel matrix anchored by said projections, said gel matrix comprising fibroblasts and keratinocytes, said gel matrix positioned above iii) a porous membrane, said membrane comprising endothelial cells in contact with iv) fluidic channels; and 2) contacting said keratinocytes with said candidate drug. 66. The method of Claim 28, wherein the fibroblasts are within the gel matrix and the keratinocytes are on top of the gel matrix. 67. The method of Claim 28, wherein said chamber lacks a covering and said candidate drug is introduced into said lumen under conditions such that said keratinocytes are contacted. 68. The method of Claim 28, wherein said candidate drug is in an aerosol. 69. The method of Claim 28, wherein said candidate drug is in a paste. 70. The method of Claim 28, wherein said microfluidic device further comprises a removable top and said method further comprises, prior to step 2), removing said removable top. 71. The method of Claim 28, wherein said microfluidic device further comprises an open region in contact with at least one of said gel matrix, said membrane, said keratinocytes or said endothelial cells. 72. A method of testing an agent comprising 1) providing a) an agent and b) microfluidic device comprising i) a chamber, said chamber comprising a lumen and projections into the lumen, said lumen comprising ii) a gel matrix anchored by said projections and comprising cells in, on or under said gel matrix, said gel matrix positioned above iii) a porous membrane and under iv) a removable cover, said membrane positioned above one or more v) fluidic channels; 2) removing said removable cover; and 3) contacting said cells in, on or under said gel matrix with said agent. 73. The method of Claim 72, wherein said agent is in an aerosol. 74. The method of Claim 72, wherein said agent is in a paste. 75. The method of Claim 72, wherein said agent is in a liquid, gas, gel, semi-solid, solid, or particulate form. Still additional embodiments are contemplated: 28. A fluidic cover comprising a fluidic channel, said fluidic cover configured to engage a microfluidic device. 29. The fluidic cover of Claim 28, wherein said microfluidic device comprises an open chamber, and wherein said fluidic cover configured to cover and close said open chamber. 30. The fluidic cover of Claim 28, further comprising one or more electrodes. 31. An assembly comprising a fluidic cover comprising a fluidic channel, said fluidic cover detachably engaged with a microfluidic device. 32. The assembly of Claim 31, wherein said microfluidic device comprises an open chamber, and wherein said fluidic cover configured to cover and close said open chamber. 33. The assembly of Claim 32, wherein said open chamber comprises a non-linear lumen. 34. The assembly of Claim 33, wherein said non-linear lumen is circular. 35. The assembly of Claim 31, wherein said fluidic cover further comprises one or more electrodes. 36. A method of making an assembly, comprising: a) providing a fluidic cover comprising a fluidic channel, said fluidic cover configured to engage b) a microfluidic device, said microfluidic device comprises an open chamber, and wherein said fluidic cover configured to cover and close said open chamber; and b) detachably engaging said microfluidic device with said fluidic cover so as to make an assembly. 37. The method of making an assembly of Claim 36, wherein said open chamber comprises a non-linear lumen. 38. The method of making an assembly of Claim 37, wherein said non-linear lumen is circular. 39. The method of making an assembly of Claim 36, wherein said fluidic cover further comprises one or more electrodes. | 154,192 |
11859166 | DESCRIPTION OF EMBODIMENTS When a culturing target material such as an animal cell or a microorganism is cultured in a culture tank, various problems occur. For example, the DO control in a culture tank is a process control with a dead time until the control target amount actually appears with respect to the target value, and it is difficult to apply PID control (Proportional-Integral-Differential Controller) generally used in control of a plant or the like. This is because PID control is a type of feedback control, and when it is applied to DO control in a culture tank, it is not suitable to cope with the oxygen consumption amount that increases with the lapse of time with one parameter. As another culture control method, ON/OFF control is known. In the ON/OFF control, for example, the DO measurement value is compared with the DO setting value in the culture tank, and when the DO measurement value does not reach the DO setting value even after a certain period of time elapses, an arbitrary amount of oxygen supply is controlled to be increased or decreased. However, in the DO control based on the ON/OFF control, the control accuracy becomes very poor, and foaming may easily occur due to excessive supply of oxygen. When foaming occurs at the culture interface due to aeration in the culture tank, it may cause deterioration of growth of microorganisms and animal cells. Foaming has an adverse effect such as inhibiting the discharge of carbon dioxide gas in the culture solution, blocking the exhaust filter when the foam leaks from the exhaust of the culture tank, floating the microcarrier in culture using the microcarrier which is a microparticle in the culture tank, separating the foam from the microorganism and the animal cell, and reducing the oxygen supply to the microorganism and the animal cell. When a defoaming agent is added in order to eliminate foaming in the culture tank, there is a possibility that the defoaming agent inhibits the growth of a culturing target material and generates a load on the purification step. Therefore, it is desirable to suppress foaming based on ventilation control without using a defoaming agent as much as possible in the culture tank. Furthermore, when a culture experiment is performed in an actual culture tank in order to calculate a control value of DO control in the culture tank, the scale of the experiment is large, a large amount of cost is required, and a long period of time is required to acquire data. The inventors have intensively studied to perform appropriate DO control in a culture tank without performing large-scale experiments. Hereinafter, a culture management apparatus according to an embodiment of the present invention will be described. As shown inFIGS.1and2, a culture system1includes a culture tank2for experiment (first culture tank), a control target culture tank20(second culture tank) for actually culturing a culturing target material, a calculation device10that executes simulation for controlling the control target culture tank20based on an experimental result of the culture tank2, and a control device30that controls the control target culture tank20. The culture tank2is, for example, a small-scale laboratory culture facility for culturing a culturing target material and measuring culture data based on DO control. The culture tank2may be an actual culture facility currently in operation, or may be the control target culture tank20. That is, any culture tank may be used as the culture tank2as long as culture data of the culturing target material can be acquired. The culture tank2includes, for example, a container that stores a culture solution for culturing a culturing target material. The culture tank2is provided with a detection unit3that detects the amount of dissolved oxygen (DO value) in the culture solution. The detection unit3includes, for example, a dissolved oxygen sensor provided in the container. In addition, the culture tank2is provided with a DO adjustment device4that supplies oxygen into the culture solution. The DO adjustment device4includes, for example, a mass flow controller (not illustrated) that supplies oxygen and a sparger (not illustrated). The mass flow controller supplies gaseous oxygen to the sparger via an oxygen supply line. The sparger diffuses gaseous oxygen into the culture solution in the culture tank2. The sparger is provided at a distal end of the oxygen supply line connected to the mass flow controller, and is disposed in the culture tank2. The sparger is formed of, for example, a porous body such as an SPG film, a ceramic film, or a sintered metal. The sparger is configured to supply fine bubbles (microbubbles) of oxygen into the solution through a myriad of pores formed in the porous body, to increase a contact area between the solution and oxygen, and to facilitate dissolution of oxygen in the solution. The control target culture tank20is a large-scale culture facility for actually culturing the culturing target material. The control target culture tank20is a newly designed culture tank or an existing culture tank in which the control system is scheduled to be renewed. The control target culture tank20is constructed in, for example, a storage container that stores a culture solution for culturing a culturing target material. The control target culture tank20is provided with a detection unit21that detects the amount of dissolved oxygen in the culture solution. The detection unit21includes, for example, a dissolved oxygen sensor provided in the container. In addition, the control target culture tank20is provided with a DO adjustment device22that supplies oxygen into water. The DO adjustment device22includes, for example, a mass flow controller that supplies oxygen and a sparger22A. The mass flow controller supplies gaseous oxygen to the sparger22A via an oxygen supply line. The sparger22A has the above-described configuration and diffuses gaseous oxygen into the culture solution in the control target culture tank20. The calculation device10is simulation device that executes simulation of virtually culturing a culturing target material in the control target culture tank20before operating the large-scale control target culture tank20. The calculation device10is realized by an information processing terminal device including a control device such as a personal computer, a tablet terminal, or a smartphone. The calculation device10is communicably connected to the culture tank2. The calculation device10may be connected to the culture tank2via a network W. The network W includes, for example, a public network and a local area network (LAN). The calculation device10may be a server device connected to the network W. The calculation device may be a system on a cloud connected to the network W. The calculation device10acquires culture data from the culture tank2. The calculation device10may not be connected to the culture tank2, and may acquire culture data from the culture tank2based on a recording medium on which data is recorded. The culture data is data over time indicating the relationship between the DO value detected at time t and the oxygen supply amount in the culture solution stored in any culture tank2in which the culturing target material is cultured. The culture data does not need to include cell growth data, and may indicate the relationship between the DO value and the oxygen supply amount controlled by any control method such as conventional ON/OFF control in culturing the culturing target material. That is, the culture data is obtained in the process of culturing the culturing target material, and can be used regardless of the control method as long as it is data in which the oxygen supply amount and the DO value at time t are continuously indicated and a state in which the oxygen supply amount increases with the lapse of time is indicated. Therefore, the culture data is not limited to the experimental data, and may be data acquired from a currently operating culture tank or data acquired from a culture tank in the past. The calculation device10acquires culture data of the culturing target material cultured in the culture tank2from an acquisition unit12. The acquisition unit12includes a communication interface for transmitting and receiving communication data, a drive device capable of reading a storage medium in which data is recorded, and the like. The data acquired from the acquisition unit12is stored in a storage unit16. The storage unit16stores data and programs used for calculation and control. The storage unit16includes a storage medium such as a hard disk drive (HDD), a flash memory, or a solid state drive (SSD). The storage unit16may be a virtual server device on a cloud connected to the network W. The data stored in the storage unit16is read by a calculation unit14, and calculation is executed. For example, the calculation unit14identifies a culture model indicating a relationship over time between the oxygen supply amount to be supplied to the culturing target material, which increases over time, and a virtual DO value based on the acquired culture data. The culture model is, for example, a temporal model approximated by a simple formula that calculates a virtual DO value detected in a first-order lag response after a predetermined time when an oxygen supply amount is input. As the culture model, any culture model may be used as long as the relationship between the oxygen supply amount and the virtual DO value can be reproduced over time. The culture model is identified by adjusting parameters included in a predetermined formula so that a virtual DO value calculated by inputting actual oxygen supply amount data approaches an actual measurement value of the DO value. The adjustment of the culture model may be performed manually based on the culture data, or may be automatically performed by the calculation unit14based on machine learning using a large number of pieces of culture data as teacher data. The calculation unit14executes simulation of virtually culturing the culturing target material in the control target culture tank20using the identified culture model and a predetermined culture control method set in advance. In the culture control method, an oxygen supply amount is adjusted so as to culture a culturing target material, and a DO value is controlled based on a predetermined algorithm so as to approach a setting value. As the culture control method, for example, PFC control (Predictive Functional Control) suitable for process control in which there is first-order lag+dead time in the culture tank is used. PFC control is one of various types of model predictive control (MPC). The PFC control is a control method in which the movement of the control target (DO value) is predicted on the assumption that the output of the control target DO value makes a temporary delay response to the input (oxygen supply amount: MV value), and the system (culture amount) is optimized to stably operate. In the PFC control, a setting value to be a control target is set. In the present embodiment, the setting value is a target DO value. Next, a curve called a reference trajectory that ideally approximates the current DO value with respect to the setting value is defined. The reference trajectory is set by, for example, an exponential function. A point called a coincidence point is set on the trajectory of the reference trajectory. The PFC control performs the optimum control based on the oxygen supply amount so as to minimize the difference between the prediction value of the control target value and the value of the reference trajectory at the coincidence point. As the culture control method, other algorithms such as ON/OFF control and PID control may be used as long as the oxygen supply amount is controlled. The calculation unit14executes simulation using a culture control method based on a control program for controlling the DO adjustment device22to adjust the oxygen supply amount. The calculation unit14executes simulation based on a setting value such as the input design value of the control target culture tank the amount of the culture solution accommodated in the control target culture tank20, and the control amount of the DO adjustment device22. The calculation unit14executes control to control the oxygen supply amount (MV value) based on the culture control method in the simulation and maintain the DO value at a target value (setting value). In the simulation, the calculation unit14calculates the DO value virtually detected with respect to the virtual oxygen supply amount (MV value) supplied to the control target culture tank based on the culture model. For the DO value virtually detected, the calculation unit14calculates a virtual oxygen supply amount so as to control the virtual DO value to a target value based on the culture control method. The calculation unit14executes simulation for supplying the calculated virtual oxygen supply amount to the control target culture tank20, and calculates a DO value virtually detected. The calculation unit14executes simulation. The calculation unit14repeatedly executes simulation for repeating the above processing, and adjusts the parameters based on the relationship between the oxygen supply amount virtually controlled based on the culture control method and the DO value detected in the control target culture tank20. According to the calculation device10, the culture prediction of the culturing target material can be performed without performing a large-scale culture experiment by performing simulation in advance before actually operating the control target culture tank20. According to the calculation device10, it is possible to execute simulation and adjust the parameters used in the culture control method without performing a large-scale culture experiment. The data of the adjusted parameters is input to the control device30, for example, in order to control the actual control target culture tank20via the network W. The control device30is connected to the control target culture tank20via, for example, the network W. The control device30is realized by, for example, an information processing terminal device including a control device such as a personal computer, a tablet terminal, a smartphone, or a programmable logic controller (PLC), a sequencer that controls a control target device, or the like. The control device30acquires the parameters from an input/output unit32. The input/output unit32includes a communication interface that transmits and receives communication data, a device that inputs and outputs a data signal, a drive device that can read a storage medium in which data is recorded, and the like. The input/output unit32also acquires data of the amount of dissolved oxygen (DO value) in the culture solution detected from the detection unit21of the control target culture tank20, and stores the data in a storage unit36. The data acquired from the input/output unit32is stored in the storage unit36. The storage unit36further stores data and programs used for calculation and control. The storage unit36includes a storage medium such as a hard disk drive (HDD), a flash memory, or a solid state drive (SSD). The data stored in the storage unit36is read by a control unit34and controls the actual control target culture tank20. A program for executing the culture control method is installed in the storage unit36. The control unit34reads the program from the storage unit36and executes the culture control method. In the actual control target culture tank20, the control unit34controls the oxygen supply amount based on the culture control method to which the input parameters are applied. The control unit34controls the oxygen supply amount based on the culture control method based on the detection value of the DO value of the detection unit21, and adjusts the DO value in the control target culture tank20to a preset setting value. The control unit34generates a control signal for controlling the DO adjustment device22. The control signal is a signal for controlling the DO adjustment device22. The control unit34outputs a control signal from the input/output unit32, controls the DO adjustment device22, and adjusts the oxygen supply amount to be supplied to the control target culture tank20. Specifically, the control unit34controls the sparger22A that releases the fine bubbles to the control target culture tank20to adjust the oxygen supply amount in the culture solution. The control unit34controls the DO adjustment device22based on the program stored in the storage unit36. The control unit34cultures the culturing target material based on the PFC control in the control target culture tank20. The control target culture tank20is provided with the detection unit21. The detection unit21measures the amount of dissolved oxygen (mg/L) in the control target culture tank20using a DO meter21A, and outputs a detection value to the calculation device10. In the calculation device10, the calculation unit14compares the set DO setting value with the DO measurement value detected by the detection unit3, and calculates the oxygen supply amount (mL/min) using the PFC control. The control unit34generates a control signal based on the calculation value of the oxygen supply amount, and outputs the control signal to the mass flow controller of the oxygen supply line provided in the DO adjustment device22. The mass flow controller supplies gaseous oxygen to the sparger22A via an oxygen supply line. The gaseous oxygen is diffused from the sparger22A into the culture solution in the control target culture tank20. In the present embodiment, in the PFC control, the calculation formula of the model predictive control is directly written into the program of the sequencer connected to the control unit34and processing is performed without using the model predictive control software. The sequencer can perform arithmetic processing at a cycle of several milliseconds. The sequencer in the present embodiment is configured to perform arithmetic processing in a cycle of one second, for example. Therefore, when the oxygen flow rate (oxygen consumption by the cells) increases according to the growth of the cells with the lapse of time in the control target culture tank20, the sequencer calculates an appropriate oxygen flow rate at a cycle of 1 second. As a result, the control unit34can output the operation amount substantially in real time to the mass flow controller that controls the oxygen flow rate. In an initial predetermined period when the culturing target material is cultured using the actual control target culture tank20, the parameters used in the culture control method are appropriately adjusted based on the detection value of the DO value. According to the culture system1, even in the culture step performed for about 7 to 10 days, control can be performed with control accuracy of about ±0.1 to 0.2 mg/L (actual value) with respect to the DO setting value continuously during the culture period. Next, each step of the culture management method in the culture system1will be described. As shown inFIG.3, when the PFC control is applied to the DO control of the control target culture tank20, culture data D indicating the relationship between the oxygen supply amount and the DO value at time t is acquired based on a culture test using the small-scale laboratory culture tank2in advance before operating the control target culture tank20. The calculation unit14identifies the culture model M for calculating the virtual DO value when the oxygen supply amount x(t) at time t is supplied. The initial parameters of the culture model M are set so as to calculate the virtual DO value based on the oxygen supply amount at time t. For example, the calculation unit14acquires the oxygen supply amount x(t) at time t based on the culture data D. The calculation unit14inputs the acquired oxygen supply amount x(t) to the culture model M in which the initial parameters are set, and calculates a virtual DO value y_sim(t). The calculation unit14adjusts the initial parameters of the culture model M so that the measured DO value y(t) detected for the oxygen supply amount x(t) at time t obtained based on the culture data D approximates the calculated virtual DO value y_sim(t), and identifies the culture model M. The initial parameters are adjusted, for example, to minimize an error between the DO value y(t) and the virtual DO value y_sim(t). Next, the calculation unit14executes simulation of virtually culturing the culturing target material based on the PFC control while reproducing the growth of the culturing target material using the culture model in the virtual control target culture tank20. The PFC control includes a plurality of parameters that can be arbitrarily set. Before actually culturing the culturing target material in the control target culture tank20, it is necessary to adjust the parameters used in the PFC control according to the target process. In normal plant facility, parameters are tuned at the time of trial operation using water or the like, and then the parameters are finely adjusted in the course of operation using the real solution to optimize the parameters. However, the dissolved oxygen control used in the culture step of culturing the culturing target material cannot reproduce the control behavior unless an experiment is performed using oxygen-consuming cells. In addition, the culture medium and the cells are very expensive, and it is difficult to tune the parameters of the culture control method while actually culturing the culturing target material. Therefore, in the culture system1, the calculation device10executes simulation for culturing the culturing target material based on the PFC control in advance, and adjusts the parameters used for the PFC control in a state where the control device30and the control target culture tank20are offline. The parameters used for the PFC control may be optimized by constructing a culture model again based on the experimental result of actually culturing the culturing target material and performing simulation based on the PFC control. FIG.4illustrates a flow of simulation based on the PFC control executed by the calculation unit14. The calculation unit14calculates the oxygen supply amount (MV value) with respect to the DO value detected at time t based on the PFC control in the virtual control target culture tank20. The calculation unit14stores the calculated MV value in the storage unit16for use in calculation in the next cycle. The calculation unit14applies the calculated MV value to the culture model M, virtually grows the culturing target material, and calculates a virtual DO value predicted to be detected by the detection unit21. The calculation unit14reads the previously-calculated MV value from the storage unit16, calculates a new MV value based on the calculated virtual DO value and the previously-calculated MV value, and applies the new MV value to the culture model to calculate a new virtual DO value. The calculation unit14repeatedly executes the above calculation in the simulation and outputs a result. It can be confirmed that the results of the simulation based on the PFC control can be stably controlled by visualizing the results with a graph. FIG.5illustrates an example of a result of simulation based on the PFC control. The simulation is performed for 8.3 hours (30,000 seconds), for example. As illustrated in the drawing, a state in which the culturing target material (cell) grows over time and the oxygen consumption increases accordingly is reproduced by simulation based on the culture model. In the case of the normal PID control, an overshoot at the rise of the oxygen flow rate and a subsequent haunting phenomenon occur. On the other hand, when applied to the control of the DO value, the PFC control can stably control the DO value near the setting value while suppressing the haunting phenomenon occurring in the PID control. The DO value is stably controlled to a value of 2.94 mg/L with respect to a target value of 3.00 mg/L after a lapse of a predetermined time from an initial state. This indicates that the control accuracy is controlled at the full scale accuracy of 0.6% with respect to the setting range of 0 to 10 mg/L. As illustrated in the drawing, the PFC control can stably control the DO value to a value close to the target value even in a case where the oxygen consumption amount increases as the control target material grows with the lapse of time. According to the PFC control, the oxygen supply amount can be optimized, and foaming based on excessive oxygen supply into the culture solution can be prevented. FIG.6illustrates each process of the simulation based on the PFC control executed in the calculation device10. The PFC control described below is executed in the simulation, and is also executed in the actual control of the control target culture tank20. The PFC control of the embodiment predicts the movement of the control target (DO value) assuming that the control target (DO value) makes a temporary delay response, repeatedly calculates the oxygen supply amount (MV value) so as to perform a stable operation, and optimizes the parameters. In the DO control of the control target culture tank20, for example, a calculation formula of the PFC control for “integral+first-order lag dead time process” is adopted. The PFC control of the embodiment repeatedly calculates the oxygen supply amount (MV value) over time as described below. In the calculation of the oxygen supply amount (MV value) based on the PFC control, the calculation unit14acquires data set with arbitrary parameters as an initial value of the MV value at the time of the first cycle calculation, and acquires data of the MV value of an immediately previous cycle when there is the MV value of the immediately previous cycle (step S100). The calculation unit14inputs the initial value of the MV value to the culture model to calculate a virtual DO value in the control target culture tank20(seeFIG.4), and calculates an oxygen supply amount (MV value) with respect to the virtual DO value based on the PFC control as the calculated MV value (step S102). The calculated MV value is not stable immediately after the activation of the PFC control or when disturbance occurs, and may excessively increase or decrease as compared with the MV value of the immediately previous cycle. Therefore, the calculation unit14does not output the calculated MV value as it is, but compares the MV upper limit (=MVmax), which is an arbitrarily settable parameter, with the calculated MV value to determine whether the calculated MV value is larger than the MV upper limit (step S104). When the calculated MV value is larger than the MV upper limit, the calculation unit14outputs the MV upper limit as the output MV value (step S106). If the calculated MV value is less than or equal to the MV upper limit (step S104: No), the calculation unit14compares the calculated MV value with the MV lower limit (=MVmin), which is an arbitrarily settable parameter, and determines whether the calculated MV value is less than the MV lower limit (step S108). When the calculated MV value is less than the MV lower limit (step S108: Yes), the calculation unit14outputs the MV lower limit as the output MV value (step S110). When the calculated MV value is greater than or equal to the MV lower limit value (step S108: No), the calculation unit14outputs the calculated MV value as the output MV value (step S112). The calculation unit14compares the difference between the output MV value and the MV value of the immediately previous cycle with the arbitrarily settable parameter DMV, and determines whether the difference is larger than the DMV (step S114). When the difference between the output MV value and the MV value of the immediately previous cycle is larger than the DMV (step S114: Yes), the calculation unit14outputs a value obtained by adding (or subtracting) the DMV to (from) the output MV value as the MV value of a current cycle (step S116). When the difference between the output MV value and the MV value of the immediately previous cycle is equal to or less than the DMV (step S114: No), the calculation unit14outputs the output MV value as the MV value of the current cycle (step S118). The calculation unit14stores the output MV value of the current cycle in the memory in the sequencer as the MV value of the immediately previous cycle and uses the MV value in the PFC control calculation formula of the next cycle (step S120). The calculation unit14returns the processing to step S100, repeatedly executes the above simulation steps, and accumulates simulation data over time (seeFIG.5). The accumulated simulation result is output to a graph in the calculation device10, visualized, and verified. In the calculation device10, the simulation result is verified, and the parameters based on the PFC control are appropriately adjusted based on the relationship between the MV value controlled based on the PFC control and the virtual DO value detected in the control target culture tank20so that the virtual DO value is stably controlled. The parameters adjusted by the calculation device10are input to the control device30after the actual control target culture tank20is constructed, and the culturing target material is actually cultured and verified in the control target culture tank20. A program for executing the PFC control is installed in the control device30. The adjusted parameters are input to the control device30. After the parameters are input, the control device30controls the sparger22A that releases fine bubbles into the control target culture tank20based on the PFC control to which the parameters are applied in the actual control target culture tank20to control the oxygen supply amount (MV value). Based on the DO value detected by the detection unit21, the control device30controls the sparger22A to adjust the oxygen supply amount (MV value), and adjusts the DO value in the control target culture tank20to a preset setting value. The detection result is stored in the storage unit36during the verification period. The accumulated experimental results are input to the calculation device10. The calculation device10outputs the experimental results to a graph and visualizes the experimental results. In the calculation device10, the experimental result is verified, and the parameters based on the PFC control are finely adjusted again based on the relationship between the MV value controlled based on the PFC control and the actual DO value detected in the control target culture tank20so that the actual DO value is stably controlled. According to the culture system1, by applying the PFC control to the culture of the culturing target material, oxygen can be stably and continuously supplied in the control target culture tank20as compared with a conventional control method such as ON/OFF control. According to the culture system1, the oxygen supply amount is gradually increased with respect to the increase in the oxygen consumption amount accompanying the increase in the number of cells and microorganisms, whereby the DO value can be controlled with high accuracy near the setting value without haunting. According to the culture system1, the oxygen supply amount can be supplied to the cells in the minimum necessary amount, and foaming in the culture tank due to oxygen supply can be suppressed. Specifically, in order to efficiently dissolve oxygen into the culture solution, oxygen is preferably supplied in the state of fine bubbles such as microbubbles. For example, in the present embodiment, oxygen is supplied from the sparger in the state of fine bubbles having a bubble diameter of 0.5 to several 100 μm. However, when oxygen is formed into fine bubbles, bubbles are easily generated. Therefore, it is more important that oxygen of the fine bubbles is supplied in a minimum necessary amount. According to the culture system1, by using the PFC control, the oxygen supply amount can be adjusted with high accuracy (the necessary amount is supplied), and thus, even if oxygen is supplied in the state of fine bubbles, foaming can be suppressed. Along with this, the oxygen consumption amount can also be suppressed. According to the culture system1, by suppressing foaming, it is possible to avoid microcarriers from being caught in bubbles even in cell culture using microcarriers. According to the culture system1, a culture model can be identified based on culture data obtained from the small-scale laboratory culture tank2. According to the culture system1, the control parameters can be examined offline by executing simulation of culturing a culturing target material based on the PFC control in the calculation device10. According to the culture system1, it is possible to perform control with high accuracy in actual culture based on parameters obtained in simulation. According to the culture system1, it is not necessary to perform an experiment in the large-scale control target culture tank20, and it is possible to greatly reduce the cost until the culturing target material is actually cultured. According to the culture system1, it is possible to identify a culture model based on culture data and execute simulation regardless of the scale of a culture tank and a control method. According to the culture system1, it is possible to design another culture tank (third culture tank) that expands the scale of the control target culture tank20currently in operation. That is, according to the culture system1, simulation in the third culture tank that expands the second culture tank can be executed based on culture data obtained by culturing a culturing target material in the second culture tank, and the performance of the third culture tank can be evaluated based on the simulation result. In this case, the third culture tank is not limited to the same scale as the second culture tank, and may be set at any scale. Some or all of the components of the calculation device10described above are realized, for example, by a hardware processor such as a central processing unit (CPU) executing a program (software). Some or all of these components may be installed in hardware (circuit unit; including circuitry) such as a large scale integration (LSI), an application specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or a graphics processing unit (GPU), or by cooperation of software and hardware. The program may be stored in advance in a storage device such as a hard disk drive (HDD), an SSD, or a flash memory, or may be stored in a removable non-transitory storage medium such as a DVD, a CD-ROM, or a flash memory, and may be installed by attaching the storage medium to the drive device. Furthermore, the computer program may be distributed to a computer via a communication line, and the computer that has received the distribution may execute the program. Although the embodiments for carrying out the present invention have been described above using the embodiments, the present invention is not limited to these embodiments at all, and various modifications and substitutions can be made without departing from the gist of the present invention. For example, the calculation unit14may control the control target culture tank20while executing simulation based on not only the PFC control but also another culture control method. The calculation unit14may perform machine learning based on culture data, execute simulation based on the learning result, and control the control target culture tank20. The control device30may control a plurality of control target culture tanks20. The calculation device10may acquire culture data from a plurality of culture tanks2or a plurality of control target culture tanks20in an integrated manner. The calculation device10may execute a plurality of simulations for the plurality of control target culture tanks20in an integrated manner. The calculation device10may appropriately execute simulation based on culture data recorded at a predetermined timing such as after the lapse of a predetermined period in which the control target culture tank20is operated, and adjust parameters used for the PFC control. The control device may be replaced with a control device of the existing control target culture tank20. | 36,497 |
11859167 | DETAILED DESCRIPTION OF THE EMBODIMENTS The inventors made an detailed analysis of a relationship between a process of diffusion of a dissolved substance in a solution which is associated with a chemical reaction occurring in a biological sample and a current flowing through an electrode on a substrate in electrochemical measurement. As a result, the inventors found the fact that arranging a biological sample at a given distance determined by an electrode surface diameter and a biological sample diameter away from an electrode surface in a direction perpendicular to the electrode surface and forming a path for free diffusion of a dissolved substance below the biological sample made the amount of current larger and measurement sensitivity higher than in a case where the biological sample was immediately above and close to the electrode surface. The inventors also found the fact that arranging a biological sample away from an electrode surface in a direction perpendicular to the electrode surface made variability in current value due to the low accuracy of controlling a position of the biological sample relative to the electrode surface smaller than that when the biological sample was immediately above and close to the electrode surface and improved measurement comparability and reproducibility. Simulation results which have led to the above-described findings will be described below. In simulations, the simulation software COMSOL Multiphysics (Japanese registered trademark) was used. An embryoid body produced from mouse ES cells was adopted as a model sample. PAP generated by an ALP enzymatic reaction at a surface of the model sample was adopted as a chemical substance to be generated in the model sample. The chemical substance (PAP) generated in the model sample diffuses, reaches an electrode surface of a working electrode, and is oxidized on the electrode surface. At this time, a value of a current generated in the working electrode is detected. The other conditions are as described below. <Enzymatic Reaction> At a surface of a model sample, an ALP enzymatic reaction using PAPP that is a dissolved substance in a solution as a substrate progresses, and PAP is generated. A rate v of reaction (generation) for the ALP enzymatic reaction follows Michaelis-Menten equation (1). v=AspVmax[S]Km+[S](1) In equation (1), ASPrepresents the surface area of the model sample, Vmaxrepresents a rate of reaction per unit surface area of the model sample when the substrate concentration is infinite, Kmrepresents the Michaelis constant for the ALP enzymatic reaction, and [S] represents the substrate concentration. Values of Vmaxand Kmwere respectively set to 2.65×10−7mol/(s·m2) and 1.7×10−3mol/L. An initial value of [S] was set to 5.0×10−3mol/L. <Electrode Reaction> A two-electron oxidation reaction of PAP generated in the model sample progresses on the electrode. An electrode potential was assumed to be sufficiently high for the reaction to be completely diffusion-controlled. A current value I during the reaction follows equation (2). I=∫Aeli(x,y)dAel(2)i(x,y)=nFDdc(x,y)dz(3) In equation (3), i(x,y) and c(x,y) respectively represent a current density and the concentration of the chemical substance to be detected, at an arbitrary point (x,y) on a surface of the electrode. Aelrepresents an electrode area, n represents the number of electrons involved in the reaction, F represents the Faraday constant, D represents a diffusion coefficient of the chemical substance to be detected in the solution, and z represents a coordinate in a direction perpendicular to the electrode surface (an x-y plane). n, F, and D were respectively set to 2, 9.64×104C/mol, and 6.47×10−10m2/s. The current value I after 200 seconds since the start of the electrode reaction was shown as a measurement result. <Others> Shape of model sample: diameter dsp=200 μm, spherical Shape of electrode surface: diameter del=20 μm, circular Position of electrode surface: a horizontal distance between central coordinates of the electrode surface and center coordinates (x,y) of the model sample is 0 Distance z: 0 to 80 μm The present inventors investigated how the current value I resulting from the oxidation reaction of the chemical substance generated in the model sample changed with the distance z between the electrode surface and a lower end of the model sample.FIG.1is a graph of a simulation result representing a relationship between the current value I and the distance z. As seen fromFIG.1, the current value I has a local maximum value at z=16 μm. Thus, it turned out that placing the model sample at an optimum position where a peak current value was obtained made measurement sensitivity much higher than the sensitivity when the distance z was 0 μm. The tendency of the distance z is independent of the electrode diameter deland the model sample diameter dsp. Further, it is clear fromFIG.1that a variation in the current value I when the distance z varies near the above-described local maximum value is much smaller than a variation in the current value I when the distance z varies near z=0 μm. If a cell, a cell aggregate, a piece of tissue, or the like is adopted as a biological sample, it is difficult to control the distance z with an accuracy of several μm because unevenness is present at a surface of the biological sample or the shape of the biological sample is not necessarily a spherical shape. However, setting the distance z to a value near the local maximum value can reduce variability in the current value I resulting from the low accuracy of controlling the distance z, resulting in improvements in the accuracy of determining a quantitative relationship between different measurement objects (comparability) and the reproducibility of a measurement result for the same measurement object. The closer to the local maximum value the distance z is, the larger the improvements in the comparability and the reproducibility becomes. The improvements are especially remarkable when the distance z is within a range where the current value is 90% or more of the peak current value. For this reason, if the distance z is set to a value within the range, potent effects are achieved not only in terms of improving the sensitivity but also in terms of improving the comparability and the reproducibility. It was found from results of various simulations performed by the inventors that the effective range for the distance z varied significantly depending on measurement conditions, especially the electrode diameter and the biological sample diameter. It is thus necessary to set an electrode having an appropriate diameter and the appropriate distance z in order to evaluate a biological sample having a particular diameter. However, in the case of biological samples, such as cells, cell aggregates, or pieces of tissue, the diameters of the biological samples vary widely depending on the types and states of cells. Even if the biological samples are harvested from the same site of the same test body or are acquired under the same culture conditions, the diameters of the biological samples vary by several μm to several hundreds of μm. It is unrealistic in terms of cost to check the diameters of all biological samples before measurement and set an appropriate electrode diameter and the appropriate distance z for each biological sample. Additionally, quantitative comparison of measurement results acquired for different electrode diameters and different values of the distance z is extremely difficult. In order to solve the above-described problems, it is effective to obtain an electrode diameter and a range for the distance z which allow production of potent effects for all biological samples having various diameters within a reasonable range and to measure various biological samples using an electrochemical measurement device with the same configuration. The inventors obtained electrode diameters and a range for the distance z which achieve high sensitivity, the effect of improving the comparability, and the effect of improving the reproducibility if the diameters of cell aggregates, which are said to more accurately reproduce a bioactivity inside a biological body, vary within the commonly used range of 100 to 600 μm. The procedure for the obtainment will be described below. First, the inventors investigated how a lower limit zminand an upper limit zmaxof an effective range for the distance z changed with the model sample diameter dspif the electrode diameter delwas 20 μm.FIG.2shows a result of the simulation. When the electrode diameter delis 20 μm and the distance z is not less than zminand not more than zmaxshown inFIG.2for each value of the model sample diameter dsp, the current value I is 90% or more of the peak current value (zoptin the drawing represents an optimum value for the distance z which gives the peak current value). Let zmax* be zmaxwhen the model sample diameter dspis 100 μm and zmin* be zminwhen the model sample diameter dspis 600 μm. If the distance z is within a shaded range inFIG.2which has a lower limit of zmin* and an upper limit of zmax* when the model sample diameter dsphas a value of 100 to 600 μm, the current value I is 90% or more of the peak current value. The inventors then investigated how zmin* and zmax* changed with the electrode diameter del.FIG.3shows a result of the simulation. If a value of the electrode diameter delis within the range of 0 to 80 μm, and the distance z is within the range not less than zmin* and not more than zmax* shown inFIG.3, the above-described potent effects are obtained for biological samples having dsp=100 to 600 μm. By fitting operation using a non-linear least-squares method, the range for the distance z is roughly represented by equation (4) that is a function of the electrode diameter del. Thus, the distance z may be set within the range represented by equation (4), where z>0. z=21.8(del+0.8)del+9.7±5[μm](4) As can be seen fromFIG.3, equation (4) cannot be used when the electrode diameter delis not less than about 80 μm. However, electrochemical measurement on minute biological samples, such as cells, commonly uses an electrode having del=50 μm or less. This is because an S/N ratio of a current value (the ratio between a Faraday current generated by an oxidation-reduction reaction of a chemical substance to be detected and a charging current generated by an electrolyte not to be detected) increases significantly at an electrode having del=50 μm or less. Thus, the distance z can be determined in accordance with equation (4). The effective range for the distance z can vary depending on the rate v of generation, at which a chemical substance is generated in a biological sample, and the diffusion coefficient D of the chemical substance, in addition to the electrode diameter deland the biological sample diameter dsp. The influences of the parameters, however, are limited. It is clear from equation (1) that, if the substrate concentration [S] is sufficiently high, the rate v of generation is virtually determined by the rate Vmaxof reaction when the substrate concentration is infinite. For this reason, the inventors investigated how the effective range for the distance z changed with the rate Vmaxof reaction.FIG.4shows a result of simulating the current value I for each of various combinations of the rate Vmaxof reaction and the distance z. The ordinate of the graph shown inFIG.4represents the current value I normalized with the rate Vmaxof reaction. It is clear fromFIG.4that a relationship between the current value I normalized with the rate Vmaxof reaction and the distance z remains almost unchanged even if the rate Vmaxof reaction changes and that the effective range for the distance z remains almost unchanged. Similarly, the inventors investigated how the effective range for the distance z changed with the diffusion coefficient D.FIG.5shows a result of simulating the current value I for each of various combinations of the diffusion coefficient D and the distance z. Values of the diffusion coefficient D for typical chemical substances to be detected used in the medical or life science field, such as PAP, iron complexes, ruthenium complexes, and hydrogen peroxide, range roughly from 1×10−10to 20×10−10m2/s. It is clear fromFIG.5that, if the diffusion coefficient D is within the range of 1×10−10to 20×10−10m2/s, the relationship between the current value I and the distance z remains almost unchanged, and the effective range for the distance z remains almost unchanged. As seen from the above-described results, equation (4) indicating a relationship between the distance z and the electrode diameter delneeded to achieve the above-described potent effects for biological samples having diameters of 100 to 600 μm is also useful for measurement systems having various values of the rate v of generation and various values of the diffusion coefficient D. First Embodiment of Present Invention Based on the simulation results described above, a first embodiment of an electrochemical measurement device according to the present invention has the configuration below. An electrochemical measurement device has a solution well60, a spacer10or50, a wall plate31, and a plurality of working electrodes21. The spacer10or50, the wall plate31, and the plurality of working electrodes21are all fixed on a flat surface20aof the solution well60. The flat surface20ais a bottom surface of the solution well60which is in contact with a solution in a state where the solution well60contains the solution and is, for example, a surface of a semiconductor chip on which an integrated circuit is formed. A surface of each working electrode21which is in contact with the solution is an electrode surface21a. The spacer10or50, the electrode surfaces21a, and the wall plate31are immersed in the solution during measurement. <Biological Sample> A biological sample40has a diameter not less than 100 μm and not more than 600 μm. The “diameter” of the biological sample40refers to the diameter of a smallest sphere which encompasses the biological sample40. <Spacer> The spacer10or50has a profile surface at which a distance in a perpendicular direction to the flat surface20afalls within a range for a distance z given by equation (4). The spacer10or50permits a dissolved substance in the solution to diffuse while inhibiting the biological sample40from entering a region on the flat surface20aside of the profile surface. In other words, the spacer10or50has a height of h1(a length from the flat surface20aalong a normal to the flat surface20a) and a structure in which an enclosed three-dimensional region is not formed by the biological sample40, the flat surface20a, and the spacer10or50while the biological sample40is in contact with the spacer10or50. h1is a predetermined value within the range for the distance z given by equation (4). The biological sample40is arranged along the profile surface of the spacer10or50. That is, the biological sample40is arranged in contact with the spacer10or50. With the above-described structure, the distance between the flat surface20aand the biological sample40can be roughly kept at h1. The “distance between the flat surface20aand the biological sample40” means a shortest distance between the flat surface20aand the biological sample40and is a minimum length of a half line connecting an intersection of the normal to the flat surface20aand the flat surface20aand an intersection of the normal and the biological sample40. The reason why the term “roughly” is used here is that the distance between the flat surface20aand the biological sample40may be strictly less than h1depending on the shape and posture of the biological sample40. Even in this case, the shapes of the individual biological samples40are strictly different from one another, and the postures of the individual biological samples40are strictly different from one another, so the above-described effect of “improving sensitivity, comparability, and reproducibility” is not lost from a statistical standpoint. On the grounds that the distance between the flat surface20aand the biological sample40may be strictly less than h1, h1may be a predetermined value within a range for the distance z given by equation (4a). Equation (4a) means that an upper limit for the distance z is 21.8(del+0.8)/(del+9.7)+5[μm] and that a lower limit for z is 21.8(del+0.8)/(del+9.7)±0 [μm]. z=21.8(del+0.8)del+9.7(+5,0)[μm](4a) h1may be a constant independent of a position on the flat surface20aor a value determined by a function with the position on the flat surface20aas a variable. Even in the latter case, h1is a value within the range for the distance z given by equation (4). That is, the height10or50of the spacer10or50need not be uniform over a whole region on the flat surface20a. For example, a region where the height of the spacer10or50is relatively low and a region where the height of the spacer10or50is relatively high may be present on the flat surface20a. Alternatively, a region where the height of the spacer10or50changes in a stepwise manner may be present on the flat surface20a. <Electrode Surface> Arrangement of an electrode surface is not particularly limited. For example, the distance between adjacent electrode surfaces is roughly not less than 120 μm. However, in actual usage, a distance L between a center of a first electrode surface corresponding to a first biological sample and a center of a second electrode surface corresponding to a second biological sample (the second biological sample is different from the first biological sample) needs to satisfy the condition that the first biological sample and the second biological sample are not contact each other while the first biological sample and the second biological sample are respectively arranged above the first electrode surface and the second electrode surface. That is, all electrode surfaces may be used or some electrode surfaces may be used in actual electrochemical measurement. The electrode surface21aof each working electrode21supplies or receives electrons to or from a chemical substance generated or consumed in the biological sample40having a diameter not less than 100 μm and not more than 600 μm. As a result, an oxidation-reduction reaction of the chemical substance progresses. All of the electrode surfaces21ahave diameters not more than 80 μm. Although the shape of each electrode surface21ais preferably circular, the shape may be elliptical or polygonal. If the shape of the electrode surface21ais non-circular, a diameter delof the electrode surface21ais 2[A/π]1/2. Note that A represents the area of the electrode surface21a. A center of the electrode surface21ais a geometrical center of the electrode surface21a. If the shape of the electrode surface21ais circular, the center of the electrode surface21ais a center of a circle. If the shape of the electrode surface21ais elliptical, the center of the electrode surface21ais an intersection of a major axis and a minor axis of an ellipse. If the shape of the electrode surface21ais rectangular, the center of the electrode surface21ais an intersection of diagonal lines. If the electrode surface21ahas a complicated shape, a center of a minimum circle which encompasses the electrode surface21ais defined as the center of the electrode surface21a. Although two electrode surfaces21aare illustrated inFIGS.6to12to be referred to in the description below, the total number of electrode surfaces21ais not limited to two. The arrangement shape of three or more electrode surfaces21ais not limited. Any arrangement shape, such as a lattice shape (a shape with electrode surfaces located on lattice points), a linear shape (a shape with electrode surfaces located on a straight line), a circular shape (a shape with electrode surfaces located on a circle), or the shape of a polygonal frame (a shape with electrode surfaces located on sides of a polygon), can be selected. <Wall Plate> The wall plate31is placed between two electrode surfaces21aadjacent to each other. The wall plate31has the property of being impervious to the dissolved substance in the solution. The wall plate31reduces crosstalk. The shape of the wall plate31when the flat surface20ais squarely viewed, that is, the shape of the wall plate31when the flat surface20ais viewed from a normal direction of the flat surface20amay be, for example, the shape of a straight line, the shape of a polygonal line, or a shape extending curvedly, or an annular shape or the shape of a polygonal frame. Note that, if the wall plate31has a closed structure as in the latter case, the wall plate31needs to be structured not to be in contact with the biological sample40while the biological sample40is in contact with the spacer10or50. The wall plate31has a height not less than the height of h1. That is, the height of the wall plate31is above the range for the distance z represented by equation (4). There is no limit to an upper limit for the height of the wall plate31. In actual usage, a solution surface exceeds the height of the wall plate31from the standpoint of uniformity of measurement conditions. Additionally, it is not required that the wall plate31is formed throughout the flat surface20a. For example, the wall plate31is unnecessary in a location sufficiently away from the electrode surface21a. The relationship between the number of electrode surfaces and the number of wall plates is not particularly limited. However, it is unnecessary to uselessly form many wall plates. A configuration in which a plurality of electrode surfaces are arranged between adjacent wall plates or between a wall plate and a wall of the solution well is admissible. <Type 1> FIGS.6A and6Bshow an example of the first embodiment of the electrochemical measurement device. The spacer10is composed of a plurality of pillar structures11standing close together. The wall plate31is placed between two adjacent electrode surfaces21a. The pillar structures11having uniform heights extend from the flat surface20aof a substrate20, on which the electrode surfaces21aare arranged, in the normal direction of the flat surface20a. In this example, the interval between any two pillar structures11is less than 100 μm. For example, when the biological sample40having a diameter of 100 μm is to be measured, an electrochemical measurement device having the pillar structures11formed at intervals of about 30 μm is used. A broken line inFIG.6Bindicates a position of the profile surface that is away by a distance of h1from the flat surface20a. A supplemental explanation of the interval between the pillar structures11will be given. If the interval between the pillar structures11is set wide (for example, if the interval is slightly smaller than the diameter of the biological sample40), although a path enough for the dissolved substance in the solution to diffuse can be secured, it is difficult to set the distance between the flat surface20aand the biological sample40within the range for the distance z given by equation (4). On the other hand, if the interval between the pillar structures11is set narrow (for example, if the interval is significantly smaller than the diameter of the biological sample40), although it is easy to set the distance between the flat surface20aand the biological sample40within the range for the distance z given by equation (4), a path enough for the dissolved substance in the solution to diffuse cannot be secured, and it is difficult to keep the biological sample40at a position immediately above the electrode surface21a. Thus, in actual usage, an electrochemical measurement device having a spacer composed of the pillar structures11that are appropriately arranged in accordance with the diameter and shape of the biological sample40is used. The intervals between the pillar structures11may not be fixed. Additionally, it is not required that the pillar structures11are formed throughout the flat surface20a. For example, a spacer is unnecessary in a location sufficiently away from the electrode surface21a. The wall plate31extends in a straight line in a direction crossing a line segment connecting the centers of the two adjacent electrode surfaces21a. In this example, the wall plate31extends in a y direction orthogonal to an array direction of the two electrode surfaces21astanding side by side in an x direction. The wall plate31is placed at a position equally distant from the two adjacent electrode surfaces21a. The wall plate31has a height not less than the heights of the pillar structures11. That is, the height of the wall plate31is above the range for the distance z represented by equation (4). The biological sample40is put above the electrode surface21aby pipetting operation using a microscope, by using a guide, or the like. That is, a horizontal distance between the electrode surface21aand the biological sample40(a distance in a direction parallel to the electrode surface21a, that is, the flat surface20a) is roughly 0. In this case, the distance z between the electrode surface21aand the biological sample40falls roughly within the range in equation (4) even without performing a special operation. Thus, a path, through which the dissolved substance in the solution diffuses, is formed between the biological sample40and the electrode surface21a. As a result, the amount of a chemical substance to be detected generated or consumed in the biological sample40increases. Additionally, since the volume of a three-dimensional region between the biological sample40and the electrode surface21aincreases, the amount of part of the generated or consumed chemical substance which stays inside the three-dimensional region increases. The two effects contribute to an increase in the amount of the chemical substance that reaches the electrode surface21a. With the presence of the spacer10, a diffusion stroke between the biological sample40and the electrode surface21ais long. The amount of the chemical substance that scatters away without reaching the electrode surface21ais thus considered to increase. However, since the distance between the biological sample40and the electrode surface21ais controlled by the spacer10so as to fall within an appropriate range, the two effects described earlier dominate, and, as a result, the amount of the chemical substance that reaches the electrode surface21ais considered to increase. <Type 2> An “inverse-cone-shaped structure” in which the height of a spacer at a position closest to the center of the electrode surface21ais lowest and increases gradually in a direction away from the center of the electrode surface21acan be adopted as a spacer structure. If the biological sample40having a specific gravity higher than that of the solution is put in an electrochemical measurement device including the spacer50with this configuration by using a pipette or the like, the biological sample40can be sunk under its own weight toward a low position of the spacer50, that is, the center of the electrode surface21awithout using any special mechanism. Thus, as for the positional relationship of the biological sample40with the electrode surface21a, not only a distance in the normal direction of the flat surface20abut also a distance in a direction parallel to the electrode surface21a, that is, the flat surface20acan be optimized. Additionally, appropriate setting of the relationship between a horizontal distance (a distance in the direction parallel to the electrode surface21a, that is, the flat surface20a) m from a given point X on the flat surface20ato the center of the electrode surface21aand the height of the spacer at the point X allows the distance between the flat surface20aand the biological sample to be set within the effective range for the distance z obtained by the above-described simulations as long as a biological sample diameter dsphas a value within the range of 100 to 600 μm. FIGS.7A and7Billustrate an electrochemical measurement device including the spacer50having an inverse-cone-shaped structure. InFIGS.7A and7B, the spacer50having the inverse-cone-shaped structure is composed of a plurality of pillar structures51different in height. The pillar structures51standing close together extend from the flat surface20aof the substrate20, on which the electrode surfaces21aare arranged, in the normal direction of the flat surface20a. In this example, the interval between any two pillar structures51is less than 100 μm. For example, when the biological sample40having a diameter of 100 μm is to be measured, an electrochemical measurement device having the pillar structures51formed at intervals of about 30 μm is used. See the supplemental explanation on the interval between the pillar structures11for the interval between the pillar structures51. FIG.7Billustrates two biological samples40different in diameter dsp. If several values are appropriately selected as values of the diameter dspfrom the range of 100 to 600 μm, and a curve circumscribing outlines of all of respective biological samples which are arranged at heights equal to optimum values of z for the values of dsp, that is, h2indicated by a broken line inFIG.7Bis obtained by fitting, h2is roughly a median in equation (5) below. m represents a distance [unit: μm] from the center of the electrode surface21ain the direction parallel to the flat surface20a. Note that, letting L be a center-to-center distance between two adjacent electrode surfaces21awhich are closest, m at least satisfies 0<m≤L/2 (h2>0). h2=√{square root over ((1.05de1+6.89)m)}−0.48de1−2.38±5[μm] (5) The spacer50in this example has an inverse-cone-shaped profile surface in which the distance h2in the normal direction of the flat surface20athat is dependent on the distance m [μm] satisfies equation (5). In a state where the biological sample40is in contact with the profile surface of the spacer50(that is, the pillar structures51) in the solution and is located immediately above the center of the electrode surface21a, electrochemical measurement is executed. The wall plate31extends in a straight line in a direction crossing a line segment connecting centers of two adjacent electrode surfaces21a. In this example, the wall plate31extends in the y direction orthogonal to an array direction of the two electrode surfaces21astanding side by side in the x direction. The wall plate31is placed at a position equally distant from the two adjacent electrode surfaces21a. The wall plate31has a height not less than a maximum height of the pillar structures51. That is, the wall plate31has a height not less than a maximum value within a range for h2given by equation (5), where m is a horizontal distance of a point on the flat surface20alocated immediately below the wall plate31from the center of the electrode surface21aclosest to the point. When the biological sample40is placed, the biological sample40can be sunk under its own weight toward a bottom portion of the inverse-cone-shaped spacer50without any special operation. At this time, a horizontal distance between the electrode surface21aand the biological sample40(a distance in the direction parallel to the electrode surface21a, that is, the flat surface20a) is 0. Note that since a position where the biological sample40is in contact with the spacer50varies depending on the sample diameter dsp, the distance z between the electrode surface21aand a lower end of the biological sample40varies depending on dsp. As in the configuration shown inFIGS.6A and6B, a path, through which the dissolved substance in the solution diffuses, is formed between the biological sample40and the electrode surface21ain the configuration shown inFIGS.7A and7B. Thus, the amount of a chemical substance to be detected generated or consumed in the biological sample40increases. Also, since the volume of a three-dimensional region between the biological sample40and the electrode surface21aincreases, the amount of part of the generated or consumed chemical substance which stays inside the three-dimensional region increases. The two effects contribute to an increase in the amount of the chemical substance that reaches the electrode surface21a. With the presence of the spacer50, a diffusion stroke between the biological sample40and the electrode surface21ais long. The amount of the chemical substance that scatters away without reaching the electrode surface21ais thus considered to increase. However, since the distance between the biological sample40and the electrode surface21ais controlled by the inverse-cone-shaped spacer50so as to fall within an appropriate range, the two effects described earlier dominate, and, as a result, the amount of the chemical substance that reaches the electrode surface21ais considered to increase. <Modification> Although a spacer is composed of a plurality of pillar structures in the above-described examples, the present invention is not limited to this configuration. For example, a thin-plate porous structure having a large number of microscopic pores, such as an agarose gel, may be used as a spacer. The diameters of the microscopic pores may not be fixed. The porous structure is placed on the flat surface20a. Additionally, it is not required that the spacer as the porous structure is formed throughout the flat surface20a. For example, the spacer (porous structure) is unnecessary in a location sufficiently away from the electrode surface21a. Second Embodiment of Present Invention Based on the simulation results described above, a second embodiment of the electrochemical measurement device according to the present invention has the configuration below. An electrochemical measurement device includes a solution well60, a plurality of wall plates32or33, and a plurality of working electrodes21. The plurality of wall plates32or33and the plurality of working electrodes21are all fixed on a flat surface20aof the solution well60. The flat surface20ais a bottom surface of the solution well60which is in contact with a solution in a state where the solution well60contains the solution and is, for example, a surface of a semiconductor chip on which an integrated circuit is formed. A surface of each working electrode21which is in contact with the solution is an electrode surface21a. The electrode surfaces21aand the wall plates32or33are immersed in the solution during measurement. <Biological Sample and Electrode Surface> A biological sample40and the electrode surfaces21aare the same as described in the above-described first embodiment. <Wall Plate and Spacer> Two or more wall plates32or33are placed between two electrode surfaces21aadjacent to each other. The wall plates32or33have the property of being impervious to a dissolved substance in the solution. The wall plates32or33reduce crosstalk. The wall plates32or33need to be shaped such that an enclosed three-dimensional region is not formed by the biological sample40, the flat surface20a, and the wall plates32or33while the biological sample40is in contact with the wall plates32or33. For example, the shape of a straight line, the shape of a polygonal line, or a shape extending curvedly can be adopted as the shape of each wall plate32or33when the flat surface20ais squarely viewed. Alternatively, the wall plates32or33may be arranged at intervals in a cylindrical shape or the shape of a polygonal frame. Alternatively, a structure in a cylindrical shape or the shape of a polygonal frame having one or more slits formed therein can be adopted as the wall plate32or33. The interval between the wall plates32or33may not be fixed. Additionally, it is not required that the wall plates32or33are formed throughout the flat surface20a. For example, the wall plate32or33is unnecessary in a location sufficiently away from the electrode surface21a. A height of h1(a length from the flat surface20aalong a normal to the flat surface20a) of each wall plate32or33has a predetermined value within a range for a distance z given by equation (4). In the second embodiment, the plurality of wall plates32or33have a function as a spacer. A spacer composed of the plurality of wall plates32or33has a profile surface at which a distance in a perpendicular direction to the flat surface20afalls within the range for the distance z given by equation (4). The spacer permits the dissolved substance in the solution to diffuse while inhibiting the biological sample40from entering a region on the flat surface20aside of the profile surface. With the above-described structure, the distance between the flat surface20aand the biological sample40can be roughly kept at h1. The “distance between the flat surface20aand the biological sample40” means a shortest distance between the flat surface20aand the biological sample40and is a minimum length of a half line connecting an intersection of the normal to the flat surface20aand the flat surface20aand an intersection of the normal and the biological sample40. The reason why the term “roughly” is used here is that the distance between the flat surface20aand the biological sample40may be strictly less than h1depending on the shape and posture of the biological sample40. Even in this case, the shapes of the individual biological samples40are strictly different from one another, and the postures of the individual biological samples40are strictly different from one another, so the above-described effect of “improving sensitivity, comparability, and reproducibility” is not lost from a statistical standpoint. On the grounds that the distance between the flat surface20aand the biological sample40may be strictly less than h1, h1may be a predetermined value within a range for the distance z given by equation (4a). h1may be a constant independent of a position on the flat surface20aor a value determined by a function with the position on the flat surface20aas a variable. Even in the latter case, h1is a value within the range for the distance z given by equation (4). That is, the height of the wall plate32or33need not be uniform over a whole region on the flat surface20a. For example, a region where the height of the wall plate32or33is relatively low and a region where the height of the wall plate32or33is relatively high may be present on the flat surface20a. Alternatively, a region where the heights of the wall plates32or33change in a stepwise manner may be present on the flat surface20a. <Type 1> In an example shown inFIGS.8A and8B, two or more wall plates32are arrayed on the flat surface20ain an x direction at intervals less than 100 μm. The wall plates32extend in a straight line in a y direction. Two electrode surfaces21aare arranged in the x direction on the flat surface20aof a substrate20, on which an x-y orthogonal coordinate system is defined. For the same reason as that in the case of a pillar structure, in actual usage, an electrochemical measurement device having a spacer composed of the wall plates32that are appropriately arranged in accordance with the diameter and shape of the biological sample40is used. In the example inFIGS.8A and8B, the plurality of wall plates32have uniform heights. A height h3of each wall plate32falls within the range for z given by equation (4). That is, the height h3satisfies the equation below. h3=21.8(del+0.8)del+9.7±5[μm] In the configuration shown inFIGS.8A and8B, the plurality of wall plates32have the same function as that of the plurality of pillar structures11shown inFIGS.6A and6B, that is, a function as a spacer and further have the same function as that of the wall plate31shown inFIGS.6A and6B, that is, a function as a wall plate which reduces crosstalk. Note that although y coordinates of centers of the two electrode surfaces21amatch up with each other in the example inFIGS.8A and8B, the y coordinates of the centers of the two electrode surfaces21aneed not match up with each other. That is, a line connecting the centers of the two electrode surfaces21aand an extension direction of the wall plates32may not be orthogonal to each other. In other words, the angle which the line connecting the centers of the two electrode surfaces21aforms with the extension direction of the wall plates32may be larger than 0 degrees and smaller than 90 degrees. <Type 2> In a configuration shown inFIGS.9A and9B, the plurality of wall plates33have the same function as that of the plurality of pillar structures51shown inFIGS.7A and7B, that is, a function as a spacer and further have the same function as that of the wall plate31shown inFIGS.7A and7B, that is, a function as a wall plate which reduces crosstalk. The plurality of wall plates33are arrayed on the flat surface20ain the x direction at intervals less than 100 μm. The wall plates33extend in a straight line in the y direction. See the description of the interval between the wall plates32for the interval between the wall plates33. A height h4of the wall plate33at a position away by a distance m from the center of the electrode surface21ain the x direction satisfies equation (5). That is, h4=h2. In this example, the heights of the wall plates33do not change in the y direction. A cross-section taken perpendicularly to a y-axis of the wall plates33has a parabolic cross-sectional shape. A profile surface of the wall plates33is a groove-like surface which extends in the y direction. One or more electrode surfaces21aare arranged along a groove (a bottom of an inverse cone). In the example inFIGS.9A and9B, y coordinates of centers of the two electrode surfaces21amatch up with each other. The y coordinates of the centers of the two electrode surfaces21a, however, need not match up with each other. That is, a line connecting the centers of the two electrode surfaces21aand an extension direction of the wall plates33may not be orthogonal to each other. In other words, the angle which the line connecting the centers of the two electrode surfaces21aforms with the extension direction of the wall plates33may be larger than 0 degrees and smaller than 90 degrees. Electrode rows preferably form an array in which the electrode surfaces21aare also arranged in the x direction. There is no limit to arrangement (y coordinates) of the electrode surfaces21ain an electrode row. A configuration in which one electrode row includes only one electrode surface21ais also admissible. The wall plates32and33inFIGS.8A and8BandFIGS.9A and9Bhave the property of being impervious to the dissolved substance in the solution. The wall plates32and33reduce crosstalk. <Modifications> FIGS.10and11show modifications of the configurations shown inFIGS.8A and8BandFIGS.9A and9B. In the modifications, each wall plate extends continuously in a straight line. Two wall plates32located next to each electrode surface21aof the wall plates32shown inFIG.10have heights which change along a y direction (an extension direction of the wall plates32). In this example, changes in the heights are noticeable near the electrode surface21a, and there are no changes in the heights except near the electrode surface21a. The height of each wall plate32is lowest (minimal) at a point where a perpendicular line from a center of the electrode surface21ato the wall plate32crosses the wall plate32when the flat surface20ais squarely viewed. Although the height of the wall plate32changes smoothly in the example shown inFIG.10, the height may change in a stepwise manner. In the example shown inFIG.10, a minimum value of the height in one of the two wall plates32located next to the electrode surface21ais almost equal to a minimum value of the height in the other. In other words, depressions32aare formed at respective upper portions of the two wall plates32located next to the electrode surface21a. Each depression32ais located above the point where a perpendicular line from the center of the electrode surface21ato the wall plate32crosses the wall plate32when the flat surface20ais squarely viewed. In the above-described example, the height of the wall plate32at a position where the spherical biological sample40is in contact with the wall plate32while the biological sample40is put in the depression32ais equal to h3described above. Similarly, the height of each wall plate33shown inFIG.11changes along a y direction (an extension direction of the wall plates33). In this example, a change in the height of each wall plate33is noticeable near the electrode surface21a, and there is no change in the height except near the electrode surface21a. The height of each wall plate33is lowest (minimal) at the point where a perpendicular line from a center of the electrode surface21ato the wall plate33away by a distance m crosses the wall plate33when the flat surface20ais squarely viewed. Although the height of the wall plate33changes smoothly in the example shown inFIG.11, the height may change in a stepwise manner. In the example shown inFIG.11, a minimum value of the height in one of two wall plates32which are located at positions symmetric with respect to the center of the electrode surface21ais almost equal to a minimum value of the height in the other. In other words, a depression33ais formed at an upper portion of each wall plate33. Each depression33ais located above the point where a perpendicular line from the center of the electrode surface21ato the wall plate33crosses the wall plate33when the flat surface20ais squarely viewed. In the above-described example, the height of the wall plate33at a position where the spherical biological sample40is in contact with the wall plate33away by the distance m from the center of the electrode surface21awhile the biological sample40is put in the depression33ais equal to h4described above. The depressions32aand33aare useful for positioning of a biological sample in the y direction. The depressions32aare provided at one pair of wall plates32, which is adjacent to the electrode surface21aand between which the electrode surface21ais sandwiched, inFIG.10while the depressions33aare provided at all the wall plates33inFIG.11. The amounts of depression of the depressions32aand33aare set within ranges where the heights h3and h4are achieved. In modifications as well, in which depressions are formed at wall plates, y coordinates of centers of two electrode surfaces21aneed not match up with each other, as in the example inFIGS.8A and8B. Alternatively, the depression32amay be formed only at the wall plate32closest or second closest to a center of each electrode surface21a(either one if two wall plates32are present at positions equally distant from the center of the electrode surface21a). If the wall plate32is located above the electrode surface21a, the depression32ais located above the electrode surface21a. The depression32amay be formed at an upper portion of each of three or more wall plates32near the electrode surface21a. In the example shown inFIG.11, letting L be a center-to-center distance between two adjacent electrode surfaces21awhich are closest, the depression33ais formed at an upper portion of the wall plate33at a position away by the distance m from a center of the electrode surface21a, where m satisfies 0<m≤L/2. However, since a maximum diameter of the biological sample40is 600 μm, a structure in which the depression33ais formed at an upper portion of the wall plate33away by the distance m, not less than 0 and not more than 300 μm, from the center of the electrode surface21amay be adopted. That is, the depression33ais formed at all of the wall plates33, widths of which are wholly included in a line segment connecting a first end point and a second end point located on two sides of each electrode surface. Note that the line segment connecting the first end point and the second end point is parallel to the x direction. The first end point and the second end point are each a closer one of a point 300 μm away from a center of the electrode surface and a midpoint of the adjacent electrode rows. Note that, if the former point and the latter point match up with each other, the first end point or the second end point is at the matching points. FIG.11represents a form in a case where x-direction distances from two electrode rows illustrated (the electrode surfaces21a) to a midpoint of the two electrode rows are less than 300 μm and no other electrode row is present outside the two electrode rows illustrated (the electrode surfaces21a) in the above-described configuration. Note that since a width of the highest wall plate33that is located right on the midpoint of the two electrode surfaces21adoes not fall wholly within ranges for the left and right electrode rows, each having the first and second end points as two ends, the depression33amay not be formed at the wall plate33or may be formed as in the illustrated example. Alternatively, the depression33amay be formed at at least one wall plate33among the wall plates33, widths of which fall at least partly within an x-direction range between first and second end points, instead of providing the depressions33aat all of the wall plates33falling within the range between the first and second end points. With a depression formed at an upper portion of a wall plate, a biological sample can be positioned in the y direction. That is, the biological sample40can be located almost immediately above each electrode surface21a. If the wall plate33is located above the electrode surface21a, the depression33ais located above the electrode surface21a. FIG.12shows another modification of the configuration shown inFIGS.8A and8B. In the modification, each wall plate32extends continuously. The interval between two wall plates32located next to each electrode surface21aof the wall plates32shown inFIG.12changes along a y direction (an extension direction of the wall plates32). In this example, a change in interval is noticeable near the electrode surface21a, and there is no change in interval except near the electrode surface21a. For this reason, an extension direction of portions without a change in interval (that is, portions except near the electrode surface21a) is regarded as an extension direction of a wall plate. The interval between the wall plates32is widest (maximal) at a point where a line extending from a center of the electrode surface21aand orthogonal to the extension direction of the wall plates32crosses the wall plate32when the flat surface20ais squarely viewed. Although the interval between the wall plates32changes smoothly in the example shown inFIG.12, the interval may change in a stepwise manner. In other words, a recess32bwhich extends in a wall-plate height direction (that is, a normal direction of the flat surface20a) is formed at each of the two wall plates32located next to the electrode surface21a. The recess32bhas a portion which opens toward the center of the electrode surface21awhen the flat surface20ais squarely viewed. With widening of a wall plate interval, a biological sample can be positioned. In the example inFIG.12, y coordinates of centers of the two electrode surfaces21amatch up with each other. However, the y coordinates of the centers of the two electrode surfaces21aneed not match up with each other. That is, a line connecting the centers of the two electrode surfaces21amay not be orthogonal to the extension direction of the wall plates32. In other words, the angle which the line connecting the centers of the two electrode surfaces21aforms with the extension direction of the wall plates32may be larger than 0 degrees and smaller than 90 degrees. Unlike the example inFIG.12, the recess32bmay be formed only at one wall plate. The one wall plate in this case may be a wall plate second closest to the center of the electrode surface21a. The shape of each recess when the flat surface20ais squarely viewed may be the shape of a polygonal line or the shape of a curved line. FIG.13shows an example in which the electrode surfaces21aare arrayed in a lattice pattern. A plurality of electrode rows22are arranged in an x direction, thereby arraying a large number of electrode surfaces21a. In each electrode row22, a plurality of electrode surfaces21aare arranged in a y direction. In this configuration, wall plates34which reduce crosstalk, for example, each extend in the y direction between adjacent ones of the electrode rows22. In this case, the effect of reducing crosstalk in the x direction is achieved. Even if the wall plates34are placed in this manner, a dissolved substance is not blocked from diffusing in a direction parallel to the wall plates34(the y direction), a reduction in the amount of the dissolved substance supplied is small. A spacer is not shown inFIG.13. In the configuration shown inFIG.13, the spacer not shown is the pillar structures11or51shown inFIGS.6A and6BorFIGS.7A and7B, a porous structure, or the like. In the configuration shown inFIG.13, the spacer not shown may be the plurality of wall plates32or33shown in one ofFIGS.8to12that have a function as a spacer. In other words, in an electrochemical measurement device having the configuration shown in one ofFIGS.8to12, the wall plate34(a partition plate) having a height above the heights of the wall plates32and33may be additionally placed. The height of the wall plate34may be above a range for the height h3or h4of the wall plate32or33. If the height of the wall plate34is sufficiently high, most of the effect of reducing crosstalk is achieved by the wall plate34. If a spacer is composed of a plurality of wall plates, a break-proof, robust electrochemical measurement device can be implemented. <Specific Example> The above-described effects are expected to be achieved as long as the condition that “a chemical substance generated or consumed in a biological sample itself is electrochemically active or is transformed to another chemical substance that is electrochemically active” is satisfied. The type of a biological sample, a reaction mechanism by which a chemical substance to be detected is generated or consumed in the biological sample, a working electrode, a substrate on which the working electrode is formed, and the like are not particularly limited. For example, the configurations below are conceivable. <Biological Sample> An embryoid body which was produced from mouse ES cells was selected as a biological sample for the simulations. However, the biological sample may be a cell aggregate, a single cell, a piece of tissue, a microorganism, a non-biological sample containing a biologically-relevant substance, or the like. <Reaction Mechanism by which Chemical Substance is Generated or Consumed in Biological Sample> An ALP enzymatic reaction on a model sample was selected as a reaction mechanism for the simulations. However, the reaction mechanism may be an enzymatic reaction of protein, peptide, RNA, or the like or a catalytic reaction with a platinum thin film, a titanium oxide thin film, or the like on a biological sample. If a biological sample is cells or the like, a chemical substance may be a substance generated or consumed through various metabolic pathways or signaling pathways in cells. For example, the chemical substance may be protons released in a metabolic pathway in a glycolytic system or dopamine released from neuron cells. <Working Electrode> The material for a working electrode was not specified in the simulations. Any material, such as a noble metal (for example, gold or platinum), an inorganic substance predominantly composed of carbon (for example, graphite, diamond doped with an impurity, or a carbon nanotube), or a conductive polymer (for example, polypyrrole, polyaniline, or polythiophene), may be used as the material for the working electrode as long as the material can be used for a working electrode for electrochemical measurement. The shape of an electrode surface of the working electrode is, for example, circular, elliptical, polygonal, or the like. <Substrate> The material for a substrate was not specified in the simulations. Any material, such as quartz, glass, silicon, or ceramic, may be used as the material for the substrate as long as the material can be used for a working electrode support for electrochemical measurement. Specific Example of Spacer Fabrication Method In order to achieve the above-described effects, a spacer is desirably fabricated by a method capable of controlling the height of the spacer on the order of μm. The spacer has a solution-permeable structure, that is, a structure which permits a dissolved substance in a solution to diffuse. Additionally, if the spacer is to come into contact with an electrode, the spacer needs to have electrical insulation. As long as these conditions are satisfied, there is no limit to a fabrication method for the spacer and the material for the spacer. Preferable spacer fabrication methods and preferable materials for the spacer will be illustrated below. Fabrication Example 1 of Spacer Composed of Plurality of Pillar Structures 1) A silicon nitride film is formed on a substrate by chemical vapor deposition (CVD). The thickness of the silicon nitride film on the substrate is uniform. 2) An etching protective layer is patterned on the silicon nitride film by photolithography. 3) The silicon nitride film in a region not coated with the etching protective layer is etched by reactive ion etching, thereby forming pillar structures. 4) The etching protective layer is removed. The material for the insulating film (the material for the pillar structures) is not limited to silicon nitride and may be, for example, silicon oxide, titanium oxide, or the like. The film formation method is not limited to CVD and may be a vacuum film formation method, such as sputtering or evaporation, spin-on glass, or the like. The patterning method for the etching protective layer is not limited to photolithography and may be screen printing, ink-jet printing, or the like. The etching method is not limited to reactive ion etching and may be plasma etching, sputter etching, ion beam etching, or wet etching. Fabrication Example 2 of Spacer Composed of Plurality of Pillar Structures 1) An LSI having a current sensing element is coated with photosensitive resin by spin coating. The current sensing element includes at least a working electrode. 2) Pillar structures are fabricated by photolithography. The photosensitive resin may be any insulating and photosensitive resin as long as the resin is used in common photolithography. A photosensitive resin necessary for achievement of resolution required to fabricate a spacer having an accurate diameter and an accurate height is desirably selected. An epoxy chemically-amplified photosensitive resin which is used as a negative permanent resist is preferable from the standpoint of the chemical stability of the pillar structures. Any coating method may be used as long as the coating method can control a film thickness on the order of μm. In view of high film-thickness controllability, the coating method is not limited to spin coating and may be spray coating, dip coating, screen coating, roll coating, or the like. <Fabrication Example of Spacer as Porous Structure> 1) After an agarose solution diluted with water is prepared, the agarose solution diluted with water is heated to 80° C. or more and is changed to a sol. 2) The agarose aqueous solution is dropped onto a substrate at 80° C., and a thin film is formed by spin coating. During the process, the temperature of the substrate is constantly kept at 80° C. or more. 3) The substrate is left and cooled to the room temperature to acquire a porous spacer made of agarose gel. Any sol may be used as the sol to be dropped onto the substrate as long as the sol changes to a porous gel after coating. The heating temperature is appropriately set depending on the type of the sol. In view of ease of preparation and high biocompatibility, agarose, polyvinyl alcohol, cellulose, or the like is preferable. Any method may be used as the coating method as long as the method can control a film thickness on the order of μm and has a mechanism for keeping the temperature of the sol constant during the coating process. In view of high film-thickness controllability, the coating method is not limited to spin coating and may be spray coating, dip coating, screen coating, roll coating, or the like. <Others> A spacer composed of pillar structures can be fabricated by molding (nanoimprinting (nanoimprint lithography) or insert molding), printing (for example, screen printing or ink-jet printing), machining, or the like. A spacer which is a porous structure can also be fabricated by placing a pre-shaped porous body, such as porous silica or a nitrocellulose membrane, on a substrate. <Specific Example of Wall Plate Fabrication Method> If wall plates have a function as a spacer as in the examples shown inFIGS.8and9, the wall plates are desirably fabricated by a method capable of controlling a wall plate height on the order of μm. If a wall plate is fabricated in contact with an electrode, the wall plate needs to have electrical insulation. In these respects, the same method as a fabrication method for a spacer composed of pillar structures can be adopted as a wall plate fabrication method. The same material as that for a spacer composed of pillar structures can be adopted as the material for a wall plate. <Specifications of Spacer Composed of Pillar Structures> If a spacer is composed of a plurality of pillar structures, an interval for and the shapes of the pillar structures are determined in the manner below. <Interval Between Pillar Structures> The interval between pillar structures is appropriately set in accordance with the diameter of a biological sample. In terms of achieving higher sensitivity, that is, minimizing blocking of diffusion of a dissolved substance around a biological sample by pillar structures, a wider interval is more preferable for the pillar structures. The intervals between pillar structures need not be uniform. There may be a region where pillar structures are densely present and a region where pillar structures are sparsely present or there may be a region where no pillar structures are present. For example, a structure in which no pillar structure is formed only in a region immediately above an electrode surface and a biological sample is held only by pillar structures around the electrode surface effectively prevents blocking of diffusion of a dissolved substance immediately below the biological sample and achieves higher sensitivity. <Diameter of Pillar Structure> The diameter of each pillar structure is large enough to secure strength that allows holding of a biological sample away from an electrode surface. Note that a smaller diameter is more preferable for pillar structures in terms of achieving higher sensitivity, that is, minimizing blocking of diffusion of a dissolved substance around a biological sample by the pillar structures. <Upper Surface Shape of Pillar Structure> There are no restrictions on the shape of an upper surface of a pillar structure. The shape of the upper surface of the pillar structure may be circular or polygonal (for example, triangular or quadrangular). In a pillar structure, the shape of an upper surface and that of a lower surface need not be identical. The area of the upper surface and that of the lower surface need not be identical in the pillar structure. For example, the area of the upper surface may be intentionally reduced (that is, a tapered pillar structure may be formed) by changing etching conditions for an insulating layer at the time of pillar structure creation. If a biological sample is cells, a piece of tissue, or the like, a tapered pillar structure can reduce a contact area and adhesivity between the biological sample and pillar structures. The tapered pillar structure reduces force needed to peel the biological sample at the time of removal of the biological sample after measurement, which results in reduction of damage to the biological sample. <Specifications of Wall Plate Functioning as Spacer> If a spacer is composed of a plurality of wall plates, an interval for and the shapes of the wall plates are determined in the manner below. <Interval Between Wall Plates> The interval between wall plates is appropriately set in accordance with the diameter of a biological sample. In terms of achieving higher sensitivity, that is, minimizing blocking of diffusion of a dissolved substance around a biological sample by wall plates, a wider interval is more preferable for the wall plates. For example, no wall plate is formed in a region immediately above an electrode surface, and the electrode surface is sandwiched between wall plates. This effectively prevents blocking of diffusion of a dissolved substance immediately below a biological sample and allows effective reduction of crosstalk. <Thickness of Wall Plate> The thickness of each wall plate is large enough to secure strength that allows holding of a biological sample away from an electrode surface. <Shape of Wall Plate> The shape of an upper surface and that of a lower surface in a wall plate need not be identical. The area of the upper surface and that of the lower surface in the wall plate need not be identical. For example, the area of the upper surface may be intentionally reduced (that is, a tapered wall plate may be formed) by changing etching conditions for an insulating layer at the time of wall plate creation. If a biological sample is cells, a piece of tissue, or the like, a tapered wall plate can reduce a contact area and adhesivity between the biological sample and wall plates. The tapered wall plate reduces force needed to peel the biological sample at the time of removal of the biological sample after measurement, which results in reduction of damage to the biological sample. <Transducer> An example of a specific configuration of a transducer according to the present invention will next be described with reference toFIGS.14and15. The transducer is used for electrochemical measurement of a chemical substance generated or consumed in a biological sample. The transducer is configured such that a solution well60is mounted on an LSI chip70. The solution well60contains a solution61and a biological sample which is immersed in the solution61. A hole62is formed at the center of the solution well60. The LSI chip70is arranged at a lower end of the hole62. The hole62is closed by the LSI chip70. The LSI chip70and the solution well60are fixed on a substrate80. A pattern81of many conductors for connection with an external device which controls the transducer is formed on the substrate80. Reference numeral90inFIG.14Bindicates bonding wires which interconnect the LSI chip70and the pattern81of conductors. A sensor region71is formed on an upper surface of the LSI chip70. InFIG.14A, the sensor region71is indicated by hatching. The sensor region71is located in the hole62at a bottom surface of the solution well60. Although not shown, a plurality of electrodes (working electrodes) are formed in the sensor region71in this example, and a spacer composed of pillar structures is also formed in the sensor region71. A wall plate is formed between adjacent ones of the working electrodes. The LSI chip70has a function of applying a voltage to the working electrodes, a function of detecting a reaction at each working electrode as a current value and amplifying the current value, and the like. The spacer and the wall plate are as described earlier. As described above, a biological sample is away by a desirable distance from a flat surface with an electrode surface arranged thereon by a spacer or a plurality of wall plates. Thus, a three-dimensional region, through which a dissolved substance in a solution can diffuse, is secured, and a sufficient amount of the dissolved substance is supplied to the biological sample. According to the present invention, the amount of a chemical substance to be detected by a working electrode increases, and measurement sensitivity is higher than conventional electrochemical measurement that performs measurement with a biological sample located close to an electrode surface. A perpendicular distance between a working electrode and a biological sample may vary depending on the shape and surface state of the biological sample. However, placing a biological sample at a desirable distance from an electrode surface allows reduction of the influence of differences in a diffusion distance of a chemical substance which are associated with differences in a perpendicular distance between a working electrode and the biological sample, which makes comparability and reproducibility of measurement higher than conventional electrochemical measurement. According to the present invention, a wall plate which is formed between adjacent electrode surfaces and is impervious to a dissolved substance in a solution reduces sensing crosstalk between electrode surfaces due to diffusion of a chemical substance to be detected in the solution. For example, in the case of simultaneous evaluation of a plurality of biological samples, the influence of a distant sample on a current value at each working electrode can be prevented. This improves the quantitativity of biological sample evaluation. Since the interval between electrode surfaces can be narrowed, the cost of substrates can be reduced. An electrochemical measurement device and a transducer according to the present invention will be described from a different standpoint in the manner below. Note that the following description does not conflict with the disclosed matters described in the “MEANS TO SOLVE THE PROBLEMS” and that the following description and the “MEANS TO SOLVE THE PROBLEMS” can refer to each other. Item 1 An electrochemical measurement device for measuring a chemical substance generated or consumed in a biological sample in a solution, including:a solution well for containing the solution and the biological sample;two or more electrode surfaces, each of the electrode surfaces being a surface of an electrode which is in contact with the solution while the solution well contains the solution, and an oxidation-reduction reaction progressing between each electrode surface and the chemical substance;a spacer; andat least one wall plate, whereinthe two or more electrode surfaces, the spacer, and the at least one wall plate are arranged on a bottom surface of the solution well,a diameter delof each of the two or more electrode surfaces is not more than 80 μm,a height of the spacer has a predetermined value within a range for h given by equation (c1): h=21.8(del+0.8)del+9.7±5[μm],(c1)the spacer has a structure in which an enclosed three-dimensional region is not formed by the biological sample, the bottom surface, and the spacer while the biological sample is in contact with the spacer,the at least one wall plate has a property of being impervious to a dissolved substance in the solution,the at least one wall plate has a height not less than the height of the spacer, andat least two electrode surfaces of the two or more electrode surfaces are separated by the at least one wall plate. Item 2 An electrochemical measurement device for measuring a chemical substance generated or consumed in a biological sample in a solution, including:a solution well for containing the solution and the biological sample;two or more electrode surfaces, each of the electrode surfaces being a surface of an electrode which is in contact with the solution while the solution well contains the solution, and an oxidation-reduction reaction progressing between each electrode surface and the chemical substance;a spacer; andat least one wall plate, whereinthe two or more electrode surfaces, the spacer, and the at least one wall plate are arranged on a bottom surface of the solution well,a diameter delof each of the two or more electrode surfaces is not more than 80 μm,a height of the spacer at a position at a distance m, which is a distance in a direction parallel to the bottom surface from a center of one electrode surface of the two or more electrode surfaces, from the center of the one electrode surface has a predetermined value within a range for h given by equation (c2): h=√{square root over ((1.05del+6.89)m)}−0.48del−2.38±5[μm] (c2) where 0<m≤L/2 and h>0 hold, L being a distance between the center of the one electrode surface and a center of a different electrode surface of the two or more electrode surfaces which is closest to the one electrode surface,the spacer has a structure in which an enclosed three-dimensional region is not formed by the biological sample, the bottom surface, and the spacer while the biological sample is in contact with the spacer,the at least one wall plate has a property of being impervious to a dissolved substance in the solution,the at least one wall plate has a height not less than the height of the spacer, andat least two electrode surfaces of the two or more electrode surfaces are separated by the at least one wall plate. Item 3 In the electrochemical measurement device according to item 1 or item 2,the two or more electrode surfaces include three or more electrode surfaces,the at least one wall plate includes two or more wall plates,at least two electrode surfaces of the three or more electrode surfaces are arranged in at least one of at least one or more portions, the at least one or more portions being at least one or more portions of the bottom surface which are located between adjacent two of the two or more wall plates or at least one or more portions of the bottom surface which are located between one wall plate of the two or more wall plates and a side wall of the solution well. Item 4 In the electrochemical measurement device according to any one of item 1 to item 3,the spacer is composed of pillar structures, andeach of pillar structures extends in a normal direction of the flat surface. Item 5 In the electrochemical measurement device according to any one of item 1 to item 3,the spacer is a porous structure. Item 6 An electrochemical measurement device for measuring a chemical substance generated or consumed in a biological sample in a solution, including:a solution well for containing the solution and the biological sample;two or more electrode surfaces, each of the electrode surfaces being a surface of an electrode which is in contact with the solution while the solution well contains the solution, and an oxidation-reduction reaction progressing between each electrode surface and the chemical substance; andtwo or more wall plates, whereinthe two or more electrode surfaces and the two or more wall plates are arranged on a bottom surface of the solution well,a diameter delof each of the two or more electrode surfaces is not more than 80 μm,respective heights of the two or more wall plates have predetermined values within a range for h given by equation (c3): h=21.8(del+0.8)del+9.7±5[μm],(c3)a structure of each of the two or more wall plates does not form an enclosed three-dimensional region together with the biological sample and the bottom surface, being in contact with the biological sample,the two or more wall plates each have a property of being impervious to a dissolved substance in the solution, andat least two electrode surfaces of the two or more electrode surfaces are separated by at least one wall pate of the two or more wall plates. Item 7 An electrochemical measurement device for measuring a chemical substance generated or consumed in a biological sample in a solution, including:a solution well for containing the solution and the biological sample;two or more electrode surfaces, each of the electrode surfaces being a surface of an electrode which is in contact with the solution while the solution well contains the solution, and an oxidation-reduction reaction progressing between each electrode surface and the chemical substance; andtwo or more wall plates, whereinthe two or more electrode surfaces and the two or more wall plates are arranged on a bottom surface of the solution well,a diameter delof each of the two or more electrode surfaces is not more than 80 μm,a height of one wall plate at a position at a distance m, which is a distance in a direction parallel to the bottom surface from a center of one electrode surface of the two or more electrode surfaces, from the center of the one electrode surface of the two or more wall plates has a predetermined value within a range for h given by equation (c4): h=√{square root over ((1.05del+6.89)m)}−0.48del−2.38±5[μm] (c4) where 0<m≤L/2 and h>0 hold, L being a distance between the center of the one electrode surface and a center of a different electrode surface of the two or more electrode surfaces which is closest to the one electrode surface,a structure of each of the two or more wall plates does not form an enclosed three-dimensional region together with the biological sample and the bottom surface, being in contact with the biological sample,the two or more wall plates each have a property of being impervious to a dissolved substance in the solution, andat least two electrode surfaces of the two or more electrode surfaces are separated by at least one wall plate of the two or more wall plates. Item 8 In the electrochemical measurement device according to item 6 or item 7,a depression is formed at an upper portion of at least one of two wall plates of the two or more wall plates, the depression being located next to one electrode surface of the two or more electrode surfaces when the bottom surface is squarely viewed, the two wall plates being located on two sides of the one electrode surface. Item 9 In the electrochemical measurement device according to any one of item 6 to item 8,a recess which increases an interval between two wall plates of the two or more wall plates is formed at at least one of the two wall plates, the recess being located next to one electrode surface of the two or more electrode surfaces when the bottom surface is squarely viewed and extending in a normal direction of the bottom surface, the two wall plates being located on two sides of the one electrode surface. Item 10 The electrochemical measurement device according to any one of item 6 to item 9, includinga partition plate which is a wall plate having a height more than a maximum value for the heights of the two or more wall plates, whereinthe partition plate has a property of being impervious to the dissolved substance in the solution, andat least two electrode surfaces of the two or more electrode surfaces are separated by the partition plate. Item 11 A transducer including:an electrochemical measurement device according to any one of item 1 to item 10; andan integrated circuit, whereinthe bottom surface of the solution well is a surface of the integrated circuit. | 79,138 |
11859168 | Reference signs on the drawings denote the following:1—the volume of media contained in the reservoir portion;2—the volume of media contained in the cell culture dish overall;3—reservoir;4—additional “feet” or “skirt”;5—inspection planes;6—electrode contact;7—catheter;8—electrode;9—needle;10—plastic flask;11—cap;12—exposition/visualisation plane;13—funnel;14—electrode plates; w—width. DETAILED DESCRIPTION The electroporation cathether of the present invention is, in some embodiments, characterized by extendable and retractable needle suitable for injection of an agent or transfectant into the target tissue, organ or cavity tissue. In some embodiments, the electroporation catheter further comprises a flexible tubing having proximal and distal ends and at least one lumen. The needle of the present invention may be of any suitable medical grade construction. The needle, which in some embodiment may double as electrode, will be electrically isolated from other electrodes and their lead wires. In some embodiments, the retractable needle extends beyond the distal end of electrode catheter tubing during injection. In some embodiments, the needle is connected to a water-tight tube which is connected proximally to an injection controller. In some embodiments, the injection controller is a syringe. In some embodiments, the injection controller is a bladder. In some embodiments, the injection controller is a mechanical pump allowing finrly-controlled and/or programmed control of the injection rate. In some embodiments, a radiologically detectable tracer or marker is injected via the needle along with a transfectant of other agent to facilitate monitoring of successful injection and electroporation. The needle may be of any size deemed suitable for practice of the invention by the medical interventionalist skilled in the art. In some embodiments, the needle features multiple opening through which a transfectant or other agent may be injected. In some embodiments, wherein the needle may double as an electrode, it is further connected to a lead wire. In some embodiments, the injection needle may function as an electroporation electrode in combination with a second coaxially located electrode. In some embodiments, the injection needle may function as an electroporation electrode in combination with multiple coaxially located electrodes. In some embodiments, the injection needle may function as an electroporation electrode in combination with a loop electrode, and/or in combination with a circular array of electrodes. In some embodiments, the injection needle may function as an electroporation electrode in combination with a circular array of needle electrodes. In some embodiments, the injection needle does not double/function as an electroporation electrode, and, instead, coaxially located electrodes perform the electrode function. In some embodiments, the injection needle does not double/function as an electroporation electrode, and, instead, a loop electrode, alone or in combination with coaxially located electrodes, performs the electrode function. In some embodiments, the injection needle does not double/function as an electroporation electrode, and, instead, the electrodes of a circular array perform the electrode function. In some embodiments, the electrodes of the electroporation catheter are retractable electrodes. In some embodiments, the retractable electrodes may be extended beyond the distal end of catheter during electroporation. In some embodiments, the electrodes are extended to prior to injection to ascertain the catheter is correctly positioned, and may be used to detect a change in impedance. In some embodiments, the electrodes and/or needle doubling as an electrode may further be used to detect and/or map electrical activity in a tissue, before injection of an agent or transfectant and electroporation, or after injection or after electroporation. In preferred embodiments, the electrodes and/or needle doubling as an electrode are connected electrically to an electrical pulse generator and power supply. In some embodiments, the entire catheter system of the present invention is connected to a computer interface and/or computer by which the system is monitored and controlled. In some embodiments, the electrodes and/or needle doubling as an electrode may be used to perform irreversible electroporation. In some embodiments, a needle hand control is provided at the proximal end of the catheter. In some embodiments, injection needle system extends from the distal end section, through the catheter tubing to a needle controller. In some embodiments, the injection needle can translocate so that its distal end can extend under the influence of the needle hand control. In some embodiments, electrodes are mounted on the distal end of the catheter as coaxially positioned electrodes, loop electrode(s) and/or circular electrodes. In some embodiments, multiple electrodes are mounted on the distal end of the catheter as a circular electrode array. In some embodiments, an electrode lead wire is electrically connected to the injection needle and to a suitable monitoring apparatus, an electrical pulse generator and a power source. In some embodiments, the invention is directed to a method for introducing an agent, especially a therapeutic, drug, transfectant or DAdC population, into the tissue of a patient. In some embodiments, the method comprises introducing the distal end of a catheter into or through the patient's body, vasculature, or orifice to reach the target tissue or space, wherein the injection needle is then extended beyond the distal end of the end section and a useful agent, especially a therapeutic, drug, transfectant or developmentally activate cell population, is then injected into the tissue, organ or cavity, optionally followed by reversible electroporation. In some embodiments, the method comprises introducing the distal end of a catheter into or through the patient's body, vasculature, or orifice to reach the target tissue or space, wherein the electrodes and/or needle doubling as an electrode are used to perform electroporation. In some embodiments, the method comprises introducing the distal end of a catheter into or through the patient's body, vasculature, or orifice to reach the target tissue or space, wherein the electrodes and/or needle doubling as an electrode are used to perform electroporation. In some embodiments, the target tissue is a tissue that has suffered ischemic damage and the agent or transfectant or cells of the present invention is capable of mitigating said damage. In some embodiments, the tissue is a tissue that is genetically compromised and the agent, transfectant or cells of the present invention is capable of mitigating said compromise. In some embodiments, the tissue of the present invention is a tissue in need of regeneration and the agent, transfectant or cells of the present invention is capable of promoting regeneration. In some embodiments, the target tissue is a tissue in need of repair and the agent, transfectant or cells of the present invention is capable of promoting said repair. In some embodiments, the target tissue is in need of differentiated cellular elements and the agent, transfectant or cells of the inevntion is capable of promoting or providing said elements. In some embodiments, the tissue of the present invention is a tissue featuring abnormal growth and/or proliferation (e.g. cancer) and the agent or transfectant, or cells of the inevntion is capable of reducing or eliminating said abnormal growth and/or proliferation. In some embodiments, the target tissue hypoplastic and the agent, transfectant, or cells of the present invention is capable of promoting growth and/or proliferation. In some embodiments, the agent or transfectant is a protein or nucleic acid and transfection is mediated by nanoparticle and/or a transfection mediating reagent, named herein. In some embodiments, the agent or transfectant is a chemical compound or extract. In some embodiments, a tissue is injected with a developmentally activate cell population, and electroporation is not applied. The current invention, therefore, provides a catheter suitable for use for injection of an agent, especially a therapeutic, drug, transfectant or cell population of the present inevntion, into a target tissue, organ or cavity—with or without electroporation (EP). In some embodiments, in vivo, irreversible electroporation is performed after injection of an agent or transfectant. In some embodiment, the catheter of the present invention comprises an catheter tubing having proximal and distal ends, an end section at the distal end of the catheter tubing, and a needle hand control and deflection controller proximal the catheter tubing. In some embodiments, the tubing may be of any suitable construction and material so long as the construction and material provide for one or more flexible lumen. In some embodiments, the tubing may be of any suitable construction and material providing for one or more substantially, non-compressible lumen. In some embodiments, polyurethane, polyether ether ketone, or nylon forms the outer wall. In some embodiments, the outer wall may further comprise a mesh of stainless steel. In some embodiments, the catheter's outer diameter no more than 8 French. The outer diameter of the catheter's end section is also preferably no greater than 8 French. In some embodiments, the catheter has an inner stiffening tube made of any suitable material. In one embodiment, the inner stiffening tube is constructed from polyimide. In a preferred embodiment, the catheter is compatible with a conventional guide sheath. In some embodiments, the catheter features a compression coil and puller wire assembly. In some embodiments, one or more coaxial electrodes are mounted directly to the distal end of the flexible tubing of the end section. In some embodiments, one or more ring electrode is mounted to the end section of the catheter and connected to lead wires. In some embodiments, the catheter end section comprises a deflectable segment and an adjustable circle or loop housing an electrode array of variable number. In some embodiments, the catheter electrodes are irrigated electrodes and perfusion fluid channels are incorporated in the main body portion, and the control handle. In some embodiments, a compression coil surrounds the puller wire from the proximal end of the catheter tubing to the proximal end of the end section. In some embodiments, the compression coil is made of stainless steel. The ability to derive proliferating, self-renewing, multipotent and pluripotent cell population(s) from otherwise non-pluripotent, non-self renewing cells may have significant positive implications for all fields utilizing cellular therapies. These fields include bone marrow transplantation, transfusion medicine, and gene therapy and enable the production of patient-specific stem cells and other desired cell types. Likewise, the ability to initiate differentiation of cells into neural, muscle, and various other desirable cell populations is and will also be of significant value to medicine and commercial processes involving animals. Accordingly, the present invention provides methods for genetic production and uses of multipotent cell populations, pluripotent cell populations, neuronal cell populations, muscle cell populations, and other desired cell populations such as, for example, HIV resistant cell populations. The invention may be used with any suitable cells, including vertebrate cells, and including fish, mammalian, avian, amphibian, and reptilian cells. A theoretical basis for the embodiments of the invention is described herein, however, this discussion is not in any way to be considered as binding or limiting on the present invention. Those of skill in the art will understand that the various embodiments of the invention may be practiced regardless of the model used to describe the theoretical underpinnings. All patents, patent applications, and publications cited in this application are hereby incorporated by reference herein in their entireties. The present invention relates to equipment and apparatuses suitable for use in electroporation, especially electroporation producing developmental activation in a cell, and especially protein electroporation producing developmental activation in a cell. The current invention teaches electroporation related equipment for electroporating cells ex vivo, in vitro, or in a tissue or organ in vivo. In one part, the present invention teaches a catheter for infusing various agents, e.g. therapeutic or diagnostic agents, into a tissue or an organ, wherein the catheter comprises an injection needle that, in some embodiments, may also serve as an electrode for electroporation, including for protein electroporation. It is a proposition of the present invention that the efficient introduction or overexpression of nucleic acids or proteins corresponding to specific transcription factors and small RNAs, alone or in combination with other cell fate determinants (e.g. notch, numb, numblike, various small RNAs, and various specific aptamers, and other cell fate determinants known to the art), enables the interconversion of what have been considered transitory (multipotent, pluripotent, and/or self-renewing) or fixed (differentiated or somatic) cellular phenotypes. The ability to reliably induce developmental activation, phenotypic conversion, or cellular reprogramming allows the production of stem-like cells, replacement cells, tissues, and organs that match individual patients or subjects. In conjunction with gene therapy techniques and cell culture techniques, cell type interconversion also provides for the production of disease-resistant and genetically-repaired cells that are suitable for transplantation. The current invention teaches that it is the particular complement of transcription factors within an individual cell that determines which cellular programs are active and which are turned off. In this capacity transcription factors play a decisive role in determining and maintaining cellular identity, as well as determining cellular vulnerability. It is a further teaching and proposition of the present invention that the efficient introduction or overexpression of specific transcription factors, alone or in combination with other cell fate determinants, such as regulatory RNAs (e.g. small RNAs) enables the production of transitory (multipotent, pluripotent, and/or self-renewing) or fixed (differentiated or somatic) cellular phenotypes. The ability to reliably developmentally activate, induce or reprogram cells using cell fate determinants allows the production of stem cells, replacement cells, tissues, and organs that match individual patients or subjects. In conjunction with gene therapy techniques and cell culture techniques, cell type interconversion also provides for the production of disease-resistant and genetically-repaired cells that are suitable for transplantation. It is an object of this invention to provide various manners of generating developmentally-activated cells (DAdC)-proliferating, self-renewing, multipotent, pluripotent and/or “pluripotent-like” cell population(s), as well as other desirable cell populations, from either dividing or non-dividing cells without requisite use of oncogenes. Differentiating cell populations (aka differentiating somatic cell populations) comprise cells expressing some, but not all markers associated with specific cell type categorization. It is disclosed herein that appropriate cell fate determinants (protein or nucleic acid) in combination with other transgenes (or their corresponding proteins), especially transcription factors and small RNAs, enables the production of dividing, pluripotent, or pluripotent-like cell populations or differentiating cell populations. Moreover, the methods (including the vectors) of the present invention may be used to produce genetic modification (e.g. expression of gene products deficient in the patient) and to transiently or permanently induce proliferation, self-renewal, or stem/progenitor cell behavior in endogenous cells in vivo, particularly those cells found in tissues which normally do not show or no longer show such behavior. Finally, the methods (and the vectors) of the present invention may be used block proliferation, self-renewal, or stem/progenitor cell behavior in cells aberrantly displaying such behavior (e.g. cancer cells). Likewise, the current invention provides for production, using various, including commercially available means, of all manner of vectors known to the art, such that they comprise and allow expression of one or more members of the transcription factors and other cell fate determinants described herein for activating, inducing or reprogramming a cell to a desired type. It is also an object of the present invention to provide therapeutic vectors and cells capable of expressing beneficial sequences (such as small RNAs or synthetic oligonucleotide sequences, or sequences coding for proteins) predicted to attenuate disease processes. For example, the current invention discloses the use of synthetic oligonucleotides to reduce gene expression critical HIV and other immunodeficiency virus infection, propagation and spread. Equipment and Apparatuses suitable for use in Electroporation The present invention relates in part to equipment and apparatuses suitable for use in electroporation, especially electroporation producing developmental activation in a cell, or that may be used in conjunction with cells activated developmentally according to the methods described herein. An electroporator uses a high-voltage electrical discharge to introduce a transfectant or transfectants into a cell. This method, commonly referred to as electroporation, typically involves suspending selected, target cells in a phosphate-buffered saline (PBS) solution to which, a nucleic acid, protein, dye, or other transfectant is also added, Electroporation can be used for both transient and stable transfection. Most commonly, the cell and transfectant suspension are transferred to an electroporation cuvette. Connected to a power supply, an electrical pulse generator subjects the cells in the cuvette to a high-voltage electrical pulse of defined magnitude and length. The high-voltage pulses cause the various membranes of the cell to lose their normal integrity to take up and/or overexpress the introduced, exogenous transfectant. The electric potential across the cell membrane drives charged molecules across the membrane through the temporary pores induced in the cell membrane, in a manner similar to electrophoresis (Shigekawa and Dower, 1988). Following electroporation, the cells are often allowed to recover briefly before they are placed in normal (non-selecting) cell growth medium. Factors that can be varied to optimize electroporation effectiveness are discussed in introduction to Section I, and protein expression strategies are discussed in Chapter 16 of Curr Protoc Mol Biol. 2003 May; doi:10.1002/0471142727.mb0903s62. The amount of voltage and current required in transfection procedures depends upon the cell type and the nature of the transfectant, A transfection high-voltage controller is taught by U.S. Pat. No. 4,750,100. The commonly employed practice of transferring cells from a first cell culture apparatus to a cuvette and then to another cell culture apparatus is attendant with contamination and infectious risks. The Neon® Transfection System is a second-generation transfection system that uses an electronic pipette as an electroporation chamber, but Neon still requires that the electroporation procedure be carried out within a sterile environment, e.g. a restrictive, cell culture hood, and does not completely eliminate the transfer related contamination and infectious risks. In contrast, the present invention provides a combined electroporation chamber/cell culture apparatus allowing electroporation and cell culture to be accomplished in the same cell culture apparatus-obviating the need for transfer from an electroporation chamber to a separate cell culture apparatus. The novel culturing apparatus (assembly) of the present invention may be termed a cell culture dish. More particularly, the present invention is a “combined cell culture dish” or “dish-in-dish” apparatus comprising at least one smaller cell culture dish fixedly positioned within a larger cell culture dish, and the number of such fixated cell culture dishes can include a multiple number of fixated cell culture compartments within one another, either concentric or eccentric, in any number of geometric shapes, and without limitation to the number of compartments included. An alternate embodiment of this invention can include a plurality of cell culture dishes juxtaposed side-by-side having common interior well walls, and the well walls may or may not be different in height depending on the application. The combined cell culture dish differs from the prior art, in part, because the walls of said combined compartments may be of different heights and made from any combination of transparent and non-transparent materials that will allow juxtaposing cultures to grow simultaneously. In some embodiments, the cell culture compartment communicating with a reservoir suitable for electroporation is a cell culture bag. In some embodiments, the cell culture compartment communicating with a reservoir suitable for electroporation is a bioreactor of variable dimensions and shapes. Accordingly, one skilled in the art will recognize that the cell culture compartment may be of any construction, shape or size, so long as it may be made to communicate with a second compartment of variable size and construction suitable for electroporation. The combined cell culture/electroporation apparatus of the present invention may or may not be fitted with single or multiple covers and may or may not be stacked. A particular embodiment of the arrangement described herein comprises one or more smaller dish or compartment (aka reservoir or reservoirs) located inferiorly, within, or adjacent to a larger cell culture dish with which it can communicate, and wherein said smaller dish (which may have dimensions akin to those of standard electroporation cuvettes) comprises electrodes or electrode plates that enable electroporation. In some embodiments, a wall of low height (aka a lip) will separate the smaller dish/compartment (e.g. the cuvette-like reservoir) from the larger dish/compartment. The low wall or lip and/or funnel demarcates and surrounds the reservoir while also demarcating the space by which these two cell cultures may communicate (e.g. if a sufficient volume of medium is added and the cuvette-like reservoir overflows). Such an arrangement of compartments allows cells to undergo electroporation and incubate in a single cell culture apparatus-obviating the need to transfer cells from a first apparatus (e.g., a first cell culture dish) to a second apparatus (e.g., an electroporation cuvette), as well as obviating the need to transfer the cells from the second electroporation chamber apparatus to a third apparatus (e.g., a second cell culture dish) for further incubation; accordingly, this particular arrangement provides a “closed system” that reduces labor, costs of materials, and infectious/contamination risks. The main compartment and the reservoir may be of various sizes and dimensions. In some embodiments, when the design features a flask-like compartment, the flask like compartment will preferably approximate standard flask dimensions while the reservoir may approximate standard electroporation cuvette sizes. In one embodiment, the flask, plate, dish, bag, etc. cell compartment takes the width of an embedded cuvette-sized reservoir. In one embodiment, the reservoirs are detachable and snap onto or slide into the larger main dish, plate or flask. Likewise, the spatial relationship between the multiple compartments taught herein allows the electroporation procedure to be performed in a non-sterile environment, e.g. outside of the tissue culture hood, at the bench. Currently, the size of a tissue culture hood limits the size of the electroporation apparatus. When performed outside of the tissue culture hood using the cell culture dishes, plates and flasks equipped with one or more electroporation reservoirs, as taught herein, the electroporation apparatus may be of unlimited size and can be used to perform electroporation of multiple (up to hundreds or thousands of) cell cultures simultaneously, thereby enabling higher throughput. Accordingly, a parallel array of electrodes suitable for high throughput, parallel electroporation is also described herein. Such an array may take the form of a slot or slots containing multiple electrode pairs spaced at distances accommodating the dimensions and spacings of the reservoirs, or a block with multiple wells, each well containing one or more electrode pairs and having dimensions accommodating the one or more reservoir portions of the “reservoir-in-dish” or “reservoir in flask”, etc., cell culture dishes, plates, flasks, bags, bioreactors, etc. In general, the cell culture/electroporation apparatus of the present invention comprises two or more compartments which create a central compartment and one or more peripheral compartments which surround the central compartment. Said central and peripheral compartments may take the form of any shape, or any geometrical realtionship including, but not limited to cylindrical, square, pentagonal, or hexagonal. The material used to construct said petri dish may include, but may not be limited to any non media-permeable form of glass, plastic or metal or combination thereof, which will sustain culture growth and permit observation and recording of said culture growth, differentiation and/or signal transduction. Separated areas created by utilizing the central compartment and one or more peripheral compartments may be geometrically concentric or eccentric. The cell culture/electroporation apparatus of the present invention may comprise one or more compartments within a compartment or may be constructed of a single compartment with a flat well bottom having one or more sets of walls that extend from said well bottom forming one or more separate enclosures having the same geometric shape or a variety of geometric shapes. In some embodiments, the walls of the combined cell culture/electroporation apparatus are arranged in a manner that allows communication of cells and/or media between and amongst the separate compartments when a sufficient volume of medium is present. For example, one compartment may be filled with cells and/or medium to a certain height, wherein the medium and/or cells remain restricted, confined or localized to a first compartment, and wherein further addition of cells and/or medium allows the contents of the first compartment to ascend above or spill over walls or lips demarcating one compartment from a second compartment, or to spread into a second compartment communicating with the first. Definitions As discussed herein, “DNA” refers to deoxyribonucleic acid and “RNA” refers to ribonucleic acid. As discussed herein, “cDNA” refers to complementary DNA; “mRNA” refers to messenger RNA; “siRNA” refers to small interfering RNA; “shRNA” refers to small hairpin RNA; “miRNA” refers to microRNA, such as single-stranded RNA molecules, typically about 20-30 nucleotides in length, which may regulate gene expression; “decoy” and “decoy RNA” and “RNA decoy” refer to an RNA molecule that mimics the natural binding domain for a ligand. As used herein, the meaning of the term “ameliorating” includes lessening an effect, or reducing damage, or minimizing the effect or impact of an action, activity, or function, and includes, for example, lessening the deleterious effects of a disease or condition. As used herein, the meaning of the term “retarding” includes slowing or lessening the progress of an effect or action, and includes, e.g., slowing the progress of disease, slowing the rate of infection, or otherwise slowing or reducing the advance or progress of a disease or condition. As used herein, an “inducing agent” is an agent that aids or is alone effective to promote an action. For example, an exogenous agent that affects a promoter, e.g., by initiating or enhancing its activity, and so affects expression of a gene under control of the promoter, may be termed an inducing agent. For example, tetracycline may be used as an inducing agent; and doxycycline may be used as an inducing agent. A nucleic acid sequence (e.g., a nucleic acid seqeuence encoding a polypeptide) is termed “operably linked” to another nucleic acid sequence (e.g., a promoter) when the first nucleic acid sequence is placed in a functional relationship with the second nuceleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. As used herein, the term “driven by” refers to a gene or coding sequence that is operably linked to a promoter sequence, and that the promoter sequence affects the transcription or expression of the coding sequence. As used herein, a “marker” is a molecule that is detectable, or codes for a detectable molecule, or acts on other molecules so that the presence of the marker is detectable. A “marker protein” or “marker polypeptide” is a protein or polypeptide that is detectable in a laboratory or clinical environment, and, in embodiments, may be detectable by eye. A “marker gene” encodes a marker protein or marker polypeptide. As used herein, “HIV” refers to human immunodeficiency virus, and includes variants such as, e.g., HIV-1, HIV-2. Other immunodeficiency viruses include simian immunodeficiency virus (SIV) and feline immunodeficiency virus (Hy). Enzymes related to HIV may be termed “HIV enzymes” and include, for example, integrase, protease, reverse transcriptase, and transactivating regulatory protein (TAT). Infection by HIV is believed to involve receptors termed “HIV receptors.” There may be multiple such receptors, some of which may be termed “HIV co-receptors.” As discussed herein, HIV co-receptors include CXCR4 and CCR5. Developmentally-active cells (DAC), as defined herein, represent a broad category of cells that are either i. “transitory-type cells” typically showing high potency, such as embryonic stem (ES) cells, very small embryonic-like (VSEL) cells, pluripotent stem cells, multipotent stem/progenitor cells), or ii. “fixed-type” cell types, which typically have low or limited potency, (e.g. differentiating somatic cell types capable of further differentiation and/or integration in vivo—usually over the course of an organism's development). While “transitory” DAC tend, in nature, to divide and/or show self-renewal, “fixed” DAC are more often post-mitotic and usually do not self-renew. Instead, fixed DAC typically show the emergence of some characteristics associated with a terminally-differentiated cell of its type. Nevertheless, both “transitory” and “fixed” cells have uses and potential uses in medicine (especially regenerative medicine, transplantation medicine, veterinary medicine, animal husbandry, drug-discovery, drug-testing, gene therapy, tissue-engineering, organ production, and biological modeling), laboratory-based food production, and various other industries. As a category, developmentally-active cells (DAC) include cells that inherently show features of developmentally active cells, as well as cells that have been “activated”, aka “made”, “forced”, “induced”, or “reprogrammed” to acquire such characteristics; this latter subset of developmentally active cells herein defined as, “developmentally-activated cells (DAdC)”. Writing with respect to with respect to B cells and T cells of the immune system, Kolanus et al., (1992) observed that a “developmentally activated cell state” results from “a change in transcriptional potential”. Likewise, as used herein, “developmentally-activated cells” (DAdC) include cells which some skilled in the art term, “reprogrammed cells”, “partially-reprogrammed cells”, “induced pluripotent cells”, “directly reprogrammed cells”, “indirectly-reprogrammed cells”, etc., e.g. induced pluripotent cells, induced multipotent cells, induced hematopoietic stem/progenitor cells, induced neurons, induced cardiac cells, induced skeletal muscle cells, induced cartilage cells, induced hematopoietic cells, induced liver cells, induced pancreatic beta cells, etc.)—as such cells show a change in transcriptional potential relative to untreated cells. As used herein, terms of art, such as “pluripotent”, “multipotent”, “self-renewing”, “differentiating”, “cardiac”, “muscle”, “neuron”, “progenitor”, “stem cell”, “osteoblast”, “chondrocyte”, etc. are understood to refer either to i. cells produced in nature (natural cells) of a type, or to ii. like cells produced through the methods described herein-cells displaying some, but not necessarily all, features marking the natural cells denoted by these terms. For example, as used herein, a “pluripotent cell” is one capable of forming embryoid in vitro and that expresses some markers of pluripotency, however it need not be capable of forming teratomas in vivo; accordingly, as used herein, “pluripotent cells” need not meet all criteria for pluripotency commonly applied by some skilled in the art. Conversely, some cells recognized as “pluripotent” by those skilled in the art are considered herein to represent inherently, developmentally-active cell types, e.g. embryonic stem cells (ES) cells, Very Small Embryonic-Like (VSEL) stem cells, mouse embryonic stem cells (mES), etc. In regard to such cells, Kim et al. (2014), write,“Pluripotent stem cells (PSCs) have been considered as the most important cells in regenerative medicine as they are able to differentiate into all types of cells in the human body. PSCs have been established from several sources of embryo tissue or by reprogramming of terminally differentiated adult tissue by transduction of so-called Yamanaka factors (Oct4, Sox2, Klf4, and cMyc). Interestingly, accumuating evidence has demonstrated the residence of PSCs in adult tissue and with the ability to differentiate into multiple types of tissue-committed stem cells (TCSCs). We also recently demonstrated that a population of pluripotent Oct4(+), SSEA-1(+), Sca-1(+), Lin(−), CD45(−) very small embryonic-like stem cells (VSELs) resides in the adult murine bone marrow (BM) and in other murine tissue. These very small (˜3-6 μm) cells express pluripotent markers such as Oct4, Nanog, and SSEA-1. VSELs could be specified into several tissue-residing TCSCs in response to tissue/organ injury, and thus suggesting that these cells have a physiological role in the rejuvenation of a pool of TCSCs under steady-state conditions. In this review article, we discuss the molecular nature of the rare population of VSELs which have a crucial role in regulating the pluripotency, proliferation, differentiation, and aging of these cells (Kim Y, Jeong J, Kang H, Lim J, Heo J, Ratajczak J, Ratajczak M Z, Shin D M. The molecular nature of very small embryonic-like stem cells in adult tissues. Int J Stem Cells. 2014 November; 7(2):55-62). It follows that the pluripotent, VSEL cells and tissue-committed stem cells TCSC's of Kim et al., represent cell types included in the category of developmentally active cells (DAC). Pluripotent cells are most commonly defined by their ability to produce cell types reflecting all three embryonic germ layers of a developing gastrula (ectoderm, endoderm and mesoderm). This ability to form all three germ layers relates both to teratoma formation in vivo and embryoid formation in vitro. As Lin and Chen (2014) write, “Embryoid bodies (EB) are the three-dimensional aggregates formed in suspensionby pluripotent stem cells (PSC), including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC).” Pettinato et al. (2015) further describe embryoid bodies as,“three-dimensional (3D) hPSC [human pluripotent stem cell] aggregates that can differentiate into cells of all three germ layers (endoderm, ectoderm, and mesoderm) [3]. Many events in the in vitro lineage-specific differentiation process within the EBs recapitulate those seen in vivo in the developing embryo [6], which justifies the uses of EBs as a model system to simuate the in vivo differentiation of hPSCs under in vitro culture conditions, and mechanistically examine hPSC differentiation programs/lineage commitment during embryogenesis as an alternative to the whole embryo approach [7]. In addition, in vitro formed EBs have opened access to early precursor cell populations that are not accessible in vivo [8]. EBs have been shown to effectively initiate lineage-specific differentiation of hPSCs toward many lineages, such as cardiac [9], neural [10,11], hematopoietic [12], and pancreatic β cells [13].” In part, the current invention is directed to the production of “transitory type” developmentally-activated cells (DAdC) that display many features associated with pluripotent, mutiptipotent, and/or self-renewing cells, e.g. the capacity to form embryoid (aka embryoid bodies) when cultured in vitro. Embryoid formation is a behavior those skilled in the art often associate exclusively with pluripotent cells. As used herein, embryoid formation by cells “developmentally-activated” according to methods described herein indicates that these newly, developmentally-activated cells (DAdC) are at least “pluripotent-like” and “VSEL-like”, if not fully pluripotent. Thus, the current invention is directed, in part, to the production of cells considered, herein, to be “pluripotent-like”, “VSEL-like”, “mutiptipotent-like”, and/or “self renewing-like” cells. In part, then, the current invention is directed, in part, to the production of cells with increased potency. Teratomas contain cells from the three germ layers: ectoderm, mesoderm, and endoderm. Teratoma formation is, however, also associated with carcinogenicity. Some skilled in the art may consider that a cell cannot be termed a “pluripotent” cell unless it forms a teratoma when injected in vivo, even if this tendency to tumorigenesis is considered highly undesirable in cells intended for clinical use, especially in regenerative medicine, The developmentally-activated cells produced according to the methods taught herein need not form teratomas in order to meet the criteria of “transitory-type, developmentally activated cells (DAdC)”. In contrast, the transitory DAdC produced according to the methods described herein, show many other desirable characteristics associated with pluripotency, such as small size, expression of pluripotent markers such as SSEA3/SSEA4, Oct4, Nanog, Sox2, as well as colony formation and embryoid formation, and therefore such transitory developmentally activated cells (DAdC) are termed “pluripotent” and/or “pluripotent-like”, herein. Similarly, the current invention is directed in part, to the production of “differentiating cells” (aka differentiating somatic cells, aka somatic differentiating cells) that display some, but not necessarily all, cell type specific markers associated with the desired cell type. Those skilled in the art may differ somewhat as to the criteria for defining a pluripotent cell as well as differentiating and/or differentiated cell types. As there exists no universally accepted terminology for cells induced or activated to differentiating somatic cell types, the applicants refer to various desired, differentiating cell types producible by the methods described herein as cardiac cells, neurons, chondrocytes, cartilage cells, bone cells, hepatocytes, etc.; however these terms, when used herein also refer to “cardiac-like cells”, “neuron-like cells”, “chondrocyte-like cells”, “cartilage-like cells”, “bone-like cells”, “hepatocyte-like cells”, etc. Thus, the criteria for defining a desired cell type or cell potency taught herein may not coincide precisely or overlap entirely with the various criteria taught by others skilled in the art for defining a cell type or a cell potency, and the invention is therefore not bound by such various definitions. Instead, the current invention is aimed at producing cells displaying certain desirable features, characteristics and behaviors which commend them for the various and specific uses taught herein. The induced pluripotent stem cells of Takahashi and Yamanaka, which were generated, in part, using oncogenes c-myc and klf4, were able to form teratomas when injected in vivo. Likewise, when Takahashi and Yamanaka produced embryos from these induced pluripotent stem cells, a large percentage of the resulting animals developed tumors postnatally (Takahashi and Yamanaka, 2006; Takahashi et al., 2007). Thus, teratoma formation and carcinogenicity are features associated with some iPSCs, but are features that some skilled in the art may consider undesirable with respect to providing sources of replacement cells, cells for transplantation, cells for gene therapy, cells for tissue engineering, and cells for various other clinical uses. In contrast, the transitory, pluripotent and pluripotent-like, developmentally-activated cells (DAdC) produced according to the methods taught herein 1. display colony formation, 2. form embryoid, 3. Express various genes and proteins associated with pluripotency, 4. cluster with ES and iPS cells in hierarchical cluster analysis based on their gene expression (e.g. global gene expression profile), 5. cluster with ES and iPS cells in Principal Component Analysis (PCA) plots based on their gene expression (e.g. global gene expression profile), but 6. may fail to demonstrate the teratoma formation and the carcinogenicity frequently associated with ES cell lines and induced pluripotent stem cells. Developmentally activated cells (DAdC), may be recognized and defined, for example, by i. gene expression analysis (e.g. by reactome overrepresentation analysis using hypergeometric distribution) that reveals overrepresentation (or enrichment) of genes associated with certain cellular pathways, especially the Cell Cycle pathways and the Developmental Biology pathways, especially the Transcriptional regulation of pluripotency sub pathway, the Axon guidance sub pathway; the Myogenesis sub pathway, the Signaling by Nodal sub pathway; the Gastrulation sub pathway; the Activation of Hox genes sub pathway, the Beta cell development sub pathway, and the Transcriptional regulation of white adipocyte differentiation sub pathway (for “transitory” type developmentally activated cells), whereas the Developmental Biology pathways, Gene Expression (Transcription) pathways, and Signal Transduction pathways are overrepresented or enriched in Reactome analyses for “fixed” type, developmentally-activated cells (DAdC). Such enriched gene expression (as demonstrated by Reactome analysis) is consistent with the adaptability of DAdCs as a source of stem-like cells, replacement cells, cells suitable for gene therapy and tissue engineering, and cells differentiable to various types (according to the methods disclosed herein, for example, mutiptipotent and pluripotent cells. The developmentally-activated cells (DAdC) of the present invention also typically display ii. microscopically-visible, induced changes in cell behavior related to colony formation, embryoid formation and cell size. Likewise, iii. immunohistochemistry may reveal the “transitory” developmentally activated cells as expressing multiple markers commonly associated with pluripotency, such as such as Oct4, Nanog, c-Myc, Notch, SSEA3/4; and TRA-1-81; while “fixed” type developmentally-activated cells will show some markers of differentiating or terminally differentiated cells. As described herein, DAdC can be produced by various methods described in the parent application including the use of nucleic acids, e.g. DNA, RNA, and protein; use of miRNA aptamers, and chemical methods are also compatible with the invention. As described in the parent application, various means of detecting, monitoring, and demonstrating induced cellular phenotype include gene reporter assays (e.g. performed with a reporter construct) wherein a reporter gene's expression (e.g. an antibiotic resistance gene or fluorescent reporter gene) is linked to the promoter of a gene upregulated in DACs (e.g. c-Myc, Nanog, Oct4, DCX, etc.), as well as other assays described herein. However, as the term is used herein, Developmentally-activated cells or DAdC need not satisfy all criteria of pluripotency to be recognized as DAdC in the current invention, and therefore may not be pluripotent in the opinions of some skilled in the art, even if they do display desirable characteristics such as very small size, embryoid formation in vitro, and pluripotency marker expression, and other features that some skilled in the art consider tantamount to pluripotency; therefore to the extent they are similar to and display features overlapping with cells which have been commonly or unambiguously recognized as pluripotent, some DAdC may be considered herein and by some skilled in the art to be “pluripotent-like”, “ES-like” or even “VSEL-like”. Accordingly, and to avoid confusion, the term, developmentally-activated cell (DAdC) is applied herein and is defined by many features these cells display morphologically, microscopically, and immuno-histologically, as well as according to their pattern of gene expression (transcriptome), their reactome, their utility, and many of their potential uses. It should be understood that a developmentally-activated cell (DAdC) may one caused to display some features consistent with either “transitory” (pluripotent-like, multipotent-like, and/or self renewing cells) or “fixed” (somatic-like, differentiated-like or differentiating-like) cellular phenotypes, as described herein, and that the present invention enables the production and interconversion of these cells by efficient introduction or overexpression of nucleic acids or proteins corresponding to specific transcription factors, cell fate determinants, small RNAs, and/or aptamers, or by chemical and/or physical means, either in vivo or in vitro, in the presence or absence of specialized cell culture conditions. By the same token, while terms for somatic, differentiated cells such as “cardiac cell”, “neuron”, “liver”, “chondrocyte”, “osteoblast”, “T cell”, “beta cell”, etc. appear herein as desirable cells producible by the methods described herein, such terms are used, herein, to describe “developmentally-activated cells” (DAdC) that are similar to corresponding cells (expressing certain cell type specific markers), i.e. cardiac-like cells, neuron-like cells, liver-like cells, chondrocyte-like cells, osteoblast-like cells, T-like cells, beta-like cells, etc. Accordingly, developmentally-activated cells (DAdC) which are of the “fixed type” i. display some but usually not all markers of the desired, cell type (as assessed by Reactome, transcriptome, gene expression and/or protein expression assays), ii. are capable of survival, further differentiation and/or integration in vivo, and iii. need not meet all criteria that those skilled in the art may sometimes apply to the mature phenotype that the DAdC approximate. Examples of published studies affirming the utility of transcription factors taught in the parent application with respect to the production of cells showing markers consistent with specific somatic phenotypes include Zhou et al. (2008); Ieda et al. (2010); Szabo et al. (2010); Vierbuchen et al. (2010); Addis et al., (2011); Huang et al., (2011); Kim et al., (2011); Pfisterer et al., (2011); Caiazzo et al. (2011); Liu et al. (2012); Outani et al., (2013); Najm et al., (2013); Mong et al., (2014); Yamamoto, et al., (2015); Xu et al., (2016); Sun et al., (2016); Vadodaria et al., (2016); Ji et al., (2016); Lee et al., (2017); Duran et al., (2018); Hirai et al., (2018); Kogut et al., (2018); McGrath et al., (2018); Stone et al., (2019); Huang et al., (2019); Lin et al., (2019); Sadahiro et al. (2019); Pereira et al., (2019a); and Kandasamy et al. (2019); it follows that the transcription factors and other cell fate determinants taught by these publications are practicable in and covered by the present invention. See also, Wazan et al. (2019), Aydin and Mazzoni (2019), and Pereira et al., (2019b), etc., for reviews. It is taught herein that any protein, nucleic acid, or other factor known to those skilled in the art as capable of successfully activating, inducing or reprogramming a cell, either directly or indirectly (Srivastava and DeWitt, 2016; Seo et al., 2017; Fan et al., 2018; Kogut et al., 2018; McGrath et al., 2018; and Aydin and Mazzoni; 2019) may be applied by electroporation (in vivo or in vitro) to achieve superior reprogramming characterized by greater speed, greater efficiency and/or greater safety than demonstrated with previously taught methods. See www.harvardapparatus.com. It should be understood that “transitory” type DAdC can also serve as selected cells and be converted to “fixed” DAdC according to the methods described herein, as well as according to methods published elsewhere and known to the art for converting pluripotent, multipotent, or pluripotent-like cells to various differentiated cell types. As was the case in the parent application, while some portions of the text herein refer either only to “introduction” or only to “overexpression”, it is to be understood that causing a cell to overexpress a gene has, in the context of the present invention, the same effect as introducing said gene or corresponding RNA or corresponding protein into said cell; and accordingly, the associated methods for introducing or overexpressing are used interchangeably herein. A number of small RNAs are suitable and compatible with use in the invention and include small RNAs useful for achieving proliferating, self renewing, pluripotent, and/or pluripotent-like cells; these small RNAs include one or more selected from the miR-302/367 cluster small RNAs (miR-302a, miR-302b, miR-302c, miR-302d, miR-367), human miR-371-373 cluster small RNAs (miR-371, miR-372, miR-373), miR-17-92, C19MC cluster members, miR-133b, miR 200a, miR 23a, and miR 743b-5p, miR-187, miR-299-3p, miR-499-5p, miR-628-5p, miR-888, let-7 (let-7-b,e,f,g), miR-30 (miR-30-a-e), the mouse miR-290-295 cluster small RNAs (miR-290, miR-291a-3p, miR-291b, miR-292, miR-294, miR-295, miR-29, miR-296, miR-106a cluster, miR-93 and other pluripotency associated small RNAs known to the art, as such small RNAs can be used in conjunction with other cell fate determinants taught herein or alone. Use of RNA and proteins, which do not integrate into the host's genome, may be considered as safer approach to developmental activation/pluripotency induction/cell reprogramming, as compared to other methods that pose the risk of genomic integration. Such vectors are considered to be non-integrating and/or episomal vectors. Accordingly, use of chemicals, compounds, extracts and drugs that induce expression of said small RNAs and other cell fate determinants is likewise suitable and compatible with the present invention-especially those chemicals, compounds, extracts and drugs taught in the priority documents associated with the present invention. Studies Relevant to Protein Transfectants and Distinguishing the Electroporation Method Although some studies (Kim et al. 2009; Zhou et al. 2009) have reported that pluripotent cells could not be produced using a single application of cell penetrating proteins corresponding to pluripotency inducing factors, the current invention, in part, teaches the one-time application of native or recombinant protein transcription factors and/or protein cell fate determinants for the production of developmentally activated cells, including pluripotent-like cells, pluripotent cells, and/or self-renewing cells, as well as differentiating cells that express one or more cell type specific markers consistent with a desired cell type. Based on the studies of Kim et al. 2009 and Zhou et al. 2009, many have concluded that cell reprogramming, direct reprogramming, pluripotency induction, or developmental activation cannot be achieved using proteins. They surmise or conclude further that proteins introduced to cells for that purpose are too quickly degraded within the cells, consistent with their short half-lives in cells under normal conditions. For example, Seo et al. (2017) write, “Cell-penetrating peptide-based reprogramming might be a safe way to induce reprogramming; however, its low efficiency compared with other methods is a significant concern. The main problem is the poor stability of the recombinant proteins and following endocytic uptake”. Dey et al., (2017) make similar observations to Seo with regard to the deficiencies associated with CPP-mediated protein delivery, stating,“Presence of CPPs in reprogramming proteins is known to interfere with proper folding inside the cells and thereby decreasing the biological activity . . . endosomal entrapment is also a common barrier and is a major challenge in efficient delivery of CPP linked molecular cargo . . . . In a cell reprogramming paradigm to generate iPS cells via CPP-mediated recombinant protein transduction, reports also show that misfolded CPP-fused recombinant reprogramming proteins after endosomal release gets localized to cytoplasm and/or have a peri-nuclear region as observed in immunostained images [11,35,44,45,47-52]. Due to this, they are unable to enter the nucleus to activate downstream target genes. Nevertheless, a small amount of the biologically active recombinant transcription factors enters the nucleus and binds to its target genes to activate the cell reprogramming machinery.” On the other hand, Bekei (2013), having carefully compared the CPP-mediated protein delivery, EP-mediated protein delivery and SLO-mediated protein delivery methods), and concluded that Electroporation-mediated protein delivery is distinct from and superior to the CPP-mediated protein delivery employed by Kim et al., (2009) and Zhou et al (2009), as well as to the SLO-mediated protein delivery utilized by Taranger et al., (2005). Bekei teaches that, “this [electroporation] method provides two main advantages over CPP- and SLO-mediated protein delivery. First, it does not require any forms of chemical modifications to proteins that are to be delivered and, second, it works without having to treat cells with potentially harmful toxins that generally lower cell viability.” Bekei, also notes that,“CPP-mediated protein delivery approaches have to overcome significant obstacles. First, the uptake efficiencies of CPP-cargo constructs are highly dependent on the choice of the CPP sequence and on the combination of CPP/cargo proteins. Membrane compositions of targeted host cells additionally affect the individual uptake efficiencies and these properties need to be considered when devising a CPP-mediated delivery experiment. Second, even if optimal combinations of CPP/cargo sequences have been found for a particular cell line that is to be targeted, efficient release from endocytotic vesicles has to be achieved.” Consequently, in the words of Bekei, “Results demonstrate that low transduction efficiencies, high cell line dependences and vesicular-like intracellular distributions strongly limited the suitability of CPP-mediated protein delivery attempts.” In contrast, Bekei reports superior results from electroporation (EP)-mediated protein delivery,“This [protein electroporation (EP)] method proved to be superior to CPP-, and toxin-mediated delivery protocols, as outlined in the first half of the thesis. Transduction efficiencies, cell viabilities and intracellular distributions of two model proteins, human alpha Synuclein (Syn) and the B1 domain of Protein G (GB1) were comparatively analyzed in different mammalian cell types and found to be generally higher using the EP-mediated delivery approach.” Most notably, Bekei makes the critical observation that,“Although SLO- and EP-procedures yielded comparable transduction efficiencies at low applied protein concentrations, EP clearly outperformed the SLO approach at higher protein concentrations, because it enabled the linear delivery of increasing concentrations of exogenous proteins, with high correlations in intracellular cellular protein levels.” Thus, the work of Bekei (2013) makes it clear that the results of CPP-mediated protein delivery cannot be extrapolated or generally applied to other distinct, protein delivery methods, and in particular, should not be generalized to electroporation-mediated protein delivery. Bekei further contrasts Cell Penetrating Peptides (CPP) with electroporation-mediated delivery and SLO-mediated Permeabilization, stating,“If one were to design an ideal method for intracellular sample delivery into mammalian cells, what needed this method to be able to do? First, it ought to be generally applicable to many different cell lines and proteins, and therefore, the uptake mechanism should preferably not require specific cell-surface receptor interactions of the protein that is to be delivered into these cells. Second, the method should be suitable to transduce ‘native’ proteins, without requirements for engineered protein tags, targeting sequences, or other chemical extensions that are necessary for cellular protein uptake. Such extensions and modifications will ultimately distort the structural and functional features of the protein. Third, the method should not require any treatment of cells with toxic compounds, which decrease cell viability and signal the activation of damage response pathways. I therefore investigated the suitability of yet a third delivery approach: protein electroporation (EP). EP fulfills many of the requirements stated above and also represents a simple and fast method that can successfully be performed by inexperienced users.” The work and writings of Bekei (2013), Seo et al. (2017) and Dey et al. (2017) are consonant and show that those skilled in the art understand that the low reprogramming efficiency reported by Kim et al. (2009) and Zhou et al. (2009) was a function of their employing the cell penetrating peptide (CPP)-protein delivery method, and that the resulting low efficiency of cell reprogramming in their studies is not necessarily generalizable to electroporation-mediated protein delivery and other protein delivery methods which are not constrained by the technical limitations of CPP, as elucidated by Seo (2017), Dey (2017) and Bekei (2013). In contrast to the experience of Kim et al., (2009) and Zhou et al., (2009), the current invention teaches that specific proteins may be delivered to the interior of cells in amounts such that the protein(s) persist in the cells long enough to cause them to become developmentally activated, reprogrammed, induced to pluripotency, directly reprogrammed, etc. This is likely achieved, in part, through saturation of protein-degradative pathways by excess transfected factors—extending protein half-lives and the time the introduced proteins persist and have access to their binding sites and interaction partners inside the treated cells. The present invention teaches that half-life of RNA may likewise be extended by means of electroporation, wherein larger amounts of RNA are introduced into a cell saturating its RNA degradation pathways, allowing RNA species to persist longer within the cell. The present invention further teaches that transfectant half lives may, likewise, be extended by other transfectant delivery methods taught herein wherein such methods are utilized to introduce saturating amounts of the transfectant into a cell. The current invention covers electroporation, and other methods known to the art, capable of delivering the desired trasfectants to the interiors of cells in amounts 1. sufficient to promote persistence of the proteins in the cells, thereby producing the desired effect, and 2. insufficient to kill the cells. Numerous such methods are known to the art and are easily adapted by (e.g. by increasing protein or nucleic acid transfectant concentration); many such methods are taught herein; they include, for example, liposomal transfection methods, fusogenic or non-fusogenic liposomes, lipofectamine, cationic lipids (e.g. Thermo Scientific Pierce Protein Transfection Reagent (formerly Pro-Ject), and use of nanocapsules or nanovaults, Previously, others have induced 293T cells to pluripotency by 1. permeabilizing the cells with streptolysin O (SLO), then 2. applying a protein extract derived from ESC or undifferentiated, human NCCIT teratocarcinoma cells (for 1 hour). Finally, 3. the pores produced by SLO are sealed by incubation (for 2 hours) in 2 mM CaCl2(Taranget et al., 2005). Critically, however, ˜100,000 pluripotent ESCs or NCCIT cells was required to produce just luL of protein extract. The present invention represents an improvement over the methods of Taranger et al., in that it 1. employs much more rapid, less labor-intensive processes and 2. does not require the availability and destruction of already pluripotent cells to induce this characteristic in others. Moreover, in embodiments involving cell permeabilization and/or cell penetration, the present invention teaches methods that are, in some instance, near instantaneous (e.g. electroporation). Likewise, none of the methods taught by the present invention require 2 hours of pore re-sealing. Indeed, when permeabilization is desirable, the permeabilization methods taught herein are faster, less tedious, and may be employed with recombinant and/or defined proteins, nucleic acids, small molecules, and/or physical means. Accordingly, in some embodiments, the proteins introduced or overexpressed in selected cells consist of recombinant proteins or nucleic acids, rather than natural protein extracts. Likewise, in some embodiments wherein a permeabilizer akin to SLO is used to permeabilize cells for introduction of protein, the proteins introduced following permeabilization consist of recombinant proteins. In some embodiments wherein a permeabilizer akin to SLO is used to permeabilize cells for introduction of protein, the proteins are not derived from cell extracts. In some embodiments wherein proteins are introduced into cells to produce developmental activation/cell reprogramming or to induce pluripotency, the proteins introduced will not comprise more than two of Oct4, Sox2, and Nanog. In some embodiments of the present invention, when proteins are introduced into cells, the proteins do not comprise a complete cellular protein extract. In some embodiments, when proteins are introduced into cells, the proteins do not comprise a complete cellular extract derived from a cancer cell or embryonic stem cell. In some preferred embodiments, when large throughput is desired, the method of electroporation is large volume flow electroporation (Li et al., 2002; Craiu et al., 2008; Parham et al., 1998; Wang et al., 2009; Wang et al., 2010; Li et al., 2013; Wei et al., 2011); Kamigaki et al., 2013; and Steger et al., 2015). The invention further covers the use of cell penetrating peptides in conjunction with electroporation or another delivery method that increases the efficiency with which the peptides enter the cell. The present invention covers the combination of various delivery methods, such as electroporation in conjunction with liposomal protein, nucleic acid or other molecule delivery; electroporation in combination with cell penetrating peptides or other recombinant proteins; electroporation in combination with viral transduction; electroporation in combination with nanoparticle, nanotube, nanocapsule or nanovault delivery; electroporation in combination with cationic lipids; electroporation in combination with non-integrating viral vectors (e.g. integrase deficient, episomal, lentiviral vectors); cationic lipids in combination with nanoparticle, nanotube, nanocapsule or nanovault delivery; cationic lipids in combination with cell penetrating peptides, etc. The invention further covers the use of other methods and reagents such as those described in U.S. Pat. No. 6,841,535 for the delivery of the protein(s) and other molecules taught herein. We have successfully induced millions of cells, at high efficiencies, to change morphology, form colonies, form embryoid, express markers of pluripotency, and display reactomes consistent with developmental activation using a single application of electroporation (see Koken et al., 1994) and other methods described herein. However, the invention in no ways precludes repeated application of proteins, nucleic acids, small RNAs or other cell fate determinants taught herein, using electroporation or sonoporation, or other methods described herein for introduction of nucleic acids or proteins. In some preferred embodiments, in order to produce developmentally-activated cells (DAdC), nucleic acids or proteins corresponding to transcription factors, small RNAs and/or other cell fate determinants, are electroporated into selected cells using voltages ranging from 100V to 500V (preferentially ˜300V) and pulses ranging from 10 to 300 pulses (preferentially 50-100 pulses), and preferably a pulse length of 5 ms with 100 ms pulse intervals. See also Koken, et al., 1994; Deora et al., 2007; Shi et al., 2018). In one preferred embodiment, in order to produce developmentally-activated cells (DAdC), protein transcription factors and small RNAs and/or other cell fate determinants are electroporated into selected cells using pulse length of ˜5 ms and pulse intervals of ˜100 ms. However, any electroporation protocol known to the art and suitable for efficiently introducing into selected cells, nucleic acids or proteins corresponding to transcription factors, small RNAs and/or other cell fate determinants, is practicable in the invention. In one embodiment, developmentally activated cells are produced by electroporation with one or more transfectant selected from DNA, RNA, protein, small molecule, chemical, compound, extract, and/or oil. In one embodiment, developmentally activated cells are produced by electroporation of one or more transfectant selected from DNA, RNA and/or protein corresponding to one or more transcriptions factors and/or cell fate determinants. In one embodiment, developmentally activated cells are produced by electroporation of one or more DNA transfectant, whether plasmid DNA, vector DNA, an aptamer, a synthetic oligonucleotide, or other source of DNA encoding or inducing or promoting expression of a transcription factor and/or other cell fate determinant. In one embodiment, developmentally activated cells are produced by electroporation of one or more RNA transfectant, whether naked RNA, an RNA virus, small RNA, miRNA, a synthetic oligonucleotide, an aptamer or other source of RNA translatable to or inducing or allowing expression of a transcription factor and/or other cell fate determinant. In one embodiment, developmentally activated cells are produced by electroporation with one or more protein transfectant, whether a peptide, full length protein, partial protein, natural protein, native protein, synthetic protein, recombinant protein, or other source of protein acting as a transcription factor or other cell fate determinant; or inducing or allowing the expression of a transcription factor and/or other cell fate determinant. In one embodiment, developmentally activated cells are produced by electroporation, albeit at lower efficiencies, in the absence of a DNA, RNA or protein transfectant. In one embodiment, the one or more transfectants is derived from a subject's or a patient's own cells, tissues, fluids or body. In one embodiment, developmentally activated cells (DAdC) are produced using sonoporation (see Delalande et al. 2015; Wang et al., 2018), gene gun (see Sanford, 1993; O'Brie, 2001; O'Brien and Lummis, 2007), or laser based transfection (see Yao et al., 2008; Kim and Eberwine, 2010; Pylaev et al., 2018), or by these and other transfection methods (see Kim and Eberwine, 2010; Parent 20192019) or their combination. In one embodiment, developmentally activated cells (DAdC) are produced by forcing the cells through filters of progressively reduced size ultimately forcing the cells through the smallest, ˜5 um filter. In one embodiment, developmentally activated cells (DAdC) are produced by incubating the cells in plant extracts diluted 1:1000 to 1:5000 (See US20140271923; WO2017161387A1; US2019224193A1; U.S. 62/918,459; and U.S. 62/918,462). In one embodiment, developmentally activated cells (DAdC) are produced by forcing the cells through filters of progressively reduced size eventually forcing the cells through Sum filter. In one embodiment, developmentally activated cells (DAdC) are produced by incubating the cells in plant extracts diluted 1:1000 to 1:5000 (See US20140271923; WO2017161387A1; US2019224193A1; U.S. 62/918,459; and U.S. 62/918,462). In one embodiment, developmentally activated cells are produced by electroporation, albeit at lower efficiencies, in the absence of any transfectant other than the salts and other components of the buffer (e.g. of phosphate buffered saline). It is the proposition of this invention that electroporation allows safer and/or faster production of developmentally activated cells, is compatible with DNA, RNA and Protein transfectants, and allows the avoidance, when desired, of integrating viruses, and/or reliance on oncogenes. It is the proposition of this invention that electroporation allows safer and/or faster production of developmentally activated cells, is compatible with DNA, RNA and protein transfectants, and that multiple rounds of electroporation are not required to achieve the desired effect, although the invention anticipates and contemplates that some practitioners of the invention may, for example, decide to apply reprogramming factors or agents via mutipltiple rounds of electroporation. It is the proposition of this invention that electroporation allows safer, more efficient and/or rapid production of developmentally-activated cells using protein and/or other transfectants without repeated application of reprogramming factors and without a requirement for special cell culture conditions; however the invention anticipates, contemplates and covers the use of special cell culture conditions that may further facilitate or enable the developmentally activated cells to acquire the desired cell phenotypes. As taught herein, reprogrammed cells represent an example of developmentally-activated cells. Likewise induced pluripotent, induced mutipotent, induced self-renewing and/or induced somatic cell types (aka differentiating cells), as described herein, further represent examples of developmentally-activated cells. It is taught herein that any protein or RNA or DNA known to those skilled in the art as capable of successfully reprogramming a cell, either directly or indirectly reprogramming said cell, may be applied by electroporation (in vivo or in vitro) to achieve reprogramming with greater speed, greater efficiency and greater safety (Srivastava and DeWitt, 2016; Seo et al., 2017; Fan et al., 2018; Kogut et al., 2018; McGrath et al., 2018; and Aydin and Mazzoni; 2019). In one embodiment, greater safety is realized when electroporation is utilized to produced the desired cell types, thereby enabling integrating viral vectors and/or oncogenes to be avoided. In one embodiment, greater efficiency is realized when electroporation is utilized, as electroporation enables varying amounts of the transfectant to be delivered to the interior of the selected target cells in a controlled and linear fashion. In one embodiment, greater speed is realized when electroporation is utilized, as electroporation enables rapid changes in cell morphology and size to be observed within 24 hours of treatment. As discussed in the parent application, there are many protocols known to those skilled in the art for successful electroporation of a wide variety of transfectants including DNA, RNA, and protein. Koken et al., 1994 demonstrated successful protein electoporation almost thirty years ago. Like Koken et al., we applied electroporation at 300V to enable protein electroporation of selected cells, nevertheless, various voltages, pulse lengths and pulse intervals are suitable for practicing the invention. See, for example, the large number of protocols for electroporation of various transfectants into various cell types archived at www.btxonline.com. Delivery of the transfectant increases predictably with pulse number. All settings that we tried were successful in delivering protein to the interior of the cells selected. FITC-conjugated albumin served as a test transfectant and offered an excellent means of immediately visualizing the extent of protein delivery to the interior of the cells in conjunction with various electroporation parameters. After electroporation, cells were collected, washed and resuspended in PBS or medium for visualization using fluorescent microscopy. With increasing pulse number ranging from 10 pulses to more than 100 pulses, the brightness of the cells increased as well. However, we noted no obvious loss of cell viability, even with >100 pulses. Efficient Activation/Induction/Reprogramming was observed using 30-70 pulses: thus 70 pulses was used in the majority of experiments. However, a widely varying number of pulses could be used to achieve the claimed effect. Note that the invention may be practiced with any electroporation parameters known to the art so long as they provide for sufficient delivery of the transfectants to achieve the claimed effect at a desirable efficiency. In one preferred embodiment, the voltage is 300V, the pulse length is 5 ms, the pulse interval is 100 ms, and the number of pulses is 70. We noted that protein electroporation and other methods of transfection are compatible in activating/inducing/reprogramming a cell. Accordingly, the invention covers the use of electroporation (with or without a transfectant) in combination with other methods taught herein as well as with methods taught by others skilled in the art for activating, inducing or reprogramming a cell to a desirable phenotype. The invention may be practiced in vivo using a variety of existing methods and equipment known to the art. However, the present invention also teaches a novel device for in vivo electroporation comprising a catheter and electrode(s). In one embodiment, the catheter and electrode(s) are combined, with or without a camera and/or light a light source, as an assembly that can be optionally mounted on a wire or flexible tube such as are used for cardiac catheterization and for endoscopy. In some embodiments, the electrodes are sharp and capable of piercing tissue. In some embodiments, the electrodes are dull. In some embodiments, said assembly may also comprise a needle enabling injection of a transfectant into a tissue and a reservoir (e.g. syringe) where the transfectant is stored immediately prior to injection. In some embodiments, said assembly may comprise electrodes taking a form akin to “tweezertrodes”. In a preferred embodiment, cells are “selected” from accessible, dividing or non-dividing cell populations for the purpose of generating the desired a) proliferating, multipotent or pluripotent cell population, or b) differentiating populations of somatic cells; moreover the desired cell population may be capable of further differentiation in vitro, further differentiation in vivo, and/or tissue-appropriate and regionally-appropriate differentiation in vivo. Sources of Cells Selected for Use in the Invention Selected cells may include any cell practicable in the present invention. Cells selected for use in the present invention (herein termed “selected cells”) may originate as endogenous cells of a subject or of a patient—including cells derived from other organ systems; or from exogenous sources (including those derived from cell lines, cryopreserved sources, banked sources, and donors). Cells may also be selected from cells genetically-modified with synthetic or natural nucleic acid sequences (or their corresponding proteins). The term “selected cells”, as used herein, does not include human embryonic stem cells. In embodiments of the present invention, in order that they may be isolated without the involvement of invasive procedures, selected cells will preferably be easily accessible cells (e.g. peripheral blood leukocytes, circulating hematopoietic stem cells, epithelial cells (e.g. buccal cheek cells (e.g. Michalczyk et al., 2004), excreted cells, adipose tissue cells (e.g. Gimble et al., 2007; Ma et al., 2007), umbilical cord blood cells (e.g. Zhao, et al., 2006; Tian et al., 2007), etc.). However, bone marrow derived cells, stem cells isolated from amniotic membranes (e.g. Ilancheran et al., 2007), or amniotic fluid (e.g. De Coppi et al., 2007), as well as cells isolated from the skin (e.g. Tumbar, 2006; Dunnwald et al., 2001; Szudal'tseva et al., 2007), etc., are also covered by the present invention. Such cells can be isolated from the tissues in which they reside by any means known to the art. The selected cells may be genetically-modified cells, especially cells that have been genetically modified by any means known to the art, to encode therapeutic or commercially useful deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences. The selected cells may be genetically-modified cells, especially cells that have been genetically modified by any means known to the art, to encode therapeutic or commercially useful deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including through the use of CRISPR/CAS9 or other methods included in the category of site-specific genetic modification. In accordance with an aspect of the present invention, there is provided a method of producing a desired, developmentally activated cell population (e.g. pluripotent, pluripotent-like, neuronal, muscle, etc.) from the selected cells. Achieving multipotent, pluripotent, self renewing, “VSEL-like” and/or “pluripotent-like”, developmentally activated cell populations: In a preferred embodiment, a population of proliferating, self renewing, pluripotent or pluripotent-like cells is derived from the selected cell(s) and/or their progeny when said selected cells are transfected with nucleotide sequence(s) or proteins including those encoding the “long” (PRR insert +) isoform(s) of the mammalian numb gene (or other cell fate determinants taught herein for producing proliferating, self renewing, pluripotent or pluripotent-like cells, see below). At about the same time the selected cells may optionally be transfected with synthetic oligonucleotides targeting the short Numb isoforms and Numblike. When performed in vitro, the cells are subsequently cultured under conditions which promote growth of the selected cells at an optimal growth rate. Selected cells are maintained under these conditions for the period of time sufficient to achieve the desired cell number. When transfection is performed in vivo, no further steps are required. Transfected cells maintained in vitro may be grown under a variety of growth conditions known to the art, and optionally at the (optimal) rate of growth achieved by incubation with LIF, steel factor, and/or equipotent concentrations of Il-6, hyper IL-6, IL-7, oncostatin-M and/or cardiotrophin-1; or optionally that growth rate achieved in the presence of other growth enhancing cytokines (e.g. those conditions described for culturing pluripotent cells e.g. Guan et al., 2006), and/or chemicals selected from VC6TFZ: VPA, 5-aza-cytidine, CHIR99021 (CHIR), 616452, Tranylcypromine, Forskolin (FSK), 2-methyl-5-hydroxytryptamine (2-Me-5HT), and D4476. The growth rate is determined from the doubling times of the selected cells in said growth culture medium. Likewise, culture conditions such as those described in U.S. Pat. Nos. 6,432,711 and 5,453,357 may also be suitable for the propagation and expansion, at an optimal growth rate, of cells transfected with the long (PRR+) Numb isoform(s). Other appropriate protocols and reference cytokine concentrations have been taught by Koshimizu et al., 1996; Keller et al., 1996; Piquet-Pellorce, 1994; Rose et al., 1994; Park and Han, 2000; Guan et al., 2006; Dykstra et al., 2006; Zhang et al., 2007). However, the practice of the present invention is not limited to the details of these teachings. The cell culture medium need not necessarily contain a cytokine and need not necessarily contain serum and many serum free cell culture media are known to the art. In a preferred embodiment, the selected cells are cultured in a standard growth medium (e.g. Minimal Essential Medium with or without supplements (e.g. glutamine, and beta.-mercaptoethanol). The medium may include basic fibroblast growth factor (bFGF), steel factor, leukemia inhibitory factor (LIF), and/or factors with LIF activity (e.g. LIF, LIF receptor (LIFR), ciliary Neurotrophic factor (CNTF), oncostatin M (OSM), OSM receptor (OSMR), cardiotrophin, interleukins (IL) such as IL-6, hyper IL-6, GP130, etc.) as well as horse serum. LIF, as well as other factors with LIF activity, prevents spontaneous differentiation of the cells. Under these conditions, selected cells transfected with the cell fate determinants taught herein and their progeny are expected to achieve multipotency, pluripotency and/or self-renewal. In a preferred embodiment, the selected cell(s) and/or their progeny are transfected with, or overexpress, nucleotide sequence(s) encoding cell fate determinants (or their corresponding proteins), as well as sequences encoding other transgenes (or their corresponding proteins). Many of those transgenes are listed below along with their corresponding identification numbers (accession numbers) in the NCBI sequence database. In another preferred embodiment, the selected cell(s) and/or their progeny are transfected with, or overexpress, nucleotide sequence(s) encoding a portion of the “long” (PRR insert +) Numb isoform(s) (or their corresponding proteins), as well as sequences encoding other transgenes (or their corresponding proteins). Many of those transgenes are listed below along with their corresponding identification (accession) numbers (codes) in the NCBI sequence database. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform encoding sequences (or their corresponding proteins), as well as sequences encoding other transgenes (or their corresponding proteins), including LIF. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including ones with LIF activity. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins), as well as sequences encoding other transgenes (or their corresponding proteins), including the LIFR. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including oncostatin M (OSM). In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including oncostatin M receptor (OSMR). In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including cardiotrophin-1. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including CNTF. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including one or more selected from Oct3/4 and/or SOX2. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including NANOG, OCT3/4 and/or SOX2. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including one or more selected from Oct3/4 and SOX2 and/or a transgene with LIF activity. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, sequences encoding other transgenes (or their corresponding proteins), including one or more selected from Oct3/4 and/or SOX2 and a transgene with LIF activity. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including Notch (e.g. Gaiano et al., 2000). In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including one or more selected from Oct3/4, SOX2 and/or Notch (e.g. notch 1 and/or notch 2). In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including one or more selected from OCT3/4, SOX2, NANOG, and/or Notch. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including one or more selected from OCT3/4, SOX2, NANOG, and/or a transgene with LIF activity. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including one or more selected from OCT3/4, SOX2, NANOG, and/or multiple transgenes (or their corresponding proteins) with LIF activity. In a preferred embodiment, the selected cells and/or their progeny are transfected with, or overexpress, long (PRR+) Numb isoform(s) encoding sequences (or their corresponding proteins) as well as sequences encoding other transgenes (or their corresponding proteins), including one or more selected from OCT3/4, Notch, HOXB4 and/or SOX2. Over time, other gene combinations differing from those described herein may be described or discovered capable of causing cells to become multipotent, pluripotent, capable of self-renewal, or to begin differentiating. However this patent application covers such “genetic reprogramming” of any nucleated cell utilizing nucleic acid or protein electroporation (see Gagne et al., 1991; Saito et al., 2001; Yuan, 2008; Huang et al., 2007; Xia and Zhang, 2007; Cemazar and Sersa 2007; Isaka and Imai, 2007; Luxembourg et al., 2007; Van Tendeloos, 2007; Takahashi, 2007; etc.), liposomes, nanocapsules, nanovaults, etc. (see Goldberg et al., 2007; Li et al., 2007), and/or another approach avoiding viral integration or other random alteration of the cell's genome, as such means increase safety and efficiency. Excluded, of course, from the category of “random alteration” are approaches involving gene-targeting and site-directed methods (e.g. CRISPR/CAS9) designed to introduce or remove DNA at specific locations in the genome; and the use of CRISPR/CAS9 to practice the invention is covered by the present invention. Likewise, this patent application covers the genetic reprogramming of any nucleated cell utilizing nucleic acid or protein electroporation, liposomes, nanocapsules, nanovaults, etc., and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome, as such means increase safety and efficiency. Such approaches and methods include all known to the art and practicable in the present invention. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to a single gene, or portion thereof, (particularly those named herein, discovered according to methods described herein, discovered according to other published methods; or known to be multipotency, pluripotency, or self-renewal inducing) are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, muiltipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to a single gene, or portion thereof, (particularly those named herein, discovered according to methods described herein, discovered according to other published methods; or known to be mutiltipotency, pluripotency, or self-renewal inducing) are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to a single gene, or portion thereof, (particularly those named herein, discovered according to methods described herein, discovered according to other published methods; or known to be multipotency, pluripotency, or self-renewal inducing) so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to a single gene, or portion thereof, (particularly those named herein, discovered according to methods described herein, discovered according to other published methods; or known to be multipotency, pluripotency, or self-renewal inducing) so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to Nanog are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to Nanog so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding viral integration or other random alteration of the cell's genome. In a separate preferred embodiment, nucleic acid(s) or protein(s) corresponding to Oct4 and Sox2 are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding viral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s) or protein(s) are utilized in concert with the nucleic acid(s) or protein(s) corresponding to Oct4/Sox2 so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding viral integration or other random alteration of the cell's genome. In a separate preferred embodiment, nucleic acid(s) or protein(s) corresponding to Nxx3-1 are utlized to produce a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a separate preferred embodiment, nucleic acid(s) or protein(s) corresponding to Long (PRR+) Numb isoforms are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding viral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s) or protein(s) are utilized in concert with the nucleic acid(s) or protein(s) corresponding to Long (PRR+) Numb isoforms so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding viral integration or other random alteration of the cell's genome. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to Nanog are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to Nanog are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to Nanog so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to Nanog so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to a gene with LIF activity are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to a gene with LIF activity are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to to a gene with LIF activity so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to a gene with LIF activity so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to Oct4 are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to Oct4 are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to Oct4 so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to Oct4 so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to Sox2 are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to Sox2 are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to Sox2 so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to Sox2 so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to lin28 are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to lin28 are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to lin28 so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to c-myc are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to c-myc are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to c-myc so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to c-myc so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, nucleic acid(s) or protein(s) corresponding Oct4 and/or Sox2 are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a separate preferred embodiment, nucleic acid(s) or protein(s) corresponding to Oct4 and/or Sox2 are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s) or protein(s) are utilized in concert with the nucleic acid(s) or protein(s) corresponding to Oct4 and/or Sox2 so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s) or protein(s) are utilized in concert with the nucleic acid(s) or protein(s) corresponding to Oct4 and/or Sox2 so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, nucleic acid(s) or protein(s) corresponding to Long (PRR+) Numb isoforms are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a separate preferred embodiment, nucleic acid(s) or protein(s) corresponding to Long (PRR+) Numb isoforms are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s) or protein(s) are utilized in concert with the nucleic acid(s) or protein(s) corresponding to Long (PRR+) Numb Isoforms so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s) or protein(s) are utilized in concert with the nucleic acid(s) or protein(s) corresponding to Long (PRR+) Numb Isoforms so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, nucleic acid(s) or protein(s) corresponding to Oct4, Sox2, and/or Nanog are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a separate preferred embodiment, nucleic acid(s) or protein(s) corresponding to Oct4, Sox2, and/or Nanog are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s) or protein(s) are utilized in concert with the nucleic acid(s) or protein(s) corresponding to Oct4, Sox2, and/or Nanog so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s) or protein(s) are utilized in concert with the nucleic acid(s) or protein(s) corresponding to Oct4, Sox2, and/or Nanog so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, nucleic acid(s) or protein(s) corresponding to Long (PRR+) Numb isoforms are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells. In a separate preferred embodiment, nucleic acid(s) or protein(s) corresponding to Long (PRR+) Numb isoforms are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to produce dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells from the selected cells and the method is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s) or protein(s) are utilized in concert with the nucleic acid(s) or protein(s) corresponding to Long (PRR+) Numb isoforms so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s) or protein(s) are utilized in concert with the nucleic acid(s) or protein(s) corresponding to Long (PRR+) Numb isoforms so long as a population of dividing, self-renewing, multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” cells is produced from the selected cells and the method is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. It is to be understood that any combination of nucleic acid or protein sequences (or their corresponding proteins) described herein can be modified by excluding those corresponding to Numb and/or Numblike so long as the desired cell population or behavior is achieved. Similarly, it should be understood that the methods described herein for initiating differentiation are applicable to any induced or non-induced multipotent, pluripotent, or self-renewing stem cells, other progenitor cells, or other somatic cells, not only those obtained in the manner described herein. It is to be understood that any combination of nucleic acid or protein sequences (or their corresponding proteins) described herein can be modified by excluding nucleic acid sequences or proteins corresponding to Numb and/or Numblike so long as the desired cell population is achieved. In another embodiment, the various nucleic acid or protein combinations described herein are employed with the exclusion of the nucleic acid or protein corresponding to the Numblike and/or Numb isoforms. In a preferred embodiment, the selected cells and/or their progeny are cells that have been genetically-modified beforehand. In a preferred embodiment, the transfection steps described herein represent transient transfection. In a further preferred embodiment such transient transfection is accomplished using viral vectors that do not integrate into the host genome. Non-integrating and episomal viral vectors are well known to the art and include 2ndand 3rdgeneration, integrase-deficient, non-integrating lentiviral vectors, including 3rdgeneration lentivectors taught herein. Such integrase-deficient vectors can be readily introduced using a variety of standard transfection techniques (e.g. electroporation, chemically mediated transfection, fusogenic or non-fusogenic liposomes, lipofectamine, nanocapsules, nanovaults, etc.)—methods which allow high capacity integrase-deficient lentiviral vectors to be utilized without genomic integration and random alteration of the genome (seeFIG.3D). Over time, other gene combinations differing from those described herein may be described or discovered capable of causing cells to become multipotent, pluripotent, capable of self-renewal or to begin differentiating. However this patent application also covers the genetic reprogramming of any nucleated cell utilizing nucleic acid or protein electroporation (for example methods see Gagne et al., 1991; Saito et al., 2001; Yuan, 2008; Huang et al., 2007; Xia and Zhang, 2007; Cemazar and Sersa 2007; Isaka and Imai, 2007; Luxembourg et al., 2007; Van Tendeloos, 2007; Takahashi, 2007; etc.) electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding viral integration or other random alteration of the cell's genome as such means increase safety and efficiency. In another preferred embodiment, transfection with (or overexpression of) long (PRR+) numb isoform encoding sequences (or their corresponding proteins) (and/or synthetic oligonucleotides targeting numblike and short numb isoforms) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding human LIF (e.g. Du and Shi, 1996) oncostatin-M, cardiotrophin-1, IL-11, IL-6, IL6R, hyper IL-6, LIFR, gp130, OCT3 (OCT4), Nanog, SOX2, and/or FGF-4. Simultaneous transfection with (or overexpression of) any subset of these distinct transgene sequences (or their corresponding proteins) can be accomplished by any means known to the art including the use of a single genetic vector, multiple genetic vectors, serial transfection and selection based on distinct marker proteins and/or antibiotic resistances. In another embodiment, the cells selected for developmental activation are cultured in a cell culture promoting an optimal growth rate, such as described above, and that includes EGF, bFGF, oncostatin, LIF (e.g. Du and Shi, 1996), steel factor, IL-11, cardiotrophin-1, IL-6, hyper-IL-6, CNTF, and/or soluble gp130. In another embodiment, when neural progenitor cells are the desired cells, the cells selected for developmental activation are incubated in a growth medium comprising one or more of VPA, BIX01294, RG108, PD0325901, CHIR99021, vitamin C, CHIR99021, RepSox, A83-01, Thiazovivin, Purmophamin, LDN193189, and RG108, Assessment of Potency and Differentiation Pluripotency and multipotency can be assessed by any means known to the art including 1) transplantation, 2) culture under conditions promoting embryoid body formation, 3) injection of cells into animal blastocyst stage embryos with subsequent development, and 4) RNA expression assays (e.g. RT-PCR and microarray based analyses) for gene expression associated with differentiation, multipotency, pluripotency, etc. (see Guan et al., 2006), 5) colony-formation, as well as by ES-like morphology. One approach disclosed herein for detecting pluripotency in selected cells and/or their progeny involves transfection with (or overexpression of) a reporter construct comprising the Nanog promoter operably linked to a fluorescent protein gene. This allows identification and enrichment of Nanog expressing cells using Fluorescence Activated Cell Sorting (FACS), etc. In a preferred embodiment, endogenous cells (e.g. cells surrounding a burn or injury site) are transfected in vivo with genetic vectors encoding the long (PRR+) numb isoform(s) alone or in conjunction with other transgenes (or their corresponding proteins) named herein to transiently promote renewed or increased cell proliferation. This approach can also be utilized clinically in the setting of hypoplastic tissues, disorders where stem/progenitor cells are abnormally depleted, and other disorders where the approach can be shown to be beneficial. Achieving Differentiating Cell Populations In order to achieve b) neural c) muscle d) and other cell populations capable of further environmentally-regulated differentiation in vivo, selected cell(s) and/or their progeny are optionally transfected with long (PRR+) Numb isoform sequence(s) and/or synthetic oligonucleotide sequences and expanded by growth for sufficient time to achieve the desirable number of cell progeny in vitro (as described above). Following this optional step, the selected cells and/or their progeny are washed free of the cytokines and agents comprising the expansion/optimal growth media, and are optionally transfected with the nucleotide sequence(s) encoding the Numblike gene and/or “short” (PRR−) Numb isoform(s) and/or synthetic oligonucleotides targeting the long (PRR+) isoforms, etc. (e.g. Zaehres et al., 2005), then cultured under conditions which promote differentiation of the selected cells into the desired cell type(s). In most instances, the cells are then cultured in the presence of 5-10% fetal bovine serum and agents(s) promoting differentiation of the selected cells and/or their progeny into a desired cell population. The presence of the fetal bovine and of the agents(s) provides for growth or proliferation at a rate that is less than the optimal (or expansion) growth rate, and favors differentiation of the cells into a desired cell population. The agents and precise culture conditions are selected according to the desired cell population as described below. Achieving Neuronal or Neural Cell Populations When the desired cell population is a neural cell population, the successfully transfected cells are cultured under conditions that promote growth at a rate which is less than the optimal rate and in the presence of agent(s) promoting differentiation of the cells into neural cells. Conditions promoting differentiation into neurons have been described in numerous publications including (Benninger et al., 2003; Chung et al. 2005; Harkany et al., 2004; Ikeda et al., 2004; Ikeda et al., 2005; Wernig et al., 2002; and Wernig et al., 2004). Furthermore, combining retinoic acid exposure with the presence of additional cytokines favors specific neuronal cell type differentiation in vitro (e.g. Soundararajan et al., 2006; Soundararajan et al., 2007; U.S. Pat. No. 6,432,711). In a preferred embodiment, in vitro differentiation of neurons or neural cells occurs in the presence of 50 ng/mL nerve growth factor (NGF). In a preferred embodiment, when a neuronal population is the desired cell population, transfection with (or overexpression of) sequences encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from miR-124, miR-128, Nurr1, REN, Neurogenin1, Neurogenin2, Neurogenin3, Mash 1, Phox2b, Phox2a, dHand, Gata3, Shh, FGF8, Lmx1a, Lmx1b, Nkx2.2, Pet1, Lbx1, Ptx-3, Pitx2, Dix1, Dlx2, Dlx5, and/or Rnx. In another preferred embodiment, when dopaminergic neurons are the desired neuronal population, transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding Mash1 (Ascl1), Ngn2, Nurr1, Lmx1a, Lmx1b, Foxa2, Brn2, Myt1l, Otx2, and/or Ptx-3. In another preferred embodiment, when serotonergic neurons are the desired neuronal population, transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding Mash1 (Ascl1), Phox2b, Lmx1b, Nkx2.2, Gata2, Gata3 and/or Pet1. In another preferred embodiment, when cholinergic neurons are the desired neuronal population, transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding MASH1 (ASCL1), Phox2a and/or REST4. In another preferred embodiment, when GABAergic neurons are the desired neuronal population, transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding MASH1 (ASCL1), Phox2a and/or REST4, followed, optionally, by culture in media supplemented with LIF, Neurotrophin 3 (NT3), and/or nerve growth factor (NGF). In another preferred embodiment, when noradrenergic neurons are the desired neuronal population, transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding Mash1 (Ascl1), dHand, Phox2a, Phox2b, Brn2, Myt1, Gata2 and/or Gata3. In another preferred embodiment, when GABAergic neurons are the desired neuronal population, transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding Mash1 Ascl1), PITX2, Dlx2, Dlx5, antisense Hes1 RNA and/or other HES1 targeting synthetic oligonucleotides. In another preferred embodiment, when a neuronal or neural cell population is the desired population, cells are cultured in a cell culture medium promoting differentiation, such as described above and that includes one or more of the following agents: retinoic acid, Forskolin, ISX9, CHIR99021, SB431542, I-BET151, Forskolin PD0325901, LDN193189, Pifithrin-α, SP600125, G06983, Y-27632, NT3, NGF, glial cell-line derived growth factor (GDNF), and interferon gamma (IFN-gamma). Achieving Muscle Cell Populations When the desired cell population is a muscle population, the successfully transfected cells are cultured in the presence of an agent promoting differentiation of the cells into muscle cells and growth at a rate less than the optimal rate. Conditions promoting differentiation into muscle cells have also been described previously (Nakamura et al., 2003; Pal and Khanna, 2005; Pipes et al., 2005; Albilez et al., 2006; Pal and Khanna, 2007; Behfar et al., 2007; U.S. Pat. No. 6,432,711). Furthermore, exposure of selected cells and/or their progeny to hexamethylene bis-acrylamide or dimethylsulfoxide in the presence of additional cytokines favors the initiation of muscle type differentiation in vitro. In a preferred embodiment, when a cardiac muscle cell population is the desired population, cells transfected with short (PRR−) numb isoforms (and/or numblike) are cultured in a cell culture medium promoting differentiation into cardiomycytes (He et al., 2003; Guan et al., 2007; etc.), or that includes specific agents at concentrations promoting cardiac cell differentiation (e.g. 0.75%-1% dimethyl sulfoxide (DMSO), 20% normal bovine serum (NBS), 10(−7) mM retinoic acid (RA) and 20% cardiomyocytes conditioned medium (Hua et al., 2006). In another preferred embodiment, when a cardiomyocyte muscle cell population is the desired population, transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from miR-1-1, miR-1-2, miR-133, miR-208, miR-499 and those encoding Gata 4, Gata 5, Gata 6, myocardin, Esrrg, Mesp1, Zfpm2 Ets2, Mesp, Myocd, Nkx2.5, Hand2, Mef2c, JAK inhibitor I and Tbx5. In a preferred embodiment, when a muscle cell population is the desired cell population, transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding muscle type specific bHLH-encoding sequences (or their corresponding proteins), MyoD, Myogenin, Myf5, Myf6, Gata 4, Gata 5, Gata 6, Mef2, Tbx5, Hand2, Myocardin, Ifrd1 and/or other muscle transcription factors and small RNAs. In a preferred embodiment, when a smooth muscle cell population is the desired cell population, transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding the muscle type specific Myocardin nucleotide sequence. In a preferred embodiment, when a skeletal muscle cell population is the desired cell population, transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding the muscle type specific MyoD and myogenin nucleotide sequences. Further, when the desired cell population is a skeletal muscle cell population, the transfected or overexpressed sequences may include one or more selected from miR-1, miR-1-1, miR-1-2, miR-206, miR-26a, miR-133, miR-133a-1 and miR-133a-2. In a preferred embodiment, when an oligodendrocyte cell population is the desired cell population, transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding the oligodendrocyte-specific OLIG1, OLIG2, Nkx2.2, Nkx6.2, Sox10, ST18, Gm98, Myt1, Zfp536 and Zfp488 nucleotide sequences. Simultaneous transfection with (or overexpression of) any subset of these distinct transgene sequences (or their corresponding proteins) listed above can be accomplished by any means known to the art including the use of multiple genetic vectors, serial transfection as well as selection based on distinct marker proteins and/or antibiotic resistance. When the desired cell population is a hematopoietic cell population, the differentiation medium may include specific agents at concentrations promoting differentiation into hematopoietic progenitor cells (e.g. vascular endothelial growth factor (VEGF), thrombopoietin, etc. (e.g. Ohmizono, 1997; Wang et al., 2005; Srivastava et al., 2007; Gupta et al., 2007) or differentiated hematopoietic cell types (according to methods known to the art for providing differentiated hematopoietic cell types from undifferentiated or pluripotent cells). When the desired cell population is a germ cell population, the differentiation medium may include specific agents at concentrations promoting differentiation into germ cells (e.g. Nayernia et al. 2006a, 2006b). When the desired cell population is a germ cell population, the differentiation medium includes specific agents at concentrations promoting differentiation into germ cells (e.g. Nayernia et al. 2006a, 2006b). In a preferred embodiment, when a germ cell population is the desired cell population, transfection/contacting with short numb isoform (and/or numblike) proteins or with sequences encoding short numb isoform proteins (and/or numblike), is accompanied or replaced by transient or permanent transfection/contacting with other proteins and/or nucleic acid sequences, including ones selected from those encoding FIGLA, FIG alpha, DAZL, STRA8, FOXL2, OOGENESIN1, OOGENESIN2, OOGENESIN3, OOGENESIN4, SYCP2, SYCP3, SPO11, REC8, DMC1, MOS, STAG3, CCNB1, FOXO1, FOXO3, SOHLH1, SOHLH2, NOBOX, OBOX1, OBOX2, OBOX3, OBOX4, OBOX6, LHX8, LHX9, OOG1, SP1, ZFP38, TRF2, TB2/TRF3, TAF4B, TAF7L, TAF71, TIA1, PHTF1, TNP2, HILS1, DAZL, BMP15, PTTG3, AURKC, OTX2, SOX15, SOX30, FOXR1, ALF, OCT4, DPPA3/STELLA, ZFP38, RPS6KA3, HINFP, NPAT, SP1, SP3, HOXA1, HOXA7, HEX, YP30, ZP1, ZP2, ZP3, SFE1, SFE9, OPO, PLN, RDV, GLD1, MMU-MiR351, MMU-MiR615, MMU-MiR592, MMU-MiR882, MMU-MiR185, MMU-MiR491, MMU-MiR326, MMU-MiR330, MMU-MiR351. For example, but not limiting, in one preferred embodiment, when a sperm or spermatocyte cell population is the desired cell population, transfection/contacting with short numb isoform (and/or numblike) proteins or with sequences encoding short numb isoform proteins (and/or numblike), is accompanied or replaced by transient or permanent transfection/contacting with other proteins and/or nucleic acid sequences, including ones selected from those encoding SYCP2, SYCP3, SPO11, REC8, DMC1, MOS, STAG3, OCT4, ALF, RPS6KA3, HINFP, SP1, SP3, TAF71, TIA1, PHTF1, TNP2, HILS1, CLGN, TEKT1, FSCN3, DNAHC8, LDHC, ADAM3, OAZ3, AKAP3, MMU-MiR351, MMU-MiR615, MMU-MiR592, MMU-MiR882, and MMU-MiR185. For example, but not limiting, in one preferred embodiment, when a oocyte cell population is the desired cell population, transfection/contacting with short numb isoform (and/or numblike) proteins or with sequences encoding short numb isoform proteins (and/or numblike), is accompanied or replaced by transient or permanent transfection/contacting with other proteins and/or nucleic acid sequences, including ones selected from those encoding MOS, CCNB1, OCT4, FIG alpha, FIGL alpha, ALF, SOHLH1, SOHLH2, LHX8, LHX9, OOG1, FIG alpha, SP1, LHX3, LHX9, TBP2/TRF3, DAZL, BMP15, GDF9, PTTG3, AURKC, OTX2, SOX15, SOX30, FOXR1, NOBOX, OBOX1, OBOX2, OBOX3, OBOX6, OOGENESIN1, OOGENESIN2, OOGENESIN3, OOGENESIN4, YP30, ZP1, ZP2, ZP3, SFE1, SFE9, OPO, PLN RDV, GLD1, DAZL, STRA8, MMU-MiR615, MMU-MiR491, MMU-MiR326, MMU-MiR330, MiR212 and MMU-MiR351. When the desired cell population is an endoderm and pancreatic islet cell population, the differentiation media may include specific agents at concentrations promoting differentiation into endoderm and pancreatic islet cells (e.g. Xu et al., 2006; Denner et al., 2007; Shim et al., 2007; Jiang et al., 2007). In a preferred embodiment, differentiation of selected cells and/or their progeny may occur in the differentiation medium in the absence of transfection with (or overexpression of) numblike, short Numb isoforms (or their corresponding proteins), although the differentiation medium may be unchanged. In embodiments, a single vector will be utilized which controls the expression of nucleotide sequence(s) encoding the “long” (PRR+) isoform(s) of the mammalian numb gene (and/or synthetic oligonucleotides targeting numblike or the short numb isoforms) under one regulable promoter (e.g. a tetracycline-regulated promoter), while the Numblike and short Numb isoforms (and/or synthetic oligonucleotides targeting the long (PRR+) isoforms) are expressed under the control of another, distinct, but also regulable promoter. Thus, the long (PRR+) numb isoform(s) can be expressed (and/or short isoforms repressed) when expansion of the selected cells is desired and an inducing agent (e.g. tetracycline) is added to the growth medium; later numblike and the short isoforms can be expressed (and/or long (PRR+) numb isoform(s) repressed) when differentiation is desired. Alternatively, proteins and peptides corresponding to Numb isoforms, Notch, OCT3/4, SOX2, and/or other DNA sequences listed herein may be applied in analogous fashion to selected cells and/or their progeny via electroporation (e.g. Koken et al., 1994; Ritchie and Gilroy, 1998), using nanoparticles, cationic lipids, fusogenic liposomes (e.g. Yoshikawa et al., 2005; 2007), etc. in lieu of, or in combination with genetic transfection. Generally, electroporation allows for high transfection efficiency (and efficient production of the desired cells) without genomic integration of the transgene and is therefore associated with increased safety. The DNA or RNA encoding protein(s) or polypeptide(s) promoting proliferation, multipotentiality, pluripotentiality or differentiation of the selected cells may be isolated in accordance with standard genetic engineering techniques (for example, by isolating such DNA from a cDNA library of the specific cell line) and placing it into an appropriate expression vector, which then is transfected into the selected cells. In another preferred embodiment, endoderm and pancreatic islet cells are the desired population, and transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding Foxa2, Sox17, HLXB9, Ngn3, Mafa, Mapk, Stat3 and/or Pdx1. In another preferred embodiment, hepatocytes are the desired population, and transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding hepatic nuclear factor (HNF)-1, HNF-3, HNF-4, HNF-6, Foxa3, Foxa1, Foxa2 or Gata4, Cebpa, Cebpb, Atf5, c-myc and Prox1. In another preferred embodiment, hematopoietic cells are the desired population, and transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from those encoding Runx1/aml1, Nov(Ccn3), Erg, Gata2, Lmo2, Runx1c, Scl, Runit1, Hlf, Prdm5, Pbx1, Zfp37, Mycn, Meis1, FOSB, GFI1, SPI1 and/or cell culture in the presence of colony stimulating factors specific for the desired cell populations. The Runx1/AML1a isoform is introduced when engraftment is desired and the b isoform when differentiation is desired (Creemers et al., 2006). Further, when the desired hematopoietic cell is a progenitor cell population, the transfected or overexpressed sequences may include one or more selected from miR-128, miR-181, miR-16, miR-103 and miR-107. Further, when the desired hematopoietic cell is a T lymphoid cell population, the transfected or overexpressed sequences may include miR-150. Further, when the desired hematopoietic cell is a B lymphoid cell population, the transfected or overexpressed sequences may include one or more selected from miR-181, miR-155, miR-24, miR-17, miR-16, miR-103 and miR-107. Further, when the desired hematopoietic cell is an erythroid cell population, the transfected or overexpressed sequences may include one or more selected from miR-150, miR-155, miR-221, miR-222, miR-451, miR-16 and miR-24. Further, when the desired hematopoietic cell is a monocyte cell population, the transfected or overexpressed sequences may include one or more selected from miR-17-5p, miR-20a, miR-106a, miR-16, miR-103 and miR-107. Further, when the desired hematopoietic cell is a granulocyte cell population, the transfected or overexpressed sequences may include one or more selected from miRNA-155, miR-24, miR-17, miR-223, miR-16, miR-103 and miR-107. Further, when the desired hematopoietic cell is a megakaryocyte cell population, the transfected or overexpressed sequences may include one or more selected from miR-155, miR-24, and miR-17. In another preferred embodiment, chondrocytes are the desired population, and transfection with (or overexpression of) sequences encoding short numb isoforms and/or numblike (or their corresponding proteins) is accompanied or replaced by transient or permanent transfection of other sequences (or their corresponding proteins) including one or more selected from ones encoding Sox9, CREB-binding protein, Gata6, Runx2, and TGF-beta. In another preferred embodiment, bone cells (especially osteoblasts) are the desired population, and transfection with (or overexpression of) sequences encoding short numb isoforms and/or numblike (or their corresponding proteins) is accompanied or replaced by transient or permanent transfection of other sequences (or their corresponding proteins) including Runx2. Further, when an osteoblast population is the desired cell population, transfection with (or overexpression of) sequences may include one or more small RNAs selected from miR-125b and miR-26a. Further, when a keratinocyte population is the desired cell population, transfection with (or overexpression of) sequences may include one or more small RNAs selected from miR-203. In a preferred embodiment, the genetic vectors encoding the long Numb isoforms (such as those described herein) are introduced transiently or under the control of a regulable promoter, into endogenous cells in vivo in order to cause those cells proliferate transiently. In another embodiment, when i. brown adipocytes, ii. astrocytes, iii. endothelial cells, iv. macrophages/mocytes v. melanocytes, vi. neural stem cells, vii. glutamatergic neurons, viii. astrocytes, ix. motor neurons, or x. nephrogenic progenitors are desired cell population and transfection with (or overexpression of) sequences (or their corresponding proteins) encoding short numb isoforms (and/or numblike) is accompanied or replaced by transient or permanent transfection with (or overexpression of) other sequences (or their corresponding proteins) including one or more selected from i. PRDM16, and CEBPP, or ii. Nfia, Nfib, and Sox9, iii. Etv2, Fli1, Erg1, Foxo1, Er71, Klf2, Tal1, and Lmo2, iv. Sox2, miR-125b, PU.1, CEBP and CEBPO, v. Mitf, Sox10, Pax3, vi. Ascl1, Ngn2, Hes1, Id1, Pax6, Sox2, c-Myc, Brn2, Brn4, Klf4, c-Myc, and E47; vii. NeuroD1, Ascl1, Myt1l, Neurod2, miR-9/9, miR-124 viii. Ascl1, Ngn2 and Dlx2, ix. Brn2, Mash1, Myt1l, Lhx3, Hb9, Isl1, Ngn2, or Six1, Six2, Osr1, Eya1, Hoxa11, Snai2, respectively. In a preferred embodiment, endogenous cells (e.g. ependymal zone cells of the central nervous system) are transfected in vivo with genetic vectors encoding either the shortest numb isoform or the numblike protein(s) alone or in conjunction with other transgenes (or their corresponding proteins) named herein, in order to transiently or permanently promote renewed or increased differentiation (especially neuronal differentiation) and migration of progenitor/ependymal cells in the central nervous system). This renewal or increase is measured in terms of the number of cells showing new-onset expression of markers associated with differentiation. This may be accomplished by introduction of the genetic vectors into the organ system using methods suitable for that purpose (see examples). In a preferred embodiment, endogenous cells (e.g. ependymal zone cells of the central nervous system) are transfected in vivo with genetic vectors encoding the long numb isoform(s) and/or other transgenes (or their corresponding proteins) named herein, in order to transiently promote renewed or increased stem cell proliferation (with subsequent differentiation of progeny cells). This renewal or increase is measured in terms of the number of cells showing new-onset expression of markers associated with dividing progenitors. This may be accomplished by introduction of the genetic vectors into the organ system using methods suitable for that purpose (see examples). Likewise, this approach is also be suitable for inducing renewed or increased differentiation from other stem cell populations in other tissues (such as the skin, etc.). This approach can be utilized, for example, clinically in the setting of central nervous system injury, disorders of other tissues where normal differentiation or migration are inadequate, dysplastic disorders and other disorders where the approach is beneficial. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to a single gene, or portion thereof, (particularly those named herein, discovered according to methods described herein, discovered according to other published methods; and/or known to be capable of initiating the desired manner of differentiation) are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to initiate differentiation in the selected cells. In a preferred embodiment, in order to produce developmental activation, nucleic acid(s) or protein(s) corresponding to a single gene, or portion thereof, (particularly those named herein, discovered according to methods described herein, discovered according to other published methods; and/or known to be capable of initiating the desired manner of differentiation) are the only nucleic acid(s) or protein(s) overexpressed and/or introduced to initiate differentiation in the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to a single gene, or portion thereof, (particularly those named herein, discovered according to methods described herein, discovered according to other published methods; and/or known to be capable of initiating the desirable manner of differentiation) so long as a population of differentiating cells is produced from the selected cells. In a separate preferred embodiment, in order to produce developmental activation, other nucleic acid(s), protein(s) or other transfectants can be utilized in concert with the nucleic acid(s) or protein(s) corresponding to a single gene, or portion thereof, (particularly those named herein, discovered according to methods described herein, discovered according to other published methods; and/or known to be capable of initiating the desirable manner of differentiation) so long as a population of differentiating cells is produced from the selected cells and the method utilized is electroporation, liposomes, nanocapsules, nanovaults, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of the cell's genome. It is to be understood that any combination of nucleic acids or proteins described herein can be modified by excluding those corresponding to Numb and/or Numblike so long as the desired cell population or behavior is achieved. Similarly, it should be understood that the methods described herein (or elsewhere) for initiating differentiation are applicable to any induced or non-induced multipotent, pluripotent, or self-renewing stem cells, or other selected cells, not only those obtained in the manner described herein. Sources of Selected Cells The population of selected cells may derive from various stem cells, progenitor cells and somatic cells. However somatic cells lacking nuclei (e.g. mature, human red blood cells) are specifically excluded. Selected stem cells may be derived from existing cell lines or isolated from stored, banked, or cryopreserved sources. Typical sources of stem cells include bone marrow, peripheral blood, placental blood, amniotic fluid (e.g. De Coppi et al., 2007), umbilical cord blood (e.g. Zhao, et al., 2006; Tian et al., 2007), adipose tissue (e.g. Gimble et al., 2007; Ma et al., 2007), non-human embryos, and others. Circulating leukocytes and other non-stem cells may likewise be selected and subjected to the same culture conditions as described above effective that they acquire multipotency, pluripotency and/or self-renewal as a result. Examples of other accessible somatic cells useful in this invention include lymphocytes and epithelial (e.g. buccal cheek) cells. Isolation and collection of cells selected for use within the present invention may be performed by any method known to the art. In embodiments involving animals, stem cells isolated from prostate, testis, embryonic brain, and intestine are also disclosed as being preferred sources of selected cells. In a preferred embodiment, the selected cells and/or their progeny are cultured in a three-dimensional format. A further aim of the present invention is to provide cells for use in the production of patient-compatible and patient-specific tissues and organs for transplantation to patients or subjects deemed to be requiring such organs or tissues. It is disclosed herein that the pluripotent, multipotent, and/or differentiating cells provided by the methods described herein (or similar methods) be utilized in conjunction with techniques aimed at the production of such organs and/or tissues (e.g. Boland et al., 2006. Xu et al., 2006; Campbell and Weiss, 2007). Such utilization is specifically covered by the present invention. For instance, pluripotent, multipotent, and/or differentiating cells produced or treated according to the methods desribed herein (or other published methods) may be grown in association with three-dimensional or two-dimensional scaffoldings engineered to replicate normal tissue structure and/or organ structures (e.g. Yarlagada et al., 2005; Kim et al, 1998; WO/2003/070084; EP1482871; WO03070084; U.S. Pat. Nos. 2,395,698; 7,297,540; 6,995,013; 6,800,753; Isenberg et al., 2006). Similarly, scaffoldings to be occupied by the pluripotent, multipotent, and/or differentiating cells may be derived from cadaveric organ(s) or tissue(s) after the cadaveric organs or tissues (e.g. bone, target tissue, organ or cavity, kidney, liver, lung, etc.) may be treated in such away that the host immune cells resident in that tissue, and other undesirable or ancillary host cells, are eliminated (e.g. by ionizing radiation, sterilization (e.g. Mroz et al., 2006), and/or various methods of decellularization (U.S. Pat. Nos. 6,734,018; 6,962,814; 6,479,064; 6,376,244; 5,032,508; 4,902,508; 4,956,178; 5,281,422, 5,554,389; 6,099,567; and 6,206,931; 4,361,552 and 6,576,618; 6,753,181; U.S. application Ser. No. 11/162,715; WO/2001/048153; WO/2002/024244; WO003002165; WO/2001/049210; WO/2007/025233; European Patents EP1482871; EP1246903; EP1244396; EP0987998; EP1244396; EP1333870; Rieder et al., 2004; Ott et al., 2008; Taylor et al., 1998)). Likewise, it is anticipated that the pluripotent, multipotent, and/or differentiating cells of the present invention may be used in applications utilizing inkjet-style printing for tissue engineering (e.g. Boland et al., 2006. Xu et al., 2006; Campbell et al., 2007). Therefore, such use of the cells produced or treated according to the methods described herein is covered. In another preferred embodiment, the selected cells and/or their progeny are cultured in hanging drops. In accordance with another aspect of the present invention, selected cells may be modified genetically beforehand. In accordance with another aspect of the present invention, selected cells may be modified with DNA or RNA encoding protein(s) or polypeptide(s) promoting differentiation of the cell into a desired cell population. Screening Cell Populations In one embodiment, the methods of this invention comprise screening cells from cell lines, donor sources, umbilical cord blood, and autologous or donor bone marrow, blood, spermatogonia, primordial germ cells, buccal cheek cells, or any other cell source effective in the current invention. Selected cells can be screened to confirm successful transfection with (or overexpression of) beneficial sequence(s) or therapeutic vector(s) as well as successful initiation of differentiation by any method known to the art (Guan et al., 2006; U.S. Pat. No. 6,432,711). In some embodiments, the cells are screened using standard PCR and nucleic acid hybridization-based methods or using rapid typing methods. In preferred embodiments, the cells are screened according to expression of reporter genes. In some embodiments, cells are screened by expression of a marker gene encoded by the transgene expressing vector(s) such as an antibiotic resistance gene or a fluorescent protein (e.g. GFP) gene. Screening for Therapeutic Vectors and Beneficial Sequences Cells can be screened for the presence of beneficial sequence(s) and therapeutic vector(s) using any method(s) known to the art for detection of specific sequences. Each cell sample can be screened for a variety of sequences simultaneously. Alternatively, multiple samples can be screened simultaneously. Cell differentiation may be monitored by several means: including (i) morphological assessment, (ii) utilizing reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot, or microarray techniques to monitor changes in gene expression, (iii) assaying cellular expression of specific markers such as beta tubulin III (for neurons) etc. (Ozawa, et al., 1985). In some embodiments, the cells are screened for successful initiation of differentiation using FACS sorting based on cell type specific markers or transgenic marker expression (e.g. antibiotic resistance or fluorescent protein expression) under the control of cell type specific promoters such as the myosin promoter in muscle cells; the human cardiac α-actin promoter in cardiomyocytes; the insulin promoter in insulin producing cells; the neuronal-specific enolase (NSE) promoter for neuronal differentiation, or neurotransmitter related promoters such as the tyrosine hydroxylase promoter in dopaminergic neurons; etc.). In some embodiments, the cells are screened using standard PCR and nucleic acid hybridization-based methods. In a particularly preferred embodiment, the cells are screened using rapid typing methods. Screening for Human Leukocyte Antigen (HLA) Type In certain embodiments, the selected cells are selected with respect to compatible HLA typing. The HLA genotype can be determined by any means known to those of skill in the art. The cells used for screening may consist of cells taken directly from a donor, or from cell lines established from donor cells, or other practicable cell sources. The cells can be screened for beneficial sequence(s), and/or therapeutic vector(s) and HLA type at once, or separately. Those cells successfully transfected with a beneficial sequence and showing an appropriate HLA genotype can be prepared for transplantation to a patient. In certain embodiments, the transfected cells are transplanted without HLA typing. In other embodiments, the cells are HLA typed for compatibility. Screening for Agents Promoting a Cellular Phenotype. The present invention also provides for a methods of screening proteins and agents for their ability to produce developmental activation of the selected cells and/or their progeny into desired cell populations. Briefly, vectors encoding complementary DNAs (cDNAs) from appropriate cDNA libraries are transfected into the selected cells/and or their progeny. Once a specific cDNA that induces differentiation or other phenotypic change is identified, such cDNA then may be isolated and cloned into an appropriate expression vector for protein production in appropriate cells (e.g. COS cells) in vitro. Later the protein containing supernatant can be applied to the selected cell cultures to determine if any secreted proteins from such cells induce differentiation Alternatively, candidate agents can be applied to the selected cell cultures to determine if any of the candidates induce developmental activation. The present invention also provides for methods of screening nucleic acids for their ability to induce multipotentiality, pluripotentiality, and/or self-renewal, or to initiate differentiation of selected cells and/or their progeny. In these methods, vectors encoding selected cDNAs (or cDNAs from appropriate cDNA libraries, or other sequences are introduced into the selected cells/and or their progeny using electroporation, nanocapsules, nanovaults, liposomes, retroviruses, lentiviruses, and/or any other practicable means of transfection. Once a specific cDNA that induces a phenotypic change, multipotentiality, pluripotentiality, and/or self-renewal, is identified, such cDNA then may be isolated and cloned into an appropriate expression vector. Assays for determining such changes include those described elsewhere herein. Likewise the protein corresponding to the identified cDNA may be produced in appropriate cells (e.g. COS cells) in vitro to determine whether the protein containing supernatant can be applied to the selected cell cultures and induce the desired changes. Finally, proteins may be introduced into the selected cells/and or their progeny using electroporation, nanocapsules, nanovaults, liposomes, retroviruses, lentiviruses, and/or any other practicable means of transfection, and the resulting cells assessed as described herein for multipotentiality, pluripotentiality, self-renewal or the initiation of differentiation. Tranplantation of Cells into Patients or Subjects After screening, selected cells and/or their progeny may be cryopreserved, maintained as cell lines in culture, or may be administered to the patient. Selected cells can be cryopreserved or maintained in culture by any means known to the art and preserved for future transplantation procedures. Preferably, the cells to be screened are obtained from accessible sources allowing easy collection. With regard to producing HIV resistant cells: targeted somatic cells and stem cells of this invention can be of any type capable of differentiating into cells that can be infected by HIV, that can sustain the transcription and/or replication of HIV, that can alter the HIV immune response, or that can retard progression to AIDS. Such stem cells include, but are not limited to, pluripotent cells derived from spermatogonia, primordial germ cells, hematopoietic stem cells, peripheral blood cells, placental blood cells, amniotic fluid cells, umbilical cord blood cells, buccal cheek cells, adipose tissue cells (including stem cells derived from those tissues), reprogrammed cells, induced multipotent cells, induced pluripotent cells, etc., non-human embryos, and/or any other cell type that can form blood and immune cells, HIV target cells, and other cells. Therapeutic vector(s) express “beneficial sequence(s)” intended to render transfected or infected cells less capable of sustaining HIV replication and transcription. The genetic vector expressing “beneficial sequence(s)” as well as any virus derived from such genetic vector, are herein termed “therapeutic vector”. After screening, cells transfected with the desired therapeutic vector(s) and expressing beneficial sequence (with or without compatible HLA genotype) may be expanded ex vivo (in vitro) using standard methods to culture dividing cells and maintained as stable cell lines (U.S. Pat. Nos. 6,432,711 and 5,453,357 herein incorporated by reference). Alternatively, these cells can be administered to the patient and expanded in vivo. Selected cells can be cryopreserved by any means known to the art and preserved for future transplantation procedures. Transplantation of Desirable Cell Populations into Patients or Subjects In certain embodiments, cell populations are enriched for stem cells prior to transplantation. Various methods to select for stem cells are well known in the art. For example, cell samples can be enriched by fluorescently labeled monoclonal antibodies recognizing cell-surface markers of undifferentiated hematopoietic stem cells (e.g., CD34, CD59, Thyl, CD38 low, C-kit low, lin-minus) for sorting via fluorescence-activated cell sorting (FACS). In other embodiments, a sample of the selected cells is transplanted, without enrichment. In some embodiments, the endogenous stem cells of the bone marrow are eliminated or reduced prior to transplantation of the therapeutic stem cells. Therapeutic stem cells are defined as those stem cells containing beneficial sequence(s) or therapeutic vector(s). In some embodiments, the transplantation process may involve the following phases: (1) conditioning, (2) stem cell infusion, (3) neutropenic phase, (4) engraftment phase, and (5) post-engraftment period. In some embodiments, the endogenous stem cells that normally produce the desired cells (e.g. bone marrow stem cells) are eliminated or reduced prior to transplantation. Chemotherapy, radiation, etc. and/or methods analogous to those described in U.S. Pat. No. 6,217,867 may be used to condition the bone marrow for appropriate engraftment of the transplant. Finally, therapeutic stem cells may be transplanted into the patient using any method known to the art. Sample Transgene Encoding Vectors In one embodiment transfection with (or overexpression of) nucleic acid sequence(s) encoding transgenes is accomplished via viral transfection. The term “transgene encoding vector(s)” refers to the vectors incorporating the nucleic acid sequence(s) encoding transgenes named herein, especially encoding one or more transgenes named herein, as well as any additional sequences, synthetic oligonucleotides, etc., and any associated viral supernatant incorporating those vector sequences. In one embodiment, the transgene encoding vector(s) comprise two or more transgenes named herein for producing developmental activation. SeeFIG.1D. The transgene encoding vector(s) may comprise an expression vector. Appropriate expression vectors are those that may be employed for transfecting DNA or RNA into eukaryotic cells. Such vectors include, but are not limited to, prokaryotic vectors such as, for example, bacterial vectors; eukaryotic vectors, such as, for example, yeast vectors and fungal vectors; and viral vectors, such as, but not limited to adenoviral (Lin et al., 2007) vectors, adeno-associated viral vectors, and retroviral vectors. Examples of retroviral vectors which may be employed include, but are not limited to, those derived from Moloney Murine Leukemia Virus, Moloney Murine Sarcoma Virus, and Rous Sarcoma Virus, FIV, HIV, SIV and hybrid vectors, including the episomal, integrase-deficient, non-integrating, 3rdgeneration engineered lentiviral vectors (seeFIG.3D), described herein and/or described in references cited herein. Such vectors can be introduced safely without genomic integration or random alteration of the genome using electroporation and other methods taught herein. It is disclosed that the transgene encoding vector(s) may be used to transfect cells in vitro and/or in vivo. Transfection can be carried out by any means known to the art, especially through virus produced from viral packaging cells. Such virus may be encapsidated so as to be capable of infecting a variety of cell types. Nevertheless, any encapsidation technique allowing infection of selected cell types and/or their progeny is practicable within the context of the present invention. Design of Human Immunodeficiency Virus (HIV) Gene Therapy Vector(s) The “therapeutic vector(s)” may incorporate an expression vector. Appropriate expression vectors are those that may be employed for transfecting DNA or RNA into eukaryotic cells. Such vectors include, but are not limited to, prokaryotic vectors such as, for example, bacterial vectors; eukaryotic vectors, such as, for example, yeast vectors and fungal vectors; and viral vectors, such as, but not limited to adenoviral (Lin et al., 2007) vectors, adeno-associated viral vectors, and retroviral vectors. Examples of retroviral vectors which may be employed include, but are not limited to, those derived from Moloney Murine Leukemia Virus, Moloney Murine Sarcoma Virus, and Rous Sarcoma Virus, feline immunodeficiency virus (FIV), HIV, simian immunodeficiency virus (SIV) and hybrid vectors, including the replication incompetent, integrase-deficient, 3rdgeneration, engineered, episomal, non-integrating lentiviral vectors (seeFIG.3D), described herein and/or described in references cited herein. Such vectors can be introduced safely without genomic integration or random alteration of the genome using electroporation and other methods taught herein. It is disclosed herein that the therapeutic vector(s) may be used to transfect target cells in vitro and/or in vivo. Transfection can be carried out by any means known to the art, especially through virus produced from viral packaging cells. Such virus may be encapsidated so as to be capable of infecting CD34+ cells and/or CD4+ cells. However, in some instances, other cell types are transfected by means not involving the CD4 or CD34 proteins. Nevertheless, any encapsidation technique allowing infection of such cell types may therefore be included in the disclosure of the present invention. Pseudotyping with different envelope proteins expands the range of host cells transducible by viral vectors and therapeutic vectors and allows the virus to be concentrated to high titers, especially when pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) (Li et al., 1998; Reiser et al., 2000). Vector Construction Viral vectors utilized in this invention may be of various RNA and DNA virus types, including hybrid vectors. Vectors may, for instance, be third-generation lentiviral vectors which include only a very small fraction of the native genome (Zufferey et al., 1998). Production of transgene encoding vector(s) may also involve self-inactivating transfer vectors (Zufferey et al., 1998; Miyoshi et al., 1998) eliminating the production of full-length vector RNA after infection of target cells. Viral vectors may be utilized which are replication-incompetent due to failure to express certain viral proteins necessary for replication. However, the possibility exists that helper virus may enable therapeutic virus replication. This likelihood can be reduced by the use of vectors that are self-inactivating, as well as replication-incompetent and non-integrating, as described in references cited herein. In a preferred embodiment, transgene sequences are driven by a ubiquitin promoter, U6 promoter, EF1alpha promoter, CMV promoter, regulable promoters and/or desired cell type specific promoters. The lack of a functional integrase gene in these vectors renders them integrase-deficient and episomal, and is the consequence of inserting the EGFP expression cassette consisting of EGFP sequences and the CMV IE promoter into the region normally occupied the gag-pol genes (FIG.6Aof Reiser et al., 2000; seeFIG.1Dherein). When cell transduction is mediated by virus (i.e. viral particles) pol deletion interferes with reverse transcription of viral RNA to DNA. Accordingly, pol deletion results in a non-integrating or integration deficient lentivector. This is because “Integrase . . . is involved in the reverse transcription of HIV-1 RNA and nuclear import of the preintegration complex (PIC) (Gallay et al., 1997; Zhu et al., 2004; Philpott and Thraser, 2007). However, when one skilled in the art chooses not to use infection and instead chooses electroporation to introduce DNA lentiviral vectors to the cells, as taught by the applicant, no reverse transcription is necessary; and because the pol gene is deleted, no integrase enzyme is present in the cell to support integration. It has been taught by Wanisch and Yanez-Munoz (2009) that,“HIV-1 circles are considerably stable after infection, with progressive vector episome dilution due to cell division. Thus, the apparent decrease in circularized HIV-1 DNA after infection of CD4+ MT-2 or SupT1 T-cells is the result of ongoing cell division causing the dilution of nonreplicating viral episomes in the cell population. Episomes are stable in macrophages for at least 21 days (ref. 36), while a turnover of episomes has been observed in vivo in human peripheral blood mononuclear cells over the course of several weeks, suggesting that they can be slowly degraded.” (Wanisch and Yanez-Munoz, 2009). Accordingly, those skilled in the art recognize that integration deficient lentivectors may persist episomally for weeks or more after electroporation. Viral Tropism In a preferred embodiment, virus derived from the transgene encoding vector(s), therapeutic vector(s) and/or other transgeneic vector(s) of this invention is pseudotyped with vesicular stomatitis virus envelope glycoprotein to enable concentration of the virus to high titers and to facilitate infection of CD34+ cells. Sequence Selection The use of any sequence with 70% or greater identity (or complementarity) to any sequence referred to a transgene sequence named herein (searchable using the Entrez-Pubmed database) is covered by the invention if utilized in the manner described in the present invention. The current invention also relates in part to a genetic vector that includes sequences capable of markedly reducing the susceptibility of mammalian cells to infection by HIV 1 and HIV-2 viruses (both together referred to herein as HIV). The current invention discloses the novel combination of synthetic oligonucleotides to reduce the expression of genes critical to the HIV/AIDS disease process. The desirability of combining synthetic oligonucleotides to effect co-receptor “knock down” with expression of TAR and RRE decoy sequences arises from the proposition, expressed herein, that combining multiple gene therapy approaches simultaneously targeting 1) HIV infection, 2) HIV transcription, and 3) HIV replication in individual cells is likely to produce superior therapeutic benefits than any of these approaches in isolation. Therapeutic vector(s) express “beneficial sequence(s)” intended to render transfected or infected cells less capable of sustaining HIV replication and transcription. The genetic vector expressing “beneficial sequence(s)” as well as any virus derived from such genetic vector, are herein termed “therapeutic vector”. The present invention is directed in part to the genetic modification of cells susceptible to infection by HIV or capable of propagating HIV. Such cells are herein termed “target cells”. In one embodiment, a cell comprising a mutation or deletion in the CCR5 and/or CCRX4 co-receptors, and/or other co-receptors is developmentally-activated according to the methods described herein to provide pluripotent cells, pluripotent like cells, multipotent cells, hematopoietic progenitors and stem cells, T cells and/or macrophages such that the resulting T cells and macrophages were HIV-resistant. In a further embodiment, the CCR5 mutation or deletion is a 32 base pair deletion or other rendering the CCR5 gene non-functional. In a further embodiment, CRISPR/CAS9 or other site-directed mutational methods known to the art to produce mutation or deletion in the CCR5 and/or CCRX4 co-receptors. The present invention also provides a composition and method for using therapeutic viral vectors to reduce the susceptibility of mature or immature target cells, leukocytes, blood cells, any stem/progenitor cells, and/or their progeny (including DAdC) to infection by HIV. It follows that the present invention also provides a composition and method for using therapeutic viral vectors to reduce the susceptibility of developmentally activated cells, induced cells, reprogrammed cells, induced multipotent cells, induced pluripotent cells, and/or their progeny to infection by HIV. It is a further objective of this invention to reduce the ability of mature or immature target cells, stem/progenitor cells, (including developmentally activated cells, induced cells, reprogrammed cells, induced multipotent cells, induced pluripotent cells) and/or their progeny to sustain immunodeficiency virus replication and transcription. It is another objective of this invention to achieve efficient, long-term expression of the therapeutic sequences in mature or immature target cells, other quiescent cells, stem/progenitor cells, and/or their progeny. In one aspect, this invention provides a method for preventing or treating HIV infection. The method involves transplanting stem cells transfected with therapeutic vector(s) or sequence(s), into patients or subjects with HIV infection. Beneficial sequence(s) may be ones that reduce the ability of HIV to infect a cell, transcribe viral DNA, or replicate within an infected cell, or which enhances the ability of a cell to neutralize HIV infection. In certain embodiments, the beneficial sequence(s) represent synthetic oligonucleotide(s) which interfere with HIV entry, including one or more selected from siRNA, shRNA, antisense RNA or miRNA directed against any of the HIV co-receptors (including, but not limited to, CXCR4, CCR5, CCR2b, CCR3, and CCR1). In a preferred embodiment, the therapeutic vector(s) includes synthetic oligonucleotides targeting one or more HIV co-receptors including CXCR4, CCR5, CCR1, CCR2, CCR3, CXCR6 and/or BOB. In another preferred embodiment the therapeutic vector(s) includes synthetic oligonucleotides targeting the major HIV co-receptors CXCR4 and CCR5 In a further preferred embodiment, the therapeutic vector(s) includes synthetic oligonucleotides targeting one or more HIV enzymes such as HIV reverse transcriptase, integrase and protease. Appropriate sequences for the synthetic oligonucleotides are those 1) predictable by computer algorithms to be effective in reducing targeted sequences, and 2) capable of successfully reduce the amount of targeted enzyme by >70% in standard quantitative RNA assays and in assays of enzymatic activity or to a lesser but therapeutic degree. The phrase “targeted sequence” indicates that a particular sequence has a nucleotide base sequence that has at least 70% identity to a viral genomic nucleotide sequence or its complement (e.g., is the same as or complementary to such viral genomic sequence), or is a corresponding RNA sequence. In particular embodiments of the present invention, the term indicates that the sequence is at least 70% identical to a viral genomic sequence of the particular virus against which the oligonucleotide is directed, or to its complementary sequence. Any of the various types of synthetic oligonucleotides may be expressed via therapeutic vector transfection, and the current invention is directed to all possible combinations of such oligonucleotides. In a preferred embodiment, the synthetic oligonucleotide sequences are driven by target cell, specific promoter(s). In another preferred embodiment, the synthetic oligonucleotide sequences are driven by U6 promoter(s). Synthetic oligonucleotides, by the same token, may be included in the same therapeutic vector(s) with decoy RNA. Decoy RNA Decoy RNA are sequences of RNA that are effective at binding to certain proteins and inhibiting their function. In a preferred embodiment, the therapeutic vector(s) comprise(s) multiple decoy RNA sequences. In a further embodiment the decoy RNA sequences are flanked by sequences that provide for stability of the decoy sequence. In another preferred embodiment the decoy RNA sequences are RRE and/or TAR decoy sequences (or their corresponding proteins). In a preferred embodiment, the RRE and TAR decoy sequences are HIV-2 derived TAR and RRE sequences (or their corresponding proteins). In another preferred embodiment the decoy sequences also include Psi element decoy sequences (or their corresponding proteins). In a preferred embodiment, the decoy sequences are each driven by a U6 promoter. In another preferred embodiment, the decoy sequences are driven by target-cell specific promoters. In a preferred embodiment, the therapeutic vector targets multiple stages of the HIV life cycle by encoding synthetic nucleotide sequence(s) in combination with HIV-2 TAR and/or RRE decoy sequences (or their corresponding proteins). In another preferred embodiment, the vector includes miRNA oligonucleotide sequences (or their corresponding proteins). In another preferred embodiment, the vector includes shRNA oligonucleotide sequences (or their corresponding proteins). In another preferred embodiment, the vector includes siRNA oligonucleotide sequences (or their corresponding proteins). In another preferred embodiment, the vector includes RNAi oligonucleotide sequences (or their corresponding proteins). In another preferred embodiment, the vector includes ribozyme sequences (or their corresponding proteins). In another preferred embodiment, the vector includes a combination of synthetic oligonucleotide classes. In a further embodiment, the synthetic nucleotide sequences target HIV co-receptors such as CCR5, CXCR4, etc. In a further embodiment, the synthetic nucleotide sequences target HIV enzymes such as integrase, protease, reverse transcriptase, TAT, etc. In a further embodiment, the ribozyme sequences target HIV co-receptors such as CCR5, CXCR4, etc., or HIV enzymes such as integrase, protease, reverse transcriptase, TAT, etc. In a preferred embodiment, virus is generated using the therapeuic vector(s) and the virus is pseudotyped. In a preferred embodiment, virus is generated using the therapeuic vector(s) and the virus is not pseudotyped and the virus shows native HIV tropism. In a preferred embodiment, the therapeutic vector(s) is a viral vector. In a preferred embodiment, the therapeutic vector(s) is a lentiviral vector. In a preferred embodiment, the therapeutic vector(s) is a third-generation lentiviral vector. In a preferred embodiment, the therapeutic vector(s) includes a combination of synthetic oligonucleotide classes. In a preferred embodiment, synthetic nucleotide sequence expression is driven by the EF-1 alpha promoter or other target-cell appropriate promoters. In a preferred embodiment, synthetic nucleotide sequence expression is driven by the U6 promoter or other target-cell appropriate promoters. In a preferred embodiment, synthetic nucleotide sequence expression is driven by a combination of EF-1 alpha and U6, and/or other target-cell appropriate promoters. In a preferred embodiment, EF-1 alpha drives miRNA expression while the U6 promoter drives RNA decoy expression. In a preferred embodiment, EF-1 alpha drives siRNA sequence expression while the U6 promoter drives RNA decoy expression. In a preferred embodiment, EF-1 alpha drives shRNA sequence expression while the U6 promoter drives RNA decoy expression. In a preferred embodiment, the therapeutic vector(s) include synthetic oligonucleotides (e.g. multiple miRNA sequences)) directed against CXCR4, multiple sequences directed against CCR5, an HIV-2 RRE decoy sequence and an HIV-2 TAR decoy sequence, and the vector is a viral vector. SeeFIG.15. In a preferred embodiment, treatment involving the therapeutic vector(s) is combined with other modes of antiretroviral therapy including pharmacological therapies. Antiretroviral therapies appropriate for combination with the therapeutic vector(s) are those that have additive or synergistic effects in combination with the therapeutic vector. Cells targeted for gene therapy in HIV may include, but are not necessarily be limited to mature peripheral blood T lymphocytes, monocytes, tissue macrophages, T cell progenitors, macrophage-monocyte progenitor cells, and/or multipotent hematopoietic stem cells, such as those found in umbilical cord blood, peripheral blood, and occupying bone marrow spaces. The present invention also relates to transfection of CD4+ T cells, macrophages, T cell progenitors, macrophage-monocyte progenitors, CD 34+ stem/progenitor cells and/or any other quiescent cell, dividing cell, stem cell or progenitor cell capable of differentiation in vitro or in vivo into HIV target cells, CD4+ T cells, macrophages, T cell progenitors, macrophage-monocyte progenitors, and/or CD 34+ stem/progenitor cells. Transfected cells, therefore, can be endogenous cells in situ, or exogenous cells derived from other body regions or even other individual donors. Cells selected for this purpose are herein termed “selected cells”. By the same token, self-renewing, multipotent and/or pluripotent stem cells (including reprogrammed and induced pluripotent cells) represent another logical target for HIV gene therapy, and their use is specifically covered by the present invention. In one embodiment of this process, selected cells (e.g. hematopoietic stem cells, skin stem cells, umbilical cord cells, primordial germ cells (PGCs), spermatogonia, any accessible somatic cell, etc.) are 1) propagated in culture using one or more cytokines such as steel factor, leukemia inhibitory factor (LIF), cardiotropin-1, IL-11, IL-6, IL-6 R, GP-130, CNTF, IGF-I, bFGF, and/or oncostatin-M and 2) transfected with the therapeutic vector(s) or beneficial sequence(s) prior to differentiation using any methods known to the art, such as those described in U.S. Pat. No. 5,677,139 herein incorporated by reference, or by methods analogous to U.S. Pat. No. 5,677,139 with respect to other target cells. In separate embodiments, it may be desirable to perform the various steps prior to transfection. In separate embodiments, for the purpose of generating pluripotent stem cell populations, it may be desirable to perform only the incubation steps above. Appropriate concentrations of LIF and steel factor for stem/progenitor cell propagation/proliferation as well as other cell culture conditions have been described previously (e.g. U.S. Pat. Nos. 6,432,711 and 5,453,357 herein incorporated by reference). Other appropriate protocols and reference cytokine concentrations have been taught by Koshimizu et al., 1996; Keller et al., 1996; Piquet-Pellorce, 1994; Rose et al., 1994; Park and Han, 2000; Guan et al., 2006; Dykstra et al., 2006). The population of target cells may include somatic cells, stem cells and progenitor cells. Stem cells may be derived from existing cell lines or isolated from stored, banked, or cryopreserved sources. Typical sources of stem cells include marrow, peripheral blood, placental blood, amniotic fluid, umbilical cord blood, adipose tissue, non-human embryos, etc. Somatic cells, especially circulating leukocytes and other non-progenitor/stem cells may likewise be subjected to the same culture conditions as described above for stem/progenitor cells effective that they acquire stem/progenitor cell properties as a result. The invention also discloses the production (e.g. US Patent Application 20030099621) of target cells from stem/progenitor cells that may be made relatively resistant to HIV infection and/or HIV replication. It is understood, however, that any method of differentiating previously propagated stem/progenitor/leukocyte cells into the desired target cells may be employed within the scope of the invention so long as functional target cells relatively resistant to HIV infection and/or HIV replication/and/or HIV transcription are produced. In a preferred embodiment, the therapeutic viral vector is packaged with one or more envelope proteins from native HIV viruses conferring upon the therapeutic virus the capacity to infect any cell that native HIV strains are capable of infecting. Cells selected for use in this invention wll be in some instances accessible (e.g. umbilical cord stem cells, bone marrow stem cells, spermatogonia and primordial germ cells of the testis, stem cells isolated from amniotic fluid, stem cells isolated from the skin, etc.). Such cells can be isolated from the tissues in which they reside by any means known to the art. Other selected cells may comprise reprogrammed cells, induced multipotent cells, induced pluripotent cells, etc. In accordance with an aspect of the present invention, there is provided a method of producing a desired cell line, cell type, or cell class from the selected cells. Generally, the method comprises culturing the selected cells and/or their progeny under conditions which promote growth of the selected cells at an optimal growth rate. The resulting cell population is then cultured under conditions which promote cell growth at a rate which is typically less than the optimal rate, and in the presence of an agent promoting differentiation of the cells into the desired cell line, cell type, or cell class (e.g. CD4+ T cells). The present invention also discloses the propagation of the selected cells and/or their progeny in culture, before or after transfection with (or overexpression of) the therapeutic vector, by any means known to the art (e.g. US Patent Application 20060099177). Such methods also include incubation with LIF, steel factor, Il-6, IL-7, oncostatin-M and/or cardiotropin-1 and other growth enhancing cytokines, etc. The present invention further discloses the directed differentiation of cells transfected with the therapeutic vector(s) into desired cell types by further incubation in media containing the appropriate cytokines and growth factors such as colony stimuating factors such as M-CSF (CSF-1), GM-CSF, IL-7, any cytokine promoting CD4+ T cell differentiation, etc. Transfection Genetic modification of selected cells and target cells, whether they be exogenous cells or endogenous cells can be performed according to any published or unpublished method known to the art (e.g. U.S. Pat. Nos. 6,432,711, 5,593,875, 5,783,566, 5,928,944, 5,910,488, 5,824,547, CRISPR/CAS9, etc.) or by other generally accepted means. Suitable methods for transforming host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks. Successfully transfected cells can be identified by selection protocols involving markers such as antibiotic resistance genes in addition to RNA expression assays and morphological analyses. Clones from successfully transfected cells, expressing the appropriate exogenous DNA at appropriate levels, can be preserved as cell lines by cryopreservation (utilizing any appropriate method of cryopreservation known to the art). Selectable markers (e.g., antibiotics resistance genes) may include those which confer resistance to drugs, such as G418, hygromycin, ampicillin and blasticidin, etc. Cells containing the gene of interest can be identified by drug selection where cells that have incorporated the selectable marker gene survive, and others die. A theoretical basis for the embodiments of the invention is described herein, however, this discussion is not in any way to be considered as binding or limiting on the present invention. Those of skill in the art will understand that the various embodiments of the invention may be practiced regardless of the model used to describe the theoretical underpinnings of the invention. The invention will now be described and illustrated with respect to the following examples; however, the scope of the present invention is not intended to be limited thereby. Example 1: Construction of the Transgenic Vectors Suitable for Use in the Present Invention Suitable EGFP-Numb and EGFP-Numblike, and EGFP-X lentiviral vectors (where X is any transgene described in the present invention) can be produced by cloning into an appropriate viral vector (e.g. the two-gene HIV-EGFP-HSA vector (Reiser et al., 2000)). Adapter primers can be selected for PCR amplification of Numblike and Numb isoform cDNAs and cloning into a genetic vector. In preparation for cloning, the gene vector is digested with enzymes. Subsequently, the cDNA for each transgene is inserted into the nef coding region previously occupied by the HSA cDNA-EGFP (enhanced green fluorescent protein) and a cell population-appropriate promoter (e.g. CMV ie or EF1alpha) having been previously inserted into the viral, gag-pol coding region. The lack of a functional integrase gene in these vectors renders them integrase-deficient and episomal, and is the consequence of inserting the EGFP expression cassette consisting of EGFP sequences and the CMV IE promoter into the region normally occupied the gag-pol genes (FIG.6Aof Reiser et al., 2000; seeFIG.3Dherein). Such integrase-deficient vectors can be readily introduced using a variety of standard transfection techniques (e.g. electroporation, chemically mediated transfection, fusogenic or non-fusogenic liposomes, lipofectamine, nanocapsules, nanovaults, etc.)—methods which allow high capacity integrase-deficient lentiviral vectors to be utilized without genomic integration and random alteration of the genome. When cell transduction is mediated by virus (i.e. viral particles) pol deletion interferes with reverse transcription of viral RNA to DNA. Accordingly, pol deletion results in a non-integrating or integration deficient lentivector. This is because “Integrase . . . is involved in the reverse transcription of HIV-1 RNA and nuclear import of the preintegration complex (PIC) (Gallay et al., 1997; Zhu et al., 2004; Philpott and Thraser, 2007). However, when one skilled in the art chooses not to use infection and instead chooses electroporation to introduce DNA lentiviral vectors to the cells, as taught by the applicant, no reverse transcription is necessary; and because the pol gene is deleted, no integrase enzyme is present in the cell to support integration. It has been taught by Wanisch and Yanez-Munoz (2009) that, “HIV-1 circles are considerably stable after infection, with progressive vector episome dilution due to cell division. Thus, the apparent decrease in circularized HIV-1 DNA after infection of CD4+ MT-2 or SupT1 T-cells is the result of ongoing cell division causing the dilution of nonreplicating viral episomes in the cell population. Episomes are stable in macrophages for at least 21 days (ref. 36), while a turnover of episomes has been observed in vivo in human peripheral blood mononuclear cells over the course of several weeks, suggesting that they can be slowly degraded.” (Wanisch and Yanez-Munoz, 2009). Accordingly, those skilled in the art recognize that integration deficient lentivectors may persist episomally for weeks or more after electroporation. Genetic constructs may include a vector backbone, and a transactivator which regulates a promoter operably linked to heterologous nucleic acid sequences (or their corresponding proteins). Examples of retroviral vectors which may be employed include, but are not limited to, those derived from Moloney Murine Leukemia Virus, Moloney Murine Sarcoma Virus, and Rous Sarcoma Virus, FIV, and HIV. Appropriate expression vectors are those that may be employed for transfecting DNA or RNA into eukaryotic cells. Such vectors include, but are not limited to, prokaryotic vectors such as, for example, bacterial vectors; eukaryotic vectors, such as, for example, yeast vectors and fungal vectors; and viral vectors, such as, but not limited to, lentiviral vectors, adenoviral (Lin et al., 2007) vectors, adeno-associated viral vectors, and retroviral vectors. SeeFIG.1as well as U.S. Provisional Application Ser. No. 60/932,020, filed May 29, 2007, U.S. Provisional Application Ser. No. 60/933,133, filed Jun. 5, 2007, U.S. Provisional Application Ser. No. 60/933,670, filed Jun. 8, 2007, U.S. Provisional Application Ser. No. 61/006,449, filed Jan. 14, 2008, U.S. Provisional Application Ser. No. 61/064,761, filed Mar. 25, 2008. The replication incompetent pcDNA 6.2/EmGFP-Bsd/V5-DEST vector is an example of an appropriate expression vector (Invitrogen) and allows expression of synthetic oligonucleotides (e.g. miRNAs) transferred from the pcDNA 6.2 GW/miR vector that have the capacity to cleave targeted sequences (or their corresponding proteins). These vectors include flanking and loop sequences from endogenous miRNA to direct the excision of the engineered miRNA from a longer Pol II transcript (pre-miRNA). Combining multiple miRNA sequences directed against specific endogenous RNA species increases the likelihood of success in reducing target sequence expression. miRNA sequences may be operably linked to regulable or tissue specific promoters. By utilizing lentiviral vectors for gene expression, the resulting transgene encoding vector(s) and/or other transgenic vector(s) of this invention, becomes capable of stably transducing both dividing and non-dividing cell types. Moreover, 2nd and 3d generation, integrase-deficient lentiviral vectors provide a non-integrating, episomal vector suitable, along with adenoviral (Lin et al., 2007), AAV, hybrid vectors, plasmid DNA, etc. for use in the present invention. (SeeFIG.1D). In a preferred embodiment, the resulting Numb/Numblike encoding vector(s), and/or other transgenic vector(s) of this invention contain multiple synthetic oligonucleotide sequences driven by one or more promoters so as to reduce expression of specific numb isoforms and/or numblike (FIG.3D). Example 2 Another example of a suitable vector is a retroviral vector. Retroviruses are RNA viruses that contain an RNA genome. The gag, pol, and env genes are flanked by long terminal repeat (LTR) sequences (or their corresponding proteins). The 5′ and 3′ LTR sequences promote transcription and polyadenylation of mRNAs. The retroviral vector may provide a regulable transactivating element, an internal ribosome reentry site (IRES), a selection marker, and a target heterologous gene operated by a regulable promoter. Alternatively, multiple sequences may be expressed under the control of multiple promoters. Finally, the retroviral vector may contain cis-acting sequences necessary for reverse transcription and integration. Upon infection, the RNA is reverse transcribed to DNA that integrates efficiently into the host genome. The recombinant retrovirus of this invention is genetically modified in such a way that some of the retroviral, infectious genes of the native virus have been removed and in certain instances replaced instead with a target nucleic acid sequence for genetic modification of the cell. The sequences may be exogenous DNA or RNA, in its natural or altered form. Example 3: Example Methods for Generation of Numb/Numblike Encoding Vector(s), and/or Other Transgenic Vector(s) of this Invention The methods for generation of the resulting Numb/Numblike encoding vector(s), and/or other transgenic vector(s) of this invention include those taught in Invitrogen's Viral Power Lentiviral Expression Systems Manual, 2007. Briefly, the EmGFP-bsd cassette is cloned as a PmlI-BlpI fragment into the pLenti6/R4R2/V5-DEST vector, while the miR-long (PRR+) numb isoform or miR-short numb isoform/numblike cassettes are simultaneously transferred by BP reaction into pDONR221. Then the regulable promoter(s) and miR-isoform cassettes are Multi-site LR crossed into the modified pLenti6/EmGFP-bsd/R4R2-DESTvector. Multiple vectors can be generated in this manner comprising different combinations of synthetic oligonucleotides and transgene cassettes. The pLenti6/R4R2/V5-DEST vector sequence corresponds to (SEQ ID NO: 1). Example 4: Additional Methods for Generation of Therapeutic Vector(s) “Packaging cell lines” derived from human and/or animal fibroblast cell lines result from transfecting or infecting normal cell lines with viral gag, pol, and env structural genes. On the other hand, packaging cell lines produce RNA devoid of the psi sequence, so that the viral particles produced from packaging cell do not contain the gag, pol, or env genes. Once the therapeutic vector's DNA containing the psi sequence (along with the therapeutic gene) is introduced into the packaging cell, by means of transfection or infection, the packaging cell may produce virions capable of transmitting the therapeutic RNA to the final target cell (e.g. a CD4+ cell). The “infective range” of the therapeutic vector(s) is determined by the packaging cell line. A number of packaging cell lines are available for production of virus suitable for infecting a broad range of human cell types. These packaging cell lines are nevertheless generally capable of encapsidating viral vectors derived from viruses that in nature usually infect different animal species. For example, vectors derived from SIV or MMLV can be packaged by GP120 encapsidating cell lines. An example protocol for producing a therapeutic viral supernatant is provided as follows: 1. Twenty micrograms of retrovirus vector are mixed with 2-3 micrograms of viral DNA containing the selectable marker gene (e.g. antibiotic resistance gene) by gentle tapping in 0.8-1 milliliter of Hepes buffered saline (pH=7.05) in a 1.5 ml plastic tube. 2. Seventy microliters of 2M CaCl2are added to the mixture by repeated gentle tapping. 3. When a blue precipitate first begins to appear within the tube, the product should be gently applied to a 30% confluent layer of packaging cells (from any number of commercial vendors). The DNA mixture should be applied only after first removing the medium from the packaging cells. 4. The packaging cells are set to incubate for 20-30 minutes at room temperature (25 degrees Celsius) before transferring them back to an incubator at 36-38 degrees Celsius for 3.5 hours. 5. Add 3.5-4 milliliters of Hepes buffered saline containing 15% glycerol for 3 minutes then wash cell with Dulbecco's Modified Eagle's Medium (DMEM)+10% FBS×2. 6. Add back DMEM +10% FBS, and incubate cells for 20 hours at 37 degrees Celsius. 7. Remove and filter medium containing therapeutic viral particles. Excess viral supernatant is immediately stored or concentrated and stored at −80 degrees Celsius). Supernatant may stored with 5-8 micrograms of polybrene to increase the efficiency of target cell infection. Otherwise polybrene may be excluded or added just before infection. 8. Stable producer lines can be established by splitting packaging cell lines 1 to 20, or 1 to 40 and subsequently incubating these cells for up to 10 days (changing medium every three days) in medium containing selective drugs (e.g. certain antibiotics corresponding to transfected resistance genes). 9. After 10 days isolated colonies are picked, grown-up aliquoted and frozen for storage. Assay of Retrovirus Infectivity/Titration is achieved by application of a defined volume of viral supernatant to a layer of confluent “test” cells such as NIH 3T3 cells plated at 20% confluence. After 2-3 cell division times (24-36 hours for NIH 3T3 cells) colonies of “test” cells incubated at 37 degrees in antibiotic-containing medium are counted. The supernatant's titer are estimated from these colony counts by the following formula: Colony Forming Units/ml=colonies identified×0.5(split factor)/volume of virus (ml) The accuracy of this estimate is increased by testing large volumes of supernatant over many plates of “test” cells. Application of the therapeutic viral supernatant to target cells may be accomplished by various means appropriate to the clinical situation. Example 5: Growth Medium for Selected Cells Selected cells can be expanded/grown in Dulbecco's modified Minimal Essential Medium (DMEM) supplemented with glutamine, beta.-mercaptoethanol, 10% (by volume) horse serum, and human recombinant Leukemia Inhibitory Factor (LIF). LIF replaces the need for maintaining selected cells on feeder layers of cells, (which may also be employed) and is essential for maintaining selected cells in an undifferentiated, multipotent, or pluripotent state, such cells can be maintained in Dulbecco's modified Minimal Essential Medium (DMEM) supplemented with glutamine, beta.-mercaptoethanol, 10% (by volume) horse serum, and human recombinant Leukemia Inhibitory Factor (LIF). The LIF replaces the need for maintaining cells on feeder layers of cells, (which may also be employed) and is essential for maintaining cells in an undifferentiated state (per U.S. Pat. No. 6,432,711). In order to initiate the differentiation of the selected cells into neuronal cells, the cells are trypsinized and washed free of LIF, and placed in DMEM supplemented with 10% fetal bovine serum (FBS). After resuspension in DMEM and 10% FBS, 1×106cells are plated in 5 ml DMEM, 10% FBS, 0.5 microM retinoic acid in a 60 mm Fisher bacteriological grade Petri dishes, where the cells are expected to form small aggregates. Aggregation aids in proper cell differentiation. High efficiency transfection with (or overexpression of) appropriate neuronal transcription factors and small RNAs can occur before or after plating in DMEM, FBS, and retinoic acid. (See U.S. Pat. Nos. 6,432,711 and 5,453,357 for additional details). Example 6 HLA matching. Selected cells (e.g. umbilical cord blood or cells from any other suitable source and/or their progeny), can be screened, genetically-modified (optional), expanded, and induced to begin differentiating into the desired cell type(s) (optional). The cells are then transplanted according to standard stem cell transplantation protocols. In certain instances, cells may be transplanted into patients or subjects without HLA matching. Example 7 In some rare instances, it may be appropriate to introduce transgene encoding vectors into patients or subjects in order to stimulate or inhibit cellular division or cellular differentiation, in vivo (e.g. in cancer). Example 8: Genetic Modification of Selected Cells In vitro genetic modification of exogenous cells or patient's endogenous cells can be performed according to any published or unpublished method known to the art (e.g. U.S. Pat. Nos. 6,432,711, 5,593,875, 5,783,566, 5,928,944, 5,910,488, 5,824,547, etc.) or by other generally accepted means. Suitable methods for transforming host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks. Successfully transfected cells are identified by selection protocols involving markers such as antibiotic resistance genes in addition to RNA expression assays and morphological analyses. Clones from successfully transfected cells, expressing the appropriate exogenous DNA at appropriate levels, can be preserved as cell lines by cryopreservation (utilizing any appropriate method of cryopreservation known to the art). Selectable markers (e.g., antibiotic resistance genes) may include those conferring resistance to drugs, such as G418, hygromycin and methotrexate. Cells containing the gene of interest can be identified by drug selection where cells that have incorporated the selectable marker gene survive, and others die. The current invention discloses the selection of genetically-modified cells as “selected cells” of the invention. The term genetic modification refers to alteration of the cellular genotype by introducing natural or synthetic nucleic acids into selected cells and/or their progeny or immortalized cell lines and/or their progeny by any means known to the art. Alternatively culture conditions that induce permanent changes in gene expression patterns are considered herein to represent genetic modification. Modification of stem cells, whether they be derived from the host brain, endogenous donor sources, exogenous donor sources, or cell lines, represents a feasible approach to the treatment of certain human diseases, especially those of the human nervous system. Genetic modifications covered by this disclosure include, but are not limited to: genetic modifications performed in vivo; modifications that alter the activity or amount of metabolic enzymes expressed by endogenous or exogenous selected cells and/or their progeny; modifications which alter the activity, amount, or antigenicity of cellular proteins; modifications which alter the activity or amount of proteins involved in signal transduction pathways; modifications which alter HLA type; modifications which alter cellular differentiation; modifications which alter neoplastic potential; modifications which alter cellular differentiation; modifications which alter the amount or activity of structural proteins; modifications which alter the amount or activity of membrane associated proteins (structural or enzymatic); modifications which alter the activity or amount of proteins involved in DNA repair and chromosome maintenance; modifications which alter the activity or amount of proteins involved in cellular transport; modifications which alter the activity or amount of enzymes; modifications which alter the activity or amount of proteins involved in synapse formation and maintenance; modifications which alter the activity or amount of proteins involved in neurite outgrowth or axon outgrowth and formation; modifications altering the amount or activity of antioxidant producing enzymes within the cell; modifications which lead to altered post-translational modification of cellular proteins; modifications which alter the activity or amount of proteins involved in other aspects of cellular repair, and alterations which increase the lifespan of the cell (such as production of telomerase). Such proteins as those mentioned above may be encoded for by DNA or RNA derived from the human genome or other animal, plant, viral, or bacterial genomes. This invention also covers sequences (or their corresponding proteins) designed de novo. In addition, this invention relates to the in situ, genetic modification of selected cells and/or their progeny cells for the treatment of disease. Endogenous stem cells may be modified in situ by direct injection or application of DNA or RNA vectors, including viruses, retroviruses, liposomes, etc., into the substance of the tissue or into the appropriate portion of the ventricular system of the brain. Since 1992, we have modified thousands of stem/progenitor cells and many thousand progeny cells in this manner. Our data shows that this manner of modifying progenitor cells results in a tremendous variety of modified cell types throughout the nervous system, and has never resulted in adverse effects. Example 9: Introduction of Genetic Vectors into the Host In a preferred embodiment, endogenous cells are transfected with vectors such as those described herein in vivo by introduction of the therapeutic vector(s) into the host blood, tissues, nervous system, bone marrow, etc. The greatest benefit may be achieved by modifying a large number of endogenous target cells. This may be accomplished by using an appropriately-sized, catheter-like device, or needle to inject the therapeutic vector(s) into the venous or arterial circulation, into a specific tissue, such as muscle tissue, or into the nervous system. In a preferred embodiment, the virus is pseudotyped with VSV-G envelope glycoprotein and native HIV-1 env proteins. Example 10: Injection into the Nervous System Transplantation of selected cells (from either the growth or differentiation media) into the fetal nervous system or genetic modification of endogenous fetal cells utilizing genetic vectors may be accomplished in the following manner: Under sterile conditions, the uterus and fetuses are visualized by ultrasound or other radiological guidance. Alternatively the uterus may be exposed surgically in order to facilitate direct identification of fetal skull landmarks. Selected cells can then be introduced by injection (using an appropriately-sized catheter or needle) into the ventricular system, germinal zone(s), or into the substance of the nervous system. Injections may be performed in certain instances, through the mother's abdominal wall, the uterine wall and fetal membranes into the fetus. The accuracy of the injection is monitored by direct observation, ultrasound, contrast, or radiological isotope based methods, or by any other means of radiological guidance known to the art. Under appropriate sterile conditions, direct identification of fetal skull landmarks is accomplished visually as well as by physical inspection and palpation coupled with stereotaxic and radiologic guidance. Following cell culture, appropriate amounts of the selected or differentiating cells can then be introduced by injection or other means into the ventricular system, germinal zones, or into the substance of the nervous system. The accuracy of the injection may be monitored by direct observation, ultrasound, or other radiological guidance. In certain, neurological diseases of the adult nervous system, such as Huntington's disease and Parkinson's disease, cells of a specific portion of the brain are selectively affected. In the case of Parkinson's disease, it is the dopaminergic cells of the substantia nigra. In such regionally-specific diseases affecting adults, localized transplantation of cells may be accomplished by radiologically-guided transplantation of differentiating cells under sterile conditions. Radiologic guidance may include the use of CT and/or Mill, and may take advantage of contrast or isotope based techniques to monitor injected materials. In certain neurologic diseases, such as some metabolic storage disorders, cells are affected across diverse regions of the nervous system, and the greatest benefit may be achieved by genetically-modifying endogenous cells or introducing selected cells of the present invention (either from the growth culture media or the differentiating medium) into the tissue in large numbers in a diffuse manner. In the nervous system, these diseases may be best approached by intraventricular injections (using an appropriately-sized, catheter-like device, or needle) (especially at early stages of development) which allows diffuse endogenous cell modification or diffuse engraftment of selected cells isolated from the growth and/or differentiation media. Nevertheless, injection of the cells into the circulatory system for the same purpose is also covered. However, with regard to any disorder affecting multiple organs or the body diffusely (e.g. lysosomal storage disorders, hemoglobinopathies, muscular dystrophy), the cells isolated from the growth and/or differentiation media may also be preferentially introduced directly into the circulation and/or visceral organs, such as the liver, kidney, gut, spleen, adrenal glands, pancreas, lungs, and thymus using endoscopic guidance and any appropriately-sized, catheter-like device, allowing diffuse engraftment of the cells throughout the body, as well as specific introduction and infiltration of the cells into the selected organs. Example 11: Delivery of Cells by Injection into the Circulatory Stream and Organs Diseases of one organ system may be treatable with genetically modified cells from a separate organ system. Also, in some instances, it may become apparent that the selected cells may integrate and differentiate on their own, in vivo, in sufficient numbers if they are injected into blood stream either arterial, venous or hepatic, after culturing in the growth and/or differentiation media. This approach is covered by the present invention. The treatment of diffuse muscle (e.g. muscular dystrophies), organ, tissue, or blood disorders (e.g. Hereditary Spherocytosis, Sickle cell anemia, other hemoglobinopathies, etc.), may, for instance, involve the injection of cells isolated from the growth media or differentiating media into the patient, especially the patient's circulation. This approach is also believed to ameliorate ischemic injuries such as myocardial infarction, stroke, etc., as well as traumatic injuries to brain and other tissues. Injection of such cells produced by the current invention, directly into the circulation, by needle or catheter, so that the cells are enabled to “home” to the bone marrow, muscle, kidneys, lungs, and/or any other other organ system, as well as injection directly into the bone marrow space is suitable for the practice of the present invention. Likewise injection of the cells directly into a lesion site with or without radiologic, ultrasonic or fluoroscopic guidance is also suitable for the practice of the present invention. Methods of isolating selected cells useful in the present invention include those described by Zhao et al., 2006. In a preferred embodiment, genetic vectors encoding numblike and/or numb isoforms comprise regulable promoters operably linked to the Numb or numblike transgenes (or their corresponding proteins). In another preferred embodiment, the mode of transfection may be selected from those modes of transfection that provide for transient rather than permanent expression of the numblike and numb isoforms. Example 12: Example Genetic Modifications Hundreds of diseases and clinical conditions are able to be treated and/or ameliorated by the methods of the present invention wherein a gene deficient in a patient is replaced or corrected by heterologous cells provided according to the present invention, or by autologous cells provided according to the present invention having the deficient gene replaced or repaired by genetic modification methods. Further, the transgenes, and vectors of the present invention may be delivered in vivo. Finally, proteins, including CRISPR/CAS9 related proteins, may be delivered by electroporation in vivo or in vitro, as taught herein. Examples of diseases amenable to such correction, replacement or repair include, but in no way are limited to, Canavan's disease (ASP); Tay-Sachs disease (HEXA); Lesch-Nyhan syndrome (HRPT); Huntington's disease (HTT); Sly syndrome; type A and type B Niemann Pick disease; Sandhoff s disease (HEXB); Fabry's disease (GLA); type C Niemann-Pick disease (NPC1); Gaucher's disease (GBA); Parkinson's disease (PARK2, etc.); Von Hippel Lindau's disease, Sickle cell anemia (HBB) and other thalassemias as well as similar diseases. These transgenes may represent the coding region or portions of the coding region of the normal genes. It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments and examples described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims. Example 13 An example sequence for a vector capable of rendering cells pluripotent and expressing a long Numb isoform, Oct-4, Sox-2, and EmGFP nucleic acid sequences under the control of tetracycline-sensitive promoters corresponds to (SEQ ID NO: 2). A schematized map corresponding to the vector sequence above is shown inFIG.3C. The vector may be constructed fully through de novo gene synthesis, or in part through the cloning of the Numb, Sox and OCT3/4 cDNA sequences into the position occupied by LacZ in the Invitrogen pcDNA4tolacZ vector. Similarly, the tetR gene is found in the Invitrogen pcDNA6/TR vector. Coding sequences of genes referenced are also appropriate for cloning into the pcDNA4lacZ vector. Alternatively, the tetR gene may be transfected into target cells separately utilizing the pcDNA6/TR vector in combination with a vector comprising the sequence here minus the tetR gene and its PCMV promoter. Likewise, multiple vectors may be employed so long as elements similar to the elements included in the above sequence are present. This may reduce the likelihood of promoter competition. It is to be understood that other conditional promoter elements may be substituted for the tetracycline sensitive promoter elements. Also depicted inFIG.3AandFIG.3B, are other lentiviral vectors expressing Oct4 and Sox2 or PRR+ Numb. Integrase-deficient 2ndand 3rdgeneration lentiviral vectors may be utilized as non-integrating lentiviral vectors can be used as episomal vectors, in like manner to adenoviral (Lin et al., 2007), AAV, hybrid vectors, plasmid DNA, and other non-integrating vectors known to the art. Such integrase-deficient vectors can be readily introduced using a variety of standard transfection techniques (e.g. electroporation, chemically mediated transfection, fusogenic or non-fusogenic liposomes, lipofectamine, nanocapsules, nanovaults, etc.)—methods which allow high capacity integrase-deficient lentiviral vectors to be utilized without genomic integration and random alteration of the genome. Example 14 It is expected that intravenous and other administration of pluripotent stem cells produced according to the methods described herein (or other published methods) one or more times can provide replacement cells to the body and that such administration may serve to extend the life or improve the health of the patient suffering age-related senescence. Example 15. Production of Germ Cells The current invention covers the derivation of germ cells from dividing multipotent, pluripotent, “VSEL-like” and/or “pluripotent-like” stem cells produced according to the methods described herein (or according to other published methods). The production of such germ cells may be suitable for treating infertility and producing embryos in vitro (e.g. Hubner et al., 2003; Kehler et al., 2005; Nayernia et al., 2006a; Nayernia et al., 2006b; Drusenheimer et al., 2007; Moore et al., 2007; etc.). Likewise, the invention further covers transient or permanent transfection/contacting with other proteins and/or nucleic acid sequences, including ones selected from those encoding FIGLA, FIG alpha, DAZL, STRA8, FOXL2, OOGENESIN1, OOGENESIN2, OOGENESIN3, OOGENESIN4, SYCP2, SYCP3, SPO11, REC8, DMC1, MOS, STAG3, CCNB1, FOXO1, FOXO3, SOHLH1, SOHLH2, NOBOX, OBOX1, OBOX2, OBOX3, OBOX4, OBOX6, LHX8, LHX9, OOG1, SP1, ZFP38, TRF2, TB2/TRF3, TAF4B, TAF7L, TAF71, TIA1, PHTF1, TNP2, HILS1, DAZL, BMP15, PTTG3, AURKC, OTX2, SOX15, SOX30, FOXR1, ALF, OCT4, DPPA3/STELLA, ZFP38, RPS6KA3, HINFP, NPAT, SP1, SP3, HOXA1, HOXA7, HEX, YP30, ZP1, ZP2, ZP3, SFE1, SFE9, OPO, PLN, RDV, GLD1, MMU-MiR351, MMU-MiR615, MMU-MiR592, MMU-MiR882, MMU-MiR185, MMU-MiR491, MMU-MiR326, MMU-MiR330, MMU-MiR351. Likewise, the invention further covers transfection/contacting with other proteins and/or nucleic acid sequences, including ones selected from those encoding SYCP2, SYCP3, SPO11, REC8, DMC1, MOS, STAG3, OCT4, ALF, RPS6KA3, HINFP, SP1, SP3, TAF71, TIA1, PHTF1, TNP2, HILS1, CLGN, TEKT1, FSCN3, DNAHC8, LDHC, ADAM3, OAZ3, AKAP3, MMU-MiR351, MMU-MiR615, MMU-MiR592, MMU-MiR882, and MMU-MiR185. Likewise, the invention further covers transfection/contacting with other proteins and/or nucleic acid sequences, including ones selected from those encoding MOS, CCNB1, OCT4, FIG alpha, FIGL alpha, ALF, SOHLH1, SOHLH2, LHX8, LHX9, OOG1, FIG alpha, SP1, LHX3, LHX9, TBP2/TRF3, DAZL, BMP15, GDF9, PTTG3, AURKC, OTX2, SOX15, SOX30, FOXR1, NOBOX, OBOX1, OBOX2, OBOX3, OBOX6, OOGENESIN1, OOGENESIN2, OOGENESIN3, OOGENESIN4, YP30, ZP1, ZP2, ZP3, SFE1, SFE9, OPO, PLN RDV, GLD1, DAZL, STRA8, MMU-MiR615, MMU-MiR491, MMU-MiR326, MMU-MiR330, MiR212 and MMU-MiR351. Example 16: Generation of Transgenic Animals The present invention covers the generation of transgenic animals. As with other pluripotent cells, the pluripotent or pluripotent-like cells produced according to the methods described herein (or other published methods) may be utilized to produce transgenic animals by any method known to the art. Example 17: Therapeutic Vector Construction Examples of retroviral vectors which may be employed include, but are not limited to, those derived from Moloney Murine Leukemia Virus, Moloney Murine Sarcoma Virus, and Rous Sarcoma Virus, FIV, and HIV. Appropriate expression vectors are that may be employed for transfecting DNA or RNA into eukaryotic cells. Such vectors include, but are not limited to, prokaryotic vectors such as, for example, bacterial vectors; eukaryotic vectors, such as, for example, yeast vectors and fungal vectors; and viral vectors, such as, but not limited to, lentiviral vectors, adenoviral (Lin et al., 2007) vectors, adeno-associated viral vectors, and retroviral vectors. The replication incompetent pcDNA 6.2 GW/miR and pcDNA 6.2/EmGFP-Bsd/V5-DEST vectors are examples of an appropriate expression vectors (Invitrogen) and allow expression of synthetic oligonucleotides (e.g. miRNAs) that have the capacity to cleave targeted sequences (or their corresponding proteins). These vectors include flanking and loop sequences from endogenous miRNA to direct the excision of the engineered miRNA from a longer Pol II transcript (pre-miRNA). Alternatively, inclusion of the HIV psi sequence allows the therapeutic vector to compete with native HIV genome for packaging into viral particles, also inhibiting HIV transmission. Combining multiple miRNA sequences directed against a single target increases the likelihood of success in reducing target sequence expression. miRNA sequences may be operably linked to tissue specific promoters such as the EF-1 alpha promoter, any T cell specific promoter, or macrophage specific promoter to ensure expression in the desired cell types. Utilizing Invitrogen's lentiviral destination (DEST) vectors for gene expression, the resulting therapeutic vector(s) becomes capable of stably transducing both dividing and non-dividing cell types. In a preferred embodiment, the therapeutic vector(s) contains multiple synthetic oligonucleotide sequences driven by one or more promoters so as to reduce expression of CXCR4, CCR5, and/or any other cellular protein known to act as a co-receptor for HIV infection in target cells. In one therapeutic vector (constructed in 2006), four miRNA sequences targeting CXCR4 and CCR5 co-receptors were cloned into the pcDNA 6.2 GW/miR vector along with decoy RNA sequences targeting HIV-2 TAR and RRE. Genetic constructs may include a vector backbone, and a transactivator which regulates a promoter operably linked to heterologous nucleic acid sequences (or their corresponding proteins). Another example of a suitable vector is a retroviral vector. Retroviruses are RNA viruses which contain an RNA genome. The gag, pol, and env genes are flanked by long terminal repeat (LTR) sequences (or their corresponding proteins). The 5′ and 3′ LTR sequences promote transcription and polyadenylation of mRNAs. The retroviral vector may provide a regulable transactivating element, an internal ribosome reentry site (IRES), a selection marker, and a target heterologous gene operated by a regulable promoter. Alternatively, multiple sequences may be expressed under the control of multiple promoters. Finally, the retroviral vector may contain cis-acting sequences necessary for reverse transcription and integration. Upon infection, the RNA is reverse transcribed to DNA which integrates efficiently into the host genome. The recombinant retrovirus of this invention is genetically modified in such a way that some of the retroviral, infectious genes of the native virus are removed and in embodiments replaced instead with a target nucleic acid sequence for genetic modification of the cell. The sequences may be exogenous DNA or RNA, in its natural or altered form. Example 18: Example Methods for Generation of the Therapeutic Vector The methods for generation of the therapeutic vector(s) include those taught in Invitrogen's Viral Power Lentiviral Expression Systems Manual (incorporated by reference herein). Briefly, the EmGFP-bsd cassette is cloned as a Pm1I-BlpI fragment into the pLenti6/R4R2/V5-DEST vector, while the miR-decoy cassette is simultaneously transferred by BP reaction into pDONR221. Then the EF1a promoter and miR-decoy are Muti-site LR crossed into the modified pLenti6/EmGFP-b sd/R4R2-DESTvector. pLenti6/R4R2/V5-DEST vector sequence (SEQ ID NO: 1), Example miR-decoy cassette sequence (SEQ ID NO: 3). Example 19: Methods for Propagating/Proliferating Stem/Progenitor Cells In Vivo In order to obtain large numbers of target cells that are relatively resistant to 1) HIV infection and/or 2) HIV replication and/or 3) HIV transcription, progenitor/stem cells can be grown in Dulbecco's modified Minimal Essential Medium (DMEM) supplemented with glutamine, beta.-mercaptoethanol, 10% (by volume) horse serum, and human recombinant Leukemia Inhibitory Factor (LIF). The LIF replaces the need for maintaining progenitor/stem cells on feeder layers of cells, (which may also be employed) and is essential for maintaining progenitor/stem cells in an undifferentiated state. Example 20 Cells are collected from individuals, developmentally-activated, transfected with the therapeutic vectors, then cultured and prepared for autologous or heterologous transplantation by standard methods, with or without HLA typing and matching. Example 21 Umbilical cord blood samples are obtained from umbilical blood cord bank. The cells are then (with or without developmental activation) transfected with the therapeutic vector of beneficial sequences (or their corresponding proteins), then prepared for transplantation by standard methods, with or without HLA typing and matching. Example 22: Examples of Synthetic Oligonucleotide Sequences Suitable for Inclusion in the Therapeutic Vector Any synthetic oligonucleotide sequences that successfully reduce the protein expression of targeted sequences >70% is covered by the present invention. SeeFIG.17D. Any synthetic oligonucleotide sequences that successfully reduce the ability of target cells to sustain HIV replication by >70% or to a lesser but therapeutic degree or HIV viral activity by >70% or to a lesser but therapeutic degree are also covered by this invention. Examples of miRNA sequences include miRNA sequences derived by IVGN algorithm(Invitrogen). miRNA sequences targeting the CXCR4 gene include top strand: 5′-TGCTGATACCAGGCAGGATAAGGCCAGTTTTGGCCACTGACTGACTGGCCTTACTGCCTGGTAT-3′ (SEQ ID NO: 4) and bottom strand: 5′-CCTGATACCAGGCAGTAAGGCCAGTCAGTCAGTGGCCAAAACTGGCCTTATCCTGCCTGGTATC-3′ (SEQ ID NO: 5); as well as top strand: 5′-TGCTGTGACCAGGATGACCAATCCATGTTTTGGCCACTGACTGACATGGATTGCATCCTGGTCA-3′ (SEQ ID NO: 6) and bottom strand: 5′-CCTGTGACCAGGATGCAATCCATGTCAGTCAGTGGCCAAAACATGGATTGGTCATCCTGGTCAC-3′ (SEQ ID NO: 7). Similarly, miRNA sequences targeting the CCR5 gene include top strand: 5′-TGCTGATCGGGTGTAAACTGAGCTTGGTTTTGGCCACTGACTGACCAAGCTCATTACACCCGAT-3′ (SEQ ID NO: 8) and bottom strand: 5′-CCTGATCGGGTGTAATGAGCTTGGTCAGTCAGTGGCCAAAACCAAGCTCAGTTTACACCCGATC-3′ (SEQ ID NO: 9); as well as top strand 5′-TGCTGATAGCTTGGTCCAACCTGTTAGTTTTGGCCACTGACTGACTAACAGGTGACCAAGCTAT-3′ (SEQ ID NO: 10) and bottom strand: 5′-CCTGATAGCTTGGTCACCTGTTAGTCAGTCAGTGGCCAAAACTAACAGGTTGGACCAAGCTATC-3′ (SEQ ID NO: 11). Example 23 Examples of Decoy RNA suitable for inclusion in the therapeutic vector. Any decoy sequences that successfully reduce the ability of target cells to sustain HIV replication by >70% or to a lesser but therapeutic degree or HIV viral activity by >70% or to a lesser but therapeutic degree are covered by this invention. An example TAR decoy sequence is (SEQ ID NO: 12) gtcgctgcggagaggctggcagattgagccctgggaggttctctccagcactagcaggtagagcctgggtgttccctgctagactctcaccagtgcttggccggcactgggcagacggctccacgcttgcttgcttaaagacctcttaataaagctgc.(Browning et al., 1999)See FIG. 17D. An example RRE decoy sequence is (SEQ ID NO: 13) tgctagggttcttgggttttctcgcaacagcaggttctgcaatgggcgcggcgtccctgaccgtgtcggctcagtccoggactttactggccgggatagtgcagcaacagcaacagagttggacgtggtcaagagacaacaagaactgttgcgactgaccgtaggggaacgaaaaacctccaggcaagagtcactgctatagagaagtacctacaggaccaggcgcggctaaattcatggggatg.(Dillon et al., 1990)See FIG. 17D. Example 24: Flanking Sequences Providing Stability for RNA Decoys Examples of appropriate flanking sequences for RNA decoys are as follows: TAR DECOY SEQ(SEQ ID NO: 14)GUGCUCGCUUCGGCAGCACGTCGAC(SEQ ID NO: 15)UCUAGAGCGGACUUCGGUCCGCUUUURRE DECOY SEQ(SEQ ID NO: 16)GUGCUCGCUUCGGCAGCACGTCGAC(SEQ ID NO: 17)UCUAGAGCGGACUUCGGUCCGCUUUU.See FIG. 17D. Previously, it was demonstrated that decoy sequences flanked by hairpins on either side, 19 nucleotides (ntds) of the U6 RNA on the 5′ side as well as a 3′ stem immediately preceding a poly U terminator for POLIII, showed greater stability. This arrangement is expected to protect against 3′-5′ exonuclease attack, and to reduce the chances of the 3′ trailer interfering with the insert RNA folding. Since only the first ¾ of the tRNA sequence is present, the 5′ end of the insert should be protected and export from the nucleus should be prevented (Good et al., 1997). Example 25: Introduction of Therapeutic Vector to the Host In a preferred embodiment, blood stem/progenitor cells, and target cells are transfected with the therapeutic vector(s) (or associated therapeutic virus) in vivo by introduction of the therapeutic vector(s) into the host blood, tissues, or bone marrow, etc. The greatest benefit may be achieved by modifying a large number of endogenous target and stem/progenitor cells. This may be accomplished by using an appropriately-sized, catheter-like device, or needle to inject the therapeutic vector(s) into the venous or arterial circulation. In a preferred embodiment, the virus is pseudotyped with VSV-G envelope glycoprotein and native HIV-1 env proteins. Example 26: Introduction of Genetically-Modified Cells into the Host Blood cells, such as mature peripheral blood T lymphocytes, monocytes, macrophages, T cell progenitors, macrophage-monocyte progenitor cells, and/or pluripotent hematopoietic stem cells (such as those found in umbilical cord blood and occupying bone marrow spaces) as well as other stem/progenitor cells can be transfected using the therapeutic vector(s) in vitro. Appropriate concentrations of the therapeutic vector(s) may be those consistent with Browning et al., 1999. Subsequently, cells are expanded (propagated) in vitro, and are then transferred to the host via introduction of the cells to the venous or arterial circulation using an intravenous needle or catheter. Subsequently, cells transfected with the therapeutic vectors are able to “home” to the bone marrow and other tissues. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Example 27 Fluorescein-conjugated albumin protein is delivered in high concentration to the interior of 3T3 cells with high efficiency using protein electroporation, according to the method of Koken et al., 1994. Protein Electroporation 3T3 cells electroporated at 300V with a varying number of 5 ms pulses showed progressively increased protein uptake (seeFIGS.4A-4D). ˜200,000 3T3 cells were electroporated in a 4 mm cuvette in the presence of FITC-conjugated albumin (150 ug/200 ul). Visual inspection and photography using a fluorescent microscope revealed progressively increased FITC-albumin uptake and fluorescence over a wide range of pulse number (10-200 pulses).FIGS.4A-4Ddepict the cells (at low power magnification, 10×) 48 hours after exposure to 20 pulse (FIG.4A), 50 pulse (FIG.4B), 100 pulse (FIG.4C) and 200 pulse (FIG.4D) electroporation, demonstrating protein uptake was a function of electroporation. Example 28 Long PRR+Numb alone, or in combination with Oct4, Sox2, Nanog and/or Notch proteins, activated/reprogrammed the cells such that they were shown to be newly-positive for SSEA3, SSEA4, and Tra-1-81 antigens, indicative of pluripotent stem cells, as well as endogenous Oct4, Sox2, Nanog and PRR+ Numb proteins. Cell Culture Prior to electroporation, 3T3 cells and 3T3-PRR+ Numb cells were maintained in growth medium (GM) containing DMEM supplemented with 10% fetal bovine serum (FBS), 20 mM L-glutamine, and 1% penicillin/streptomycin. Protein Electroporation 3T3 cells and 3T3-PRR+ Numb cells were harvested and electroporated using the BTX ECM 830 electroporation machine according to the methods of Koken et al., 1994. Briefly, ˜200,000 cells were transferred to 4 mm cuvettes and electroporated in the presence of either 50 ug Notch protein or 150 ug of oct4/sox2/nanog protein cocktail. Immediately following electroporation, the cells were transferred to Dulbecco's Modified Eagle Medium (DMEM) in standard cell culture plates with or without 20 ng/ml EGF, and incubated at 37 degrees Celsius. Immunohistochemistry In all of these experimental conditions, long PRR+ Numb alone or in combination with Oct4, Sox2, Nanog and/or Notch proteins activated/reprogrammed the cells such that they were shown to be newly-positive for SSEA3, SSEA4, and Tra-1-81 antigens, indicative of pluripotent stem cells, as well as endogenous Oct4, Sox2, Nanog and PRR+ Numb proteins. SeeFIGS.5A-5D and6A-6D. Example 29 Non-pluripotent Murine Cells electroporated with Oct4, Sox2, Nanog and Notch-1 proteins (300V, 70 pulses, 50 ug per protein), or overexpressing the long PRR+Numb isoform, cluster hierarchically and in Principal Component Analysis (PCA) plots amongst published, pluripotent ES and iPS cells. Cell Culture Prior to electroporation, 3T3 cells and 3T3-PRR+Numb cells were maintained in growth medium (GM) containing DMEM supplemented with 10% fetal bovine serum (FBS), 20 mM L-glutamine, and 1% penicillin/streptomycin. Protein Electroporation 3T3 cells and 3T3-PRR+ Numb cells were harvested and electroporated using the BTX ECM 830 electroporation apparatus according to the methods of Koken et al., 1994. Briefly, 200,000 −1M cells were transferred to 4 mm cuvettes and electroporated in the presence of 200 ug/ml oct4/sox2/nanog/notch protein cocktail. Immediately following electroporation, the cells were a) returned to growth medium at 37 degrees. Resulting embryoid bodies, floating colonies and cells adherent 4 days post-electroporation were collected and their RNA extracted for gene array analysis using the Affymetrix GeneChip HTA 2.0 chip. Murine Cells electroporated with Oct4, Sox2, Nanog and Notch-1 proteins (300V, 70 pulses, 50 ug per protein), or overexpressing the long PRR+Numb isoform, clustered hierarchically in Heatmaps (FIG.7A) and in Principal Component Analysis (PCA) plots (FIG.7B) amongst published, pluripotent ES and iPS cells (GSE53299 and GSE61403). Reactome analysis reactome.org was further performed based on the results of the gene array analysis and revealed that treated cells showed enriched or overexpressed genes in, for example the Cell Cycle pathways and Developmental pathways (including the Transcriptional regulation of pluripotent stem cells sub-pathway). Example 30 After electroporation with Oct4, Sox2, and Nanog proteins, human buccal cheek cells are induced to pluripotent-like, Developmentally-Activated Cells: They Divide, Form Colonies, Form Embryoid composed of VSEL cells, and Express Oct4, Sox2, and Nanog Proteins. 200,000 human buccal cheek cells were electroporated in a 4 mm cuvette with 300V in the presence of FITC-conjugated albumin (150 ug/200 ul). Approximately 200,000 human buccal cheek cells were electroporated in a 4 mm cuvette in the presence of FITC-conjugated albumin (150 ug/200 ul). Increasing number of pulses led to progressively increased FITC-albumin uptake and fluorescence. SeeFIGS.8A,8B. Increasing number of pulses led to progressively increased FITC-albumin uptake and fluorescence. Photomicrographs show a small colony (FIG.9A) and a much larger colony of proliferating epithelioid cells (FIG.9B) induced 6 days after electroporation in the presence of 150 ug Oct4, Sox2 and Nanog protein. By 46 days post-electroporation with Oct4, Sox2, and Nanog proteins, colonies visible at low power (10×) mostly comprised darker appearing embryoid (asterisk) composed of VSEL-like cells (FIG.9C). Immunohistochemistry showed electroporated cheek cells expressed Oct4 (FIG.10A), Nanog (FIG.10B), and Sox2 (FIG.10C). Dense, darkly stained embryoid was composed of VSEL-like cells (*) are apparent onFIG.10B. Example 31 Protein Electroporation “flips the Switch”. After electroporation in PBS with 50 ug, each, of Oct4, Sox2, and Nanog proteins, human buccal cheek cells are induced to pluripotent-like, Developmentally-Activated Cells (DAdC). 14 days post-electroporation, erstwhile epithelial, buccal cheek cells show altered morphologies, divide, form colonies, form embryoid composed of VSEL cells, and express Oct4, Sox2, and Nanog Proteins. (FIGS.11A-11I). Embryoid positively-stained after immunohistochemistry using antibodies against Nanog, Sox2 and Oct4, well-known markers of pluripotency. The embryoid was formed by buccal cheek cells following electroporation with Oct4 (FIG.12C), Sox2 (FIG.12B) and Nanog (FIG.12A) proteins in PBS (300V, 70 pulses), and consists of VSEL-like cells (Ratajczak, et al., 2008; Kuruca, et al., 2019). Example 32 Human hepatocyte cells transduced with lentivirally-encoded i. PRR+Numb, ii. Oct4/Sox2, or iii. PRR+ Numb, Oct4, and Sox2, cluster hierarchically and in Principal Component Analysis (PCA) plots with published pluripotent ES and IPS cells. Human hepatic cells were transduced with pLenti-SFFV-Oct4-2A-Sox2 lentivirus (108cfu/ml), with doxycycline inducible, pReceiver-Lv 113-PRR+ Numb lentivirus, or with both lentiviruses. 4 days and 11 days after transduction with PRR+ Numb, and 1 week after transduction with Oct4/Sox2, the cells were collected, their RNA extracted and analyzed using qt-PCR and gene array (using the Human Genome U133 Plus 2.0 Array). Human hepatic cells that were electroporated with Oct4, Sox2, Nanog and Notch-1 proteins (300V, 70 pulses, 50 ug per protein), or overexpressing the long PRR+ Numb isoform, clustered hierarchically in Heatmaps (FIG.13A) and in Principal Component Analysis (PCA) plots (FIG.13B) amongst published, pluripotent ES and iPS cells (GSE76830 and GSE88963). Reactome analysis reactome.org was further performed based on the results of the gene array analysis and revealed that treated cells showed enriched or overexpressed genes in, for example the Cell Cycle pathways and Developmental pathways (including the Transcriptional regulation of pluripotent stem cells sub-pathway). Example 33 Protein electroporation according to the Method of Koken et al., 1994 (300V) provides delivery of protein at high concentrations to the interior of 3T3 cells for rapid (24-72 hr), efficient (100%) and durable (>60 days), cell reprogramming. Seventy, 300V pulses were delivered for 5 ms at 100 ms intervals. (FIGS.14A-14D,FIGS.15A-15D,FIGS.16A-16D). Less than 24 hours after protein electroporation with Oct4, Sox2, and Nanog proteins, approximately three percent of cells showed pluripotency induction (GFP reporter expression under the control of the c-MYC promoter).FIGS.14A,14Bshow corresponding 40× brightfield and Fluorescence images. In contrast, 72 hours after protein electroporation (FIGS.14C,14D), >95% cells show pluripotency induction (c-MYC/GFP stem reporter expression (ABM)). Thirty days after Oct4/Sox2/Nanog Protein Electroporation of 3T3 Cells, the resulting Embryoid bodies showed positive reactivity with anti-Oct4 (FIG.15A), anti-Nanog (FIG.15B), anti-Numb (FIG.15C) and anti-Notch (FIG.15D) antibodies. After electroporation with Oct4, Sox2 and Nanog proteins (50 ug each) in PBS with seventy 5 ms pulses at 300V, 100% of mouse 3T3 cells were activated to adopted rounded, stem cell morphologies and and form small colonies by day six (FIG.16A), large embryoid bodies on day 40 (FIG.16BandFIG.16C), and large rafts of embryoid by day 57 (FIG.16D). Example 34 Construction of the pLenti6-MSGW/EmGFP-Bsd/EF1a/miR-decoy HIV Gene Therapy Vector. Subcloning of the EmGFP-Bsd cassette from pcDNA™6.2/EmGFP-Bsd/V5-GW/CAT into the final vector was confirmed by Restriction Digestion (FIG.17A). The pLenti6-MSGW/EmGFP-Bsd/EF1a/miR-decoy vector comprises HIV RRE and TAR decoy sequences, miRNA sequences directed against HIV co-receptors, CCR5 and CXCR4 and the HIV-2 psi sequence, all of which confer resistance to various human and animal immunodeficiency viruses. Virus stock was prepared from transfected 293FT cells. Successful transfection was confirmed by visualizing syncitia formation at 72 hours (FIG.17B) versus control (FIG.17C). Example 35 Combination of Long PRR+Numb transfection with Notch and/or Oct4/Sox2/Nanog Protein electroporation (per Koken et al., 1994, 300V, 70 pulses, 5 ms pulse length, 100 ms pulse interval) produces the claimed effect (FIGS.18A-18F,19). Protein Electroporation Equal numbers (˜200,000) of 3T3 cells and 3T3-PRR+Numb overexpressing cells were electroporated only with: i) Notch protein; ii) Oct4, Sox2, and Nanog proteins; or iii) Notch, Oct4, Sox2, and Nanog proteins. On day thirty (30), non-adherent and floating, reprogrammed cell colonies were collected and resuspended in equal volumes of medium for low power (10×) visual comparison of cell reprogramming efficiencies: Control (FIG.18A), Numb (FIG.18B), Numb/Oct4/Sox2/Nanog (FIG.18C), Numb/Notch (FIG.18D), as well as, Numb/Notch/Oct4/Sox2/Nanog (FIG.18E), and Notch/Oct4/Sox2/Nanog (FIG.18F). In all of the experimental conditions, Oct4, Sox2, Nanog and/or Notch proteins (alone or in combination with transfected PRR+Numb) reprogrammed the cells such that they were shown to be newly-positive for SSEA3, SSEA4, and Tra-1-81 antigens, indicative of pluripotent stem cells, as well as endogenous Oct4, Sox2, Nanog and PRR+Numb proteins. FIG.19illustrates an embryoid bodies 35 days post electroporation. A single round of electroporation according to the method of Koken et al. (1994), in the presence of Oct4, Sox2, Nanog, and Notch proteins (50 ug each) (Abcam), consistently reprogrammed cells with high efficiency (˜100% in some experiments) to form colonies, embryoid, and VSEL-like cells, consistent with pluripotency or a “pluripotent-like” state. Example 36 A catheter style electroporation apparatus suitable for in vivo electroporation with protein and other transfectants consisting of a needle through which a protein, nucleic acid, drug or other transfectant may be administered to a tissue (FIG.2A). This apparatus may include some variants of catheter7(FIG.2BandFIG.2C).FIG.2Ashows the variant of catheter style electroporation apparatus with variant of catheter7with needle9and two electrodes8(seeFIG.2B).FIG.2Dillustrates a loop or circular electrode8array which is used in catheter7(showed onFIG.2C) suitable for in vivo electroporation. The needle9is i) situated between two electrode8prongs (which may be either sharp or dull) as shown inFIG.2B, or the needle is ii) accompanied by a single internal electrode8(FIG.2C) that is used in conjunction with an external electrode (as commonly occurs with cardiac ablation), or the needle doubles as a first electrode iiia. and a second electrode is located alongside it, or iiib. a second electrode is located externally, or iiic. a second electrode is connected to a separate accompanying catheter. The separate accompanying catheter may also comprise a needle for delivering transfectant that doubles as a second electrode. The setup overall is akin to the setup uses for cardiac ablation, except that voltage is applied locally for cellular permeabilization and uptake of the transfectant. FIG.1Eillustrates an assembly of sample cell culture dishes (number of dishes may vary for example from 1 to 1,600) wherein each has a “reservoir”3portion that functions like a typical electroporation cuvette and broader portion (“inspection plane”)5which functions like a traditional cell culture plate. An assembly may feature one or more plate covers. The dimensions (including width) of the reservoir3may vary and approximate the dimensions and materials of traditional electroporation cuvettes allowing for example, for 1 mm, 2 mm, 4 mm, 6 mm gaps, etc. In some embodiments, the electrode contact6is visible along the side of the reservoir3. “w” indicates width of reservoir3of dish. The reservoirs3and inspection planes5may take various shapes (FIGS.1A-1D) and be positioned centrically or eccentrically relative to one another.FIGS.1A,1C and1Dshow additional “feet” 4 along the edge of the plates that may or may not be detachable, so that the dishes will be able to stand on a flat surface.FIG.1Bshows “skirt”4along the edge of the plates that may or may not be detachable, so that the dishes will be able to stand on a flat surface. The various dishes may feature detachable base/stand with foot processes or skirts to provide stability. Alternatively, the dish may be manufactured with base/stand incorporated with the dish as a single piece.FIGS.1C,1D1E show electrode contact6for electroporation. Electroporation will typically occur in the reservoir3. Additional media may be added before or after electroporation allowing cells to be incubated in larger volumes of media than are accommodated by the reservoir alone. The volume of media contained in the reservoir portion is designated1. The volume of media contained in cell culture dish overall is designated2. FIGS.1F,1G,1Iillustrate “Dish-in-Dish”, cell culture flask with electroporation reservoir in which3—reservoir,10—plastic flask,11—cap,12—exposition/visualization plane,14—electrode plates, w—gap width,13—funnel.FIG.1Hillustrates “Dish-in-Dish”, cell culture flask with multiple electroporation reservoirs in which10—plastic flask,11—cap,12—exposition/visualization plane,3—reservoir,14—electrode plates, w—gap width,13—funnel.FIG.1Jillustrates “Dish-in-Dish”, cell culture dish with electroporation reservoir in which15—dish,12—Exposition/Visualization plane,3—reservoir,14—electrode plates, w—gap width. Example 37 Structural comparison between mouse Numblike and its mammalian Numb homologues and construction of integrase-deficient, transgene expressing lentivectors. FIG.20Aillustrates that Numblike shows greater than 70% sequence identity in its amino terminal half to the shortest Numb homologue, but less than 50% identity in its cytoplasmic half where a unique 15 amino acid polyglutamine domain (purple) is found. The longest Numb isoform contains an 11 amino acid insert (white) within its phosphotyrosine binding (PTB) domain (black), as well as a 49 amino acid insert (gray) adjacent to a proline rich region (PRR). Two intermediate sized isoforms contain either the PTB or PRR inserts, but not both. The shortest Numb isoform lacks both inserts.FIG.20Billustrates the HIV-EGFP Numblike and HIV-EGFP-NumbPTB+/PRR+vectors constructed from the two-gene HIV-EGFP-HSA vector (Reiser et al., 2000) by cloning the transgene cDNAs into nef coding region previously occupied by the mouse HSA cDNA. Abbreviations: Rev-response element (RRE), slice donor site (SD), splice acceptor site (SA). Example 38: In Vivo Injection of the HIV-EGFP-Numblike Transfectant into the Lateral Ventricle and Subsequent Electroporation per Saito et al., (2001) a) 72 hours after transient transfection, pairs and clusters of EGFP-positive cells were detected migrating radially in the mouse forebrain.FIG.21Aillustrates high power photograph depicting a cluster of EGFP-positive cells migrating ventrolaterally, away from the third ventricle within the developing thalamus. One cell from this cluster (FIG.21B) displays many of the classic features associated with newly-generated, migrating neurons including bipolar morphology and a leading process with apparent pseudopodia.FIG.21Cillustrates low power image depicting a pair of EGFP-positive cells which appear to be exiting the intermediate zone (iz) and entering the cortical plate (cp). DAPI stained nuclei are depicted in blue. These highly similar cells expressed EGFP (green) in their cell bodies as well as their pial directed, leading processes (arrowheads) (FIG.21D). This pair also expressed HuC/D (red), a marker of newly generated, migrating and immature neurons, in their cell bodies and processes (FIG.21E). Higher magnification of inset depicted onFIG.21Cis shown onFIG.21F(Scale bar=10 um). b)FIG.22Adepicts a 3D reconstruction of the E18 cortical plate derived from high power z series images. Numerous EGFP-positive cells (green) demonstrate morphologies and location consistent with differentiating neurons.FIGS.22B and22Cshow higher magnifications of insets illustrated onFIG.22A. These cells were identified as neurons according to their co-expression of the neuronal class III beta-tubulin (red). DAPI stained nuclei are shown in blue. A similar 3D reconstruction is shown onFIG.22D. Higher magnification of insets illustrated onFIG.22Dis shown onFIG.22EandFIG.22F. (Scale bar=50 um). Example 39: In Vivo Injection Followed by Electroporation of Mouse Ventricular Zone Cells at P0 with HIV-EGFP Numblike Versus HIV-EGFP Control Vector Upper left corner ofFIG.23Ashows a low power image of the hypothalamic third ventricle (Hy 3V) rotated so that the electroporated portion of the ventricular neuroepithelium is upwards, and the superior portion of the ventricle is to the right. Radially-oriented, EGFP-positive cells (radial glia transfected with control) are seen lining the ventricle and represent the majority of cells labeled by control (˜80%). Their long EGFP-positive processes are observed to extend to the pia within the plane of section. A smaller proportion of cells were located at or near the pial margin-always closely associated with labeled radial processes. Middle section ofFIG.23Adepicts cells transfected with HIV-EGFP-Numblike at P0. Forty-eight hours later, virtually all of the cells have migrated away from the ventricle consistent with their new identity as differentiating neurons. Many of the cells dispersing away from the ventricle showed morphologies and trajectories consistent with the classical appearance of radially migrating neurons. Lower right corner ofFIG.23Ashows a higher magnification of the inset from middle section ofFIG.23Aand depicts an EGFP-positive cell with features characteristic of migrating neurons, including bipolar morphology, a thick leading process with pseudopodia, and a thin lagging process. Abbreviations: Th3V=thalamic portion of the third ventricle.FIG.23Bshows a 3D reconstruction derived from 180 high power, z-series images of the thalamic third ventricle. P2 germinal zone cells, including those transfected with control vector and displaying radial glial morphology, consistently expressed GLAST (glial glutamate transporter) in their cell membranes.FIG.23Cshows the radial glial cell depicted in the inset illustrated onFIG.23Bat higher magnification (scale Bars=50 um onFIG.23Band 100 um on middle section ofFIG.23A). Example 40: Intraventricular Injection of the HIV-EGFP-Numblike Transfectant Followed by In Vivo Electroporation Upregulates Numb Expression FIG.24Aillustrates a 3D reconstruction depicting a section 50 um. EGFP labeled cells, both within and beyond the germinal zone, showed increased Numb immunoreactivity (red) relative to non-transfected cells in the same section (FIG.24B). A portion of the germinal zone is shown at higher magnification in lower right corner onFIGS.24A,24B. The insets again show a relatively disorganized ventricular zone following transfection. This disorganization may have been related to the emigration of cells previously lining the ventricle, but might also reflect tissue injury due to electroporation alone (scale bar=100 um onFIGS.24A,24Band 100 um in lower right corner onFIGS.24A,24B. Example 41: In Vivo Injection of the HIV-EGFP-NumbPTB−/PRR−Transfectant Followed by Electroporation Promotes Neuronal Differentiation in Postnatal Mice FIG.25Adepicts a coronal section through dorsal neocortex in a P3 mouse transfected with the HIV-EGFP-NumbPTB−/PRR−forty-eight hours earlier. Dapi-stained nuclei in the region of electroporation indicated large numbers of cells (arrows) migrating radially through the various layers of the cerebral cortex including the subventricular zone (SVZ), corpus callosum (CC), subplate (SP), and cortical plate (CP).FIG.25Billustrates EGFP expressing cells (triangles) also appeared to migrate laterally in the intermediate zone (IZ) as is known to occur during normal development. Most EGFP-positive cells also expressed high levels of Hu C/D, indicating they were newly-generated neurons (not shown). A 3D reconstruction from confocal z-series images shows HIV-EGFP-NumbPTB−/PRR−transfected cells in the P3 thalamus (FIG.25C). Most of the cells are located outside the germinal zone 48 hours later, and can be recognized as migrating neurons by their morphologies and increased expression of Hu C/D—having been induced to begin differentiating simultaneously following electroporation and to migrate as a cohort. Other cells nearer the ventricle show migratory profiles and appear to be exiting the VZ, but have not yet begun to express Hu C/D (arrowheads). Scale bar=100 um onFIG.25Aand 40 um onFIG.25C. Example 42: Transiently Expressed EGFP Strongly Correlates with Markers of Neuronal Differentiation in Cells Transfected with Numblike Following In Vivo Injection All EGFP positive cells were analyzed immunohistochemically. A discrete cluster of EGFP-positive cells located in the thalamus, 600-700 microns dorsolateral to the germinal zone is depicted. Consecutive sections containing cells from this cluster were stained for markers of neural differentiation including GLAST (FIG.26A), Numb (FIG.26B), TUJ (FIG.26E), and DCX (FIG.26F). Subsequent pixel-by-pixel analysis of these images demonstrated strong correlation between EGFP intensity (green) and markers of neuronal differentiation (red) (R-squared values≥0.76). On the other hand, EGFP expression was not correlated with expression of the immature marker, GLAST (FIG.26C: R-squared value=0.1079). DCX (FIG.26H) showed the highest correlation (R-squared value=0.909), while Numb reactivity was also strongly correlated with EGFP expression in Numblike transfected cells (FIG.26D: R-squared value=0.76). Brains of animals injected with transfectants and electroporated in vivo at PO were sectioned to completion and inspected microscopically. While large clusters of neurons transfected at PO with Numb or Numblike were detected within each of the brains, having migrated and differentiated as a cohort, they no longer expressed EGFP-evidence that the integrase deficient lentivectors remained episomal (did not integrate) and produced only transient transfection. Example 43: HIV-EGFP-NumbPRR−/PTB−and HIV-EGFP-Numblike Lentiviruses Reduce Proliferation and Promote Differentiation in Ras +, Breast Cancer Cells At 5 days post-transduction/post-plating, Ras+ cancer cells transduced with control HIV-EGFP lentivirus showed rapid proliferation and chaotic morphologies (FIG.27A). The inset shows three round, brightly fluorescing cells whose appearance was consistent with cancer stem cells. In contrast, cells transduced with HIV-EGFP-NumbPRR−/PTB−showed evidence of symmetrical, terminal divisions (cell pairs) on day 5, as well as reduced proliferation (FIG.27B). In addition to blocking proliferation, transduction with HIV-EGFP-Numblike induced Ras+ cancer cells to adopt a phenotype consistent with normal breast epithelial cells (FIG.27C). At 10 days post-plating/post-transduction, Ras+cancer cells transduced with control virus fluoresced more brightly than on day 5, but otherwise, continued to show the disorganization characteristic of breast cancer cells, in vitro (FIG.27D). In contrast, on day 10, few, mostly small cells were present in with HIV-EGFP-NumbPRR−/PTB−transduced culture (FIG.27E). Meanwhile, additional cells reverting to a normal, breast epithelial phenotype were identifiable in HIV-EGFP-Numblike transduced cultures (FIG.27F). Example 44: Examples of Expressed or Targeted Transgenes Utilized in the Present Invention Any transgene sequences (or their corresponding proteins) effective in fulfilling the present invention is suitable for use in the present invention. Suitable nucleotide sequences (or their corresponding proteins) may be drawn from any species so long as the desired cells or behavior is achieved. Likewise, the method of naming such sequences (or their corresponding proteins), either in lower case or upper case letters herein, does not imply a particular species. The sequences included in the accompanying sequence listing and stored in the NCBI database (listed by accession number) represent examples of sequences referenced above in the present application. They are also examples of specific transgene encoding sequences (cds) suitable for use in the present invention, but do not in any way limit the practice of the invention. cardiotrophin1:U43030 (SEQ ID NO: 18). CNTF:BC074964 (SEQ ID NO: 19). GP130:NM_175767 (SEQ ID NO: 20). IL6:BC015511 (SEQ ID NO: 21); AB107656. HOXB4:NM_024015 (SEQ ID NO: 22); NM_010459. IL6R:NM_000565 (SEQ ID NO: 23); NM_181359. IL11:NM_133519 (SEQ ID NO: 24); NM_008350. LIF:NM_002309 (SEQ ID NO: 25); NM_008501; BB235045. LIFR:NM_002310 (SEQ ID NO: 26). STAT3:NM_003150 (SEQ ID NO: 27); NM_213662; NM_139276. NUMB: AF171938 (SEQ ID NO: 28); AF171939 (SEQ ID NO: 29). AF171940 (SEQ ID NO: 30); AF171941 (SEQ ID NO: 31); NM_010949; NM_133287; BB483123; NM_010950; NM_010949; NM_004756; DQ022744. Numblike:NM_00475 (SEQ ID NO: 32); U96441; NM_010950; DQ022744. NANOG:NM_024865 (SEQ ID NO: 33); BC137873; NM_028016; OncostatinM(OSM):NM_020530 (SEQ ID NO: 34). OSMR:NM_003999 (SEQ ID NO: 35); NP_003990.1 OCT3/4(POU5F1):NM_203289 (SEQ ID NO: 36); NM_002701 (SEQ ID NO: 37). SOX2:NM_003106 (SEQ ID NO: 38). FGF4:NM_002007 (SEQ ID NO: 39); NP_604391 Gata2:NM_032638 (SEQ ID NO: 40). Gata3:NM_001002295 (SEQ ID NO: 41). Gata4:BC101580 (SEQ ID NO: 42). Gata5:BC117356 (SEQ ID NO: 43). Gata6:NM_005257 (SEQ ID NO: 44). HNF1:NM_000458 (SEQ ID NO: 45); NM_012669 (SEQ ID NO: 46). HNF3:X74936 (SEQ ID NO: 47). HNF3gammaX74938M (SEQ ID NO: 48). HNF3betaX74937 (SEQ ID NO: 49). HNF3G:AH008133 (SEQ ID NO: 50). HNF3A:AH008132 (SEQ ID NO: 51). HNF4alpha:NM_008261 (SEQ ID NO: 52). HNF4a:NM_022180 (SEQ ID NO: 53). HNF6:U95945 (SEQ ID NO: 54). HLXB9:NM_001096823 (SEQ ID NO: 55); NM_019944. (SEQ ID NO: 56). NM_005515 (SEQ ID NO: 57). Lbx1:NM_006562 (SEQ ID NO: 58); NM_010691. Lmx1b (SEQ ID NO: 59); NM_010725 Neurogenin(NEUROG1):NM_006161 (SEQ ID NO: 60); BQ169355. Neurogenin2(NEUROG2):NM_024019 (SEQ ID NO: 61); DR001447. Neurogenin3(NEUROG3) (SEQ ID NO: 62); NM_009719. MASH1:NM_004316 (SEQ ID NO: 63). MyoD:NM_010866 (SEQ ID NO: 64); NM_002478 (SEQ ID NO: 65). Myf5:NM_005593 (SEQ ID NO: 66); NM_131576. Myf6:NM_002469 (SEQ ID NO: 67). NM_008657; NM_008657; NM_013172. Ifrd1:NM_001007245 (SEQ ID NO: 68). Mef2A:NM_013172 (SEQ ID NO: 69). Myogenin:NM_002479 (SEQ ID NO: 70). Nkx2.2:NM_002509 (SEQ ID NO: 71). Notch. Notch1:NM_017617 (SEQ ID NO: 72). NOTCH2:NM_024408; NM_010928. NOTCH3:NM_000435 (SEQ ID NO: 73). Nurr1:NM_006186 (SEQ ID NO: 74). NOV(CCN3):NM_002514 (SEQ ID NO: 75). OLIG1:NM_138983 (SEQ ID NO: 76); OLIG2:NM_005806 (SEQ ID NO: 77). Pdx1:NM_000209 (SEQ ID NO: 78); Pet1(FEV):BC138435; NM_017521 (SEQ ID NO: 79). Phox2a:NM_005169 (SEQ ID NO: 80). Phox2b:NM_003924 (SEQ ID NO: 81). Pit1:NM_000306 (SEQ ID NO: 82). PITX3:NM_005029 (SEQ ID NO: 83); RUNX1:NM_001001890 (SEQ ID NO: 84). Runx2:NM_001015051 (SEQ ID NO: 85); Shh:NM_000193 (SEQ ID NO: 86). Sox9:NM_000346 (SEQ ID NO: 87). Sox17:NM_022454 (SEQ ID NO: 88); BC140307; NM_011441. DLX2:NM_004405 (SEQ ID NO: 89); NP_004396.1; NM_010054. DLX5:NM_005221 (SEQ ID NO: 90); NM_005221; NP_005212. HES1:NM_005524 (SEQ ID NO: 91); NP_005515.1; NM_008235; NP_032261. FGF8:NM_006119 (SEQ ID NO: 92); NM_010205; NP_034335; NM_010205; NP_034335; NP_006110. NM_033163; NP_149353; NM_033164; NP_149354; NM_033165; NP_149355. PITX2:NM_000325 (SEQ ID NO: 93); NM_000325; NP_000316; NM_153426; NP_700475; NM_153427; NP_700476; NM_001042502; NP_001035967; NM_001042504; NP_001035969. REST4:DQ644039 (SEQ ID NO: 94). CREB_binding_protein:NM_134442 (SEQ ID NO: 95); NM_004379; NP_004370; NP_604391. Zfp488:NM_001013777 (SEQ ID NO: 96); BC089025; XM_224697; XP_224697. Foxa2:NM_021784 (SEQ ID NO: 97); NP_068556; NM_012743; NP_036875; NM_010446; NP_034576. Rnx REN:NM_000537 (SEQ ID NO: 98); dHAND(HAND2):NM_021973 (SEQ ID NO: 99); NM_010402; aspartoacylase (Canavan disease) (ASPA):NM_000049 (SEQ ID NO: 100); NM_023113. hexosaminidaseA(HEXA):NM_000520 (SEQ ID NO: 101). Lesch_Nyhan_syndrome(HRPT):NM_000194 (SEQ ID NO: 102); NM_204848. Huntingtin; NM_010414; GUSB; NM_000181 (SEQ ID NO: 103); NM_010368. NPC1:NM_000271; NM_006432. hexosaminidaseB:NM_000521 (SEQ ID NO: 104). galactosidase,alpha(GLA):NM_000169 (SEQ ID NO: 105). glucosidase_beta_acid(GBA):NM_000157 (SEQ ID NO: 106); NM_008094. von_Hippel_Lindau_tumor_suppressor(VHL):NM_000551 (SEQ ID NO: 107). Beta_globin(HBB):NM_000518 (SEQ ID NO: 108). PARK2:NM_013988 (SEQ ID NO: 109); NM_004562; NM_020093. The contents of all parenthetically cited publications and the following United States Patents, are noted and incorporated by reference in their entireties, including: U.S. Pat. Nos. 7,211,247, 5,677,139, 6,432,711 and 5,453,357, 5,593,875, 5,783,566, 5,928,944, 5,910,488, and 5,824,547. | 261,995 |
11859169 | DETAILED DESCRIPTION Hereinafter, in order to describe the present invention in more specifically, an exemplary embodiment of the present invention will be described in more detail with reference to the accompanying drawings. However, the present invention is not limited to the exemplary embodiment described herein, and may also be specified in other forms. Macrophages dynamically interact with the continuously remodeled extracellular matrix (ECM), thereby giving rise to spatially and temporally disparate macroscale ligand distribution in vivo. A nano-ligand for promoting cell adhesion and regeneration of macrophages according to the present invention allows the reversible remote control by spatially and temporally varying the macroscale nano-ligand distribution, so that it is possible to emulate ECM remodeling and regulate the adhesion and polarization of macrophages to spatially and temporally control host responses. In the nano-ligand of the present invention, the negatively charged sliding nano-ligand is coupled with a positively charged substrate through electrostatic interaction, and the slidability of nano-ligand was optimized by utilizing magnetic core nanoparticles that were coated with polymer linker and negatively charged RGD ligand. The present characterized the macroscale and in situ nanoscale nano-ligand sliding under an external magnetic field, which spatiotemporally and reversibly altered the macroscale nano-ligand density. Further, the present invention presents unprecedented in situ manipulation of the macroscale ligand density by magnetically attracting the slidable nano-ligand to regulate the adhesion and polarization phenotypes of host macrophages in vivo. Specifically, the time-regulated magnetic attraction of the slidable nano-ligand inhibited inflammatory M1 phenotype of macrophages but stimulated regenerative M2 phenotype. Furthermore, the magnetic attraction of the slidable nano-ligand of the present invention facilitates the assembly of adhesion structures in macrophages, thereby stimulating polarization of the macrophages to the M2 phenotype. Therefore, the nano-ligand of the present invention enables the spatiotemporal regulation of immunomodulatory tissue-regenerative responses to implants in vivo through remote, spatiotemporal, and reversible controllability of the macroscale ligand density. The present invention provides a nano-ligand for promoting cell adhesion and regeneration of macrophages, including: a core including magnetic nanoparticles; and a coating layer provided so as to surround the core and including an integrin-specific ligand peptide, in which the integrin-specific ligand peptide is negatively charged. FIG.1is a schematic diagram illustrating a nano-ligand for promoting cell adhesion and regeneration of macrophages and a method of promoting cell adhesion and generation of macrophages by using the same according to an exemplary embodiment of the present invention. Referring toFIG.1, it can be seen that the nano-ligand of the present invention includes: a core including magnetic nanoparticles; and a coating layer coupled to a core and including an integrin-specific ligand peptide, in which the integrin-specific ligand peptide is a negatively charged integrin-specific peptide. In particular, the integrin-specific ligand peptide coupled to the core may have the form surrounding the core, like a micelle structure. Accordingly, a surface charge of the nano-ligand may represent a negative charge. For example, a superparamagnetic core iron oxide nanoparticle in the slidable nano-ligand is first synthesized, the superparamagnetic core nanoparticle is functionalized with functional amino-silica shell, and then coated with a polyethylene glycol (PEG) linker to enhance the slidability of nano-ligand to which a negatively charged RGD peptide ligand (CDDRGD) is grafted. Further,FIG.3is a Transmission Electron Micrograph (TEM) image of the nano-ligand for promoting cell adhesion and regeneration of macrophages according to the present invention, and a size of the nano-ligand can be recognized. In particular, the nano-ligand may have a diameter of 30 to 60 nm. When the diameter of the nano-ligand is less than 30 nm, it is difficult to control a movement of the nano-ligand, and when the diameter of the nano-ligand is larger than 60 nm, cell adhesion efficiency of the macrophages may be degraded. In more particular, the nanobarcode may have a diameter of 30 nm to 50 nm, or 35 nm to 45 nm. As long as the magnetic nanoparticles are nanoparticles having magnetic properties, the magnetic nanoparticles are not particularly limited. For example, the magnetic nanoparticles may have a diameter of 5 to 30 nm. When the diameter of the nanoparticle is less than 5 nm, the particle is too small, resulting in large loss and reducing efficiency, and when the diameter of the nanoparticle is larger than 30 nm, the diameter of the nano-ligand increases, resulting in degrading cell adhesion efficiency of macrophages. More particularly, the magnetic nanoparticle may have a diameter of 5 nm to 15 nm, or 10 nm to 20 nm. The nano-ligand includes the magnetic nanoparticles as described above, so that the nano-ligand of the present invention may promote cell adhesion and regeneration of macrophages by using a magnetic field. Further, in the magnetic nanoparticle, silica may be coated to a surface. In particular, in the magnetic nanoparticle, amino-silica may be coated to the surface. The kind of the silica may be any one or more of tetraethyl orthosilicate (TEOS) and (3-Aminopropyl)triethoxysilane (APTES). For example, the nano-ligand of the present invention has a structure in which the core and the coating layer are connected by the linker, and the linker may be a polyethylene glycol (PEG)-based linker. In particular, the polyethylene glycol (PEG) linker may be maleimide-poly(ethylene glycol)-NHS ester (Mal-PEG-NHS ester). The present invention includes the linker, thereby improving coupling force between the core and the coating layer and improving durability of the nano-ligand. The coating layer is coupled to the core or the linker coupled with the core, and has the form surrounding the core. In particular, the coating layer includes the integrin-specific ligand peptide (RGD), and the integrin-specific ligand peptide may have the negatively charged form and include a negatively charged thiolated integrin-specific ligand peptide. The present invention includes the negatively charged thiolated integrin-specific ligand peptide, so that the surface of the nano-ligand of the present invention has the negatively charged form, and accordingly, the nano-ligand may freely move on a substrate through the electrostatic coupling with the substrate. By the characteristic, the nano-ligand is also referred to as the “slidable nano-ligand”, and may promote cell adhesion and regeneration of macrophages through sliding of the nano-ligand on the substrate. Further, the present invention provides a method of preparing the nano-ligand for promoting cell adhesion and regeneration of macrophages, the method including: preparing a core including magnetic nanoparticles; preparing a core coupled with a linker by mixing the core and a first suspension including the linker; and mixing the core coupled with the linker and a second suspension including an integrin-specific ligand peptide (RGD). The preparing of the core may include forming the silane-coated core by stirring the magnetic nanoparticles with a silane solution. In particular, the preparing of the core may include forming an amino-silane coated core by stirring the magnetic nanoparticles with an amino-silane solution. The kind of the silane included in the silane solution may be any one or more of tetraethyl orthosilicate (TEOS) and (3-Aminopropyl)triethoxysilane (APTES). In particular, the preparing of the core coupled with the linker may be performed by stirring the core in a suspension including the linker for 10 to 20 hours or 10 to 15 hours under the dark condition. Accordingly, the linker-coupled core may be obtained. In this case, the linker-coupled core may be obtained by washing the core mixed with the suspension with a solvent two or more times by using the permanent magnet. The solvent may contain any one or more of dimethylformaldehyde (DMF) and dimethyl sulfoxide (DMSO). In this case, the linker may be a polyethylene glycol (PEG) linker. In particular, the polyethylene glycol (PEG) linker may be maleimide-poly(ethylene glycol)-NHS ester (Mal-PEG-NHS ester). By coupling the linker to the core, it is possible to improve coupling force between the core and the coating layer and improve durability of the nano-ligand. Further, the mixing of the core with the second suspension may be performed by stirring the core coupled with the linker in a suspension including the integrin-specific ligand peptide (RGD) for 10 to 20 hours or 10 to 15 hours under the dark condition. In this case, the magnetic nanoparticles (nano-ligands) coupled with the negatively charged integrin-specific ligand peptide may be obtained by using the solvent using the permanent magnet. The solvent may contain any one or more of dimethylformaldehyde (DMF) and dimethyl sulfoxide (DMSO). Herein, the coating layer may be formed on the core through the process of stirring the integrin-specific ligand peptide. In particular, the integrin-specific ligand peptide may be the negatively charged form, and may be the negatively charged thiolated integrin-specific ligand peptide. The coating layer is formed on the core with the negatively charged integrin-specific ligand peptide, so that the surface of the nano-ligand of the present invention may have the negatively charged form, resulting in the free movement of the nano-ligand on the substrate through the electrostatic coupling with the substrate. By the characteristic, the nano-ligand is also referred to as the “slidable nano-ligand”, and may promote cell adhesion and regeneration of macrophages through sliding of the nano-ligand on the substrate. Further, the present invention provides a method of promoting cell adhesion and regeneration of macrophages, including: manufacturing a nano-ligand presenting substrate by putting a substrate, of which a surface is positively charged, in a solution including the nano-ligand for promoting cell adhesion and regeneration of macrophages; and controlling cell adhesion and regenerative polarization of macrophages by treating the nano-ligand presenting substrate with a culture medium and then applying an external magnetic field. FIGS.1and2are diagrams illustrating the method of promoting cell adhesion and regeneration of macrophages according to the exemplary embodiment of the present invention. Referring toFIGS.1and2, it can be seen that the nano-ligand, of which the surface is negatively charged, is electrostatically coupled onto the positively charged substrate, following by applying a magnetic field, so that cell adhesion of macrophages is promoted, inflammatory M1 phenotype is inhibited, and regenerative M2 phenotype is activated in the part to which the magnetic field is applied. In particular, the substrate and the nano-ligand are coupled through the electrostatic coupling, so that the nano-ligand moves (slides) along the location to which the magnetic field is applied, and thus it is possible to promote cell adhesion and regeneration of macrophages in a desired region by regulating a density of the nano-ligand in the portion to which the magnetic field is applied. In particular, the manufacturing of the nano-ligand presenting substrate includes: soaking the surface of the substrate in an acid solution; activating the surface of the substrate so that the surface of the substrate is positively charged by putting the soaking-completed substrate in an amino-silane solution; and treating the substrate, of which the surface is positively charged, by using ultrasonic waves at a room temperature. The soaking of the surface of the substrate in the acid solution may include soaking the surface of the substrate in an acid solution containing any one or more of hydrochloric acid and sulfuric acid for 30 minutes to 2 hours or 30 minutes to 1 hour. Through this, bonding with an amino group is facilitated by bonding a hydroxyl group to the surface of the substrate, thereby effectively performing activation of the surface of the substrate. The activating of the surface of the substrate may include activating the surface of the substrate so that the surface of the substrate exhibits positive charges by putting the substrate in the amino-silane solution under the dark condition. The amino-silane solution may include (3-aminopropyl)triephoxysilane (APTES). In this case, the activation of the surface of the substrate means that the surface of the substrate is positively charged, and particularly, the surface of the substrate may be activated by binding an amine group onto the substrate. The surface of the substrate is positively charged by activating the surface of the substrate by soaking the substrate in the amino-silane solution, so that the substrate may be coupled with the nano-ligand by electrostatic attraction. Further, the treating of the substrate, of which the surface is positively charged, by using ultrasonic waves may include manufacturing the nano-ligand presenting substrate by putting the substrate, of which the surface is positively charged, in the solution including the nano-ligand. In particular, the treating of the activated substrate by using ultrasonic waves was performed by putting the substrate, of which the surface is positively charged, in the solution including the nano-ligand under ultrasonic-wave treatment in purified water for 30 minutes to 2 hours or 30 minutes to 1 hour at a room temperature. The controlling the cell adhesion and regenerative polarization of macrophages may be performed by positioning the nano-ligand presenting substrate in vivo or ex vivo and then applying a magnetic field of 100 to 700 mT for 12 to 48 hours. In particular, the controlling the cell adhesion and regenerative polarization of macrophages may be performed by locating the nano-ligand presenting substrate in vivo or ex vivo and then applying a magnetic field of 100 to 600 mT, 200 to 600 mT, or 300 to 550 mT for 12 to 36 hours, 24 to 26 hours, or 12 to 24 hours. By applying the magnetic field to the nano-ligand presenting substrate, it is possible to promote cell adhesion of macrophages to the nano-ligand located on the substrate, and also inhibit polarization of the inflammatory M1 phenotype of the adhered macrophages and promote polarization of the regenerative M2 phenotype. Further, the controlling of the cell adhesion and regenerative polarization of macrophages may be performed by changing the location in the substrate to which the magnetic field is applied. In particular, the cell adhesion and regenerative polarization of macrophages may be spatially controlled by changing the location in the substrate, to which the magnetic field is applied, while applying the magnetic field of 100 to 600 mT, 200 to 600 mT, or 300 to 550 mT. For example, it is possible to promote the cell adhesion and regeneration of macrophages only in a desired portion of the substrate by regulating the density of nano-ligands on the substrate by applying the magnetic field to a part of the substrate, and inhibit polarization of the inflammatory M1 phenotype of the adhered macrophages and promote polarization of the regenerative M2 phenotype. In addition, the controlling the cell adhesion and regeneration of macrophages may be performed by changing the location of the magnetic field applied to a lower end of the substrate over time. In particular, the cell adhesion and phenotype of the macrophages may be temporally and spatially controlled by changing the location in the substrate to which the magnetic field is applied while applying the magnetic field of 100 to 600 mT, 200 to 600 mT, or 300 to 550 mT. More particular, it is possible to control the degree of promotion of the cell adhesion and regenerative M2 polarization of the macrophages in each portion on the substrate by regulating the density of the nano-ligands located on the substrate over time by individually applying the magnetic field to each portion of the substrate. For example, in the case where the magnetic field is applied to the left side of the substrate for 12 to 24 hours and the magnetic field is applied to the right side of the substrate for 24 to 36 hours, the amount of macrophages adhered to the left side and the right side of the substrate or the macrophages of the regenerative M2 polarization may be varied Hereinafter, examples of the present invention will be described. However, the examples below are merely preferable examples of the present invention, and the scope of the present invention is not limited by the examples. Preparation Example Preparation Example 1 Prepare Slidable Nano-Ligand 1) Prepare Magnetic Core (MNP) For the magnetic control of a slidable nano-ligand, a magnetic core of a slidable nano-ligand was prepared as described below. About 80 mL of ethanol, 60 mL of deionized (DI) water, and 140 mL of heptane were first mixed. To this mixture, 120 mmol of sodium oleate and 40 mmol of iron (III) chloride hexahydrate were added to a solvent mixture of 80 ml of ethanol, 60 mL of deionized (DI) water, and 140 mL of heptane at 70° C. for 4 hours under an inert environment. The heptane layer including an iron-oleate was collected and washed with DI water. The heptane was then evaporated off to collect the dried iron oleate, to which 20 mmol of oleic acid and 200 g of 1-octadecene were added to approximately 40 mmol of the dried iron-oleate. This solution was stirred at 100° C. for approximately 5 minutes. The temperature was then raised to 320° C. and maintained for approximately 30 minutes. The mixture solution was suspended under air, allowed to be cool to room temperature and then washed with ethanol three times using a permanent magnet to collect the nanoparticle. In order to store this magnetic core nanoparticle (MNP), the solution was stored in heptane until used. 2) Functionalization of Amino-Silica of Magnetic Core (Amino-Silica Coated MNP) The magnetic core nanoparticle was coated with an amino-silica shell for the nanoassembly of slidable nano-ligand. The magnetic core nanoparticle (30 mg) was dispersed in cyclohexane (25 mL). To this suspension, triton-X (5 mL), 1-hexanol (5 mL), ammonium hydroxide (0.5 mL), and DI water (1 mL) were serially added, which was stirred for 30 minutes. After stabilizing the emulsion, tetraethyl orthosilicate (TEOS, 12.5 μL) was slowly mixed and stirred for 10 minutes. For amino-functionalization, (3-aminopropyl)triethoxysilane (APTES, 6.25 μL) was mixed and stirred overnight. Following amino-functionalization, the amino-silica shell coated MNP was washed with acetone and dimethylformamide (DMF), three times each, which was collected with a magnet. 3) Prepare Slidable Nano-Ligand the sliding property of nano-ligand was enhanced by using a polyethylene glycol (PEG) linker, and then the negatively charged RGD peptide ligands were grafted onto the surface amino-silica shell coated MNP. The PEG linker was used in the nanoassembly of the slidable nano-ligand to prevent cellular uptake. Amino-silica shell coated MNP in DMF (1 mL) was used to dissolve 5 mg of Maleimide-poly(ethylene glycol)-NHS ester (Molecular weight: 5 kDa, Biochempeg). N,N-Diisopropylethylamine (DIPEA, 2 μl) was added to this suspension and stirred overnight in the dark, which was subsequently washed with DMF three times and dimethyl sulfoxide (DMSO) three times, after which it was collected using a permanent magnet. The PEG-amino-silica shell coated MNP in DMSO (1 mL) was added to dissolve the negatively charged thiolated RGD peptides (CDDRGD, GL Biochem, 0.5 mg). DIPEA (2 μl) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 10 mM) were added to this suspension to avoid the formation of disulfide bonds and the mixture was stirred overnight in the dark. The suspension was washed with DMSO and the slidable nano-ligand was collected by using a permanent magnet prior to coupling with the substrate that is coupled via electrostatic interaction. Comparative Preparation Example 1 A “No RGD” nano-ligand was prepared by the same method as that of Preparation Example 1 except that a negatively charged thiolated RGD peptide (CDDRGD, GL Biochem) was not added. EXAMPLE Example 1 Slidable Nano-Ligand and Coupling of Slidable Nano-Ligand with Substrate In order to reversibly couple the slidable nano-ligand prepared in the Preparation Example to the substrate, culture-grade glass coverslips (22 mm×22 mm) were used. The substrates were soaked in the 1:1 mixture of hydrochloric acid and methanol for 30 minutes to remove any organic impurities and then washed with DI water. The substrate was soaked in sulfuric acid for one hour and washed with deionized water. The substrates were subjected to the soaking in 1:1 mixture of 3-aminopropyltriethoxysilane (APTES) and ethanol for 1 hour in the dark condition to achieve the amination of the substrates. The substrates were then washed with ethanol and dried at 100° C. for 1 hour. To facilitate the electrostatic interactions, the negatively charged slidable nano-ligand (RGD-PEG-amino silica shell-coated magnetic nanoparticles) in 1 mL of DMSO was diluted with DMSO (1:20) and then incubated with the positively charged aminated substrates for 1 hour under the sonication. The substrates were washed with DI water to yield the slidable nano-ligand existing substrates. Experimental Example Experimental Example 1 In order to check the form of the slidable nano-ligand according to the present invention, Transmission Electron Micrograph (TEM), dynamic light scattering, and High-Angle Annular Dark-Field Scanning TEM (HAADF-STEM) analysis were performed on the slidable nano-ligand, and the result of the analysis is represented inFIGS.3and4. Further, in order to check the property and a chemical bonding characteristic of the slidable nano-ligand, Vibrating-Sample Magnetometry and Fourier Transform Infrared Spectroscopy (FTIR) were performed on the slidable nano-ligand, and the result thereof is represented inFIGS.5and6. In particular, in the TEM experimental, TEM imaging was performed by using Tecnai 20 (FEI, USA) in order to check a size and a shape characteristic of the slidable nano-ligand. Further, High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy (HAADF-STEM) is to characterize the size and shape characteristic of the representative slidable nano-ligand, and HAADF-STEM imaging was carried out by using JEOL 2100F with 1 nm probe size, 20 μm condenser aperture, and 80 to 150 mrad collection angle for Z contrast. In addition, in Dynamic Light Scattering (DLS) analysis, in order to quantify the size distribution profile (hydrodynamic diameter) in the assembly process of sliding nano-ligand, DLS measurement (Zetasizer Nano ZS90 Malvern Panalytical, Malvern, UK) was carried out. Further, the FT-IR was carried out by using GX1 (Perkin Elmer Spectrum, USA) in order to confirm the serial chemical changes in the modification of slidable nano-ligand. The samples subjected to the analysis of changes in chemical bond characteristics were lyophilized and densely packed into KBr pellet prior to the analysis. In order to characterize the reversible slidable (superparamagnetic) property of the nano-ligand, the magnetic nanoparticle core in the slidable nano-ligand was subjected to the VSM measurement (EV9; Microsense) at a room temperature under the applied magnetic field. The corresponding magnetic moment was presented in a hysteresis loop after normalization to the dry weight with the magnetic core in the slidable nano-ligand. FIG.3is a Transmission electron micrograph (TEM) image of a nanoscale image of the slidable nano-ligand and in this case, a scale bar indicates 20 nm. a ofFIG.4is a result of dynamic light scattering of magnetic nanoparticles (MNPs) and amino-silica-coated MNPs with size distribution, and b ofFIG.4is an HAADF-STEM image of the amino-silica-coated MNP, and in this case, a scale bar indicates 20 nm. Referring toFIG.3, the slidable nano-ligand was visualized by transmission electron micrograph (TEM), which revealed uniform morphology of sphere of the slidable nano-ligand including a superparamagnetic core (16±2 nm) and a slidable nano-ligand core-shell (42±5 nm). Further, referring toFIG.4, as determined by dynamic light scattering, the uniform diameter of the superparamagnetic core of 15±4 nm and slidable nano-ligand core-shell of 41±5 nm, which was consistent with TEM and high-angle annular dark-field scanning TEM (HAADF-STEM) images. FIG.5is a Fourier transform infrared spectra image of the slidable nano-ligand according to the exemplary embodiment of the present invention. In particular,FIG.5is the Fourier transform infrared spectra image of the MNP, the silica-coated MNP, and the RGD ligand-presenting PEG grafted silica-coated MNP (RGD-PEG-silica-coated MNP, slidable nano-ligand).FIG.6is a vibrating sample magnetometer hysteresis of the slidable nano-ligand. Referring toFIG.5, the change in the chemical bonding in the process of preparing the slidable nano-ligand can be recognized through the FTIR. In particular, Fe—O binding was detected at the absorption peak value of 699 cm−1in superparamagnetic iron oxide core nanoparticles. Si—O binding was detected at the absorption peak value of 1168 cm−1in the silica shell. In the slidable nano-ligand, the PEG linker (Mn=5,000 Da) improve the sliding property and inhibits uptake by cells as demonstrated in previous literature, and CDDRGD represented C═O bonding at the absorption peak of 1152 cm−1and amide bonding at the absorption peak of 1635 cm−1. The FTIR analysis confirmed the successful assembly of the slidable nano-ligand. Referring toFIG.6, the result of the analysis confirmed the superparamagnetic property of 20 emu/g Ms. Through this, the slidable nano-ligand according to the present invention exhibits the superparamagnetic property to be reversibly slidable, so that the superparamagnetic property is very important to magnetically manipulating the sliding of the nano-ligand temporally and reversibly. Experimental Example 2 In order to verify in situ reversible spatiotemporal control of the slidable nano-ligand according to the present invention, the slidable nano-ligand was photographed with the SEM, and AFM imaging was carried out, and the result thereof is represented inFIGS.8and9. FIG.7is a diagram illustrating electrostatic coupling of the slidable nano-ligand to a substrate according to the exemplary embodiment of the present invention. Referring toFIG.7, magnetic nanoparticles (core) were PEGylated and subsequently coated with negatively charged RGD peptide ligand (CDDRGD) to form slidable nano-ligand, which was coupled to the positively charged substrate via an electrostatic interaction. In particular, as illustrated inFIG.7, In particular, as illustrated inFIG.7, in order to electrostatically control the macroscale nano-ligand presentation, in the present invention, the slidable ligand was coupled to the positively charged substrate for in situ spatiotemporally controlling the sliding of the nano-ligand. Through the electrostatic coupling of the nano-ligand and the substrate, reversible movement of the sliding of the nano-ligand was allowed. Herein, the SEM is for the purpose of confirming the negatively charged slidable nano-ligand coupled to the positively charged substrate via an electrostatic interaction, and Field emission-SEM imaging (FE-SEM, FEI, Quanta 250 FEG) was conducted on the dried and platinum-coated substrate. The density of the substrate-conjugated slidable nano-ligand was determined with Image J using 10 different images. SEM imaging was also conducted to characterize reversible and spatiotemporal manipulation of macroscale ligand density. The position of a permanent magnet (150 mT) was switched from the bottom left side of the substrate to the bottom right side, and then to the bottom left side back again every 12 hours. The corresponding changes in the macroscale ligand density were calculated using Image J, and the result of the calculation is represented inFIG.8. Further, in order to confirm the characteristics of in situ 2 and 3D images of slidable nano-ligand on the substrate, in situ magnetic Atomic Force Microscopy (AFM) imaging (Asylum Research, XE-100 System) was carried out in AC in air mode at 25° C. The imaging was carried out in AC by using AFM cantilever (Nanosensors, SSS-SEIHR-20) with a spring constant of 5-3 N/m and a resonance frequency of 96-175 kHz. For static serial imaging in the absence of a magnet near the substrate, the imaging was conducted on the same scanning area of the substrate to examine the non-magnet-mediated displacement of the slidable nano-ligand. For in situ magnetic imaging, the imaging was first conducted and then a permanent magnet was placed at the bottom of the substrate but at the opposite side of the scanning area, and subsequently, the imaging was conducted in the same scanning area for characterizing in situ nanoscale nano-ligand sliding. FIG.8is an image for in situ reversible spatiotemporal manipulation of sliding of macroscale and nanoscale nano-ligands. Referring to a and b ofFIG.8, the positively charged amino-functionalized substrate was optimally and homogeneously coupled with the nano-ligand as evidenced by the SEM and the 3D AFM. Further, the macroscale density of the nano-ligand was calculated as 20±3 nano-ligand particles/μm2. It can be seen that this density was optimal for the effective control of slildability of the nano-ligand, which did not cause the aggregation of the slidable nano-ligand. Accordingly, the nano-ligand of the present invention is reversibly slidable (movable) on the substrate, so that it is possible to spatiotemporally and reversibly control cell adhesion and phenotype of the macrophages. c and d ofFIG.8are SEM imaging results of the spatiotemporal control experiment, and it can be seen that it is possible to control slidability by regulating the density of the macroscale nano-ligand by using the magnetic control. In particular, in the SEM imaging, a permanent magnet was positioned under the left side of the substrate to attract the slidable nano-ligand toward the left side, switched to the right side, and reverted to the left side each 12 hours. In the time-lapse SEM imaging, compared to the right side, the nano-ligand density on the left side was significantly higher by 81% after 12 hours, lower by 31% after 24 hours, but higher by 82% after 36 hours. e ofFIG.8is a comparative example of the spatiotemporal control experiment, and shows nanoscale displacement of nano-ligand sliding through serial in situ magnetic AFM scanning on the identical area regardless of the existence of an external magnetic field in the scanning area. In e ofFIG.8, the magnetic field was generated by placing a magnet at the bottom of the slidable nano-ligand presenting substrate (the opposite side of the scanning area), and the slidable nano-ligand was clearly identified in the absence of the magnet, but disappeared from the scanning area in the presence of the magnet, indicative of nanoscale nano-ligand sliding. FIG.9is an image of a comparative example experiment, and is an in situ AFM image of the nano-ligand sliding in the absence of the magnet in the identical scanning. Referring toFIG.9, black dotted lines are illustrated along the slidable nano-ligands in two different images. A scale bar indicates 50 nm. It was characterized repeatable and stable imaging in the absence of magnet near the substrate and found that the movement of the slidable nano-ligand was negligible within 4 nm displacement in the serial scanning on the identical area of the substrate. Through this, it can be seen that it is possible to reversibly control nano-ligand sliding by spatiotemporally and reversibly alter the macroscale ligand density. Experimental Example 3 In the method of promoting cell adhesion and regeneration of macrophages by using the slidable nano-ligand according to the present invention, the following experiment was performed to confirm the effect of controlling the density of the macroscale nano-ligand on the control of cell adhesion of the macrophages. The binding experiment of integrin β1 to the slidable nano-ligand was carried out as described below. The substrate presenting slidable nano-ligand was used to quantify the binding efficacy of integrin β1 to the slidable nano-ligand. The substrate was immersed in integrin β1 (50 μg/mL) in phosphate-buffered saline (PBS) at 4° C. for approximately 12 hours by positioning a permanent magnet at the bottom “left” side of the substrate. The treated substrate was used for immunofluorescent staining for against integrin β1 (Santa Cruz Biotechnology) and subsequent confocal imaging to quantify integrin β1 bound to the slidable nano-ligand in situ, and a result thereof is represented inFIG.10. FIG.10is a diagram illustrating temporal modulation of integrin β1 binding of in situ control of the sliding nano-ligand. a ofFIG.10is a schematic diagram illustrating the sliding nano-ligand with a permanent magnet positioned under the “left” side of the substrate, and b ofFIG.10is confocal microscope images of immunofluorescence for integrin β1 clusters bound to the sliding nano-ligand at the “left”, “middle”, and “right” side of the substrate, which are indicated by green arrows, and in this case, a scale bar indicates 50 μm. c ofFIG.10illustrates quantification of staining intensity of the integrin β1 clusters at the “magnet”, “medium”, and “non-magnet”side of the substrate. Data is displayed as mean±standard error (n=30). Statistically significant differences are indicated by different alphabet letters. FIG.11is an image representing a result of the in situ control experimental of the nano-ligand sliding according to the exemplary embodiment of the present invention, and represents the promotion of adhesion of macrophages by modulating the macroscale ligand density by magnetic attraction of the slidable nano-ligand. a ofFIG.11is schematic presentation of manipulating the slidable nano-ligand by positioning a permanent magnet at the bottom of the substrate (“magnet” side) with corresponding confocal microscope images of immunofluorescent staining against vinculin, actin, and nuclei after 24 hours of culturing macrophages subjected to a magnet placed at the bottom of the substrate (“magnet” side). The images of adherent macrophages were taken at the center of “magnet”, “medium”, and “non-magnet” sides of the substrate (at the red dotted rectangles in the schematic representation). A scale bar is 20 μm. Control groups include “No RGD” and “No magnet” groups. b ofFIG.11is a graph illustrating quantification of the macrophage density, cell area, and cell aspect ratio. Data is displayed as mean±standard error (n=30). Statistical significances are indicated by different alphabet letters. FIG.12is an image illustrating a result of adhesion experiment of the macrophages according to a comparative example, and a ofFIG.12is a confocal microscope image of immunofluorescent staining of the macrophages for vinculin, F-actin, and nuclei after culturing the macrophages for 24 hours in the absence of an RGD peptide ligand or a magnetic field. The images of adherent macrophages were taken at the center of “left”, “medium”, and “right” sides of the substrate. A scale bar is 20 μm. b ofFIG.12is a graph illustrating quantification of macrophage density, area, and aspect ratio. Data is displayed as mean±standard error (n=30). Statistically significant differences are indicated by different alphabet letters. Referring to a ofFIG.10, whether the ligation of integrin 131 can be maneuvered by the slidable nano-ligand in situ was investigated, and the substrate presenting the slidable nano-ligand was incubated with integrin β1 for 12 hours whereas a permanent magnet was positioned at the bottom of the substrate (“magnet” side) Referring to b and c ofFIG.10, confocal microscope images of the immunofluorescence revealed significantly higher binding efficiency of the integrin (31 to the nano-ligand that was drawn to the magnet side of the substrate by 30% and 55%, as compared to the medium and non-magnet sides of the substrate, respectively Since integrin ligation facilitates the adhesion of macrophages, this finding suggests that macrophages could adhere more strongly to the slidable nano-ligand, which moves toward the magnet side to elevate the macroscale nano-ligand density on the magnet side. Further, in the present invention, the adhesion of macrophages under the control of the macroscale ligand density in situ was investigated. Referring to a ofFIG.11, a magnet was positioned at the bottom of the substrate (“magnet” side) during the culturing and the adhesion of macrophages imaged at the center of “magnet”, “medium”, and “non-magnet” sides of the substrate was observed. Also, control experiments were conducted with the substrate presenting the slidable nano-ligand, but without the magnet (“No magnet”) or the substrate with the nanoparticles without conjugated RGD peptide ligand (“No RGD”). The results showed that macrophages adhered more robustly to the “magnet” side of the substrate in significantly higher density and cell area with the pronounced actin filament assembly and vinculin expression in more elongated morphology than those adhered to the “medium” and “non-magnet” sides of the substrate. Quantitatively, the density of adherent macrophages on the “magnet” side was 59% and 82% higher than those on the “medium” and “non-magnet” sides, respectively. Similarly, the area of adherent macrophages on the “magnet” side was 57% and 60% higher than those on the “medium” and “non-magnet” sides, respectively. In addition, the aspect ratio (elongation shape factor) of adherent macrophages was 54% and 50% higher on the “magnet” side, than those on the “medium” and “non-magnet” sides, respectively. Referring to a and b ofFIG.12, this regulation of cell adhesion of the macrophage adhesion based on the nano-ligand-sliding-mediated control was found to be inefficient for the “No RGD” and “No magnet” groups exhibiting similar adherent macrophage density and area in the “left”, “middle”, and “right” sides of the substrate The “No RGD” group showed minimal cell adhesion of the macrophages, thereby indicating effective blocking. These results prove that increasing macroscale ligand density with magnetic attraction of the slidable nano-ligand in situ efficiently facilitates integrin ligation-mediated adhesion of macrophages. Experimental Example 4 An experiment about the control of reversible adhesion of macrophages by the regulation of macroscale nano-ligand density according to the present invention was conducted as described below. Macrophages exhibit dynamic adhesion and polarization on the ECM, which is continuously being remodeled with spatially and temporally varying macroscale ligand distribution. Therefore, remote control of spatially, temporally, and reversibly varying macroscale nano-ligand distribution may emulate ECM remodeling that regulates the cell adhesion of macrophages. To this end, in the present invention, an experiment about whether temporal conversion of macroscale nano-ligand presentation by attracting the slidable nano-ligand can alter the adhesion of macrophages was conducted, and the result thereof is represented inFIGS.13to15. FIG.13is a diagram illustrating a result of an experiment of controlling adhesion of macrophages through the control of the macroscale nano-ligand. a ofFIG.13is an immunofluorescent confocal microscope image against vinculin with F-actin and nuclei after 12 hours or 24 hours of culturing macrophages subjected to placing a permanent magnet at the bottom of the substrate [“magnetic field (MF)”] or withdrawing the permanent magnet from the substrate [“no magnetic field (NMF)”]. “MF” or “NMF” were applied throughout 24 hours period of culture or alternately applied after 12 hours. The images of adherent macrophages were taken at the center of “left” or “right” side of the substrate. A scale bar is 20 μm. b ofFIG.13is a graph illustrating a calculation of the macrophage density, cell area, and cell aspect ratio for the “left” side of the substrate. Data is displayed as mean±standard error (n=30). Statistical significances are indicated by different alphabet letters. FIG.14is a graph illustrating quantification of the macrophage density, cell area, and cell aspect ratio for the “right” side of the substrate in the experiment illustrated inFIG.13. A permanent magnet was placed at the bottom “left” side of the substrate [“magnetic field (MF)”] or withdrawn from the substrate [“no magnetic field (NMF)”] during the culture of macrophages for 12 hours or 24 hours. “MF” or “NMF” were applied throughout 24 hours of culture or alternately applied after 12 hours. Data is displayed as mean±standard error (n=30). Statistically significant differences are indicated by different alphabet letters. FIG.15is a time-resolved confocal microscope images of immunofluorescent staining against vinculin, actin, and nuclei after 12 hours, 24 hours, and 36 hours of culturing macrophages in the “left” and “right” sides of the substrate subjected to the switching the position of a permanent magnet between two opposite sides at the bottom “left” side of the substrate to the bottom “right” side and then to the bottom “left” side for every 12 hours. The slidable nano-ligands were imaged at the center of “left” and “right” side of the substrate (at the red dotted rectangles in the schematic diagram). A scale bar is 20 μm. b ofFIG.15is a graph illustrating calculation of the density, cell area, and cell aspect ratio of the adherent macrophages in the “left” side of the substrate. Data is displayed as mean±standard error (n=30). Statistical significances are indicated by different alphabet letters. Referring toFIG.13, a permanent magnet was continuously positioned at the bottom left side of the substrate (magnetic field “MF”) for 24 hours. A group without a magnet near the substrate for 24 hours was also included (No magnetic field “NMF”). Further, the present invention included a group without a magnet placed nearby for the initial 12 hours, but with the magnet placed at the bottom left side of the substrate after 12 hours (“NMF-MF”). After 24 hours, it was found that the “MF” group exhibited remarkably more pronounced macrophage adhesion, particularly higher adherent cell area by 63% and aspect ratio by 66%, on the left side (magnet side) than “NMF” group (a and b ofFIG.13). This trend was also reflected in the “NMF-MF” group that exhibited a low extent of macrophage adhesion at 12 hours without the magnet, but cell adhesion of macrophages was strikingly pronounced after 24 hours after the magnet-mediated attraction of slidable nano-ligand toward the left (magnet) side. Once the slidable nano-ligand had been attracted to the magnet (left) side, it appears to have remained even after withdrawing the magnet after 12 hours, as evidenced by maintaining macrophage adhesion after 24 hours in the “MF-NMF” group. The dramatic time-regulated switching of nano-ligand sliding was effective in regulating the cell adhesion of macrophages on the left side (magnet side), but, referring toFIG.14, was rather ineffective in modulating the cell adhesion of macrophages on the right side (non-magnet side), thereby confirming the magnet-mediated in situ control of nano-ligand sliding. Furthermore, in the present invention, an experiment for the effect of spatiotemporally tuning of nano-ligand sliding on the reversible adhesion of macrophages was conducted. Referring toFIG.14, a permanent magnet was disposed at the bottom left side of the substrate to attract the slidable nano-ligand toward the left side for 12 hours. Subsequently, the position of the magnet was kept at the bottom right side of the substrate for 12 hours and then kept at the bottom left side for 12 hours. The corresponding time-resolved macrophage adhesion on the left and right side was investigated (a ofFIG.15). Referring toFIG.15, it can be seen that the temporal regulation of the nano-ligand sliding controls reversible adhesion of macrophages. At 12 hours, the left side of the substrate showed a significantly increased adherent macrophage area by 60% and aspect ratio by 44% compared to the right side (a and b ofFIG.15). At 24 hours, the right side of the substrate showed a rather higher adherent macrophage area and aspect ratio, by 67% and 70%, respectively, than the left side. After 36 hours, reversibly, the left side of the substrate exhibited higher adherent macrophage density, cell area, and aspect ratio by 51%, 67%, and 68%, respectively, than the right side. Therefore, this remote and spatiotemporal manipulation of the macroscale nano-ligand distribution presents a powerful control in the reversible regulation of the cell adhesion of macrophages. Experimental Example 5 In order to confirm that the slidable nano-ligand according to the present invention alters the adhesion-dependent polarization of macrophages through the time-regulated tuning, the following experiment was conducted, and a result thereof is represented inFIGS.16to20. Dynamic ECM remodeling exhibits temporally varying heterogeneous ligand distribution, which regulates the development of adhesion structures of macrophages that modulate their functional polarization phenotypes, thereby spatially and temporally regulating the host response to implants. It is known that macrophages, which develop adhesion structures with cytoskeletal actin assembly and elongated morphology, are functionally activated to the regenerative M2 phenotypes. The previous reports suggest that prior findings suggest that temporal tuning of the macroscale nano-ligand presentation may alter the adhesion-dependent polarization of macrophages. a ofFIG.16is a graph illustrating quantitative gene expression of M1 phenotype markers (iNOS and TNF-α) for macrophages cultured under M1-polarizating medium or M2 phenotype markers (Arginase-1 and Ym1) for macrophages cultured under M2-polarizating medium for 36 hours on the “left” and “right” side of the substrate subjected to a magnet placed at the bottom “left” side of the substrate. Data is displayed as mean±standard error (n=30). b and c ofFIG.16are confocal microscope images of immunofluorescence against iNOS with Arg-1 and nuclei of cultured macrophages by positioning a permanent magnet at the bottom of the substrate [“magnetic field (MF)”] on the “left” side or not positioning a permanent magnet [“no magnetic field (NMF)”]. “MF” or “NMF” were applied throughout 36 hours period of culture or alternately applied after 12 hours. The images of polarized macrophages were taken at the center of “left” or “right” side of the substrate. A scale bar is 20 μm. Different alphabet letters signify statistical significances. FIG.17is a graph illustrating M2 phenotype of macrophages in the M1 polarizing medium of the slidable nano-ligand according to the exemplary embodiment of the present invention.FIG.17illustrates ineffective control of M2 phenotype of macrophages under the slidable nano-ligand in the M1-polarizing medium. Quantitative gene expression of M2 markers (Arginase-1 and Ym1) for macrophages cultured in M2-polarizating medium on the “left” and “right” side of the substrate for 36 hours subjected to a permanent magnet placed at the bottom “left” substrate (“magnet” side). Data is displayed as mean±standard error (n=30). FIG.18is a graph illustrating M1 phenotype of macrophages in the M2 polarizing medium of the slidable nano-ligand according to the exemplary embodiment of the present invention.FIG.18illustrates that control of M1 phenotype of macrophages under the slidable nano-ligand is inefficient in the M2-polarizing medium.FIG.18illustrates gene expression profiles of M1 markers (iNOS and TNF-α) for macrophages cultured in M2-polarizating medium on the “left” and “right” sides of the substrate for 36 hours subjected to a permanent magnet placed at the bottom “left” substrate (“magnet” side). Data is displayed as mean±standard error (n=30). FIG.19is a confocal microscope image of immunofluorescence of an M2 phenotype experiment of macrophages for magnetic attraction of the slidable nano-ligand according to the exemplary embodiment of the present invention. Magnetic attraction of the slidable nano-ligand promotes ROCK2 expression in developing adhesion structures of macrophages to promote their M2 phenotypes.FIG.19is a confocal microscope image of immunofluorescence staining against ROCK2 and nuclei after culturing macrophages in a basal or M2-polarizing medium for 36 hours on the “magnet” and “non-magnet” side of the substrate subjected to a permanent magnet placed at the bottom of the substrate (“magnet” side). A scale bar is 20 μm. FIG.20is a confocal microscope image of immunofluorescence (a) of a magnetic attraction regulating experiment of the nano-ligand according to the exemplary embodiment of the present invention, and b ofFIG.20is a graph illustrating calculation of the density, cell area, and cell aspect ratio of the adherent macrophages or the area, aspect ratio, and Arg-1 staining intensity after culturing in M2 medium. Magnetic manipulation of attracting the slidable nano-ligand promotes the assembly of adhesion structures of macrophages in stimulating their M2 phenotype. a ofFIG.20is a confocal microscope image of immunofluorescence for actin and nuclei after culturing macrophages in a basal medium for 36 hours and Arg-1 with actin and nuclei after culturing macrophages in the M2-polarizing medium for 36 hours under ROCK signal inhibition (with Y27632), myosin II forming inhibition (with blebbistatin), or actin polymerization inhibition (with cytochalasin D) on the “magnet” and “non-magnet” side of the substrate subjected to a magnet placed at the bottom of the substrate (“magnet” side). A scale bar is 20 μm. b ofFIG.20is graph illustrating calculation of the density, cell area, and cell aspect ratio of the adherent macrophages after basal medium culture or the area, aspect ratio, and Arg-1 staining intensity after culturing in M2 medium at the “magnet” and “non-magnet” sides of the substrate. Data is displayed as mean±standard error (n=30). Statistical significances are indicated by different alphabet letters. Referring toFIG.16, it can be seen that the time-regulated magnetic attraction of the slidable nano-ligand suppresses the M1 phenotype of macrophages while stimulating the M2 phenotype. Phenotype presentation of the macrophages under time-regulated manipulation of the slidable nano-ligand was examined with the substrate presenting the slidable nano-ligand subjected to placing a permanent magnet at the bottom left side of the substrate (“MF” group) continuously for 36 hours, not placing a permanent near the substrate (“NMF” group) continuously for 36 hours, or switching from “NMF” to “MF” at 12 hours. After culturing macrophages in M1-polarizing medium, gene expression profiles revealed that in the “MF” group, the expression of the M1 markers iNOS (inducible nitric oxide synthase) and TNF-α (tumor necrosis factor-α) were significantly lower by 192% and 231%, respectively, on the left side (magnet side) than on the right side (non-magnet side). In contrast, after M2-polarizing medium culture in the “MF” group, the expression of M2 markers Arg-1 (arginasae-1) and Ym1 (chitinase-like 3) were significantly higher by 715% and 383%, respectively, on the left side (magnet side) than on the right side (non-magnet side). Referring toFIGS.17and18, in the “MF” group, the expression of M1 markers after M2-polarizing medium culture or M2 markers after M1-polarizing medium culture, did not differ between the left and right sides, which indicates the requirement of polarizing soluble stimuli with magnetic control of nano-ligand sliding to modulate macrophage polarization. Referring to b and c ofFIG.16, the confocal microscope images of immunofluorescence corroborated the findings of gene expression profiles. In the “MF” group, iNOS fluorescence was considerably lower after M1-polarizing medium culturing, but Arg-1 immunofluorescence was remarkably higher after M2-polarizing medium culturing on the left side (magnet side), as compared to the right side (non-magnet side). This trend was also consistent in the “NMF-MF” group exhibiting lower iNOS immunofluorescence after M1-polarizing medium culture but higher Arg-1 immunofluorescence after M2-polarizing medium culture on the magnet side, which suggests that the slidable nano-ligand may be attracted to the magnet side at any prescribed time to activate M2 polarization. In the “NMF” group, no considerable difference in iNOS and Arg-1 immunofluorescence was observed between the two sides. These findings indicate that magnetic attraction of the slidable nano-ligand, which enhanced the development of pervasive adhesion structures on the magnet side polarized the macrophages to the regenerative M2 phenotype while suppressing the inflammatory M1 polarization. Next, an experiment was conducted on how the assembly of adherent structures in macrophages facilitates their polarization to the M2 phenotypes by the magnetic attraction of the slidable nano-ligand. Referring toFIG.19, the adherent structures of macrophages including an elongated shape, actin cytoskeletal organization, and contractility, and ROCK are known to mediate the M2 phenotype. In basal medium and M2-polarizing medium cultures, macrophages exhibited considerably higher ROCK2 immunofluorescence on the magnet side than on the non-magnet side. Strikingly, ROCK signal inhibition with Y27632 during M2-polarizing medium culturing significantly reduced the adherent macrophage area and aspect ratio by 32% and 33%, respectively, as well as their Arg-1 immunofluorescence by 29% on the magnet side. Consistently, under myosin II forming inhibition with blebbistatin during M2-polarizing medium culturing decreased the area, aspect ratio, and Arg-1 immunofluorescence of the adherent macrophages by 33%, 31%, and 29% on the magnet side, respectively. These decreases in the area, aspect ratio, and Arg-1 fluorescence of the adherent macrophages were also clearly observed with actin polymerization inhibition by cytochalasin D. On the non-magnet side, inhibition of ROCK, myosin II, and actin polymerization did not result in significant changes in the adherent structures and production of the M2 phenotype. Taken together, these findings collectively indicate that the magnetic control of attracting slidable nano-ligand efficiently promotes the assembly of adhesion structures of macrophages in stimulating the production of the M2 phenotype. Experimental Example 6 The following experiment was conducted in order to confirm that the slidable nano-ligand according to the present invention spatially regulates adhesion and phenotype of host macrophages in vivo through in-situ control, and a result thereof is represented inFIGS.21to23. Material implants cause host responses and it is the most important to control the adhesion and functional phenotypes of macrophages in order to elicit host responses to implants. Remote control of spatiotemporal, reversible, and macroscale nano-ligand variation may emulate dynamic and heterogenous ECM remodeling in order to spatially regulate host responses to implants. In particular, it is remarkably beneficial to regulate the cell adhesion of macrophages and production of the regenerative and anti-inflammatory M2 phenotype to mediate tissue repair while suppressing inflammation. To this end, the present invention explored the in vivo translation of the adhesion structure assembly-mediated M2 phenotype of adherent macrophages in the spatially varying regulation of the regenerative and anti-inflammatory immune responses to implants. It has recently been shown that the spatially heterogeneous modulation of macrophages adhesion by UV light is feasible. However, in the present invention, a tissue-penetrative and cytocompatible alternative using a magnetic field was used in order to regulate not only the macrophage adhesion but also the functional phenotype of adherent macrophages. FIG.21is a diagram illustrating an experiment of adherent and inflammatory M1 phenotype of host macrophages in vivo against magnetic attraction of the slidable nano-ligand according to the exemplary embodiment of the present invention. a ofFIG.21is a schematic diagram of magnetic control of the slidable nano-ligand in vivo. IL-4 and IL-13 were both injected onto the substrate presenting a slidable nano-ligand substrate after subcutaneous implantation. b ofFIG.21is a confocal microscope image of immunofluorescence against iNOS, F-actin, and nuclei of cells adhered to the “magnet” and “non-magnet” sides of the substrate after 24 hour were analyzed. A permanent magnet was continuously placed at the bottom of the substrate (“magnet” side) for 24 hours. The images of cells were taken at the center of the “magnet” side or “non-magnet” side of the substrate. A scale bar is 20 μm. c ofFIG.21is a graph illustrating calculation of the density, cell area, and cell aspect ratio (n=30) as well as gene expression profiles (n=3) of M1 phenotype markers (iNOS and TNF-α) of the in vivo adhered cells. Data are expressed as mean±standard error. Different alphabet letters signify statistical significances. FIG.22is a diagram illustrating a result of an experiment of in vivo adhesion of host neutrophils for the slidable nano-ligand according to the exemplary embodiment of the present invention. a ofFIG.22is a confocal microscope images of immunofluorescent staining against NIMP-R14, F-actin, and nuclei of host cells adhered to the “magnet” and “non-magnet” sides of the substrate presenting the slidable nano-ligand after 24 hours of subcutaneous implantation with an injection of IL-4 and IL-13 onto the substrate. A permanent magnet was continuously placed at the bottom of the substrate (“magnet” side) for 24 hours. The images of cells were taken at the center of “left side” or “right side” of the substrate. A scale bar is 20 μm. b ofFIG.22is a graph illustrating quantification of the density of the in vivo adhered NIMP-R14-positive host neutrophils. Data is displayed as mean±standard error (n=30). Different alphabet letters signify statistical significances. FIG.23is a diagram illustrating a result of an experiment of adhesive and regenerative M2 phenotype of host macrophages in vivo for magnetic attraction of the slidable nano-ligand according to the exemplary embodiment of the present invention. a ofFIG.23is a confocal microscope image of immunofluorescence against Arg-1, F-actin, and nuclei of cells adhered to the “magnet” and “non-magnet” sides of the substrate presenting the slidable nano-ligand after 24 hours of subcutaneous implantation with an injection of IL-4 and IL-13 onto the substrate. A permanent magnet was continuously placed at the bottom of the substrate (“magnet” side). The images of cells were taken at the center of the “magnet” side or “non-magnet” side of the substrate. A scale bar is 20 μm. b ofFIG.23is a graph illustrating calculation of the density, cell area, and cell aspect ratio (n=30) as well as gene expression profiles (n=3) of M2 phenotype markers (Arg-1 and Ym1) of the in vivo adhered cells. Referring toFIG.21, the nano-ligand-presenting substrate was used for subcutaneous implantation into balb/c mice. Following implantation, M2-polarizing soluble factors (interleukin-4 and -13) were injected onto the substrate (FIG. a ofFIG.21). A permanent magnet was attached to the bottom of the substrate (“magnet” side) on the abdomen side of the mice to promote the sliding of the nano-ligand toward the magnet side of the substrate. The substrate was collected 24 hours after implantation for confocal imaging of immunofluorescence and gene expression analysis. Confocal microscope images of immunofluorescence for iNOS and F-actin showed that pronounced colocalization was considerably more dominant on the non-magnet side than on the magnet side (b ofFIG.21). Concomitantly, more pronounced development of adhesion structures with significantly higher macrophage density, area, and aspect ratio (by 67%, 36%, and 53%, respectively) was observed on the magnet side, which promoted significantly lower expression of both iNOS and TNF by 33% and 60%, respectively, in host macrophages than on the non-magnet side (b and c ofFIG.21). Referring toFIG.22, not only host macrophages, but also host neutrophils were observed via NIMP-R14 immunofluorescence during the acute inflammation period. Concurrently, confocal microscope images of immunofluorescence for Arg-1 and F-actin revealed the highly pronounced assembly of adhesion structures by F-actin that positively led to colocalization with regenerative Arg-1 expression on the magnet side considerably more than on the non-magnet side. Referring to a and b ofFIG.23, this observation was also confirmed with significantly higher expression of both Arg-1 and Ym1 (regenerative M2 markers) by 27% and 60%, respectively, in the host macrophages at the magnet side, compared to the non-magnet side. Through this, it can be seen that remote control through the magnetic attraction of slidable nano-ligand promotes the regenerative M2 phenotype of host macrophages but suppresses the inflammatory M1 phenotype in vivo in a spatially regulated fashion. Tight control of early acute inflammation and regenerative responses is known to govern the long-term host responses to implants. It can be seen that it is possible to adjust immunomodulation of implants in the clinical environment through the experiment on magnetic field-mediated spatiotemporal and reversible long-term regulation in promoting tissue repair and inhibiting inflammation and fibrous capsule formation in deep interior tissues. In the experiment example, an experiment on the adhesion and phenotype of macrophages in the culture under the in situ control of nano-ligand sliding was conducted as follows. The effect of nano-ligand sliding in vitro on the adhesion and phenotype of macrophages was examined. The substrate presenting the nano-ligand was sterilized under UV light illumination for 1 hour. The sterile substrate was subjected to blocking with 1% bovine serum albumin (BSA) for minimizing non-nano-ligand-specific adhesion of macrophages. Macrophages (RAW 264.7, passage 5, ATCC) at 50 k cells/cm2were plated on the treated substrate. Macrophages were cultured in a basal medium including high glucose DMEM supplemented with 10% (v/v) heat inactivated fetal bovine serum and 50 U/mL penicillin/streptomycin at 37° C. and 5% CO2. Cells were subjected to a magnet (270 mT) placed at the bottom of the substrate (“magnet” or “left” side) and their adhesion at the center of various sides (magnet, medium, and non-magnet side) was examined. The substrates presenting nanoparticles but without RGD peptide ligand or nano-ligand but without an application of a magnet were used as control groups. The adhesion of macrophages under temporal manipulation of slidable nano-ligand was examined with a substrate presenting nano-ligand subjected to placing a permanent magnet at the bottom of the substrate [“magnetic field (MF)”] or withdrawing it from the substrate [“no magnetic field (NMF)”]. Temporal switching of a permanent magnet was also applied between two opposite sides at the bottom “left” side of the substrate to the bottom “right” side and then to the bottom “left” side back again. Polarization phenotypes of macrophages under time-regulated manipulation of the slidable nano-ligand was examined using a substrate presenting nano-ligand subjected to placing a permanent magnet at the bottom of the substrate [“magnetic field (MF)”] or withdrawing it from the substrate [“no magnetic field (NMF)”] with their switching between “NMF” and “MF”. The M1-polarizing medium included the basal medium supplemented with lipopolysaccharide (LPS, 10 ng/mL) and recombinant interferon-gamma (IFN-γ, 10 ng/mL). The M2-polarizing medium included the basal medium supplemented with interleukin-4 (IL-4, 20 ng/mL) and interleukin-13 (IL-13, 20 ng/mL). The adhesion structure assembly-mediated M2 phenotype of macrophages was examined after subjecting the substrate to a magnet placed at the bottom of the substrate (“magnet” side) under ROCK signal inhibition (with 50 μM Y27632), myosin II forming inhibition (with 10 μM blebbistatin), or actin polymerization inhibition (with 2 μg/mL cytochalasin D). Macrophage adhesion under the slidable nano-ligand in situ by immunofluorescence and confocal imaging was analyzed as follows. Macrophage cultures were immersed in a fixing solution of 4% (w/v) paraformaldehyde for 10 minutes, which were then washed with PBS. The cultures were incubated in a blocking buffer of 3% BSA and 0.1% Triton-X in PBS for 30 minutes. The cultures were soaked in the solution with primary antibodies against integrin (31, vinculin, iNOS, Arginase-1, ROCK2, and NIMP-R14 at 4° C. overnight, followed by washing with PBS. The cultures were immersed in the solution with secondary antibodies, phalloidin, and DAPI at room temperature for 30 minutes, followed by washing with PBS. The cultures were mounted on a microscope slide and imaged with confocal microscope (LSM700, Carl Zeiss) using the same exposure conditions for all of the compared groups, which were then examined with ImageJ software. The confocal microscope images were used to quantify the adhesion of macrophages. Integrin β1 binding intensities were calculated with histogram function. The adherent macrophage density was calculated by counting the DAPI-stained nuclei. The adherent macrophage area and aspect ratio were calculated by analyzing the F-actin staining, as reported previously. Macrophage polarization under slidable nano-ligand in situ with reverse transcription-polymerase chain reaction was analyzed as follows. Macrophages were cultured in M1- or M2-polarizing medium and harvested in 1 mL of Trizol per sample. The substrate was divided into two halves (magnet and non-magnet side). For each sample, 900 ng of extracted RNA was subjected to reverse transcription to cDNA by using High-Capacity RNA-to-cDNA Kit. The cDNA with Sybr Green assays was used to run real-time PCR reactions (StepOne Plus Real-Time PCR System, Applied Biosystems). The relative fold expressions of the target genes (iNOS, TNF-α, Arginase-1, and Ym1) were presented following their normalization to GAPDH expression. Further, an experiment on in vivo remote control of the nano-ligand sliding for regulating the adhesion and phenotype of host macrophages was conducted as follows. To investigate the effect of nano-ligand sliding in vivo on the adhesion and phenotype of host macrophages, the nano-ligand-presenting silicon substrate was used for subcutaneous implantation. 16 two month-old balb/c mice were utilized after the approval of the Institutional Animal Care and Use Committee of Korea University. The mixture of 2 mL of alfaxan and 1 mL of rompun was used for an intraperitoneal injection. The back of the mice was incised approximately in 2.5 cm length. Following implantation, IL-4 (50 ng) and IL-13 (50 ng) was injected onto the substrate. The adhesion of mouse macrophages to the substrate was tested. A permanent magnet attached to the bottom (abdomen side) of the substrate (“magnet” side) promoted the sliding of the nano-ligand toward the magnet side. At 24 hours after implantation, the substrate was retrieved for confocal imaging of the immunofluorescence and RT-PCR analysis. | 68,338 |
11859170 | Top panel represents the locus amy2 with the integration of the FLP landing pad composed of FRT-F and FRT-F3 the FLPase recognition site, as well as the amdS (acetamide) selection marker and the FLPase expression cassette. A split PyrG marker has been used and at the amy2 locus the 5′ end of the pyrG marker is inserted. Middle panel represents the transforming DNA, in particular the region that is integrated at the FLP landing pad by site specific recombination mediated by FLPase. The plasmid or PCR product must contain FRT-F and F3 sites as well as the remaining 3′ part of the pyrG marker. Bottom panel represents the resulting amy2 locus after site specific integration of the transforming DNA between the FRT sites. The amdS and FLP cassettes have been exchanged with the GOI expression cassette and the 3′ part of the pyrG marker reconstituting a fully functional selection marker. Definitions Animal: The term “animal” refers to any animal except humans. Examples of animals are monogastric animals, including but not limited to pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks, quail, guinea fowl, geese, pigeons (including squabs) and chicken (including but not limited to broiler chickens (referred to herein as broiles), chicks, layer hens (referred to herein as layers)); horses (including but not limited to hotbloods, coldbloods and warm bloods) crustaceans (including but not limited to shrimps and prawns) and fish (including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish). Animal feed: The term “animal feed” refers to any compound, preparation, or mixture suitable for, or intended for intake by a monogastric animal. Animal feed for a monogastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbial, amino acids and/or other feed ingredients (such as in a premix). Antimicrobial activity: The term “antimicrobial activity” is defined herein as an activity that kills or inhibits the growth of microorganisms, such as, algae, archea, bacteria, fungi and/or protozoans. The antimicrobial activity can, for example, be bactericidal meaning the killing of bacteria or bacteriostatic meaning the prevention of bacterial growth. The antimicrobial activity can include catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins. Antimicrobial activity can also include the LYS polypeptide binding to the surface of the microorganism and inhibiting its growth. The antimicrobial effect can also include the use of the LYS polypeptides of the present invention for activation of bacterial autolysins, as an immunostimulator, by inhibiting or reducing bacterial toxins and by an opsonin effect. Body Weight Gain: The term “body weight gain” means an increase in live weight of an animal during a given period of time e.g. the increase in weight from day 1 to day 21. cDNA: The term “cDNA” means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA. Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof. Concentrates: The term “concentrates” means feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from e.g. corn, oats, rye, barley, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)). Control sequences: The term “control sequences” means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide. European Production Efficacy Factor (EPEF): The “European Production Efficacy Factor” is a way of comparing the performance of animals. This single-figure facilitates comparison of performance within and among farms and can be used to assess environmental, climatic and managemental variables. The EPEF is calculated as [(liveability (%)×Liveweight (kg))/(Age at depletion (days)×FCR)]×100, wherein livability is the percentage of animals alive at slaughter, Liveweight is the average weight of the animals at slaughter, age of depletion is the age of the animals at slaughter and FCR is the feed conversion ratio at slaughter. Expression: The term “expression” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression vector: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. Feed Conversion Ratio (FCR): FCR is a measure of an animal's efficiency in converting feed mass into increases of the desired output. Animals raised for meat—such as swine, poultry and fish—the output is the mass gained by the animal. Specifically, FCR is calculated as feed intake divided by weight gain, all over a specified period. Improvement in FCR means reduction of the FCR value. A FCR improvement of 2% means that the FCR was reduced by 2%. Feed efficiency: The term “feed efficiency” means the amount of weight gain per unit of feed when the animal is fed ad-libitum or a specified amount of food during a period of time. By “increased feed efficiency” it is meant that the use of a feed additive composition according the present invention in feed results in an increased weight gain per unit of feed intake compared with an animal fed without said feed additive composition being present. Forage: The term “forage” as defined herein also includes roughage. Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are Alfalfa (lucerne), birdsfoot trefoil, brassica (e.g. kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g. alsike clover, red clover, subterranean clover, white clover), grass (e.g. Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, orchard grass, ryegrass, Timothy-grass), corn (maize), millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets. Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains); residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries. Fragment: The term “fragment” means a LYS polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has lysozyme activity. In one aspect, the fragment comprises at least 90% of the length of the mature polypeptide, such as at least 203 amino acids of SEQ ID NO: 2, at least 203 amino acids of SEQ ID NO: 3, at least 203 amino acids of SEQ ID NO: 5, at least 203 amino acids of SEQ ID NO: 6, at least 200 amino acids of SEQ ID NO: 8, at least 200 amino acids of SEQ ID NO: 9, at least 273 amino acids of SEQ ID NO: 11, at least 273 amino acids of SEQ ID NO: 12, at least 205 amino acids of SEQ ID NO: 14, at least 205 amino acids of SEQ ID NO: 15, at least 207 amino acids of SEQ ID NO: 17, at least 207 amino acids of SEQ ID NO: 18, at least 207 amino acids of SEQ ID NO: 20, at least 207 amino acids of SEQ ID NO: 21, at least 208 amino acids of SEQ ID NO: 23, at least 208 amino acids of SEQ ID NO: 24, at least 205 amino acids of SEQ ID NO: 26, at least 205 amino acids of SEQ ID NO: 27, at least 205 amino acids of SEQ ID NO: 29, at least 205 amino acids of SEQ ID NO: 30, at least 203 amino acids of SEQ ID NO: 32, at least 203 amino acids of SEQ ID NO: 33, at least 202 amino acids of SEQ ID NO: 35, at least 202 amino acids of SEQ ID NO: 36, at least 202 amino acids of SEQ ID NO: 38, at least 202 amino acids of SEQ ID NO: 39, at least 273 amino acids of SEQ ID NO: 41, at least 273 amino acids of SEQ ID NO: 42, at least 204 amino acids of SEQ ID NO: 44, or at least 204 amino acids of SEQ ID NO: 45. In one aspect, the fragment comprises at least 92% of the length of the mature polypeptide, such as at least 207 amino acids of SEQ ID NO: 2, at least 207 amino acids of SEQ ID NO: 3, at least 207 amino acids of SEQ ID NO: 5, at least 207 amino acids of SEQ ID NO: 6, at least 205 amino acids of SEQ ID NO: 8, at least 205 amino acids of SEQ ID NO: 9, at least 279 amino acids of SEQ ID NO: 11, at least 279 amino acids of SEQ ID NO: 12, at least 209 amino acids of SEQ ID NO: 14, at least 209 amino acids of SEQ ID NO: 15, at least 211 amino acids of SEQ ID NO: 17, at least 211 amino acids of SEQ ID NO: 18, at least 211 amino acids of SEQ ID NO: 20, at least 211 amino acids of SEQ ID NO: 21, at least 213 amino acids of SEQ ID NO: 23, at least 213 amino acids of SEQ ID NO: 24, at least 209 amino acids of SEQ ID NO: 26, at least 209 amino acids of SEQ ID NO: 27, at least 209 amino acids of SEQ ID NO: 29, at least 209 amino acids of SEQ ID NO: 30, at least 207 amino acids of SEQ ID NO: 32, at least 207 amino acids of SEQ ID NO: 33, at least 207 amino acids of SEQ ID NO: 35, at least 207 amino acids of SEQ ID NO: 36, at least 207 amino acids of SEQ ID NO: 38, at least 207 amino acids of SEQ ID NO: 39, at least 279 amino acids of SEQ ID NO: 41, at least 279 amino acids of SEQ ID NO: 42, at least 208 amino acids of SEQ ID NO: 44, or at least 208 amino acids of SEQ ID NO: 45. In one aspect, the fragment comprises at least 94% of the length of the mature polypeptide, such as at least 212 amino acids of SEQ ID NO: 2, at least 212 amino acids of SEQ ID NO: 3, at least 212 amino acids of SEQ ID NO: 5, at least 212 amino acids of SEQ ID NO: 6, at least 209 amino acids of SEQ ID NO: 8, at least 209 amino acids of SEQ ID NO: 9, at least 285 amino acids of SEQ ID NO: 11, at least 285 amino acids of SEQ ID NO: 12, at least 214 amino acids of SEQ ID NO: 14, at least 214 amino acids of SEQ ID NO: 15, at least 216 amino acids of SEQ ID NO: 17, at least 216 amino acids of SEQ ID NO: 18, at least 216 amino acids of SEQ ID NO: 20, at least 216 amino acids of SEQ ID NO: 21, at least 218 amino acids of SEQ ID NO: 23, at least 218 amino acids of SEQ ID NO: 24, at least 214 amino acids of SEQ ID NO: 26, at least 214 amino acids of SEQ ID NO: 27, at least 214 amino acids of SEQ ID NO: 29, at least 214 amino acids of SEQ ID NO: 30, at least 212 amino acids of SEQ ID NO: 32, at least 212 amino acids of SEQ ID NO: 33, at least 211 amino acids of SEQ ID NO: 35, at least 211 amino acids of SEQ ID NO: 36, at least 211 amino acids of SEQ ID NO: 38, at least 211 amino acids of SEQ ID NO: 39, at least 285 amino acids of SEQ ID NO: 41, at least 285 amino acids of SEQ ID NO: 42, at least 213 amino acids of SEQ ID NO: 44, or at least 213 amino acids of SEQ ID NO: 45. In one aspect, the fragment comprises at least 96% of the length of the mature polypeptide, such as at least 216 amino acids of SEQ ID NO: 2, at least 216 amino acids of SEQ ID NO: 3, at least 216 amino acids of SEQ ID NO: 5, at least 216 amino acids of SEQ ID NO: 6, at least 214 amino acids of SEQ ID NO: 8, at least 214 amino acids of SEQ ID NO: 9, at least 291 amino acids of SEQ ID NO: 11, at least 291 amino acids of SEQ ID NO: 12, at least 218 amino acids of SEQ ID NO: 14, at least 218 amino acids of SEQ ID NO: 15, at least 220 amino acids of SEQ ID NO: 17, at least 220 amino acids of SEQ ID NO: 18, at least 220 amino acids of SEQ ID NO: 20, at least 220 amino acids of SEQ ID NO: 21, at least 222 amino acids of SEQ ID NO: 23, at least 222 amino acids of SEQ ID NO: 24, at least 218 amino acids of SEQ ID NO: 26, at least 218 amino acids of SEQ ID NO: 27, at least 218 amino acids of SEQ ID NO: 29, at least 218 amino acids of SEQ ID NO: 30, at least 216 amino acids of SEQ ID NO: 32, at least 216 amino acids of SEQ ID NO: 33, at least 216 amino acids of SEQ ID NO: 35, at least 216 amino acids of SEQ ID NO: 36, at least 216 amino acids of SEQ ID NO: 38, at least 216 amino acids of SEQ ID NO: 39, at least 291 amino acids of SEQ ID NO: 41, at least 291 amino acids of SEQ ID NO: 42, at least 217 amino acids of SEQ ID NO: 44, or at least 217 amino acids of SEQ ID NO: 45. In one aspect, the fragment comprises at least 98% of the length of the mature polypeptide, such as at least 221 amino acids of SEQ ID NO: 2, at least 221 amino acids of SEQ ID NO: 3, at least 221 amino acids of SEQ ID NO: 5, at least 221 amino acids of SEQ ID NO: 6, at least 218 amino acids of SEQ ID NO: 8, at least 218 amino acids of SEQ ID NO: 9, at least 297 amino acids of SEQ ID NO: 11, at least 297 amino acids of SEQ ID NO: 12, at least 223 amino acids of SEQ ID NO: 14, at least 223 amino acids of SEQ ID NO: 15, at least 225 amino acids of SEQ ID NO: 17, at least 225 amino acids of SEQ ID NO: 18, at least 225 amino acids of SEQ ID NO: 20, at least 225 amino acids of SEQ ID NO: 21, at least 227 amino acids of SEQ ID NO: 23, at least 227 amino acids of SEQ ID NO: 24, at least 223 amino acids of SEQ ID NO: 26, at least 223 amino acids of SEQ ID NO: 27, at least 223 amino acids of SEQ ID NO: 29, at least 223 amino acids of SEQ ID NO: 30, at least 221 amino acids of SEQ ID NO: 32, at least 221 amino acids of SEQ ID NO: 33, at least 220 amino acids of SEQ ID NO: 35, at least 220 amino acids of SEQ ID NO: 36, at least 220 amino acids of SEQ ID NO: 38, at least 220 amino acids of SEQ ID NO: 39, at least 297 amino acids of SEQ ID NO: 41, at least 297 amino acids of SEQ ID NO: 42, at least 222 amino acids of SEQ ID NO: 44, or at least 222 amino acids of SEQ ID NO: 45. In one aspect, the fragment comprises at least 99% of the length of the mature polypeptide, such as at least 223 amino acids of SEQ ID NO: 2, at least 223 amino acids of SEQ ID NO: 3, at least 223 amino acids of SEQ ID NO: 5, at least 223 amino acids of SEQ ID NO: 6, at least 220 amino acids of SEQ ID NO: 8, at least 220 amino acids of SEQ ID NO: 9, at least 300 amino acids of SEQ ID NO: 11, at least 300 amino acids of SEQ ID NO: 12, at least 225 amino acids of SEQ ID NO: 14, at least 225 amino acids of SEQ ID NO: 15, at least 227 amino acids of SEQ ID NO: 17, at least 227 amino acids of SEQ ID NO: 18, at least 227 amino acids of SEQ ID NO: 20, at least 227 amino acids of SEQ ID NO: 21, at least 229 amino acids of SEQ ID NO: 23, at least 229 amino acids of SEQ ID NO: 24, at least 225 amino acids of SEQ ID NO: 26, at least 225 amino acids of SEQ ID NO: 27, at least 225 amino acids of SEQ ID NO: 29, at least 225 amino acids of SEQ ID NO: 30, at least 223 amino acids of SEQ ID NO: 32, at least 223 amino acids of SEQ ID NO: 33, at least 222 amino acids of SEQ ID NO: 35, at least 222 amino acids of SEQ ID NO: 36, at least 222 amino acids of SEQ ID NO: 38, at least 222 amino acids of SEQ ID NO: 39, at least 300 amino acids of SEQ ID NO: 41, at least 300 amino acids of SEQ ID NO: 42, at least 224 amino acids of SEQ ID NO: 44, or at least 224 amino acids of SEQ ID NO: 45. Fusion polypeptide: The term “fusion polypeptide” is a polypeptide in which one polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J.12: 2575-2583; Dawson et al., 1994, Science266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol.3: 568-576; Svetina et al., 2000, J. Biotechnol.76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.63: 3488-3493; Ward et al., 1995, Biotechnology13: 498-503; and Contreras et al., 1991, Biotechnology9: 378-381; Eaton et al., 1986, Biochemistry25: 505-512; Collins-Racie et al., 1995, Biotechnology13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics6: 240-248; and Stevens, 2003, Drug Discovery World4: 35-48. Host cell: The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. Hybrid polypeptide: The term “hybrid polypeptide” means a polypeptide comprising domains from two or more polypeptides, e.g., a binding domain from one polypeptide and a catalytic domain from another polypeptide. The domains may be fused at the N-terminus or the C-terminus. Isolated: The term “isolated” means a substance in a form that does not occur in nature or in an environment in which the substance does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). Lysozyme activity: The term “lysozyme activity” means the hydrolysis of the 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan, resulting in bacteriolysis. Lysozyme belongs to the enzyme class EC 3.2.1.17. Lysozyme activity is typically measured by the lytic action of the lysozyme onMicrococcus luteusATCC 4698. In appropriate experimental conditions these changes are proportional to the amount of lysozyme in the medium (c.f. INS 1105 of the Combined Compendium of Food Additive Specifications of the Food and Agriculture Organisation of the UN (www.fao.org)). For the purpose of the present invention, lysozyme activity is determined according to the reducing-ends assay described in Example 1 (“Determination of Lysozyme Activity using reducing ends assay”). The polypeptide has lysozyme activity if it shows activity againstMicrococcus luteus ATCC4698. In one aspect, the polypeptides of the present invention have at least 50%, e.g., preferably at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or most preferably at least 100% of the lysozyme activity of SEQ ID NO: 12, preferably wherein lysozyme activity is determined as described in Example 1. In one aspect, the polypeptides of the present invention have at least 50%, e.g., preferably at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or most preferably at least 100% of the lysozyme activity of SEQ ID NO: 12 wherein lysozyme activity is determined as follows: LYS polypeptide (50 μL of 0.7 μg/mL LYS polypeptide in phosphate buffer (5 mM citrate, 5 mM K2HPO4, 0.01% TritonX-100, pH 5.0)) is mixed withMicrococcus lysodeikticussolution (450 μL of 1% lyophilizedMicrococcus lysodeikticusATCC No. 4698 in milli-Q water) and incubated at 40° C. with shaking (500 rpm) for 45 min; the sample is centrifuged (4000 g, 5 min); supernatant (100 μL) is mixed with HCl (50 μL 3.2M) and incubated at 95° C. for 80 min; NaOH (50 μL, 3.5 M) is added and 150 μL of the sample is added to 4-hydroxybenzhydrazide in K—Na tartrate/NaOH buffer (75 μL of 50 g/L K—Na tartrate+20 g/L NaOH); the mixture is incubated at 95° C. for 10 min; and the optical density is measured at 405 nm. Mature polypeptide: The term “mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 1 to 226 of SEQ ID NO: 2 and amino acids −19 to −1 of SEQ ID NO: 2 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 226 of SEQ ID NO: 3. In one aspect, the mature polypeptide is amino acids 1 to 226 of SEQ ID NO: 5 and amino acids −19 to −1 of SEQ ID NO: 5 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 226 of SEQ ID NO: 6. In one aspect, the mature polypeptide is amino acids 1 to 223 of SEQ ID NO: 8 and amino acids −20 to −1 of SEQ ID NO: 8 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 223 of SEQ ID NO: 9. In one aspect, the mature polypeptide is amino acids 1 to 304 of SEQ ID NO: 11 and amino acids −20 to −1 of SEQ ID NO: 11 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 304 of SEQ ID NO: 12. In one aspect, the mature polypeptide is amino acids 1 to 228 of SEQ ID NO: 14 and amino acids −19 to −1 of SEQ ID NO: 14 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 228 of SEQ ID NO: 15. In one aspect, the mature polypeptide is amino acids 1 to 230 of SEQ ID NO: 17 and amino acids −20 to −1 of SEQ ID NO: 17 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 230 of SEQ ID NO: 18. In one aspect, the mature polypeptide is amino acids 1 to 230 of SEQ ID NO: 20 and amino acids −21 to −1 of SEQ ID NO: 20 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 230 of SEQ ID NO: 21. In one aspect, the mature polypeptide is amino acids 1 to 232 of SEQ ID NO: 23 and amino acids −22 to −1 of SEQ ID NO: 23 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 232 of SEQ ID NO: 24. In one aspect, the mature polypeptide is amino acids 1 to 228 of SEQ ID NO: 26 and amino acids −20 to −1 of SEQ ID NO: 26 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 228 of SEQ ID NO: 27. In one aspect, the mature polypeptide is amino acids 1 to 228 of SEQ ID NO: 29 and amino acids −20 to −1 of SEQ ID NO: 29 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 228 of SEQ ID NO: 30. In one aspect, the mature polypeptide is amino acids 1 to 226 of SEQ ID NO: 32 and amino acids −19 to −1 of SEQ ID NO: 32 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 226 of SEQ ID NO: 33. In one aspect, the mature polypeptide is amino acids 1 to 225 of SEQ ID NO: 35 and amino acids −20 to −1 of SEQ ID NO: 35 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 225 of SEQ ID NO: 36. In one aspect, the mature polypeptide is amino acids 1 to 225 of SEQ ID NO: 38 and amino acids −19 to −1 of SEQ ID NO: 38 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 225 of SEQ ID NO: 39. In one aspect, the mature polypeptide is amino acids 1 to 304 of SEQ ID NO: 41 and amino acids −19 to −1 of SEQ ID NO: 41 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 304 of SEQ ID NO: 42. In one aspect, the mature polypeptide is amino acids 1 to 227 of SEQ ID NO: 44 and amino acids −19 to −1 of SEQ ID NO: 44 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 227 of SEQ ID NO: 45. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide. Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having lysozyme activity. Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences. Obtained or obtainable from: The term “obtained or obtainable from” means that the polypeptide may be found in an organism from a specific taxonomic rank. In one embodiment, the polypeptide is obtained or obtainable from the kingdom Fungi, wherein the term kingdom is the taxonomic rank. In a preferred embodiment, the polypeptide is obtained or obtainable from the phylum Ascomycota, wherein the term phylum is the taxonomic rank. In another preferred embodiment, the polypeptide is obtained or obtainable from the subphylum Pezizomycotina, wherein the term subphylum is the taxonomic rank. If the taxonomic rank of a polypeptide is not known, it can easily be determined by a person skilled in the art by performing a BLASTP search of the polypeptide (using e.g. the National Center for Biotechnology Information (NCIB) website http://www.ncbi.nlm.nih.gov/) and comparing it to the closest homologues. An unknown polypeptide which is a fragment of a known polypeptide is considered to be of the same taxonomic species. An unknown natural polypeptide or artificial variant which comprises a substitution, deletion and/or insertion in up to 10 positions is considered to be from the same taxonomic species as the known polypeptide. Operably linked: The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence. Roughage: The term “roughage” means dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste). Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labelled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment) For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labelled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment) Subsequence: The term “subsequence” means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5′ and/or 3′ end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having lysozyme activity. Substantially pure polypeptide: The term “substantially pure polypeptide” means a preparation that contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. Preferably, the polypeptide is at least 92% pure, e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the polypeptide by well known recombinant methods or by classical purification methods. Variant: The term “variant” means a polypeptide having lysozyme activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, of one or more (several) amino acid residues at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1, 2, or 3 amino acids adjacent to and immediately following the amino acid occupying the position. In one aspect, the variant according to the invention may comprise from 1 to 5; from 1 to 10; from 1 to 15; from 1 to 20; from 1 to 25; from 1 to 30; from 1 to 35; from 1 to 40; from 1 to 45; or from 1-50, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 alterations. In one aspect, the variant of the present invention has at least 50%, e.g., preferably at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or most preferably at least 100% of the lysozyme activity of SEQ ID NO: 12, preferably wherein lysozyme activity is determined as described in Example 1. In one aspect, the variant of the present invention has at least 50%, e.g., preferably at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or most preferably at least 100% of the lysozyme activity of SEQ ID NO: 12 wherein lysozyme activity is determined as follows: LYS polypeptide (50 μL of 0.7 μg/mL LYS polypeptide in phosphate buffer (5 mM citrate, 5 mM K2HPO4, 0.01% TritonX-100, pH 5.0)) is mixed withMicrococcus lysodeikticussolution (450 μL of 1% lyophilizedMicrococcus lysodeikticusATCC No. 4698 in milli-Q water) and incubated at 40° C. with shaking (500 rpm) for 45 min; the sample is centrifuged (4000 g, 5 min); supernatant (100 μL) is mixed with HCl (50 μL 3.2M) and incubated at 95° C. for 80 min; NaOH (50 μL, 3.5 M) is added and 150 μL of the sample is added to 4-hydroxybenzhydrazide in K—Na tartrate/NaOH buffer (75 μL of 50 g/L K—Na tartrate+20 g/L NaOH); the mixture is incubated at 95° C. for 10 min; and the optical density is measured at 405 nm. In one aspect, the variant according to the invention may comprise from 1 to 5; from 1 to 10; from 1 to 15; from 1 to 20; from 1 to 25; from 1 to 30; from 1 to 35; from 1 to 40; from 1 to 45; or from 1-50, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 alterations and has at least 50%, e.g., preferably at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or most preferably at least 100% of the lysozyme activity of SEQ ID NO: 12, preferably wherein lysozyme activity is determined as described in Example 1. Nomenclature For purposes of the present invention, the nomenclature [E/Q] means that the amino acid at this position may be a glutamic acid (Glu, E) or a glutamine (Gln, Q). Likewise the nomenclature [V/G/A/I] means that the amino acid at this position may be a valine (Val, V), glycine (Gly, G), alanine (Ala, A) or isoleucine (Ile, I), and so forth for other combinations as described herein. Unless otherwise limited further, the amino acid X is defined such that it may be any of the 20 natural amino acids. DETAILED DESCRIPTION OF THE INVENTION The inventors have discovered a completely novel class of polypeptides having lysozyme activity. Said polypeptides are structurally quite different from known lysozymes. As shown in the sequence identity matrix below, the polypeptides of the present invention all have a sequence identity less than 45% to the prior art sequences disclosed in WO2013/076259, suggesting that these novel polypeptides may have a different folding pattern to known lysozymes. GHclassSEQ3SEQ2SEQ4SEQ6SEQ8HEWLSEQ ID NO: 3 ofNot1002733.343.823.330.93present inventiondefinedSEQ ID NO: 2 ofGH23271002733.33221.3WO2013/076259SEQ ID NO: 4 ofGH2433.3271007932.733.33WO2013/076259SEQ ID NO: 6 ofGH2543.833.37910034.144.78WO2013/076259SEQ ID NO: 8 ofGH2523.33232.734.110028.97WO2013/076259Hen Egg WhiteGH2230.921.333.344.829100(SwissprotP00698) The polypeptides of the present invention demonstrate typical lysozyme activity such as activity in the traditional OD drop assay againstMicrococcus lysodeikticus(see example 14) or a reducing ends assay usingMicrococcus lysodeikticusas substrate (see example 13). The polypeptides of the invention having lysozyme activity are herein named LYS polypeptides and comprise one or more LAD (Lysozyme Active Domain) catalytic domains and optionally one or more lysozyme enhancing domains (LED). Compositions Comprising Polypeptides Having Lysozyme Activity In the first aspect, the invention relates to a composition comprising at least 0.01 mg of LYS polypeptide per kilogram of composition, wherein the polypeptide (a) has lysozyme activity and (b) comprises one or more LAD catalytic domains; wherein the LAD catalytic domain gives a domT score of at least 180 when queried using a Profile Hidden Markov Model (HMM) prepared using SEQ ID NOs: 46 to 187 and hmmbuild software program, and wherein the query is carried out using hmmscan software program by the Method of Determining the LAD Catalytic Domain by HMM. In an embodiment, the polypeptide further comprises one or more lysozyme enhancing domains (LED). Thus, the invention further relates to a composition comprising at least 0.01 mg of LYS polypeptide per kilogram of composition, wherein:(a) the LYS polypeptide has lysozyme activity;(b) the LYS polypeptide comprises one or more LAD catalytic domains; wherein the LAD catalytic domain gives a domT score of at least 180 when queried using a Profile Hidden Markov Model (HMM) prepared using SEQ ID NOs: 46 to 187 and hmmbuild software program, and wherein the query is carried out using hmmscan software program by the Method of Determining the LAD Catalytic Domain by HMM;(c) the polypeptide comprises one or more LED domains, wherein the LED gives a domT score of at least 100 when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 188 to 316 and hmmbuild software program, and wherein the query is carried out using the hmmscan software program. The theory behind Profile HMMs as described in Durbin et al. (Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998) and Krogh et al. (1994J. Mol. Biol.235:1501-1531), both incorporated herein by reference, is characterization of a set of proteins based on the probability of each amino acid occurring at each position in the alignment of the proteins of the set. Specifically, profile HMMs are statistical models of multiple sequence alignments, or even of single sequences. They capture position-specific information about how conserved each column of the alignment is, and which residues are likely. All profile methods are more or less statistical descriptions of the consensus of a multiple sequence alignment. They use position-specific scores for amino acids or nucleotides (residues) and position specific penalties for opening and extending an insertion or deletion. Traditional pairwise alignment (for example, BLAST, FASTA or the Smith/Waterman algorithm) uses position-independent scoring parameters. This property of profiles captures important information about the degree of conservation at various positions in the multiple alignment, and the varying degree to which gaps and insertions are permitted. The advantage of using HMMs is that HMMs have a formal probabilistic basis. Probability theory is used to guide how all the scoring parameters should be set. One of the most important aspect is that HMMs have a consistent theory for setting position-specific gap and insertion scores. The methods are consistent and therefore highly automatable, allowing hundreds of profile HMMs to be applied to e.g. whole genome analysis. An example of a protein domain model database is Pfam (Sonnhammer et al., 1997, ‘A comprehensive database of protein families based on seed alignments’, Proteins, 28:405-420; Finn et al., 2010, ‘The Pfam protein families database’, Nucl. Acids Res., 38:D211-D222), which is a significant part of the Interpro protein domain annotation system. The construction and use of Pfam is tightly tied to the HMM ER software package (see https://en.wikipedia.org/wiki/HMMER). The LAD catalytic domain is defined in the following manner. SEQ ID NOs: 46 to 187, which are partial sequences of the Uniprot entries as explained in the ‘overview of sequence listing’ section herein, are aligned using the software program MUSCLE v3.8.31 with the default settings. Using this alignment, a hidden Markov model (HMM) is built for the LAD catalytic domain. The HMM is constructed using the software program ‘hmmbuild’ from the package HMMER 3.0 (March 2010) (http://hmmer.org/) and the software is invoked using the default settings. A LAD catalytic domain is defined to match the above mentioned HMM using the software program ‘hmmscan’ from the package HMMER 3.0 (March 2010) (http://hmmer.org/) using the default settings if the domT score is at least 170. In a preferred embodiment, the domT score is at least 175, preferably at least 180, more preferably at least 185, even more preferably at least 190, even more preferably at least 195, or most preferably at least 200. The HMM profile of the LAD catalytic domain as generated using SEQ ID NOs: 46 to 187 according to the procedure above is given in example 10. The HMM profile can be copied into a text file which is subsequently loaded into the software program ‘hmmscan’ so that other polypeptides can be tested to see whether said polypeptide comprises one or more LAD catalytic domains. The Lysozyme Enhancing Domain (LED) is defined in the following manner. SEQ ID NOs: 188 to 316, which are partial sequences of the Uniprot entries as explained in the ‘overview of sequence listing’ section herein, are aligned using the software program MUSCLE v3.8.31 with the default settings. Using this alignment, a hidden Markov model (HMM) is built for the LED. The HMM is constructed using the software program ‘hmmbuild’ from the package HMMER 3.0 (March 2010) (http://hmmer.org/) and the software is invoked using the default settings. A LED is defined to match the above mentioned HMM using the software program ‘hmmscan’ from the package HMMER 3.0 (March 2010) (http://hmmer.org/) using the default settings if the domT score is at least 100. In a preferred embodiment, the domT score is at least 103, preferably at least 106, more preferably at least 109, more preferably at least 112, more preferably at least 115, more preferably at least 118, even more preferably at least 121, or most preferably at least 124. The HMM profile of the LED as generated using SEQ ID NOs: 188 to 316 according to the procedure above is given in example 11. The HMM profile can be copied into a text file which is subsequently loaded into the software program ‘hmmscan’ so that other polypeptides can be tested to see whether said polypeptide comprises one or more LED. In an embodiment, the LAD catalytic domain gives a domT score of at least 175 and the LED gives a domT score of at least 100. In an embodiment, the LAD catalytic domain gives a domT score of at least 180 and the LED gives a domT score of at least 100. In an embodiment, the LAD catalytic domain gives a domT score of at least 185 and the LED gives a domT score of at least 100. In an embodiment, the LAD catalytic domain gives a domT score of at least 190 and the LED gives a domT score of at least 100. In an embodiment, the LAD catalytic domain gives a domT score of at least 195 and the LED gives a domT score of at least 100. In an embodiment, the LAD catalytic domain gives a domT score of at least 200 and the LED gives a domT score of at least 100. In an embodiment, the LAD catalytic domain gives a domT score of at least 175 and the LED gives a domT score of at least 103. In an embodiment, the LAD catalytic domain gives a domT score of at least 180 and the LED gives a domT score of at least 103. In an embodiment, the LAD catalytic domain gives a domT score of at least 185 and the LED gives a domT score of at least 103. In an embodiment, the LAD catalytic domain gives a domT score of at least 190 and the LED gives a domT score of at least 103. In an embodiment, the LAD catalytic domain gives a domT score of at least 195 and the LED gives a domT score of at least 103. In an embodiment, the LAD catalytic domain gives a domT score of at least 200 and the LED gives a domT score of at least 103. In an embodiment, the LAD catalytic domain gives a domT score of at least 175 and the LED gives a domT score of at least 106. In an embodiment, the LAD catalytic domain gives a domT score of at least 180 and the LED gives a domT score of at least 106. In an embodiment, the LAD catalytic domain gives a domT score of at least 185 and the LED gives a domT score of at least 106. In an embodiment, the LAD catalytic domain gives a domT score of at least 190 and the LED gives a domT score of at least 106. In an embodiment, the LAD catalytic domain gives a domT score of at least 195 and the LED gives a domT score of at least 106. In an embodiment, the LAD catalytic domain gives a domT score of at least 200 and the LED gives a domT score of at least 106. In an embodiment, the LAD catalytic domain gives a domT score of at least 175 and the LED gives a domT score of at least 109. In an embodiment, the LAD catalytic domain gives a domT score of at least 180 and the LED gives a domT score of at least 109. In an embodiment, the LAD catalytic domain gives a domT score of at least 185 and the LED gives a domT score of at least 109. In an embodiment, the LAD catalytic domain gives a domT score of at least 190 and the LED gives a domT score of at least 109. In an embodiment, the LAD catalytic domain gives a domT score of at least 195 and the LED gives a domT score of at least 109. In an embodiment, the LAD catalytic domain gives a domT score of at least 200 and the LED gives a domT score of at least 109. In an embodiment, the LAD catalytic domain gives a domT score of at least 175 and the LED gives a domT score of at least 112. In an embodiment, the LAD catalytic domain gives a domT score of at least 180 and the LED gives a domT score of at least 112. In an embodiment, the LAD catalytic domain gives a domT score of at least 185 and the LED gives a domT score of at least 112. In an embodiment, the LAD catalytic domain gives a domT score of at least 190 and the LED gives a domT score of at least 112. In an embodiment, the LAD catalytic domain gives a domT score of at least 195 and the LED gives a domT score of at least 112. In an embodiment, the LAD catalytic domain gives a domT score of at least 200 and the LED gives a domT score of at least 112. In an embodiment, the LAD catalytic domain gives a domT score of at least 175 and the LED gives a domT score of at least 115. In an embodiment, the LAD catalytic domain gives a domT score of at least 180 and the LED gives a domT score of at least 115. In an embodiment, the LAD catalytic domain gives a domT score of at least 185 and the LED gives a domT score of at least 115. In an embodiment, the LAD catalytic domain gives a domT score of at least 190 and the LED gives a domT score of at least 115. In an embodiment, the LAD catalytic domain gives a domT score of at least 195 and the LED gives a domT score of at least 115. In an embodiment, the LAD catalytic domain gives a domT score of at least 200 and the LED gives a domT score of at least 115. In an embodiment, the LAD catalytic domain gives a domT score of at least 175 and the LED gives a domT score of at least 118. In an embodiment, the LAD catalytic domain gives a domT score of at least 180 and the LED gives a domT score of at least 118. In an embodiment, the LAD catalytic domain gives a domT score of at least 185 and the LED gives a domT score of at least 118. In an embodiment, the LAD catalytic domain gives a domT score of at least 190 and the LED gives a domT score of at least 118. In an embodiment, the LAD catalytic domain gives a domT score of at least 195 and the LED gives a domT score of at least 118. In an embodiment, the LAD catalytic domain gives a domT score of at least 200 and the LED gives a domT score of at least 118. In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In one embodiment of the first aspect, the invention relates to a composition comprising one or more LYS polypeptides having lysozyme activity, wherein the polypeptide is dosed at least 0.01 mg of polypeptide per kilogram of composition and is selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 42;(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 45;(p) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 positions;(q) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;(r) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and(s) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having lysozyme activity and having at least 90% of the length of the mature polypeptide. In one embodiment of the first aspect, the invention relates to a composition comprising one or more LYS polypeptides having lysozyme activity, wherein the LYS polypeptide is dosed at least 0.01 mg of polypeptide per kilogram of composition and comprises a LAD catalytic domain that is selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 81 to 220 of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 304 of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 88 to 230 of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 87 to 230 of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 90 to 232 of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 83 to 222 of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 82 to 225 of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 303 of SEQ ID NO: 42; and(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 227 of SEQ ID NO: 45. In one embodiment of the first aspect, the invention relates to a composition comprising one or more LYS polypeptides having lysozyme activity, wherein the LYS polypeptide is dosed at least 0.01 mg of polypeptide per kilogram of composition and comprises a LAD catalytic domain that is selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 81 to 220 of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 304 of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 88 to 230 of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 87 to 230 of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 90 to 232 of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 83 to 222 of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 82 to 225 of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 303 of SEQ ID NO: 42; and(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 227 of SEQ ID NO: 45; and wherein the LYS polypeptide comprises a LED domain that is selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 72 of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 72 of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 42;(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 45;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 96 to 167 of SEQ ID NO: 12; and(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 96 to 168 of SEQ ID NO: 42. In one embodiment to any part of the first aspect, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In one embodiment to any part of the first aspect, the polypeptide is of fungal origin. In an embodiment, the polypeptide is obtained or obtainable from the taxonomic phylum Ascomycota, preferably the taxonomic subphylum Pezizomycotina. In one embodiment to any part of the first aspect, the composition comprises at least 0.01 mg of polypeptide (enzyme protein) per kilogram of composition, such as at least 0.02 mg, 0.05 mg, 0.10 mg, 0.2 mg, 0.5 mg, 1.0 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1.0 g, 2.5 g, 5 g, 7.5 g, 10 g, 25 g, 50 g, 75 g or 100 g per kilogram of composition. In one embodiment, the composition comprises at most 250 g of polypeptide per kilogram of composition, such as at most 150 g, 100 g, 50 g, 40 g, 30 g, 20 g, 10 g, 7.5 g, 5 g, 2.5 g, 1.0 g, 750 mg, 500 mg, 250 mg, 100 mg, 50 mg, 25 mg, 10 mg, 5 mg, 2.5 mg or 1 mg per kilogram of composition. In one embodiment, the composition comprises between 0.01 mg and 250 g of polypeptide (enzyme protein) per kilogram of composition, such as between 0.02 mg, 0.05 mg, 0.10 mg, 0.2 mg, 0.5 mg, 1.0 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1.0 g, 2.5 g, 5 g, 7.5 g, 10 g, 25 g, 50 g, 75 g or 100 g per kilogram of composition and 150 g, 100 g, 50 g, 40 g, 30 g, 20 g, 10 g, 7.5 g, 5 g, 2.5 g, 1.0 g, 750 mg, 500 mg, 250 mg, 100 mg, 50 mg, 25 mg, 10 mg, 5 mg, 2.5 mg or 1 mg per kilogram of composition, or any combination thereof. In one embodiment to any part of the first aspect, the composition comprises one or more formulating agents (such as those described herein), preferably a formulating agent selected from the list consisting of glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin, maltodextrin, cyclodextrin, wheat, PVA, acetate, phosphate and cellulose, preferably selected from the list consisting of 1, 2-propylene glycol, 1, 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment to any part of the first aspect, the composition comprises one or more additional enzymes. The one or more additional enzymes is preferably selected from the group consisting of acetyl xylan esterase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lipase, lysophospholipase, lysozyme, mannanase, alpha-mannosidase, beta-mannosidase, phytase, phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, protease, pullulanase, pectinase, pectin lyase, xylanase, beta-xylosidase or any combination thereof. In one embodiment to any part of the first aspect, the composition comprises one or more probiotics. The one or more probiotics is preferably selected from the group consisting ofBacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacteriumsp.,Carnobacteriumsp.,Clostridium butyricum, Clostridiumsp.,Enterococcus faecium, Enterococcussp.,Lactobacillussp.,Lactobacillus acidophilus, Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactococcus lactis, Lactococcussp.,Leuconostocsp.,Megasphaera elsdenii, Megasphaerasp.,Pediococcus acidilactici, Pediococcussp.,Propionibacterium thoenii, Propionibacteriumsp. andStreptococcussp. or any combination thereof. Granules Comprising Polypeptides Having Lysozyme Activity In a second aspect, the invention relates to a granule comprising a LYS polypeptide, wherein the polypeptide (a) has lysozyme activity and (b) comprises one or more LAD catalytic domains; wherein the LAD catalytic domain gives a domT score of at least 180 when queried using a Profile Hidden Markov Model (HMM) prepared using SEQ ID NOs: 46 to 187 and hmmbuild software program, and wherein the query is carried out using hmmscan software program by the Method of Determining the LAD Catalytic Domain by HMM. In one embodiment, the granule comprises a core particle and one or more coatings. In a preferred embodiment, the coating comprises salt and/or wax and/or flour. Preferred formulations are disclosed in the formulation section below. In an embodiment, the polypeptide further comprises one or more lysozyme enhancing domains (LED). Thus, the invention further relates to a granule comprising a LYS polypeptide, wherein:(a) the LYS polypeptide has lysozyme activity;(b) the LYS polypeptide comprises one or more LAD catalytic domains, wherein the LAD catalytic domain gives a domT score of at least 170 when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 46 to 187 and hmmbuild software program, and wherein the query is carried out using hmmscan software program by the Method of Determining the LAD Catalytic Domain by HMM;(c) the polypeptide comprises one or more LED domains, wherein the LED gives a domT score of at least 100 when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 188 to 316 and hmmbuild software program, and typically wherein the query is carried out using the hmmscan software program by the Method of Determining the Lysozyme Enhancing Domain by HMM. In one embodiment, the granule comprises a core particle and one or more coatings. In a preferred embodiment, the coating comprises salt and/or wax and/or flour. Preferred formulations are disclosed in the formulation section below. In an embodiment, the domT score of the LAD catalytic domain is at least 175, preferably at least 180, more preferably at least 185, even more preferably at least 190, even more preferably at least 195, or most preferably at least 200. In an embodiment, the domT score of the LED is at least 103, preferably at least 106, more preferably at least 109, more preferably at least 112, more preferably at least 115, more preferably at least 118, even more preferably at least 121, or most preferably at least 124. Preferred combinations of domT scores are as disclosed in the first aspect of the invention. In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In one embodiment of the second aspect, the invention relates to a granule comprising one or more LYS polypeptides having lysozyme activity, wherein the LYS polypeptide is selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 42;(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 45;(p) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 positions;(q) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;(r) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and(s) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having lysozyme activity and having at least 90% of the length of the mature polypeptide. In one embodiment, the granule comprises a core particle and one or more coatings. In a preferred embodiment, the coating comprises salt and/or wax and/or flour. Preferred formulations are disclosed in the formulation section below. In one embodiment of the second aspect, the invention relates to a granule comprising one or more LYS polypeptides having lysozyme activity, wherein the LYS polypeptide comprises a LAD catalytic domain that is selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 81 to 220 of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 304 of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 88 to 230 of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 87 to 230 of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 90 to 232 of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 83 to 222 of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 82 to 225 of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 303 of SEQ ID NO: 42; and(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 227 of SEQ ID NO: 45. In one embodiment of the second aspect, the invention relates to a granule comprising one or more LYS polypeptides having lysozyme activity, wherein the LYS polypeptide comprises a LAD catalytic domain that is selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 81 to 220 of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 304 of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 88 to 230 of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 87 to 230 of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 90 to 232 of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 83 to 222 of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 82 to 225 of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 303 of SEQ ID NO: 42; and(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 227 of SEQ ID NO: 45; and wherein the LYS polypeptide comprises a LED domain that is selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 72 of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 72 of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 42;(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 45;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 96 to 167 of SEQ ID NO: 12; and(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 96 to 168 of SEQ ID NO: 42. In one embodiment, the granule comprises a core particle and one or more coatings. In a preferred embodiment, the coating comprises salt and/or wax and/or flour. Preferred formulations are disclosed in the formulation section below. In one embodiment to any part of the second aspect, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In one embodiment to any part of the second aspect, the polypeptide is of fungal origin. In an embodiment, the polypeptide is obtained or obtainable from the taxonomic phylum Ascomycota, preferably the taxonomic subphylum Pezizomycotina. In one embodiment to any part of the first aspect, the composition comprises at least 0.01 mg of polypeptide (enzyme protein) per kilogram of composition, such as at least 0.02 mg, 0.05 mg, 0.10 mg, 0.2 mg, 0.5 mg, 1.0 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1.0 g, 2.5 g, 5 g, 7.5 g, 10 g, 25 g, 50 g, 75 g or 100 g per kilogram of composition. In one embodiment, the composition comprises at most 250 g of polypeptide per kilogram of composition, such as at most 150 g, 100 g, 50 g, 40 g, 30 g, 20 g, 10 g, 7.5 g, 5 g, 2.5 g, 1.0 g, 750 mg, 500 mg, 250 mg, 100 mg, 50 mg, 25 mg, 10 mg, 5 mg, 2.5 mg or 1 mg per kilogram of composition. In one embodiment, the composition comprises between 0.01 mg and 250 g of polypeptide (enzyme protein) per kilogram of composition, such as between 0.02 mg, 0.05 mg, 0.10 mg, 0.2 mg, 0.5 mg, 1.0 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1.0 g, 2.5 g, 5 g, 7.5 g, 10 g, 25 g, 50 g, 75 g or 100 g per kilogram of composition and 150 g, 100 g, 50 g, 40 g, 30 g, 20 g, 10 g, 7.5 g, 5 g, 2.5 g, 1.0 g, 750 mg, 500 mg, 250 mg, 100 mg, 50 mg, 25 mg, 10 mg, 5 mg, 2.5 mg or 1 mg per kilogram of composition, or any combination thereof. In one embodiment to any part of the second aspect, the granule comprises one or more formulating agents (such as those described herein), preferably a formulating agent selected from the list consisting of glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin, maltodextrin, cyclodextrin, wheat, PVA, acetate, phosphate and cellulose, preferably selected from the list consisting of 1, 2-propylene glycol, 1, 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment to any part of the second aspect, the granule comprises a core particle and one or more coatings. In a preferred embodiment, the coating comprises salt and/or wax and/or flour. Preferred formulations are disclosed in the formulation section below. In one embodiment to any part of the second aspect, the granule comprises one or more additional enzymes. The one or more additional enzymes is preferably selected from the group consisting of acetyl xylan esterase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lipase, lysophospholipase, lysozyme, mannanase, alpha-mannosidase, beta-mannosidase, phytase, phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, protease, pullulanase, pectinase, pectin lyase, xylanase, beta-xylosidase or any combination thereof. In one embodiment to any part of the second aspect, the granule comprises one or more probiotics. The one or more probiotics is preferably selected from the group consisting ofBacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacteriumsp.,Carnobacteriumsp.,Clostridium butyricum, Clostridiumsp.,Enterococcus faecium, Enterococcussp.,Lactobacillussp.,Lactobacillus acidophilus, Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactococcus lactis, Lactococcussp.,Leuconostocsp.,Megasphaera elsdenii, Megasphaerasp.,Pediococcus acidilactici, Pediococcussp.,Propionibacterium thoenii, Propionibacteriumsp. andStreptococcussp. or any combination thereof. Liquid Formulations Comprising Polypeptides Having Lysozyme Activity In a third aspect, the invention relates to liquid formulations, wherein the liquid formulation comprises:(a) 0.01% to 25% w/w of LYS polypeptide wherein:(i) the LYS polypeptide has lysozyme activity;(ii) the LYS polypeptide comprises one or more LAD catalytic domains, wherein the LAD catalytic domain gives a domT score of at least 170 when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 46 to 187 and hmmbuild software program, and wherein the query is carried out using hmmscan software program by the Method of Determining the LAD Catalytic Domain by HMM;(b) 20% to 80% w/w of polyol;(c) 0.01% to 2.0% w/w preservative; and(d) water. In an embodiment, the domT score of the LAD catalytic domain is at least 175, preferably at least 180, more preferably at least 185, even more preferably at least 190, even more preferably at least 195, or most preferably at least 200. In an embodiment, the domT score of the LED is at least 103, preferably at least 106, more preferably at least 109, more preferably at least 112, more preferably at least 115, more preferably at least 118, even more preferably at least 121, or most preferably at least 124. Preferred combinations of domT scores are as disclosed in the first aspect of the invention. In an embodiment, the polypeptide further comprises one or more lysozyme enhancing domains (LED). Thus, the invention further relates to a liquid formulation, wherein the liquid formulation comprises:(a) 0.01% to 25% w/w of LYS polypeptide wherein:(i) the LYS polypeptide has lysozyme activity;(ii) the LYS polypeptide comprises one or more LAD catalytic domains, wherein the LAD catalytic domain gives a domT score of at least 170 when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 46 to 187 and hmmbuild software program, and wherein the query is carried out using hmmscan software program by the Method of Determining the LAD Catalytic Domain by HMM;(iii) the LYS polypeptide comprises one or more LED domains, wherein the LED gives a domT score of at least 100 when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 188 to 316 and hmmbuild software program, and wherein the query is carried out using the hmmscan software program by the Method of Determining the Lysozyme Enhancing Domain by HMM;(b) 20% to 80% w/w of polyol;(c) 0.01% to 2.0% w/w preservative; and(d) water. In an embodiment, the domT score of the LAD catalytic domain is at least 175, preferably at least 180, more preferably at least 185, even more preferably at least 190, even more preferably at least 195, or most preferably at least 200. In an embodiment, the domT score of the LED is at least 103, preferably at least 106, more preferably at least 109, more preferably at least 112, more preferably at least 115, more preferably at least 118, even more preferably at least 121, or most preferably at least 124. Preferred combinations of domT scores are as disclosed in the first aspect of the invention. In one embodiment of the third aspect, the invention relates to a liquid formulation comprising one or more LYS polypeptides having lysozyme activity, wherein the liquid formulation comprises:(A) 0.01% to 25% w/w of LYS polypeptide wherein the LYS polypeptide is selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 42;(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 45;(p) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 positions;(q) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;(r) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and(s) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having lysozyme activity and having at least 90% of the length of the mature polypeptide;(B) 20% to 80% w/w of polyol;(D) 0.01% to 2.0% w/w preservative; and(D) water. In one embodiment of the third aspect, the invention relates to a liquid formulation comprising one or more LYS polypeptides having lysozyme activity, wherein the liquid formulation comprises:(A) 0.01% to 25% w/w of LYS polypeptide wherein the LYS polypeptide comprises a LAD catalytic domain that is selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 81 to 220 of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 304 of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 88 to 230 of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 87 to 230 of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 90 to 232 of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 83 to 222 of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 82 to 225 of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 303 of SEQ ID NO: 42; and(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 227 of SEQ ID NO: 45;(B) 20% to 80% w/w of polyol;(C) 0.01% to 2.0% w/w preservative; and(D) water. In one embodiment of the third aspect, the invention relates to a liquid formulation comprising one or more LYS polypeptides having lysozyme activity, wherein the liquid formulation comprises:(A) 0.01% to 25% w/w of LYS polypeptide wherein the LYS polypeptide comprises a LAD catalytic domain that is selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 81 to 220 of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 304 of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 88 to 230 of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 87 to 230 of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 90 to 232 of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 228 of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 84 to 226 of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 83 to 222 of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 82 to 225 of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 161 to 303 of SEQ ID NO: 42; and(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 85 to 227 of SEQ ID NO: 45;(B) the LYS polypeptide comprises a LED domain selected from the group consisting of:(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 3;(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 6;(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 72 of SEQ ID NO: 12;(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 15;(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 24;(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 27;(j) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 36;(m) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 72 of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 42;(o) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 1 to 73 of SEQ ID NO: 45;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 96 to 167 of SEQ ID NO: 12; and(n) a polypeptide having at least 80%, e.g., at least 85%, at least 90% or at least 95% sequence identity to amino acids 96 to 168 of SEQ ID NO: 42;(C) 20% to 80% w/w of polyol;(D) 0.01% to 2.0% w/w preservative; and(E) water. In one embodiment to any part of the third aspect, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In one embodiment to any part of the third aspect, the polypeptide is of fungal origin. In an embodiment, the polypeptide is obtained or obtainable from the taxonomic phylum Ascomycota, preferably the taxonomic subphylum Pezizomycotina. In one embodiment to any part of the third aspect, the liquid formulation comprises one or more formulating agents (such as those described herein), preferably a formulating agent selected from the list consisting of glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the list consisting of 1, 2-propylene glycol, 1, 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment to any part of the third aspect, the liquid formulation comprises one or more polyols, preferably a polyol selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600, more preferably selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG) or any combination thereof. In one embodiment to any part of the third aspect, the liquid formulation comprises 20%-80% polyol (i.e. total amount of polyol), preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol. In one embodiment to any part of the third aspect, the liquid formulation comprises 20%-80% polyol, preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600. In one embodiment to any part of the third aspect, the liquid formulation comprises 20%-80% polyol (i.e. total amount of polyol), preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol wherein the polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG). In one embodiment to any part of the third aspect, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02% to 1.5% w/w preservative, more preferably 0.05% to 1.0% w/w preservative or most preferably 0.1% to 0.5% w/w preservative. In one embodiment, the liquid formulation comprises 0.01% to 2.0% w/w preservative (i.e. total amount of preservative), preferably 0.02% to 1.5% w/w preservative, more preferably 0.05% to 1.0% w/w preservative or most preferably 0.1% to 0.5% w/w preservative wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment to any part of the third aspect, the liquid formulation comprises 0.05% to 20% w/w LYS polypeptide, more preferably 0.2% to 15% w/w LYS polypeptide, more preferably 0.5% to 15% w/w LYS polypeptide or most preferably 1.0% to 10% w/w LYS polypeptide. In one embodiment to any part of the third aspect, the liquid formulation comprises one or more additional enzymes. The one or more additional enzymes is preferably selected from the group consisting of acetyl xylan esterase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lipase, lysophospholipase, lysozyme, mannanase, alpha-mannosidase, beta-mannosidase, phytase, phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, protease, pullulanase, pectinase, pectin lyase, xylanase, beta-xylosidase or any combination thereof. In one embodiment to any part of the third aspect, the liquid formulation comprises one or more probiotics. The one or more probiotics is preferably selected from the group consisting ofBacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacteriumsp.,Carnobacteriumsp.,Clostridium butyricum, Clostridiumsp.,Enterococcus faecium, Enterococcussp.,Lactobacillussp.,Lactobacillus acidophilus, Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactococcus lactis, Lactococcussp.,Leuconostocsp.,Megasphaera elsdenii, Megasphaerasp.,Pediococcus acidilactici, Pediococcussp.,Propionibacterium thoenii, Propionibacteriumsp. andStreptococcussp. or any combination thereof. Polypeptides Having Lysozyme Activity In a fourth aspect, the invention relates to polypeptides having lysozyme activity having at least 95%, e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2. In one embodiment, the polypeptides differ by up to 11 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids from the mature polypeptide of SEQ ID NO: 2. In a continuation of the fourth aspect, the invention relates to polypeptides having lysozyme activity having at least 95%, e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3. In one embodiment, the polypeptides differ by up to 11 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids from SEQ ID NO: 3. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 3 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 3. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 2. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 3; comprises the amino acid sequence of SEQ ID NO: 3 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 3 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 3. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 226 of SEQ ID NO: 3. In one embodiment, the polypeptide has been isolated. In a continuation of the fourth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 of at least 95%, e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the fourth aspect, the invention relates to variants of SEQ ID NO: 3 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 3 is not more than 11, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 3 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 3 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 3 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In an embodiment of the fourth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 3. In one embodiment, lysozyme activity is determined as described in example 1. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly. Other examples of conservative substitutions are G to A; A to G, S; V to I, L, A, T, S; I to V, L, M; L to I, M, V; M to L, I, V; P to A, S, N; F to Y, W, H; Y to F, W, H; W to Y, F, H; R to K, E, D; K to R, E, D; H to Q, N, S; D to N, E, K, R, Q; E to Q, D, K, R, N; S to T, A; T to S, V, A; C to S, T, A; N to D, Q, H, S; Q to E, N, H, K, R. Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for lysozyme activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem.271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labelling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science255: 306-312; Smith et al., 1992, J. Mol. Biol.224: 899-904; Wlodaver et al., 1992, FEBS Lett.309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide. Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR,phagedisplay (e.g., Lowman et al., 1991, Biochemistry30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene46: 145; Ner et al., 1988, DNA7: 127). Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide. The polypeptide may be a hybrid polypeptide or a fusion polypeptide. In a fifth aspect, the invention relates to polypeptides having lysozyme activity having at least 94%, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 5. In one embodiment, the polypeptides differ by up to 13 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids from the mature polypeptide of SEQ ID NO: 5. In a continuation of the fifth aspect, the invention relates to polypeptides having lysozyme activity having at least 94%, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6. In one embodiment, the polypeptides differ by up to 13 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids from SEQ ID NO: 6. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 6 of at least 94% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 6. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 6 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 6. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 5. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 6; comprises the amino acid sequence of SEQ ID NO: 6 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 6 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 6. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 226 of SEQ ID NO: 6. In one embodiment, the polypeptide has been isolated. In a continuation of the fifth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 4 of at least 94%, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the fifth aspect, the invention relates to variants of SEQ ID NO: 6 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 6 is not more than 13, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 6 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 6 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 6 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the fifth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 6. In one embodiment, lysozyme activity is determined as described in example 1. In a sixth aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 8. In one embodiment, the polypeptides differ by up to 44 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids from the mature polypeptide of SEQ ID NO: 8. In a continuation of the sixth aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9. In one embodiment, the polypeptides differ by up to 44 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids from SEQ ID NO: 9. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 9 of at least 80% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 9. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 9 of at least 85% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 9. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 9 of at least 90% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 9. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 9 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 9. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 8. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 9; comprises the amino acid sequence of SEQ ID NO: 9 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 9 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 9. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 223 of SEQ ID NO: 9. In one embodiment, the polypeptide has been isolated. In a continuation of the sixth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 7 of at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the sixth aspect, the invention relates to variants of SEQ ID NO: 9 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 9 is not more than 44, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 9 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 9 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 9 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the sixth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 9. In one embodiment, lysozyme activity is determined as described in example 1. In a seventh aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 11. In one embodiment, the polypeptides differ by up to 50 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids from the mature polypeptide of SEQ ID NO: 11. In a continuation of the seventh aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12. In one embodiment, the polypeptides differ by up to 50 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids from SEQ ID NO: 12. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 12 of at least 80% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 12. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 12 of at least 85% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 12. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 12 of at least 90% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 12. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 12 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 12. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 11. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 12; comprises the amino acid sequence of SEQ ID NO: 12 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 12 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 12. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 304 of SEQ ID NO: 12. In one embodiment, the polypeptide has been isolated. In a continuation of the seventh aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 10 of at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the seventh aspect, the invention relates to variants of SEQ ID NO: 12 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 12 is not more than 50, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 12 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 12 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 12 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the seventh aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 12. In one embodiment, lysozyme activity is determined as described in example 1. In a eighth aspect, the invention relates to polypeptides having lysozyme activity having at least 87%, e.g., at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 14. In one embodiment, the polypeptides differ by up to 29 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids from the mature polypeptide of SEQ ID NO: 14. In a continuation of the eighth aspect, the invention relates to polypeptides having lysozyme activity having at least 87%, e.g., at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15. In one embodiment, the polypeptides differ by up to 29 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids from SEQ ID NO: 15. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 15 of at least 87% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 15. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 15 of at least 90% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 15. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 15 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 15. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 14. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 15; comprises the amino acid sequence of SEQ ID NO: 15 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 15 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 15. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 228 of SEQ ID NO: 15. In one embodiment, the polypeptide has been isolated. In a continuation of the eighth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 13 of at least 87%, e.g., at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the eighth aspect, the invention relates to variants of SEQ ID NO: 15 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 15 is not more than 29, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 15 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 15 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 15 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the eighth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 15. In one embodiment, lysozyme activity is determined as described in example 1. In a ninth aspect, the invention relates to polypeptides having lysozyme activity having at least 81%, e.g., at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 17. In one embodiment, the polypeptides differ by up to 43 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43 amino acids from the mature polypeptide of SEQ ID NO: 17. In a continuation of the ninth aspect, the invention relates to polypeptides having lysozyme activity having at least 81%, e.g., at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18. In one embodiment, the polypeptides differ by up to 43 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43 amino acids from SEQ ID NO: 18. In a continuation of the ninth aspect, the invention relates to polypeptides having lysozyme activity having at least 81%, e.g., at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18. In one embodiment, the polypeptides differ by up to 28 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 amino acids from SEQ ID NO: 239. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 18 of at least 81% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 18. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 18 of at least 85% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 18. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 18 of at least 90% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 18. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 18 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 18. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 17. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 239. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 18; comprises the amino acid sequence of SEQ ID NO: 18 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 18 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 18. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 230 of SEQ ID NO: 18. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 146 of SEQ ID NO: 18. In one embodiment, the polypeptide has been isolated. In a continuation of the ninth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 16 of at least 81%, e.g., at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the ninth aspect, the invention relates to variants of SEQ ID NO: 18 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 18 is not more than 43, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 18 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 18 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 18 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the ninth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 18. In one embodiment, lysozyme activity is determined as described in example 1. In a tenth aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 20. In one embodiment, the polypeptides differ by up to 45 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids from the mature polypeptide of SEQ ID NO: 20. In a continuation of the tenth aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21. In one embodiment, the polypeptides differ by up to 45 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids from SEQ ID NO: 21. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 21 of at least 80% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 21. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 21 of at least 85% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 21. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 21 of at least 90% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 21. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 21 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 21. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 20. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 21; comprises the amino acid sequence of SEQ ID NO: 21 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 21 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 21. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 230 of SEQ ID NO: 21. In one embodiment, the polypeptide has been isolated. In a continuation of the tenth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 19 of at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the tenth aspect, the invention relates to variants of SEQ ID NO: 21 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 21 is not more than 45, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 21 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 21 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 21 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the tenth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 21. In one embodiment, lysozyme activity is determined as described in example 1. In a eleventh aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 23. In one embodiment, the polypeptides differ by up to 46 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 amino acids from the mature polypeptide of SEQ ID NO: 23. In a continuation of the eleventh aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24. In one embodiment, the polypeptides differ by up to 46 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 amino acids from SEQ ID NO: 24. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 24 of at least 80% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 24. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 24 of at least 85% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 24. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 24 of at least 90% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 24. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 24 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 24. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 23. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 24; comprises the amino acid sequence of SEQ ID NO: 24 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 24 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 24. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 232 of SEQ ID NO: 24. In one embodiment, the polypeptide has been isolated. In a continuation of the eleventh aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 22 of at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the eleventh aspect, the invention relates to variants of SEQ ID NO: 24 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 24 is not more than 46, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 24 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 24 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 24 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the eleventh aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 24. In one embodiment, lysozyme activity is determined as described in example 1. In a twelfth aspect, the invention relates to polypeptides having lysozyme activity having at least 87%, e.g., at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 26. In one embodiment, the polypeptides differ by up to 29 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids from the mature polypeptide of SEQ ID NO: 26. In a continuation of the twelfth aspect, the invention relates to polypeptides having lysozyme activity having at least 87%, e.g., at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27. In one embodiment, the polypeptides differ by up to 29 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids from SEQ ID NO: 27. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 27 of at least 87% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 27. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 27 of at least 90% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 27. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 27 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 27. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 26. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 27; comprises the amino acid sequence of SEQ ID NO: 27 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 27 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 27. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 228 of SEQ ID NO: 27. In one embodiment, the polypeptide has been isolated. In a continuation of the twelfth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 25 of at least 87%, e.g., at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the twelfth aspect, the invention relates to variants of SEQ ID NO: 27 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 27 is not more than 29, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 27 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 27 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 27 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the twelfth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 27. In one embodiment, lysozyme activity is determined as described in example 1. In a thirteenth aspect, the invention relates to polypeptides having lysozyme activity having at least 96%, e.g., at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 29. In one embodiment, the polypeptides differ by up to 8 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from the mature polypeptide of SEQ ID NO: 29. In a continuation of the thirteenth aspect, the invention relates to polypeptides having lysozyme activity having at least 96%, e.g., at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30. In one embodiment, the polypeptides differ by up to 8 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from SEQ ID NO: 30. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 30 of at least 96% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 30. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 29. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 30; comprises the amino acid sequence of SEQ ID NO: 30 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 30 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 30. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 228 of SEQ ID NO: 30. In one embodiment, the polypeptide has been isolated. In a continuation of the thirteenth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 28 of at least 96%, e.g., at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the thirteenth aspect, the invention relates to variants of SEQ ID NO: 30 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 30 is not more than 8, e.g. 1, 2, 3, 4, 5, 6, 7, or 8. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 30 is not more than 8, e.g. 1, 2, 3, 4, 5, 6, 7, or 8. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 30 is not more than 8, e.g. 1, 2, 3, 4, 5, 6, 7, or 8. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the thirteenth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 30. In one embodiment, lysozyme activity is determined as described in example 1. In a fourteenth aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 32. In one embodiment, the polypeptides differ by up to 45 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids from the mature polypeptide of SEQ ID NO: 32. In a continuation of the fourteenth aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33. In one embodiment, the polypeptides differ by up to 45 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids from SEQ ID NO: 33. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 33 of at least 80% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 33. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 33 of at least 85% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 33. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 33 of at least 90% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 33. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 33 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 33. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 32. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 33; comprises the amino acid sequence of SEQ ID NO: 33 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 33 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 33. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 226 of SEQ ID NO: 33. In one embodiment, the polypeptide has been isolated. In a continuation of the fourteenth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 31 of at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the fourteenth aspect, the invention relates to variants of SEQ ID NO: 33 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 33 is not more than 45, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 33 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 33 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 33 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the fourteenth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 33. In one embodiment, lysozyme activity is determined as described in example 1. In a fifteenth aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 35. In one embodiment, the polypeptides differ by up to 44 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids from the mature polypeptide of SEQ ID NO: 35. In a continuation of the fifteenth aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36. In one embodiment, the polypeptides differ by up to 44 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids from SEQ ID NO: 36. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 36 of at least 80% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 36. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 36 of at least 85% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 36. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 36 of at least 90% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 36. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 36 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 36. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 35. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 36; comprises the amino acid sequence of SEQ ID NO: 36 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 36 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 36. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 225 of SEQ ID NO: 36. In one embodiment, the polypeptide has been isolated. In a continuation of the fifteenth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 34 of at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the fifteenth aspect, the invention relates to variants of SEQ ID NO: 36 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 36 is not more than 44, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 36 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 36 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 36 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the fifteenth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 36. In one embodiment, lysozyme activity is determined as described in example 1. In a sixteenth aspect, the invention relates to polypeptides having lysozyme activity having at least 81%, e.g., at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 38. In one embodiment, the polypeptides differ by up to 42 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 amino acids from the mature polypeptide of SEQ ID NO: 38. In a continuation of the sixteenth aspect, the invention relates to polypeptides having lysozyme activity having at least 81%, e.g., at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39. In one embodiment, the polypeptides differ by up to 42 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 amino acids from SEQ ID NO: 39. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 39 of at least 81% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 39. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 39 of at least 85% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 39. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 39 of at least 90% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 39. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 39 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 39. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 38. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 39; comprises the amino acid sequence of SEQ ID NO: 39 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 39 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 39. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 225 of SEQ ID NO: 39. In one embodiment, the polypeptide has been isolated. In a continuation of the sixteenth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 37 of at least 81%, e.g., at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the sixteenth aspect, the invention relates to variants of SEQ ID NO: 39 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 39 is not more than 42, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 39 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 39 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 39 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the sixteenth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 39. In one embodiment, lysozyme activity is determined as described in example 1. In a seventeenth aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 41. In one embodiment, the polypeptides differ by up to 50 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids from the mature polypeptide of SEQ ID NO: 41. In a continuation of the seventeenth aspect, the invention relates to polypeptides having lysozyme activity having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42. In one embodiment, the polypeptides differ by up to 50 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids from SEQ ID NO: 42. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 42 of at least 80% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 42. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 42 of at least 85% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 42. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 42 of at least 90% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 42. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 42 of at least 95% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 42. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 41. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 42; comprises the amino acid sequence of SEQ ID NO: 42 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 42 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 42. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 304 of SEQ ID NO: 42. In one embodiment, the polypeptide has been isolated. In a continuation of the seventeenth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 40 of at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the seventeenth aspect, the invention relates to variants of SEQ ID NO: 42 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 42 is not more than 50, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 42 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 42 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 42 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the seventeenth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 42. In one embodiment, lysozyme activity is determined as described in example 1. In a eighteenth aspect, the invention relates to polypeptides having lysozyme activity having at least 100%, e.g., or 100% sequence identity to the mature polypeptide of SEQ ID NO: 44. In one embodiment, the polypeptides differ by up to 0 amino acids, e.g., or 1 amino acids from the mature polypeptide of SEQ ID NO: 44. In a continuation of the eighteenth aspect, the invention relates to polypeptides having lysozyme activity having at least 100%, e.g., or 100% sequence identity to SEQ ID NO: 45. In one embodiment, the polypeptides differ by up to 0 amino acids, e.g., or 1 amino acids from SEQ ID NO: 45. In one embodiment, the invention relates to polypeptides having lysozyme activity and having a sequence identity to SEQ ID NO: 45 of at least 100% and wherein the polypeptide has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 45. In one embodiment, lysozyme activity is determined as described in example 1. In one embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 44. In one embodiment, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 45; comprises the amino acid sequence of SEQ ID NO: 45 and a N-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the amino acid sequence of SEQ ID NO: 45 and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereof having lysozyme activity and having at least 90% such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of SEQ ID NO: 45. In one embodiment, the polypeptide comprises or consists of amino acids 1 to 227 of SEQ ID NO: 45. In one embodiment, the polypeptide has been isolated. In a continuation of the eighteenth aspect, the invention relates to polypeptides having lysozyme activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 43 of at least 100%, e.g., or 100%. In a further embodiment, the polypeptide has been isolated. In a continuation of the eighteenth aspect, the invention relates to variants of SEQ ID NO: 45 having lysozyme activity comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof at one or more (e.g., several) positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 45 is not more than 0, e.g. or 1. In one embodiment, the number of substitutions and/or deletions and/or insertions in SEQ ID NO: 45 is not more than 0, e.g. or 1. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 45 is not more than 0, e.g. or 1. Examples of amino acid changes and conservative substitutions are described in the fourth aspect of the invention. In an embodiment of the eighteenth aspect, the variant has at least 50%, such as at least 75%, at least 90%, at least 95% or at least 100% of the lysozyme activity of SEQ ID NO: 45. In one embodiment, lysozyme activity is determined as described in example 1. Taxonomic and Structural Families In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In one embodiment, the polypeptide having lysozyme activity is obtained or is obtainable from the taxonomic phylum Ascomycota, preferably the taxonomic subphylum Pezizomycotina and is preferably is selected from the group selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45. In one embodiment, the polypeptide having lysozyme activity is obtained or is obtainable from the taxonomic class Eurotiomycetes, preferably the taxonomic order Eurotiales and is more preferably selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30 and SEQ ID NO: 36. In one embodiment, the polypeptide having lysozyme activity is obtained or is obtainable from the taxonomic order Eurotiales, preferably the taxonomic family Aspergillaceae and is more preferably selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27 and SEQ ID NO: 30. In one embodiment, the polypeptide having lysozyme activity is obtained or is obtainable from the taxonomic order Eurotiales, preferably the taxonomic family Trichocomaceae and is more preferably selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 36. In one embodiment, the polypeptide having lysozyme activity is obtained or is obtainable from the taxonomic class Sordariomycetes and is preferably selected from the group selected from SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45. In one embodiment, the polypeptide having lysozyme activity is obtained or is obtainable from the taxonomic order Sordariales, preferably the taxonomic family Chaetomiaceae and is more preferably selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 39 and SEQ ID NO: 45. In one embodiment, the polypeptide having lysozyme activity is obtained or is obtainable from the taxonomic order Hypocreales, preferably the taxonomic family Clavicipitaceae and is more preferably selected from the group consisting of SEQ ID NO: 42. Sources of Polypeptides Having Lysozyme Activity A polypeptide having lysozyme activity of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly. The polypeptide may be a fungal polypeptide. In one aspect, the polypeptide is a polypeptide having lysozyme activity from a fungus of the class Eurotiomycetes, such as from the order Eurotiales, or from the family Aspergillaceae, or from the genusPenicillium, or from the speciesPenicillium simplicissimum, Penicillium vasconiae, Penicillium antarcticum, Penicillium wellingtonense, Penicillium roseopurpureumorPenicillium virgatum. The polypeptide may be a fungal polypeptide. In one aspect, the polypeptide is a polypeptide having lysozyme activity from a fungus of the class Eurotiomycetes, such as from the order Eurotiales, or from the family Aspergillaceae, or from the genusAspergillus, or from the speciesAspergillussp. XZ2668 orAspergillus niveus. The polypeptide may be a fungal polypeptide. In one aspect, the polypeptide is a polypeptide having lysozyme activity from a fungus of the class Eurotiomycetes, such as from the order Eurotiales, or from the family Trichocomaceae, or from the genusTalaromyces, or from the speciesTalaromyces proteolyticusorTalaromyces atricola. The polypeptide may be a fungal polypeptide. In one aspect, the polypeptide is a polypeptide having lysozyme activity from a fungus of the class Sordariomycetes, such as from the order Hypocreales, or from the family Clavicipitaceae, or from the genusMetarhizium, or from the speciesMetarhizium carneum. The polypeptide may be a fungal polypeptide. In one aspect, the polypeptide is a polypeptide having lysozyme activity from a fungus of the class Sordariomycetes, such as from the order Sordariales, or from the family Chaetomiaceae, or from the genusOvatospora, or from the speciesOvatospora brasiliensis. The polypeptide may be a fungal polypeptide. In one aspect, the polypeptide is a polypeptide having lysozyme activity from a fungus of the class Sordariomycetes, such as from the order Sordariales, or from the family Chaetomiaceae, or from the genusChaetomium, or from the speciesChaetomiumsp. ZY369. The polypeptide may be a fungal polypeptide. In one aspect, the polypeptide is a polypeptide having lysozyme activity from a fungus of the class Sordariomycetes, such as from the order Sordariales, or from the family Chaetomiaceae, or from the genusTrichocladium, or from the speciesTrichocladium asperum. The polypeptide may be a fungal polypeptide. In one aspect, the polypeptide is a polypeptide having lysozyme activity from a fungus of the class Sordariomycetes, such as from the order Sordariales, or from the family Chaetomiaceae, or from the genusThielavia, or from the speciesThielavia terrestris. It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL). The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra). Polynucleotides The present invention also relates to polynucleotides encoding a polypeptide of the present invention, as described herein. In an embodiment, the polynucleotide encoding the polypeptide of the present invention has been isolated. The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used. The polynucleotides may be cloned from a strain ofTrichophaeaor a strain ofTrichoderma, or a related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the polynucleotide. Modification of a polynucleotide encoding a polypeptide of the present invention may be necessary for synthesizing polypeptides substantially similar to the polypeptide. The term “substantially similar” to the polypeptide refers to non-naturally occurring forms of the polypeptide. Nucleic Acid Constructs The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. The polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art. The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from theBacillus amyloliquefaciensalpha-amylase gene (amyQ),Bacillus licheniformisalpha-amylase gene (amyL),Bacillus licheniformispenicillinase gene (penP),Bacillus stearothermophilusmaltogenic amylase gene (amyM),Bacillus subtilislevansucrase gene (sacB),Bacillus subtilisxyIA and xyIB genes,Bacillus thuringiensiscryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology13: 97-107),E. colilac operon,E. colitrc promoter (Egon et al., 1988, Gene69: 301-315),Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Gilbert et al., 1980, Scientific American242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835. Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes forAspergillus nidulansacetamidase,Aspergillus nigerneutral alpha-amylase,Aspergillus nigeracid stable alpha-amylase,Aspergillus nigerorAspergillus awamoriglucoamylase (glaA),Aspergillus oryzaeTAKA amylase,Aspergillus oryzaealkaline protease,Aspergillus oryzaetriose phosphate isomerase,Fusarium oxysporumtrypsin-like protease (WO 96/00787),Fusarium venenatumamyloglucosidase (WO 00/56900),Fusarium venenatumDania (WO 00/56900),Fusarium venenatumQuinn (WO 00/56900),Rhizomucor mieheilipase,Rhizomucor mieheiaspartic proteinase,Trichoderma reeseibeta-glucosidase,Trichoderma reeseicellobiohydrolase I,Trichoderma reeseicellobiohydrolase II,Trichoderma reeseiendoglucanase I,Trichoderma reeseiendoglucanase II,Trichoderma reeseiendoglucanase III,Trichoderma reeseiendoglucanase V,Trichoderma reeseixylanase I,Trichoderma reeseixylanase II,Trichoderma reeseixylanase III,Trichoderma reeseibeta-xylosidase, andTrichoderma reeseitranslation elongation factor, as well as the NA2-tpi promoter (a modified promoter from anAspergillusneutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from anAspergillustriose phosphate isomerase gene; non-limiting examples include modified promoters from anAspergillus nigerneutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from anAspergillus nidulansorAspergillus oryzaetriose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Pat. No. 6,011,147. In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiaeenolase (ENO-1),Saccharomyces cerevisiaegalactokinase (GAL1),Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiaetriose phosphate isomerase (TPI),Saccharomyces cerevisiaemetallothionein (CUP1), andSaccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast8: 423-488. The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3′-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention. Preferred terminators for bacterial host cells are obtained from the genes forBacillus clausiialkaline protease (aprH),Bacillus licheniformisalpha-amylase (amyL), andEscherichia coliribosomal RNA (rrnB). Preferred terminators for filamentous fungal host cells are obtained from the genes forAspergillus nidulansacetamidase,Aspergillus nidulansanthranilate synthase,Aspergillus nigerglucoamylase,Aspergillus nigeralpha-glucosidase,Aspergillus oryzaeTAKA amylase,Fusarium oxysporumtrypsin-like protease,Trichoderma reeseibeta-glucosidase,Trichoderma reeseicellobiohydrolase I,Trichoderma reeseicellobiohydrolase II,Trichoderma reeseiendoglucanase I,Trichoderma reeseiendoglucanase II,Trichoderma reeseiendoglucanase III,Trichoderma reeseiendoglucanase V,Trichoderma reeseixylanase I,Trichoderma reeseixylanase II,Trichoderma reeseixylanase III,Trichoderma reeseibeta-xylosidase, andTrichoderma reeseitranslation elongation factor. Preferred terminators for yeast host cells are obtained from the genes forSaccharomyces cerevisiaeenolase,Saccharomyces cerevisiaecytochrome C (CYC1), andSaccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra. The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene. Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensiscryIIIA gene (WO 94/25612) and aBacillus subtilisSP82 gene (Hue et al., 1995, Journal of Bacteriology177: 3465-3471). The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5′-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used. Preferred leaders for filamentous fungal host cells are obtained from the genes forAspergillus oryzaeTAKA amylase andAspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiaeenolase (ENO-1),Saccharomyces cerevisiae3-phosphoglycerate kinase,Saccharomyces cerevisiaealpha-factor, andSaccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP). The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used. Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes forAspergillus nidulansanthranilate synthase,Aspergillus nigerglucoamylase,Aspergillus nigeralpha-glucosidaseAspergillus oryzaeTAKA amylase, andFusarium oxysporumtrypsin-like protease. Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol.15: 5983-5990. The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5′-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5′-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used. Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes forBacillusNCIB 11837 maltogenic amylase,Bacillus licheniformissubtilisin,Bacillus licheniformisbeta-lactamase,Bacillus stearothermophilusalpha-amylase,Bacillus stearothermophilusneutral proteases (nprT, nprS, nprM), andBacillus subtilisprsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews57: 109-137. Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes forAspergillus nigerneutral amylase,Aspergillus nigerglucoamylase,Aspergillus oryzaeTAKA amylase,Humicola insolenscellulase,Humicola insolensendoglucanase V,Humicola lanuginosalipase, andRhizomucor mieheiaspartic proteinase. Useful signal peptides for yeast host cells are obtained from the genes forSaccharomyces cerevisiaealpha-factor andSaccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra. The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes forBacillus subtilisalkaline protease (aprE),Bacillus subtilisneutral protease (nprT),Myceliophthora thermophilalaccase (WO 95/33836),Rhizomucor mieheiaspartic proteinase, andSaccharomyces cerevisiaealpha-factor. Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence. It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, theAspergillus nigerglucoamylase promoter,Aspergillus oryzaeTAKA alpha-amylase promoter, andAspergillus oryzaeglucoamylase promoter,Trichoderma reeseicellobiohydrolase I promoter, andTrichoderma reeseicellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked to the regulatory sequence. Expression Vectors The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used. The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers areBacillus licheniformisorBacillus subtilisdal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in anAspergilluscell areAspergillus nidulansorAspergillus oryzaeamdS and pyrG genes and aStreptomyces hygroscopicusbar gene. Preferred for use in aTrichodermacell are adeA, adeB, amdS, hph, and pyrG genes. The selectable marker may be a dual selectable marker system as described in WO 2010/039889. In one aspect, the dual selectable marker is an hph-tk dual selectable marker system. The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination. For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication inE. coli, and pUB110, pE194, pTA1060, and pAMß1 permitting replication inBacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene98: 61-67; Cullen et al., 1987, Nucleic Acids Res.15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883. More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra). Host Cells The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. In some embodiments, the polypeptide is heterologous to the recombinant host cell. In some embodiments, at least one of the one or more control sequences is heterologous to the polynucleotide encoding the polypeptide. In some embodiments, the recombinant host cell comprises at least two copies, e.g., three, four, or five, of the polynucleotide of the present invention. The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote. The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, andUreaplasma. The bacterial host cell may be anyBacilluscell including, but not limited to,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, andBacillus thuringiensiscells. The bacterial host cell may also be anyStreptococcuscell including, but not limited to,Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, andStreptococcus equisubsp.Zooepidemicuscells. The bacterial host cell may also be anyStreptomycescell including, but not limited to,Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, andStreptomyces lividanscells. The introduction of DNA into aBacilluscell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet.168: 111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol.81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol.56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol.169: 5271-5278). The introduction of DNA into anE. colicell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res.16: 6127-6145). The introduction of DNA into aStreptomycescell may be effected by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol.(Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol.171: 3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA98: 6289-6294). The introduction of DNA into aPseudomonascell may be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods64: 391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol.71: 51-57). The introduction of DNA into aStreptococcuscell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun.32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol.65: 3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev.45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used. The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell. The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi,8th edition, 1995, CAB International, University Press, Cambridge, UK). The fungal host cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors,Soc. App. Bacteriol. Symposium Series No.9, 1980). The yeast host cell may be aCandida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowiacell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, orYarrowia lipolyticacell. The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiaeis by budding of a unicellular thallus and carbon catabolism may be fermentative. The filamentous fungal host cell may be anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, orTrichodermacell. For example, the filamentous fungal host cell may be anAspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, orTrichoderma viridecell. Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation ofAspergillusandTrichodermahost cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA81: 1470-1474, and Christensen et al., 1988, Bio/Technology6: 1419-1422. Suitable methods for transformingFusariumspecies are described by Malardier et al., 1989, Gene78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors,Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol.153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA75: 1920. Methods of Production The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide. In one aspect, the cell is aPenicillium simplicissimumcell. In one aspect, the cell is aPenicillium vasconiaecell. In one aspect, the cell is aTalaromyces proteolyticuscell. In one aspect, the cell is anAspergillussp. XZ2668 cell. In one aspect, the cell is aPenicillium antarcticumcell. In one aspect, the cell is aOvatospora brasiliensiscell. In one aspect, the cell is aPenicillium wellingtonensecell. In one aspect, the cell is aPenicillium roseopurpureumcell. In one aspect, the cell is aPenicillium virgatumcell. In one aspect, the cell is anAspergillus niveuscell. In one aspect, the cell is aChaetomiumsp. ZY369 cell. In one aspect, the cell is aTalaromyces atricolacell. In one aspect, the cell is aTrichocladium asperumcell. In one aspect, the cell is aMetarhizium carneumcell. In one aspect, the cell is aThielavia terrestriscell. The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide. The host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates. The polypeptide may be detected using methods known in the art that are specific for the polypeptides. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide. The polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the fermentation medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a fermentation broth comprising the polypeptide is recovered. The polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides. Plants The present invention also relates to isolated plants, e.g., a transgenic plant, plant part, or plant cell, comprising a polynucleotide of the present invention so as to express and produce a polypeptide or domain in recoverable quantities. The polypeptide or domain may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the polypeptide or domain may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor. The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass,Poa), forage grass such asFestuca, Lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn). Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. Plant cells and specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part. Also included within the scope of the present invention are the progeny of such plants, plant parts, and plant cells. The transgenic plant or plant cell expressing the polypeptide or domain may be constructed in accordance with methods known in the art. The present invention also relates to methods of producing a polypeptide or domain of the present invention comprising (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the polypeptide or domain under conditions conducive for production of the polypeptide or domain; and (b) recovering the polypeptide or domain. Fermentation Broth Formulations or Cell Compositions The present invention also relates to a fermentation broth formulation or a cell composition comprising a polypeptide of the present invention. The fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding the polypeptide of the present invention which are used to produce the polypeptide of interest), cell debris, biomass, fermentation media and/or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium. The term “fermentation broth” as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification. For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells. In some embodiments, the fermentation broth formulation and cell compositions comprise a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof. In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing. In one aspect, the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris. In some embodiments, the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components. The fermentation broth formulations or cell compositions may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art. The cell-killed whole broth or composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis. In some embodiments, the cell-killed whole broth or composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art. A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition. The whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673. Enzyme Compositions The present invention also relates to compositions comprising a polypeptide of the present invention. Preferably, the compositions are enriched in the polypeptide of the invention. The term “enriched” indicates that the lysozyme activity of the composition has been increased, e.g., with an enrichment factor of at least 1.1, such as at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 10. In a preferred embodiment, the composition comprises one or more LYS polypeptides having lysozyme activity selected from the list consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45. In an embodiment, the composition comprises the polypeptide of the invention and one or more formulating agents, as described below. The compositions may further comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of phytase, xylanase, galactanase, alpha-galactosidase, beta-galactosidase, protease, phospholipase A1, phospholipase A2, lysophospholipase, phospholipase C, phospholipase D, amylase, lysozyme, arabinofuranosidase, beta-xylosidase, acetyl xylan esterase, feruloyl esterase, cellulase, cellobiohydrolases, beta-glucosidase, pullulanase, and beta-glucanase or any combination thereof. The compositions may further comprise one or more probiotics. In an embodiment, the probiotic is selected from the group consisting ofBacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacteriumsp.,Carnobacteriumsp.,Clostridium butyricum, Clostridiumsp.,Enterococcus faecium, Enterococcussp.,Lactobacillussp.,Lactobacillus acidophilus, Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactococcus Lactis, Lactococcussp.,Leuconostocsp.,Megasphaera elsdenii, Megasphaerasp.,Pediococsus acidilactici, Pediococcussp.,Propionibacterium thoenii, Propionibacteriumsp. andStreptococcussp. or any combination thereof. In an embodiment, the composition comprises one or more formulating agents as disclosed herein, preferably one or more of the compounds selected from the list consisting of glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin, maltodextrin, cyclodextrin, wheat, PVA, acetate, phosphate, kaolin and cellulose. In an embodiment, the composition comprises one or more components selected from the list consisting of vitamins, minerals and amino acids. Formulation The enzyme of the invention may be formulated as a liquid or a solid. For a liquid formulation, the formulating agent may comprise a polyol (such as e.g. glycerol, ethylene glycol or propylene glycol), a salt (such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or a sugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, and sorbitol). Thus in one embodiment, the composition is a liquid composition comprising the polypeptide of the invention and one or more formulating agents selected from the list consisting of glycerol, ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose, and sorbitol. The liquid formulation may be sprayed onto the feed after it has been pelleted or may be added to drinking water given to the animals. For a solid formulation, the formulation may be for example as a granule, spray dried powder or agglomerate (e.g. as disclosed in WO2000/70034). The formulating agent may comprise a salt (organic or inorganic zinc, sodium, potassium or calcium salts such as e.g. such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol). In one embodiment, the composition is a solid composition, such as a spray dried composition, comprising the LYS polypeptide of the invention and one or more formulating agents selected from the list consisting of sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin, maltodextrin, cyclodextrin, wheat, PVA, acetate, phosphate and cellulose. In a preferred embodiment, the formulating agent is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate, magnesium sulfate and calcium carbonate. The present invention also relates to enzyme granules/particles comprising the LYS polypeptide of the invention optionally combined with one or more additional enzymes. The granule is composed of a core, and optionally one or more coatings (outer layers) surrounding the core. Typically the granule/particle size, measured as equivalent spherical diameter (volume based average particle size), of the granule is 20-2000 μm, particularly 50-1500 μm, 100-1500 μm or 250-1200 μm. The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation. Methods for preparing the core can be found in Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Volume 1; 1980; Elsevier. Preparation methods include known feed and granule formulation technologies, e.g.:a) spray dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme-containing particulate material;b) layered products, wherein the enzyme is coated as a layer around a pre-formed inert core particle, wherein an enzyme-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the enzyme-containing solution adheres to the core particles and dries up to leave a layer of dry enzyme on the surface of the core particle. Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found. This type of product is described in, e.g., WO 97/23606;c) absorbed core particles, wherein rather than coating the enzyme as a layer around the core, the enzyme is absorbed onto and/or into the surface of the core. Such a process is described in WO 97/39116.d) extrusion or pelletized products, wherein an enzyme-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the enzyme paste, which is harmful to the enzyme;e) prilled products, wherein an enzyme-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomiser, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor);Powdered detergents; Surfactant Science Series; 1998; vol. 71; page 140-142; Marcel Dekker). The product obtained is one wherein the enzyme is uniformly distributed throughout an inert material instead of being concentrated on its surface. Also U.S. Pat. Nos. 4,016,040 and 4,713,245 are documents relating to this technique;f) mixer granulation products, wherein a liquid is added to a dry powder composition of, e.g., conventional granulating components, the enzyme being introduced either via the liquid or the powder or both. The liquid and the powder are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme. Such a process is described in U.S. Pat. No. 4,106,991 and related documents EP 170360, EP 304332, EP 304331, WO 90/09440 and WO 90/09428. In a particular product of this process wherein various high-shear mixers can be used as granulators, granulates consisting of enzyme as enzyme, fillers and binders etc. are mixed with cellulose fibres to reinforce the particles to give the so-called T-granulate. Reinforced particles, being more robust, release less enzymatic dust.g) size reduction, wherein the cores are produced by milling or crushing of larger particles, pellets, tablets, briquettes etc. containing the enzyme. The wanted core particle fraction is obtained by sieving the milled or crushed product. Over and undersized particles can be recycled. Size reduction is described in (Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons);h) fluid bed granulation, which involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them and form a granule;i) the cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or detergent industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90° C. For some enzymes it is important the cores comprising the enzyme contain a low amount of water before coating. If water sensitive enzymes are coated before excessive water is removed, it will be trapped within the core and it may affect the activity of the enzyme negatively. After drying, the cores preferably contain 0.1-10% w/w water. The core may include additional materials such as fillers, fibre materials (cellulose or synthetic fibres), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances. The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate. The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend. In one embodiment, the core comprises a material selected from the group consisting of salts (such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and minerals and clay minerals (also known as hydrous aluminium phyllosilicates). In one embodiment, the core comprises a clay mineral such as kaolinite or kaolin. The core may include an inert particle with the enzyme absorbed into it, or applied onto the surface, e.g., by fluid bed coating. The core may have a diameter of 20-2000 μm, particularly 50-1500 μm, 100-1500 μm or 250-1200 μm. The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt and/or wax and/or flour coating, or other suitable coating materials. The coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, 1% or 5%. The amount may be at most 100%, 70%, 50%, 40% or 30%. The coating is preferably at least 0.1 μm thick, particularly at least 0.5 μm, at least 1 μm or at least 5 μm. In some embodiments the thickness of the coating is below 100 μm, such as below 60 μm, or below 40 μm. The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit is encapsulated or enclosed with few or no uncoated areas. The layer or coating should in particular be homogeneous in thickness. The coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc. A salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight. The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 μm, such as less than 10 μm or less than 5 μm. The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility at least 0.1 g in 100 g of water at 20° C., preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water. The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminium. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, sorbate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used. The salt in the coating may have a constant humidity at 20° C. above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in WO1997/05245, WO1998/54980, WO1998/55599, WO2000/70034, WO2006/034710, WO2008/017661, WO2008/017659, WO2000/020569, WO2001/004279, WO1997/05245, WO2000/01793, WO2003/059086, WO2003/059087, WO2007/031483, WO2007/031485, WO2007/044968, WO2013/192043, WO2014/014647 and WO2015/197719 or polymer coating such as described in WO 2001/00042. Specific examples of suitable salts are NaCl (CH20° C.=76%), Na2CO3 (CH20° C.=92%), NaNO3 (CH20° C.=73%), Na2HPO4 (CH20° C.=95%), Na3PO4 (CH25° C.=92%), NH4Cl (CH20° C.=79.5%), (NH4)2HPO4 (CH20° C.=93.0%), NH4H2PO4 (CH20° C.=93.1%), (NH4)2504 (CH20° C.=81.1%), KCl (CH20° C.=85%), K2HPO4 (CH20° C.=92%), KH2PO4 (CH20° C.=96.5%), KNO3 (CH20° C.=93.5%), Na2SO4 (CH20° C.=93%), K2504 (CH20° C.=98%), KHSO4 (CH20° C.=86%), MgSO4 (CH20° C.=90%), ZnSO4 (CH20° C.=90%) and sodium citrate (CH25° C.=86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, magnesium acetate, calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, sodium acetate, sodium benzoate, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate and zinc sorbate. The salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfate heptahydrate (MgSO4.7H2O), zinc sulfate heptahydrate (ZnSO4.7H2O), sodium phosphate dibasic heptahydrate (Na2HPO4.7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate. Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed. A wax coating may comprise at least 60% by weight of a wax, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight. Specific examples of waxes are polyethylene glycols; polypropylenes; Carnauba wax; Candelilla wax; bees wax; hydrogenated plant oil or animal tallow such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC), polyvinyl alcohol (PVA), hydrogenated ox tallow, hydrogenated palm oil, hydrogenated cotton seeds and/or hydrogenated soy bean oil; fatty acid alcohols; mono-glycerides and/or di-glycerides, such as glyceryl stearate, wherein stearate is a mixture of stearic and palmitic acid; micro-crystalline wax; paraffin's; and fatty acids, such as hydrogenated linear long chained fatty acids and derivatives thereof. A preferred wax is palm oil or hydrogenated palm oil. The granule may comprise a core comprising the LYS polypeptide of the invention, one or more salt coatings and one or more wax coatings. Examples of enzyme granules with multiple coatings are shown in WO1993/07263, WO1997/23606 and WO2016/149636. Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. The coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. The granulate may further comprise one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D. Another example of formulation of enzymes by the use of co-granulates is disclosed in WO 2013/188331. The present invention also relates to protected enzymes prepared according to the method disclosed in EP 238,216. Thus, in a further aspect, the present invention provides a granule, which comprises:(a) a core comprising a LYS polypeptide having lysozyme activity according to the invention, and(b) a coating consisting of one or more layer(s) surrounding the core. In one embodiment, the coating comprises a salt coating as described herein. In one embodiment, the coating comprises a wax coating as described herein. In one embodiment, the coating comprises a salt coating followed by a wax coating as described herein. Animal Feed Additives The present invention also relates to animal feed additives comprising one or more LYS polypeptides having lysozyme activity. Thus, in one embodiment, the invention relates to an animal feed additive comprising a LYS polypeptide, wherein:(a) the polypeptide has lysozyme activity;(b) the polypeptide comprises one or more LAD catalytic domains; and(c) the LAD catalytic domain gives a domT score of at least 170 when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 46 to 187 and hmmbuild software program, and wherein the query is carried out using hmmscan software program by the Method of Determining the Lysozyme Enhancing Domain by HMM. In an embodiment, the polypeptide further comprises one or more lysozyme enhancing domains, wherein the lysozyme enhancing domain gives a domT score of at least 100 when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 188 to 316 and hmmbuild software program, and wherein the query is carried out using the hmmscan software program. In an embodiment, the domT score of the LAD catalytic domain is at least 175, preferably at least 180, more preferably at least 185, even more preferably at least 190, even more preferably at least 195, or most preferably at least 200. In an embodiment, the domT score of the LED is at least 103, preferably at least 106, more preferably at least 109, more preferably at least 112, more preferably at least 115, more preferably at least 118, even more preferably at least 121, or most preferably at least 124. Preferred combinations of domT scores are as disclosed in the first aspect of the invention. In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In another aspect, the invention relates to animal feed additives comprising one or more LYS polypeptides having lysozyme activity, wherein the polypeptide is selected from the group consisting of:(a) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 3;(b) a polypeptide having at least 84%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 6;(c) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 9;(d) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 12;(e) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 15;(f) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 18;(g) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 21;(h) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 24;(i) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 27;(j) a polypeptide having at least 84%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 30;(k) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 33;(l) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 36;(m) a polypeptide having at least 84%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 39;(n) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 42;(o) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 45;(p) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 positions;(q) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;(r) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and(s) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having lysozyme activity and having at least 90% of the length of the mature polypeptide. In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In one embodiment, the polypeptide is of fungal origin. In an embodiment, the polypeptide is obtained or obtainable from the taxonomic phylum Ascomycota, preferably the taxonomic subphylum Pezizomycotina. In an embodiment, the amount of enzyme in the animal feed additive is between 0.001% and 10% by weight of the composition. In an embodiment, the animal feed additive comprises one or more formulating agents, preferably as described herein above. In an embodiment, the animal feed additive comprises one or more additional enzymes, preferably as described herein below. In an embodiment, the animal feed additive comprises one or more probiotics, preferably as described herein below. In an embodiment, the animal feed additive comprises one or more vitamins, preferably as described herein below. In an embodiment, the animal feed additive comprises one or more minerals, preferably as described herein below. In an embodiment, the animal feed additive comprises one or more amino acids, preferably as described herein below. In an embodiment, the animal feed additive comprises one or more prebiotics, preferably as described herein below. In an embodiment, the animal feed additive comprises one or more organic acids, preferably as described herein below. In an embodiment, the animal feed additive comprises one or more phytogenics, preferably as described herein below. Animal Feed The present invention also relates to animal feed compositions comprising one or more lysozymes of the invention. In one embodiment, the invention relates to an animal feed comprising the granule as described herein and plant based material. In one embodiment, the invention relates to an animal feed comprising the animal feed additive as described herein and plant based material. Animal feed compositions or diets have a relatively high content of protein. Poultry and pig diets can be characterised as indicated in Table B of WO 01/58275, columns 2-3. Fish diets can be characterised as indicated in column 4 of this Table B. Furthermore such fish diets usually have a crude fat content of 200-310 g/kg. An animal feed composition according to the invention has a crude protein content of 50-800 g/kg, and furthermore comprises at least one polypeptide having lysozyme activity as claimed herein. Furthermore, or in the alternative (to the crude protein content indicated above), the animal feed composition of the invention has a content of metabolisable energy of 10-30 MJ/kg; and/or a content of calcium of 0.1-200 g/kg; and/or a content of available phosphorus of 0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or a content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50 g/kg. In particular embodiments, the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (R. 2-5). Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, i.e. Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC). Metabolisable energy can be calculated on the basis of the NRC publication Nutrient requirements in swine, ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen by, Wageningen. ISBN 90-71463-12-5. The dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7. In a particular embodiment, the animal feed composition of the invention contains at least one vegetable protein as defined above. The animal feed composition of the invention may also contain animal protein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal, typically in an amount of 0-25%. The animal feed composition of the invention may also comprise Dried Distillers Grains with Solubles (DDGS), typically in amounts of 0-30%. In still further particular embodiments, the animal feed composition of the invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or 0-20% whey. The animal feed may comprise vegetable proteins. In particular embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof. In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. In another particular embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed, and cabbage. In another particular embodiment, soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum. Animal diets can e.g. be manufactured as mash feed (non-pelleted) or pelleted feed. Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. Enzymes can be added as solid or liquid enzyme formulations. For example, for mash feed a solid or liquid enzyme formulation may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) lysozyme/enzyme preparation may also be added before or during the feed ingredient step. Typically a liquid lysozyme/enzyme preparation comprises the polypeptide having lysozyme activity of the invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets. The enzyme may also be incorporated in a feed additive or premix. Alternatively, the polypeptide having lysozyme activity can be prepared by freezing a mixture of liquid enzyme solution with a bulking agent such as ground soybean meal, and then lyophilizing the mixture. The final enzyme concentration in the diet is within the range of 0.01-200 mg enzyme protein per kg diet, preferably between 0.05-100 mg/kg diet, more preferably 0.1-50 mg, even more preferably 0.2-20 mg enzyme protein per kg animal diet. It is at present contemplated that the enzyme is administered in one or more of the following amounts (dosage ranges): 0.01-200; 0.05-100; 0.1-50; 0.2-20; 0.1-1; 0.2-2; 0.5-5; or 1-10; —all these ranges being in mg LYS polypeptide protein per kg feed (ppm). For determining mg LYS polypeptide protein per kg feed, the LYS polypeptide is purified from the feed composition, and the specific activity of the purified LYS polypeptide is determined using a relevant assay (see under lysozyme activity). The lysozyme activity of the feed composition as such is also determined using the same assay, and on the basis of these two determinations, the dosage in mg lysozyme protein per kg feed is calculated. In a particular embodiment, the animal feed additive of the invention is intended for being included (or prescribed as having to be included) in animal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05 to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This is so in particular for premixes. The same principles apply for determining mg LYS polypeptide protein in feed additives. Of course, if a sample is available of the LYS polypeptide used for preparing the feed additive or the feed, the specific activity is determined from this sample (no need to purify the LYS polypeptide from the feed composition or the additive). Thus in a further aspect, the present invention also relates to an animal feed comprising one or more LYS polypeptides having lysozyme activity and plant based material. In another aspect, the present invention also relates to an animal feed comprising the animal feed additive of the invention (as described herein above) and plant based material. In one embodiment, the invention relates to an animal feed comprising plant based material and a LYS polypeptide, wherein the polypeptide (a) has lysozyme activity and (b) comprises one or more LAD catalytic domains; wherein the LAD catalytic domain gives a domT score of at least 180 when queried using a Profile Hidden Markov Model (HMM) prepared using SEQ ID NOs: 46 to 187 and hmmbuild software program, and wherein the query is carried out using hmmscan software program by the Method of Determining the LAD Catalytic Domain by HMM. In an embodiment, the polypeptide further comprises one or more lysozyme enhancing domains, wherein the lysozyme enhancing domain gives a domT score of at least 100 when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 188 to 316 and hmmbuild software program, and wherein the query is carried out using the hmmscan software program by the Method of Determining the Lysozyme. In an embodiment, the domT score of the LAD catalytic domain is at least 175, preferably at least 180, more preferably at least 185, even more preferably at least 190, even more preferably at least 195, or most preferably at least 200. In an embodiment, the domT score of the LED is at least 103, preferably at least 106, more preferably at least 109, more preferably at least 112, more preferably at least 115, more preferably at least 118, even more preferably at least 121, or most preferably at least 124. Preferred combinations of domT scores are as disclosed in the first aspect of the invention. In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In another aspect, the invention relates to an animal feed comprising plant based material and one or more LYS polypeptides having lysozyme activity, wherein the polypeptide is selected from the group consisting of:(a) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 3;(b) a polypeptide having at least 84%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 6;(c) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 9;(d) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 12;(e) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 15;(f) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 18;(g) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 21;(h) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 24;(i) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 27;(j) a polypeptide having at least 84%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 30;(k) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 33;(l) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 36;(m) a polypeptide having at least 84%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 39;(n) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 42;(o) a polypeptide having at least 80%, such as at least 85%, at least 90% or at least 95% sequence identity to the polypeptide of SEQ ID NO: 45;(p) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 positions;(q) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;(r) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and(s) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having lysozyme activity and having at least 90% of the length of the mature polypeptide. In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In an embodiment, the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In one embodiment, the polypeptide is of fungal origin. In an embodiment, the polypeptide is obtained or obtainable from the taxonomic phylum Ascomycota, preferably the taxonomic subphylum Pezizomycotina. In an embodiment, the plant based material is selected from the group consisting of legumes, cereals, oats, rye, barley, wheat, maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet, soybean, wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, rapeseed (oilseed rape), rice, beet, cabbage, sugar beet, spinach, quinoa, or pea, in a processed form thereof (such as soybean meal, rapeseed meal) or any combination thereof. In a further embodiment, the animal feed has been pelleted. Additional Enzymes In another embodiment, the compositions described herein optionally include one or more enzymes. Enzymes can be classified on the basis of the handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also the ENZYME site at the internet: http://www.expasy.ch/enzyme/. ENZYME is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc., 1992, and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch A. The ENZYME database, 2000, Nucleic Acids Res28:304-305). This IUB-MB Enzyme nomenclature is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural features of these enzymes. Another classification of certain glycoside hydrolase enzymes, such as endoglucanase, xylanase, galactanase, mannanase, dextranase, lysozyme and galactosidase is described in Henrissat et al, “The carbohydrate-active enzymes database (CAZy) in 2013”, Nucl. Acids Res. (1 Jan. 2014) 42 (D1): D490-D495; see also www.cazy.org. Thus the composition of the invention may also comprise at least one other enzyme selected from the group comprising of phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1); arabinofuranosidase (EC 3.2.1.55); beta-xylosidase (EC 3.2.1.37); acetyl xylan esterase (EC 3.1.1.72); feruloyl esterase (EC 3.1.1.73); cellulase (EC 3.2.1.4); cellobiohydrolases (EC 3.2.1.91); beta-glucosidase (EC 3.2.1.21); pullulanase (EC 3.2.1.41), alpha-mannosidase (EC 3.2.1.24), mannanase (EC 3.2.1.25) and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or any mixture thereof. In a particular embodiment, the composition of the invention comprises a phytase (EC 3.1.3.8 or 3.1.3.26). Examples of commercially available phytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P, Ronozyme® NP and Ronozyme® HiPhos (DSM Nutritional Products), Natuphos™ (BASF), Natuphos™ E (BASF), Finase® and Quantum® Blue (AB Enzymes), OptiPhos® (Huvepharma), AveMix® Phytase (Aveve Biochem), Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytases include those described in e.g. WO 98/28408, WO 00/43503, and WO 03/066847. In a particular embodiment, the composition of the invention comprises a xylanase (EC 3.2.1.8). Examples of commercially available xylanases include Ronozyme® WX (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium), Hostazym® X (Huvepharma), Axtra® XB (Xylanase/beta-glucanase, DuPont) and Axtra® XAP (Xylanase/amylase/protease, DuPont), AveMix® XG 10 (xylanase/glucanase) and AveMix® 02 CS (xylanase/glucanase/pectinase, Aveve Biochem), and Naturgrain (BASF). In a particular embodiment, the composition of the invention comprises a protease (EC 3.4). Examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products). In a particular embodiment, the composition of the invention comprises an alpha-amylase (EC 3.2.1.1). Examples of commercially available alpha-amylases include Ronozyme® A and RONOZYME® RumiStar™ (DSM Nutritional Products). In one embodiment, the composition of the invention comprises a multicomponent enzyme product, such as FRA® Octazyme (Framelco), Ronozyme® G2, Ronozyme® VP and Ronozyme® MultiGrain (DSM Nutritional Products), Rovabio® Excel or Rovabio® Advance (Adisseo). Eubiotics Eubiotics are compounds which are designed to give a healthy balance of the micro-flora in the gastrointestinal tract. Eubiotics cover a number of different feed additives, such as probiotics, prebiotics, phytogenics (essential oils) and organic acids which are described in more detail below. Probiotics In an embodiment, the animal feed composition further comprises one or more additional probiotic. In a particular embodiment, the animal feed composition further comprises a bacterium from one or more of the following genera:Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, ClostridiumandMegasphaeraor any combination thereof. In a preferred embodiment, animal feed composition further comprises a bacterium from one or more of the following strains:Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Enterococcus faecium, Enterococcusspp, andPediococcusspp,Lactobacillusspp,Bifidobacteriumspp,Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillus farciminus, Lactobacillus rhamnosus, Clostridium butyricum, Bifidobacterium animalisssp.animalis, Lactobacillus reuteri, Lactobacillus salivariusssp.salivarius, Megasphaera elsdenii, Propionibacteriasp. In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains ofBacillus subtilis:3A-P4 (PTA-6506), 15A-P4 (PTA-6507), 22C-P1 (PTA-6508), 2084 (NRRL B-500130), LSSA01 (NRRL-B-50104), BS27 (NRRL B-501 05), BS 18 (NRRL B-50633), BS 278 (NRRL B-50634), DSM 29870, DSM 29871, DSM 32315, NRRL B-50136, NRRL B-50605, NRRL B-50606, NRRL B-50622 and PTA-7547. In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains ofBacillus pumilus: NRRL B-50016, ATCC 700385, NRRL B-50885 or NRRL B-50886. In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains ofBacillus licheniformis: NRRL B 50015, NRRL B-50621 or NRRL B-50623. In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains ofBacillus amyloliquefaciens: DSM 29869, DSM 29869, NRRL B 50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B-50606, NRRL B-50013, NRRL B-50151, NRRL B-50141, NRRL B-50147 or NRRL B-50888. The bacterial count of each of the bacterial strains in the animal feed composition is between 1×104and 1×1014CFU/kg of dry matter, preferably between 1×106and 1×1012CFU/kg of dry matter, and more preferably between 1×107and 1×1011CFU/kg of dry matter. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed composition is between 1×108and 1×1010CFU/kg of dry matter. The bacterial count of each of the bacterial strains in the animal feed composition is between 1×105and 1×1015CFU/animal/day, preferably between 1×107and 1×1013CFU/animal/day, and more preferably between 1×108and 1×1012CFU/animal/day. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed composition is between 1×109and 1×1011CFU/animal/day. In one embodiment, the amount of probiotics is 0.001% to 10% by weight of the composition. In another embodiment, the one or more bacterial strains are present in the form of a stable spore. Examples of commercial products are Cylactin® (DSM Nutritional Products), Alterion (Adisseo), Enviva PRO (DuPont Animal Nutrition), Syncra® (mix enzyme+probiotic, DuPont Animal Nutrition), Ecobiol® and Fecinor0 (Norel/Evonik) and GutCare® PY1 (Evonik). Prebiotics Prebiotics are substances that induce the growth or activity of microorganisms (e.g., bacteria and fungi) that contribute to the well-being of their host. Prebiotics are typically non-digestible fiber compounds that pass undigested through the upper part of the gastrointestinal tract and stimulate the growth or activity of advantageous bacteria that colonize the large bowel by acting as substrate for them. Normally, prebiotics increase the number or activity of bifidobacteria and lactic acid bacteria in the GI tract. Yeast derivatives (inactivated whole yeasts or yeast cell walls) can also be considered as prebiotics. They often comprise mannan-oligosaccharids, yeast beta-glucans or protein contents and are normally derived from the cell wall of the yeast,Saccharomyces cerevisiae. In one embodiment, the amount of prebiotics is 0.001% to 10% by weight of the composition. Examples of yeast products are Yang® and Agrimos (Lallemand Animal Nutrition). Phytogenics Phytogenics are a group of natural growth promoters or non-antibiotic growth promoters used as feed additives, derived from herbs, spices or other plants. Phytogenics can be single substances prepared from essential oils/extracts, essential oils/extracts, single plants and mixture of plants (herbal products) or mixture of essential oils/extracts/plants (specialized products). Examples of phytogenics are rosemary, sage, oregano, thyme, clove, and lemongrass. Examples of essential oils are thymol, eugenol, meta-cresol, vaniline, salicylate, resorcine, guajacol, gingerol, lavender oil, ionones, irone, eucalyptol, menthol, peppermint oil, alpha-pinene; limonene, anethol, linalool, methyl dihydrojasmonate, carvacrol, propionic acid/propionate, acetic acid/acetate, butyric acid/butyrate, rosemary oil, clove oil, geraniol, terpineol, citronellol, amyl and/or benzyl salicylate, cinnamaldehyde, plant polyphenol (tannin), turmeric and curcuma extract. In one embodiment, the amount of phytogeneics is 0.001% to 10% by weight of the composition. Examples of commercial products are Crina® (DSM Nutritional Products); Cinergy™, Biacid™, ProHacid™ Classic and ProHacid™ Advance™ (all Promivi/Cargill) and Envivo EO (DuPont Animal Nutrition). Organic Acids Organic acids (C1-C7) are widely distributed in nature as normal constituents of plants or animal tissues. They are also formed through microbial fermentation of carbohydrates mainly in the large intestine. They are often used in swine and poultry production as a replacement of antibiotic growth promoters since they have a preventive effect on the intestinal problems like necrotic enteritis in chickens andEscherichia coliinfection in young pigs. Organic acids can be sold as mono component or mixtures of typically 2 or 3 different organic acids. Examples of organic acids are propionic acid, formic acid, citric acid, lactic acid, sorbic acid, malic acid, acetic acid, fumaric acid, benzoic acid, butyric acid and tartaric acid or their salt (typically sodium or potassium salt such as potassium diformate or sodium butyrate). In one embodiment, the amount of organic acid is 0.001% to 10% by weight of the composition. Examples of commercial products are VevoVitall® (DSM Nutritional Products), Amasil®, Luprisil®, Lupro-Grain®, Lupro-Cid®, Lupro-Mix® (BASF), n-Butyric Acid AF (OXEA) and Adimix Precision (Nutriad). Premix The incorporation of the composition of feed additives as exemplified herein above to animal feeds, for example poultry feeds, is in practice carried out using a concentrate or a premix. A premix designates a preferably uniform mixture of one or more microingredients with diluent and/or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix. A premix according to the invention can be added to feed ingredients or to the drinking water as solids (for example as water soluble powder) or liquids. Amino Acids The composition of the invention may further comprise one or more amino acids. Examples of amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan. In one embodiment, the amount of amino acid is 0.001% to 10% by weight of the composition. Vitamins and Minerals In another embodiment, the animal feed may include one or more vitamins, such as one or more fat-soluble vitamins and/or one or more water-soluble vitamins. In another embodiment, the animal feed may optionally include one or more minerals, such as one or more trace minerals and/or one or more macro minerals. Usually fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed. Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3. Non-limiting examples of water-soluble vitamins include vitamin C, vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g., Ca-D-panthothenate. Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, iodine, selenium and zinc. Non-limiting examples of macro minerals include calcium, magnesium, phosphorus, potassium and sodium. In one embodiment, the amount of vitamins is 0.001% to 10% by weight of the composition. In one embodiment, the amount of minerals is 0.001% to 10% by weight of the composition. The nutritional requirements of these components (exemplified with poultry and piglets/pigs) are listed in Table A of WO 01/58275. Nutritional requirement means that these components should be provided in the diet in the concentrations indicated. In the alternative, the animal feed additive of the invention comprises at least one of the individual components specified in Table A of WO 01/58275. At least one means either of, one or more of, one, or two, or three, or four and so forth up to all thirteen, or up to all fifteen individual components. More specifically, this at least one individual component is included in the additive of the invention in such an amount as to provide an in-feed-concentration within the range indicated in column four, or column five, or column six of Table A. In a still further embodiment, the animal feed additive of the invention comprises at least one of the below vitamins, preferably to provide an in-feed-concentration within the ranges specified in the below Table 1 (for piglet diets, and broiler diets, respectively). TABLE 1Typical vitamin recommendationsVitaminPiglet dietBroiler dietVitamin A10,000-15,000 IU/kg feed8-12,500 IU/kg feedVitamin D31800-2000 IU/kg feed3000-5000 IU/kg feedVitamin E60-100 mg/kg feed150-240 mg/kg feedVitamin K32-4 mg/kg feed2-4 mg/kg feedVitamin B12-4 mg/kg feed2-3 mg/kg feedVitamin B26-10 mg/kg feed7-9 mg/kg feedVitamin B64-8 mg/kg feed3-6 mg/kg feedVitamin B120.03-0.05 mg/kg feed0.015-0.04 mg/kg feedNiacin (Vitamin B3)30-50 mg/kg feed50-80 mg/kg feedPantothenic acid20-40 mg/kg feed10-18 mg/kg feedFolic acid1-2 mg/kg feed1-2 mg/kg feedBiotin0.15-0.4 mg/kg feed0.15-0.3 mg/kg feedCholine chloride200-400 mg/kg feed300-600 mg/kg feed Other Feed Ingredients The composition of the invention may further comprise colouring agents, stabilisers, growth improving additives and aroma compounds/flavourings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, antioxidants, anti-microbial peptides, anti-fungal polypeptides and mycotoxin management compounds. Examples of colouring agents are carotenoids such as beta-carotene, astaxanthin, and lutein. Examples of aroma compounds/flavourings are creosol, anethol, deca-, undeca- and/or dodeca-lactones, ionones, irone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannin. Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A, Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity. Examples of antifungal polypeptides (AFP's) are theAspergillus giganteus, andAspergillus nigerpeptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384. Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid. Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase. Antioxidants can be used to limit the number of reactive oxygen species which can be generated such that the level of reactive oxygen species is in balance with antioxidants. Mycotoxins, such as deoxynivalenol, aflatoxin, zearalenone and fumonisin can be found in animal feed and can result in negative animal performance or illness. Compounds which can manage the levels of mycotoxin, such as via deactivation of the mycotoxin or via binding of the mycotoxin, can be added to the feed to ameliorate these negative effects. Examples of mycotoxin management compounds are Vitafix®, Vitafix Ultra (Nuscience), Mycofix®, Mycofix® Secure, FUMzyme®, Biomin® BBSH, Biomin® MTV (Biomin), Mold-Nil®, Toxy-Nil® and Unike® Plus (Nutriad). Uses Use in Animal Feed A LYS polypeptide of the invention may also be used in animal feed, wherein the term “animal” refers to all animals except humans. Examples of animals are mono-gastric animals, e.g. pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry (including but not limited to poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick); fish (including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish); and crustaceans (including but not limited to shrimps and prawns). In the use according to the invention the LYS polypeptide can be fed to the animal before, after, or simultaneously with the diet. The latter is preferred. In a particular embodiment, the LYS polypeptide, in the form in which it is added to the feed, or when being included in a feed additive, is well-defined. Well-defined means that the LYS polypeptide preparation is at least 50% pure as determined by Size-exclusion chromatography (see Example 12 of WO 01/58275). In other particular embodiments the LYS polypeptide preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by this method. A well-defined LYS polypeptide preparation is advantageous. For instance, it is much easier to dose correctly to the feed a LYS polypeptide that is essentially free from interfering or contaminating other lysozymes. The term dose correctly refers in particular to the objective of obtaining consistent and constant results, and the capability of optimizing dosage based upon the desired effect. For the use in animal feed, however, the LYS polypeptide need not be pure; it may e.g. include other enzymes, in which case it could be termed a LYS polypeptide preparation. The LYS polypeptide preparation can be (a) added directly to the feed, or (b) it can be used in the production of one or more intermediate compositions such as feed additives or premixes that is subsequently added to the feed (or used in a treatment process). The degree of purity described above refers to the purity of the original LYS polypeptide preparation, whether used according to (a) or (b) above. Methods of Improving Animal Performance In an embodiment, the present invention also relates to a method of improving the performance of an animal comprising administering to the animal the animal feed or the animal feed additive of the invention. In a preferred embodiment, the method of improving the performance of an animal comprises administering to the animal the animal feed or the animal feed additive comprising the LYS polypeptide of the invention. In one embodiment, the LYS polypeptide is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45. In an embodiment, the present invention also relates to the use of the animal feed or an animal feed additive of the invention for improving the performance of an animal. In another embodiment, the invention relates to the use of one or more lysozymes of the invention for improving the performance of an animal. In one embodiment, ‘improving the performance of an animal’ means that there is an increase in body weight gain. In another embodiment, ‘improving the performance of an animal’ means that there is an improved feed conversion ratio. In a further embodiment, ‘improving the performance of an animal’ means that there is an increased feed efficiency. In a further embodiment, ‘improving the performance of an animal’ means that there is an increase in body weight gain and/or an improved feed conversion ratio and/or an increased feed efficiency. In an embodiment, the animal feed comprises plant based material selected from the group consisting of legumes, cereals, oats, rye, barley, wheat, maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet, soybean, wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, rapeseed (oilseed rape), rice, beet, cabbage, sugar beet, spinach, quinoa, or pea, in a processed form thereof (such as soybean meal, rapeseed meal) or any combination thereof. Methods of Preparing an Animal Feed In an embodiment, the present invention provides a method for preparing an animal feed comprising adding one or more LYS polypeptide of the invention to one or more animal feed ingredients. Animal feed ingredients include, but are not limited to concentrates (as defined herein), forage (as defined herein), enzymes, probiotic, vitamins, minerals and amino acids. In a preferred embodiment, the method of preparing an animal feed comprises mixing plant based material with the LYS polypeptide of the invention. In one embodiment, the LYS polypeptide is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45. In an embodiment, the plant based material is selected from the group consisting of legumes, cereals, oats, rye, barley, wheat, maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet, soybean, wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, rapeseed (oilseed rape), rice, beet, cabbage, sugar beet, spinach, quinoa, or pea, in a processed form thereof (such as soybean meal, rapeseed meal) or any combination thereof. Preferred Embodiments Herein follows a list of preferred embodiments of the invention.1. A composition comprising at least 0.01 mg of LYS polypeptide per kilogram of composition, wherein the polypeptide (a) has lysozyme activity and (b) comprises one or more LAD catalytic domains; wherein the LAD catalytic domain gives a domT score of at least 180 when queried using a Profile Hidden Markov Model (HMM) prepared using SEQ ID NOs: 46 to 187 and hmmbuild software program, suitably wherein the query is carried out using hmmscan software program by the Method of Determining the LAD Catalytic Domain by HMM.2. The composition of item 1, wherein the polypeptide further comprises one or more lysozyme enhancing domains, wherein the lysozyme enhancing domain gives a domT score of at least 100 when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 188 to 316 and hmmbuild software program, and wherein the query is carried out using the hmmscan software program by the Method of Determining the Lysozyme Enhancing Domain.3. The composition of any of items 1 to 2, wherein(a) the LAD catalytic domain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and/or one or more motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318); and/or(b) the lysozyme enhancing domain comprises one or more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319).4. A composition comprising one or more LYS polypeptides having lysozyme activity, wherein the polypeptide is dosed at least 0.01 mg of polypeptide per kilogram of composition and is selected from the group consisting of:(a) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 3;(b) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 6;(c) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 9;(d) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 12;(e) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 15;(f) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 18;(g) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 21;(h) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 24;(i) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 27;(j) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 30;(k) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 33;(l) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 36;(m) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 39;(n) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 42;(o) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 45;(p) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 positions;(q) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;(r) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and(s) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having lysozyme activity and having at least 90% of the length of the mature polypeptide.5. The composition of item 4, wherein the polypeptide is selected from the group consisting of:(a) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 3;(b) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 6;(c) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 9;(d) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 12;(e) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 15;(f) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 18;(g) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 21;(h) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 24;(i) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 27;(j) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 30;(k) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 33;(l) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 36;(m) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 39;(n) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 42;(o) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 45; and(p) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 positions.6. The composition of item 4, wherein the polypeptide is selected from the group consisting of:(a) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 3;(b) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 6;(c) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 9;(d) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 12;(e) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 15;(f) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 18;(g) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 21;(h) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 24;(i) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 27;(j) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 30;(k) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 33;(l) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 36;(m) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 39;(n) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 42;(o) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 45; and(p) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 positions.7. The composition of item 4, wherein the polypeptide is selected from the group consisting of:(a) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 3;(b) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 6;(c) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 9;(d) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 12;(e) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 15;(f) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 18;(g) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 21;(h) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 24;(i) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 27;(j) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 30;(k) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 33;(l) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 36;(m) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 39;(n) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 42;(o) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 45; and(p) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 positions.8. The composition of any of items 4 to 7, wherein the LYS polypeptide comprises one or more motifs selected from the group consisting of (a) motif I:(SEQ ID NO: 317)AG[I/L]AT[A/G][I/L][T/V]ES;(b) motif II(SEQ ID NO: 318)V[G/A]XLCQXVQXSAYP;and(c) motif III:(SEQ ID NO: 319)[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN].9. The composition of any of items 1 to 8, wherein the polypeptide is of fungal origin.10. The composition of any of items 1 to 9, wherein the polypeptide is obtained or obtainable from the taxonomic phylum Ascomycota, preferably the taxonomic subphylum Pezizomycotina.11. The composition of any of items 1 to 10, wherein the polypeptide comprises or consists of amino acids1 to 226 of SEQ ID NO: 2, amino acids 1 to 226 of SEQ ID NO: 3, amino acids 1 to 226 of SEQ ID NO: 5, amino acids 1 to 226 of SEQ ID NO: 6, amino acids 1 to 223 of SEQ ID NO: 8, amino acids 1 to 223 of SEQ ID NO: 9, amino acids 1 to 304 of SEQ ID NO: 11, amino acids 1 to 304 of SEQ ID NO: 12, amino acids 1 to 228 of SEQ ID NO: 14, amino acids 1 to 228 of SEQ ID NO: 15, amino acids 1 to 230 of SEQ ID NO: 17, amino acids 1 to 230 of SEQ ID NO: 18, amino acids 1 to 230 of SEQ ID NO: 20, amino acids 1 to 230 of SEQ ID NO: 21, amino acids 1 to 232 of SEQ ID NO: 23, amino acids 1 to 232 of SEQ ID NO: 24, amino acids 1 to 228 of SEQ ID NO: 26, amino acids 1 to 228 of SEQ ID NO: 27, amino acids 1 to 228 of SEQ ID NO: 29, amino acids 1 to 228 of SEQ ID NO: 30, amino acids 1 to 226 of SEQ ID NO: 32, amino acids 1 to 226 of SEQ ID NO: 33, amino acids 1 to 225 of SEQ ID NO: 35, amino acids 1 to 225 of SEQ ID NO: 36, amino acids 1 to 225 of SEQ ID NO: 38, amino acids 1 to 225 of SEQ ID NO: 39, amino acids 1 to 304 of SEQ ID NO: 41, amino acids 1 to 304 of SEQ ID NO: 42, amino acids 1 to 227 of SEQ ID NO: 44, or amino acids 1 to 227 of SEQ ID NO: 45.12. The composition of any of items 1 to 11 further comprising one or more formulating agents.13. The composition of item 12, wherein the one or more formulating agent is selected from the group consisting of glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin, maltodextrin, cyclodextrin, wheat, PVA, acetate, phosphate and cellulose or any combination thereof.14. The composition of any of items 1 to 13 further comprising one or more additional enzymes.15. The composition of item 14 wherein the one or more additional enzymes is selected from the group consisting of acetyl xylan esterase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lipase, lysophospholipase, lysozyme, mannanase, alpha-mannosidase, beta-mannosidase, phytase, phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, protease, pullulanase, pectinase, pectin lyase, xylanase, beta-xylosidase, or any combination thereof.16. The composition of any of items 1 to 15 further comprising one or more microbes.17. The composition of item 16, wherein the one or more microbes is selected from the group consisting ofBacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacteriumsp.,Carnobacteriumsp.,Clostridium butyricum, Clostridiumsp.,Enterococcus faecium, Enterococcussp.,Lactobacillussp.,Lactobacillus acidophilus, Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactococcus Lactis, Lactococcussp.,Leuconostocsp.,Megasphaera elsdenii, Megasphaerasp.,Pediococsus acidilactici, Pediococcussp.,Propionibacterium thoenii, Propionibacteriumsp. andStreptococcussp. or any combination thereof.18. The composition of any of items 1 to 17 further comprising one or more components selected from the list consisting of:one or more vitamins;one or more minerals;one or more amino acids;one or more phytogenics;one or more prebiotics;one or more organic acids; andone or more other feed ingredients.19. A granule comprising the composition of any of items 1 to 18.20. The granule of item 19 wherein the granule is coated.21. The granule of item 20 wherein the coating comprises a salt and/or wax and/or a flour.22. An animal feed additive comprising the composition of any of items 1 to 18 or the granule of any of items 19 to 21.23. An animal feed comprising plant based material and the composition of any of items 1 to 18, the granule of any of items 19 to 21 or the animal feed additive of item 22.24. The animal feed of item 23, wherein the plant based material is selected from the group consisting of legumes, cereals, oats, rye, barley, wheat, maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet, soybean, wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, rapeseed (oilseed rape), rice, beet, cabbage, sugar beet, spinach, quinoa, or pea, in a processed form thereof (such as soybean meal, rapeseed meal) or any combination thereof.25. A pelleted animal feed comprising plant based material and the composition of any of items 1 to 18, the granule of any of items 19 to 21 or the animal feed additive of item 22.26. The pelleted animal feed of item 25, wherein the plant based material is selected from the group consisting of legumes, cereals, oats, rye, barley, wheat, maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet, soybean, wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, rapeseed (oilseed rape), rice, beet, cabbage, sugar beet, spinach, quinoa, or pea, in a processed form thereof (such as soybean meal, rapeseed meal) or any combination thereof.27. A liquid formulation comprising the composition of any of items 1 to 18.28. The liquid formulation of item 27, wherein the LYS polypeptide is dosed between 0.01% to 25% w/w of liquid formulation, preferably 0.05% to 20% w/w LYS polypeptide, more preferably 0.2% to 15% w/w LYS polypeptide, more preferably 0.5% to 15% w/w LYS polypeptide or most preferably 1.0% to 10% w/w LYS polypeptide.29. The liquid formulation of any of items 27 to 28, wherein the formulation further comprises 20% to 80% w/w of polyol.30. The liquid formulation of item 29, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600 or any combination thereof.31. The liquid formulation of any of items 27 to 30, wherein the formulation further comprises 0.01% to 2.0% w/w preservative.32. The liquid formulation of item 31, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.33. The liquid formulation of any of items 27 to 32 further comprising one or more components selected from the list consisting of:one or more enzymes;one or more microbes;one or more vitamins;one or more minerals;one or more amino acids;one or more phytogenics;one or more prebiotics;one or more organic acids; andone or more other feed ingredients.34. A method of preparing an animal feed comprising applying the liquid formulation of any of items 27 to 33 onto plant based material.35. The method of item 34, wherein the liquid formulation is applied via a spray.36. The method of any of items 34 to 35, wherein the plant based material is selected from the group consisting of legumes, cereals, oats, rye, barley, wheat, maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet, soybean, wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, rapeseed (oilseed rape), rice, beet, cabbage, sugar beet, spinach, quinoa, or pea, in a processed form thereof (such as soybean meal, rapeseed meal) or any combination thereof.37. The method of any of items 34 to 36, wherein the plant based material is in pelleted form.38. A pelleted animal feed prepared using the method of any of items 34 to 37.39. A method of improving one or more performance parameters of an animal comprising administering to one or more animals the composition of any of items 1 to 18, the granule of any of items 19 to 21, the animal feed additive of item 22, the animal feed of any of items 23 to 24, the pelleted animal feed of any of items 25 to 26 or 38 or the liquid formulation of any of items 27 to 33.40. The method of item 39 wherein the performance parameter is selected from the list consisting of body weight gain (BWG), European Production Efficiency Factor (EPEF) and Feed Conversion Ratio (FCR) or any combination thereof.41. A method of preparing an animal feed, comprising mixing the composition of any of items 1 to 18, the granule of any of items 19 to 21, the animal feed additive of item 22, the animal feed of any of items 23 to 24, the pelleted animal feed of any of items 25 to 26 or 38 or the liquid formulation of any of items 27 to 33 with plant based material.42. The method of item 41, wherein the plant based material is selected from the group consisting of legumes, cereals, oats, rye, barley, wheat, maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet, soybean, wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, rapeseed (oilseed rape), rice, beet, cabbage, sugar beet, spinach, quinoa, or pea, in a processed form thereof (such as soybean meal, rapeseed meal) or any combination thereof.43. Use of composition of any of items 1 to 18, the granule of any of items 19 to 21, the animal feed additive of item 22, the animal feed of any of items 23 to 24, the pelleted animal feed of any of items 25 to 26 or 38 or the liquid formulation of any of items 27 to 33:in animal feed;in animal feed additives;in the preparation of a composition for use in animal feed;for improving the nutritional value of an animal feed;for increasing digestibility of the animal feed; and/orfor improving one or more performance parameters in an animal.44. An isolated polypeptide having lysozyme activity, selected from the group consisting of:(a) a polypeptide having at least 95%, e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 3;(b) a polypeptide having at least 94%, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 6;(C) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 9;(d) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 12;(e) a polypeptide having at least 87%, e.g., at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 15;(f) a polypeptide having at least 81%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 18;(g) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 21;(h) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 24;(i) a polypeptide having at least 87%, e.g., at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 27;(j) a polypeptide having at least 96.2%, e.g., at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 30;(k) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 33;(l) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 36;(m) a polypeptide having at least 81%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 39;(n) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 42;(o) a variant of the polypeptide of SEQ ID NO: 3, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 positions;(p) a variant of the polypeptide of SEQ ID NO: 6, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 positions;(q) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 36, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 positions;(r) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 33 and SEQ ID NO: 42, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 positions;(s) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO: 27, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 positions;(t) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 39, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 positions;(u) a variant of the polypeptide of SEQ ID NO: 30, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7 or 8 positions;(v) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (r), (s), (t) or (u) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;(w) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (r), (s), (t) or (u) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and(x) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (r), (s), (t) or (u) having lysozyme activity and having at least 90% of the length of the mature polypeptide.45. The polypeptide according to item 44, wherein the polypeptide comprises or consists of amino acids 1 to 316 of SEQ ID NO: 2, amino acids 1 to 316 of SEQ ID NO: 3, amino acids 1 to 322 of SEQ ID NO: 4, amino acids 1 to 318 of SEQ ID NO: 6, amino acids 1 to 318 of SEQ ID NO: 7, amino acids 1 to 326 of SEQ ID NO: 8, amino acids 1 to 316 of SEQ ID NO: 10, amino acids 1 to 316 of SEQ ID NO: 11, amino acids 1 to 324 of SEQ ID NO: 12, amino acids 1 to 316 of SEQ ID NO: 14, amino acids 1 to 316 of SEQ ID NO: 15, amino acids 1 to 324 of SEQ ID NO: 16, amino acids 1 to 316 of SEQ ID NO: 18, amino acids 1 to 316 of SEQ ID NO: 19, amino acids 1 to 324 of SEQ ID NO: 20, amino acids 1 to 316 of SEQ ID NO: 22, amino acids 1 to 316 of SEQ ID NO: 23, amino acids 1 to 324 of SEQ ID NO: 24, amino acids 1 to 516 of SEQ ID NO: 26, amino acids 1 to 516 of SEQ ID NO: 27, amino acids 1 to 524 of SEQ ID NO: 28, amino acids 1 to 317 of SEQ ID NO: 30, amino acids 1 to 317 of SEQ ID NO: 31, amino acids 1 to 325 of SEQ ID NO: 32, amino acids 1 to 316 of SEQ ID NO: 34, amino acids 1 to 316 of SEQ ID NO: 35, amino acids 1 to 324 of SEQ ID NO: 36, amino acids 1 to 316 of SEQ ID NO: 38, amino acids 1 to 316 of SEQ ID NO: 39 or amino acids 1 to 324 of SEQ ID NO: 40.46. A polynucleotide encoding the polypeptide of any of items 44 to 45.47. A nucleic acid construct or expression vector comprising the polynucleotide of item 46 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.48. A recombinant host cell comprising the polynucleotide of item 46 operably linked to one or more control sequences that direct the production of the polypeptide.49. The recombinant host cell of item 48, wherein the host is a filamentous fungus, such asAspergillus, TrichodermaorFusarium, or a yeast, such asPichiaorSaccharomyces.50. The recombinant host cell of item 49, wherein the host is anAspergillus, such asAspergillusawamori,Aspergillusfoetidus,Aspergillusjaponicus,Aspergillusnidulans,Aspergillus nigerorAspergillusoryzae.51. The recombinant host cell of item 49, wherein the host is aTrichoderma, such asTrichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reeseiorTrichoderma viride.52. The recombinant host cell of item 48, wherein the host is aBacillussuch asBacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Geobacillus stearothermophilus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, orBacillus thuringiensis.53. A method of producing the polypeptide of any of items 44 to 45, comprising:(a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conductive for production of the polypeptide; and(b) recovering the polypeptide.54. A method of producing the polypeptide of any of items 44 to 45, comprising:(a) cultivating the recombinant host cell of any of items 48 to 52 under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.55. A transgenic plant, plant part or plant cell transformed with a polynucleotide encoding the polypeptide of any of items 44 to 45.56. A whole broth formulation or cell culture composition comprising a polypeptide of any of items 44 to 45. The present invention is further described by the following examples that should not be construed as limiting the scope of the invention. Examples Strains Escherichia coliTop-10 strain purchased from Invitrogen (Life Technologies, Carlsbad, CA, USA) was used to propagate our expression vectors encoding for LYS polypeptides. Aspergillus oryzaestrain MT3568 was used for heterologous expression of the LYS polypeptide encoding sequences.A. oryzaeMT3568 is an amdS (acetamidase) disrupted gene derivative ofAspergillus oryzaeJaL355 (WO 2002/40694) in which pyrG auxotrophy was restored by disrupting theA. oryzaeacetamidase (amdS) gene with the pyrG gene. The fungal strain NN044175 was isolated from soil samples collected from China, in 1998 by the dilution plate method with PDA medium, pH7, 25 C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN044175 was identified asPenicillium simplicissimum, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN053742 was isolated from a soil sample collected from Hubei province, China, in 2011 by the dilution plate method with PDA medium, at pH3, 25 C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN053742 was identified asPenicillium vasconiae, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN058285 was isolated from soil samples collected from Guizhou Province, China, in 2014 by the dilution plate method with PDA medium pH3. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN058285 was identified asTalaromyces proteolyticus, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN053333 was isolated from soil samples collected from Hunan province, China, in 2010 by the dilution plate method with PDA medium, pH7, 25 C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN053333 was identified asAspergillussp. XZ2668, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN058605 was from CBS with access number as CBS100492. The strain NN058605 was identified asPenicillium antarcticum, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN047528 was isolated from soil samples collected from China, in 1998 by the dilution plate method with YG medium, pH7, 37C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN047528 was identified asOvatospora brasiliensis, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN054749 was isolated from soil samples collected from Tibet, China, in 2012 by the dilution plate method with PDA medium, pH7, 10C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN054749 was identified asPenicillium wellingtonense, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN054129 was isolated from soil samples collected from Gotland, Sweden in 2011 by the dilution plate method with Water agar, 24C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN054129 was identified asPenicillium roseopurpureum, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN058650 was isolated from soil samples collected from Guizhou Province, China, in 2014 by the dilution plate method with PDA medium pH3. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN058650 was identified asPenicillium virgatum, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN046949 was isolated from soil samples collected from China, in 1998 by the dilution plate method with YG medium, pH7, 37C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN046949 was identified asAspergillus niveus, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN057921 was obtained through a collaboration with Professor Cai Lei in Institute of Microbiology, CAS, in 2014. The strain was collected from China. It was identified asChaetomiumsp. ZY369, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN058427 was isolated from soil samples collected from Guizhou Province, China, in 2014 by the dilution plate method with PDA medium pH3, 25C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain N NN058427 was identified asTalaromyces atricola, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN053773 was obtained through a collaboration with Institute of Microbiology, CAS, in 2011. The strain was collected from China and isolated by the dilution plate method with PDA medium pH7, 10C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN053773 was identified asTrichocladium asperum, based on both morphological characteristics and ITS rDNA sequence. The fungal strain NN058086 was isolated from soil samples collected from Guizhou Province, China, in 2014 by the dilution plate method with PDA medium pH3, 25C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN058086 was identified asMetarhizium carneum, based on both morphological characteristics and ITS rDNA sequence. StrainThielavia terrestrisstrain NRRL 8126 was purchased ATCC, and inoculated onto a PDA plate and incubated for 7 days at 37° C. in the darkness. Mycelia and spores from the plate were inoculated into 500 ml shake flasks containing 100 mls of YPG medium. The flasks were incubated for 6 days at 37° C. with shaking at 150 rpm. Media and Solutions DAP4C-1 medium was composed of 0.5 g yeast extract, 10 g maltose, 20 g dextrose, 11 g magnesium sulphate heptahydrate, 1 g dipotassium phosphate, 2 g citric acid monohydrate, 5.2 g potassium phosphate tribasic monohydrate, 1 mL Dowfax 63N10 (antifoaming agent), 2.5 g calcium carbonate, supplemented with 1 mL KU6 metal solution, and deionised water to 1000 mL. KU6 metal solution was composed of 6.8 g ZnCl2, 2.5 g CuSO4·5H2O, 0.13 g NiCl2, 13.9 g FeSO4·7H2O, 8.45 g MnSO4·H2O, 3 g C6H8O7·H2O, and deionised water to 1000 mL. YP 2% glucose medium was composed of 10 g yeast extract, 20 g Bacto-peptone, 20 g glucose, and deionised water to 1000 mL. LB plates were composed of 10 g of Bacto-tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of Bacto-agar, and deionised water to 1000 mL. LB medium was composed of 10 g of Bacto-tryptone, 5 g of yeast extract, and 10 g of sodium chloride, and deionised water to 1000 mL. COVE-Sucrose-T plates were composed of 342 g of sucrose, 20 g of agar powder, 20 mL of COVE salt solution, and deionised water to 1000 mL. The medium was sterilized by autoclaving at 15 psi for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). The medium was cooled to 60° C. and 10 mM acetamide, Triton X-100 (50 μL/500 mL) were added. COVE-N-Agar tubes were composed of 218 g Sorbitol, 10 g Dextrose, 2.02 g KNO3, 25 g agar, 50 mL Cove salt solution, and deionised water up to 1000 mL. COVE salt solution was composed of 26 g of MgSO4·7H2O, 26 g of KCL, 26 g of KH2PO4, 50 mL of COVE trace metal solution, and deionised water to 1000 mL. COVE trace metal solution was composed of 0.04 g of Na2B4O7·10H2O, 0.4 g of CuSO4·5H2O, 1.2 g of FeSO4·7H2O, 0.7 g of MnSO4·H2O, 0.8 g of Na2MoO4·2H2O, 10 g of ZnSO4·7H2O, and deionised water to 1000 mL·YPM medium contained 1% of Yeast extract, 2% of Peptone and 2% of Maltose. Example 1: Determination of Lysozyme Activity Using Reducing Ends Assay The LYS polypeptide was diluted in phosphate buffer (5 mM citrate, 5 mM K2HPO4, 0.01% TritonX-100, pH 5.0) to 50 μg/mL in polypropylene tubes. The diluted LYS polypeptide was further diluted in a 96-well polypropylene microtiter plate to a concentration of 5.0 or 0.7 μg/mL in phosphate buffer (5 mM citrate, 5 mM K2HPO4, 0.01% TritonX-100, pH 5.0). In a polypropylene deepwell plate 50 μL of the LYS polypeptide dilution was mixed with 450 μL 1%Micrococcus lysodeikticussolution (lyophilizedMicrococcus lysodeikticusATCC No. 4698 (Sigma M3770) in milli-Q water) and incubated at 40° C. with shaking (500 rpm) for 45 min. After incubation the deepwell plate was centrifuged (4000 g, 5 min) to pellet insoluble material and 100 μL of the supernatant was mixed with 50 μL 3.2M HCl in a 96-well PCR plate and incubated at 95° C. for 80 min. 50 μL of 3.5 M NaOH was added to each well of the PCR plate, and 150 μL of each sample was transferred to a new PCR plate containing 75 μL/well 4-hydroxybenzhydrazide solution in K—Na tartrate/NaOH buffer (50 g/L K—Na tartrate+20 g/L NaOH). The plate was incubated at 95° C. for 10 min before 100 μL/sample was transferred to a clear flat-bottomed microtiter plate for optical density (OD) measurement at 405 nm. OD measurements were performed on three times diluted samples (50 μL sample diluted in 100 μL in Milli-Q water). Example 2: Determination of Lysozyme Activity Using OD Drop Assay Freeze-driedMicrococcus lysodeikticusATCC No. 4698 (Sigma) was washed and suspended in 60 mM KH2PO4buffer at pH6.0 with final concentration of 1% (w/v) as substrate stock. The concentration of the strain was adjusted by adding citric acid-Na2HPO4buffer until OD450 reach approximately 1. Citric acid-Na2HPO4pH4 buffer were prepared by adding 61.45 ml 0.1M citric acid and 38.55 ml 0.2M Na2HPO4for pH4. 20 μL enzyme at 50 μg/mL and 200 μL of diluted bacterial strain solution in citric acid-Na2HPO4buffer at pH4 were added to a 96 well plate, mixed and the OD450 was read. Then the plate was incubated at 37° C., 300 rpm for 1 hour and the OD450 was read. The OD difference between the 1 hour time point to the initial read showed the OD drop activity for the LYS polypeptide. Blank was set by adding 20 ul MQ water or the corresponding buffer, and each sample was measured in triplicate. Example 3: Genomic DNA Extraction Penicillium simplicissimumstrain NN044175 was inoculated onto a PDA plate and incubated for 7 days at 25° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 9 days at 25° C. with shaking at 160 rpm. Penicillium vasconiaestrain NN053742 was inoculated onto a PDA plate and incubated for 7 days at 25° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 3 days at 25° C. with shaking at 160 rpm. Talaromyces proteolyticusstrain NN058285 was inoculated onto a PDA plate and incubated for 7 days at 25° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25° C. with shaking at 160 rpm. Aspergillussp. strain NN053333 was inoculated onto a PDA plate and incubated for 7 days at 25° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 5 days at 25° C. with shaking at 160 rpm. Penicillium antarcticumstrain NN058605 was inoculated onto a PDA plate and incubated for 7 days at 25° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25° C. with shaking at 160 rpm. Ovatospora brasiliensisstrain NN047528 was inoculated onto a PDA plate and incubated for 7 days at 37° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 2 days at 37° C. with shaking at 160 rpm. Penicillium wellingtonensestrain NN054749 was inoculated onto a PDA plate and incubated for 7 days at 25° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 11 days at 25° C. with shaking at 160 rpm. Penicillium roseopurpureumstrain NN054129 was inoculated onto a PDA plate and incubated for 7 days at 25° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25° C. with shaking at 160 rpm. Penicillium virgatumstrain NN058650 was inoculated onto a PDA plate and incubated for 7 days at 25° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25° C. with shaking at 160 rpm. Aspergillus niveusstrain NN046949 was inoculated onto a PDA plate and incubated for 7 days at 25° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 3 days at 25° C. with shaking at 160 rpm. Chaetomiumsp. strain NN057921 were inoculated onto a PDA plate and incubated for 7 days at 37° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 8 days at 37° C. with shaking at 160 rpm. The mycelia ofPenicillium antarcticumstrain NN058605 were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using MP Fast DNA spin kit for soil (MP Biomedicals, Santa Ana, California, USA) following the manufacturer's instruction. Talaromyces atricolastrain NN058427 was inoculated onto a PDA plate and incubated for 7 days at 25° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 3 days at 25° C. with shaking at 160 rpm. Trichocladium asperumstrain NN053773 was inoculated onto a PDA plate and incubated for 7 days at 15° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 15° C. with shaking at 160 rpm. Metarhizium carneumstrain NN058086 was inoculated onto a PDA plate and incubated for 7 days at 25° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25° C. with shaking at 160 rpm. The mycelia ofThielavia terrestriswere collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer's instructions. The mycelia of all the other strains were collected by filtration through MIRACLOTH® and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer's instruction. Example 4: Genome Sequencing, Assembly and Annotation The extracted genomic DNA samples ofPenicillium simplicissimumstrain NN044175 were delivered to Exiqon A/S (Denmark) for genome sequencing using an ILLUMINA® MiSeq System (Illumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program ldba (Peng, Yu et al., 2010, Research in Computational Molecular Biology,6044:426-440. Springer Berlin Heidelberg). The extracted genomic DNA samples ofTalaromyces proteolyticusstrain NN058285, Penicillium antarcticumstrain NN058605,Penicillium roseopurpureumstrain NN054129,Penicillium virgatumstrain NN058650,Aspergillus niveusstrain NN046949, Metarhizium carneumstrain NN058086 were delivered to Exiqon A/S for genome sequencing using an ILLUMINA® MiSeq System. The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et al., 2012, Journal of Computational Biology,19(5): 455-477). The extracted genomic DNA samples ofPenicillium vasconiaestrain NN053742, Ovatospora brasiliensisstrain NN047528, Trichocladium asperumstrain NN053773 were delivered to Fasteris (Switzerland) for genome sequencing using an ILLUMINA® HiSeq 2000 System (Illumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program ldba. The extracted genomic DNA samples ofAspergillussp. strain NN053333,Chaetomiumsp. strain NN057921 andTalaromyces atricolastrain NN058427 were delivered to Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System. The raw reads were assembled at Novozymes Denmark using program Spades. The extracted genomic DNA samples ofPenicillium wellingtonensestrain NN054749 were delivered to Novozymes Davis for genome sequencing using an ILLUMINA® MiSeq System. The raw reads were assembled at Novozymes Denmark using program ldba. The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et al., 2008, Genome Research18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.10 (Altschul et al., 1990, Journal of Molecular Biology.215(3): 403-410, ftp://ftp.ncbi.nlm.nih.gov/blast/executables/release/2.2.10/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The NZ5 family was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics7: 263) and SignalP program (Nielsen et al., 1997, Protein Engineering10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends in Genetics. 16(6): 276-277) was used to predict isoelectric points and molecular weights. Example 5: Cloning, Expression and Fermentation of Fungal NZ5 Genes (SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37 and 40) Fourteen fungal LYS wild type genes, LYS_Pesi (SEQ ID NO:1), LYS_Pv (SEQ ID NO:4), LYS_Tapr (SEQ ID NO:7), LYS_Asp2668 (SEQ ID NO:10), LYS_Pean (SEQ ID NO:13), LYS_chbr (SEQ ID NO:16), LYS_Pewe (SEQ ID NO:19), LYS_Pr (SEQ ID NO:22), LYS_Pevir (SEQ ID NO:25), LYS_asni (SEQ ID NO:28), LYS_ch369 (SEQ ID NO: 31), LYS_Taat (SEQ ID NO:34), LYS_Tras (SEQ ID NO: 37), LYS_Meca2 (SEQ ID NO:40) were cloned fromPenicillium simplicissimumstrain NN044175, Penicillium vasconiaestrain NN053742,Talaromyces proteolyticusstrain NN058285,Aspergillussp. strain NN053333,Penicillium antarcticumstrain NN058605, Ovatospora brasiliensisstrain NN047528, Penicillium wellingtonensestrain NN054749, Penicillium roseopurpureumstrain NN054129, Penicillium virgatumstrain NN058650,Aspergillus niveusstrain NN046949,Chaetomiumsp. strain NN057921, Talaromyces atricolastrain NN058427, Trichocladium asperumstrain NN053773, Metarhizium carneumstrain NN058086 respectively. The fungal LYS genes were cloned into anAspergillus oryzaeexpression vector pCaHj505 as described in WO2013029496. The transcription of the LYS coding sequence with the native secretion signal was under the control of anAspergillus oryzaealpha-amylase gene promoter. The final expression plasmids, p505-LYS_Pesi, p505-LYS_Pv, p505-LYS_Tapr, p505-LYS_Asp2668, p505-LYS_Pean, p505-LYS_chbr, p505-LYS_Pewe, p505-LYS_Pr, p505-LYS_Pevir, p505-LYS_asni, p505-LYS_ch369, p505-LYS_Taat, p505-LYS_Tras and p505-LYS_Meca2, were individually transformed into anAspergillus oryzaeexpression host. The LYS genes were integrated by homologous recombination into theAspergillus oryzaehost genome upon transformation. Four transformants of each transformation were selected from the selective media agar plate and inoculated to 3 ml of YPM or Dap4C medium in 24-well plate and incubated at 30° C., 150 rpm. After 3 days incubation, 20 μl of supernatant from each transformant were analyzed on NuPAGE Novex 4-12% Bis-Tris Gel w/MES according to the manufacturer's instructions. The resulting gel was stained with Instant Blue. SDS-PAGE profiles of the cultures showed that all genes were expressed with 1 protein band detected at approximately 28 kDa, 25 kDa, 25 kDa, 35 kDa, 25 kDa, 25 kDa, 25 kDa, 25 kDa, 25 kDa, 25 kDa, 25 kDa, 25 kDa, 25 kDa, 30 kDa. The recombinantAspergillus oryzaestrains with the strongest protein band were selected for shaking flask culturing. The recombinant strains were inoculated on slant made of slant medium and incubated at 37C for 6-7 days. When strains were well grown to fully sporulated, they were inoculated to 2 L shaking flasks each containing 400 ml of YPM or DAP4C, 5-6 flasks for each strain. Flasks were shaking at 80 rpm, 30C. Cultures were harvested on day 3 or day 4 and filtered using a 0.22 μm DURAPORE Membrane and were purified as described in example 9. Example 6: Construction of the Improved Split-MarkerAspergillus oryzaeHost An improvedAspergillus oryzaehost/vector system comparable to the one described in example 5 disclosed in WO 2016026938A1 was constructed. The improvement was made to reduce the size of the transforming DNA by moving the FLPase expression cassette located on PART-11 of the plasmid pDAu724 (see page 34 in WO2016/026938,FIG.7and SEQ ID NO:30) to the integration locus amy2 in the genome of the host strain. The cloning of the FLPase expression cassette into pDAu703 (WO2016/026938 page 32 andFIG.6and SEQ ID:29) in done by amplification of the FLPase expression cassette from pDAu724 and cloning in between FRT-F3 and the amdS selection marker of pDAu703 to give the plasmid pDAu770 (FIG.1, SEQ ID NO: 320). The same protocol as described in WO2016/026938 page 33 was used to transform the linearized plasmid pDAu770 into protoplasts ofA. oryzaestrain Jal1338 (disclosed in WO2012/160097). Transformants were selected on AmdS selection plates to obtain strain DAu785. The resulting recombinant host strain DAu785 has a modified amy2 locus comparable to the one in DAU716 (WO2016/026938) with the addition of the FLPase expression cassette (FIG.2, top panel). The host strain DAu785 is now constitutively expressing the FLPase site specific recombinase allowing the integration at the FRT sites of the transforming DNA in this case the PCR fragments obtained by Overlap Extension PCR reaction (FIG.2, middle and bottom panels) and described in Example 7. Example 7: Overlap Extension PCR Cloning (SEQ ID NO: 43) A PCR amplification of SEQ ID NO: 43 encoding the LYS polypeptide was carried out using Phusion High-Fidelity DNA polymerase (New England Biolabs, BioNordika Denmark A/S, Herlev, Denmark) in a 50 μL volume reaction and the primers disclosed in table 2. TABLE 2PCR primersPrimerSEQPrimer*ID NO:SequenceKKSC0972-3215'-CTATATACACAACTGGGGATCCACCFATGCAGCTCTCCCTCCTCGTKKSC0972-3225'-TAGAGTCGACCCAGCCGCGCCGGCCARTTACAACCCACCAGCCTGGC*-F-forward primer; -R-reverse primer; Bold letters represent coding sequence. The PCR reaction mix consisted of 10 μL Phusion reaction buffer HF (5×); 1 μL of PCR nucleotide Mix (10 mM); 2 μL forward cloning primers (2.5 mM); 2 μL reverse cloning primers (2.5 mM); 1 μL Phusion High-Fidelity DNA Polymerase #M0530L (2000U/mL); and PCR grade water up to 50 μL. PCR reaction was incubated on a thermocycler T100 (Biorad, Hercules, California, USA) using the following program: initial denaturation of 2 min at 98° C. followed by 30 cycles of 10 sec at 98° C., 2 min at 72° C. and ending up by a final elongation of 10 min at 72° C. The PCR amplicon was purified using AM Pure XP beads system kit (Agencourt, Beverly, Massachusetts, USA) adapted on a Biomek FXp Liquid handler (Beckman Coulter, Brea, California, USA). pDAu724 plasmid was used as DNA template to amplify two PCR products (F1 and F3) in reactions composed of 10 μL of KAPA polymerase buffer 5×, 1 μL 10 mM KAPA PCR Nucleotide Mix, 1 μL of 10 μM of the appropriate forward primers (SEQ ID NO: 323 for F1 and SEQ ID NO: 325 for F3, table 3), 1 μL of 10 μM of the appropriate reverse primers (SEQ ID NO: 324 for F1 and SEQ ID NO: 326 for F3, table 3), 1 to 10 ng of pDAu724 plasmid, 1 μL of KAPA Biosystems polymerase KK2502 (1unit) and PCR-grade water up to 50 μL. PCR amplification reactions were carried out on a DYAD® Dual-Block Thermal Cycler (MJ Research Inc., Waltham, MA, USA) programmed for 2 min. at 98° C. and followed by 35 cycles of 10 sec. at 98° C. and 2 min. at 72° C. and one final cycle of 10 min. at 72° C. Five μl of the PCR reaction were analyzed by 1% agarose gel electrophoresis using TAE buffer where DNA bands of the appropriate size were observed. The remaining PCR reactions were purified using an ILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to the manufacturer's instructions. TABLE 3PCR primersPrimerSEQPrimerID NO:SequenceForward323GAATTCGAGCTCGGTACCTTGAAGTTCprimer F1Reverse324GGTGGATCCCCAGTTGTGTATATAGAGGATTprimer F1Forward325TGCGCGGCGCGGCTGGGTCGACTCTAprimer F3Reverse326TTCACACAGGAAACAGCTATGACCATGprimer F3 Overlap Extension PCR reaction for cloning the LYS polypeptide gene amplified fromThielavia terrestrisgDNA was composed of 10 μL KAPA polymerase buffer (5×), 1 μL 10 mM KAPA PCR Nucleotide Mix, 50 ng of PCR fragment F1 and equimolar amounts of PCR fragment F3 and LYS polypeptide gene encoding for SEQ ID NO: 45, 1 μl KAPA Biosystems polymerase KK2502 (1unit) and PCR-grade water up to 48 μL. Reaction was incubated on a DYAD® Dual-Block Thermal Cycler (MJ Research Inc., Waltham, MA, USA) using a program composed of 2 min. at 98° C.; followed by 5 cycles each composed of 10 sec. at 98° C., 30 sec. at 68° C., and 5 min. at 72° C. and completed by a final extension of 8 min. at 72° C. During the OE PCR reaction, annealing between fragment F1 and the LYS polypeptide gene encoding for SEQ ID NO: 45 was ensured by the overlap in SEQ ID NO: 327 included in the forward cloning primer (KKSC0972-F) and annealing between fragment F3 and the LYS polypeptide gene encoding for SEQ ID NO: 45 was ensured by the overlapping SEQ ID NO: 328 included in the reverse cloning primer (KKSC0972-R). One μL of 10 mM primer SEQ1 and 1 μL of 10 mM primer SEQ4 were added to the OE PCR reaction and the reaction was incubated a second time on a DYAD® Dual-Block Thermal Cycler (MJ Research Inc., Waltham, MA, USA) using a program composed of 2 min at 98° C.; followed by 25 cycles each composed of 10 sec. at 98° C., and 4 min. at 72° C. and completed by a final extension of 10 min. at 72° C. Five μl of the PCR reaction was analysed by 1% agarose gel electrophoresis using TAE buffer where an DNA band of the appropriate size was observed. The remaining PCR reaction was up-concentrated to 20 μL by heating the tube at 60° C. 10 μL of this reaction was used forAspergillus oryzaeDAu785 protoplasts transformation. Primer bind forward SEQ ID NO: 327:CTATATACACAACTGGGGATCCACCPrimer bind reverse SEQ ID NO: 328:TAGAGTCGACCCAGCCGCGCCGGCCA Example 8: Preparation ofAspergillusprotoplasts Protoplasts ofAspergillus oryzaeMT3568 were prepared according to WO 95/002043. One hundred μl of protoplasts were mixed with OE PCR fragment KKSC0972 and 250 μL of 60% PEG 4000 (Applichem, Darmstadt, Germany) (polyethylene glycol, molecular weight 4,000), 10 mM CaCl2, and 10 mM Tris-HCl pH 7.5 and gently mixed. The mixtures were incubated at 37° C. for 30 minutes and the protoplasts were spread onto COVE plates for selection. After incubation for 4-7 days at 37° C. spores of four transformants were inoculated into 0.2 mL of YP+2% glucose or DAP4C-1 medium in 96 well microtiter plates. After 4 days cultivation at 30° C., the culture broths were analysed by SDS-PAGE to identify transformants producing the highest amounts of LYS polypeptide. Spores of the best transformant from the transformation were spread onto COVE plates containing 0.01% TRITON® X-100 in order to isolate single colonies. The spreading was repeated twice in total on COVE plates containing 10 mM sodium nitrate. Spores were then inoculated into 500 mL shake flasks containing 100 mL of YP+2% glucose and incubated for 4 days at 30° C. with shaking at 100 rpm. Culture broths were harvested by filtration using a 0.2 μm filter device and purified as described in Example 9. Example 9: Purification of LYS Polypeptides Activity Detection for Purification Procedure Freeze-dried bacterial strainsMicrococcus lysodeikticusATCC No. 4698 (Sigma) andExiguobacteriumsp. (isolated from soil) were separately washed and suspended in 60 mM KH2PO4buffer at pH6.0 with final concentration of 1% (w/v) as substrate stock. Before activity detection, the concentration of substrate was diluted into 0.035% which correlates to OD450 about 0.7 by 60 mM KH2PO4 buffer at pH6.0 or pH 4.0. 10 ul of polypeptide sample (or 5 ul of sample with 5 ul of MQ water if containing high concentration of salt) and 190 ul of 0.035% substrate were added into 96-well plate, and then read OD450. The plate was incubated for 30 or 60 minutes, 300 rpm at room temperature or 37° C. in the thermomixer. The plate was shaked 10 seconds and read OD450 again. The OD drop showed lysozyme activity. Blank is added 10 μl of 60 mM KH2PO4at pH 6.0 or pH4.0 buffer, and each sample was measured in duplicate if necessary. Purification of SEQ ID NO: 3 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20 mM NaAc at pH4.5. The solution was filtered with 0.45 um filter and then loaded into SP Fast Flow column (GE Healthcare) equilibrated with 20 mM NaAc at pH4.5. A gradient increase of NaCl concentration was applied as elution buffer from zero to 1M, and then the elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, and then concentrated for further evaluation. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 6 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then the precipitation was added water to adjust conductance to about 140 mS/cm. The solution was filtered with 0.45 um filter and then loaded into HIC High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH8.0 with 1.2M (NH4)2SO4added. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.2M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The flow-through and Fractions from 1 to 15 were collected and conductance was adjusted to 180 mS/cm, then reloaded into HIC column equilibrated with 20 mM PBS at pH8.0 with 1.8M (NH4)2SO4added. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.8M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, pooled together, and diafiltrated with 20 mM PBS at pH6.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 9 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then the precipitation was added water to adjust conductance to about 170 mS/cm. The solution was filtered with 0.45 um filter and then loaded into HIC High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.5M (NH4)2SO4added. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.5M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, pooled together, and diafiltrated with 20 mM PBS at pH6.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 12 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then the precipitation was added water to adjust conductance to about 185 mS/cm. The solution was filtered with 0.45 um filter and then loaded into HIC High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH6.0 with 1.8M (NH4)2SO4added. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.8M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, pooled together, and diafiltrated with 20 mM Bis-Tris at pH6.0. The sample was loaded into a Mono Q column (GE Healthcare) equilibrated with 20 mM Bis-Tris at pH6.0. A gradient increase of NaCl concentration was applied as elution buffer from zero to 1M, and then the elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, and then concentrated for further evaluation. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 15 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then the precipitation was added water to adjust conductance to about 170 mS/cm. The solution was filtered with 0.45 um filter and then loaded into Phenyl Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH6.0 with 1.5M (NH4)2SO4added. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.5M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The flow-through and Fractions with lysozyme activity were collected and conductance was adjusted to 190 mS/cm, then reloaded into HIC column equilibrated with 20 mM PBS at pH6.0 with 1.5M (NH4)2SO4added again. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.5M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, pooled together, and diafiltrated with 20 mM PBS at pH6.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 18 The culture supernatant from the expression of LYS_chbr (SEQ ID NO:16) was firstly precipitated with ammonium sulfate (80% saturation), then the precipitation was added water to adjust conductance to about 170 mS/cm. The solution was filtered with 0.45 um filter and then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH6.0 with 1.8M (NH4)2SO4added. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.8M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, and fractions were pooled together, and diafiltrated with 20 mM PBS at pH6.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Analysis by intact molecular weight (MAXIS II electrospray mass spectrometer (Bruker Daltonik GmbH, Bremen, DE)) showed that the major product corresponded to amino acids 1 to 230 of SEQ ID NO: 18 (detected mass 24128.35 Da, predicted mass 24128.21 Da) with a minor product corresponded to amino acids 4 to 230 of SEQ ID NO: 18 (detected mass 23768.79 Da, predicted mass 23768.16 Da). Purification of SEQ ID NO: 329 The culture supernatant from the expression of LYS_chbr (SEQ ID NO:16) was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20 mM PBS at pH6.5. The solution was filtered with 0.45 um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20 mM PBS at pH6.5. A gradient increase of NaCl concentration was applied as elution buffer from zero to 1M, and then the elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, and then concentrated for further evaluation. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Analysis by N-terminal sequencing (Applied Biosystems Precise Amino Acid Sequencer Model 494) and intact molecular weight (MAXIS II electrospray mass spectrometer (Bruker Daltonik GmbH, Bremen, DE)) showed that the N-terminal LED domain had been cleaved off leaving the LAD catalytic domain and that the molecule had a heterogeneous N-terminal (see table 4). The major product corresponded to residues 85-230 which is disclosed as SEQ ID NO: 329. TABLE 4N-terminal and intact molecular weigh determinationIntact Molecular WeightApplied BiosystemsResidues ofN-terminal sequenceSEQ ID NO: 18M.Wt CalculatedM.Wt. ObservedIDGNLPGLN88-23015167.31 Da15167.92 DaOKGKGNLPG86-23015352.54 Da15353.04 DaOKGGKGNLP85-23015409.59 Da15410.06 DaOK Purification of SEQ ID NO: 21 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then the precipitation was added water to adjust conductance to about 140 mS/cm. The solution was filtered with 0.45 um filter and then loaded into Phenyl Fast Flow column (GE Healthcare) equilibrated with 20 mM NaAc at pH4.5 with 2M NaCl added. A gradient decrease of NaCl concentration was applied as elution buffer from 2M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The flow-through and Fractions with lysozyme activity were collected and conductance was adjusted to 180 mS/cm, then reloaded into HIC column equilibrated with 20 mM NaAc at pH4.5 with 4M NaCl added again. A gradient decrease of NaCl concentration was applied as elution buffer from 4M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity and unbound sample were analyzed by SDS-PAGE, pooled together, and concentrated. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 24 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then the precipitation was added water to adjust conductance to about 170 mS/cm. The solution was filtered with 0.45 um filter and then loaded into HIC High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH6.0 with 4M NaCl added. A gradient decrease of NaCl concentration was applied as elution buffer from 4M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The flow-through and unbound sample with lysozyme activity were collected and conductance was adjusted to 190 mS/cm, then reloaded into HIC column equilibrated with 20 mM PBS at pH6.0 with 1.8M (NH4)2SO4added again. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.8M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, pooled together, and diafiltrated with 20 mM PBS at pH6.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 27 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20 mM NaAc at pH5.5. The solution was filtered with 0.45 um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20 mM NaAc at pH5.5. A gradient increase of NaCl concentration was applied as elution buffer from zero to 1M, and then the elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE. The fractions with lysozyme activity were pooled and concentrated, but degradation of sample was found. The conductance of sample was adjusted to 200 mS/cm, then reloaded into Phenyl High Performance column equilibrated with 20 mM PBS at pH6.0 with 2.0M (NH4)2SO4added again. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 2.0M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, pooled together, and diafiltrated with 20 mM PBS at pH6.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 30 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20 mM NaAc at pH4.5. The solution was filtered with 0.45 um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20 mM NaAc at pH4.5. A gradient increase of NaCl concentration was applied as elution buffer from zero to 1M, and then the elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, and then concentrated for further evaluation. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 33 The culture supernatant of 033X73 was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20 mM NaAc at pH4.5. The solution was filtered with 0.45 um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20 mM NaAc at pH4.5. A gradient increase of NaCl concentration was applied as elution buffer from zero to 1M, and then the elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, and then concentrated for further evaluation. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 36 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20 mM PBS at pH7.0. The solution was filtered with 0.45 um filter and then loaded into Capto Q column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0. A gradient increase of NaCl concentration was applied as elution buffer from zero to 1M, and then the elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE. The flow-through fraction with lysozyme activity was picked up for further purification. The pH of flow-through fraction was adjusted to pH4.5, then reloaded into Capto SP column equilibrated with 20 mM NaAC at pH4.5. A gradient increase of NaCl concentration was applied as elution buffer from zero to 1M, and then the elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, and pooled together. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 39 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then the precipitation was added water to adjust conductance to about 170 mS/cm. The solution was filtered with 0.45 um filter and then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH6.0 with 1.5M (NH4)2SO4added. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.5M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The lysozyme activity still was found in flow-through fraction and fractions 1 to 12, and they were pooled together for further purification. The conductance of samples with lysozyme activity was adjusted to 190 mS/cm, then reloaded into Phenyl Sepharose High Performance column equilibrated with 20 mM PBS at pH6.0 with 1.8M (NH4)2SO4added again. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.8M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, pooled together, and diafiltrated with 20 mM PBS at pH6.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of SEQ ID NO: 42 The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then the precipitation was added water to adjust conductance to about 170 mS/cm. The solution was filtered with 0.45 um filter and then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH6.0 with 1.5M (NH4)2SO4added. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.5M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE, but with two bands found. The fractions with lysozyme activity were pooled together for further purification. The conductance of the fractions was adjusted to 140 mS/cm, then reloaded into Phenyl Sepharose High Performance column equilibrated with 20 mM PBS at pH6.0 with 1.2M (NH4)2SO4added again. A gradient decrease of (NH4)2SO4concentration was applied as elution buffer from 1.2M to zero, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were analyzed by SDS-PAGE. Fractions 29 to 37 have lower molecular weight, were pooled together, and diafiltrated with 20 mM PBS at pH6.0. Fraction 43 to 45 have higher molecular weight, were pooled together, and diafiltrated with 20 mM PBS at pH6.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Analysis by N-terminal sequencing (Applied Biosystems Precise Amino Acid Sequencer Model 494) showed that the product began with the N-terminal sequence YPIKDNN, corresponding to amino acids 1 to 7 of SEQ ID NO: 42. Analysis by intact molecular weight (MAXIS II electrospray mass spectrometer (Bruker Daltonik GmbH, Bremen, DE)) showed that the major product corresponded to amino acids 1 to 304 of SEQ ID NO: 42 (detected mass 31755.59 Da, predicted mass 31754.97 Da). There was also a small amount of a secondary product corresponding to amino acids 76 to 304 of SEQ ID NO: 42 (detected mass 23617.23 Da, predicted mass 23617.15 Da) due to the first LED domain being cleaved off the N-terminal. Purification of SEQ ID NO: 45 The fermentation supernatant with the lysozyme was filtered through a Fast PES Bottle top filter with a 0.22 μm cut-off. 250 ml filtered fermentation samples was diluted with 250 ml MilliQ water and pH was adjusted to 4.5. The lysozyme containing solution was purified by chromatography on Capto S, approximately 30 ml in a XK16 column, using as buffer A 50 mM Na-acetate pH 4.5, and as buffer B 50 mM Na-acetate+2 M NaCl pH 4.5 using a 0-100% gradient over ca. 10CV. The fractions from the column were pooled based on the chromatogram (absorption at 280 and 254 nm) and SDS-PAGE analysis. The molecular weight was estimated to 25 kDa from SDS-PAGE and the purity was >90%. Example 10: Method of Determining the LAD Catalytic Domain by HMM SEQ ID NOs: 46 to 187 were aligned using the software program MUSCLE v3.8.31 with the default settings. Using this alignment, the HMM was constructed using the software program ‘hmmbuild’ from the package HMMER 3.0 (March 2010) (http://hmmer.org/) and the software was invoked using the default settings by the command: hmmscan3—tblout output.dat model.hmm sequences.fasta. The LAD catalytic domain HMM profile thereby generated for subsequent loading into the software program ‘hmmscan’ is given below. HMMER3/b [3.0 | March 2010]NAME LAD catalytic domainLENG 136ALPH aminoRF noCS noMAP yesDATE Fri Apr 21 12:03:08 2017NSEQ 142EFFN 1.547058CKSUM 201442427STATS LOCAL MSV −10.1515 0.71110STATS LOCAL VITERBI −10.6276 0.71110STATS LOCAL FORWARD −4.1803 0.71110HMM A C D E F G H I K L M NP Q R S T V W Ym−>m m−>i m−>d i−>m i−>i d−>m d−>dCOMPO 2.28000 4.46955 2.96306 2.70047 3.44014 2.89264 3.73492 2.959022.72837 2.64684 3.53697 3.08243 3.38858 2.79348 2.98339 2.54635 2.85094 2.678604.50931 3.453442.68610 4.42256 2.77533 2.73152 3.46377 2.40496 3.72526 3.293722.67763 2.69331 4.24673 2.90332 2.73683 3.18173 2.89805 2.37875 2.77520 2.985324.58508 3.615120.86176 1.29948 1.18774 1.49367 0.25431 0.00000 *1 2.70450 4.96091 2.44483 2.25748 4.38595 1.70411 3.76708 3.831552.65982 3.40989 4.23378 2.69810 3.83638 2.89968 3.15311 2.53822 2.77031 3.446135.62694 4.24971 17 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.02516 4.09093 4.81328 0.61958 0.77255 0.70021 0.686132 2.95798 4.37838 4.41979 3.85216 2.84679 4.06442 4.15724 2.492603.70306 1.06749 3.04032 4.02131 4.37743 3.87638 3.84752 3.36755 3.18313 2.464093.85059 2.85522 18 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.02269 4.19301 4.91535 0.61958 0.77255 0.78684 0.607493 2.64712 5.07695 2.31050 2.33245 4.40298 2.91636 3.64509 3.865962.37831 3.34917 4.16311 2.30868 3.81995 2.76486 2.86800 2.40011 2.43728 3.465755.56134 4.16658 19 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.02259 4.19722 4.91957 0.61958 0.77255 0.69335 0.692944 2.14335 5.06169 2.79203 2.15230 4.37029 3.13800 3.41689 3.831592.36004 3.35168 4.11530 2.86703 3.32033 2.51530 2.75969 2.34749 2.87496 3.401785.51777 4.12854 20 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.02150 4.24624 4.96858 0.61958 0.77255 0.57423 0.828135 2.32752 4.64440 3.12310 2.59357 3.83630 3.40168 3.74641 3.186482.50736 2.57706 3.73330 3.10307 3.85311 2.95065 2.73902 2.58368 2.21457 2.535795.18615 3.89627 21 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01979 4.32837 5.05072 0.61958 0.77255 0.64493 0.743806 2.80872 5.24372 2.92193 2.38964 4.59162 3.53977 3.72591 4.020132.14929 3.51532 4.32009 2.91909 3.77704 1.36571 2.64059 2.86001 3.11361 3.644125.64559 4.31503 22 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01973 4.33131 5.05366 0.61958 0.77255 0.64820 0.740217 2.62542 4.63457 2.55319 2.60028 3.82280 3.52902 3.75230 3.038152.62398 2.57975 2.90066 3.12772 3.91645 2.93691 3.05484 2.18187 2.58195 2.788755.17871 3.89049 23 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01973 4.33131 5.05366 0.61958 0.77255 0.62772 0.763158 2.35670 5.14104 2.59023 2.29666 4.47219 2.93010 3.62820 3.947102.04764 3.43912 4.18729 2.84545 3.83264 2.55019 2.41265 2.56554 2.74894 3.524575.58079 4.17986 24 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01956 4.34008 5.06243 0.61958 0.77255 0.52419 0.896579 2.70329 4.59946 3.33128 2.77671 3.58063 3.60472 2.72765 2.940062.73677 2.77463 3.69458 2.14343 3.98982 3.07351 3.13881 2.83571 2.93476 2.525315.01301 2.57944 25 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01861 4.38940 5.11175 0.61958 0.77255 0.57190 0.8311510 0.58061 4.39851 3.89198 3.70803 4.66940 3.05741 4.71068 4.002803.76833 3.77981 4.64326 3.71756 3.68666 4.04155 4.00008 2.69179 3.01786 3.441766.02272 4.86740 26 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01861 4.38940 5.11175 0.61958 0.77255 0.57190 0.8311511 2.28535 5.06565 2.88471 2.36748 4.19761 3.27471 3.62729 3.533222.10760 3.18026 4.11808 2.80247 3.71214 2.51474 2.31369 2.57926 2.86084 3.387945.52458 3.98224 27 - 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-2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510111 1.15417 2.40052 3.43268 3.31342 3.94825 3.05617 4.23524 3.291343.27802 2.88728 3.90490 3.55144 3.99881 3.56368 3.59756 2.72050 2.93186 2.964895.37506 4.15892 190 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510112 3.26861 5.44238 2.97663 2.64398 4.75892 3.69012 4.06288 4.301482.66362 3.78982 4.71915 3.28643 4.23921 0.76296 2.96287 3.24618 3.53605 3.964095.86880 4.56259 191 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510113 1.91522 4.95047 2.97467 2.39757 4.20895 3.50202 3.70112 3.613092.14870 3.19155 3.18272 3.01748 3.89338 2.73359 2.84536 2.53242 2.55518 3.293915.43928 4.09037 192 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510114 3.29634 4.59962 5.14480 4.67781 3.93848 4.66858 5.40358 1.713824.56710 2.45181 3.72266 4.84032 5.02031 4.85191 4.75526 4.08284 3.33697 0.676555.91411 4.69288 193 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510115 3.15567 5.44348 2.80271 2.49407 4.78415 3.60247 3.94943 4.289792.58206 3.78846 4.65575 2.96667 4.14360 0.91366 2.91447 3.11238 3.41462 3.918705.87574 4.52658 194 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510116 2.71563 4.90409 3.12545 2.55928 4.14921 2.85289 3.44680 3.058622.22913 3.15932 3.97583 3.07155 3.92680 2.53911 2.16870 2.75050 2.94327 2.446865.39558 4.07226 195 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510117 2.19207 4.40484 3.86126 3.64147 4.76639 3.17370 4.68117 4.153673.70231 3.88127 4.70113 3.67352 3.97014 3.97418 3.96685 0.66193 2.77617 3.522346.10860 4.92572 196 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510118 1.23716 4.99590 2.82777 2.30137 4.34451 3.20483 3.85483 3.768232.68928 3.36557 4.18982 3.06501 3.97138 2.91804 3.03321 2.75915 3.06515 3.418935.59981 4.25545 197 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510119 2.63152 4.27604 4.03648 3.47068 1.69703 3.81555 3.85315 2.684133.36748 2.44928 3.39118 3.31828 4.18646 3.53607 3.59165 3.09668 2.86423 2.469014.64999 2.19303 198 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510120 3.10050 4.93865 3.89438 3.80685 5.08138 2.93213 4.95457 4.685243.99850 4.30514 5.22080 4.02495 0.41952 4.31180 4.23729 3.27299 3.60547 4.111296.18106 5.18331 199 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510121 2.63821 5.16765 1.71131 2.06185 4.48005 3.20764 3.58490 3.948542.45253 3.45931 4.22038 2.80491 3.88069 2.79852 2.94579 2.60137 2.82264 3.541454.92801 3.93756 200 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510122 1.86376 4.90237 3.28241 2.68163 4.15144 3.62338 3.62925 3.542902.36576 2.72221 3.98901 3.18063 4.00712 2.90768 1.74335 2.86644 3.03069 3.245615.38278 4.04754 201 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510123 4.19177 5.36263 4.97401 4.78151 2.11394 4.76326 3.76878 3.893684.60093 3.15403 4.48829 4.44462 5.10497 4.55202 4.59461 4.21077 4.43309 3.830393.88657 0.47676 202 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510124 1.79635 5.14532 2.26839 2.39026 4.45744 3.24099 3.34639 3.840572.35322 3.43625 4.19753 2.81236 3.87618 2.60394 2.87095 2.67589 2.92156 3.518695.59711 4.13300 203 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.02128 4.45077 4.66879 0.61958 0.77255 0.48576 0.95510125 2.49141 5.20122 2.74864 2.18402 4.54180 3.39680 3.60493 4.018981.78979 3.49991 4.24589 2.89607 3.84714 2.16259 2.63607 2.67773 2.87195 3.590045.63139 4.23074 204 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.02321 4.44704 4.48894 0.61958 0.77255 0.49180 0.94553126 2.70259 4.78288 3.10316 2.62084 3.23014 3.55717 2.77689 3.393282.58605 3.02278 3.86371 3.04868 3.94378 2.53415 2.11061 2.76991 2.93283 3.101803.35180 3.35576 205 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01765 4.44148 5.16383 0.61958 0.77255 0.50070 0.93168127 2.64699 4.39217 3.56865 2.05763 3.29481 3.66295 3.91739 2.605382.95345 2.50897 2.59486 3.40402 4.04141 3.00636 3.30409 2.90867 2.78869 2.225614.97634 3.56924 206 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01765 4.44148 5.16383 0.61958 0.77255 0.50070 0.93168128 2.15651 5.08647 2.66218 2.31635 4.38993 2.79836 3.66501 3.847432.38128 3.37411 4.13923 2.89371 2.92678 2.67029 2.83170 2.46722 2.70744 3.454744.50984 4.16121 207 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01765 4.44148 5.16383 0.61958 0.77255 0.50070 0.93168129 2.47139 5.13557 2.73837 1.94974 4.45770 3.46490 3.60456 3.856232.21863 2.88369 4.18268 2.94198 3.82238 2.40411 2.53877 2.63956 2.79959 3.314205.58153 4.18624 208 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.01765 4.44148 5.16383 0.61958 0.77255 0.50070 0.93168130 0.54170 4.41455 3.99487 3.83758 4.67181 3.21702 4.80217 3.941603.87765 3.78720 4.68767 3.78860 4.02574 4.15301 4.08310 2.61480 3.04914 3.408786.07441 4.88693 209 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.04858 4.44148 3.33421 0.61958 0.77255 0.50070 0.93168131 2.47060 5.10222 2.92302 2.23845 4.41518 3.31163 3.65256 3.833002.30764 3.39268 4.15289 2.86002 3.85601 2.39911 2.32759 2.51827 2.29611 3.305965.55412 4.16668 210 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.03356 4.41110 3.86983 0.61958 0.77255 0.54715 0.86416132 2.26873 5.14785 2.83342 2.18702 4.47827 3.38243 3.63990 3.952732.07133 3.41537 4.19342 2.78217 3.84499 2.33511 2.64759 2.45499 2.67363 3.498655.58841 4.18815 211 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.03225 4.39603 3.94192 0.61958 0.77255 0.56894 0.83500133 3.29475 4.59909 5.27958 4.75843 3.67853 4.82300 5.34776 1.055874.65842 1.73667 3.38036 4.93089 5.04836 4.83561 4.78636 4.20753 3.60004 1.396035.69975 4.55067 212 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.19685 4.38251 1.79460 0.61958 0.77255 0.58780 0.81091134 2.59525 2.26255 4.26037 3.68225 3.24006 3.81148 4.16702 2.189273.54565 2.25647 3.24248 3.85052 4.18879 3.76123 3.70265 3.11792 2.92528 1.724614.80046 2.95881 213 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.04263 4.20800 3.61724 0.61958 0.77255 0.77092 0.62099135 2.12293 5.06795 2.53477 2.27785 4.37789 3.30834 3.61326 3.840722.22403 3.35911 4.12421 2.69439 3.80736 2.57158 2.81142 2.37052 2.76704 3.413965.52424 4.13208 214 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.615030.05033 4.19589 3.38058 0.61958 0.77255 0.78725 0.60714136 1.41298 4.54007 3.16784 2.67479 3.96764 3.28514 3.85979 3.324962.60972 2.92660 3.87024 3.16231 3.88002 3.04176 3.09126 2.66015 2.58267 3.009865.31055 4.04058 215 - -2.68596 4.42247 2.77539 2.73143 3.46365 2.40403 3.72516 3.293582.67762 2.69335 4.24680 2.90368 2.73761 3.18168 2.89806 2.37907 2.77541 2.985314.58498 3.614910.51189 0.91469 * 1.07030 0.41993 0.00000 *// Example 11: Method of Determining the Lysozyme Enhancing Domain by HMM SEQ ID NOs: 188 to 316 were aligned using the software program MUSCLE v3.8.31 with the default settings. Using this alignment, the HMM was constructed using the software program ‘hmmbuild’ from the package HMMER 3.0 (March 2010) (http://hmmer.org/) and the software was invoked using the default settings by the command: hmmscan3—tblout output.dat model.hmm sequences.fasta. The lysozyme enhancing domain HMM profile thereby generated for subsequent loading into the software program ‘hmmscan’ is given below. HMMER3/b [3.0 | March 2010]NAME lysozyme_enhancing_domainLENG 73ALPH aminoRF noCS noMAP yesDATE Tue Feb 3 15:29:15 2015NSEQ 129EFFN 1.263702CKSUM 3302514446STATS LOCAL MSV −9.1036 0.71868STATS LOCAL VITERBI −9.7357 0.71868STATS LOCAL FORWARD −3.7686 0.71868HMM A C D E F G H I K L M NP Q R S T V W Ym−>m m−>i m−>d i−>m i−>i d−>m d−>dCOMPO 2.64236 3.16005 2.87141 2.79417 3.60706 2.63596 3.86157 2.942292.65279 2.95816 3.97690 3.11757 3.46392 3.12498 3.11011 2.56828 2.58627 2.580864.17029 3.042962.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.17958 4.32413 1.88959 0.61958 0.77255 0.00000 *1 3.80107 5.04040 4.67499 4.39045 1.81828 4.48873 3.56285 3.509914.23379 2.82560 4.11380 4.16131 4.81340 4.22617 4.28030 3.87997 4.03091 3.435773.70270 0.73371 1 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02327 4.16783 4.89017 0.61958 0.77255 0.67437 0.712282 2.33952 4.44593 3.23890 3.02979 4.35083 3.16321 4.20776 3.676033.02584 3.40031 4.31198 3.32021 1.10553 3.46227 3.39562 2.64660 2.86963 3.245035.70138 4.44714 2--2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02327 4.16783 4.89017 0.61958 0.77255 0.58149 0.818863 2.99224 4.47028 5.00531 4.50059 3.72470 4.59877 5.18674 1.107014.40105 2.19903 3.51509 4.69439 4.89181 4.65354 4.58483 3.98375 3.44715 1.218385.67709 4.47722 3--2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02218 4.21548 4.93783 0.61958 0.77255 0.62351 0.768004 2.67119 4.76820 3.04982 2.54195 4.12069 3.43519 3.75916 3.511492.19874 3.13703 3.97428 2.96845 3.89558 2.93360 2.89915 2.57877 1.61288 3.102815.38435 4.08520 4--2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02491 4.21548 4.62159 0.61958 0.77255 0.52151 0.900485 2.37234 5.08464 2.49824 2.21575 4.41120 1.99198 3.58111 3.866772.52882 3.41126 4.20120 2.81325 3.86140 2.84200 3.02691 2.42476 2.83819 3.480015.60144 4.21122 5--2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02114 4.26301 4.98536 0.61958 0.77255 0.56183 0.844366 2.63301 5.17310 2.09015 2.17272 4.49463 3.27178 3.65545 3.970972.38584 3.47241 4.23433 2.52660 3.34330 2.76874 2.94084 2.41690 2.44683 3.553935.62559 4.21427 6--2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.07099 4.26555 2.90975 0.61958 0.77255 0.56422 0.841197 2.61111 4.85146 2.70126 2.41033 4.02944 2.66646 3.65077 3.525142.43178 3.11934 3.92649 2.77281 3.83428 2.77300 2.91547 2.42718 2.41739 2.796855.35415 3.75427 7--2.68619 4.42226 2.77521 2.73124 3.46355 2.40511 3.72496 3.29355 2.677422.69356 4.24691 2.90348 2.73741 3.18147 2.89802 2.37888 2.77504 2.98519 4.584783.615040.09494 2.48394 4.93906 0.38374 1.14353 0.52245 0.899108 3.16563 4.52406 5.00410 4.47860 3.59477 4.61318 5.11093 1.751304.36399 1.66390 3.39589 4.68330 4.88103 4.58179 4.52924 3.98371 3.48258 0.986545.56065 4.39116 9--2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02108 4.26555 4.98790 0.61958 0.77255 0.56422 0.841199 2.74568 5.18103 2.62071 2.37499 4.50785 3.44148 3.43386 3.970092.14487 3.46738 4.24084 1.77122 3.86903 2.77625 2.44752 2.65464 2.97757 3.566155.60976 4.22640 10 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02108 4.26555 4.98790 0.61958 0.77255 0.43965 1.0335610 3.21633 0.35479 4.79708 4.65548 4.57914 3.72194 5.20787 3.829384.49870 3.70850 4.85167 4.53077 4.45916 4.82584 4.53365 3.48556 3.72159 3.535055.86530 4.85631 11 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551011 3.30119 5.31389 3.73668 3.09114 4.51121 3.86969 3.13217 4.160182.10415 3.58751 4.49408 3.45992 4.26052 2.99924 0.83233 3.31264 3.47550 3.857055.50391 4.26079 12 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551012 2.24421 4.51545 3.30473 2.81428 4.39051 3.23670 4.14928 3.774913.04692 3.44962 4.27960 3.30747 3.89875 3.36729 3.42822 1.05895 2.51437 3.318595.70279 4.43964 13 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551013 2.69468 4.75034 2.89429 2.56341 4.66767 0.94912 4.20148 4.128223.15097 3.75743 4.60332 3.20872 3.96757 3.42394 3.55601 2.47312 3.12631 3.624105.94365 4.63448 14 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551014 2.67424 4.69741 3.65691 3.53445 4.63811 3.40395 4.65858 4.094863.63150 3.77181 4.76683 3.75211 0.59051 3.99224 3.88152 2.99980 3.31792 3.644805.92609 4.77080 15 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551015 2.26323 4.51933 3.21961 2.94036 4.47411 1.38422 4.14822 3.912453.06085 3.54037 4.34588 2.97288 3.87499 3.35219 3.46854 1.96711 2.52323 3.403505.76384 4.49002 16 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551016 2.20668 4.36145 3.82003 3.56662 4.41076 3.20360 4.55272 3.515973.53768 3.41304 4.35967 3.64187 3.96341 3.86648 3.78744 2.68079 0.81042 3.108315.83931 4.64399 17 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551017 2.61570 5.10849 2.62653 2.38205 4.43252 2.78078 3.60811 3.890182.47426 3.42628 4.20945 2.71231 3.87390 2.61734 2.99949 1.64993 2.94705 3.499365.60799 4.21799 18 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551018 2.58292 4.58689 3.02231 2.68583 3.00245 3.32285 2.97170 3.145642.67302 2.81683 3.67939 2.99852 3.79428 3.00246 3.09283 2.65865 2.82740 2.884735.11794 2.25478 19 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551019 2.02971 5.05961 2.68453 2.36019 4.37471 3.26819 3.65420 3.830902.10672 3.36092 4.13132 2.92980 3.45060 2.77060 2.82369 2.16905 2.88648 3.438865.53518 4.15201 20 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551020 3.16684 4.47509 4.98151 4.48359 3.78153 4.52597 5.13073 1.233574.37208 2.38350 3.60515 4.64947 4.86068 4.63385 4.54770 3.62858 3.43521 1.038965.65865 4.43929 21 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551021 2.67985 4.50015 3.40866 2.83947 3.66095 3.60615 3.61051 2.500382.12953 2.71143 3.59755 3.27678 3.98763 2.81347 3.06211 2.85030 2.83675 1.922945.06195 3.81377 22 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551022 2.57540 5.16214 3.25351 2.63376 4.54584 3.62294 3.69980 3.939391.30635 3.43432 4.24813 3.13299 4.00544 2.78831 2.11440 2.89381 2.74985 3.583265.55169 4.27942 23 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551023 2.54455 4.73655 3.02733 2.59505 4.05985 3.44683 3.70095 3.464662.60056 3.09418 3.92399 3.08417 3.89235 2.67009 3.04116 2.11836 1.78482 2.959745.35882 4.04770 24 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551024 3.40982 4.75103 4.67192 4.22624 1.97308 4.35966 3.81417 3.048884.07862 2.38151 3.72579 4.16721 4.67838 4.14717 4.17038 3.68839 3.63576 2.473654.02768 0.99393 25 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551025 2.33607 5.05721 2.94517 2.24042 4.36369 3.44673 3.55543 3.817541.82729 3.31948 4.11325 2.80831 3.17764 2.75227 2.78214 2.58400 2.65726 3.320875.51479 4.13659 26 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551026 2.36555 5.04925 3.04993 2.35049 4.35720 3.51989 3.68369 3.715511.45376 3.06828 4.12214 3.00561 3.91721 2.74243 2.72409 2.73518 2.95391 3.420665.49677 4.16815 27 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551027 2.61202 4.80098 2.75023 2.62858 4.42906 1.32629 3.93435 3.864832.78346 3.46292 4.27988 3.09911 3.91159 3.10786 2.85440 2.43819 2.91018 3.444685.68001 4.35178 28 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551028 2.39518 5.06692 2.42469 2.35324 4.15968 3.22653 3.04826 3.831882.39351 3.35617 4.12096 2.82671 3.83411 2.52507 2.87979 2.43622 2.35726 3.410275.52582 4.13893 29 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551029 2.56098 5.10975 1.84700 2.22610 4.41970 3.43432 3.64564 3.820902.31577 3.39892 4.16246 2.88269 3.84241 2.68525 2.69153 2.62766 2.81515 3.481935.56120 3.47482 30 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551030 2.71289 4.20867 4.05384 3.08944 3.32534 3.70711 4.11635 2.104603.37170 2.31376 3.31881 3.74286 4.18005 3.62760 3.59433 3.09939 2.94743 1.408214.85949 3.10435 31 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551031 2.56066 3.98356 2.83917 2.26935 4.26245 3.45019 3.65582 3.702071.96206 3.26063 4.04582 2.95679 3.84786 2.75603 2.89654 2.30158 2.29348 3.337285.46026 4.09624 32 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551032 2.98261 4.34033 4.74407 4.16222 2.74015 4.19374 4.48870 1.337224.00888 1.70186 3.16247 4.27271 4.50036 4.14592 4.09048 3.51345 3.21313 1.977554.93581 3.15765 33 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551033 2.50714 4.52249 3.32761 2.73441 3.99413 2.87824 3.93263 3.376112.83014 3.05077 3.89907 3.20556 3.90546 3.15647 3.23473 2.09891 1.66153 2.588875.34950 4.08041 34 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551034 2.73372 0.86170 4.21080 3.82223 3.72250 3.51687 4.43487 3.147323.50989 2.93675 3.97687 3.88251 4.17078 3.93344 3.44432 2.99564 3.14856 2.896525.25169 3.70577 35 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551035 2.78221 4.93521 3.15849 2.72873 3.66991 3.61431 3.78500 3.572062.57323 3.13265 4.05241 3.20038 4.05162 1.36867 2.92156 2.93601 3.12692 3.298345.09768 2.57435 36 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551036 2.39232 4.68655 3.16574 2.46000 3.89951 3.51768 3.74742 2.862232.34831 2.93724 3.78511 3.11031 3.91433 2.90991 3.00803 2.69966 1.85059 2.951705.23226 3.93826 37 - 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-2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02007 4.31435 5.03670 0.61958 0.77255 0.49963 0.9333364 1.89508 4.31720 3.70048 3.48388 4.63634 0.95179 4.55159 4.008073.58485 3.73760 4.55934 3.55250 3.87506 3.84242 3.86415 2.56592 2.50453 3.402045.97883 4.79808 69 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.03953 4.31435 3.67355 0.61958 0.77255 0.49963 0.9333365 2.63597 3.39258 3.14566 2.45530 3.91792 3.49097 3.54341 3.312882.40023 2.95698 3.79792 3.08785 3.89316 2.92081 3.00148 1.97899 2.29007 2.969155.24220 3.94276 70 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02046 4.29528 5.01763 0.61958 0.77255 0.52575 0.8943266 2.59137 4.97540 2.68695 2.29850 4.05453 3.34590 3.64946 3.593802.42308 3.26160 4.04572 2.57932 3.83844 2.77492 2.90216 1.98570 2.44689 3.225305.46068 4.09334 71 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02046 4.29528 5.01763 0.61958 0.77255 0.52575 0.8943267 2.36239 3.82492 3.07537 2.52645 4.05085 1.94430 3.77137 3.458532.53920 3.08721 3.91418 2.86394 3.87403 2.94691 3.05153 2.46988 2.65768 3.129235.35184 4.04208 72 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02046 4.29528 5.01763 0.61958 0.77255 0.52575 0.8943268 2.51473 4.23017 3.73065 3.15738 2.96324 3.66009 3.93184 2.626173.08387 2.43082 2.74387 3.49391 3.33993 3.35931 3.14679 2.64784 2.86016 2.493024.81896 2.22311 73 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02046 4.29528 5.01763 0.61958 0.77255 0.52575 0.8943269 2.33893 4.26290 4.21737 3.66773 3.27773 3.87203 4.32579 2.122333.56960 2.41061 3.43058 3.89614 4.28394 3.82133 3.79298 2.87144 3.03565 1.214535.06701 3.86005 74 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02293 4.29528 4.70670 0.61958 0.77255 0.52575 0.8943270 2.49593 4.60476 3.23381 2.52485 3.79749 3.53767 3.76569 3.142972.26081 2.40764 3.70182 3.15412 3.92810 2.98301 3.03430 2.76728 2.09050 2.558465.16115 3.88294 75 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02051 4.29287 5.01521 0.61958 0.77255 0.52898 0.8896771 2.60687 5.12624 2.52016 2.23382 4.44833 2.55208 3.64005 3.918842.12896 3.37075 4.18386 2.86694 3.03933 2.74997 2.89501 2.33562 2.83629 3.506725.58015 4.17955 76 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.02051 4.29287 5.01521 0.61958 0.77255 0.52898 0.8896772 2.57816 5.12677 2.87095 2.24649 4.45454 3.41210 3.40077 3.910861.59193 3.41242 4.17909 2.90547 3.85681 2.60853 2.68264 2.65653 2.67555 3.509605.55782 4.18309 77 - -2.68618 4.42225 2.77520 2.73117 3.46354 2.40513 3.72495 3.29354 2.677412.69355 4.24690 2.90347 2.73740 3.18147 2.89801 2.37887 2.77520 2.98519 4.584773.615030.09497 3.41028 2.85473 0.52137 0.90068 0.52898 0.8896773 3.08258 0.42515 4.66004 4.49479 4.42412 3.61311 5.06351 3.648684.33208 3.53771 4.67608 4.38809 4.34603 4.66309 4.38494 3.35266 3.58099 3.363155.74041 4.70439 79 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.615030.01461 4.23357 * 0.61958 0.77255 0.00000 *// Example 12: Determination of DomT Scores The DomT scores for the LAD domain and the LED domain of the LYS polypeptides of the invention were determined using the LAD Catalytic Domain HMM from Example 10 and the Lysozyme Enhancing Domain HMM from Example 11 as described herein are presented in table 5 below. TABLE 5DomT scores for LAD and LED domainsLAD domainLED domainAmino acidDomTAmino acidDomTSequencenumbersscorenumbersscoreSEQ ID NO: 384 to 226202.51 to 73118.3SEQ ID NO: 684 to 226195.41 to 73118.5SEQ ID NO: 981 to 220199.21 to 73116.7SEQ ID NO: 12161 to 304193.41 to 72108.776 to 147104.6SEQ ID NO: 1585 to 228200.11 to 73125.2SEQ ID NO: 1888 to 230205.31 to 73119.7SEQ ID NO: 2187 to 230201.41 to 73116.9SEQ ID NO: 2490 to 232201.51 to 73119.1SEQ ID NO: 2785 to 228199.81 to 73123SEQ ID NO: 3085 to 228202.81 to 73122.6SEQ ID NO: 3384 to 226198.21 to 73115.2SEQ ID NO: 3683 to 222194.91 to 73113.1SEQ ID NO: 3982 to 225203.01 to 72117.4SEQ ID NO: 42161 to 303192.61 to 73115.877 to 149111.2SEQ ID NO: 4585 to 227208.31 to 73124.9SEQ ID NO: 3294 to 146205.3—— All of the claimed LYS polypeptides have a LAD DomT score of at least 170, indicating good homology to the LAD HMM model. Likewise all claimed LYS polypeptides have a LED, had a LED DomT score of at least 100, indicating good homology to the LED HMM model. Example 13: Activity of LYS Polypeptides as Determined Using Reducing Ends Assay The LYS polypeptides of the invention were tested according to Example 1 at two enzyme concentrations and the results are shown in tables 6 to 8 below. TABLE 6OD Drop of SEQ ID NO: 3OD DropOD DropLYS polypeptide(5.0 μg/ml)1(0.7 μg/ml)1SEQ ID NO: 35.42.41enzyme concentration TABLE 7OD Drop of SEQ ID NO: 6 to 45OD DropOD DropLYS polypeptide(5.0 μg/ml)1(0.7 μg/ml)1SEQ ID NO: 64.42.0SEQ ID NO: 95.22.7SEQ ID NO: 122.41.4SEQ ID NO: 156.73.2SEQ ID NO: 213.92.2SEQ ID NO: 243.11.8SEQ ID NO: 277.84.6SEQ ID NO: 308.76.0SEQ ID NO: 338.65.7SEQ ID NO: 365.42.9SEQ ID NO: 397.84.8SEQ ID NO: 425.13.1SEQ ID NO: 458.53.9SEQ ID NO: 3295.02.31enzyme concentration As can be seen, the LYS polypeptides of the invention display lysozyme activity as determined using the reducing ends assay. Example 14: Activity of LYS Polypeptides as Determined Using OD Drop Assay The LYA polypeptides of the invention were tested according to Example 2 at pH4 and the results are shown in tables 8 and 9 below. TABLE 8OD Drop againstM. luteusOD DropM. luteusLYS polypeptide1 h, pH 4SEQ ID NO: 30.116SEQ ID NO: 60.151SEQ ID NO: 90.121SEQ ID NO: 120.177SEQ ID NO: 150.125SEQ ID NO: 210.113SEQ ID NO: 240.121SEQ ID NO: 270.071SEQ ID NO: 300.081SEQ ID NO: 330.052SEQ ID NO: 360.171SEQ ID NO: 390.154SEQ ID NO: 420.162 TABLE 9OD Drop againstM. luteusOD DropM. luteusLYS polypeptide1 h, pH 4SEQ ID NO: 180.078SEQ ID NO: 3290.063 As can be seen, the LYS polypeptides of the invention display lysozyme activity as determined using the traditional OD drop assay againstM luteus. Example 19: Animal Feed and Animal Feed Additives Comprising a LYS Polypeptide Animal Feed Additive A formulation comprising the LYS polypeptide of the invention (e.g. SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 or 239) containing 0.01 g to 10 g enzyme protein is added to the following premix (per kilo of premix): 5000000IEVitamin A1000000IEVitamin D313333mgVitamin E1000mgVitamin K3750mgVitamin B12500mgVitamin B21500mgVitamin B67666mcgVitamin B1212333mgNiacin33333mcgBiotin300mgFolic Acid3000mgCa-D-Panthothenate1666mgCu16666mgFe16666mgZn23333mgMn133mgCo66mgI66mgSe5.8%Calcium25%Sodium Animal Feed This is an example of an animal feed (broiler feed) comprising the animal feed additive as described above:62.55% Maize33.8% Soybean meal (50% crude protein)1.0% Soybean oil0.2% DL-Methionine0.22% DCP (dicalcium phosphate)0.76% CaCO3(calcium carbonate)0.32% Sand0.15% NaCl (sodium chloride)1% of the above Premix The ingredients are mixed, and the feed is pelleted at the desired temperature, e.g. 60, 65, 75, 80, 85, 90 or even 95° C. The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. | 476,875 |
11859171 | DETAILED DESCRIPTION The present invention applies the concept of barcoding to generate long sequence reads by providing a technical advance in juxtaposing the assigned barcode to random overlapping segments of the original template. The present invention relies on assigning barcodes to individual template molecules, allowing for unambiguous assembly of template sequences even for molecules with high sequence similarity. This also means that the present invention will work for sequencing targeted genomic regions or viral genomes. The current maximum read length of next-generation sequencing technologies, such as those developed by Illumina® and Life Technologies™, is around 250 bases. The present invention, also known as “Barcode-directed Assembly for Extra-long Sequences (BAsE-Seq)” provides an approach that can: 1) increase the effective read length of these commercially available sequencing platforms to several kilobases and 2) be broadly applied to obtain long sequence reads from mixed template populations. In brief, our method relies on assigning random DNA barcodes to long template molecules (FIG.1A), followed by a library preparation protocol that juxtaposes the assigned barcode to random short segments of the original template (FIGS.1B and2). The resulting molecules are ligated with platform-specific adaptors for next-generation sequencing. Sequence reads are de-multiplexed using the barcode sequence and used to assemble long-range haplotypes that were present on the original template. In practice, we have applied this technology to perform single virion sequencing on the Hepatitis B virus, a DNA virus with a 3.2 kb genome. In general, we anticipate that this technology can be broadly applied to generate extended sequence reads and will be useful for long-range haplotype analysis on targeted genomic regions, or for improving de novo genome and transcriptome assemblies. A detailed description of our protocol is described in the following paragraphs. There is described a method for generating extended sequence reads of long DNA molecules (>3 kb), in a sample. The method comprises the steps of: (i) assigning a specific barcode sequence to each template DNA molecule in a sample to obtain barcode-tagged molecules; (ii) amplifying the barcode-tagged molecules; (iii) fragmenting the amplified barcode-tagged molecules to obtain barcode-containing fragments; (iv) juxtaposing the barcode-containing fragments to random short segments of the original DNA template molecule during the process of generating a sequencing library to obtain demultiplexed reads; and (v) assembling the demultiplexed reads to obtain extended sequence reads for each DNA template molecule. a) Barcode Assignment. In the first step, individual template molecules are assigned with a unique DNA barcode. In our example, two rounds of PCR amplification are performed using primers with template-specific sequence from opposite ends of the molecule (FIG.1A). This will generate uniquely tagged template molecules for preparing libraries and can be broadly applied for assigning barcodes to targeted genomic regions. Both primers contain a universal sequence on their 5′-ends and one of them contains a barcode, i.e., a string of 20 random nucleotides (encodes for >1012sequences). To ensure that each template molecule is uniquely assigned, the template should be diluted to obtain a relatively small number of genomes (<109) compared to unique barcode sequences. Subsequently, barcode-tagged molecules can be clonally amplified by PCR using universal primers and the PCR product can be used to prepare sequencing libraries. In other manifestations where the template sequence is unknown, the barcode can be assigned by ligation of double- or single-stranded DNA linkers carrying a random string of nucleotides flanked by universal sequences. The use of unique barcodes to tag individual template molecules has been shown to greatly reduce the error rate of massively parallel sequencing. Using this strategy, mutations that pre-existed on the template and errors introduced during barcode assignment will be found in all daughter molecules. In contrast, errors introduced in subsequent steps of library preparation, sequencing, or base-calling can be easily removed because they will only be present in a minority of daughter molecules (FIG.1A). Based on the published error rate of the DNA polymerase used in our protocol, this translates to one error in every 50 template sequences for template molecules that are 3 kb in size. Furthermore, by using barcodes as unique identifiers for individual genomes, sequences associated with each barcode can be assembled into a complete template sequence. b) Library Preparation. The goal of library preparation is to tag overlapping fragments of each template molecule with its assigned barcode in order to obtain uniform sequence coverage. This concept is illustrated inFIG.1Band a detailed outline of the protocol is shown inFIG.2. Firstly, clonally amplified barcode-tagged molecules are deleted from the barcode-distal end to achieve a broad size distribution of fragments ranging from ˜300 bp to N bp, where N equals the length of the template molecule. Unidirectional deletion can be achieved by protecting the barcode-proximal end with nuclease-resistant nucleotides or a 3′-protruding overhang, and performing time-dependent digestion from the barcode-distal end using a 3′ to 5′ exonuclease (such as Exonuclease III), followed by treatment with an endonuclease (such as S1 Nuclease or Mung Bean Nuclease) to generate blunt-ends. Barcode-containing fragments are purified using streptavidin-coated beads, and these biotinylated fragments will be dissociated and subjected to end repair, such that both ends of the molecules are blunt and 5′-phosphorylated. The end-repaired molecules are circularized by intramolecular ligation using a DNA ligase (such as T4 DNA ligase). Uncircularized molecules will be removed by nuclease treatment (such as a combination of Exonuclease I and Lambda Exonuclease). After circularization, different regions from the original template will be juxtaposed to its barcode. The circularized molecules will be used as template for random fragmentation and adaptor tagging using a transposome-based method, such as the Nextera XT kit (Illumina®). Importantly, the primers used during PCR enrichment of the sequencing library will be designed such that the second sequencing read will be anchored by the barcode sequence. Thus, this PCR generates double-stranded DNA molecules that are “sequencing-ready”. Finally, the PCR products are subjected to size selection before sequencing. A custom sequencing primer that anneals to the forward priming sequence is used for the second sequencing read. There are several alternative approaches to generate a broad distribution of barcode-tagged molecules before circularization. One approach involves creating a nick at the barcode-distal end using a nicking endonuclease, nick translation towards the barcode-proximal end using DNA polymerase I, followed by treatment with endonuclease to generate a blunt end. Another approach involves performing random fragmentation using a mechanical method, such as using the Covaris instrument for focused-ultrasonication, or an enzymatic method, such as using the NEBNext dsDNA Fragmentase, followed by purification of barcode-containing fragments using streptavidin-coated paramagnetic beads. An alternative, PCR-free approach to clonal amplification is contemplated, such as circularizing the barcoded template and performing rolling circle amplification using phi29 polymerase. Barcodes can be assigned by linker ligation. Both linkers will contain universal sequences on their 5′-end to facilitate clonal amplification in the next step. The barcode-containing linker will also contain a unique universal sequence on its 3′-end for primer annealing during the PCR step at the end of the protocol. Software packages for obtaining extended or extra-long sequence reads by reference-assisted assembly and for obtaining extended or extra-long sequence reads from template molecules of an unknown sequence are illustrated inFIGS.3and4, respectively. There is described hereinafter a system for obtaining extended sequence reads from template molecules of a DNA sequence. The system comprises (i) a quality filtering module for filtering raw paired-end sequence reads from a sequencer by removing read-pairs with low quality scores, removing read-pairs with missing barcode sequences and trimming platform-specific adaptor sequences; (ii) a barcode analysis module for identifying highly-represented barcodes and re-assigning sequences associated with poorly-represented barcodes; (iii) a demultiplexing module for using barcode sequences as identifiers to obtain reads associated with individual template molecules and removing duplicate read-pairs; and (iv) an assembly module for assembling demultiplexed reads to obtain extended sequence reads for each template molecule (FIG.4). The template molecules are long, preferably >3 kb. Where the DNA sequence is a known sequence, the system further comprises (i) a sequence alignment module for performing paired-end alignment to a reference sequence and removing disconcordant alignments; (ii) a demultiplexing module for using barcode sequences as identifiers to obtain alignments to individual template molecules and removing duplicate read-pairs in place of the demultiplexing module shown inFIG.4; and (iii) a haplotyping module for obtaining pileup of aligned reads at each position along the reference sequence, determining consensus base-call at each position and assembling base-calls to obtain extended sequence reads for each template molecule in place of the assembly module shown inFIG.4(FIG.3). There is also disclosed a computer-readable medium with an executable programme stored thereon, the programme comprising instructions for obtaining extended sequence reads from template molecules of a DNA sequence, wherein the programme instructs a microprocessor to perform the following steps of (i) filtering raw paired-end sequence reads from a sequencer by removing read-pairs with low quality scores, removing read-pairs with missing barcode sequences and trimming platform-specific adaptor sequences; (ii) identifying highly-represented barcodes and re-assigning sequences associated with poorly-represented barcodes; (iii) using barcode sequences as identifiers to obtain reads associated with individual template molecules and removing duplicate read-pairs; and (iv) assembling demultiplexed reads to obtain extended sequence reads for each template molecule. Where the DNA sequence is a known sequence, the programme instructs the microprocessor to further perform the following steps of (i) performing paired-end alignment to a reference sequence and removing disconcordant alignments at the step of identifying highly-represented barcodes and re-assigning sequences associated with poorly-represented barcodes; (ii) replacing the step of using barcode sequences as identifiers to obtain reads associated with individual template molecules and removing duplicate read-pairs described above with the step of using barcode sequences as identifiers to obtain alignments to individual template molecules and removing duplicate read-pairs; and (iii) replacing the step of assembling demultiplexed reads described above with the step of obtaining pileup of aligned reads at each position along the reference sequence, determining consensus base-call at each position and assembling base-calls to obtain extended sequence reads for each template molecule. Examples Hepatitis B virus (HBV), which contains a 3.2 kb dsDNA genome, was used as a template for methodology development and generating proof-of-concept data. The results presented below demonstrate the use of BAsE-Seq to obtain long (˜3.2 kb) sequence reads from individual template molecules, thereby achieving single virion sequencing of HBV. HBV DNA was isolated from a chronically infected patient, PCR-amplified to obtain full-length viral genomes, and cloned into a TOPO pCR2.1 vector (Life Technologies™). Sanger sequencing was performed across each clone to obtain full-length sequences, and two clones (Clone-1 and Clone-2) with 17 single nucleotide polymorphisms (SNPs) between them were used as input for barcode assignment. In the results presented hereafter, barcode-tagged whole-genome amplicons from 20,000 template molecules (HBV genomes) were used as input for library preparation using the BAsE-Seq protocol described above. Summary statistics from a typical single virion sequencing experiment of HBV are shown in Table 1, and coverage data per template molecule are illustrated inFIGS.5and6. In this library, 18,143,186 read-pairs were obtained from the MiSeq sequencer (Illumina®), from which Ser. No. 12/004,237 read-pairs contained the barcode in the expected orientation. After trimming for adaptor, barcode tag and universal sequences, and removing reads shorter than 15 bp, 7,336,915 pass-filter read-pairs were used for alignment to a HBV reference genome. From these read-pairs, 97% read-pairs aligned concordantly, and were distributed across 4,294 individual template molecules, 2,717 of which were identified as “high coverage” and were used for constructing long reads. TABLE 1Summary statistics from a BAsE-Seq run of HBV.MiSeq run (2 × 150 bp)Genomes as input20,000Sequencing read-pairs18,143,186Barcode-associated read-pairs112,004,237 (66%)Pass-filter read-pairs27,366,915Concordantly aligned to HBV genome7,151,142 (97% of pass-filter)Unique barcodes observed34,294High coverage HBV genomes42,7171Read-pairs that contain the barcode in the expected orientation2Read-pairs that are ≥15 bp after removal of adaptor and universal sequences3Barcodes associated with at least 50 read-pairs4≥5 unique reads per base position across ≥85% of the genome To test the sensitivity and accuracy of our methodology in generating long sequence reads, Clone-1 and Clone-2 were mixed at different ratios to generate a mixed template population where Clone-1 is present at approximately 1% or 10% frequency in the sample. BAsE-Seq was performed on each mixed-template pool. Firstly, barcodes were removed from each read-pair prior to alignment and the resulting data was treated as a “bulk” sequencing experiment to determine overall allele frequencies at the SNP positions. The minor allele frequencies in both libraries were very close to the mixing ratio—0.98% for the “1% pool” (Lib_1:99) and 13.44% for the “10% pool” (Lib_1:9)—indicating that the mixed template pool was generated correctly and PCR bias was negligible (Table 2 andFIG.7). Subsequently, the “bulk” sequence data was de-multiplexed using barcode sequences and sequence reads from individual template molecules were analyzed to obtain ˜3.2 kb reads. Using the long sequence reads, 17-SNP haplotypes were generated for each template molecule. In Lib_1:9, 240 molecules carried a Clone-1 haplotype and 1,639 molecules carried a Clone-2 haplotype, corresponding to a 12.77% minor haplotype frequency. In Lib_1:99, 20 molecules carried a Clone-1 haplotype and 1,912 molecules carried a Clone-2 haplotype, corresponding to a 1.04% minor haplotype frequency. Importantly, chimeric sequences where Clone-1 and Clone-2 SNPs were found on the same molecule were present at ≤0.1% frequency. Furthermore, the use of barcodes to correct for sequencing errors resulted in a very low error rate for BAsE-Seq, allowing for significant separation of true sequence variants from background noise in Lib_1:99 (Table 2 andFIG.8). TABLE 2Detection of low frequency haplotypes by BAsE-SEqLib_1:99Lib_1:9Mixing ratio (Clone-11:9910:90vs. Clone-2)Expected minor clone0.98%13.44%frequency1Observed minor clone1.04% (20/1912)12.77% (240/1639)haplotypes(Clone-1/Clone-2)2Chimeric haplotypes0.10% (2/1912)0.06% (1/1639)1Based on average allele frequency of Clone-1 SNPs from “bulk” sequencing analysis2Based on 17-SNP haplotypes observed in the data from individual template molecules Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features. Each document, reference, patent application or patent cited in this text, if any, is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein. The invention described herein may include one or more range of values (e.g. size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention. | 19,404 |
11859172 | DETAILED DESCRIPTION The disclosed systems, components, kits, and methods provides methods for transcriptional modification and identification of transcriptional effectors. Disclosed herein is a high-throughput platform to screen and select for bacterial CRISPR-Cas transcriptional modifiers, e.g., bacterial CRISPR-Cas transcriptional activators (CasTAs). A number of natural bacterial and phage regulatory effectors were screened and a phage protein that induced gene activation when fused to dCas9 was identified. The targeting window of this CasTA was characterized and further rounds of directed evolution were performed using the screening platform to yield higher functioning variants, which mediated both CRISPRi and CRISPRa of genomic and plasmid targets. This activator system was applied to a metagenomic promoter library mined from diverse bacteria to build a library of CasTA-inducible promoters of varying basal and induced expression levels that are useful as a resource for the synthetic biology research community. Successful transfer of the CRISPRa system to other bacterial species of clinical and bioindustrial importance was also achieved. Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting. Definitions The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of cell culture, molecular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and/or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non-nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acid or amino acid sequence “identity,” as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (e.g., that are identical) as between the sequence of interest and the reference sequence divided by the length of the longest sequence (e.g., the length of either the sequence of interest or the reference sequence, whichever is longer). A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3×, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al.,J. Molecular Biol.,215(3): 403-410 (1990), Beigert et al.,Proc. Natl. Acad. Sci. USA,106(10): 3770-3775 (2009), Durbin et al., eds.,Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding,Bioinformatics,21(7): 951-960 (2005), Altschul et al.,Nucleic Acids Res.,25(17): 3389-3402 (1997), and Gusfield,Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)). A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell. A cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. As used herein, the terms “providing”, “administering,” “introducing,” are used interchangeably herein and refer to the placement of the systems of the disclosure into a cell, organism, or subject by a method or route which results in at least partial localization of the system to a desired site. The systems can be administered by any appropriate route which results in delivery to a desired location in the cell, organism, or subject. Systems Disclosed herein are systems comprising: a conjugate comprising Cas9 protein linked to a transcriptional effector or variant or fragment thereof and/or a first nucleic acid encoding the fusion protein; and at least one guide RNA (gRNA) and/or at least one second nucleic acid encoding the guide RNA sequence, wherein the gRNA is complementary to a target DNA sequence. In some embodiments, the system further comprises at least one reporter gene and/or at least one third nucleic acid encoding the reporter gene. The Cas9 protein can be obtained from any suitable microorganism, and a number of bacteria express Cas9 protein orthologs or variants (see, e.g., U.S. Pat. No. 10,266,850 incorporated herein by reference) and may be used in connection with the present disclosure. The amino acid sequences of Cas proteins from a variety of species are also publicly available through the GenBank and UniProt databases. In some embodiments, the Cas9 protein is a catalytically-dead Cas9. Catalytically-dead Cas9 is essentially a DNA-binding protein due to, typically, two or more mutations within its catalytic nuclease domains which renders the protein with very little or no catalytic nuclease activity. For example,Streptococcus pyogenesCas9 may be rendered catalytically dead by mutations of D10 and at least one of E762, H840, N854, N863, or D986, typically H840 and/or N863A (see, e.g., U.S. Pat. No. 10,266,850, incorporated herein by reference). Mutations in corresponding orthologs are known. Oftentimes, such mutations cause catalytically-dead Cas9 to possess no more than 3% of the normal nuclease activity. The transcriptional effector may be linked to the Cas9 protein at the N or C terminus. In some embodiments, the transcriptional effector is linked to the C-terminal end of the Cas9 protein. In some embodiments, a linker (e.g., a peptide linker) is used to link the Cas9 protein and the transcriptional effector. The linkers may comprise any amino acid sequence of any length. The linkers may be flexible such that they do not constrain either of the two components they link together in any particular orientation. The linkers may essentially act as a spacer. In select embodiments, the linker links the C-terminus of the Cas9 protein to the N-terminus of the transcriptional effector. In some embodiments, the linker comprises an amino acid sequence of SAGGGGSGGGGS (SEQ ID NO:1) or CAGGGGSGGGGS (SEQ ID NO:2). Transcriptional effectors are proteins or protein domains that can be used to control gene expression. Transcriptional effectors may bind to and regulate promoters, promoter elements, or RNA polymerases. The transcriptional effector may be a transcriptional activator. Transcriptional activators may increase or start transcription resulting in an increased expression of a gene or gene product over time. The transcriptional effector may be a transcriptional repressor. Transcriptional repressors may decrease or stall transcription resulting in decreased expression of a gene or gene product over time. The present system may be used with transcriptional effectors known in the art or to screen putative transcriptional effectors, as described elsewhere herein. The transcriptional effector of the present system may be selected from the group consisting of: B42 transactivation domain (B42), BTAD domain-containing protein 1 (BTAD1), BTAD domain-containing protein 2 (BTAD2), transcription elongation factor GreA (GreA), RNA polymerase-binding transcription factor DksA (DksA), regulatory protein SoxS (SoxS), N4 single stranded binding protein, Motility Protein A (MotA), 10 kDa anti-sigma factor (AsiA), omega subunit of DNA-dependent RNA polymerase (w), or a fragment or variant thereof. In some embodiments, the transcriptional effector comprises AsiA, or a fragment or variant thereof. In some embodiments, the transcriptional effector comprises an amino acid sequence of wild-type AsiA (SEQ ID NO: 80). In select embodiments, the transcriptional effector comprises a variant of AsiA having mutations in any or all of Q51, V58, and E60 of SEQ ID NO: 80. In some embodiments, the transcriptional effector comprises an amino acid sequence of SEQ ID NO: 80 with a Q51R mutation, V58I mutation, E60K mutation, or any combination thereof. In select embodiments, the transcriptional effector comprises an amino acid sequence of SEQ ID NO:95 or SEQ ID NO: 96. The system comprises at least one guide RNA (gRNA) and/or at least one second nucleic acid encoding the guide RNA sequence, wherein the gRNA is complementary to a target DNA. The guide RNA sequence specifies the target site with an approximate 20-nucleotide guide sequence followed by a protospacer adjacent motif (PAM) that directs Cas9 via Watson-Crick base pairing to a target sequence. The gRNA may be a non-naturally occurring gRNA. The terms “target DNA sequence,” “target nucleic acid,” “target sequence,” and “target site” are used interchangeably herein to refer to a polynucleotide (nucleic acid, gene, chromosome, genome, etc.) to which a guide sequence (e.g., a guide RNA) is designed to have complementarity, wherein hybridization between the target sequence and a guide sequence promotes the formation of a Cas9 complex, provided sufficient conditions for binding exist. A general theme in transcription factor regulation of gene expression is that all that is generally required is simple association with the promoter and sufficient proximity. The distance is not very important as long as it facilitates the correct position and orientation to the promoter or the transcription start site. Thus, the target site recognized by the gRNA may be various distance from the transcription start site, in an upstream or downstream region of a target gene. In some embodiments, the target DNA sequence is upstream of the transcription start site (TSS) of a reporter gene. The target DNA sequence may be greater than 10 base pairs, greater than 50 base pairs, greater than 100 base pairs, greater than 150 base pairs, greater than 200 base pairs, or greater than 250 base pairs upstream of the TSS. In some embodiments, the target DNA sequence is 50-300 base pairs (e.g., 50-200 base pairs, 50-100 base pairs, 100-300 base pairs, or 100-200 base pairs) upstream of the TSS. In some embodiments, the target DNA sequence is near (within 50 base pairs) of the transcription start site (TSS) of a reporter gene. In some embodiments, the target DNA sequence is within the gene body of a reporter gene. In some embodiments, the target DNA is a DNA sequence in a host cell. In some embodiments, the target DNA sequence comprises DNA endogenous to the host cell. In some embodiments, the endogenous DNA is a genomic DNA sequence. The term “genomic,” as used herein, refers to a nucleic acid sequence (e.g., a gene or locus) that is located on a chromosome in a cell. In some embodiments, the target DNA sequence comprises DNA exogenous to the host cell. DNA exogenous to the host cell is DNA which does not naturally occur in the cells, such as a transgene and recombinant DNAs. In some embodiments, the exogenous DNA is on a plasmid or stably integrated into the genome of the host cell from an exogenous source. In some embodiments, whether endogenous or exogenous, the target DNA is upstream or in proximity to a target gene encoding for a gene product. For example, in some embodiments, the target DNA is greater than 50 base pairs upstream of the transcription start site of a target gene. In some embodiments, the target DNA is less than 50 base pairs upstream of the transcription start site of a target gene. In some embodiments, the target DNA is within the gene body of the target gene. The target gene product may be any gene product endogenous to the cell or provided exogenously as described above. In some embodiments, the gene product comprises a reporter gene. In some embodiments, the host cell is a bacterial cell. As used herein, the term “reporter gene” refers to a polynucleotide that encodes a reporter molecule that can be detected, either directly or indirectly, when expressed under control of its promoter. The reporter gene includes all the required sequence elements required for synthesis of the reporter molecule. Reporter genes facilitate the rapid analysis of a large number of cells by allowing selective measurement of the reporter gene product. Any number of reporter genes and the means of measuring or detecting the gene product of the reporter gene are known in the art. In some embodiments, the reporter gene may encode any one or combinations of fluorescent proteins, bioluminescent proteins, enzymes, antigenic epitopes, growth selection markers, and the like. The target sequence and guide sequence need not exhibit complete complementarity, provided that there is sufficient complementarity to cause hybridization and promote binding and association with the Cas9-transcriptional effector conjugate. A target sequence may comprise any polynucleotide, such as DNA or RNA. Suitable DNA/RNA binding conditions include physiological conditions normally present in a cell. Other suitable DNA/RNA binding conditions (e.g., conditions in a cell-free system) are known in the art; see, e.g., Sambrook, referenced herein and incorporated by reference. The strand of the target DNA that is complementary to and hybridizes with the DNA-targeting RNA is referred to as the “complementary strand” and the strand of the target DNA that is complementary to the “complementary strand” (and is therefore not complementary to the DNA-targeting RNA) is referred to as the “noncomplementary strand” or “non-complementary strand.” The gRNA may be a crRNA, crRNA/tracrRNA (or single guide RNA, sgRNA). The terms “gRNA,” “guide RNA” and “CRISPR guide sequence” may be used interchangeably throughout and refer to a nucleic acid comprising a sequence that determines the binding specificity of the CRISPR-Cas system. A gRNA hybridizes to (complementary to, partially or completely) a target nucleic acid sequence (e.g., the genome) in a host cell. The system may further comprise a target nucleic acid. The gRNA or portion thereof that hybridizes to the target nucleic acid (a target site) may be between 15-40 nucleotides in length. gRNAs or sgRNA(s) can be between about 5 and 100 nucleotides long, or longer. To facilitate gRNA design, many computational tools have been developed (See Prykhozhij et al. (PLoS ONE, 10(3): (2015)); Zhu et al. (PLoS ONE, 9(9) (2014)); Xiao et al. (Bioinformatics. Jan. 21, 2014); Heigwer et al. (Nat Methods, 11(2): 122-123 (2014)). Methods and tools for guide RNA design are discussed by Zhu (Frontiers in Biology, 10 (4) pp 289-296 (2015)), which is incorporated by reference herein. Additionally, there are many publicly available software tools that can be used to facilitate the design of sgRNA(s); including but not limited to, Genscript Interactive CRISPR gRNA Design Tool, WU-CRISPR, and Broad Institute GPP sgRNA Designer. There are also publicly available predesigned gRNA sequences to target many genes and locations within the genomes of many species (human, mouse, rat, zebrafish,C. elegans), including but not limited to, IDT DNA Predesigned Alt-R CRISPR-Cas9 guide RNAs, Addgene Validated gRNA Target Sequences, and GenScript Genome-wide gRNA databases. To construct cells that express the present system, expression vectors for stable or transient expression of the present system may be constructed via conventional methods and introduced into host cells. For example, nucleic acids encoding the components of the present system may be cloned into a suitable expression vector, such as a plasmid in operable linkage to a suitable promoter. The first nucleic acid, the at least one second nucleic acid, and the at least one third nucleic acid may be provided on a single vector or different vectors. For example, each of the first nucleic acid, the at least one second nucleic acid, and the at least one third nucleic acid may be provided on a first, second and third vector (e.g., plasmid), respectively. Any of the vectors comprising a nucleic acid sequence that encodes the components of the present system is also within the scope of the present disclosure. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding components of the present system into cells. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), and a nucleic acid. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors. Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Examples of delivery vehicles include ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan. 1; 459(1-2):70-83), incorporated herein by reference. When introduced into the host cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA. Promoters for the expression of the components that may be used include T7 RNA polymerase promoters, constitutiveE. colipromoters, and promoters that could be broadly recognized by transcriptional machinery in a wide range of bacterial organisms. The system may be used with various bacterial hosts. Drug selection strategies may be adopted for positively selecting for cells that underwent successful introduction into a cell or cells. Plasmids that are non-replicative, or plasmids that can be cured by high temperature may be used. The present disclosure also provides for DNA segments encoding the proteins disclosed herein, vectors containing these segments and host cells containing the vectors. The vectors may be used to propagate the segment in an appropriate host cell and/or to allow expression from the segment (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a cloned DNA sequence. In one embodiment, a DNA segment encoding the present protein(s) is contained in a plasmid vector that allows expression of the protein(s) and subsequent isolation and purification of the protein produced by the recombinant vector. Accordingly, the proteins disclosed herein can be purified following expression, obtained by chemical synthesis, or obtained by recombinant methods. In some embodiments, the system is a cell-free system. Cas9-Transcription Effector Fusion Proteins Also disclosed herein are fusion proteins comprising a Cas9 protein linked to a transcriptional effector. The Cas9 protein can be obtained from any suitable microorganism, and a number of bacteria express Cas9 protein orthologs or variants (see, e.g., U.S. Pat. No. 10,266,850 incorporated herein by reference) and may be used in connection with the present disclosure. The amino acid sequences of Cas proteins from a variety of species are also publicly available through the GenBank and UniProt databases. In some embodiments, the Cas9 protein is a catalytically-dead Cas9. Catalytically-dead Cas9 is essentially a DNA-binding protein due to, typically, two or more mutations within its catalytic nuclease domains which renders the protein with very little or no catalytic nuclease activity. For example,Streptococcus pyogenesCas9 may be rendered catalytically dead by mutations of D10 and at least one of E762, H840, N854, N863, or D986, typically H840 and/or N863A (see, e.g., U.S. Pat. No. 10,266,850, incorporated herein by reference). Mutations in corresponding orthologs are known. Oftentimes, such mutations cause catalytically-dead Cas9 to possess no more than 3% of the normal nuclease activity. The transcriptional effector may be linked to the Cas9 protein at the N or C terminus. In some embodiments, the transcriptional effector is linked to the C-terminal end of the Cas9 protein. In some embodiments, a linker (e.g., a peptide linker) is used to link the Cas9 protein and the transcriptional effector. The linkers may comprise any amino acid sequence of any length. The linkers may be flexible such that they do not constrain either of the two components they link together in any particular orientation. The linkers may essentially act as a spacer. In select embodiments, the linker links the C-terminus of the Cas9 protein to the N-terminus of the transcriptional effector. In some embodiments, the linker comprises an amino acid sequence of SAGGGGSGGGGS (SEQ ID NO:1) or CAGGGGSGGGGS (SEQ ID NO:2). The transcriptional effector may include a transcriptional activator and/or a transcriptional repressor. The transcriptional effector may be selected from the group consisting of B42 transactivation domain (B42), BTAD domain-containing protein 1 (BTAD1), BTAD domain-containing protein 2 (BTAD2), transcription elongation factor GreA (GreA), RNA polymerase-binding transcription factor DksA (DksA), regulatory protein SoxS (SoxS), N4 single stranded binding protein, Motility Protein A (MotA), 10 kDa anti-sigma factor (AsiA), omega subunit of DNA-dependent RNA polymerase (w), or a fragment or variant thereof. The transcriptional effector may be a putative transcriptional effector. The putative transcription effector may be confirmed or identified by the methods described elsewhere herein. In some embodiments, the transcriptional effector comprises AsiA, or a fragment or variant thereof. In some embodiments, the transcriptional effector comprises an amino acid sequence of wild-type AsiA (SEQ ID NO: 80). In select embodiments, the transcriptional effector comprises a variant of AsiA having mutations in any or all of Q51, V58, and E60 of SEQ ID NO: 80. In some embodiments, the transcriptional effector comprises an amino acid sequence of SEQ ID NO: 80 with a Q51R mutation, V58I mutation, E60K mutation, or any combination thereof. In select embodiments, the transcriptional effector comprises an amino acid sequence of SEQ ID NO:95 or SEQ ID NO: 96. Also provided for herein are nucleic acids encoding the fusion protein and cells (e.g., bacterial cells) comprising the nucleic acids and/or fusions proteins. The nucleic acids may be contained on a vector (e.g., an expression plasmid or vector with a promoter, as described herein). Methods for Altering Transcription Also disclosed herein are methods for altering transcription in a bacteria by introducing into a bacterial cell the system disclosed herein. The descriptions and embodiments provided above for the system components (gRNA, Cas9-transcriptional effector fusion, target DNA, and bacteria) as well as methods of delivery the components provided elsewhere herein are applicable to the methods for altering transcription in a host cell. In some embodiments, the introduction of the at least one guide RNA (gRNA) and/or at least one second nucleic acid encoding the guide RNA sequence, the fusion protein comprising Cas9 protein linked to a transcriptional effector or variant or fragment thereof and/or a first nucleic acid encoding the fusion protein and the at least one reporter gene and/or at least one third nucleic acid encoding the reporter gene, if applicable is simultaneous or nearly simultaneous. In some embodiments, all the components may be introduced, in any order, with a time period separating each introduction. Identifying and Screening for Putative Transcriptional Effectors Also disclosed herein are methods for screening for or identifying a putative transcriptional effector. The methods may comprise introducing into a bacterial host cell a plurality of putative transcriptional effectors linked to a Cas9 protein or a first nucleic acid encoding a putative transcriptional effector linked to a Cas9 protein, at least one guide RNA (gRNA) and/or at least one second nucleic acid encoding the at least one guide RNA sequence, wherein the at least one gRNA is complementary to a target DNA sequence, and a third nucleic acid comprising the target DNA sequence adjacent to at least one reporter gene encoding a gene product; determining the presence or relative quantity of the gene product in the bacterial host cell; isolating bacterial host cells showing a change in quantity of the gene product relative to those host cells lacking the putative transcriptional effector or the gRNA; and identifying the putative transcriptional effector by isolating DNA and/or RNA from the isolated bacterial host cells and sequencing the isolated DNA and/or RNA. The descriptions and embodiments provided above for the second nucleic acid, the gRNA, the third nucleic acid, the target DNA sequence and the bacterial host cell provided elsewhere herein are applicable to the methods for screening for or identifying a putative transcriptional effector. The introduction of the a plurality of putative transcriptional effectors linked to a Cas9 protein or a first nucleic acid encoding a putative transcriptional effector linked to a Cas9 protein, at least one guide RNA (gRNA) and/or at least one second nucleic acid encoding the at least one guide RNA sequence, wherein the at least one gRNA is complementary to a target DNA sequence, and a third nucleic acid comprising the target DNA sequence adjacent to at least one reporter gene encoding a gene product is simultaneous or nearly simultaneous. In some embodiments, all the components may be introduced, in any order, with a time period separating each introduction. The Cas9 protein can be obtained from any suitable microorganism, and a number of bacteria express Cas9 protein orthologs or variants (see, e.g., U.S. Pat. No. 10,266,850, incorporated herein by reference in its entirety) and may be used in connection with the present disclosure. The amino acid sequences of Cas proteins from a variety of species are also publicly available through the GenBank and UniProt databases. In some embodiments, the Cas9 protein is a catalytically-dead Cas9. Catalytically-dead Cas9 is essentially a DNA-binding protein due to, typically, two or more mutations within its catalytic nuclease domains which renders the protein with very little or no catalytic nuclease activity. For example,Streptococcus pyogenesCas9 may be rendered catalytically dead by mutations of D10 and at least one of E762, H840, N854, N863, or D986, typically H840 and/or N863A (see, e.g., U.S. Pat. No. 10,266,850, incorporated herein by reference in its entirety). Mutations in corresponding orthologs are known. Oftentimes, such mutations cause catalytically-dead Cas9 to possess no more than 3% of the normal nuclease activity. The transcriptional effector may be linked to the Cas9 protein at the N or C terminus. In some embodiments, the transcriptional effector is linked to the C-terminal end of the Cas9 protein. In some embodiments, a linker (e.g., a peptide linker) is used to link the Cas9 protein and the transcriptional effector. The linkers may comprise any amino acid sequence of any length. The linkers may be flexible such that they do not constrain either of the two components they link together in any particular orientation. The linkers may essentially act as a spacer. In select embodiments, the linker links the C-terminus of the Cas9 protein to the N-terminus of the transcriptional effector. In some embodiments, the linker comprises an amino acid sequence of SAGGGGSGGGGS (SEQ ID NO:1) or CAGGGGSGGGGS (SEQ ID NO:2). As described above, cells that contain the first nucleic acid, the at least one second nucleic acid, and the at least one third nucleic acid can be constructed using expression vectors for stable or transient expression of the components via conventional methods for vector construction and introduction into the host bacterial cell. For example, nucleic acids encoding the components of the present system may be cloned into a suitable expression vector, such as a plasmid in operable linkage to a suitable promoter. The first nucleic acid, the at least one second nucleic acid, and the at least one third nucleic acid may be provided on a single vector or different vectors. For example, each of the first nucleic acid, the at least one second nucleic acid, and the at least one third nucleic acid may be provided on a first, a second, and a third vector (e.g., plasmid), respectively. The descriptions and embodiments provided above for the reporter gene are applicable to theses methods as well. In some embodiments, the reporter gene encodes a fluorescent protein, a selection marker, or a combination thereof. In some embodiments, the selection marker comprises a degradation tag. The degradation tag may comprise an amino acid sequence of AANDENYALAA (SEQ ID NO: 66). Thus, the methods for determining the presence or relative quantity of the gene product in the bacterial host cell and/or isolating bacterial host cells showing a change in quantity of the gene product relative to those host cells lacking the putative transcriptional effector or the gRNA may comprise fluorescence detection (fluorescence-activated cell sorting (FACS), fluorescence microscopy, or the like) and or antibiotic or drug selection (colony selection by plate based methods), for example. The methods may also be used to screen for variants of the identified putative transcriptional effectors. In some embodiments, the methods further comprise mutating the putative transcriptional effector to create a library of mutant transcriptional effectors and repeating the method with the library of mutant transcriptional effectors. Methods for mutating protein sequences are well known in the art, including for example, error prone PCR of the nucleic acid sequence encoding the putative transcription factor as described herein. Kits Also within the scope of the present disclosure are kits that include the components of the present system. The kit may include instructions for use in any of the methods described herein. The instructions can comprise a description of the system, components, and/or related methods. Kits optionally may provide additional components such as buffers, selection antibiotics or drugs, host cells or bacteria clones, plasmids, or vectors without the components, etc. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above. The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. EXAMPLES The following are examples of the present invention and are not to be construed as limiting. Materials and Methods Strains and Culturing Conditions E. colistrains and other bacterial species herein are listed in Table 1 and allE. colistrains were derived from the MG1655 parental background. Cells were grown in rich LB medium at 37° C. with agitation unless stated otherwise. For plasmid transformation, general protocols were followed, and plasmids were maintained under antibiotics selection at all times. For constructing genomic insertions, GFP expression cassette amplified from pWJ89 was cloned between multiple cloning sites of pOSIP-Kan and inserted chromosomally following the clonetegration method (St-Pierre et al., ACS Synth Biol 2: 537-541, incorporated herein by reference in its entirety). For the antibiotic selection and induction of target genes, the following concentrations were used: Carbenicillin (Carb) 50 μg/ml, Chloramphenicol (Cam) 20 μg/ml, Kanamycin (Kan) 50 μg/ml, Spectinomycin (Spec) 50 μg/ml, Bleocin (Bleo) 5 μg/ml, Anhydrotetracycline (aTc) 100 ng/ml. For induction of target genes, aTc was added to the culture at the exponential growth phase for 4 hours before cells were harvested for characterization. TABLE 1Bacterial strains and speciesStrainSpeciesNameGenotypeNoteEscherichiaBW25113F-, DE(araD-araB)567,Wild-type cellcolilacZ4787(del)::rrnB-3,LAM-, rph-1,DE(rhaD-rhaB)568,hsdR514EscherichiaWT-GFPF-, Δ(araD-araB)567,Wild-type cellcoliΔlacZ4787(::rrnB-3), λ-,chromosomallyrph-1, DE(rhaD-rhaB)568,inserted withhsdR514,GFP cassetteatt::[ϕ21 Wj89-GFP]EscherichiaJEN202F-, ΔrpoZDeletion of omegacolisubunit of RNAPSalmonellaSerovarSource: ATCCentericaTyphi Ty2700931KlebsiellaM5A1Source: DSMoxytoca7342 Construction of Plasmids The dCas9 fusion library was constructed based on the pdCas9-bacteria plasmid (Addgene #44249). Linker sequences (SAGGGGSGGGGS (SEQ ID NO: 1)) and fusion candidates were either amplified from DNA synthesized de novo (IDT Gblocks®) orE. coligenomic DNA and subcloned after the dCas9 sequence in the pdCad9-bacteria plasmid (Addgene #44249). All guide RNA plasmids (pgRNA-H1 to pgRNA-H21) were constructed from a pgRNA-bacteria plasmid (Addgene #44251) using inverted PCR and blunt-end ligation to modify the N20 seed sequences. For dual gRNA plasmids (pgRNA-H4H5, pgRNA-H4H11), each gRNA was built separately and jointed subsequently. GFP reporter plasmids (pWJ89, pWJ96, pWJ97) were provided by the Marraffini lab at Rockefeller University. The promoter region upstream of the GFP reporter in pWJ89 was amplified for constructing other antibiotic reporter plasmids (01E134-37). The GFP-mScarlet reporter plasmid (01E139) was constructed by cloning the mScarlet gene from pEB2-mScarlet-I (Addgene #104007) under WJ89 promoter and joined with the constitutive GFP expression cassette from pWJ97. For screening inducible metagenomic promoter library (RS7003), gRNA-H22 and gRNA-H23 expression cassettes were jointed with dCas9-AsiA_m2.1 separately, resulting 01E140, 01E141. Cloning was done by Gibson assembly if not otherwise noted in all cases. Plasmids used and associated details were listed in Table 2. Development of CasTA Screening Platform dCas9 fusion library, gRNAs, and reporter genes were built on 3 different compatible plasmids (dCas9: p15A, Cam resistance; gRNA: ColE1, Carb resistance; reporter: SC101, Kan resistance), for transformation and propagation within the same cell (FIG.6). To use an antibiotic resistance gene as a reporter, different antibiotic genes were tested and degradation rate (fusion with ssrA tag: AANDENYALAA (SEQ ID NO: 66)) was modulated for selective stringency (FIGS.7A and7B). Dual selective reporters (Kan and Bleo) were constructed, which decreased the escape rate by 10 fold (FIGS.7C and7D). Directed Evolution of dCas9-AsiA Using CasTA Screening Platform The wild-type AsiA region of dCas9-AsiA was mutated using the GeneMorph II EZClone Domain Mutagenesis Kit (Agilent Technologies), following manufacture's protocol. In brief, 50 ng of parental template DNA was used for amplification with error prone DNA polymerase (Mutazyme II). Under this condition, the AsiA region contains on average ˜2 nucleotides changes per variant after PCR mutagenesis (FIG.9). In the first round of directed evolution, dCas9-AsiA mutant library was transformed to the cells expressing gRNA-H4 and dual selective reporters (01E137 and pHH38). Approximately 5×108transformants were grown under 0.2× regular Kan concentration and 2× regular Bleo concentration. Grown colonies were harvested and propagated together with Cam selection to maintain solely the dCas9-AsiA variant plasmids. The dCas9-AsiA plasmids were subsequently extracted and transformed to cells containing pgRNA-H4 and pWJ89. Individual colonies were Sanger sequenced to identify the mutations in AsiA and characterized based on GFP intensity (Table 3). The dCas9-AsiA_m1.1 plasmid from the most abundant mutant variant was extracted and transformed to GFP reporter strain (containing pgRNA-H4 and pWJ89) again to verify fluorescent intensity (FIG.2C). In the second directed evolution round, the dCas9_AsiA_m1.1 variant was used as a template to generate additional variants following the same conditions. The second generation of dCas9-AsiA mutant library was transformed to GFP reporter cells, containing pgRNA-H4 and pWJ89 as described above. The top 0.1% of highest GFP expression were enriched from the population of 1×107transformants using fluorescence activated cell soring (BD FACS Aria II). Post-sorted cell population was plated on selective LB again to obtain clonal colonies, and individual colony was picked for Sanger sequencing and measurement of GFP intensity. TABLE 2PlasmidsPlasmidPromoter forAntibioticReplicationNameDescriptionGOIResistanceOriginpdCas9-linkerFor constructing dCas9 fusionpTetOCamp15Acandidate librarypgRNA-For constructing different gRNAJ23119CarbCOlE1bacteriaplasmidspWJ89Expressing GFP under weakJ23117KanSc101promoterpWJ96Expressing GFP under mediumJ23116KanSc101promoterpWJ97Expressing GFP under strongJ23110KanSc101promoterpdCas9-AsiAExpressing dCas9 fusion AsiApTetOCamp15ApdCas9-Expressing dCas9 fusion AsiApTetOCamp15AAsiA_m1.1variant 1.1pdCas9-Expressing dCas9 fusion AsiApTetOCamp15AAsiA m2.1variant 2.1pdCas9-AsiA-pdCas9-AsiA with modified RBSpTetOCamp15AwRBSsequence from B0034 to B0033pdCas9-pdCas9-AsiA_m2.1 with modifiedpTetOCamp15AAsiA_m2.1-RBS sequence from B0034 towRBSB0033pHH34Expressing Spec resistance geneJ23117KanSc101under weak promoterpHH35Expressing Bleo resistance geneJ23117KanSc101under weak promoterpHH36Expressing Kan resistance geneJ23117KanSc101under weak promoterpHH37Expressing KanR-ssrA under weakJ23117KanSc101promoterpHH38Constitutively expressed gRNA-H4J23119 (gRNA-CarbColE1and Bleo resistance gene, servingH4), J23117for dual antibiotic selection(BleoR)pHH39Expressing mScarlet-I under strongJ23110KanSc101promoter and GFP under weak(mScarlet-I),promoterJ23119 (GFP)pHH40Expressing dCas9-AsiA_m2.1 andpTetI (dCas9-CamColE1gRNA-H22AsiA_m2,1),J23199 (gRNA-H22)pHH41Expressing dCas9-AsiA_m2.1 andpTetI (dCas9-CamColE1gRNA-H23AsiA_m2,1),J23199 (gRNA-H23)pHH42Expressing dCas9-AsiA_m2.1 andpTetI (dCas9-CamColE1gRNA-H24AsiA_m2,1),J23199 (gRNA-H24) TABLE 3Candidates characterized from dCas9-AsiA directed evolutionCycleMutationsFrequencyNote1stV58I, E60K, linker S1C0.76dCas9-AsiA_m1.11stA15V0.04dCas9-AsiA_m1.21stLinker S1C0.04dCas9-AsiA_m1.31stE45K0.021stI70T, linker S1C0.021stL84S, linker S1C0.021stE28D0.021stD6E, I12V, F77S0.021stWT0.042ndQ51R, V58I, E60K, linker S1C0.5dCas9-AsiA_m2.12ndI40V, V58I, S59R, E60K,0.08E85V, linker S1C2ndR23H, Q51P, V58I, E60K,0.08Y81N, linker S1C2ndN29K, V58I, E60K, T88N,0.08plasmid unstablelinker S1C2ndV58I, E60K, L61Q, linker S1C0.082ndN4I, N32K, V58I, E60K,0.08plasmid unstablelinker S1C2ndV58I, E60K, linker S1C0.08dCas9-AsiA_m1.1 Quantification of Gene Expression Induced by CasTA To examine CRISPRa on genomic targets, pdCas9-AsiA_m2.1 was transformed along with gRNA constructs (gRNA-H12 to gRNA-H21, Table 4) designed for each gene (Table 5). Cells expressing dCas9-AsiA_m2.1 and a non-specific gRNA (gRNA-H4) were used as controls. After CRISPRa induction with 100 ng/ml aTc, cells were harvested for RNA extraction following the RNAsnap protocol (Stead et al, 2012). After column purification (RNA Clean & Concentrator Kits, Zymo Research), total RNA was reverse transcribed into cDNA using random hexamers (SuperScript III Reverse Transcriptase, Invitrogen). Quantitative PCR was performed on each sample with gene specific primers (Table 5) using the KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems). The rrsA gene was selected as the housekeeping gene to normalize expression between samples. TABLE 4N20 of gRNAsIDTargetN20SEQ ID NO:H1WJ89ATGTAACACCGTGCGTGTTG4H2WJ89GAAGATCCGGCCTGCAGCCA5H3WJ89GGCTCGAGTCGACAGTTCAT6H4WJ89CTACGGAACTCTTGTGCGTA7H5WJ89GCAAAAGCTCATTTCTGAAG8H6WJ89AACTCTTGTGCGTA9H7WJ89-GFPTTGACAGCTAGCTCAGTCCT10H8WJ89-GFPGCTAGCGAATTCCTTTAAAG11H9WJ89-GFPCCATCTAATTCAACAAGAAT12H10WJ89-GFPGAATTAGATGGTGATGTTAA13H11m Scarlet-ITCTGGGTGCCTTCATACGGA14H13cadBTTTATGTAATAAAAATTATG15H15zraPGCTGTCAGAAAGGGATGAGC16H19iraMTGCCAATTTGCTAAACATTA17H20iraDATAATACATGGCTGATTATG18H21ycgZTTTTTATCAATGTAAAGAAA19H22RS7003AATAATGGTTTCTTAGACGT20promoterlibraryH23RS7003AAAAGGGAATAAGGGCGACA21promoterlibraryH24GenomicAAGCTGAAGAAAAATGAGCA22controlH25dxsCAATTTAATGATAAACTTCA23H26ffhAGTCTTGCGCTGATTGTTCC24H27yehAATACCGATCAGCGCAAGCCA25H28ydiNTTTTTACTGGCACTGTTTAT26H29idiCTGATAAAGATTTAAAAGTC27H30WJ89CGGTGTTACATTAGGCATAC28H31WJ89AACACGCACGGTGTTACATT29H32WJ89CGTGCGTGTTGTGGAAGATC30H33WJ89CGGATCTTCCACAACACGCA31H34WJ89GCCAAGGTGATAATCCATAG32H35WJ89TTATCACCTTGGCTGCAGGC33H36WJ89TGGATTATCACCTTGGCTGC34H37WJ89GCCTCTATGGATTATCACCT35H38WJ89ACTGTCGACTCGAGCCTCTA36H39WJ89CAGTTCATAGGTGATTGCT37H40WJ89CTCAGGACATTTCTGTTAGA38H41WJ89CTTGTGCGTAAGGAAAAGTA39H42WJ89AACACAAACTTGAACAGCTA40H43WJ89TTTCTGAAGAGGACTTGTTG41 TABLE 5Genomic TargetsGeneGeneIDnameForward PrimerReverse Primer945729iraMATTTCTCCCTCCTGGCAGTATGGAGGACACTCTTGACTGC(SEQ ID NO: 42)(SEQ ID NO: 43)948851iraDAACCCGAGCGACAAACATCTGAGTGTGGCAGTACGCTTCT(SEQ ID NO: 44)(SEQ ID NO: 45)945885ysgZCTCAGCAGGAAACTCTCGGGCTGTTCCTCTTCCCCAGTCG(SEQ ID NO: 46)(SEQ ID NO: 47)948654cadBCGGGTATCGCCTGTATTGCTCAAACCAATGCCAGCCAACA(SEQ ID NO: 48)(SEQ ID NO: 49)948507zraPGACAGCGTGGCAGAAAATCCCTTTGGCGACCGCGTTAATT(SEQ ID NO: 50)(SEQ ID NO: 51)945060DxsAAGGCCCGCAGTTCCTGCATGGCAAACCGCCGCTACTTTTC(SEQ ID NO: 52)(SEQ ID NO: 53)947102FfhCTGCAAGGTGCCGGTAAAACTCAAGCTGTTTGATTGCCGC(SEQ ID NO: 54)(SEQ ID NO: 55)946642yehATGGCAAGTCATGGGATGCATAATCGTCCGGTTTGCAGGTT(SEQ ID NO: 56)(SEQ ID NO: 57)946198ydiN TTTCCTGCACGGCATTAGTGTATCAATCGCCCCAAACCGAT(SEQ ID NO: 58)(SEQ ID NO: 59)949020IdiATCTCGCGTTCTCCAGTTGGGATCACTGCGTCTTCGTTGC(SEQ ID NO: 60)(SEQ ID NO: 61)948332rrsACTCTTGCCATCGGATGTGCCCACCAGTGTGGCTGGTCATCCTCTSEQ ID NO: 62)CA (SEQ ID NO: 63) For whole-transcriptome analysis of CRISPRa specificity, total RNA from the samples was extracted as described above and rRNAs were depleted using Ribo-Zero rRNA removal-Bacteria kit (Illumina). RNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs) and sequenced on the Illumina NextSeq platform (Mid-Output Kit, 150 cycles). The raw reads were aligned to the reference genome (BW25113) using Bowtie 2, and the read counts of each gene were quantified by HTseq. Expression level of individual gene was normalized by total read counts within each sample. Screening for CRISPRa Mediated Inducible Promoters Metagenomic promoter library (RS7003) was derived from Johns et al. (Nat Methods 15: 323 (2018), incorporated herein by reference in its entirety). About 8,000 regulatory elements were transformed to cells expressing dCas9-AsiA_m2.1 and either gRNA-H22, gRNA-H23 or genomic targeting gRNA-H24 (Table 4). After CRISPRa induction, four biological replicates were harvested to measure promoter activity. A constitutive promoter without CRISPRa induction (ID:14076, Table 6) was spiked in the cell populations for normalizing expression levels between samples. Total RNA was extracted and purified as previously described. Gene specific primers were used for cDNA generation (Maxima reverse transcriptase, Thermo Scientific), and an RNA sequencing library was prepared by ligation with the common adaptor primer for downstream sequencing. To quantify abundance of each promoter in the library, plasmid DNA from each sample was also extracted using PrepGem bacteria kit (Zygem) and used to generate a DNA amplicon sequencing library. Both RNA and DNA libraries were sequenced on the Illumina NextSeq platform (Mid-output kit, 300 cycles). TABLE 6SequencesSequenceLinkerSAGGGGSGGGGS (SEQ ID NO: 1)MS2 HairpinGCGCACATGAGGATCACCCATGTGCT (SEQ ID NO: 64)MCP-AsiAMASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSAGGGGSGGGGSGGGGSMNKNIDTVREIITVASILIKFSREDIVENRANFIAFLNEIGVTHEGRKLNQNSFRKIVSELTQEDKKTLIDEFNEGFEGVYRYLEMYTNK (SEQ ID NO: 65)DegradationAANDENYALAA (SEQ ID NO: 66)TagWeak RBSTCACACAGGAC (SEQ ID NO: 67)Strong RBSAAAGAGGAGAAA (SEQ ID NO: 68)ConstitutiveGTATACTTTTTTTAAAGAAAAGATTTACAAGCGCACTTTTCTTTAAPromoterTATCTTACAATAATGTAAGTTTGAACAGGAGAATGTAAGCCAAAGCGATGGCTACGCATTCTCTTTCTTTGTTATACTAACACCATATTCGAGGTAGAAAATTATTTAGGAGGATAGAT (SEQ ID NO: 69) Sequencing reads from DNA and RNA libraries were merged by BBmerge and low quality reads were filtered out. Custom pipelines that were previously described (Yim et al., (2019) Mol Syst Biol 15: e8875, incorporated herein by reference in its entirety) were adopted to identify sequencing reads corresponding to each promoter. Expression level of each promoter was quantified by determining the ratio of RNA abundance over DNA abundance. To compare across samples, expression levels were normalized to the same spiked-in control promoter in each sample. Fold change in CRISPRa induced gene expression was calculated by dividing by the reporter expression of control cells containing dCas9-AsiA_m2.1 and a genomic targeting gRNA-H24. Example 1 A Screening-Selection Platform for Bacterial CRISPRa Development To expedite the discovery of bacterial CRISPRa components, a screening-selection platform inEscherichia coliwas developed to identify candidate dCas9-mediated transcription activators. In the CRISPRa design, aS. pyogenesdCas9 was C-terminally fused with candidate transcription activation domains or proteins via a previously described flexible peptide linker (SAGGGGSGGGGS—SEQ ID NO: 1). This CasTA then used a specific gRNA to target to the regulatory region of a reporter gene for transcriptional activation, gene expression, and production of reporter products (FIG.1A). The three components of the platform (dCas9-activator fusion, the guide RNA, and the reporter gene) were separated into 3 compatible plasmids (FIG.1B). The dCas9-activator was regulated by a PtetO induction system with anhydrotetracycline (aTc) on a p15A medium copy plasmid, while the gRNA was constitutively expressed from a strong promoter (BBa_J23119) on a high copy ColE1 plasmid, and the reporter was placed behind a very weak promoter (BBa_J23117) on a low copy SC101 plasmid (FIG.6). Since different dCas9 activators may have their own respective optimal gRNA binding windows and possible biases toward targetable promoter sequences, the screening-selection platform was designed to be highly modular to facilitate combinatorial assessment of system components. As library-scale screening for transcription activators can often be hampered by auto-activators in the population, a dual screening-selection reporter design using both fluorescent protein and antibiotic resistance genes was employed to eliminate potential false positive clones. The selective reporter was engineered to contain multiple separate antibiotic genes with degradation tags (BBa_M0050) to increase the rate of turnover to reach higher stringency and specificity of the selection (see Methods,FIG.7). Using this platform, a list of transcriptional activator candidates, including phage proteins, transcription factors, and RNAP interacting proteins (Table 7), paired with different gRNAs (gRNA-H1, gRNA-H2, gRNA-H3) targeted to different spacing distances to transcriptional start site (TSS) of the reporter gene (60 bp, 85 bp, 120 bp, respectively), were screened for potential dCas9-activators. Among the transcription activation modules screened, a phage protein, AsiA, was found that upregulated the reporter gene expression to a level comparable to the previously identified dCas9-ω activator, although at a different optimal spacing distance (FIGS.1C-1D). AsiA (Audrey Stevens' inhibitor A) is a 90 amino acid anti-σ70 protein from T4 bacteriophage that binds to the host σ70 subunit and suppresses endogenous gene expression. In combination with another T4 phage protein, MotA, the σ70-AsiAMotA complex specifically binds to T4 phage promoters and activates phage transcription during the T4 viral life cycle. TABLE 7dCas9 Fusion CandidatesBindingpartner ofCandidateCategoryRNAPSequenceNotesB42RNAPUnspecifiedGINKDIEECNAIIEQFIDYLRTbindingGQEMPMEMADQAINVVPGMTPKTILHAGPPIQPDWLKSNGFHEIEADVNDTSLLLSGDAS(SEQ ID NO: 70)BTAD1RNAPUnspecifiedAEGALDLARAQDLASAAEKABacterialbindingRSAGDLCHARDLLRRALDLWtranscriptionalDGEVLAGVPGPYAQTQRVRLactivationGEWRLQLLETRLDMDLDQGdomain fromCHAEAVSELTALTAAHPLREStreptomycesRLRELLMLALYRSGRQAEALantibioticAVYADTRRLLADELGVDPRPregulatoryGLQELQQRILQADPALAprotein(SEQ ID NO: 71)BTAD2RNAPUnspecifiedPPSTVDVNRFERDADDGQELBacterialbindingLQRGDAAGGTKLGHALALWtranscriptionRGPALADVVASGRLFSYVTRactivationLEELRFRILELRIEADLATGRHdomain fromRELVSELKSLVLAHPLHEHLHStreptomycesGLLMLALHRSGRPHEALEVYantibioticRSVRHKMIEDLALEPAQDFAregulatoryTLHHTLLSDSPPEAprotein(SEQ ID NO: 72)GreATranscriptionBeta andMQAIPMTLRGAEKLREELDFType IIfactorbeta'LKSVRRPEIIAAIAEAREHGDLtranscriptionsubunitKENAEYHAAREQQGFCEGRIfactorKDIEAKLSNAQVIDVTKMPNNGRVIFGATVTVLNLDSDEEQTYRIVGDDEADFKQNLISVNSPIARGLIGKEEDDVVVIKTPGGEVEFEVIKVEY(SEQ ID NO: 73)DksATranscriptionUnspecifiedMQEGQNRKTSSLSILAIAGVEType IIfactorPYQEKPGEEYMNEAQLAHFRtranscriptionRILEAWRNQLRDEVDRTVTHfactorMQDEAANFPDPVDRAAQEEEFSLELRNRDRERKLIKKIEKTLKKVEDEDFGYCESCGVEIGIRRLEARPTADLCIDCKTLAEIREKQMAG (SEQ ID NO: 74)DksATranscriptionUnspecifiedMQEGQNRKTSSLSILAIAGVEDksA mutantD74EfactorPYQEKPGEEYMNEAQLAHFRwith higherRILEAWRNQLRDEVDRTVTHbindingMQDEAANFPDPVDRAAQEEEaffinity toFSLELRNRDRERKLIKKIEKTLRNAPKKVEDEDFGYCESCGVEIGIRRLEARPTADLCIDCKTLAEIREKQMAG (SEQ ID NO: 75)DksATranscriptionUnspecifiedMQEGQNRKTSSLSILAIAGVEDksA mutantD74NfactorPYQEKPGEEYMNEAQLAHFRwith higherRILEAWRNQLRDEVDRTVTHbindingMQDEAANFPDPVDRAAQEEEaffinity toFSLELRNRDRERKLIKKIEKTLRNAPKKVEDEDFGYCESCGVEIGIRRLEARPTADLCIDCKTLAEIREKQMAG (SEQ ID NO: 76)SoxSTranscriptionAlphaMSHQKIIQDLIA WIDEHIDQPLSoxS variantG32AfactorsubunitNIDVVAKKSAYSKWYLQRMwith defectiveFRTVTHQTLGDYIRQRRLLLADNA bindingAVELRTTERPIFDIAMDLGYVabilitySQQTFSRVFRRQFDRTPSDYRHRL (SEQ ID NO: 77)N4SSBPhageBeta andMSNLFGNLAGQAAKAEKATproteinbeta'DNLGGGFGAKESDIYLATLKsubunitVAYAGKAASGANFIQIIADLTDLDGHSAGEYREQLYITSGTEKGCKCTYEKNGKEYFLPGYTVINDILVMTSGETIPEAVFEEKVVNVYDFDEKKEVAKSVMVPVNAIGGKFAVAILKSEEDKQTKDGSGNYVSTGETRFTNTIEKVFHPDLHLTVVEAEELTERGKELTVEEAVFWDKWLEKNKGVTRDKTTKGGASGKAGQPPKPGATNTGAGASAAKSLFGKK (SEQ ID NO: 78)MotA-NPhageSigmaDLGNAVVNSNIGVLIKKGLVMotA variantproteinfactorEKSGDGLIITGEAQDIISNAATwith truncationLYAQENAPELLKKRATRKARof DNAEITSDMEEDKDLMLKLLDKNbindingGFVLKKVEIYRSNYLAILEKRdomain at theTNGIRNFEINNNGNMRIFGYKC-terminusMMEHHIQKFTDIGMSCKIAKNGNVYLDIKRSAENIEAVITVA (SEQ ID NO: 79)AsiAPhageSigmaMNKNIDTVREIITVASILIKFSHighlightedproteinfactorREDIVENRANFIAFLNEIGVTHresidues areEGRKLN NSFRKI S LTQEDthose mutatedKKTLIDEFNEGFEGVYRYLEin m1.1 (V58I,MYTNK (SEQ ID NO: 80)E60K) andm2.1 variant(Q51R, V58I,E60K)ωRNAPSigmaMARVTVQDAVEKIGNRFDLVsubunitfactorLVAARRARQMQVGGKDPLVPEENDKTTVIALREIEEGLINNQILDVRERQEQQEQEAAELQAVTAIAEGRR (SEQ ID NO:81) When directly fused to dCas9 with a peptide linker, AsiA upregulated gene expression of a GFP reporter, with the magnitude of activation tunable via design of the gRNA. Transcriptional activation by dCas9-AsiA (dubbed CasTA1.0) was seen across a wide window along the target regulatory region, reaching up to 12-fold at ˜200 base pairs (bp) from the TSS (FIG.1E). In contrast, the optimal gRNA targeting positions for other dCas9 activators (e.g., dCas9-ω and dCas9-MS2/MCP-SoxS) was 100 bp or less from the TSS with a narrower targetable window, possibly suggesting a distinct mechanism of activation by dCas9-AsiA. Unlike other dCas9 activators that mediate activation with re-engineered endogenous transcription factors, AsiA is an anti-σ70 protein that has evolved to outcompete host transcriptional machinery. The strong interaction between AsiA and σ70 may result in a different mode of activation from other systems. Simultaneously targeting with multiple gRNAs furthered increase transcriptional activation (FIG.1F), although no synergistic enhancement was observed in contrast to eukaryotic CRISPRa systems. Based on different CRISPRa architectures that have been described in literature, tethering of AsiA to other parts of the dCas9 complex was explored. The MS2 hairpin RNA has been engineered in the gRNA to enable recruitment of transcription activation domains linked to a MCP domain, such as in the bacterial dCas9-MS2/MCP-SoxS system and the eukaryotic Synergistic Activation Mediator (SAM) system. CasTA-AsiA where the gRNA contains a MS2 domain in different stem loops and where AsiA is tethered to MCP (e.g., dCas9-MS2/MCP-AsiA) was tested. While the MS2 hairpins did not affect the gRNA performance, it was not found that the SAM implementation of AsiA could activate gene activation (FIG.8). These results were in agreement with a previous observation that dCas9-MS2/MCP-AsiA system was not a functional activator. It was also not found that a G32A mutant (DNA binding disruption) of the previously described SoxS activator in the dCas9-MS2/MCP-SoxS system to be functional as a direct dCas9 fusion (e.g., dCas9-SoxSG32A) (FIG.1C), potentially due to the instability of the G32A mutant. These results highlighted potential mechanistic and performance differences between CRISPRa systems where the activation domain is directly fused to dCas9 versus tethered via the MS2-MCP system. Example 2 Directed Evolution and Characterization of the dCas9-AsiA Transcriptional Activator To increase the dynamic range and performance of dCas9-AsiA-mediated transcriptional activation, a series of directed evolution studies using our screening-selection platform were performed. A dCas9-AsiA variant library was constructed by error-prone PCR of AsiA, with each AsiA variant having on average two randomly distributed residue mutations (FIG.9). Approximately 5×108AsiA mutant variants were screened for improved transcriptional activation on antibiotic selection plates (FIGS.2A and7). The resulting colonies were individually isolated and plasmids encoding the dCas9-AsiA variants were extracted and transformed into cells expressing a gRNA and GFP reporter for re-validation (Table 3). Of 47 colonies isolated and characterized, one variant (m1.1) was found most enriched (>75% of the time), while several other variants (m1.2, m1.3) were also identified at lower frequency (FIG.2A-B). The most abundant variant m1.1 after selection also mediated the highest GFP activation (FIG.2C). The m1.1 variant contained two key mutations on AsiA (V58I, E60K). An additional mutation (S1C) on the peptide linker was also found, which likely arose during the cloning steps of the directed evolution protocol. Interestingly, the AsiA mutations occurred within the middle of the anti-σ factor protein and are structurally away from the interface that binds to σ70 (FIG.2B). AsiA binds to sigma factors through the first helix structure (residues 1 to 20), suggesting that the mutations in m1.1 may not affect direct binding to sigma factors. This m1.1 variant significantly increased the transcriptional activation to ˜70 fold compared to ˜10 fold using the wild-type AsiA (FIG.2C). Another round of directed evolution was performed on m1.1 and the resulting clones were screened for additional mutants with further improvements (FIG.2A). From 107 variants, validation and characterization of the resulting colonies revealed an additional mutant (m2.1) to be significantly enriched in the population with >135-fold activation (FIGS.2B and2C). The m2.1 variant contained an additional Q51R mutation, which also faced away from σ70 similar to the other m1.1 mutations. The activation potential of dCas9-AsiA-m2.1 (CasTA2.1) for targeting promoters with different basal expression levels and at different CasTA2.1 expression levels was explored. Transcriptional activation across weak to strong promoters reached similar saturating levels and at the same optimal gRNA targeting distance (FIGS.2D,10A, and10B). The fold induction inversely correlated with the basal promoter strength. To investigate the rules for gRNA designs at finer resolution, gRNA targeting all NGG positions in the weak promoter (BBa_J23117) except for ones overlapping with σ70 binding sites were constructed and paired with CasTA2.1 to mediate gene activation. An additional peak of activation was found at around 100 bps to TSS (FIG.10D). Similar periodicity of optimal gRNA targeting was recently observed in the dCas9-MS2/MCP-SoxS system. However, CasTA2.1 has a generally broader activation window. gRNAs tested with distances of more than 100 bp from the TSS, all led to gene activation from 10- to 288-fold. These 10 gRNAs targeted promoter regions across more than 150 bps, suggesting a flexible window from effective gRNA designs. Transcriptional or translational enhancement of the expression of CasTA1.0 or 2.1 could also increase activation of the target gene (FIG.10C), thus providing different options to tuning the overall system. Since AsiA binds and sequesters the host σ70, overexpression of AsiA may become toxic to the cell. The toxicity of dCas9-AsiA was quantified in the system. Overexpression of CasTA1.0 or 2.1 under aTc induction did not have significant impact on cellular growth rate beyond the basal fitness burden of dCas9 overexpression alone (FIG.11). Doubling times during exponential growth were generally unaffected under CasTA overexpression, while stationary cell density was somewhat impacted. To gain a higher resolution of the effects of CasTA on the endogenous transcriptome, RNAseq was performed on cells with CasTA1.0 and CasTA2.1, relative to ancestral control cells (FIG.12). CasTA2.1 mediated higher gene activation on the GFP target without loss of specificity genome-wide compared to cells with CasTA1.0 (FIG.12A) or ancestral cells (FIG.12B). Upon overexpression of CasTA2.1, upregulation of some low-expression endogenous genes was observed (FIG.12C). These off-target gene activations may be the result of non-specific dCas9 binding to other genomic loci, which has been reported previously. This was supported by the fact that strong off-targets (fold change >30) were regulated by not just σ70 but also other a factors (FIG.12C). Notably, the fold induction of the GFP targets was also higher under significant CasTA2.1 overexpression (FIG.12C), which highlights a trade-off between higher target activation and increased off-targets in this CRISPRa system. Example 3 Utility of dCas9-AsiA for Multi-Gene and Library Scale Transcriptional Regulation To explore whether CasTA can be used to regulate endogenous genomic targets, a GFP reporter was inserted into the genome and CasTA2.1 upregulated the expression of this chromosomal reporter (FIG.3A). Five genes in the genome could be upregulated (by up to 200-fold) using CasTA2.1 (FIGS.3B and14; Table 5). One gRNA was designed for each gene using a search window of 200±20 bp from the TSS. Optimization of gRNA designs may be used for different genomic targets. gRNAs (gRNA-H7 to gRNA-H10) positioned near the TSS or within the gene body of the target GFP reporter efficiently inhibited gene expression using the CasTA2.1 protein, including both strands of the target DNA (FIG.3C). When two different gRNAs were designed to target two reporter genes for concurrent activation and repression, simultaneous CRISPRa and CRISPRi was observed using CasTA2.1 at efficiencies similar to applying CRISPRa or CRISPRi separately (FIG.3D), which highlighted the systems potential utility for multiplexed gene modulation of regulatory networks in a single cell. Development of complex synthetic genetic circuits requires diverse regulatory parts with tunable dynamic rage. However, the number of inducible promoters with defined expression ranged is limited for many applications in synthetic biology. A promoter library from metagenomic sequences with varying species-specific constitutive expression levels was previously developed (Johns et al., 2018 Nat Methods 15: 323, incorporated herein by reference in its entirety). Whether such a constitutive promoter library could be turned into an inducible promoter library was explored using the present CRISPRa system (FIG.4A). Two gRNAs spaced ˜150 bp apart targeting the constant regulatory region upstream of the variable regulatory sequences of each promoter were designed and a screen identified subsets of promoters that could be upregulated by CasTA2.1. The expression level from all promoters in the library with and without CasTA2.1 was quantified by targeted RNAseq (to obtain RNA transcript for each promoter) and DNAseq (to normalize for plasmid copy numbers across the library) as previously described (Yim et al., (2019) Mol Syst Biol 15: e8875, incorporated herein by reference in its entirety) (FIG.13A, Methods). Of approximately 8,000 promoters characterized, thousands of promoters that were activated by CasTA2.1 with at least one of the gRNAs were identified (FIGS.4B and13B). Among them, several hundred had a high level of induction (>10-fold) across 2-orders of magnitude in basal expression level (FIG.4C). In general, more promoters were activated with the distal gRNA (gRNA-H23), although interestingly the proximal gRNA (gRNA-H22) also resulted in CRISPRi activity in some promoters (FIG.13B). The phylogenetic origin and sequence composition of these inducible promoters were diverse, which may facilitate their use for assembly of large genetic circuits with minimal recurrent sequence motifs (FIG.13C). This library of CasTA-inducible promoters greatly expands the repertoire of regulatory parts that can be activated with one or two gRNAs by CRISPRa for more complex genetic circuits in various synthetic biology applications. Example 4 Portability of dCas9-AsiA to Other Bacteria Species Since homologs of the T4 AsiA protein are widely found in many different phages that infect diverse bacteria (FIG.5A), it was hypothesized that the dCas9-AsiA system could be ported to other bacteria with greater possibility of success and minimal re-optimization. Two bacterial speciesSalmonella entericaandKlebsiella oxytocaof clinic and bioindustrial significance were chosen to test the CasTA system. Each of the three plasmids (CasTA, gRNA, reporter) was transformed into the two species. dCas9 was functional in these two species, as confirmed by using a gRNA targeting for repression of a reporter GFP gene (e.g., CRISPRi) activity (FIG.5B). CRISPRa was tested using the CasTA1.0 and 2.1 systems with the appropriate gRNA and GFP reporter. CasTA2.1 showed significant GFP activation in both species, but CasTA1.0 did not. It is interesting to note that AsiA fromSalmonellaphage SG1 shares the same residues at positions 50-61 as theE. coliT4 phage, while theKlebsiellaphage F48 had some differences especially at residues 51-53, 57, and 59, which all face away from the binding surface to σ70. Notably, residues 51-53 and 57-61 of AsiA appear to be more variable across phylogenetically diverse phages (FIG.5A), which are also the key residue regions mutated in m2.1 (Q51R, V58I, E60K) from our directed evolution experiments. In fact, some of the mutant residues in CasTA2.1 are also found in natural AsiA variants, suggesting that the mutations identified might mediate conserved molecular interactions leading to improved gene activation. Together, these results demonstrate that the CasTA system can be ported into other bacteria. The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety. | 69,080 |
11859173 | DETAILED DESCRIPTION The present disclosure relates, at least in part, to living materials fabricated solely from microbial cells at ambient conditions that are stiff, strong and can self-regenerate. Living systems have not only the exemplary capability to fabricate materials (e.g. skin, wood, bone) at ambient conditions but also consist of living cells that enable them properties like growth and self-regeneration. In some aspects of the invention, disclosed herein is the fabrication of stiff living materials (SLMs) produced entirely from microbial cells without the incorporation of any structural biopolymers (e.g., cellulose, chitin, or collagen) or biominerals (e.g., hydroxyapatite, calcium carbonate) that are known to impart stiffness to biological materials. Notably, such SLMs are also lightweight, strong, resistant to organic solvents, and can self-regenerate. The living materials technology disclosed herein can serve as a powerful bio-manufacturing platform to design and develop sustainable structural materials, biosensors, self-regulators, self-healing, and environment-responsive smart materials. In this light, disclosed herein is the fabrication of the stiffest living materials to date, which can also self-regenerate, thereby serving as a unique example of circular material economy (FIG.1A). Most of the biomaterials (e.g., wood, silk) found in nature are formed at ambient conditions and degrade naturally to enable a sustainable eco-system. In contrast, the production of human-made materials (e.g., cement, plastics) require high temperatures or harsh chemical treatments, while their non-biodegradability unfavorably affects the environment. With the growing concerns on the global climate changes, there is an ever-increasing need to design materials for a sustainable world. This sustainability issue can be addressed by employing living cells as factories to produce materials at ambient conditions. Herein, living microbial cells, soft living cells, are utilized to produce stiff and strong materials. Advantages Include: Unlike, typical human-made materials that follow linear material economy (make-use-dispose practices), SLMs represent a unique example of a circular material economy. SLMs are not only biodegradable but can also regenerate. SLMs may be as stiff and strong as plastics, wood, bone. SLMs may be resistant to organic solvents. In some embodiments, SLMs can harness the unique features of living cells such as self-regeneration, self-healing, self-regulation and environmental responsiveness. SLMs may be fabricated at room temperature without using any harmful chemicals. SLMs are cheap and easy to manufacture. SLMs comprise of benign cellular components and thereby offer sustainable solutions. Potential Uses Include: Provided herein are stiff and strong materials, solvent-resistant materials, self-regenerating materials, and methods of making said materials. The technology disclosed herein provides materials that have stiffness and strength similar to plastics, wood, silk, bone and concrete. In some aspects, the invention provides limitless opportunities to design and develop sustainable structural materials, biosensors, self-regulators, self-healing and environment-responsive smart materials. Also provided herein is basis (e.g., a platform) for future materials manufacturing technologies that inevitably rely on living cells. Cost Advantages Include: SLM may be fabricated from living microbial cells that can be grown at large-scale in bioreactors very easily and cheaply. SLM may be fabricated at room temperature by ambient drying and therefore do not involve any expensive processes. SLM can also regenerate. Performance Advantages Include: SLM falls into the circular material economy model, as they can self-regenerate and biodegrade completely under all environmental conditions. Living microbial cells are employed as factories to produce the material, thus the invention disclosed herein provides scalability and ease of fabrication. In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description. The term “extremophile” as disclosed herein refers to a microorganism (i.e., microbial cell) that exhibits optimal growth under extreme environment conditions. Extremophiles include acidophiles, alkaliphiles, halophiles, thermophiles, metalotolerant organisms, osmophiles, and xerophiles. The terms “microbial cell,” “microorganism,” and “microbe” are used interchangeably and should be interpreted to encompass microscopic organism, particularly those commonly studied by microbiologists. Such organisms may include, but are not limited to, bacteria, fungi, and other single-celled organisms including the non-limiting examples of archaea, protozoa, fungi, algae, green algae, rotifers, planarians, and parasitic pathogens. Preferably, the microbes may be bacteria. As used herein, the term “engineered microbial cell,” “engineered microorganism,” and “engineered microbe” refer to a microbial cell that has been genetically modified from its native state. For instance, an engineered microbial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be stably incorporated into the genome of the microbe (e.g., present in the chromosome of a bacteria or bacterial cell), or on an exogenous nucleic acid, such as a plasmid in a bacteria or bacterial cell. Accordingly, engineered microbial cells of the disclosure may comprise exogenous nucleotide sequences on plasmids. Alternatively, recombinant microbial cells may comprise exogenous nucleotide sequences stably incorporated into their genome. In some embodiments, the engineered microbe is non-pathogenic. In some embodiments, the engineered microbe is pathogenic. As used herein, the term “expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, to translation of an mRNA into a polypeptide, and/or the final product encoded by a gene or fragment thereof. The term “genetic modification,” as used herein, refers to any genetic change. Exemplary genetic modifications include those that increase, decrease, or abolish the expression of a gene, including, for example, modifications of native chromosomal or extrachromosomal genetic material. Exemplary genetic modifications also include the introduction of at least one plasmid, modification, mutation, base deletion, base addition, and/or codon modification of chromosomal or extrachromosomal genetic sequence(s), gene over-expression, gene amplification, gene suppression, promoter modification or substitution, gene addition (either single or multi-copy), antisense expression or suppression, or any other change to the genetic elements of a host cell, whether the change produces a change in phenotype or not. Genetic modification can include the introduction of a plasmid, e.g., a plasmid comprising at least one amino acid catabolism enzyme operably linked to a promoter, into a bacterial cell. Genetic modification can also involve a targeted replacement in the chromosome, e.g., to replace a native gene promoter with an inducible promoter, regulated promoter, strong promoter, or constitutive promoter. Genetic modification can also involve gene amplification, e.g., introduction of at least one additional copy of a native gene into the chromosome of the cell. Alternatively, chromosomal genetic modification can involve a genetic mutation. In some aspects of the invention, disclosed herein are engineered living material (ELMs). In some embodiments, said ELMs comprise a plurality of microbial cells, wherein the ELMs have a Young's Modulus of at least 5 Gpa. In some embodiments, the disclosed ELM has a Young's Modulus of at least 5 Gpa to 42 Gpa. In some embodiments, the ELM has a hardness of at least 0.2 Gpa to 2.4 Gpa. In certain embodiments, the ELM has a yield strength of at least 60-800 MPa. Preferably, the ELMs disclosed herein consist essentially of microbial cells. Most preferably, said ELMs consist of microbial cells. Said microbial cells may be prokaryotic or eukaryotic. In some embodiments, the microbial cells are bacterial or fungal cells. In some embodiments, the microbial cells areEscherichia coli, Lactobacillus rhamnosus, orSaccharomyces cerevisiae. In certain embodiments, the microbial cells are engineered to be incapable of producing extracellular components. In some such embodiments, the microbial cells areEscherichia colistrain PQN4. The ELMs disclosed herein may comprise microbial cells which are xerotolerant. In some embodiments, the microbial cells are engineered to have enhanced xerotolerance. In certain embodiments, the microbial cells are extremophilic. In some embodiments, the microbial cells are a xerophilic. In some embodiments, The ELMs disclosed herein comprise an outer surface of lysed and/or desiccated cells; and a core of living cells. Said core of living cells may have a planar packing density of at least 5-7 cells. In some embodiments, the core of living cells has a planar packing density of at least 6 cells. In some embodiments, the cells in the core of living cells die at an exponential rate. In some such embodiments, the core of living cells has a calculated cell death rate of less than 1 cell per day or less than 0.5 cells per day. Preferably, the core of living cells has a calculated cell death rate of about 0.43 cells per day. In some embodiments, the ELMs disclosed herein are capable of self-regeneration. In other embodiments, the ELMs may be fully desiccated; and do not comprise living cells. In some embodiments, said ELMs are resistant to organic solvents. In some aspects of the invention, provided herein are method of fabricating ELMs, wherein said ELMs have a Young's Modulus of at least 5 Gpa. Such methods may comprise proliferating a plurality of engineered xerotolerant microbial cells to produce a population of engineered living xerotolerant microbial cells; isolating and casting said living xerotolerant microbial cells onto a substrate; and allowing the isolated cells to dry; thereby forming an ELM. Said microbial cells may be prokaryotic or eukaryotic. In some embodiments, the microbial cells are bacterial or fungal cells. In some embodiments, the microbial cells areEscherichia coli, Lactobacillus rhamnosus, orSaccharomyces cerevisiae. In certain embodiments, the microbial cells are engineered to be incapable of producing extracellular components. In some such embodiments, the microbial cells areEscherichia colistrain PQN4. The ELMs disclosed herein may be fabricated from microbial cells engineered to have enhanced xerotolerance. In certain embodiments, the microbial cells are extremophilic. In some embodiments, the microbial cells are a xerophilic. In some such embodiments of the invention, the substrate is porous. In preferred embodiments, the substrate comprises polyvinylidene fluoride (PVDF). In some embodiments, the isolated cells are dried at 25° C. for up to 24 hours. Said isolated cells may be drawn down onto the substrate by applying vacuum suction. The dried living material may be removed from the substrate using an organic solvent, preferably dimethylformamide (DMF). In some embodiments, the ELM comprises an outer surface of lysed and/or desiccated cells; and a core of living cells. The core of living cells has a planar packing density of at least 5-7 cells. Preferably, the core of living cells has a planar packing density of at least 6 cells. In some embodiments, the core of living cells has a cell death rate of less than 1 cell per day or less than 0.5 cells per day. Preferably, the core of living cells has a cell death rate of about 0.43 cells per day. In some embodiments, said ELM is capable of self-regeneration. In some such embodiments, said ELM is resistant to organic solvents. In certain aspects of the invention, disclosed herein are engineered biomaterials, comprising a plurality of microbial cells. In some embodiments, the contemplated biomaterials have a Young's Modulus of at least 5 Gpa. In some such embodiments, the biomaterial does not comprise extracellular components. In some embodiments, the contemplated biomaterials do not comprise living microbial cells. In some embodiments, the biomaterial has a Young's Modulus of 5 Gpa to 42 Gpa. In some embodiments, the biomaterial has a hardness of 0.2 Gpa to 2.4 Gpa. In some embodiments, the biomaterial has a yield strength of 60-800 MPa. The biomaterials contemplated herein may consist essentially of microbial cells. In other embodiments, the biomaterial consists of microbial cells. In some such embodiments, the microbial cells are prokaryotic or eukaryotic. In some embodiments, the microbial cells are bacterial or fungal cells. In some such embodiments, the microbial cells areEscherichia coli, Lactobacillus rhamnosus, orSaccharomyces cerevisiae. The microbial cells of the biomaterial may be engineered to be incapable of producing extracellular components. In some embodiments, the microbial cells areEscherichia colistrain PQN4. In some embodiments, the microbial cells are xerotolerant. In some embodiments, the microbial cells are engineered to have enhanced xerotolerance. The microbial cells may be extremophilic. In some embodiments, the microbial cells are xerophilic. In some embodiments, the biomaterial disclosed herein may comprise an outer surface of lysed and/or desiccated microbial cells; and a core of intact desiccated microbial cells. In some such embodiments, the core of intact desiccated microbial cells may have a planar packing density of at least 5-7 cells. In some embodiments, core of intact desiccated microbial cells has a planar packing density of at least 6 cells. In other embodiments, the outer surfaces of the biomaterial comprises an array of desiccated intact microbial cells; and an amorphous core comprising lysed and/or desiccated cells. In some embodiments, biomaterial is resistant to organic solvents. In some aspects of the invention, provided herein are methods of fabricating a biomaterial, wherein the biomaterial has a Young's Modulus of at least 5 Gpa. The disclosed methods may comprise proliferating a plurality of engineered xerotolerant microbial cells to produce a population of engineered living xerotolerant microbial cells; isolating and casting said living xerotolerant microbial cells onto a substrate; and desiccating the isolated cells; thereby forming the biomaterial. Said microbial cells may be prokaryotic or eukaryotic. In some embodiments, the microbial cells are bacterial or fungal cells. In some embodiments, the microbial cells areEscherichia coli, Lactobacillus rhamnosus, orSaccharomyces cerevisiae. In certain embodiments, the microbial cells are engineered to be incapable of producing extracellular components. In some such embodiments, the microbial cells areEscherichia colistrain PQN4. The biomaterials disclosed herein may be fabricated from microbial cells engineered to have enhanced xerotolerance. In certain embodiments, the microbial cells are extremophilic. In some embodiments, the microbial cells are a xerophilic. In some such embodiments of the invention, the substrate is porous. In preferred embodiments, the substrate comprises polyvinylidene fluoride (PVDF). In some embodiments, the methods disclosed herein may further comprise applying vacuum suction to draw down the isolated cells onto the substrate. In some embodiments, the methods further comprise removing from the substrate desiccated biomaterial using an organic solvent, preferably dimethylformamide (DMF). In some embodiments, the fabricated biomaterial comprises an outer surface of lysed and/or desiccated cells; and a core of intact desiccated cells. The core of intact desiccated cells may have a planar packing density of at least 5-7 cells. The core of intact desiccated cells may have a planar packing density of at least 6 cells. In other embodiments, the outer surface of the fabricated biomaterial comprises an array of desiccated intact microbial cells; and an amorphous core comprising lysed and/or desiccated cells. In some embodiments, said biomaterials are resistant to organic solvents. Definitions of common terms in cell biology and molecular biology can be found in The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds. Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); and Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), which are all incorporated by reference herein in their entireties. The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims. Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure. The following examples are set forth as being representative of the present disclosure. These examples are not to be construed as limiting the scope of the present disclosure as these and other equivalent embodiments will be apparent in view of the present disclosure, figures and accompanying claims. Example 1: Materials and Methods Cell Strains and Plasmids SLMs were fabricated from the following cell strains; PQN4,Escherichia colicell strain derived from LSR10 (MC4100, ΔcsgA, λ(DE3), CamR). pET-21d(+) plasmid (Novagen 67743-3) was transformed to PQN4 to get ampicillin resistance.Lactobacillus rhamnosus(ATCC® 27773™)Saccharomyces cerevisiae(ATCC® 9763™) Fabrication of SLMs E. coli(lysogeny broth with carbenicillin, 37° C., 24 h),L. rhamnosus(MRS broth with chloramphenicol, 37° C., 48 h) andS. cerevisiae(YPD broth, 30° C., 24 h) were cultured (500 ml media) in an incubator.E. coliandS. cerevisiaecells were pelletized at 3000 rpm, whereas 8000 rpm was employed forL. rhamnosus. The microbial cells were then washed twice (250 ml and 50 ml) with milli-Q water to remove the culture media. (FIG.2) The resulting microbial pellet was casted on a PVDF (polyvinylidene fluoride; Millipore Immobilon-P, IPVH09120) membrane, which was firmly sandwiched between polypropylene molds (inner dimensions 2 cm×2 cm×1.6 mm) by adhesive tapes. The microbial pellet was air-dried at ambient conditions (25° C. & 40±5% relative humidity) for 24 h. PVDF membrane strongly adheres to EC-SLM and LR-SLM, but did not stick to SC-SLM. By gently wiping the PVDF membrane with DMF (dimethylformamide) solvent and after 5-10 min, it can be easily peeled off. The SLMs were stored at ambient conditions and used as needed. Colony Forming Unit (CFU) Analysis of SLMs 5-10 mg of SLM or 20-100 mg of the microbial pellet (water washed) was subjected to serial dilutions and each dilution was plated onto a selective agar plate. The resulting colonies were counted to obtain the CFU count. For time-dependent CFU analysis, the SLMs stored at ambient conditions were utilized at day 0, 15 and 30. Thermal Gravimetric Analysis (TGA) TGA experiments were performed using a TA Q5000 IR instrument. SLMs (5-10 mg) were run at 5° C. min−1under N2purging at 50 mL min−1in platinum pans. Dynamic Scanning Calorimetry (DSC) DSC measurements were done using a TA Q200 instrument. Measurements were run under N2purging at 40 mL min−1and at 2° C. min−1with ˜5 mg of SLM. Each measurement was performed in aluminum pans in the range of −10 to 100° C. with successive heat-cool cycles. UV-Vis Absorption Spectroscopy UV-Vis spectra were recorded on a Cary 5000 UV-Vis-NIR spectrophotometer (Agilent Technologies) in the range of 400 to 800 nm to obtain their percentage transparency. X-Ray Diffraction (XRD): XRD experiments on SLMs were performed using a Bruker D2 Phaser equipped with a beam of λCuKα=0.15418 nm. The diffraction intensity of SLMs were recorded for 20 in the range of 4° to 80°. Nanoindentation Nanoindentation studies were performed on the samples using the Agilent Technologies G200 Nanoindenter. The machine continuously monitors the load, P, and the depth of the penetration, h, of the indenter with the resolutions of 1 nN and 0.2 nm, respectively. A Berkovich diamond tip indenter with the tip radius of ˜100 nm is used for the indentation. A peak load, Pmaxof 1 mN with the loading and unloading rates of 0.2 mN s−1, Poisson's ratio of 0.3 and a hold time (at Pmax) of 10 s was employed. A minimum of 125 indentations are performed in each case. The P-h curves were analyzed using the Oliver-Pharr method to extract the Young's modulus (E), and the hardness (H) of the samples. The yield strength, σywas estimated using the relation σy=H/3. Field Emission Scanning Electron Microscope (FESEM) FESEM samples were prepared by sputtering a 10-20 nm layer of Pt/Pd/Au. Images were acquired using a Zeiss Ultra55/Supra55VP FESEM equipped with a field emission gun operating at 5-10 kV. Solvent Resistance of SLMs EC-SLMs (˜10 mg) were fully immersed in 2 ml of hexane, chloroform, ethyl acetate, acetonitrile, absolute ethanol, methanol, dimethylformamide (DMF) or milli-Q water. After 24 h incubation, the SLMs immersed in solvents were removed and air-dried overnight to remove any traces of solvent. The weight of EC-SLMs before and after the incubation was noted. As EC-SLM disperses in water, its weight could not be obtained after the incubation. Self-Regeneration of SLMs 5-10 mg of EC-SLM (first generation, Gen I) was added to 500 ml of lysogeny broth supplemented with carbenicillin incubated at 37° C. for 24 h. The cells were pelletized, casted on to the mold and air-dried to obtain the second generation, Gen II of EC-SLM (same fabrication protocol as described above). Similarly, a 5-10 mg fragment of Gen II was utilized to obtain the third generation, Gen III of EC-SLM. Example 2: Evolution and Fabrication of SLMs Engineered Living Materials (ELMs) may be defined as engineered materials composed of living cells that form or assemble the material itself, or modulate the functional performance of the material in some manner. All the ELMs reported so far are essentially soft materials in the form of biofilms, semi-solids or hydrogels that are produced by genetically engineering the extra-cellular matrix of living cells.(12-27) Notably, the stiff structural characteristic of the extra-cellular matrix (viz. curli fibers) can be exploited to fabricate a macroscopic stiff (2-4 GPa) plastic.(28) In contrast to these approaches, it was investigated whether the living cells (without the extra-cellular matrix) alone can be employed to produce a material, and in doing so, may be able to effectively incorporate life-like properties (e.g., self-regeneration, self-regulation, self-healing, environmental responsiveness and self-sustainability) into the resulting material. For this reason, theEscherichia colistrain PQN4 was cultured; the bacteria developed from LSR10 that has been shown to not produce extracellular components such as curli fibers, flagella or cellulose.(12) After culturing for 24 h in lysogeny broth media,E. coliwas pelletized and washed with milli-Q water to remove the nutrient media. The so obtained pellet when drop-casted on a glass slide, upon ambient drying, resulted in a fragmented transparent living material that indicated its brittleness and the ability to form a cohesive material from cells (FIG.3A). Increasing the amount of the pellet within a mold was found to slightly minimize the fragmentation of the living material. However, unlike the top surface (FIG.3F), the bottom surface (FIG.3G) of the living material was found to have patches of cells that were not dried effectively, inhibiting the formation of a cohesive glossy material. It was reasoned that the non-porous nature of the glass substrate might be contributing to ineffective drying of the bottom surface of the living material. Drop casting on porous substrates like copper or stainless-steel mesh circumvented the cell patches but left imprint on the bottom surface of the living material (FIG.3K). In case of nylon membrane, the living material was found to adhere to the substrate, which upon peeling off manually, left ineffectively dried cell patches all along the point of contact with the nylon matrix. On the hydrophobic surface of polytetrafluoroethylene (PTFE) coated stainless-steel, the living material deformed to a curved architecture. Although, the hydrophobic nature of the substrate prevents adhesion to living material, it was reasoned that adhesion to substrate and/or low vacuum suction might aid the fabrication of fragmentation-free and flat living material. Hydrophobic polyvinylidene fluoride (PVDF) membrane that is typically used in western blotting to bind proteins was employed as a substrate. By drop castingE. colicell pellet on PVDF membrane mounted on a Millipore SNAP i.d. Mini Blot Holder, connected to a low vacuum suction, a flat living material was achieved, but with few fragments (FIGS.3U,3V). In the same latter set up, in the absence of low vacuum suction, fragmentation-free flat living materials were also fabricated by ambient drying on PVDF membranes. The strong adhesion of living material with the PVDF membrane did not facilitate its manual peeling but can be easily removed by gently wiping the membrane with dimethylformamide (DMF) solvent. Instead of ambient drying, use of higher temperature (50/75/100° C.) speeds up the formation of living materials but with extensive cracks and discoloration or charring (FIGS.3N-3T). TABLE 1List of parameters and conditionsemployed during the fabrication of SLM.Types/ParameterConditionsRemarksSubstrateGlassThe bottom surface of SLM had scale-like architectures of patches of cellspossibly due to ineffective dryingon the non-porous nature of glasssurface. SLM cracks extensively.CopperThe bottom surface of SLM lackedMeshscale-like architectures.SLM cracks and it is non-flat.The mesh pattern gets imprintedon the bottom surface of SLM.StainlessThe bottom surface of SLM lackedSteel Meshscale-like architectures. SLMcracks and it is non-flat. The meshpattern gets imprinted on the bottomsurface of SLM.NylonAdheres to nylon and the bottomMembranesurface of SLM had tiny scale-likearchitectures when peeled off fromnylon. SLM Cracks.The mesh pattern gets imprintedon the bottom surface of SLM.Polytetra-SLM does not stick to PTFE. SLMfluoro-Cracks and it is deformed to aethylenenon-flat (curvy) shape.(PTFE)CoatedSteel MeshPolyvinyl-SLM adheres to PVDF that cannot beidenepeeled off manually but can be removedfluorideby gently wiping with dimethyl-(PVDF)formamide (DMF) solvent. Flat andfragmentation-free SLMs can be obtained.Tem-25/50/75/Higher temperature speeds up the SLMperature100 □formation but have disadvantages likeextensive cracks, charring/discoloration(depending on temperature and duration)and enhanced cell death. At 25 □, theSLM formation takes up to 24 h.LowMilliporeSLM formation speeds up when vacuumVacuumSNAPsuction (unidirectional;Suctioni.d. Minifrom bottom side of the cellBlot Holderpellet) is applied via a blot holder. BlotVacuumholder set up can facilitate flat SLMs.DesiccatorThe three-dimensional suction in avacuum desiccator results in potholeson SLM that cracks and it is non-flat (highly curvy). Vacuum desiccatortakes longer time for SLM formation.Duration12/24/48 h24 h duration was found to be optimal for(at 25 □)SLM formation at 25 □, while at 100 □,1/2/3/6/24 h1-3 h was sufficient but suffer from(atcracking, charring and/or higher cell deaths.50/75/100 □)Lysing70% EthanolEthanol treatment was found to be moreCellTreatmentconvenient and effective than the otherUltra-methods.sonicationFreeze-Thawing An optimized fabrication of the SLM involved firmly sandwiching the PVDF membrane between two polypropylene molds (FIG.1A). Casting theE. colicell pellet on top of PVDF membrane and drying at ambient conditions (25° C. and 40±5% relative humidity) for 24 h resulted in the fragmentation-free glossy flat SLM (FIG.1B). Given that the SLM was fabricated fromE. colicells (denoted by EC-SLM), whether any of them were alive was investigated. Notably, one milligram of EC-SLM was found to have 1.0±0.5*107colony forming units (CFUs), while its precursor, the wet cell pellet had a CFU count of 1.5±0.04*108mg−1(FIG.1E). The same protocols were employed for the Gram-positiveLactobacillus rhamnosusand the yeastSaccharomyces cerevisiaeto investigate whether other microbes can also form SLMs similarly. Interestingly,L. rhamnosusresulted in a SLM (denoted by LR-SLM) with highly wrinkled top surface, while that fromS. cerevisiae(denoted by SC-SLM) had extensive cracks and a non-glossy texture (FIGS.1C,1D). CFU analysis revealed that SC-SLM had 2.7±0.2*105mg−1, but no cell was found to be alive in the LR-SLM (FIG.1E). Further, in order to more accurately evaluate the relative abundances of live and dead cells in the EC-SLM, LR-SLM and SC-SLM, their weight was analyzed before and after the drying procedure (FIG.7). From this analysis, it was estimated that the CFU counts for pellets ofE. coli, L. rhamnosusandS. cerevisiaethat were corrected for their dry weights (FIG.8). It was notable to find that 35.1%, 0% and 50.3% of the cells were alive in EC-SLM, LR-SLM and SC-SLM, respectively (FIG.1F). However, the SLM fabricated from lysedE. colicells treated with 70% ethanol were found to fragment extensively and the CFU analysis expectedly did not show any living cells in the SLM (FIG.9). Thus, the very first examples of living bulk materials fabricated entirely from viable microbial cells were demonstrated. Example 3: Physical Characteristics of SLMs SLMs were first subjected to X-ray diffraction (XRD) to decipher any order arising due to self-assembly of cellular components. XRD spectra shown inFIG.10Aindicate that both EC-SLM and LR-SLM have a main diffraction peak corresponding to a d-spacing value of 0.44 nm, while EC-SLM has two additional ordering of 0.88 nm and 0.23 nm (FIG.10A). Although, it is difficult to assign the identity of these peaks, XRD spectra do establish that SLMs are amorphous materials. Thermal gravimetric analysis (TGA) of SLMs showed that the material degrades above 130° C., while the earlier weight loss could be attributed to loss of water (FIG.11). Differential scanning calorimetry (DSC) investigation of EC-SLM showed a glass-transition-like second-order transition (50-60° C.) during the first cycle of the heating curve (FIG.12). However, the successive second and third cycles of DSC did not reveal the presence of such transitions, which can be attributed to the probable role of water acting as a plasticizer. Similar features were also observed for the DSC traces of LR-SLM and SC-SLM (FIG.13). EC-SLM appeared to be transparent but the absorption spectra recorded in the visible range clearly showed that SLMs have less than 10% transparency (FIG.15). Example 4: Mechanical Characteristics of SLMs The mechanical properties of the SLMs were investigated by using the nanoindentation technique, as it offers small loads that are suitable for molecular materials and enables probing of microscopic dimensions as well as heterogeneity.(29, 30) SLMs were indented (n≥125) with a Berkovich diamond tip to obtain the continuous load, P, verses depth of penetration, h, curves. Nanoindentation experiments showed smooth P-h curves, which were analyzed using the Oliver-Pharr method to extract Young's modulus, E, and hardness, H, of the SLMs (FIG.16). EC-SLM was found to have E ranging from 5 to 42 GPa, while their H were about 0.2 to 2.4 GPa (FIGS.10B,10C). LR-SLM (E=6-31 GPa, H=0.2-2 GPa) and SC-SLM (E=1-32 GPa, H=0.02-1 GPa) also showed stiffness and hardness in the similar range of EC-SLM (FIGS.10B,10C). However, a careful examination revealed that nearly 44% of the E values of EC-SLM were 6±0.5 GPa and 35% were 7±0.5 GPa. On the other hand, LR-SLM had a broader distribution of E values with about 39% of 8±0.5 GPa, while SC-SLM had an even broader distribution. This stiffness distribution of SLMs was consistently observed across different samples, which could be attributed to the packing of heterogenous components (FIG.17). Interestingly, the SLM obtained from lysedE. coli(70% ethanol treatment) also exhibited similar E and H values, which further supports that cellular components can self-assemble, albeit heterogeneously, to form stiff materials (FIG.18). Example 5: Structural and Morphological Characteristics of SLMs As SLMs are formed exclusively from microbial cells, their organization in the material that not only enables them to be alive but also stiff was investigated. Field emission scanning electron microscopy (FESEM) imaging of the top surface of EC-SLM revealed closely packedE. colicells that appear to be ruptured (FIG.10D). But from CFU analysis, it was determined thatE. colicells are alive in EC-SLM, which prompted investigation of the core of the material. Cross-sectional imaging of EC-SLM showed ordering of cells into tightly packed domains amidst loosely bound cells (FIG.10E). Each domain could comprise of anywhere between 3 to nearly 500 cells, spanning up to a width of 30 μm. Notably,E. coliis a rod-shaped cell but, in these domains, transforms to a polygonal prism with a planar packing density (η, number of surrounding cells within the same plane; as seen from the images) of predominantly 6. The cells in the loosely bound regions may have greater survivability compared to the tightly packed domains. On the other hand, the top surface of LR-SLM was found to have an array ofL. rhamnosuscells, whose rod-shape structure appeared to be intact (FIG.10F). Herein, it is difficult to ascertain the η ofL. rhamnosusdue to their known inherent tendency to form chains. Further, the cross-sectional images of LR-SLM revealed that the cells were lysed to form an amorphous heterogenous solid (FIG.10G). These FESEM images of LR-SLM provide additional evidence for their CFU data of no living cells (FIGS.1E,1F). In contrast, SC-SLM was found to form a close packing of spherical shapedS. cerevisiaecells with η of 6, while 5 and 7 were also observed (FIG.10H). Interestingly, the cross section of SC-SLM showed,S. cerevisiaecells were packed less densely at the core but formed tightly compressed layers both on the top and bottom surfaces (FIG.10I,FIG.21). Thus, it appears that lysis ofS. cerevisiaecells forms a hard-protective shell on the outer surface and thereby enables cells at the core to survive to a greater extent. Example 6: Self-Regeneration of SLMs The living cells embedded in the SLMs were then exploited to develop a self-regenerating material. When a fragment of EC-SLM was introduced into selective lysogeny broth media, the SLM started to disperse and the cells self-replicated to form the turbid culture. After 24 h of culture, the cells were pelletized and casted onto the mold as per the same fabrication protocol described above. Ambient drying of the pellet for 24 h resulted in the second generation (denoted by Gen II) of EC-SLM fabricated from its first generation (denoted by Gen I,FIG.22A). Similarly, a tiny fragment (5-10 mg) of Gen II was utilized to fabricate the third generation (denoted by Gen III) of EC-SLM. Both Gen II and Gen III were found to have a CFU count of around 107 mg-1, which is almost same as that of Gen I (FIG.22B). Moreover, nanoindentation studies showed that E (5-41 GPa) and H (0.2-2.5 GPa) of self-regenerated EC-SLM (Gen II and Gen III) were also similar to that of the parent EC-SLM (FIGS.22C,22D). Further, in order to understand the survivability of the cells in the EC-SLM, time-dependent CFU analysis was performed (FIG.22E). At day 15, the CFU count of EC-SLM was reduced to ˜104 mg-1 and at day 30, it was about 21 mg-1. From this exponential decay data, the calculated cell death rate was found to be 0.43 per day. Example 7: Robustness of SLMs During the fabrication of SLMs, it was learned that PVDF membrane can be removed by gently wiping with DMF solvent. It was also noticed that EC-SLM did not disperse even when submerged in DMF. EC-SLM was then incubated in different solvents viz., hexane, chloroform, ethyl acetate, acetonitrile, absolute ethanol, methanol, DMF and milli-Q water (FIG.23). Notably, EC-SLM dispersed only in water and not in any other solvents, whose polarity index spans the entire spectrum. After 24 h of incubation in solvents, they were subjected to CFU analysis and greater than 106mg−1of CFU count was found in all solvents, except chloroform and methanol, in which all cells in the SLM were dead (FIG.24A). When repeated using the cell pellet instead, the CFU analysis withE. colishowed that the cells were dead in all solvents, except for hexane and milli-Q water (FIG.25). The latter can be attributed to hexane's non-polarity that phase-separates the cell pellet. While in milli-Q water the cells settle at the bottom of the test tube and create an intermediate phase, most likely comprising some lysed cells (FIG.25). Notably, EC-SLM is stable in both water-miscible and -immiscible organic solvents (FIG.24B). EC-SLM showed no significant weight loss after the 24 h incubation in solvents, which further supports their stability. The robustness of EC-SLM was also tested by incubating at 100° C. for 1 h and a mean CFU count of over 700 mg−1was observed. These robust characteristics of EC-SLM may be due to a protective outer layer formed from the lysedE. colicells, as seen inFIG.10D. Example 8: Mechanical Landscape of SLMs Based on the nanoindentation studies, it is evident that SLMs are both stiff and hard. To put things into perspective, a comparison of the mechanical properties of SLMs to other biomaterials and various types of human-made materials—metals, polymers, composites, ceramics, elastomers and foams is provided. Material properties charts, commonly known as Ashby plots, are presented inFIG.26A, depicting the plot of Young's modulus, E and density, p, for SLMs and other classes of materials, e.g., metals, polymers, composites, ceramics, elastomers, foams and biomaterials.(31) The SLMs are both light and stiff, which are comparable to biomaterials, polymers and composites. The yield strength, σy(estimated using the relation σy=H/3) of SLMs was also obtained, which was found to be about 60-800 MPa.(32, 33) InFIG.26B, the Ashby plot of specific modulus (ratio of E and ρ) and specific strength (ratio of σyand p) is shown, which indicates that the specific properties of SLMs are comparable to metals and ceramics, due to their low density.(31) Further, provided herein are specific examples of materials that are categorized into biomaterials (e.g. silk, collagen, cellulose etc.), biomaterials with cells (e.g. wood, skin, ligament etc.), non-biological materials (e.g. steel, glass, concrete, plastics etc.) and SLMs in an Ashby plot of E and strength, σ, inFIG.26C.(31, 34) Notably, the stiffness and strength of SLMs are superior to actin, balsa, cancellous bone, skin and plastics amongst others, and they are comparable to structural materials such as silk, collagen, wood and concrete. Example 9: Discussion Microbial cells have been subjected to desiccation under various environmental constraints over millions of years, which has enabled them to develop tolerance to different levels.(35) Xerotolerance of microbes has been studied and widely used (e.g. dry yeast) for nearly a century that has provided interesting insights on the molecular, structural, metabolic and physiological adaptations which keep them alive.(36-38) However, these studies were usually carried out in small volumes (e.g. microliters of microbial culture) that focused on either deciphering the mechanisms of xerotolerance or enhancing the survivability of microbes.(37) On the other hand, the large-scale production of dried microbes were mostly formulated in powder form, which often involves additives, emulsifiers etc.(38) In spite of all the above detailed fundamental and technological advancements, microbes have not been exploited earlier to fabricate a stiff living material and couple their biological properties with physicochemical properties. The living cell is a heterogeneous mixture of proteins, nucleic acids, sugars etc. and to comprehend their relative amounts in making the SLM, a Voronoi tree diagram shows the composition of a dryE. colicell (FIG.26D, Table 2).(39) Although, it is difficult to ascertain the role of each of these cellular components in the formation of SLM, efforts to fabricate SLM from ethanol treatedE. colicells, wherein the cellular membrane was disrupted, does indicate that the cellular integrity is essential to form a cohesive fragmentation-free material. Accordingly, the biomaterials contemplated herein include those comprising a fully desiccated biomass. Without being bound by theory, the ELMs of the invention (e.g., the SLMs disclosed herein), may be fully desiccated, taking advantage of the assembly, organization, and cellular integrity of the ELMs to fabricate stiff, cohesive, fragmentation-free material that do not comprise living cells. The latter-type disintegration approaches may help to understand the roles of components, but it has limited scope for the goal of incorporating life-like properties in materials. On the other hand, the various xerotolerance mechanisms (e.g. production of trehalose, extracellular polymeric substances, hydrophilins etc.) are expected to have a significant impact on the self-assembly and survival of cells in SLMs. (36-38, 40) Accordingly, the SLMs disclosed herein may comprise engineered microbes with enhanced or exogenous xerotolerance mechanisms which may include, without limitation and solely for the purpose of exemplification, modified, enhanced or exogenous production of trehalose, extracellular polymeric substances, hydrophilins, and the like as are known in the art (36-38, 40). 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11859174 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will now be described with reference to the drawings, but the scope of the present invention is not limited to the disclosed embodiments. The present invention will now be described in detail. <Klebsiella pneumoniaeStrain> An aspect of the present invention relates to aKlebsiella pneumoniaestrain inducing inflammation in the liver. The inflammation of livers includes not only inflammation of livers but also inflammation of intra- and extra-hepatic bile ducts, and examples thereof include hepatitis and cholangitis. Examples of hepatitis include viral hepatitis, drug-induced hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, and autoimmune hepatitis. Examples of cholangitis include sclerosing cholangitis (such as primary sclerosing cholangitis, IgG4-related sclerosing cholangitis, and secondary sclerosing cholangitis), primary biliary cholangitis (primary biliary cirrhosis), ascending cholangitis, secondary sclerosing cholangitis, and recurrent pyogenic cholangitis. TheKlebsiella pneumoniaestrain of the present invention is preferably a strain inducing Th17 cells in the liver. The induction of Th17 cells can be verified by, for example, treating mononuclear cells of the liver with a fluorescence labeled anti-CD4 antibody and an anti-IL-17 antibody and verifying a significant increase in IL-17-producing CD4 positive helper T (Th17) cells by flow cytometric analysis. TheKlebsiella pneumoniaestrain of the present invention preferably has the ability to form pores which is capable of forming pores on the large intestinal epithelia. The pores may have any shape and size allowing enteric bacteria to leak through the pores. For example, the diameter may be 0.1 to 20 μm, more preferably 0.5 to 15 μm, in observation of large intestinal epithelium with a scanning electron microscope. The leakage of enteric bacteria through the pores can be verified by, for example, hybridizing the DNA of the enteric bacteria with a fluorescent probe, staining the large intestinal epithelial cells with a fluorescent dye that emits fluorescence having a color different from that of the fluorescence of the probe, and observing the section of the large intestinal epithelium with a fluorescence microscope. Although the details of the mechanism of forming pores are unclear, it is inferred that theKlebsiella pneumoniaestrain of the present invention comes into direct contact with large intestinal epithelium to induce apoptosis, resulting in the formation of pores. TheKlebsiella pneumoniaestrain of the present invention preferably has a type 6 secretion system (T6SS). The type 6 secretion system can be verified by, for example, the presence of a gene in comparative analysis of whole genome sequencing of the strain. TheKlebsiella pneumoniaestrain of the present invention is preferably derived from a patient suffering from both primary sclerosing cholangitis and ulcerative colitis described below. The strain can be isolated from a fecal sample of the patient by a known method using a generic growth medium. Examples of the medium include a brain heart infusion (BHI) medium, a MacConkey agar medium, and a VRBG medium. TheKlebsiella pneumoniaestrain of the present invention preferably has a DNA consisting of the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM385182v1, and theKlebsiella pneumoniaestrain is more preferably one whose deposit number is NITE BP-02879. TheKlebsiella pneumoniaestrain of the present invention preferably has a DNA consisting of the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM386511v1. <Primary Sclerosing Cholangitis> Primary sclerosing cholangitis can be diagnosed in accordance with known clinical guidelines. For example, as the diagnosis items, when “bile duct image” (A) and “increase in alkaline phosphatase level” (B) are defined as major items; “complication of inflammatory bowel disease” (a) and “liver tissue image (fibrous cholangitis/onion skin lesion)” (b) are defined as minor items; and the bile duct image (A) is classified into “recognition of findings of bile duct image characteristic to primary sclerosing cholangitis” (A1) and “no recognition of findings of bile duct image characteristic to primary sclerosing cholangitis” (A2), only definite diagnosis and probable diagnosis in the following Table 1 can be treated as primary sclerosing cholangitis. TABLE 1Major itemMinor item(A1)(A2)(B)(a)(b)Diagnosis∘—∘——Definite diagnosis∘——∘—Definite diagnosis∘———∘Definite diagnosis—∘∘∘∘Definite diagnosis∘————Probable diagnosis—∘∘∘—Probable diagnosis—∘∘—∘Probable diagnosis—∘—∘∘Probable diagnosis—∘—∘—Possible diagnosis—∘——∘Possible diagnosis The alkaline phosphatase level can be measured by a known method. For example, it can be measured in accordance with the common standard method proposed by the Japan Society of Clinical Chemistry (JSCC). When the measured value is, for example, 2 to 3 times as high as the reference value, the value can be determined as an abnormal value. The bile duct image can be judged to be (A1) or (A2) mentioned above based on, for example, findings of diffuse wall irregularities and strictures associated with inflammation of intra- and extra-hepatic bile ducts characteristic to primary sclerosing cholangitis by performing an endoscopic retrograde cholangiography (ERC) or magnetic resonance cholangiopancreatography (MRCP) test. The above-mentioned diagnosis of primary sclerosing cholangitis needs to exclude malignant tumors such as cholangiocarcinoma, IgG4-related sclerosing cholangitis, and secondary sclerosing cholangitis. The IgG4-related sclerosing cholangitis can be diagnosed based on, for example, combination of four criteria ((1) finding of characteristic bile duct image, (2) increase in serum IgG4 level, (3) complication of IgG4-related disease of an organ other than biliary tract, and (4) characteristic histopathological findings) in Clinical diagnostic criteria of IgG4-related sclerosing cholangitis, 2012 (Journal of Hepato-Biliary-Pancreatic Sciences, vol. 19, pp. 536-542). Examples of the secondary sclerosing cholangitis include congenital diseases, such as Caroli's disease and cystic fibrosis; chronic obstructive diseases, such as choledocholith, biliary stricture, Mirizzi syndrome, anastomotic stenosis after liver transplantation, and tumors; infectious diseases, such as bacterial cholangitis, recurrent pyogenic cholangitis, parasitic infection, and cytomegalovirus infection; poisoning diseases, such as erroneous intrabiliary injection of alcohol, formaldehyde, or hypertonic saline; immune abnormalities, such as eosinophilic cholangitis and those with AIDS; ischemic diseases, such as those related to vascular injury, post-traumatic sclerosing cholangitis, hepatic artery embolism after liver transplantation, rejection after liver transplantation, or coronary arterial anticancer drug infusion; and invasive lesions, such as systemic vasculitis, amyloidosis, sarcoidosis, systemic mastocytosis, eosinophilia, Hodgkin's disease, and xanthogranulomatous cholangitis. <Ulcerative Colitis> Ulcerative colitis can be diagnosed in accordance with known clinical guidelines. For example, ulcerative colitis can be clearly diagnosed when the following item (B-1) or (B-2) is satisfied, in addition to the items (A) and (C), and the diseases (D) are excluded. (A) Clinical symptom: Persistent or recurrent mucous or bloody stool or medical history thereof is observed. (B-1) Endoscopic examination: (1) The mucous membrane is diffusely affected, the visible vascular pattern disappears, and coarse or fine granular form is observed. Furthermore, friability and hemorrhage-prone properties (contact bleeding) cause attachment of mucous and bloody purulent secretion; or (2) multiple erosion, ulcer, or pseudopolyposis is observed. (3) In general, lesions are observed continuously from the rectum. (B-2) Enema X-ray examination: (1) Diffuse change like a coarse or fine granular form of the surface of the mucosal membrane, (2) multiple erosion or ulcer, and (3) pseudopolyposis are observed. In addition, disappearance of Haustra coli (lead pipe appearance) or narrowing or shortening of the intestine is observed. (C) Biopsy histologic examination: In the active phase, diffuse inflammatory cell infiltration, cryptic tumor, and a severe decrease in goblet cells are observed in the entire mucosal layer. Since all of them are nonspecific findings, comprehensive judgment is performed. In the remission phase, abnormal arrangement of glands (meander, bifurcation) and atrophy remain. These changes are generally observed continuously from the rectum to the oral side. (D) Infectious enteritis, such as bacterial andClostridium difficileenteritis, amebic colitis,Salmonellaenteritis,Campylobacterenteritis, colonic tuberculosis, andChlamydiaenteritis; Crohn's disease; radiation irradiated colitis; drug-induced colitis; lymphoid follicular hyperplasia; ischemic colitis; intestinal Behcet, and so on. In addition to the above definite diagnosis cases, when the items (B-1) or (B-2) and (C) are satisfied multiple times, ulcerative colitis can be clearly diagnosed when findings grossly and histologically characteristic to ulcerative colitis are observed by excision surgery or autopsy. <Use for Prediction of Development of Primary Sclerosing Cholangitis> Another aspect of the present invention relates to use of theKlebsiella pneumoniaestrain only or use of theKlebsiella pneumoniaestrain, aProteus mirabilisstrain, and anEnterococcus gallinarumstrain for predicting the development of primary sclerosing cholangitis. As described above, for example, aKlebsiella pneumoniaestrain derived from a patient suffering from both primary sclerosing cholangitis and ulcerative colitis is characterized by (1) having a type 6 secretion system, (2) having an ability to form a pore on large intestinal epithelia, and (3) inducing Th17 cells in the liver. This suggests that in a patient suffering from ulcerative colitis, theKlebsiella pneumoniaestrain having a type 6 secretion system forms pores on the large intestinal epithelium to leak enteric bacteria, such as aProteus mirabilisstrain and anEnterococcus gallinarumstrain, and induce Th17 cells in the liver, resulting in complication of primary sclerosing cholangitis. Accordingly, if genome sequencing has revealed that theKlebsiella pneumoniaestrain in a fecal sample of a patient suffering from ulcerative colitis has a type 6 secretion system, the development of primary sclerosing cholangitis can be predicted. In addition, if formation of pores is recognized on the large intestinal epithelium when a biopsy tissue sample of the large intestine is subsequently observed with a scanning electron microscope, the prediction accuracy is increased. Furthermore, if an increase in Th17 cells in the liver is recognized in, for example, flow cytometry of a biopsy sample of the liver, the prediction accuracy is further increased. In addition, a fecal sample of a patient is cultured, and the presence or absence of aProteus mirabilisstrain or anEnterococcus gallinarumstrain, in addition to theKlebsiella pneumoniaestrain, can be used as a predictor. TheProteus mirabilisstrain is preferably aProteus mirabilisstrain whose accession number is NITE ABP-02923 and theEnterococcus gallinarumstrain is preferably anEnterococcus gallinarumstrain whose accession number is NITE ABP-02922. <Method for Producing Mouse Model Suffering from Both Primary Sclerosing Cholangitis and Ulcerative Colitis> Another aspect of the present invention relates to a method for producing a mouse model suffering from both primary sclerosing cholangitis and ulcerative colitis. Specifically, the method for producing a mouse model suffering from both primary sclerosing cholangitis and ulcerative colitis includes a step of administering a bacterial solution containing theKlebsiella pneumoniaestrain described in the paragraph <Klebsiella pneumoniaestrain> described above to a mouse and a step of administering 3,5-dicarbetoxy-1,4-dihydrocollidine to a mouse. Although the step of administering a bacterial solution containing theKlebsiella pneumoniaestrain to a mouse and the step of administering 3,5-dicarbetoxy-1,4-dihydrocollidine to a mouse may be performed in any order, it is preferred to perform the step of administering a bacterial solution containing theKlebsiella pneumoniaestrain to a mouse and then administering 3,5-dicarbetoxy-1,4-dihydrocollidine to the mouse. Although the original mouse to be used for producing the mouse model may be any mouse that can suffer from both primary sclerosing cholangitis and ulcerative colitis, a germ-free mouse is preferred. Examples of the germ-free mouse include mice produced from strains such as BALB/c, C57BL/6, and ICR. In order to produce the mouse model, for example, a suspension is prepared by suspending fecal samples collected from primary sclerosing cholangitis and ulcerative colitis patients in, for example, a phosphate-buffered saline (PBS) solution, and the suspension is orally administered to original mice to produce humanized gnotobiotic mice. Subsequently, the humanized gnotobiotic mice 19 to 23 days after the administration can be given free access to a diet containing 0.01% to 0.1% 3,5-dicarbetoxy-1,4-dihydrocollidine (DDC) for 12 to 16 days to produce a mouse model suffering from both primary sclerosing cholangitis and ulcerative colitis. Another aspect of the present invention relates to theProteus mirabilisstrain whose accession number is NITE ABP-02923. Another aspect of the present invention relates to theEnterococcus gallinarumstrain whose accession number is NITE ABP-02922. Although the embodiments of the present invention have been described in detail, they are merely examples, and the present invention is not limited thereto. The scope of the present invention should be interpreted by the claims and includes all modifications within the meaning and scope equivalent to the claims. EXAMPLES The present invention will now be further specifically described based on Examples, but is not limited to these Examples. [Example 1] Comparison of Intestinal Bacterial Floras of Human and Humanized Gnotobiotic Mouse (1) Collection of Fecal Sample Subjects were 14 patients suffering from both primary sclerosing cholangitis and ulcerative colitis (hereinafter referred to as “Psc/Uc” in some cases), 8 patients suffering from ulcerative colitis only (hereinafter referred to as “Uc” in some cases), and 10 healthy subjects (hereinafter referred to as “Hc” in some cases). Primary sclerosing cholangitis was diagnosed based on clinical guidelines and findings by cholangiography and liver biopsy. Ulcerative colitis was diagnosed by combination of endoscopic examination, histopathologic examination, and radiographic and serological examinations. Fecal samples were collected using feces collection tubes and were suspended in a solution containing 40% glycerol and an equivalent amount (w/v) of PBS, and the resulting suspensions were rapidly frozen and were stored at −80° C. until use. (2) Production of Humanized Gnotobiotic Mouse The frozen stock of each fecal sample of the above (1) was thawed and suspended in 6 volumes of PBS, and the suspension was passed through a 70-μm cell strainer to prepare a suspension for oral administration. 200 μL of the suspension for oral administration was orally administered to 6 to 8-week old male germ-free mice (GF mice, available from Sankyo Laboratories) using a stainless steel oral gavage needle to produce humanized gnotobiotic mice. Fecal samples were collected from the humanized gnotobiotic mice 3 to 4 weeks after the administration. (3) Collection of DNA in Fecal Sample DNAs of the bacteria in the human fecal samples in the above (1) and the humanized gnotobiotic mice fecal samples in the above (2) were isolated by an enzymatic dissolution method using lysozyme (manufactured by Sigma-Aldrich Co. LLC) and achromopeptidase (manufactured by FUJIFILM Wako Pure Chemical Corporation). The resulting DNA samples were treated with Ribonuclease A (manufactured by FUJIFILM Wako Pure Chemical Corporation) for purification and were then precipitated with 20% polyethylene glycol solution (PEG 6000, 2.5 M sodium chloride aqueous solution). Each precipitate was collected by centrifugation and was then washed with 75% ethanol and dissolved in a Tris-EDTA buffer. (4) 16S rRNA Metagenomic Analysis The hypervariable region V3-V4 of 16S rRNA gene of each of the DNAs obtained in the above (3) was amplified with TaKaRa Ex Taq (R) Hot Start Version (manufactured by Takara Bio Inc.), and the amplicon was purified with AMPure XP (manufactured by Beckman Coulter, Inc.). A mixture sample was prepared from the resulting DNA, and sequencing was performed with Miseq Reagent Kit V3 and Miseq Sequencer (manufactured by Illumina, Inc.) in accordance with the product manual. The sequence analysis was performed with QIIME software package ver. 1.9.1. Paired-end sequences were joined using a fastq-join tool in the EA-Utils software package. From the quality filter-passed sequences, 15,000 high-quality sequences were chosen for each sample. Chimera sequences were detected by the USEARCH, and the primer sequences were removed using cutadapt. Assignment of OTUs was performed using the UCLUST algorism with a sequence similarity of 96%. The assignment of OTUs was performed using the GLSEARCH program by similarity searching against the 16S (RDP version 10.27 and CORE update 2 Sep. 2012) and the NCBI genome database. The data were rarefied to 10,000 sequences per sample, as determined by the rarefaction curves, and the relative abundances of the bacteria were determined. The unweighted UniFrac analysis was performed in accordance with the method described in the document (Tsuda, A., et al., Influence of Proton-Pump Inhibitors on the Luminal Microbiota in the Gastrointestinal Tract, Clin. Transl. Gastroenterol., 6, e89 (2015)). The results are shown inFIGS.1aand1b. The results demonstrated that the main bacterial flora was conserved in human and humanized gnotobiotic mice. [Example 2] Measurement of Gene Expression Levels of Serum Amyloid Protein A (SAA) and IL-1β in Organ The liver, colon, and spleen of the humanized gnotobiotic mouse produced in Example 1 were homogenized, and total RNA of each organ was extracted using RNeasy Mini Kit (manufactured by QIAGEN N.V.). Complementary DNA was synthesized from 1 μg of the resulting total RNA by reverse transcription, and each target gene was amplified by PCR using AmpliTaq Gold Fast PCR MasterMix (manufactured by Applied Biosystems, Inc.) and the following designed primers (manufactured by Thermo Fisher Scientific Inc.). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh): Mm03302249_g1 Saa1: Mm00656927_g1 Saa2: Mm04208126_mH Saa3: Mm00441203_m1 IL-1β: Mm01336189_g1 The amplicons were quantitatively measured by real-time PCR using TaqMan Universal Master Mix and StepOne Plus systems (manufactured by Applied Biosystems, Inc.). In each sample, the target gene expression level was standardized using Gapdh. The results are shown inFIGS.2ato2c. The results demonstrated that in the liver (FIG.2a) and the colon (FIG.2b) of humanized gnotobiotic mice (PSCUC in the graphs) administered a fecal sample derived from Psc/Uc, the gene expression of serum amyloid protein A and IL-1β was increased and that bacteria involved in hepatic and large intestinal diseases were present in the intestinal bacterial flora of Psc/Uc. [Example 3] Production of Mouse Model Suffering from Both Primary Sclerosing Cholangitis and Ulcerative Colitis (1) Measurement of Serum Alkaline Phosphatase and Serum Total Bilirubin GF mice and the humanized gnotobiotic mice produced in Example 1 (2) by orally administering feces of Hc or Psc/Uc 21 days after the administration were given free access to a diet containing 0.05% 3,5-dicarbetoxy-1,4-dihydrocollidine (DDC) for 14 days, and serum alkaline phosphatase (ALP) and total bilirubin (TB) were measured by an LDH-UV kinetic method (manufactured by SRL, Inc.). The results are shown inFIG.3a. The humanized gnotobiotic mice administered the fecal sample derived from Psc/Uc (PSCUC in the graphs) showed high expression levels of both ALP (left inFIG.1a) and TB (right inFIG.1a), compared to those in GF mice (GF in the graphs) and the humanized gnotobiotic mice administered the fecal sample derived from Hc (HC in the graphs). (2) Histochemical Observation of Liver Livers were collected from the DDC-treated humanized gnotobiotic mice and were fixed in 10% formalin and embedded in paraffin according to a usual method to produce paraffin blocks. Sections were cut from the paraffin blocks and were stained with hematoxylin-eosin and Masson-trichrome or stained with Sirius red to prepare specimen samples. The results of microscopic observation of the specimen samples are shown inFIG.3b. The upper part ofFIG.3bshows the results of Sirius red staining, and the lower part ofFIG.3bshows the results of co-staining with hematoxylin-eosin and Masson-trichrome. In the liver of the humanized gnotobiotic mice administered the fecal sample derived from Psc/Uc, hepatic disorder due to fiber formation in the liver was observed. (3) Immunological Analysis Livers were collected from the DCC-treated mice and were perfused with PBS from the portal vein. After the perfusion, the livers were chopped and passed through a 100-μm nylon mesh to obtain cells of the livers. The resulting cells were suspended in 40% Percoll solution and then overlaid in 75% Percoll fraction, followed by density gradient centrifugal separation at 840×g for 20 minutes at room temperature. Mononuclear cells were collected from the intermediate layer. The resulting mononuclear cells were washed and were suspended in FACS buffer. The mononuclear cells were subjected to blocking treatment using an anti-Fc antibody (CD16/32, manufactured by BD Pharmingen) and were then subjected to intracellular staining with an anti-CD4 antibody (APC-cy7, BV510) and an anti-CD11b antibody (APC-cy7). The intracellular stained cells were treated with Ionomycine (500 ng/mL) or Golgistop (10 μg/mL, manufactured by BD Biosciences) in the presence of lipopolysaccharide (derived fromEscherichia coliB5, manufactured by Sigma-Aldrich Co. LLC) or PMA (50 ng/mL, manufactured by Sigma-Aldrich Co. LLC) and brefeldin A (10 μg/mL, manufactured by BD Biosciences). Subsequently, an anti-IFN-γ antibody, an anti-TNF-α antibody, an anti-IL-1β antibody, and an anti-IL-17 antibody (manufactured by BD Pharmingen) were added thereto, followed by co-culture at 4° C. for 20 minutes. The cells after the culture were washed with PBS and were measured with a cell sorter (“FACS CantoII,” manufactured by Becton, Dickinson and Company) and analyzed using FlowJo software (manufactured by Tree Star, Inc.). The results are shown inFIG.3c. The results demonstrated that in DDC-treated mice administered a fecal sample derived from Psc/Uc, the development of hepatic disorder and recruitment of Th17 cells and IL-1β+CD11b+macrophages were observed, and a mouse model suffering from both primary sclerosing cholangitis and ulcerative colitis was provided. [Example 4] Identification of Intestinal Bacterial Flora Derived from Psc/Uc (1) Analysis of Intestinal Bacterial Flora in Mouse Fecal Sample In order to identify the bacteria that induce hepatitis, the livers, mesenteric lymph nodes, and spleens were collected from humanized gnotobiotic mice orally administered a fecal sample of Hc or Psc/Uc and SPF mice (6- to 8-week old C57BL/6 mice, available from CLEA Japan, Inc.) as a control group on the 21st day from the administration, and the bacteria were cultured on agar plates. As a result, no colonies were observed in the liver and spleen of any of the mice. In the mesenteric lymph nodes, colonies were observed in only that from the humanized gnotobiotic mice administered a fecal sample derived from Psc/Uc. Bacterial flora analysis of 16S rRNA of the colonies demonstrated that the bacteria of the colonies wereKlebsiella pneumoniae(KP),Proteus mirabilis(PM), andEnterococcus gallinarum(EG). (2) Analysis of Intestinal Bacterial Flora in Human Fecal Sample Fecal samples derived from Hc, Psc/Uc, and Uc were anaerobically cultured in BHI medium (manufactured by Becton, Dickinson and Company), and the KP, PM, and EG cells were counted. The results are shown inFIG.4a. It was demonstrated that in the fecal sample derived from Psc/Uc, KP was remarkably present, and PM and EG were also present. The KP included a KP strain (deposit number: NITE BP-02879) having a DNA consisting of the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM385182v1 and a KP strain having a DNA consisting of the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM386511v1. The PM included a strain whose accession number is NITE ABP-02923, and the EG included a strain whose accession number is NITE ABP-02922. (3) Correlation Analysis of KP in Fecal Samples of Human and Humanized Gnotobiotic Mice As in Example 2 (3), correlations between the proportion of Th17 cells or Th1 cells in the liver of the humanized gnotobiotic mice administered the fecal sample derived from Psc/Uc and the number of KP cells in the fecal sample were verified by flow cytometric analysis using an anti-CD3e antibody (FITC), an anti-CD4 antibody (APC-cy7, BV510), an anti-IFN-γ antibody, and an anti-IL-17 antibody, and the significance was verified by a Spearman rank-order correlation test. The results are shown inFIG.4b. A correlation was found between KP and Th17 cells (left inFIG.4b), but no correlation was found between KP and Th1 cells (right inFIG.4b). The results demonstrated that KP derived from Psc/Uc contributed to induction of Th17 cells in the liver. [Example 5] Evaluation of Properties of KP, PM, and EG (1) Evaluation of Induction of Th17 Cell in Liver by Single Strain and Mixed Strain Administration In order to investigate contribution of the above-mentioned three strains to Th17 cell induction, 1×108CFU/200 μL of KP, PM, and EG were orally administered to GF mice by (1) KP alone (KP), (2) combination of PM and EG (PM+EG), or (3) combination of three strains (3-mix) each twice per week. On the 21st day from the administration, as in Example 2 (3), the liver, colon, and mesenteric lymph node were analyzed by flow cytometry using an anti-CD4 antibody (APC-cy7, BV510), an anti-IL-17 antibody, and an anti-RORγt antibody (manufactured by BD Pharmingen). The results are shown inFIG.5a. In all organs of the liver (left inFIG.5a), the colon (center inFIG.5a), and the mesenteric lymph node (right inFIG.5a), Th17 cell induction was recognized in the KP administration groups, in particular, in the 3-mix administration group, compared to those in GF mice (GF) and SPF mice (SPF) not administered the strains. (2) Histochemical Observation of Large Intestine Ileum tissues including fecal materials of humanized gnotobiotic mice prepared by administering KP alone, PM and EG, and three strains of KP, PM, and EG to GF mice (KP, PM+EG gnoto, and 3-mix gnoto) were fixed in Carnoy's solution for 3 hours and embedded in paraffin to produce paraffin blocks. Tissue sections were cut from the blocks and were hybridized at 50° C. overnight using EUB338 probe (ALEXA555 label), hybridizing with the DNA of the above-mentioned strains, prepared such that the final concentration in hybridization buffer (20 mM Tris-HCl (pH 7.4), 0.9 M NaCl, 0.1% SDS, 20% formamide) was 10 μg/mL. The tissue sections were washed with washing buffer (20 mM Tris-HCl (pH 7.4), 0.9 M NaCl) for 10 minutes and with PBS for 10 minutes three times and were then stained with Phalloidin-iFluor (manufactured by Abcam plc.). After washing with PBS for 10 minutes three times, the sections were mounted in Prolong anti-fade mounting media with DAPI (manufactured by Life Technologies). Microscopic observation was performed with a BIO-REVO BZ-9000 fluorescence microscope (manufactured by Keyence Corporation). The results are shown inFIG.5b. It was observed that the bacteria stained in red in the micrographs invaded the large intestinal epithelium mucous membrane in the 3-mix gnoto and KP gnoto, and some of the bacteria further invaded the large intestinal epithelium. In particular, the invasion was remarkable in the 3-mix gnoto. In contrast, the invasion was only slight in the PM+EG gnoto and was not observed in the control SPF. [Example 6] Evaluation 1 of Ability to Form Pore on Large Intestinal Epithelium of KP Stain (1) Production of Monolayer Organoid Co-Culture System Healthy human colon organoids were embedded in Matrigel (Corning) and were three-dimensionally cultured in Advanced DMEM/F12 culture solution (containing penicillin/streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1×B27 (manufactured by Life Technologies), 1 mM N-acetylcysteine (manufactured by FUJIFILM Wako Pure Chemical Corporation), 10 nM Gastrin I (manufactured by Sigma-Aldrich Co. LLC), 50 ng/mL human recombinant EGF, 0.5 μM A83-01, 3 μM SB202190, 50% Afamin-Wnt3a (Afm-W) complex condition medium (CM, v/v), R-spondin 1 (R)-CM (10% v/v), and Noggin (N)-CM (10% v/v)). The three-dimensionally cultured human colon organoids were cultured for at least one day in a medium containing 10 μM Y-27632 and containing Afm-Wnt, R-spondin 1, Noggin, EGF, A83-01, and SB202190 and were then separated and seeded in a ThinCert 24-well plate (manufactured by Greiner Bio-One International GmbH) coated with 10% Cellmatrix type I-C (manufactured by Nitta Gelatin Inc.) and having a pore size of 0.4 μm. 2 to 3 days after the seeding, the medium was replaced by a condition medium not containing Afm-Wnt and SB202190Y-27632. The colonic epithelium was washed with Advanced DMEM/F12 culture solution before seeding of the strain, and antibiotic-free DM was added thereto to produce a monolayer organoid co-culture system. (2) Co-Culture of Strain and Large Intestinal Epithelial Cell and Morphological Analysis KP strains (KP-P1 and KP-P5) isolated from the mesenteric lymph nodes of mice administered a fecal sample derived from Psc/Uc, enterohemorrhagicEscherichia coliO-157:H7, and a KP strain (KP JCM1662) obtained from Riken BioResource Research Center were added to the monolayer organoid co-culture systems each at a concentration of 1×105CFU, followed by culturing for 8 hours. As a control, PBS was used. The co-cultured large intestinal epithelium was collected and pre-fixed in 5% glutaraldehyde (manufactured by TCI Chemicals) dissolved in PBS at 4° C. overnight. The pre-fixed sample was post-fixed in 1% osmium tetraoxide dissolved in PBS for 1 hour. The post-fixed sample was dehydrated with ethanol and coated by gold sputtering and was then observed with a scanning electron microscope (“VHX-D510 scanning electron microscope,” manufactured by Keyence Corporation)” in a high-vacuum mode. The results are shown inFIG.6a. In addition, the pores having a pore size of 10 μm or more on the large intestine were counted by observation of four independent positions of the large intestinal epithelium. The experiment was repeated 3 to 6 times. The results are shown inFIG.6b. The results demonstrated that the KP strains (KP-P1 and KP-P5) of mice receiving a fecal sample derived from Psc/Uc formed pores on the large intestinal epithelium, but the JCM1662 strain formed no pores. In addition, a same test using other KP strains demonstrated that seven KP strains (JCM20034, ATCC BAA1705, ATCC BAA2552, ATCC700721, JCM20348, JCM20694, and JCM20507) formed pores on the large intestinal epithelium, but four strains (JCM1662, JCM1663, JCM1664, and ATCC700603) formed no pores on the large intestinal epithelium. Furthermore, the co-cultured large intestinal epithelial cells were triple-stained with an anti-cleaved caspase-3 antibody (manufactured by Cell Signaling Technology, Inc.), a filamentous actin stain phalloidin (manufactured by Thermo Fisher Scientific Inc.), and a DNA stain Hoechst 33324 (manufactured by Thermo Fisher Scientific Inc.), and apoptosis cells were observed with a confocal laser microscope (SP5, manufactured by Leica). The results are shown inFIG.6c. It is inferred from the results that KP-P1 comes into direct contact with the large intestinal epithelium and induces apoptosis. (3) Genome Sequencing of KP Strain The whole genome sequencing of the KP strain having or not having an ability to form a pore on the large intestinal epithelium was performed by whole-genome shotgun sequencing using PacBio RSII and Illumina MiSeq sequencers, and comparative analysis was performed. The results are shown inFIG.7. The results demonstrated that in 97 orthologous genes, the KP strain having an ability to form a pore on the large intestinal epithelia includes genes involved in a type 6 secretion system (T6SS) and reactive oxygen species (ROS) decomposition. [Example 7] Evaluation 2 of Ability to Form a Pore on the Large Intestinal Epithelium of KP Stain (1) Histochemical Observation of Large Intestine Humanized gnotobiotic mice (3-mix gnoto) were produced by administering to GF mice three strains: PM, EG, and KP-P1 which is a KP strain forming pores on the large intestinal epithelia, and humanized gnotobiotic mice (m3-mix gnoto) were produced by administering to mice three strains: PM, EG, and KP JCM1662 which is a KP strain not forming pores on the large intestinal epithelia. On the 21st day from the administration, the ileum was collected from each mouse and was subjected to histochemical observation in the same manner as in Example 5 (2). The results are shown inFIG.8b. The results demonstrated that 3-mix gnoto invaded the large intestine mucous membrane and the epithelial tissue, whereas m3-mix gnoto only slightly invaded the large intestine mucous membrane. (2) Measurement of Serum LPS Level Blood was collected from GF mice, SPF mice, 3-mix gnoto, and m3-mix gnoto, and samples were prepared using ToxinSensor Chromogenic Limulus Amebocyte Lysate (LAL) Endotoxin Assay Kit (manufactured by GenScript Biotech Corporation) in accordance with the manual of the product. The absorbance at 540 nm was measured using FilterMax F3 Multi-Mode Microplate Reader (manufactured by Molecular Devices, LLC.). The results are shown inFIG.8c. The results demonstrated that the serum LPS level in 3-mix significantly increased compared to those in the GF mice, SPF mice, and m3-mix groups. (3) Evaluation of induction of Th17 cells The livers of GF mice, 3-mix gnoto, and m3-mix gnoto were analyzed by flow cytometry using an anti-CD4 antibody (APC-cy7, BV510), an anti-IL-17 antibody, and an anti-RORγt antibody (manufactured by BD Pharmingen) in the same manner as in Example 2 (3). The results are shown inFIG.8d. The results demonstrated that 3-mix gnoto significantly induced Th17 cells compared to GF mice and m3-mix gnoto. [Example 8] Evaluation of Pathogenicity of KP Strain-Induced Th17 Cells (1) Evaluation of Induction of Th17 Cells by RORγt Inverse Agonist Treatment 3-mix gnoto was fed on a diet containing an RORγt inverse agonist (RORγt IA) or water as a control and DDC every day for 2 weeks 14 days after the administration of the strain (FIG.9a). Subsequently, the liver of each mouse was subjected to flow cytometric analysis using an anti-CD4 antibody (APC-cy7, BV510), an anti-IL-17 antibody, an anti-IFN-γ antibody, and an anti-RORγt antibody (manufactured by BD Pharmingen) in the same manner as in Example 2 (3). The results are shown inFIG.9b. In addition, serum alkaline phosphatase and serum total bilirubin were measured. The results are shown inFIG.9cin the same manner as in Example 3 (1). Furthermore, sections of the liver were stained with hematoxylin-eosin and Masson-trichrome and were subjected to microscopic observation in the same manner as in Example 3 (2). The results are shown inFIG.10. It is inferred from these results that Th17 cells induced by strains in the liver play a pathogenetic role in the development of bile duct disorder induced by DDC. The contents of Japanese Patent Application No. 2018-082192 (application date: Apr. 23, 2018), including the specification, claims, drawings, and abstract, are incorporated herein by reference in its entirety. | 36,708 |
11859175 | DESCRIPTION OF EMBODIMENTS Hereinafter, configurations of the present invention will be described in detail. The present invention provides a cell reprogramming method including subjecting a mixture of differentiated or non-differentiated cells and a culture medium to physical stimulation which can promote an environmental influx, and culturing the mixture subjected to the physical stimulation for a predetermined time to obtain reprogrammed cells. The present invention is characterized in that the differentiated or non-differentiated cells are cultured in any medium capable of inducing desired reprogrammed cells while subjecting differentiated or non-differentiated cells to physical stimulation which can promote an environmental influx such as ultrasonic waves, laser, heat shock, etc. to induce reprogramming of cells into pluripotent cells; or arbitrary differentiated cells having a different expression type from the differentiated or non-differentiated cells, for example, hepatocytes, osteoblasts, adipocytes, myocytes, neurons, astrocytes, keratinocytes, hair follicle cells, pancreatic beta cells or cardiomyocytes. For example, if pluripotent cells are intended as reprogrammed cells, the differentiated cells may be reprogrammed into pluripotent cells by mixing the differentiated cells with a stem cell culture medium and culturing the mixture for a predetermined time by subjecting the mixture to physical stimulation. As another example, when arbitrary differentiated cells having an expression type different from that of the differentiated cells are intended as the reprogrammed cells, the differentiated cells may be reprogrammed into arbitrary differentiated cells having a different expression type by mixing the differentiated cells with a differentiation-inducing medium of desired differentiated cells and culturing the mixture for a predetermined time by subjecting the mixture to physical stimulation. As yet another example, the differentiated cells may be reprogrammed into desired differentiated cells with improved differentiation rate as compared with the related art by mixing the non-differentiated cells such as induced pluripotent stem cells or embryonic stem cells with a differentiation-inducing medium of desired differentiated cells and culturing the mixture for a predetermined time by subjecting the mixture to physical stimulation. In the cell reprogramming method of the present invention, the reprogramming of the differentiated or the non-differentiated cells may be induced according to an environmental influx other than the cells through physical stimulation to the differentiated or non-differentiated cells. Such an environmental influx means an influx into the adjacent differentiated or non-differentiated cells of genetic materials, chemicals, small molecules, exosomes, or extracellular vesicles containing exosomes released from the differentiated cells subjected to the physical stimulation; or culture medium components. According to the cell reprogramming method of the present invention, the environmental influx into the differentiated or non-differentiated cells may determine reprogramming directivity into pluripotent cells stably expressing a pluripotent marker or a triploblastic marker and differentiated cells having a different expression type from the differentiated or non-differentiated cells. In addition, the reprogramming directivity may be determined by a kind of culture medium. That is, as described above, the reprogramming from the differentiated or non-differentiated cells into pluripotent cells may be induced by subjecting the mixture of the differentiated cells and the stem cell culture medium to the physical stimulation, and the reprogramming from the differentiated cells into arbitrary differentiated cells having a different expression type may be induced by subjecting the mixture of the differentiated cells and the differentiation-inducing medium of the arbitrary differentiated cells to the physical stimulation, and the non-differentiated cells may be reprogrammed into arbitrary differentiated cells by subjecting the mixture of the non-differentiated cells and the differentiation-inducing medium of the arbitrary differentiated cells to the physical stimulation. With regard to the environmental influx into the differentiated or non-differentiated cells, the present inventors have particularly considered cell membrane damage by physical stimulation and cellular secretion materials (exosomes or extracellular vesicles containing exosomes). That is, the ultrasonic waves, laser, heat shock, etc. induce temperature rise by energy, oscillation of microbubbles generated by ultrasonic waves, and induction of liquid flow generation, that is, generation of microstream along the cell membrane to apply minute damage to the cell membrane due to such an effect and induce generation of holes so that absorption of external materials is increased. It is confirmed that in a change of cytosol Ca2+concentration, that is, analysis of a change of cytosol Ca2+concentration, when the damage to the cell membrane or cell membrane fluidity is increased, a cytosol Ca2+concentration is instantaneously increased and thus the cell membrane fluidity is increased. According to one embodiment of the present invention, it can be seen that the Ca2+concentration immediately after ultrasonic wave treatment is rapidly increased and then gradually decreased to be decreased to a level of a control group not treated with ultrasonic waves and restored after the damage to the cell membrane is induced. It is also known that ATP generation and increase due to ultrasonic waves induce response on various cellular stresses and endocytosis by reacting with ATP receptors in the cell membrane. In other words, there is a relation between ATP concentration and cell damage and intracellular substance influx, and in order to verify the relation, as a result of analyzing ATP concentration in cells after ultrasonic wave treatment, the ATP concentration was higher than that in the untreated control group. In addition, expression of ionic P2X receptors and metabolic P2Y receptors in ATP-affected cell membranes is also activated in the cells treated with ultrasonic waves compared to the control group. These results indicate the possibility of influx of extracellular environment as well as intracellular damage by ultrasonic waves. Meanwhile, it is known that the exosomes or the extracellular vesicles containing exosomes include genetic information materials (DNA, mRNA, microRNA, protein) therein, and when the exosomes or the extracellular vesicles containing exosomes released outside the cell membrane through the cell membrane damage enter other neighboring cells again, the genetic information materials in the exosomes or the extracellular vesicles containing exosomes may be delivered. Accordingly, due to ultrasonic wave stimulation, expression of pluripotent markers, triploblastic markers, or differentiated cell markers which have been maintained in a low expression state or expression-suppressed state in the cells is induced and promoted and simultaneously, the damage to the cell membrane occurs, and thus, the exosomes or the extracellular vesicles containing exosomes present in the cells including the pluripotent markers, triploblastic markers, or differentiated cell markers of which the expression is induced or promoted are released outside to be delivered to the neighboring cells. Since the neighboring cells are also in a state where the cell membrane is partially damaged, the cell membrane fluidity is increased and thus it is estimated that the efficiency in which the exosomes or the extracellular vesicles containing exosomes enter the inside of the cells is higher than that in a normal state, and it is considered that the expression-induced and promoted pluripotency, generation, differentiation-related genetic information present in the exosomes or the extracellular vesicles containing exosomes is delivered so that pluripotent cells or arbitrary differentiated cells are produced. In one embodiment of the present invention, during a pluripotent cell inducing process, the culture medium is recovered, the exosomes or the extracellular vesicles containing exosomes in the medium are extracted, and then it is confirmed whether the pluripotent cell-related pluripotent markers or differentiation markers are present therein, and as a result, it is confirmed that known pluripotent markers and differentiation markers exhibit a high expression degree and thus it is considered that the hypothesis of the present inventors is supported. In addition, it has also been shown that even in ultrasonic waves, laser, or heat shock, the exosomes or the extracellular vesicles containing exosomes are normal without malformation of karyotypes. This hypothesis makes it possible to produce pluripotent cells or differentiated cells by inducing the release of exosomes or extracellular vesicles containing exosomes due to cell membrane damage. As the differentiated cells, somatic cells including mammalian-derived dermal fibroblasts, skin fibroblasts, and the like; cancer cells including uterine cancer cells (HeLa), liver cancer cells (Hep3B), and the like; or endotracheal cells including pulmonary epithelial cells (L132 cells), and the like may be used. In this specification, the term “somatic cell” refers to a cell constituting an adult and having limited differentiation potency and autopoiesis. According to one embodiment, the somatic cells may be somatic cells constituting the skin, hair, and fat of a mammal, preferably, mammalian-derived fibroblasts, but are not limited thereto. In this specification, the term “non-differentiated cell” refers to a cell having differentiation potency and autopoiesis. Examples of the non-differentiated cells may include induced pluripotent stem cells, embryonic stem cells, progenitor cells, and the like. In this specification, the term “pluripotent cells” refer to cells having pluripotency after physical stimulation, strictly, ultrasonic waves, laser, magnetic fields, plasma, light-emitting diodes, electrical stimulation, chemical exposure, heat shock, or acid treatment. In this specification, the pluripotency refers to a state in which pluripotent markers expressed in stem cells comprehensively are stably expressed. In addition, the pluripotency refers to a state in which triploblastic markers of endoderm, ectoderm, and mesoderm are expressed. The pluripotent cells may be used as “embryonic stem cell media-based environmental transition-guided cellular reprogramming (es/ENTER) cells. The pluripotent cells according to the present invention are differentiated from known induced pluripotent stem cells in that the differentiation is induced well according to an external environment and a property of progenitor cell having a higher differentiation property than the property of a stem cell is higher. That is, when embryonic stem cells such as induced pluripotent stem cells are used as a cell therapeutic agent, a preparation step is required to undergo a certain degree of differentiation, and a risk factor that can be transformed into cancer is implicated, and a safety problem from using viral vectors to introduce a reprogramming inducing factor is raised. However, since the pluripotent cells of the present invention are induced without introducing a reprogramming inducing factor for genetic mutation or a reprogramming inducing substance such as a chemical material, culture through co-culture with different types of cells is not required, and thus, there is no cell contamination (problem of mixing with other cells) problem, and there is no problem in cancer generation without forming teratoma similar to cancer cells in an in-vivo experiment, thereby ensuring safety. In other words, the pluripotent cells of the present invention have an advantage that the induction process is simple and short, and the time for transplantation may be drastically shortened by treating autologous cells. The pluripotent cell is characterized to stably express a pluripotent marker of any one of Oct3/4, S0X2, NANOG, c-MYC, KLF4, TDGF1, SSEA4, TRA-1-60, PAX6, Nestin, Brachyury, SMA, GATA4, or AFP or a triploblastic marker gene consisting of mesoderm or endoderm. In this specification, the term “reprogramming” means a process of restoring or converting differentiated cells present in different types such as cells having no differentiation potency or cells having partial differentiation potency to final new type of cells or a state having new type of differentiation potency. In addition, a process of converting cells having differentiation potency to final new type of cells is also included. According to the present invention, when the differentiated cells are subjected to the physical stimulation which can promote the environmental influx, the differentiated cells may be reprogrammed to pluripotent cells or desired arbitrary differentiated cells having an expression type different from differentiated cells. Further, the non-differentiated cells may be reprogrammed to arbitrary differentiated cells having significantly excellent differentiation rate when being subjected to the physical stimulation which can promote the environmental influx. Examples of the differentiated cells may include neurons (referred to as “neuronal stem cell media-based ENTER, n/ENTER”) expressing any one of PAX6, SOX1, SOX2, Nestin, MAP2, TuJ1, GFAP, or 04; myocytes (referred to as “muscle differentiation media-based ENTER, m/ENTER”) expressing any one of Desmin, Pax3, Actinin, SMA, GATA4, or NKX2-5; hepatocytes (referred to as “hepatocyte differentiation media-based ENTER, h/ENTER”) expressing any one of AFP, HNF1a, HNF4a, CK18, or ALB, and adipocytes (referred to as “adipocyte differentiation media-based ENTER, a/ENTER”) expressing any one of Pparc2, C/ebpa, aP2, or Fabp4, but are not limited thereto. In this specification, the “culture medium” is a medium used for cell culture in vitro in a comprehensive sense, and in the present invention, the “culture medium” means a stem cell culture medium or a differentiation-inducing medium, and the stem cell culture medium more particularly means an embryonic stem cell culture medium. In addition, the “differentiation-inducing medium” is a medium used for induction to differentiated cells of general stem cells, and for example, may be a multipotent cell differentiation-inducing medium, a hepatocyte differentiation-inducing medium, an osteogenic differentiation-inducing medium, an adipocyte differentiation-inducing medium, a myocyte differentiation-inducing medium, an astrocyte differentiation-inducing medium, a neuronal cell differentiation-inducing medium, an endothelial cell differentiation-inducing medium, a keratinocyte differentiation-inducing medium, a pancreatic beta cell differentiation-inducing medium, a cardiomyocyte differentiation-inducing medium, or the like, but is not limited thereto. The cell reprogramming method of the present invention will be described in detail with reference toFIG.1. First, the culture medium is mixed with differentiated or non-differentiated cells, and the mixture is subjected to the physical stimulation. The reprogramming efficiency of the cells may be enhanced by subjecting the culture medium to the physical stimulation before subjecting the mixture including the differentiated or non-differentiated cells to the physical stimulation. The physical stimulation may be any one of ultrasonic waves, laser, plasma, light-emitting diodes, electrical stimulation, chemical exposure, heat shock, or acid treatment. The ultrasonic wave treatment for the culture medium may be performed by applying ultrasonic waves having an output intensity of 1 W/cm2to 20 W/cm2for 1 to minutes, specifically ultrasonic waves having an output intensity of 2 W/cm2to 10 W/cm2for 5 to 15 minutes, and more specifically ultrasonic waves having an output intensity of 3 W/cm2to 7 W/cm2for 7 to 13 minutes. The laser treatment for the culture medium may be performed by irradiating a pulsed laser beam with a wavelength band of 300 to 900 nm for 1 minute to 20 minutes, more specifically the pulsed laser beam with the wavelength band for 3 minutes to 10 minutes, and much more specifically the pulsed laser beam with the wavelength band for 4 to 6 minutes. The wavelength band may use, for example, wavelengths of 400 nm, 808 nm, and 880 nm. The heat shock for the culture medium may be performed at a temperature of to 50° C. for 5 to 20 minutes. When the differentiated or non-differentiated cells are subjected to the physical stimulation, it is preferable to exposure the differentiated or non-differentiated cells at a predetermined intensity, and a cell survival rate may be reduced out of the above range. Accordingly, the ultrasonic wave treatment for the mixture of the culture medium and the differentiated or non-differentiated cells may be performed by applying ultrasonic waves having an output intensity of 0.5 W/cm2to 3 W/cm2for 1 to 5 seconds, specifically ultrasonic waves having an output intensity of 0.7 W/cm2to 2 W/cm2for 1 to 5 seconds, and more specifically ultrasonic waves having an output intensity of 0.8 W/cm2to 1.5 W/cm2for 1 to 5 seconds. The laser treatment for the mixture of the culture medium and the differentiated or non-differentiated cells may be performed by irradiating a pulsed laser beam with a wavelength band of 300 to 900 nm for 1 second to 20 seconds, more specifically the pulsed laser beam with the wavelength band for 3 seconds to 10 seconds, and much more specifically the pulsed laser beam with the wavelength band for 4 to 6 seconds. The wavelength band may use, for example, wavelengths of 400 nm, 808 nm, and 880 nm. The heat shock for the mixture of the culture medium and the differentiated or non-differentiated cells may be performed by exposure for 1 to 10 minutes at a temperature condition of 40 to 50° C. and then exposure for 5 to 10 seconds at a temperature condition of 0 to 4° C. Next, the mixture subjected to the physical stimulation is cultured for a predetermined time to obtain reprogrammed cells. The culture of the mixture subjected to the physical stimulation may be performed for a period during which spheroid stably expressing the pluripotent marker or the differentiation marker is formed through a suspended culture or monolayer culture method, that is, for 2 to 10 days, but is not particularly limited thereto. According to one embodiment of the present invention, the suspended culture exhibits efficiency of spheroid formation higher than that of the monolayer culture. In addition, the suspended culture has a larger number and size of spheroid than that of the monolayer culture and exhibits a constant size distribution. According to one embodiment of the present invention, the expression of the pluripotent marker or the differentiation marker is increased or stabilized from about 3 days during the suspended culture of ultrasonic waves or laser-treated human skin fibroblasts, and reprogramming is started from this point. In addition, the expression of the pluripotent marker is increased or stabilized at about 8 days during the suspended culture of heat-treated human skin fibroblasts, and reprogramming is started from this period. The pluripotency of the spheroid can be confirmed by expression of the pluripotent marker such as Oct3/4, SOX2, NANOG, c-MYC, KLF4, TDGF1, SSEA4, and TRA-1-60. The confirmation of the pluripotency marker may be analyzed through RT-PCR or immunocytochemistry, but is not particularly limited thereto. In addition, the pluripotent cells of the present invention have a feature of a high level of expression of triploblastic markers, that is, ectodermal (PAX6, Nestin), mesenchymal (Brachyury, SMA), and endodermal (GATA4, AFP) markers. In another embodiment, when the skin fibroblasts are subjected to the physical stimulation in the differentiation-inducing medium, the spheroid may be formed between about 1 to 20 days after the culture. The differentiation marker may be at least one of PAX6, SOX1, SOX2, Nestin, MAP2, TuJ1, GFAP, or 04 when reprogrammed into neurons. The differentiation marker may be at least one of Desmin, Actinin, Pax3, SMA, GATA4, or NKX2-5 when reprogrammed into myocytes. The differentiation marker may be at least one of AFP, HNF1a, HNF4a, CK18, or ALB when reprogrammed into hepatocytes. The differentiation marker may be stained with oil red O and may be at least one of Pparc2, C/ebpa, aP2, or Fabp4 when reprogrammed into adipocytes. Further, the pluripotent cells of the present invention are characterized by having proliferation ability by expressing a proliferation marker protein, Ki-67. In addition, when the reprogrammed pluripotent cells are co-cultured with nutritious cells, proliferation of the pluripotent cells may be increased. Further, the cell reprogramming method of the present invention may further include culturing the pluripotent cells in the differentiation-inducing medium. Depending on a type of differentiation-inducing medium, the pluripotent cells may be differentiated into desired differentiated cells. Examples of the differentiation-inducing medium may include a multipotent cell differentiation-inducing medium, a hepatocyte differentiation-inducing medium, an osteogenic differentiation-inducing medium, an adipocyte differentiation-inducing medium, a myocyte differentiation-inducing medium, an astrocyte differentiation-inducing medium, a neuronal cell differentiation-inducing medium, an endothelial cell differentiation-inducing medium, a keratinocyte differentiation-inducing medium, a pancreatic beta cell differentiation-inducing medium, a cardiomyocyte differentiation-inducing medium, or the like, but are not particularly limited thereto. The present invention provides a cell reprogramming method including subjecting a mixture of differentiated or non-differentiated cells and a culture medium to physical stimulation which can promote an environmental influx, culturing the mixture subjected to the physical stimulation for 1 day to 6 days, and mixing the differentiated or non-differentiated cells with extracellular vesicles containing exosomes isolated from the culture medium and culturing the mixture for a predetermined time to obtain reprogrammed cells. The cell reprogramming method of the present invention is characterized in that culturing the extracellular vesicles containing exosomes isolated from the differentiated or non-differentiated cells subjected to the physical stimulation with the differentiated or non-differentiated cells for a predetermined time may reprogram the extracellular vesicles containing exosomes to arbitrary differentiated cells. The extracellular vesicles containing exosomes may be recovered through centrifugation by subjecting the mixture of the differentiated or non-differentiated cells and the culture medium to the physical stimulation which can promote an environmental influx and culturing the mixture subjected to the physical stimulation for 1 to 6 days. The physical stimulation of the differentiated or non-differentiated cells is described above and will be omitted to avoid the duplicated disclosure. The extracellular vesicles containing exosomes may express any one pluripotent marker or a triploblastic marker of Oct3/4, SOX2, NANOG, c-MYC, KLF4, TDGF1, SSEA4, TRA-1-60, PAX6, Nestin, Brachyury, SMA, GATA4, or AFP; any one neuronal cell marker of PAX6, Nestin, Sox1, Sox2, MAP2, TuJ1, GFAP, or O4; any one of myocyte marker of Desmin, Pax3, Actinin, SMA, GATA4, or NKX2-5; any one hepatocyte marker of AFP, HNF1a, HNF4a, CK18, or ALB; or may be stained with oil red O and express any one adipocyte marker of Pparc2, C/ebpa, aP2, or Fabp4. For example, when the extracellular vesicles containing exosomes contain a pluripotent marker, the differentiated cells may be reprogrammed into pluripotent cells when cultured with the differentiated cells. In addition, when the extracellular vesicles containing exosomes contain a differentiation marker, the differentiated cells may be reprogrammed into arbitrary differentiated cells having a different expression type when cultured with the differentiated cells. In addition, when the extracellular vesicles containing exosomes contain the differentiation marker, the differentiated cells may be reprogrammed into arbitrary differentiated cells when cultured with the non-differentiated cells. According to an embodiment of the present invention, the expression of various pluripotent markers in extracellular vesicles (EVs) stained with CD63, which was an exosomal marker recovered upon induction of es/ENTER, was confirmed, and in EV-treated normal human somatic cells, after 3 days of culture, pluripotent markers Oct4, Sox2, and Nanog are expressed, and thus the cell reprogramming is confirmed. In addition, the expression of markers of neural stem cells such as Pax6 was confirmed in EVs stained with CD63, which was an exosomal marker recovered in the induction of n/ENTER, and in EV-treated normal human somatic cells, after 3 days of culture, the expression of neural stem cell markers Sox1, Sox2, Pax6, and Nestin was confirmed. In addition, expression of a myocyte marker such as Pax3 was confirmed in EVs stained with CD63, which was an exosomal marker recovered in the induction of m/ENTER, and expression of a hepatocyte marker such as HNF1a was confirmed in EVs stained with CD63, which was an exosomal marker recovered in the induction of h/ENTER. Thus, it can be seen that the differentiated or non-differentiated cells subjected to the physical stimulation secrete the extracellular vesicles containing the reprogramming factor. The differentiated or non-differentiated cells are treated with the extracellular vesicles and cultured for 1 to 20 days through a suspended culture or monolayer culture method. Thus they can be reprogrammed into arbitrary pluripotent or differentiated cells. The cells that can be reprogrammed by the cell reprogramming method of the present invention may be the above-mentioned kinds of pluripotent cells or differentiated cells, and the disclosure thereof will be omitted to avoid the duplicated disclosure. Hereinafter, the present invention will be described in detail by Examples below. However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples. EXAMPLES <Example 1> Experiment for Verifying Intracellular Environmental Influx by Physical Stimulation This Example is an experiment for verifying intracellular environmental influx by physical stimulation, and to this end, the cells, primary HDF cells purchased from Invitrogen, were cultured in a DMEM added with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco), the ultrasonic wave treatment for the culture medium was performed at 5 W/cm2for 10 minutes, the cell treatment was performed in 1×106cells at 1 W/cm2for 5 seconds, and then 2×105cells were cultured in a 35 mm culture dish with the treated culture medium. For SEM image analysis, untreated HDF cells and cells immediately after the above treatment and cultured for 2 hours in a 5% CO2incubator at 37° C. were fixed with 4% paraformaldehyde at 4° C. for 12 hours, and then treated with a 0.1% tannic acid solution for 1 hour and a 1% osmium tetroxide solution for 2 hours and dehydrated with acetone for each concentration step. Thereafter, the cells were dried with liquid CO2and fixed on a surface coated with gold-palladium to be observed by an electron microscope (1555 VP-FESEM, Carl Zeiss). For live/dead image analysis, untreated HDF and cells immediately after ultrasonic wave treatment and cultured for 2 hours in a 5% CO2incubator at 37° C. were stained with a live/dead viability/cytotoxicity assay kit (Molecular Probes, Eugene, OR, USA). In the straining process, after 2 μM calcein (live cell staining dye) and 4 μM ethidium homodimer-1 (EthD-1, dead cell staining dye) were added to a cell culture medium, living cells were cultured at 37° C. in a 5% CO2incubator for 30 minutes, and then red fluorescence (EthD-1 staining, dead or damaged cells, excitation/emission, 528/617 nm) and green fluorescence (calcein staining, living cells, excitation/emission, 494/517 nm) were analyzed by a fluorescence microscope (IX3-ZDC, Olympus). As shown inFIG.2A, the cell membrane was damaged by the ultrasonic waves and a hole capable of introducing the external environment was formed, and such damage was recovered after 2 hours. The cells were stained using a live/dead kit, which was used to analyze cell death, in order to confirm the damaged cells after ultrasonic wave stimulation and recovery of the cells. As shown inFIG.2B, the cells were treated with ultrasonic waves, stained immediately after treatment, and stained 2 hours later. As a result, the cells with both green fluorescence and red fluorescence were observed immediately after treatment, and although the number of cells showing red fluorescence after 2 hours was significantly reduced, the red fluorescence was reduced since the cell membrane was recovered as shown inFIG.2A. It is considered that the cell damage is generated by the ultrasonic wave stimulation, but can be restored, and a medium environmental influx can be possible due to the cell membrane damage by such stimulation, and a phenomenon caused by influx of intracellular substances was analyzed. On the other hand, inFIG.2, ultrasound-exposed medium and cells (usMC) refers to a case in which ultrasonic waves are treated to each of the cells and the culture medium, and usMC-S means usMC in the suspended culture. <Example 2> Experiment for Verifying Influx of External Substances to Cells by Physical Stimulation A change in intracellular calcium concentration sensitive to the influx of the external substances in the cells was measured, and in order to confirm the possibility of substance influx according to ATP generation by ultrasonic waves, the expression of ATP receptors, which have been known that the receptor in the cell membrane opens an external substance influx passage on a cell membrane by the ATP measurement and the ATP reaction in the cells, was analyzed by RT-PCR. Calcium concentration was analyzed using a Fluo-4 NW Calcium Assay Kit (Molecular Probes). Untreated HDF cells and cells (usMC-S) exposed to a medium treated with ultrasonic waves after being directly treated with ultrasonic waves (1 W/cm2, 5 seconds) were mixed with an assay buffer among the components in the kit, respectively, divided with 3×104cells per well of a 96-well plate, and then mixed with 50 μl of Fluo-4 NW per well, and thereafter, fluorescence in a range of an excitation wavelength of 494 nm and an emission wavelength of 516 nm was measured at 10-second intervals for 15 minutes by a Varioskan flash fluorescent microplate fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). ATP was measured using an adenosine 5′-triphosphate (ATP) bioluminescent assay kit. After the untreated cells and the cells (usMC-S) exposed to a medium treated with ultrasonic waves after being directly treated with ultrasonic waves (1 W/cm2, 5 seconds) were divided with 3×104cells per well of a 96-well plate, the cells were cultured at room temperature for 3 minutes by dividing 100 μl of an ATP assay mix and ATP standard material per well, and thereafter, the luminescence intensity was measured by a Varioskan flash fluorescent microplate fluorometer (Thermo Fisher Scientific). For the RT-PCR for ATP receptor expression analysis, RNA was extracted from the treated cells by using an RNeasy plus mini kit (Qiagen, Hilden, Germany) and cDNA was synthesized by a Super ScripII kit (Invitrogen, Carlsbad CA, USA). The PCR was performed, after mixing cDNA and primers with a PCR premix (Bioneer, Daejeon, Korea), under conditions of denaturation at 95° C. for 5 min, 35 cycles at 95° C. for 30 sec, and gradient (50 to 65° C.) for 30 sec, and at 72° C. for 1 min, and at 72° C. for 15 min using a thermal cycler dice PCR machine (TP600, TAKARA, Otsu, Japan). TABLE 1PCR primer list of P2 receptorsGenePrimer sequence (5′-3′)codeForwardBackwardP2X4TCTCAACAGGCAGGTGCGTAGGCTCAACGTCCCGTGTATCGACTT (SEQ ID NO: 1)GG (SEQ ID NO: 65)P2X7CAGAAGGCCAAGAGCAGCGGGGACACGTTGGTGGTCTTGTCTT (SEQ ID NO: 2)GTCA (SEQ ID NO: 66)P2Y1CTTGGTGCTGATTCTGGGCTGGCTCGGGAGAGTCTCCTTCTG(SEQ ID NO: 3)(SEQ ID NO: 67)P2Y2CCGCTCGCTGGACCTCAGCTGCTCACTGCTGCCCAACACATC(SEQ ID NO: 4)(SEQ ID NO: 68)P2Y11GAGGCCTGCATCAAGTGTCTGACGTTGAGCACCCGCATGATG(SEQ ID NO: 5)(SEQ ID NO: 69) As shown inFIG.3, the intracellular calcium influx after the ultrasonic wave stimulation was increased up to 60 seconds, the intracellular ATP concentration was maximally increased at 60 minutes, and the expression of the ATP receptors in the cell membrane was also increased at 1 hour and 4 hours, respectively. It was confirmed by the increase of calcium concentration that the external substance was introduced into the early cells by the ultrasonic wave stimulation, and it can be seen that the ATP was generated by the ultrasonic wave and as a result, the ATP receptor reacted, and then the cell membrane passage was opened and thus, the external substances can be introduced. <Example 3> Experiment for Verifying Influx of External Substances into Cells by Physical Stimulation Using QD605 QD605 was set as an external substance and whether the QD605 was introduced into the cells by ultrasonic waves was confirmed. QD605 is a fluorescent nano-material that is known to be poorly permeable in living cells, and the influx of the external substance into the cells by the ultrasonic waves using QD605 was confirmed. To this end, like Example 1, HDF was subjected to the ultrasonic wave stimulation and treated with 100 pmol of QD605, and presence of QD605 in a single cell and spheroid was confirmed after 24 hours. As shown inFIG.4, no fluorescence was observed in HDF that was not subjected to ultrasonic wave stimulation. However, fluorescence was observed in the HDF subjected to ultrasonic wave stimulation. In order to confirm the possibility of cell changes due to the influx of external substances, 100 pmol of QD605 was added after treating ultrasonic waves in each medium environment (ES, neuroprogenitor, hepatocyte, muscle), and after 24 hours, expression of transcription factors (ES: Oct4, Neuroprogenitor: Pax6, Hepatocyte: HNF1a, Muscle: Pax3) during each differentiation process was confirmed through ICC. As shown inFIGS.4B and4C, the transcription factors were observed in each of the cells and spheroid containing the external substance (QD605). This is a result indicating the possibility of cell reprogramming due to the influx of external substances. During the experiment, only es/ENTER and n/ENTER among four environmental influx samples formed spheroids. However, m/ENTER and h/ENTER did not form spheroids. The reason lies in the characteristics of the cells and the medium composition. In the case of ES and neuroprogenitors, spheroid or sphere was formed in the suspended culture process, but in the case of myocytes or hepatocytes, the spheroid was not formed. This is because the cells were adhered and cultured in a coated culture dish during the differentiation induction process, and particularly, FBS was contained in the medium of myocytes, but since the FBS increased cell adhesion, the spheroid was not formed. <Example 4> Analysis of Exosomes in Cell Culture Medium Subjected to Physical Stimulation Since the cell stimulation by the ultrasonic waves is not equally stimulated in all cells, reprogramming of the cells may occur in some cells, and the possibility of cell exchange between these changed cells and non-changed cells was considered. Recently, with reference to the possibility of intercellular material exchange by exosomes, there is a possibility that the exosomes in the culture media released from the cells treated with ultrasonic waves contain genetic materials. There is a possibility that the material secreted from the reprogrammed cells contains a genetic material which plays an important role in the reprogramming, and the exosomes in the culture medium cultured after ultrasonic wave treatment were recovered during medium exchange for each culture time, RNAs of the exosomes in the culture medium were extracted by Amicon Ultra-0.5 kit (Millipore), and the cDNA synthesis was performed by Super ScripII kit (Invitrogen, Carlsbad CA, USA). The PCR was performed, after mixing cDNA and primers with a PCR premix (Bioneer, Daejeon, Korea), under conditions of denaturation at 95° C. for 5 min, 35 cycles at 95° C. for 30 sec, and gradient for 30 sec, and at 72° C. for 1 min, and at 72° C. for 15 min using a thermal cycler dice PCR machine (TP600, TAKARA, Otsu, Japan) (Table 2). As shown inFIG.5, the expression of pluripotent RNA in RNAs of the exosomes was confirmed. Meanwhile, inFIG.5, usMC refers to a case where the cells and the culture medium are treated with ultrasonic waves, respectively, and usMC-A means monolayer-cultured usMC. TABLE 2AnnealingGenePrimer sequence (5′-3′)temperaturecodeForwardBackward(° C.)Oct4GACAGGGGGAGGGGAGGCTTCCCTCCAACCAGTTGC60(POU5F1)AGCTAGGCCCAAAC(SEQ ID NO: 6)(SEQ ID NO: 70)Sox2GGGAAATGGGAGGGGTGCTTGCGTGAGTGTGGATGG63AAAAGAGGGATTGGTG(SEQ ID NO: 7)(SEQ ID NO: 71)NanogCAGCCCCGATTCTTCCACCCGGAAGATTCCCAGTCGG64AGTCCCGTTCACC(SEQ ID NO: 8)(SEQ ID NO: 72)Utf1CCGTCGCTGAACACCGCCCGCGCTGCCCAGAATGAA65CTGCTGGCCCAC(SEQ ID NO: 9)(SEQ ID NO: 73)Lin28aAGCGCAGATCAAAAGGAGCCTCTCGAAAGTAGGTTG50ACA (SEQ ID NO: 10)GCT (SEQ ID NO: 74)Rex1CAGATCCTAAACAGCTCGGCGTACGCAAATTAAAGTC52CAGAATCAGA(SEQ ID NO: 11)(SEQ ID NO: 75)Fgf4CTACAACGCCTACGAGTCGTTGCACCAGAAAAGTCA55CTACAGAGTTG(SEQ ID NO: 12)(SEQ ID NO: 76)Foxd3AAGCTGGTCGAGCAAACTCTCCCATCCCCACGGTACT50CA (SEQ ID NO: 13)A (SEQ ID NO: 77)Esg1ATATCCCGCCGTGGGTGAACTCAGCCATGGACTGGA60AAGTTCGCATCC(SEQ ID NO: 14)(SEQ ID NO: 78)Tdgf1CTGCTGCCTGAATGGGGGGCCACGAGGTGCTCATCCA65AACCTGCTCACAAGG(SEQ ID NO: 15)(SEQ ID NO: 79)c-MycAATGAAAAGGCCCCCAAGGTCGTTTCCGCAACAAGTC50GTAGTTATCCCTCTTC(SEQ ID NO: 16)(SEQ ID NO: 80)Klf4CCCACATGAAGCGACTTCCAGGTCCAGGAGATCGTT54CC (SEQ ID NO: 17)GAA (SEQ ID NO: 81) <Example 5> Experiment for Verifying Material Delivery by Exosomes Since the expression of a pluripotent marker was confirmed in exosomes in the culture medium of cells treated with ultrasonic wave treatment in Example 4 above, whether a genetic material and a protein were delivered by the exosomes was confirmed. As a result of photographing images of living cells by adding QD605 after ultrasonic wave treatment, as shown inFIG.6A, it was confirmed that QD605 of the cells introduced with QD605 on 7 hours and 45 minutes was separated with a part of the cytoplasm to be moved to other cells. The separated part of the cytoplasm was expected to be exosomes, and the usMC-treated cells were exposed to various medium environments, immobilized in 4% paraformaldehyde for 10 minutes, and embedded in PBS containing 0.1% Triton X-100 for 40 minutes. The cells were blocked with a PBS solution containing 5% (v/v) goat serum for 1 hour and an exosome marker CD63 (1:100, Santa Cruz Biotechnology) and initial expression markers of cells induced by each differentiation-inducing medium, such as embryonic stem cells (Oct4 1:200; Nanog 1:200; abeam), neural stem cells (Pax6, 1:200; abeam), myocytes (Pax3, 1:200; abeam), and hepatocytes (HNF1a, 1:200; Cell Signaling Technology) were stained overnight at 4° C. with primary antibodies. The cells were washed with a PBS buffer containing 0.03% Triton X-100, stained with secondary antibodies, Alexa-488 or -594 binding anti-rabbit, and anti-mouse antibodies (1:1000, Thermo, excitation/emission, 495/519 nm, excitation/emission, 590/617 nm) at room temperature for about 1 hour and 30 minutes, washed with a PBS buffer containing 0.03% Triton X-100, and then mounted on a mounting sol containing DAPI (Vector Laboratories, Inc., Burlingame, CA, excitation/emission, 420/480 nm), and images were analyzed with a confocal laser fluorescence microscope (LSM 700; Carl Zeiss). As shown inFIG.6B, those estimated to be a part of the cytoplasm that was released around the cells were stained with CD63, and the expression of the pluripotent cell marker such as Oct4 and Nanog was confirmed in extracellular vesicles (EVs) stained with CD63, which was an exosome marker, during es/ENTER induction, the expression of the neural stem cell marker such as Pax6 was confirmed in the EVs stained with CD63, which was the exosome marker, during n/ENTER induction, the expression of the myocyte marker such as Pax3 was confirmed in the EVs stained with CD63, which was the exosome marker, during m/ENTER induction, and the expression of the hepatocyte marker such as Hnf1a was confirmed in the EVs stained with CD63, which was the exosome marker, during h/ENTER induction. From the above results, it was hypothesized that the exosomes were separated from the cytoplasm and contained genetic materials and proteins to be delivered to the surrounding cells so as to induce changes in surrounding cells. In order to prove this hypothesis, it was considered that the poly(A)27-Cy5.5 may be delivered by the exosomes when the exosomes were extracted and stained with CD63 (stained to distinguish the newly injected exosomes because the exosomes existed even in the cultured cells), introduced with a genetic material expressed by poly(A)27-Cy5.5 and cultured with untreated HDF. As shown inFIG.6C, it was confirmed that the exosomes stained with CD63 were found in the cells, and cy5.5 was expressed like CD63. This means that the gene (poly-A) injected by the exosomes was delivered. <Example 7> Co-Culture of Cells Subjected to Physical Stimulation and Normal Cells Since the genetic material may be delivered by the exosomes, it has been hypothesized that the exosomes secreted from the cells cultured in a human ES medium may change the properties of surrounding cells or untreated cells. To verify this, the exosomes were extracted from the 2-day cultured medium of cells treated with ultrasonic wave cultured in the human ES medium environment, and the exosome extract was mixed and cultured for 6 days in a process of culturing the untreated cells in the human ES medium and a fibroblast culture medium, DMEM. As a result, spheroid was produced in a group added with exosomes (FIG.7A), and as a result of verifying the Oct4 expression in the cells, the expression of a pluripotent marker, Oct4, was observed (FIG.7B). This indicates that the delivery of the genetic material by the exosomes may induce the cell reprogramming. <Example 8> Direct Reprogramming of Fibroblasts In Examples 1 to 7 above, a possibility of cell reprogramming and reprogramming up to surrounding cells by a change in medium environment was verified, and based on this, the reprogramming of cells was confirmed by applying various medium environments. To this end, as shown inFIG.8, human fibroblasts were collected into 1×106cells in a 1 mL differentiation-inducing medium, treated with ultrasonic waves at an intensity of 1 W/cm2for 5 seconds, divided into 2×105/Well in a 35 mm culture dish or 6-well plate, and then cultured in a 2 mL differentiation-inducing medium treated with the ultrasonic waves at an intensity of 10 W/cm2for 10 minutes. Although there is a difference depending on a type of differentiation-inducing medium, spheroid was formed between about 2 days and 6 days after culturing. In addition, it was observed that intracellular bubbles were formed by culturing for 20 days after usMC treatment using the adipocyte differentiation-inducing medium, and oil red O, a lipid staining reagent for discriminating adipocytes, was stained by analysis of the bubbles. This is an indicator that cells produce fat (FIG.9A). The expression of adipocyte marker genes, Pparc2, C/ebpa, aP2, and Fabp4 was confirmed by RT-PCR after the cell RNA was extracted, and as a result, the expression after differentiation induction was increased (FIG.9B). In addition, in order to confirm the differentiation of HDF into neuroprogenitors by a neural stem cell (neuroprogenitor) differentiation-inducing medium and ultrasonic waves, the expression of neuroprogenitor markers, Oct4, Sox2, Pax6, and Nestin were confirmed by staining by immunocytochemistry in spheroids produced on day 3 after differentiation induction and attached cells. FIG.10Ashows the morphology of the differentiated cells andFIG.10Bshows the neuroprogenitor markers in the spheroid, in which it was confirmed that if differentiation was induced, the expression of Oct4 was reduced and the expression of Sox2, Pax6, and Nestin was high.FIG.10Cshows the expression in the attached cells, and is the same expression pattern as above. At this time, the markers of neuroprogenitors and neural stem cells are Sox2, Pax6, and Nestin. Oct4 is a pluripotent marker, and the expression of Oct4 is decreased in adult stem cells and progenitor cells. TABLE 3Composition of differentiation-inducing mediumComponentsContentNeuroprogenitorDMEM F12differentiation-bFGF20 ng/mlinducing mediumEGF20 ng/mlB27 supplement (×50)1/50N2 supplement (×100)1/100 FIG.11shows a result of analyzing an expression pattern of Pax6/Nestin in the cells differentiation-induced for 7 days after induction of differentiation through flow cytometry, in which the expression of Pax6 and Nestin was relatively 50% or more on day 1 after the treatment, and the expression of Pax6 and Nestin was the highest on day 3. FIG.12shows the expression of ki67 to confirm the proliferation of cells expressing the neuroprogenitor marker (Pax6/nestin) on day 3 after induction of differentiation, in which in cells indicated by a white arrow, the expression of ki67 in the cells where Nestin was expressed may be confirmed. This result indicates that the differentiation-induced cells have proliferation ability. InFIG.12, the arrow is a marker indicating that the Nestin-stained cells are proliferating. FIG.13shows an experimental result of confirming self-renewal of differentiation-induced cells (n/ENTER cells), and inFIG.13A, it was confirmed through a video that the cells proliferated from one spheroid express Pax6 and Nestin and thus it can be seen that the properties of neuroprogenitors are transmitted to the proliferated cells. Next, differentiation-induced cells (n/ENTER cells) were injected into the brain of 5-week-old mice, and after 4 weeks, the brain was recovered and the differentiation of the injected cells into surrounding cells was confirmed, and the function of the differentiated cells was confirmed. As shown inFIG.14A, when the cells injected into the brain (n/ENTER cells) were stained with a human nuclear antigen (HNA) and marked, and the marked cells were stained with a Gfap antibody, it was confirmed that Gfap was expressed in the injected cells. In order to confirm whether the cells expressing Gfap have a normal function, the cells were stained with a synapsin 1 antibody (1:500, R&D system) to confirm whether synapsin was secreted. As a result, the expression of synapsin 1 was observed in the cells expressing Gfap among the cells expressing HNA (FIG.14B). Next, the expression of neuroprogenitor markers (Oct4, Sox2, Pax6, and Nestin) in the spheroid generated on day 3 after induction of differentiation was confirmed by immunocytochemical staining. To this end, the spheroid and attached cells were immobilized in 4% paraformaldehyde for 10 minutes and embedded in PBS containing 0.1% Triton X-100 for 40 minutes. The cells were blocked with a PBS containing 5% (v/v) goat serum for 1 hour and Oct4 (1:200), Sox2 (1:200), Pax6 (1:200), Nestin (1:200, Cell Signaling Technology), and the like were stained with a primary antibody overnight at 4° C. The cells were washed with a PBS buffer containing 0.03% Triton X-100, stained with secondary antibodies, Alexa-488 or -594 binding anti-rabbit, and anti-mouse antibodies (1:1000, Thermo, excitation/emission, 495/519 nm, excitation/emission, 590/617 nm) at room temperature for about 1 hour and 30 minutes, washed with a PBS buffer containing 0.03% Triton X-100, and then mounted on a mounting sol containing DAPI (Vector Laboratories, Inc., Burlingame, CA, excitation/emission, 420/480 nm), and images were analyzed with a confocal laser fluorescence microscope (LSM 700; Carl Zeiss). InFIG.15A, the spheroid formation was confirmed and the expression of neuroprogenitor makers Pax6 and Nestin was highly expressed in spheroid (FIG.15B). FIG.15Cshows a result of analyzing an expression pattern of Pax6/Nestin in the cells differentiation-induced for 3 days after induction of differentiation through flow cytometry, in which the expression of Pax6 and Nestin was relatively 70% or more on day 1 after the treatment, and the expression of Pax6 and Nestin was the highest on day 3. FIG.16shows a result of confirming that the expression of neuroprogenitor markers (Sox2, Pax6 and Nestin) is confirmed in cells after 20 days after induction of differentiation by immunocytochemical staining, in which the top and middle photographs show that the expression of Sox2, Pax6 and Nestin is high and the bottom photograph shows the same expression pattern as above as a result of confirming expression of an oligodendrocyte marker. This result indicates that differentiation-induced cells have differentiation potency similar to neuroprogenitor differentiation potency. <Example 9> Direct Hepatocyte Differentiation Like the schematic diagram ofFIG.17, HDF cells, HeLa cells, and Hep3B cells were collected into 1×106cells in a 1 mL differentiation-inducing medium, treated with ultrasonic waves at an intensity of 1 W/cm2for 5 seconds, divided into 2×105in a 35 mm Laminin coating culture dish, treated with ultrasonic waves at an intensity of 10 W/cm2for 10 minutes, and then cultured in a 2 mL hepatocyte differentiation-inducing medium. Differentiation-induced cells using a hepatocyte differentiation-inducing medium were named as h/ENTER. FIG.18induced differentiation of HDF treated with a hepatocyte differentiation-inducing medium and ultrasonic waves into hepatocytes (h/ENTER).FIG.18shows a change in cell appearance after 20 days of induction of HDF differentiation, and as a result of confirming hepatocyte markers (AFP, HNF4a, CK18, and ALB) after 20 days of induction of HDF differentiation through immunocytochemistry, the expression of the hepatocyte markers was confirmed in the h/ENTER cells. Next, differentiation of HeLa cells treated with a hepatocyte differentiation-inducing medium and ultrasonic waves into hepatocytes (HeLa h/ENTER) was induced. A change in cell (HeLa h/ENTER) appearance after 19 days of the induction of HeLa cell differentiation, and hepatocyte markers (ALB, HNF4a, CYP3A4F, CYP3A7F, AIAT, SOX7, and GATA6) were confirmed through (HeLa h/ENTER) qPCR after 20 days of the induction of HeLa cell differentiation. As shown inFIG.19A, it was confirmed that most of hepatocyte markers were increased in differentiation-induced HeLa cells compared to HeLa cells. In addition, hepatocyte markers (HNF4a, CK18, and ALB) were confirmed by immunocytochemistry after 3 weeks of the induction of HeLa cell differentiation, and as a result, it was confirmed that the expression of the hepatocyte markers was increased in differentiated HeLa cells (HeLa h/ENTER) compared with the HeLa cells (FIG.19B). The hepatocyte markers (HNF4a, CK18, and ALB) were confirmed by immunocytochemistry after 3 weeks of the induction of HeLa cell differentiation, and as a result, it was confirmed that the expression of the hepatocyte markers was increased in differentiated HeLa cells (HeLa h/ENTER) compared with the HeLa cells (FIG.19C). Next, differentiation of Hep3B cells treated with a hepatocyte differentiation-inducing medium and ultrasonic waves into hepatocytes (Hep3B h/ENTER cell) was induced. A change in cell appearance after 19 days of induction of Hep3B cell differentiation is shown, and expression of hepatocyte markers (HNF4a, CK18, and ALB) after 3 weeks of induction of Hep3B cell differentiation by immunocytochemistry were confirmed. As shown inFIG.20B, it was confirmed that the expression of the hepatocyte markers was increased in the differentiated Hep3B cells (Hep3B h/ENTER) compared with the Hep3B cells. Next, differentiation of HDF cells treated with a human ES culture medium and ultrasonic waves into es/ENTER cells was induced.FIG.21Ashows a change of cell morphology according to a culture time, andFIG.21Bshows a result of having a difference in Oct4 expression according to a culture time, in which spheroid was formed on day 1 after induction of differentiation by the human ES medium, and expression of a pluripotent marker, Oct4, was increased according to a culture time. Spheroids formed after culturing for 6 days were recovered and the expression of the pluripotent marker was confirmed by RT-PCR and ICC. As a result, in the es/ENTER cells, expression of a (A) pluripotent marker gene and (B) protein was confirmed. As a result of analyzing a pluripotent property in the es/ENTER cells, expression of SSEA4 and TRA-1-60 of es/ENTER cells was confirmed using flow cytometry (FIG.23A), and as a result of analyzing a pattern of pluripotent gene expression by a microarray (affymetrix chip), a pattern similar to the ES cells instead of HDF was shown (FIG.23B). In addition, as a result of confirming whether methylation of a promoter region at which the synthesis in a DNA gene expressed by bisulfate sequencing starts is loosened, it was confirmed that important pluripotent genes Oct4 and Nanog were opened. From these results, it can be seen that es/ENTER differentiation-induced by the human ES medium has pluripotent properties (FIG.23C). TABLE 4List of bisulfite sequencing primers for methylation analysisAnnealingGenePrimer sequence (5′-3′)temperaturecodeForwardBackward(° C.)Oct4CCAGGTTCAATGGATTCTCGTATCCGACCAGGGTTAG58C (SEQ ID NO: 18)GG (SEQ ID NO: 82)NanogTTCTCTCCTCCTCCCTCTCCTCCCAAAATGCTGGGAT56C (SEQ ID NO: 19)TA (SEQ ID NO: 83)Tdgf11GTGGGTCCTCTTCAGTGCGCTGCTGGAGAGGTGCTT60AT (SEQ ID NO: 20)AG (SEQ ID NO: 84)2GACCCTCGCCTTATCCTTTCACTGCCCTACTGCTTGG60C (SEQ ID NO: 21)TT (SEQ ID NO: 85)3GCACAGAGGGTGTCCATCCTGCCCCTCTCACTCATCT60TT (SEQ ID NO: 22)C (SEQ ID NO: 86)AfpCAGTCCAGCAACAAGCCTACTGGAGTCACTGGGAGG58TT (SEQ ID NO: 23)AA (SEQ ID NO: 87)Gata4TAGGATGCCTGCTGGATTTCATTCATTCGCCCTCTCTT58C (SEQ ID NO: 24)C (SEQ ID NO: 88)Acta2GGAGCACTTGAGAAGCACTCAGGAAAGCCTCCCTC60AAGA (SEQ ID NO: 25)TT (SEQ ID NO: 89)Msx1GTAGACGCGGTTTGTGGATTGGGGCTCTGTTTTTAAC60AC (SEQ ID NO: 26)G (SEQ ID NO: 90)Pax6GTTGCAGCTGGTGTGTTGGCATTGTTGTGAATGCTGC60AC (SEQ ID NO: 27)T (SEQ ID NO: 91)NestinGGGTCAAGTGGACTTTCCCACCCTCCTTGTCACTCCT60TG (SEQ ID NO: 28)C (SEQ ID NO: 92) Next, the differentiation marker was confirmed in the es/ENTER cells.FIG.24shows (A) expression of a triploblastic marker gene and (B) expression of proteins in the es/ENTER cells,FIG.25shows a change in expression of Oct4 and the triploblastic marker gene in the es/ENTER cells for each culture time, andFIG.26shows (A) expression of a triploblastic marker protein in the monolayer-cultured es/ENTER cells and (B) a result of triploblastic marker DNA methylation analysis of es/ENTER cells using bisulfate sequencing. TABLE 5PCR primer list of differentiation marker genesAnnealingGenetemperaturecodePrimer sequence (5′-3′)(° C.)EndodermAfpAGCAGCTTGGTGGTGGATCCTGAGCTTGGCACAG63GA (SEQ ID NO: 29)ATCC(SEQ ID NO: 93)Foxa2TTCAGGCCCGGCTAACTCCCTTGCGTCTCTGCAAC58TG (SEQ ID NO: 30)ACC (SEQ ID NO: 94)Gata6TGTGCGTTCATGGAGAAGTTTGATAAGAGACCTCA60ATCA (SEQ ID NO: 31)TGAACCGACT(SEQ ID NO: 95)EctodermNestinGAAACAGCCATAGAGGGTGGTTTTCCAGAGTCTT50CAAACAGTGA(SEQ ID NO: 32)(SEQ ID NO: 96)Pax6ACCCATTATCCAGATGTGATGGTGAAGCTGGGCAT58TTTGCCCGAGAGGCGGCAG(SEQ ID NO: 33)(SEQ ID NO: 97)Mesoderm,Acta2CTATGAGGGCTATGCCTTGCTCAGCAGTAGTAAC50cardio-(a-GCC (SEQ ID NO: 34)GAAGGAmyocyteSMA)(SEQ ID NO: 98)MesodermBrachy-GCCCTCTCCCTCCCCTCCCGGCGCCGTTGCTCAC66ury (T)ACGCACAGAGACCACAGG(SEQ ID NO: 35)(SEQ ID NO: 99)Msx1CGAGAGGACCCCGTGGAGGCGGCCATCTTCAGCT58TGCAGAGTCTCCAG(SEQ ID NO: 36)(SEQ ID NO: 100)Cardio-My17GGGCCCCATCAACTTCACTGTAGTCGATGTTCCCC58myocyteCGTCTTCCGCCAGGTCC(SEQ ID NO: 37)(SEQ ID NO: 101)Nkx2-5CCAAGGACCCTAGAGCCATAGGCGGGGTAGGCG50GAA (SEQ ID NO: 38)TTAT(SEQ ID NO: 102)TnTcATGAGCGGGAGAAGGAGTCAATGGCCAGCACCTT63CGGCAGAACCCTCCTCTC(SEQ ID NO: 39)(SEQ ID NO: 103)NeuronsMap2CAGGTGGCGGACGTGTGCACGCTGGATCTGCCTG66.5AAAATTGAGAGTGGGGACTGTG(SEQ ID NO: 40)(SEQ ID NO: 104)TuJ1GAGCGGATCAGCGTCTACGATACTCCTCACGCACC52TACAATTGCT(SEQ ID NO: 41)(SEQ ID NO: 105)GfapCCTCTCCCTGGCTCGAATGGAAGCGAACCTTCTC52G (SEQ ID NO: 42)GATGTA(SEQ ID NO: 106)Vglut1CGACGACAGCCTTTTGTGGCCGAGACGTAGAAAA50GT (SEQ ID NO: 43)CAGAG(SEQ ID NO: 107)Vmat2CTTTGGAGTTGGTTTTGCGCAGTTGTGGTCCATGA43G (SEQ ID NO: 44)(SEQ ID NO: 108)AdipocytePparc2ATTGACCCAGAAAGCGATCAAAGGAGTGGGAGTG52.7TC (SEQ ID NO: 45)GTCT(SEQ ID NO: 109)C/ebpaGCAAACTCACCGCTCCAATTAGGTTCCAAGCCCCA56.7TG (SEQ ID NO: 46)AGTC(SEQ ID NO: 110)aP2AACCTTAGATGGGGGTGTTCGTGGAAGTGACGCC57.2CCTGTTTC(SEQ ID NO: 47)(SEQ ID NO: 111)Fabp4ACTGGGCCAGGAATTTGCTCGTGGAAGTGACGC55ACG (SEQ ID NO: 48)CTT(SEQ ID NO: 112)HepatocyteAlbAGCTGTTATGGATGATTTCCTCGGCAAAGCAGGT60CGCAGCTC(SEQ ID NO: 49)(SEQ ID NO: 113)Cyp3a4GTGACTTTGCCCATTGTTCAGGCGTGAGCCACTG60TAGAAAGTG (SEQ ID NO: 114)(SEQ ID NO: 50)Cyp3a7GATTCTGTACGTGCATTGATTTGGTCATCTCCTCTA60TGCTCTATTACCAAGT(SEQ ID NO: 51)(SEQ ID NO: 115)TatCCACACCCACACTCAGATATTAGTGAGTCACTCTA60CCT (SEQ ID NO: 52)GCAGCGC(SEQ ID NO: 116)A1ATGGTCACAGAGGAGGCACAGTCCCTTTCTCGTCGA60CC (SEQ ID NO: 53)TGGT(SEQ ID NO: 117)Sox7TGCCCACTTCATGCAACTAGGTACCCTGGGTCTTT60CC (SEQ ID NO: 54)GGTCA(SEQ ID NO: 118)Housebeta-CATGTACGTTGCTATCCACTCCTTAATGTCACGCA50keepingactinGGC (SEQ ID NO: 55)CGAT(SEQ ID NO: 119)geneGapdhATGGGGAAGGTGAAGGTGGGTCATTGATGGCAAC60CG (SEQ ID NO: 56)AATATC(SEQ ID NO: 120) The expression of the triploblastic marker in es/ENTER spheroid was confirmed by RT-PCR and ICC, and such a result indicates that the es/ENTER cells have multi-differentiation properties. Accordingly, the expression pattern of the pluripotent marker Oct4 according to a culture time was confirmed, and as a result, it was confirmed that after 6 days, the expression of Oct4 as the pluripotent property was decreased and the expression of the triploblastic marker was increased. Such a result shows that the es/ENTER cells are differentiated in the pluripotent property. As a result, it was confirmed that the cells released from the spheroid cultured for 2 days by attaching the es/ENTER spheroid expressed the triploblastic marker, and as a result of analyzing the DNA methylation of a representative triploblastic marker gene, it was confirmed that the triploblastic marker gene was opened. FIG.27shows a result of an in-vitro differentiation experiment of es/ENTER cells into (A) neurons, (B) cardiomyocyte, and (C) hepatocytes. The es/ENTER cells were differentiation-induced using a medium differentiation-inducing into cells differentiated from each of triploblasts for 4 weeks, and as a result, the es/ENTER cells were differentiation-induced into neurons (ectoderm), cardiomyocyte (mesoderm), and hepatocytes (endoderm), and respective differentiation markers were expressed. FIG.28shows a result of confirming expression of differentiation markers of (A) neuron, (B) cardiomyocyte, and (C) hepatocyte in HDF, and no differentiation marker was expressed in HDF. FIG.29shows a result of RT-PCR analysis showing the expression of differentiation marker genes of (A) neuron, (B) cardiomyocyte, and (C) hepatocyte of differentiation-induced es/ENTER cells, in which the expression of the differentiation marker genes was increased in the differentiation-induced es/ENTER. These results are results that verifies multi-differentiation ability of es/ENTER. FIG.30shows karyotypes of the es/ENTER cells by chromosome G-band analysis, and as a result of analyzing the mutation of the cell chromosomes due to ultrasonic wave stimulation in the es/ENTER production, it was normal. Next, es/ENTER cells were transplanted into leg muscles of 5-week-old SCID mice and after 4 weeks, the transplanted cells were confirmed using HNA. As shown inFIG.31, it was confirmed that the es/ENTER cells were differentiated into a skeletal muscle. It was also confirmed that Oct4 was not expressed and proliferated in the transplanted cells. Next, the es/ENTER cells were transplanted into the brain of mice to confirm in vivo differentiation. To this end, the es/ENTER cells were transplanted into the brain of 5-week-old SCID mice and after 4 weeks, the transplanted cells were confirmed using HNA. As a result, it was confirmed that the es/ENTER cells were differentiated into astrocytes (Gfap) and synapsin and a vesicular glutamate transpoter were secreted. This indicates that the transplanted cells are normally differentiated and perform functions. In addition, it was confirmed that Oct4 was not expressed in the transplanted cells and not proliferated (FIG.32). Next, MEF (mouse embryo fibroblast) was induced to differentiate into mouse es/ENTER cells using a hES medium in the same manner as HDF. The MEF used in this experiment was OG2-MEF and was performed with an embryo fibroblast of mouse transfected with an Oct4 promoter vector. These cells express GFP fluorescence when Oct4 is expressed and were used to observe the expression of Oct4. As shown inFIG.33, the number and sizes of spheroids and GFP expression were increased over time in the cells induced by ultrasonic wave treatment. Next, pluripotent properties were analyzed in the mouse es/ENTER cells. As shown inFIG.34, ICC, RT-PCR, flow cytometry, and AP staining of mouse es/ENTER showed similar tendency to mouse ES. Next, triploblastic properties were analyzed in the mouse es/ENTER cells. As a result, the triploblastic properties shown in the human es/ENTER were also observed even in the mouse es/ENTER cells, and a difference in expression was shown over time through RT-PCR and ICC analysis for each culture time. This result was the same as the result of human es/ENTER (FIG.35). FIG.36shows in-vitro differentiation of mouse es/ENTER into (A) neuron and (B) cardiomyocyte, andFIG.36Cshows a karyotype analysis result by chromosome G band analysis of mouse es/ENTER. The karyotype analysis was performed using a GTG banding chromosome analysis (GenDix, Inc. Seoul, Korea). As an experimental result, the differentiation markers of neurons and cardiomyocytes were confirmed in mouse es/ENTER cells to be differentiated, and as a result of karyotype analysis of chromosomal mutations by ultrasonic waves, it was confirmed that there was no mutation. <Example 10> Experiment for Differentiation Induction of Es/ENTER Using Other Cells These results show that this method is applicable to cells of other individuals as well as HDF, and applied to various cells (L132, MSC, patient skin fibroblasts). Cell differentiation was induced in the same manner as es/ENTER using L132 (pulmonary epithelial cells), mesenchymal stem cells (MSCs), and skin fibroblasts (patient-derived skin fibroblasts), and as a result, it was shown that cell spheroids were formed, and the pluripotent markers and the triploblastic markers were expressed similar to es/ENTER. FIG.37shows a result of the differentiation of L132 cells into L132 es/ENTER cells by a human ES culture medium and ultrasonic wave stimulation, in whichFIG.37Ashows a change in cell morphology according to a culture time, andFIGS.37B and37Cshow changes in (B) pluripotent and (C) triploblastic properties of the L132 es/ENTER cells. FIG.38shows a result of the differentiation of MSC into MSC es/ENTER cells by a human ES culture medium and ultrasonic wave stimulation, in whichFIG.38Ashows a change in cell morphology according to a culture time, andFIGS.38B and38Cshow changes in (B) pluripotent and (C) triploblastic properties of the MSC es/ENTER cells. FIG.39shows a result of the differentiation of human skin fibroblasts into SF es/ENTER cells by a human ES culture medium and ultrasonic wave stimulation, in whichFIG.39Ashows a change in cell morphology according to a culture time, andFIGS.39B and39Cshow changes in (B) pluripotent and (C) triploblastic properties of the SF es/ENTER cells. <Example 11> Differentiation Induction into Es/ENTER Cells Using Other Physical Stimulation Heat shock and laser were used as physical stimulation for induction of differentiation into the same medium as the human ES medium. First, differentiation of HDF into es/ENTER cells was induced by heat shock and a hES medium. For heat shock, HDF was exposed at 42° C. for 2 minutes and then left for about 5 seconds on ice.FIG.40Ashows differentiation-induced HDF spheroid, andFIGS.40B and40Cshow (B) pluripotent and (C) triploblastic properties of es/ENTER cells. Next, differentiation of es/ENTER cells of HDF was induced by laser stimulation and a hES medium. As the laser treatment condition, an Ocla treatment laser (Ndlux) was used, and the cells were cultured after irradiating a laser at 808 nm for 5 seconds.FIG.41Ashows differentiation-induced HDF spheroid, andFIGS.41B and41Cshow (B) pluripotent and (C) triploblastic properties of es/ENTER cells. As shown inFIGS.40and41, it was confirmed that cell spheroids were formed by both stimulations similarly to the effects by ultrasonic waves, and the pluripotent and triploblastic markers were expressed. These results indicate that cell reprogramming due to a medium environmental influx is applicable to various cells and that various methods are possible as the physical stimulation for environmental influx with the medium environment, and like the previous result, the properties of the cells may be changed by the environment. <Example 12> Reprogramming of Cells Using Extracellular Vesicles (EVs) The cells, primary HDFs purchased from Invitrogen, were cultured in a DMEM added with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco), ultrasonic wave treatment to the culture medium was performed at 5 W/cm2, for 10 minutes, the cell treatment was performed in 1×10 6 HDFs at 1 W/cm2for 5 seconds, and then 2×105cells were cultured in a 35 mm culture dish together with the ultrasonic wave-treated culture medium under conditions of 37° C. and 5% CO2for 1 day. The culture medium was recovered, put in an Amicon Ultra centrifugal filter (Millipore), and centrifuged at 14000 rpm for 20 minutes, and then the EVs in the culture medium were filtered and recovered by a filter. Next, the HDF was cultured in a culture dish so as to be filled with about 70 to 80%, and the culture medium was recovered and washed twice with D-PBS. Thereafter, 10 μl/mL (v/v) of concentrated EVs recovered from the culture medium on day 1 of es/ENTER and n/ENTER were added to an embryonic stem cell medium or a neural stem cell differentiation medium (Gibco), mixed with the HDF washed above, and then cultured for 3 days. <Example 13> Experiment of Delivery of EVs in Normal Somatic Cells (HDF) In order to confirm reprogramming of somatic cells using EVs, EVs obtained after one day of culturing in cells subjected to physical stimulation as in Example 12 were concentrated, the EVs were labeled using Did dye, and the EVs was delivered to normal somatic cells, and the expression of Oct4, a pluripotent marker, and Pax6, a neural stem cell marker, was confirmed in the delivered cells. To this end, 50 μl of EVs obtained in Example 12 was mixed with 450 μl of D-PBS and diluted, and 2.5 μl of a Vybrant DiD cell-labelling solution (molecular probe, excitation/emission, 644/667 nm) was added thereto and exosomes were stained at 37° C. for 30 minutes. After staining, the Did-stained EVs were concentrated by centrifugation at 14,000 rpm for 20 minute by an Amicon Ultra centrifugal filter (Millipore), diluted with D-PBS twice, added to 3 mL of a HDF medium (DMEM (Gibco) culture medium containing 5% FBS), and then cultured at 37° C. and 5% CO2for 24 hours. The HDF cultured for 24 hours was immobilized with 4% paraformaldehyde for 10 minutes and permeabilized with a 0.2% triton X100 in PBS buffer for 10 minutes. Thereafter, the cells were blocked with 3% BSA in PBS buffer for 1 hour, stained overnight at 4° C. with primary antibodies, anti-rabbit Oct4 (1:250, abeam) and Pax6 (1:200, abeam), and then stained with a secondary antibody, an anti-rabbit conjugated Alexa-488 (1:1000, Thermo, excitation/emission, 495/519 nm) for 1 hour. The images of samples stained with secondary antibodies were analyzed by a confocal laser scanning microscope (LSM 700; Carl Zeiss) using a mounting solution containing DAPI (4′,6-diamidino-2-phenylindole dihydrochloride, Vector Laboratories, excitation/emission, 420/480 nm), and the results are shown inFIG.42. In the drawing, green is Oct4, and red is EVs stained with Did dye. As shown inFIG.42, it was confirmed that EVs secreted from cells after physical stimulation include genes and proteins of various pluripotent markers according to a cell culture medium environment, and these factors may be delivered to adjacent cells by the EVs. These results suggest that EVs secreted from cells subjected to the physical stimulation in various medium conditions have a possibility to induce reprogramming of normal somatic cells. <Example 14> Reprogramming Effect of Human Fibroblasts by EVs Since the EVs secreted from the cells subjected to the physical stimulation in various media environments in Example 13 above have a possibility to induce reprogramming of normal somatic cells, in order to verify the possibility, the experiment was performed using a DMEM medium, which is a human fibroblast culture medium, and a hESC medium, which is a culture medium of human embryonic stem cells or iPS cells. A control group was cultured for 3 days in each medium without adding EVs, and a treated group was cultured for 3 days by adding 10 μl/mL (v/v) of EVs. The cultured cells were stained with a primary antibody, rabbit-anti-Oct4 (1:250, abeam) and a secondary antibody, anti-rabbit conjugated Alexa-488 (1:1000, Thermo excitation/emission, 495/519 nm), mounted with a mounting solution containing DAPI, then images were analyzed with a confocal laser microscope, and the results are shown inFIG.43. InFIG.43, hESC indicates a human ESC medium, DMEM indicates a fibroblast culture medium, and EVs indicates EVs recovered upon es/ENTER induction. As shown inFIG.43, the expression of Oct4 was not observed in the control group, but the expression of Oct4 and the formation of spheroid from the cells were observed in the treated group. These results indicate that cell reprogramming was induced only by EVs, not by culture medium. <Example 15> Change in Cell Morphology of EVs-Treated Human Fibroblasts for Each Culture Time Human fibroblasts were treated with 10 μl/mL (v/v) of EVs recovered during es/ENTER induction and cultured for 6 days, and then changes in morphology of cells were observed. As shown inFIG.44, the morphology of the cells varied according to a culture time, and the formation of spheroid was observed on day 3. <Example 16> Experiment for Verifying Expression of Pluripotent Marker of HDF Cultured for 6 Days According to EV-Adding Amount Recovered During Es/ENTER Induction To determine an appropriate concentration of EVs for cell reprogramming, the cells were treated with HDF and cultured for 6 days by varying the adding amount of EVs. In addition, since the EVs recovered during es/ENTER induction are used, cells expressing the pluripotent marker Oct4 were analyzed by flow cytometry. To this end, the EVs recovered during es/ENTER induction were added during fibroblast culturing at concentrations of 0, 5, 12.5, 25, 50, and 100 μl/mL (v/v) and cultured under conditions of 37° C. and 5% CO2for 6 days. The cultured cells were stained with a primary antibody, rabbit-anti-Oct4 (1:250, abeam) and a secondary antibody, anti-rabbit conjugated Alexa-488 (1:1000, Thermo excitation/emission, 495/519 nm) like Example 13 and analyzed with a BD Accuri™ C6 flow cytometry (BD biosciences). As shown inFIG.45, when the EVs of 12.5 μl/mL (v/v) were treated, the expression of Oct4 was confirmed in 84.6% of the cells, which was the most. <Example 17> Experiment for Verifying Expression of Pluripotent Marker of HDF Treated with EVs Recovered During Es/ENTER Induction Cultured for 3 Days Human fibroblasts were treated with 10 μl/mL (v/v) of EVs recovered during es/ENTER induction and cultured for 3 days, and a cell reprogramming effect was confirmed. For ICC analysis, like Example 13, the cultured cells used rabbit-anti-Oct4 (1:250, abeam), Sox2 (1:250, abeam), and Nanog (1:250, abeam) as primary antibodies and anti-rabbit conjugated Alexa-488 (1:1000, Thermo excitation/emission, 495/519 nm) as a secondary antibody, mounted with a mounting solution containing DAPI, and then images were analyzed with a confocal laser microscope. For qPCR analysis, total RNA was recovered using Trizol (Takara) in cells cultured for 3 days, and then cDNA was synthesized with Superscrip 2 kit (Invitrogen). PCR analysis was performed with real time PCR instrument (ab step one plus, AB) with respect to the pluripotent markers Oct4, Sox2, and Nanog. As shown inFIG.46, the ICC analysis result shows that the expression of the pluripotent markers Oct4, Sox2 and Nanog was observed in the human fibroblast nucleus, and the qPCR analysis result of gene expression by real time PCR shows that the genes Oct4, Sox2 and Nanog were overexpressed by about 50 times as much as normal fibroblasts which were not treated with EVs. <Example 18> Experiment for Verifying Expression of Neural Stem Cell Marker of HDF Treated with EVs Recovered During n/ENTER Induction and Cultured for 3 Days Human fibroblasts were treated with 10 μl/mL (v/v) of EVs recovered upon n/ENTER induction and cultured for 3 days, and a cell reprogramming effect was confirmed. For ICC analysis, like Example 13 above, the cultured cells used rabbit-anti-Sox1 (1:200, abeam), Sox2 (1:250, abeam), Pax6 (1:200, abeam), and mouse-anti-Nestin (1:250, Thermo Scientific) as primary antibodies and anti-rabbit conjugated Alexa-488 (1:1000, Thermo excitation/emission, 495/519 nm) and anti-mouse conjugated Alexa-594 (1:1000, Thermo, alexa 488 excitation/emission, 495/519 nm; alexa 594 excitation/emission, 590/617 nm) as secondary antibodies, mounted with a mounting solution containing DAPI, and then images were analyzed with a confocal laser microscope. For qPCR analysis, total RNA was recovered using Trizol (Takara) in cells cultured for 3 days, and then cDNA was synthesized with Superscrip 2 kit (Invitrogen). PCR analysis was performed with real time PCR instrument (ab step one plus, AB) with respect to the neural stem cell markers Sox1, Sox2, Pax6 and Nestin. As shown inFIG.47, the expression of the neural stem cell markers Sox1, Sox2, and Pax6 was observed in the human fibroblast nucleus, and the expression of Nestin was observed in the cytoplasm through ICC analysis. As the qPCR analysis result of gene expression by real time PCR, Sox1, Sox2, Pax6 and Nestin genes were overexpressed about 200 times as compared to normal fibroblasts which were not treated with EVs. TABLE 6qPCR primer listGenePrimer sequence (5′-3′)codeForwardBackwardPluripotentOct4GGGTTTTTGGGATTAAGTTGCCCCCACCCTTTGTGTTmarkerCTTCA(SEQ ID NO: 121)(SEQ ID NO: 57)Sox2CAAAAATGGCCATGCAGGAGTTGGGATCGAACAAAATT (SEQ ID NO: 58)GCTATT(SEQ ID NO: 122)NanogACAACTGGCCGAAGAATAGGTTCCCAGTCGGGTTCAGCA (SEQ ID NO: 59)C (SEQ ID NO: 123)NeuralSox1TCTGTTAACTCACCGGGAACTCCAGGGTACACACAGstem cellCC (SEQ ID NO: 60)GG (SEQ ID NO: 124)markerSox2GGAGTGCAATAGGGCGGACCAGTTGTAGACACGCACAT (SEQ ID NO: 61)CT (SEQ ID NO: 125)Pax6GTCCATCTTTGCTTGGGAATAGCCAGGTTGCGAAGAAA (SEQ ID NO: 62)CT (SEQ ID NO: 126)NestinCTCCAGAAACTCAAGCACTCCTGATTCTCCTCTTCCAC (SEQ ID NO: 63)(SEQ ID NO: 127)GapdhATGGGGAAGGTGAAGGTCATGGGGAAGGTGAAGGTCG (SEQ ID NO: 64)G (SEQ ID NO: 128) INDUSTRIAL APPLICABILITY The present invention can be used for a cell therapeutic agent field. | 76,407 |
11859176 | DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS FIG.1is a flowchart of a method for in vitro activation and/or expansion of immune cells of an embodiment of the disclosure.FIG.2is a schematic diagram of a magnetic particle for a method for in vitro activation and/or expansion of immune cells of an embodiment of the disclosure. Referring toFIG.1andFIG.2simultaneously, first, step S10is performed to provide magnetic particles100. The surface of the magnetic particle100is multi-protrusive and modified with at least one type of immuno-inducing substance102aand102b, in which the magnetic particle100includes a copolymer core110, a polymer layer120, a magnetic substance layer130, and a silicon-based layer140from the inside to the outside. In an embodiment, the magnetic particle100has an average diameter D, that is, an average value of the shortest diameter (e.g., D1) and the longest diameter (e.g., D2) of the magnetic particle100, and may range from 1 μm to 50 μm. In an embodiment, the average diameter of the magnetic particle100may be 2 μm to 40 μm. In another embodiment, the average diameter of the magnetic particle100may be 3 μm to 30 μm. In yet another embodiment, the average diameter of the magnetic particle100may be 4 μm to 20 μm. In still yet another embodiment, the average diameter of the magnetic particle100may be 2 μm to 10 μm. The aforementioned knobby copolymer core110is spherical and the surface thereof has a plurality of protrusions112. An average height h of the protrusions112, that is, the average value of the vertical distance of the top side of each protrusion112to the connection line between two bottom sides of each protrusion112(for example, heights h1, h2, and h3), and may range from 100 nm to 5000 nm, such as 100 nm to 500 nm, 500 nm to 1000 nm, 1000 nm to 1500 nm, 1500 nm to 2000 nm, 2000 nm to 2500 nm, 2500 nm to 3000 nm, 3000 nm to 3500 nm, 3500 nm to 4000 nm, 4000 nm to 4500 nm, or 4500 nm to 5000 nm. In an embodiment, an average height h of the protrusions112may be 300 nm to 4000 nm. In another embodiment, the average height h of the protrusions112may be 500 nm to 3000 nm. In still another embodiment, the average height h of the protrusions112may be 800 nm to 2000 nm. In still yet another embodiment, the average height h of the protrusions112may be 1000 nm to 1800 nm. In an embodiment, the protrusions112may be uniformly or non-uniformly distributed on the surface, and as a whole, the protrusions112are substantially irregular protrusions. For example, the protrusions112may include, but are not limited to, a papillary shape or a spherical shape. In an embodiment, the method for forming the aforementioned knobby copolymer core110may include, but is not limited to, a dispersion polymerization method, a suspension polymerization method, or an emulsion polymerization method, that is, at least two monomers are polymerized into a copolymer via the above polymerization methods. In an embodiment, the at least two monomers may be lipid-soluble monomers, and may include, for example, monofunctional monomers, bifunctional monomers, or a combination thereof. In an embodiment, in the case of a combination of a monofunctional monomer and a bifunctional monomer, the volume percentage of the bifunctional monomer relative to the monofunctional monomer may be 0.4% to 2%, such as 0.5% to 1.8% or 0.6% to 1.5%. Further, the monofunctional monomer may be a monovinyl monomer such as styrene, methyl methacrylate, vinyl chloride, or other monofunctional monomers. The bifunctional monomer may be a divinyl monomer such as divinylbenzene, ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, or other bifunctional monomers. In an embodiment, the copolymer may include a styrene/divinylbenzene copolymer, a methyl methacrylate/triethylene glycol dimethacrylate copolymer, a methyl methacrylate/ethylene glycol dimethacrylate copolymer, a styrene/triethylene glycol dimethacrylate copolymer, a styrene/ethylene glycol dimethacrylate copolymer, or a methyl methacrylate/divinylbenzene copolymer, but is not limit thereto. In an embodiment, the aforementioned polymer layer120may cover the knobby copolymer core110, and the polymer layer120includes at least one functional group. In particular, the material of the polymer layer120is different from the knobby copolymer core110, and the functional group may carry a charge, so that the surface of the knobby copolymer core110is charged to facilitate the subsequent adsorption of the magnetic substance precursor to form the magnetic substance layer130. In an embodiment, the functional group may include, but not limited to, carrying a negative charge. Further, the functional group may be a carboxyl group, an amine group, or a combination thereof, but is not limited thereto. It should be mentioned that, although the polymer layer120and the knobby copolymer core110are depicted as distinguishable layers inFIG.2, the polymer layer120and the knobby copolymer core110actually do not exist clear boundaries. Further, in the disclosure, a film layer having a functional group and located on the surface of the knobby copolymer core is collectively referred to as a polymer layer, in practice, however, the polymer layer120may be a surface of the knobby copolymer core modified by a functional group, rather than specifically forming a film layer. The aforementioned magnetic substance layer130may cover the polymer layer120. In an embodiment, the knobby copolymer core110covered with the polymer layer120may adsorb the magnetic substance precursor to form the magnetic substance layer130on the polymer layer120. In an embodiment, the magnetic substance precursor may include, but is not limited to, an iron ion (Fe2+), a cobalt ion (Co2+), a nickel ion (Ni2+), or a combination thereof. Specifically, the magnetic substance precursor may be a salt of the aforementioned metal ions such as ferrous chloride, cobalt chloride, nickel chloride, or the like. In an embodiment, the surface of the magnetic substance layer130may have small protrusions, that is, have a rough surface. Further, the magnetic substance layer130may include, but is not limited to, a paramagnetic substance, a superparamagnetic substance, a ferromagnetic substance, a ferrite magnetic substance, or a combination thereof. In an embodiment, the magnetic substance layer130may be a ferromagnetic substance. Also, in an embodiment, the magnetic substance layer130may have a substantially uniform thickness and entirely cover the copolymer core110. For example, the thickness of the magnetic substance layer130may be 20 nm to 200 nm, and may be, for example, 40 nm to 100 nm. The aforementioned silicon-based layer140may cover the magnetic substance layer130. In an embodiment, the material of the silicon-based layer140may include, but is not limited to, siloxane, silicon glass, silicon oxide, silicate, or a combination thereof. Specifically, the silicon-based layer140may include a material such as tetramethoxysilane (TMOS), tetraethoxysilane (TEOS), 3-aminopropyltriethoxysilane (APTES), and 3-glycidoxypropyltrimethoxysilane (GOPTS), or the like. Moreover, in an embodiment, the thickness of the silicon-based layer140may be 1 nm to 50 nm, and may be 5 nm to 40 nm, 10 nm to 35 nm, 15 nm to 30 nm, or 10 nm to 20 nm. In an embodiment, since the polymer layer120, the magnetic substance layer130, and the silicon-based layer140sequentially formed on the knobby copolymer core110do not substantially change the morphology of the knobby copolymer core110, the resulting magnetic particles100still have multi-protrusive appearance. As a result, the surface area of the magnetic particles100which may be in contact with the immune cells may be greatly increased. In addition, the multi-protrusive appearance facilitates the close packing of the magnetic particles100. In an embodiment, the aforementioned immuno-inducing substances102aand102bmay be any substance that may elicit immune response, and may include, but are not limited to, a particular type of peptide, protein, or fragment. In an embodiment, a particular type of antibody may be selected as the immune-inducing substances102aand102bfor a corresponding type of immune cell to be expanded and/or activated. For example, the immuno-inducing substances102aand102bmay be anti-CD3 antibodies, anti-CD28 antibodies, anti-TCR γ/δ antibodies, anti-CD83 antibodies, anti-CD137 antibodies, 4-1BBL (4-1BB ligand, or CD137 ligand), anti-CD2 antibodies, anti-CD335 antibodies, or a combination thereof. In an embodiment, in order to activate T cells, at least two different antibodies, such as anti-CD3 antibodies and anti-CD28 antibodies, may be selected as the immuno-inducing substances102aand102b, but is not limited thereto. It should be noted that although two different types of immuno-inducing substances102aand102bare shown inFIG.2, in other embodiments, the outermost surface of the magnetic particles100may include the same type of immuno-inducing substance or two or more different types of immuno-inducing substances. Further, the arrangement of the immuno-inducing substances102aand102binFIG.2is merely an exemplification, and is not intended to limit the arrangement of the immuno-inducing substances. In an embodiment, the immune-inducing substances102aand102bmay be uniformly distributed on the outermost surface of the magnetic particles100to facilitate contacting with the immune cells. In another embodiment, the immuno-inducing substances102aand102bmay also be modified on the surface of the silicon-based layer140. The method by which the immuno-inducing substances102aand102bare modified to the surface of the magnetic particles100may include non-covalent binding, covalent binding, avidin-biotin interaction, electrostatic adsorption, hydrophobic adsorption, or a combination thereof. In addition, other suitable binding methods may also be used. Further, in another embodiment, the immuno-inducing substances102aand102bmay be modified on the surface of the silicon-based layer140by a coupling reagent. The coupling reagent may include, but is not limited to, 3-(3-glycidyloxypropyl)trimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, triethoxysilane-polyethylene-glycol-N-hydroxysuccinimide, or a combination thereof. Next, referring toFIG.1andFIG.2, step S20is performed to provide a cell solution, and the cell solution includes at least one type of the immune cell. In an embodiment, the immune cell may include, but is not limited to, an αβ T cell, a γδ T cell, a regulatory T cell, a NK cell, or a combination thereof. In another embodiment, the immune cell is, for example, a γδ T cell. Thereafter, please continue to refer toFIG.1andFIG.2. Step S30is performed to bring the magnetic particles100in contact with the cell solution such that at least one type of the immuno-inducing substance102aand102bon the surface of the magnetic particles100may activate and expand the at least one type of the immune cell in the cell solution. In an embodiment, the immune cell is exemplified by a γδ T cell, and the immune-inducing substances102aand102bon the surface of the magnetic particles100may be anti-TCR γ/δ antibodies and 4-1BBL. In an embodiment, the aforementioned contact may be achieved by mixing approximately equal numbers of the magnetic particles100and the immune cells. The cell expansion ratio of the number of living cells of the aforementioned immune cell varies depending on the cell type, the inter-individual difference, and the culture environment. In terms of the culture environment, the operator may select the desired culture scale according to actual needs. For example, the cell expansion ratio of the number of living immune cells of a small-scale culture environment (for example, a 6-well culture dish) may be 20 to 120, such as 30 to 110, 40 to 100, etc., but is not limited thereto. The cell expansion ratio of the number of living immune cells of a large-scale culture environment (for example, a bioreactor) may be 100 to 8000, such as 500 to 7000, 800 to 6000, etc., but is not limited thereto. In a particular embodiment, the cell expansion ratio of the number of living cells of certain immune cells may even reach tens of thousands. In general, the average expansion ratio of the number of living immune cells may be 20 to 1000. In an embodiment, the average expansion ratio of the number of living immune cells may be 30 to 900. In another embodiment, the average expansion ratio of the number of living immune cells may be 40 to 800. Further, in an embodiment, the magnetic particles100may have residual magnetism, that is, after the applied magnetic field is removed, the magnetic properties of the magnetic particles100do not disappear, but tend to stay to some extent. As a result, in an embodiment, when the magnetic particles100are dispersed in the cell solution, the magnetic particles100may be arranged in a row via the residual magnetism, but are not limited to only a single row, and the chance of contacting with the immune cells by the row(s) of magnetic particles100is increased due to the larger overall surface area. Also, in an embodiment, a plurality of rows of the magnetic particles100may be aggregated with the immune cells to facilitate the activation and/or expansion of the immune cells. The aforementioned magnetic particles have multi-protrusive surface similar to the most potent antigen presenting cells (APCs) in the human body, and even similar to dendritic cells (DCs). Therefore, under the concept of biomimetics, it can be speculated that the knobby magnetic particles may be in contact with the immune cells through larger surface area, thereby facilitating the activation and/or expansion of the immune cells. Specific experimental examples are exemplified below to specifically describe the method for in vitro activation and/or expansion of immune cells. Experimental Example 1: Modification of an Immuno-Inducing Substance to the Surface of Knobby Magnetic Particles (1) Surface Functionalization of Knobby Magnetic Particles First, three types of knobby magnetic particles of the disclosure were provided, which had an average diameter of 2.5 μm, 4.5 μm, and 8.5 μm, respectively, and the morphologies illustrated by electron microscope (10000×) were as shown inFIG.3AtoFIG.3C. Further, taking the knobby magnetic particles having an average diameter of 4.5 μm as an example, the hysteresis curve analysis chart thereof is as shown inFIG.4. Generally, when the magnetic field strength is 0, if the hysteresis curves are coincident and the magnetization is 0, paramagnetism is observed, and if the hysteresis curves do not overlap, ferromagnetism is observed. According toFIG.4, it can be known that the knobby magnetic particles are ferromagnetic. 1 gram of the knobby magnetic particles, 100 mL of deionized water, 500 mL of ethanol, 30 mL of ammonia (NH4OH), and 0.1 mL of 3-aminopropyltriethoxysilane (APTES) were performed to place into a reactor and stirred for reaction at room temperature for 1 hour. After the reaction was completed, the knobby magnetic particles were collected by a magnet. Next, the reaction solution was removed, and the knobby magnetic particles were cleaned by adding deionized water. After cleaning repeatedly with deionized water three times, knobby magnetic particles having a surface modified with an amine group was obtained. (2) Method for Modifying with Immuno-Inducing Substances First, about 7×107of the aforementioned knobby magnetic particles having a surface modified with an amine group were cleaned three times with 100 μL of 4-morpholineethanesulfonic acid (MES) buffer solution (25 mM, pH 5.0). Next, 20 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 20 mg of N-hydroxysulfosuccinimide sodium salt (NHS), and 4 mg of polyacrylic acid (PAA, 15 kDa) were dissolved in 400 μL of an MES buffer solution. The mixture was then mixed with the cleaned knobby magnetic particles, and the resulting solution was reacted at room temperature for 30 minutes. After the reaction was completed, the knobby magnetic particles were collected by a magnet. Next, the reaction solution was removed and the knobby magnetic particles were cleaned three times with 100 μL of MES buffer solution, and then a mixture containing 10 μL of anti-CD3 antibodies (manufacturer: ebioscience), 60 μL of anti-CD28 antibodies (manufacturer: ebioscience), and 130 μL of MES buffer solution were added, and the mixture was subjected to overnight reaction at 4° C. Thereafter, 200 μL of a human serum albumin (HSA, manufacturer: Sigma) solution (10 mg/mL, prepared with MES buffer solution) was added, and an overnight reaction was again performed at 4° C. After the reaction was completed, the knobby magnetic particles were collected by a magnet. Next, the reaction solution was removed, and the knobby magnetic particles were cleaned three times with a PBS buffer solution containing 0.1% HSA and 2 mM ethylenediaminetetraacetic acid (EDTA) for at least 5 minutes each time, and then the knobby magnetic particles were cleaned three times with a PBS buffer containing 0.1% HSA. Lastly, the knobby magnetic particles modified with immuno-inducing substances were dispersed in 1.75 mL of PBS buffer solution containing 0.1% HSA to obtain a solution, which includes knobby magnetic particles having a surface modified with immuno-inducing substances and has a concentration of 4×107particles/mL. Experimental Example 2: Activation and/or Expansion of T Cells by Knobby Magnetic Particles Modified with Immuno-Inducing Substances (1) Separation of T Cells from Blood 10 mL of blood and 0.5 mL of T cell separation reagent (named RosetteSep Human T Cell enrichment cocktail, manufacturer: STEMCELL Technologies Inc.) were mixed in a ratio recommended in the product manual and reacted for 20 minutes. Then, a PBS buffer solution containing 2% fetal bovine serum was added via a pipette and mixed in equal volume to obtain a cell solution. Next, the cell solution was added to a gradient density centrifuge tube containing 15 mL of a T cell separation solution and centrifuged at a speed of 1200 rcf for 20 minutes. Thereafter, the cell supernatant was transferred to a new centrifuge tube and centrifuged at a speed of 300 rcf for 10 minutes. Next, after the supernatant was removed, the cells were reconstituted with 5 mL of a PBS buffer solution containing 2% fetal bovine serum, and 20 mL of a red blood cell lysis solution (named RBC lysis buffer, manufacturer: STEMCELL Technologies Inc.) was added to react at 4° C. for 10 minutes, and then centrifugation was performed at a speed of 300 rcf for 8 minutes. Afterward, the cells were reconstituted with 5 mL of a cell culture medium (RPMI 1640 culture medium containing 10% fetal bovine serum (FBS, manufacturer: BI), manufacturer: Gibco), and centrifugation was performed at a speed of 300 rcf for 8 minutes, and then cell cleaning was performed once. Lastly, the cells were reconstituted using a cell culture medium to obtain a high-purity (such as 90% to 97%) T cell solution. (2) Stimulation and Culture of T Cells 5×105of the knobby magnetic particles having a surface modified by the immuno-inducing substances and obtained in experimental example 1 were cleaned three times with 0.2 mL of a cell culture medium, and then the knobby magnetic particles were dispersed in 0.15 mL of the cell culture medium. Next, 5×105of the T cells obtained in (1) of the aforementioned experimental example 2 were mixed with the knobby magnetic particles dispersed in a culture medium and having a surface modified with immuno-inducing substances, and the total volume was made up to 0.5 mL with the cell culture medium to obtain a T cell solution. At the same time, commercially available spherical magnetic particles (Dynabeads®) and 5×105of the T cells obtained in (1) of the aforementioned experimental example 2 were mixed to obtain a T cell solution served as a comparison group. Then, the T cell solutions of the experimental group and the comparison group were respectively performed to place into a 24-well culture plate (manufacturer: Corning), in which the T cells were stimulated and cultured in a 37° C. incubator containing 5% carbon dioxide (at this time, it is called day 0 of culture). Then, 0.5 mL of the cell culture solution was added on day 2 of the culture, and the T cells and the magnetic particles were dispersed using a pipette on day 5 of the culture. Thereafter, the mixture of the T cells and the magnetic particles was transferred to a microtube, and the microtube was placed on a magnetic base to attract the magnetic particles to the wall of the microtube, thus a cell suspension stimulated by the immuno-inducing substances but not containing the magnetic particles was transferred to a new 6-well culture dish (manufacturer: Corning). Next, the total volume and cell concentration of the aforementioned stimulated cell suspension which is free of magnetic particles were measured. After the concentration of the cell solution was adjusted to 0.5 to 1×106cells/mL, the cell suspension was placed in a new culture dish and returned to a 37° C. incubator containing 5% carbon dioxide to continue the culture. Thereafter, on day 6 to day 8 of the culture, the following steps were repeated every day: the T cells were uniformly dispersed using a pipette and the total volume and the cell concentration of the cell solution were measured, and after the concentration of the stimulated T cell solution was adjusted to 0.5 to 1×106cells/mL, the stimulated T cell solution was returned to the incubator to continue the culture. On day 9 to day 12 of the culture, the following steps were repeated every day: the T cells were uniformly dispersed using a pipette, and the total volume and cell concentration of the cell solution were measured, and the average expansion ratio of the T cells on days 9 to 12 was calculated using the cell concentration on day 0 as a reference. FIGS.5A to5Drespectively show the cell number on different culture days for T cell activation and/or expansion using commercially available magnetic particles and knobby magnetic particles having an average diameter of 2.5 μm, 4.5 μm, and 8.5 μm, in which the commercially available magnetic particles were served as a comparison group. According to the results shown inFIG.5AtoFIG.5D, on day 9, compared to the average expansion ratio of 26.64 of the T cells of the comparison group, the average expansion ratios of the T cells of the experimental group treated with the knobby magnetic particles having an average diameter of 2.5 μm, 4.5 μm, and 8.5 μm were 29.20, 31.39, and 29.89, respectively. In addition, after day 9, the average expansion ratio of the T cells of the comparison group was gradually decreased or tended to be gentle, but the average expansion ratio of the T cells of the experimental group continued to increase, in which the expansion efficiency achieved by the 4.5 μm magnetic particles (that is, as shown inFIG.5C) was the most remarkable, reaching an expansion ratio of 90 or more. Experimental Example 3: Activation and/or Expansion of T Cells by the Knobby Magnetic Particles Modified with Two Antibodies in Different Ratios The operation procedure in experimental example 3 was the same as that of experimental example 2, and the main difference was that in experimental example 3, the magnetic particles of 4.5 μm were used as a test object, and in the modification ratio of the immuno-inducing substance, anti-CD3 antibodies and anti-CD28 antibodies were prepared as antibody mixtures at ratios of 1:2, 1:6, and 1:10, and the antibody mixtures were used to modify the aforementioned 4.5 μm magnetic particles, respectively. In the present experimental example, commercially available spherical magnetic particles (Dynabeads®) were simultaneously served as a comparison group, but the ratio of antibodies on the commercially available spherical magnetic particles could not be confirmed. FIGS.6A to6Drespectively show the cell number on different culture days for T cell activation and/or expansion using commercially available magnetic particles (Dynabeads®) and knobby magnetic particles for which the surface was modified with the antibodies in which the ratios of anti-CD3 antibodies and anti-CD28 antibodies were respectively 1:2, 1:6, and 1:10. According toFIG.6AtoFIG.6D, it can be known that, compared to the comparison group (FIG.6A, Dynabeads®), the magnetic particles of the experimental group with surface modified by different ratios of antibodies (FIG.6B, anti-CD3 antibodies: anti-CD28 antibodies=1:2;FIG.6C, anti-CD3 antibodies: anti-CD28 antibodies=1:6;FIG.6C, anti-CD3 antibodies: anti-CD28 antibodies=1:10) all may effectively activate and/or expand the T cells. Experimental Example 4: Characteristic Analysis of Activated and/or Expanded T Cells Characteristic analysis was performed on the aforementioned T cells of the disclosure activated and/or expanded by the knobby magnetic particles by flow cytometer (SONY SA3800), and the results were simultaneously compared with that of T cells (the comparison group 1 and the comparison group 2) activated and/or expanded by two commercial products (including magnetic particles and modified antibodies, in which the products only disclose that the modified antibodies thereof were anti-CD3 antibodies and anti-CD28 antibodies, but the ratios of the antibodies were not provided). In particular, in the comparison group 1, the T cells were activated and/or expanded with a commercially available product TransAct™ T Cell reagent (Miltenyi Biotec), and in the comparison group 2, the T cells were activated and/or expanded with a commercially available product Dynabeads® Human T-Activator CD3/CD28 (ThermoFisher). In the experimental group, the T cell were activated and/or expanded with the knobby magnetic particles for which the surface was modified with the antibodies in which a ratio of anti-CD3 antibodies and anti-CD28 antibodies is 1:6, and the untreated initial cells of day 0 were served as a control group. The results are as shown inFIG.7AtoFIG.7C. According toFIGS.7A to7C, it can be known that, compared to the comparison group 1 (TransAct™) and the comparison group 2 (Dynabeads®), the T cells of the experimental group (i.e., T cells activated and/or expanded by the surface-modified knobby magnetic particles of the disclosure) have the following characteristics: (1) the ratio of CD4+T cells to CD8+T cells is closest to 1:1 (FIG.7A); (2) central memory T cells (TCM) occupy a higher ratio than effector memory T cells (TEM) (FIG.7B); and (3) the sum of the ratios of T cells that expressed four different immunosuppressive molecules (PD-1 (programmed cell death-1), CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4), TIM-3 (T-cell immunoglobulin and mucin-domain containing-3), and LAG-3 (lymphocyte-activation gene 3)) is minimum (FIG.7C). Based on the above, the magnetic particles of the disclosure have multi-protrusive surface similar to the most potent antigen-presenting cells in the human body, even similar to dendritic cells. Therefore, under the concept of biomimetics, it can be speculated that the knobby magnetic particles may be in contact with the immune cells in larger surface area, thereby contributing to the efficiency of activating immune cells and/or expanding the number of immune cells. In addition, a specific type of immuno-inducing substance may be designed and modified on the surface of the magnetic particles of an embodiment of the disclosure such that the magnetic particles may be applied for the activation and expansion of γδ T cells to overcome the current issue of a lack of the related products applicable to γδ T cells. For example, in an embodiment, a peripheral blood mononuclear cell (PBMC) separated from whole blood is used as a source, and the knobby magnetic particles modified with the immuno-inducing substances may enhance the expansion ratio of γδ T cells by about 1.2 to 3.5 compared with magnetic particle-free conditions. Furthermore, since the magnetic particles have magnetic properties and specific immuno-inducing substances, they have the advantage of being able to rapidly separate and induce immune response, and thus, the immune cells may be applied to the research and development of techniques such as cancer immunotherapy. It will be apparent to those skilled in the art that various modifications and variations may be made to the structure of the disclosed embodiments without departing from the scope or spirit of the disclosure. In view of the foregoing, it is intended that the disclosure cover modifications and variations of this disclosure provided they fall within the scope of the following claims and their equivalents. | 28,872 |
11859177 | DESCRIPTION The present disclosure relates to the field of medical therapies employing nanoparticles and nanobubbles. More specifically, the present disclosure relates to methods, systems, and apparatus for employing nanobubbles for theranostic purposes. In general, the present disclosure aims, at least in part, to improve the efficacy of the diagnosis and treatment of malaria. Rapid, accurate, and non-invasive detection of low levels of malaria parasites in blood is critical for surveillance, treatment, and elimination of malarial infection. In addition, innovative methods are required to combat growing drug resistance of malaria parasites. Both detection and parasite destruction ultimately need single infected cell sensitivity and specificity, robust inexpensive devices, and minimal dependence upon chemical reagents. None of the existing technologies can rapidly and non-invasively detect and destroy the parasite in a single red blood cell. Thus, the present disclosure aims, at least in part, to improve the efficacy of the diagnosis and treatment of malaria by generating laser-induced photothermal nanobubbles (PTNBs) around malaria-specific nanoparticles. A PTNB may act as a diagnostic and/or parasiticidal agent and may cause destruction of the Hz nanocrystal, the malaria parasite, the malaria infected red blood cell (MIRBC), or a combination thereof. The present disclosure is based, at least in part, on the photoexcitation of a MIRBC by a short laser pulse causing selective transient heating of a malaria-specific nanoparticle (e.g., a Hz nanocrystal) and resulting in the creation of a transient, water vapor nanobubble, a PTNB, surrounding the malaria-specific nanoparticle. Such bubbles are generated by the nanocrystal's absorption of optical light energy and the resulting overheating and evaporation of the surrounding solvent. The bubbles are termed photothermal nanobubbles due to their optical and thermal origin. The expanding PTNB creates an impact similar to an explosion and can be controlled at nanoscale. This mechanical impact allows for the destruction of the Hz nanocrystal, the malaria-specific parasite, the MIRBC, or a combination thereof. In addition, PTNBs may be detected by one or more optical or acoustic detectors, allowing for the detection of MIRBCs and affording real-time guidance of the application of destructive PTNBs to eliminate the malaria-specific parasite. In certain embodiments, the present disclosure provides methods for detecting the presence of a malaria-specific nanoparticle, destroying the malaria-specific parasite, and receiving real-time guidance on the destruction of the malaria-specific parasite. In certain embodiments, the present disclosure provides systems comprising one or more optical detectors capable of detecting the presence of a malaria-specific nanoparticle and a laser capable of generating a short laser pulse sufficient to create a PTNB around the malaria-specific nanoparticle. Some embodiments utilize an acoustic detector in place of any optical detectors, while various embodiments use one or more optical detectors in combination with an acoustic detector. In certain embodiments, the present disclosure provides an apparatus comprising a means for detecting the presence of a malaria-specific nanoparticle, a means for destroying the malaria-specific parasite, and a means for receiving real-time guidance on the destruction of the malaria-specific parasite. As used herein, the term malaria-specific nanoparticle refers to a nanoparticle associated with a malaria-specific parasite (e.g.Plasmodium falciparum, and other types) having a dimension (e.g., a diameter) of about 1,000 nm or less, and capable of converting electromagnetic radiation into thermal energy. The nanoparticle may have any shape or structure (e.g., spherical, tubular, shell-like, elongated, etc.). In certain embodiments, malaria-specific nanoparticles may be Hz nanocrystals, the tightly packed nanocrystals produced endogenously by the malaria parasite through the parasite's digestion of hemoglobin. Hz nanocrystals have a high optical absorbance, which is significantly higher than that of a normal red blood cell (RBC) and of normal hemoglobin, the major RBC protein. As a result, a Hz nanocrystal can convert the optical energy associated with a short laser pulse into heat and can generate a localized transient PTNB within a malaria parasite located in a MIRBC. Thus, in certain embodiments, unlike many current malaria treatments that combat a parasite by preventing Hz formation, Hz nanocrystals may be used as an “Achilles heel” to facilitate parasite detection and destruction. In some embodiments, the malaria-specific nanoparticles may be exogenously added nanoparticles with appropriate photothermal properties (e.g., gold nanoparticles) conjugated to malaria-specific antibodies. As used herein, the terms nanobubble and PTNB refer to the transient vapor bubble that emerges around a nanoparticle when it is locally and transiently heated by exposure to electromagnetic radiation. The nanoparticle itself may not evaporate, instead acting as a heat source and heat accumulator in an intricate process of heat transfer and phase transition in the nanoparticle environment at nanoscale. The PTNB expands rapidly to its maximal diameter and then collapses with its lifespan being longer than the duration of radiation pulse that feeds the energy to the bubble through the nanoparticle. Thus, a PTNB results when a nanoparticle evaporates a very thin volume (nanometer size) of the surrounding medium, creating a PTNB that expands and collapses within a short nanosecond. The PTNB's rapid expansion produces a localized mechanical and non-thermal impact that may result in damage or destruction to cellular components or to the cell itself. By way of explanation, PTNBs allowed for, among other things, higher parasiticidal efficacy, shorter treatment time, and lower optical dose of the treatment as compared to a hyperthermia approach. Thus, PTNBs are particularly suited for treatment of MIRBCs because they allow for parasiticidal efficacy while minimizing destruction of uninfected RBCs, due, for example, to delocalized photothermal heating. In certain embodiments, malaria-specific nanoparticle (e.g., Hz nanocrystals) act as photothermal targets within MIRBCs or other malaria-infected tissues and cells. In particular embodiments, selective laser pulse-induced heating of a malaria-specific nanoparticle causes generation of a PTNB. Generation of a PTNB around optically absorbing objects, such as Hz nanocrystals, assumes a transient localized evaporation of the liquid media around the object. Rapid heat transfer from the laser-excited optical absorber raises the temperature of the surrounding solvent layer above its evaporation threshold, with the simultaneous buildup of the internal vapor pressure. When the pressure inside the evaporated layer exceeds the external pressure of the surface tension at the boundary of the vapor inside and bulk liquid outside, the PTNB begins to expand rapidly, with speeds ranging from 10 meters per second to 100 meters per second, until the bubble reaches a maximal diameter that corresponds to a transient equilibrium, when the internal and external pressures are equal. Because, in some embodiments, PTNB generation is induced by a single short pulse, the bubble has no continuing source of internal energy, and will therefore eventually depressurize and collapse back to the nanocrystal that generated it. The maximal size of the PTNB is determined by the thermal energy that is generated from light absorption by the Hz nanocrystals. In certain embodiments, a PTNB diameter may be sufficient to destroy a malaria-specific parasite. For example, the PTNB diameter may range in size from 100 nanometers to tens of micrometers. The duration of the expansion-collapse cycle determines the lifetime of the PTNB, from 10 nanoseconds to microseconds, and is proportional to its maximal diameter, which is used as the main metric of the PTNB. Efficient and ultrafast heating of the liquid surrounding the malaria-specific nanoparticle is required to minimize energy dissipation by thermal diffusion. Efficient nanobubble formation is achieved through a fast deposition of light energy into the strongly absorbing malaria-specific nanoparticle (e.g., Hz nanocrystals) with a short laser pulse. In certain embodiments, the PTNB may be formed through a short laser pulse. The laser pulse should be of sufficient energy and duration to form a photothermal nanobubble with a diameter sufficient to cause mechanical destruction of a malaria-specific parasite. Suitable laser pulses may be delivered using, for example, high energy pulsed picosecond laser. In certain embodiments, the laser pulse may have a duration of from 1 picosecond to 100 nanoseconds. The particular laser pulse duration may depend on, among other things, the particular laser chosen. In certain embodiments, suitable laser pulses may be determined with reference to the characteristic cooling time due to the thermal diffusion is determined by the diameter d of the heated object: τ=d227a where a is the thermal diffusivity of the environment of the object. Here, we assume that a equals the thermal diffusivity of water, 1.4×105μm2/second. The sizes of Hz nanocrystals are reported to range between 50 nanometers and 1000 nanometers with the smallest crystals being formed during the early ring stage of the malaria parasite. This reported size range predicts cooling times for the Hz absorbers between 0.5 nanoseconds and 26 nanoseconds. Therefore, to ensure rapid enough energy deposition to create a PTNB, and to minimize thermal diffusive losses, rather than simple heating, in certain embodiments a 70 picosecond pulsed laser (e.g., PL-2250, Ekspla, Vilnius, Lithuania) and/or a 14 nanosecond pulsed laser (e.g., Nd-YAG laser LS-2145T, Lotis TII, Minsk, Belarus) may be employed. An optical microscope-based experimental set up, known in the art, may be used to mount and position samples of malaria-specific nanoparticles with a motorized microscope stage (e.g., 8MT167-100, Standa Ltd., Vilnius, Lithuania), operated via custom-made LabView modules (e.g., National Instruments Corporation, Austin, TX). In single cell experiments performed in accordance with certain embodiments, the excitation laser pulse may focused down to a 15 μm area in the sample plane, providing uniform exposure of the entire RBC (diameter 7 μm). In bulk, cultured cells experiments performed in accordance with certain embodiments, the diameter of the excitation laser beam may be increased to 210 μm, providing simultaneous exposure of a monolayer of 600-800 cells by a single laser pulse. Spatial intensity profiles of both beams are Gaussian and their fluence may be measured at the sample plane. The fluence of each single laser pulse may be controlled with a polarizing attenuator and may be measured by registering the size of the image of the laser beam at the sample plane with an EM CCD camera (e.g., Luka model, Andor Technology, Northern Ireland). The pulse energy may be assessed with an energy meter (e.g., Ophir Optronics, Ltd., Israel). The fluence may be calculated using the pulse energy and the laser beam image size, with the beam diameter measured at the level of 1/e2relative to the maximum. In accordance with various embodiments, each MIRBC may be positioned into the center of laser beam and may be exposed to a single pulse at a specific fluence. The duration of a 70 picosecond pulse is much shorter than the estimated cooling times (due to, e.g., thermal dissipation), and, therefore, such pulse durations should provide very localized heating with minimal dissipation (due to, e.g., diffusion) of heat during the deposition of optical energy into the Hz nanocrystal. In certain embodiments, the excitation wavelength is a wavelength where a malaria-specific nanoparticle shows relatively high optical absorbance (See e.g.,FIG.1). For example, the excitation wavelength may be approximately 532 nanometers, a wavelength where Hz shows relatively high optical absorbance. In other examples, the excitation wavelength may have a value in the range from 400 to 1000 nanometers. For example, the excitation wavelength may have a value of 650 nanometers. Unlike hemoglobin, Hz does not have sharp specific spectral peaks, but nevertheless, its optical absorbance is five- to seven-fold higher than that of hemoglobin in RBCs. This large difference enables selective photothermal generation of PTNB around Hz nanocrystals in accordance with particular embodiments, without inducing vapor bubbles or significant heating in uninfected RBCs. In various embodiments, the maximal size of a PTNB is determined by the optical energy transmitted to a malaria-specific nanoparticle by a laser. Increasing the optical energy increases the maximal size of the PTNB. Mechanical destruction caused by the PTNB depends on its maximal size. In certain embodiments, this rapid expansion and collapse may destroy the nanoparticle, the food vacuole of the malaria parasite, the malaria parasite itself, or the MIRBC depending on the maximal diameter of the PTNB. The maximal diameter of the PTNB corresponds to the energy received by the malaria-specific nanoparticle from a laser or other source of electromagnetic radiation. Thus, the generation of PTNB around the malaria-specific nanoparticle requires a small energy pulse, destruction of the food vacuole requires an increase in the energy of the laser pulse, destruction of the parasite itself requires another energy increase, and destruction of the MIRBC requires an even higher energy pulse. Destruction of the MIRBC assumes that all internal components are also destroyed. In certain embodiments, malaria may be diagnosed through one or more optical detectors, an acoustic detector, or both, by detecting the presence of PTNBs generated around Hz nanocrystals present in malaria-specific parasite. A PTNB generated by the short laser pulse may be detected with a low intensity continuous probe laser that measures the strong optical scattering produced by the expansion and collapse of nanobubbles using a photodetector. Optical scattering changes will only occur in MIRBCs containing malaria-specific nanoparticles (e.g., Hz nanocrystals) and thus, are diagnostic of malarial infection. Optical scattering signals of PTNB may be registered in various embodiments in several ways, including, as a time-resolved optical scattering image that will show the presence of transient PTNBs and as an optical scattering time-response that will measure the maximal diameter and lifetime of the PTNB. The maximal diameter determines the optical properties of the PTNB. In certain embodiments, the generation of even a single PTNB in a single MIRBC may be detected acoustically, because the PTNB emits a pressure pulse that may be detected independently or in parallel with an optical signal of the bubble from an ultrasound transducer. Thus, certain embodiments of the present disclosure provide at least three independent techniques for a real time detection of Hz nanocrystals with cell sensitivity. In particular embodiments, the diagnostic sensitivity of these embodiments may range from detecting 1 MIRBC in 104uninfected RBCs to 1 MIRBC in 108uninfected RBCs, and, in particular, may range from detecting 1 MIRBC in 106uninfected RBCs to 1 MIRBC in 108uninfected RBCs, thus outperforming current methods of diagnosis. In addition, the PTNB diagnostics method of particular embodiments may employ real time signal detection, and thus diagnosis may take only seconds. As a result, advantages of certain embodiments over previous diagnostic attempts using Hz nanocrystals may include heightened sensitivity and the ability to conduct in vivo or single cell testing, even in the early ring stage of the malaria parasite, using a rapid label- and needle-free procedure. In certain embodiments, optical or acoustic signals, or both, may also guide the therapeutic use of PTNB generation. From a therapeutic perspective, the bulk laser pulse treatment of human blood in accordance with various embodiments results in PTNB-induced explosive mechanical destruction of up to 95% of malaria parasites, while leaving uninfected cells undamaged. This provides a significant advantage over previous attempts to use photothermal destruction of MIRBCs that relied on pre-treating MIRBCs with an absorbing dye and used a much longer pulse and 1000-fold higher energy, resulting in low selectivity of MIRBCs for destruction and damage to uninfected RBCs. The disclosed embodiments also provide advantages over previous attempts to use magnetic heating of Hz to destroy malaria parasites, which suffered from significant thermal diffusive losses due to long excitation times leading to reduced efficacy and selectivity. In contrast, the short, low energy laser pulses disclosed herein, in accordance with particular embodiments, provide only localized mechanical impact and single cell selectivity without heating or damaging uninfected cells. Since diagnostics and therapeutics are supported by the same PTNB-based process, in particular embodiments, they may be united into one connected and fast theranostic procedure that may detect, destroy and simultaneously guide in real time the destruction of malaria parasites with single cell selectivity and nanosecond speed. In various embodiments, such a theranostic protocol includes: detection of Hz nanocrystals, which are indicative of the presence of the malaria parasite, by generating PTNB-specific optical and acoustic signals for diagnosis of malaria infection; selective destruction of the parasite using a short laser pulse to locally destroy the parasite as a therapy; and real time guidance of the destructive PTNBs with the optical and acoustic signals coming solely from MIRBCs. In certain embodiments, the device that supports a theranostic method may comprise an optically transparent cuvette of specific dimensions in combination with a pump that provides the flow of blood cells through the cuvette in such a way that all cells form a two-dimensional monolayer that can be exposed by a pulsed laser radiation. By means of example, and not limitation, such cuvette may include an optically transparent segment 2 cm wide, 10 cm long and 200 μm high, while the pump provides the blood flow speed in the range from 1 cm/c to 10 m/s. Certain embodiments may comprise an excitation pulsed laser with the pulse duration below 20 ns, wavelength ranging from 400 nm to 1200 nm, pulse fluence that can be tuned in the range from 10 mJ/cm2to 500 mJ/cm2, and pulse repetition rate in the range from 1 hertz to 10 kilohertz. Various embodiments may comprise a continuous probe laser of any wavelength with the power being low enough to avoid heating any Hz nanocrystals, but sufficient to provide the detection of a portion of the optical radiation being scattered by a single PTNB. The probe laser may illuminate the same area of the cuvette as the excitation pulsed laser beam. Certain embodiments may comprise an optical detector of any type that can detect the portion of the radiation of the probe laser being scattered by a single PTNB. Speed (temporal resolution) of such photodetector and associated signal analyzer should provide the detection of a single signal pulse with duration from 10 ns to 1000 ns. Particular embodiments may comprise an acoustic detector of any type that can detect a pressure pulse emitted by at least a single PTNB in the area exposed to the excitation pulsed laser. In various embodiments, the device comprises an optical fiber probe capable of delivering an excitation laser pulse from the pulsed laser and collecting the light of the probe laser after it is scattered by PTNBs. In various embodiments, the optical fiber probe also comprises a photodetector capable of detecting the collected scattered light. In particular embodiments, PTNBs may be detected in parallel with an ultrasound detector. Certain embodiments may count and analyze output signals of the photodetector and ultrasound detector through a computer algorithm that delivers the diagnostic data. Aspects of these embodiments may be used together or separately and may be appropriate for in vivo application. In certain embodiments, the malaria-specific nanoparticle may be an exogenously added photothermal agent, such as a gold nanoparticle conjugated to a malaria-specific antibody. Malaria-specific antigens expressed at the membrane of MIRBCs may be used to selectively target gold nanoparticles to MIRBCs. Such short pre-treatment of blood opens the following opportunities for improving the treatment of malaria by generating laser-induced generation of PTNBs that will be large enough to destroy the parasite in MIRBCs selectively and rapidly during single pulse treatment. In some embodiments, laser-induced generation of small PTNBs could also be used for intracellular delivery of anti-malaria drugs that otherwise have limited targeting efficacy against malaria by selectively opening liposome vesicles containing the drugs and attached gold nanoparticles. In certain embodiments, malaria parasites may be detected and destroyed in vivo. In some cases MIRBCs with parasites may adhere to blood vessel walls (due to the interaction of adhesive nobs with endothelial receptors) and as a consequence, these MIRBCs cannot be accessed via extra-corporeal treatment making in vivo detection and destruction advantageous. The mechanism of PTNB-based theranostics can be employed in vivo as well as ex vivo and by using a fiber optical catheter for delivery and collection of laser radiation. The level of laser fluence required for PTNB generation is within the safety limits (25-40 mJ/cm2) established for in vivo use of pulsed laser radiation. In certain embodiments, the performance of PTNB in vivo may be further improved by optimizing the excitation wavelength in the Hz-specific range, approximately 640-660 nanometers, where blood and tissues have better transparency than at 532 nanometers. In some embodiments, an optical catheter may be used for the delivery of the excitation and probe laser radiation and for collection of the light scattered by PTNBs. In particular embodiments, the PTNB diagnostic mode may utilize acoustic detection of PTNBs with a sensor attached outside to the body of a patient. In various embodiments, the optical fiber may be employed only for the delivery of the excitation laser radiation. Besides intravascular delivery, in certain embodiments, the fiber may be directly brought to specific localized target by using a biopsy needle as a guide for optical fiber. Further, in certain embodiments, PTNBs may be generated around Hz nanocrystals and detected in vivo in a non-invasive way for the purpose of diagnostics alone. In cases where a blood vessel is located very close to a surface (e.g., in the ears, eyes, lips, etc.) the excitation laser radiation may be delivered from an external source through the skin and through a vessel wall. A PTNB may be generated when a MIRBC flows into the irradiated zone and emits an acoustic pulse that may be detected by an acoustic sensor attached to the skin. In various embodiments, delivery of laser radiation may occur through a free space set up or with a fiber optical system that includes a fiber probe whose tip is brought into a contact with skin at the point closest to the target blood vessel. Optical and acoustic transmittance between the probe, sensor and skin may be enhanced by using existing transparent gels. Signals associated with Hz-generated PTNBs may be detected and counted over a specific time. In particular embodiments, such signals may detect a single MIRBC. Small blood vessels have blood flows of over 109RBCs per minute (less than 1 mL of blood). Therefore, by detecting, for example, 100 PTNB signals, various embodiments may achieve a diagnostic sensitivity of 1 MIRBC per 107normal RBCs over a 1 minute period. These parameters significantly surpass the performance of many current diagnostic methods. In addition, due to the small laser-irradiated volume required for various embodiments, the energy required for a laser pulse may be reduced resulting in much lower price to create an embodiment. Various embodiments of the present disclosure present technical advantages over current malarial diagnostic and treatment procedures by detecting and/or destroying any stage (including gametocytes) and any type of malaria parasite that contains Hz nanocrystals. The present disclosure thus supports early-stage diagnosis, fast screening, and monitoring of residual parasites. In particular, from a diagnostic perspective, various embodiments may detect minor amounts of Hz nanocrystals in individual cells and may significantly improve the sensitivity and specificity of malaria diagnosis, detecting 1 MIRBC among 104-8normal (non-infected) RBCs. Moreover, as discussed previously, the time required to diagnosis malaria utilizing various embodiments is meaningfully reduced. The increase in sensitivity and reduction in time for certain embodiments provides an improvement over existing technology. Various embodiments may provide significant therapeutic advantages as well. To date there is no absolutely efficient drug that cures malaria, given at least the problems associated with drug resistance, non-specific targeting of drugs, intracellular location of the malaria parasite, toxicity of the drugs and lack of understanding of all biological malaria-related mechanisms that are targeted by drug therapies. The technical advantages of certain embodiments of the present disclosure may include the ability to combine diagnostics and therapeutics into one connected theranostic procedure. Particular embodiments may include a field diagnostic device that operates in a “one button-one reading” mode, for example by delivering results in seconds by trans-cutaneous generation and detection of PTNB in blood vessels, and that does not require high technical expertise or use any reagents or needle. This embodiment may allow for increased screening of at-risk populations “in the field,” i.e., in settings remote from established health care facilities. The present disclosure may also allow for non-invasive monitoring of traditional treatments and/or the in vivo monitoring of the efficacy of new drugs and vaccines. To facilitate a better understanding of the present disclosure, the following examples of certain aspects of some embodiments are given. In no way should the following examples be read to limit, or define, the entire scope of the disclosure. EXAMPLES Optical absorbance. MIRBCs contain a malaria-specific photothermal target, Hz nanocrystals, that have a significantly higher optical absorbance than that for normal (i.e., uninfected) RBC and normal hemoglobin (Hb), the major RBC protein (FIG.1). As a result, the Hz nanocrystal may be used as localized optical nano-target for selective laser pulse-induced heating and PTNB generation, resulting in localized and selective destruction of the target itself, the plasmodium parasite and the MIRBC without damage to normal RBCs that may be exposed to identical treatment. Laser pulse heating of hemozoin. Photo-excitation of the MIRBCs by a short laser pulse causes selective transient heating of Hz crystals due to its high optical absorbance (compared to any other molecular optical absorbers in normal blood) and formation of localized PTNB (FIG.2). Short pulse excitation of Hz will prevent heat losses from the crystal and damage to the host RBC and its environment. Instead, the short mechanical explosive action of the PTNB will, depending upon the maximal diameter of PTNB, locally disrupt and destroy the Hz crystal (smallest PTNB), the food vacuole in which the crystals are found (larger PTNB), and then the malaria parasite itself (PTNB), providing a therapeutic effect, without damaging the host RBC (FIG.2a-d). The maximal diameter of the PTNB is determined by the energy (fluence) of the excitation laser pulse (FIG.3). Larger PTNBs generated by more intense laser pulses will destroy all the above-mentioned components and the MIRBC itself. Destruction of either the intracellular parasite or the infected cell will provide a therapeutic effect. Detection of hemozoin: optical and acoustic signals. Optical scattering and acoustical emission by laser induced PTNBs will allow highly sensitive detection of Hz nanocrystals (FIG.4). Response of individual Hz crystals to single short laser pulses was studied in standard phosphate buffer suspension (PBS) of Hz (#tlrl-hz, InvivoGen, San Diego, California) prepared at the concentration of 10 μg/mL. Individual crystals were identified though optical scattering images and were positioned into the center of the excitation and probe laser beams. Each Hz crystal (FIG.5a) was exposed to a single excitation pulse at specific fluence and the data for 30 different crystals (exposed to identical laser pulses) were averaged and analyzed. We observed PTNB-specific optical scattering images (FIG.5c) and time-response (FIG.5d) at fluences greater than 10 mJ/cm2. Therefore, Hz crystals were able to generate PTNBs even at low optical energies (fluences). Some Hz crystals survived the first pulse and were able to generate the bubbles after being exposed to additional pulses. However, as a rule we observed the destruction and disappearance of the Hz crystal after the first laser pulse (FIG.5b). In addition to optical detection of PTNBs generated by Hz crystals, we registered acoustic time responses of PTNBs (FIG.5e). Detection, imaging, and quantification. In some of our experimental work, detection, imaging, and quantification of PTNBs were performed simultaneously with the excitation pulse using three independent methods. Time-resolved optical scattering imaging (FIG.6c) shows the PTNB and its spatial location, while optical scattering (FIG.6b) and acoustic (FIG.6d) traces are employed to measure the lifetime of the PTNB. The lifetime of the PTNB is proportional to its maximal diameter. In previous work, we have shown that PTNB lifetimes correlate with favorable diagnostic and therapeutic effects where similar PTNBs were generated in cancer cells targeted with gold nanoparticles. Optical detection is based on the excellent light scattering properties of the PTNBs. Acoustic detection is based on the generation of the pressure transients during the bubble expansion and collapse, complements light scattering detection, and, most importantly for diagnostic application, can be used for in vivo detection of PTNBs in opaque tissue. Light scattering time-responses were measured as integral scattering effects of the PTNB on the continuous probe laser beam that was focused onto the sample collinearly to the excitation laser beam (FIG.6b). A continuous probe laser beam of very low power (633 nm, <0.1 mW, 05-STP-901, CVI Meller Griot, Albuquerque, NM) was focused at the sample (FIG.6b) and its axial intensity was monitored with a high-speed photodetector (FPD510-FV, Thorlabs Inc., Newton, NJ) connected to a digital oscilloscope (X42, Lecroy Corporation, Chestnut Ridge, NY) that was synchronized with the excitation lasers. The scattering of the probe laser beam by the PTNB reduces the axial intensity of the probe laser and results in a dip-shaped trace that showed the expansion and collapse of the PTNB as a bubble-specific time course (FIG.6b). The duration of scattering trace is measured at the half level of its minimum with 0.4 ns resolution and is defined as a lifetime of the PTNB. The probability of PTNB generation is measured as the ratio of PTNB-positive events (objects) (M) to the total number of the objects (N) exposed to the laser pulse: PRB=MN The level of laser pulse fluence that corresponds to the PRB of 0.5 was determined as the threshold of the PTNB generation. Time-resolved scattering images (FIG.6c) were obtained with a short laser pulse (576 nm) delayed for 10 ns relative to the excitation pulse to allow formation and expansion of the PTNB (FIG.6c). This probe laser side-illuminates the sample so that only light scattered by the PTNB is collected by the microscope objective lens and projected onto an image detector (Luka model, Andor Technology, Northern Ireland). The image of the PTNB is then used to determine the location of the PTNB relative to the malaria parasite whose location is determined with fluorescent microscopy imaging using a parasite-specific SYBR green I fluorescence dye as discussed herein. Acoustic traces (FIG.6d) were detected at the distance of several millimeters from the sample with an ultrasound transducer XMS-310 (Olympus NDT Inc., Waltham, MA) coupled to the oscilloscope (X42, Lecroy Corporation, Chestnut Ridge, NY) through an amplifier (Ultrasonic Preamp 5676, Olympus NDT Inc., Waltham, MA). The transducer head was immersed into the cell suspension and was directed toward the exposed area at the distance of approximately 2-3 mm. Pressure transients generated during the expansion and collapse of the PTNBs produce compression-rarefaction type traces that are quantified from their maximal amplitudes. All three types of signals were recorded simultaneously during exposure of each object to a single laser pulse. The study of each individual cell or the ensemble of the static cells involved the following protocol:A cell (a field) was positioned into the center of laser beam.A bright field image of the cell was obtained.A SYBR green I fluorescent image was obtained.A single laser pulse was applied at specific duration and fluence.The three PTNB signals were simultaneously recorded by using the excitation laser pulse to trigger the image detector (see below) and the oscilloscope to record the light scattering and acoustic signals.Ten nanoseconds after the trigger pulse, a bright field image of the cell was obtained using the CCD detector attached microscopic objective lens For experiments with individual cells, this protocol allows correlations of the spatial locations of the Hz crystals in the parasite with the PTNB and of parameters of the PTNB with the parasite stage in each infected cell. For bulk ensemble cells experiments, this protocol also allows counting of MIRBCs and uninfected RBCs in each laser-exposed area. The operation of the motorized microscope stage, lasers, oscilloscope and the image detector was controlled by custom-made program modules assembled using the LabView 8 platform (National Instruments Corporation, Austin, TX). Malaria parasite infection model. Suspensions of Hz were prepared by adding 5 mg Hz crystals (InvivoGen, #HMZ-33-04) into 1 mL of sterile phosphate buffered saline (pH 7.4). This suspension was sonicated for 5 minutes at room temperature to obtain a more homogenous dispersion of the crystals. The sample for studying individual Hz crystals was prepared by diluting of the stock suspension 1000-fold and then dispersing 5 μL of this working suspension on standard microscope slides and coverslips. P. falciparum, strain 3D7, was obtained from RBC stabilates preserved in liquid nitrogen (the level of parasitemia during storage is ≥10%). Cultures were maintained on plates at 37° C. at 5% parasitemia in RPMI 1640 (#31800-022, Gibco-Life Technology, Rockville, MD) supplemented with 0.5% Albumax II (#11021-029, Gibco-Life Technology, Rockville, MD) under a 5% O2/5% CO2/90% N2atmosphere as previously described by Trager and Jensen. Prior to laser treatment, the level of parasitemia of an aliquot of stock culture was measured by light microscopy using Giemsa staining and SYBR green I (#S7563, Molecular Probes, Eugene, OR) fluorescence. Cells, approximately 2-5×103, were examined for determining the percentage of infected cell (defined as parasitemia). Both staining techniques were used also for analyzing the percentage of infected cells 24 hours after laser treatment and 48 hours after laser treatment (FIG.7d). The level of parasitemia was adjusted prior to laser treatment in asynchronous culture. Ring, trophozoite and schizont stages of intraerythrocyticPlasmodium falciparumwere included in the samples. For fluorescent imaging of the parasites, a solution of SYBR green I (diluted to 10× concentration in complete medium) was added to an aliquot of a stock culture, the suspension was mixed, and the sample placed in the dark for 5 minutes. Cells were washed twice with complete medium to remove unbound SYBR green I before imaging. RBC concentrations were counted for each sample with a hemocytometer before treatment (0 hours), 24 hours after laser treatment, and 48 hours after laser treatment. Cell concentration was adjusted to 7×105cells/mL for the experiments with individual cells, 1×107cells/mL for static bulk exposure of cell mixtures and 3×106cells/mL for the flow experiments. For the experiments with individual cells, RBC suspensions were placed on Ibidi 6-channel plates (μ-Slide VI 0.4, #80606, Ibidi, LLC., Verona, WI). For the static exposure of cell mixtures, 35 mm Petri dishes were used, and for the flow experiments, an Ibidi 1 mm flow cuvette (μ-Slide VI 0.1, #80666, Ibidi, LLC., Verona, WI) was used. Experiments with individual cells were repeated three times under identical conditions. Bulk laser scans of blood samples were also performed three to four times under identical conditions. Flow treatment of infected blood was repeated four times under identical conditions, but while using new stocks of cultured parasites. Microscopy-based imaging and counts of the cells stained with the two methods were used to detect and quantify infection. First, Giemsa staining (FIG.7-I and -II) was used as a standard approach to identify ring and schizont stages of malaria parasite development and to measure the level of parasitemia, that is, the ratio of the MIRBCs to the total number of cells. Second, fluorescent staining with SYBR green I (FIG.7-III) was used as additional independent method to identify MIRBCs and specific stages of the parasite development. SYBR green I staining was also used to identify viable parasites. Since the SYBR green 1 dye does not absorb the excitation laser radiation (532 nm), the dye was used in the PTNB experiments for identifying infected cells before and after their exposure to the laser excitation pulses. We used a continuous 473 nm laser (RGBLase LLC, Fremont, CA) for excitation of the SYBR green I fluorescence. The spectral properties of this dye excluded absorption of the excitation laser pulse at 532 nm. To improve the accuracy of the identification and counts of MIRBCs and the developmental stage of the parasites, we employed laser scanning confocal microscopy (LSM 710, Carl Zeiss Inc.), which enabled much higher quality bright field (FIG.7-II) and fluorescent (FIG.7-III) images as compared to standard microscopy imaging. Depending on the level of parasitemia, we collected 10 to 20 frames for the images of 2500-5000 cells and used the two staining methods (Giemsa- and SYBR green I) to identify uninfected cells (FIG.7a) and MIRBCs in early ring (FIG.7b) and mature schizont (FIG.7c) stages. We observed good correlation between the SYBR green I- and Giemsa-based counts for all three groups of cells (FIG.7d). This correlation validates our use of the SYBR green I fluorescent method for real time monitoring of individual cells before and after exposure to single excitation laser pulses (FIG.8). We observed that the PTNB, generated by the excitation of Hz, lyses the MIRBC but its membrane was apparently not fully destroyed and appeared to envelope the destroyed parasite fragments within the original location several hours after the single pulse treatment (FIG.8). PTNB generation. The ability of Hz to generate transient PTNBs was explored with isolated Hz nanocrystals in water (FIG.9a). Single excitation laser pulses of specific fluence (70 ps or 14 ns, 532 nm) were applied and the generation of PTNB was detected by three distinct methods (seeFIG.6). Time-resolved optical scattering images, optical scattering and acoustic traces all showed the transient PTNB of nanosecond duration around Hz nanocrystals in response to single laser excitation pulse (FIG.9a). A bright flash is seen in the scattering image (FIG.9a-III), the expansion and collapse of the PTNB is reported in the optical scattering trace (FIG.9a-IV), and a pressure transient induces a specific acoustic trace (FIG.9a-V). The duration of the optical trace reports the PTNB lifetime and is a metric of PTNB maximal size. PTNB lifetime increased with the energy (fluence) of the laser pulse and also depended upon its duration (FIG.10a). The longer 14 ns pulse showed much lower efficacy for the PTNB generation, likely due to diffusive thermal losses from nanocrystal during slower optical excitation. These results show that Hz nanocrystals efficiently convert the optical energy of a short laser pulse into a localized, tunable and transient PTNB. We next cultured malaria parasites,Plasmodium falciparum(strain 3D7), in human blood and exposed individual MIRBCs to single laser pulses (70 ps or 14 ns, 532 nm). Generation of PTNBs in MIRBCs was monitored with the three independent signals described above (seeFIG.6). The presence and stage of the parasite in each cell were verified with the two independent microscopy methods (FIG.7), Giemsa staining with bright field imaging (FIGS.9cand9c-I) and SYBR green I staining with fluorescent imaging (FIGS.9band9c-II). Using laser fluences similar to those in the isolated Hz experiments (40 mJ/cm2), we detected PTNB in individual MIRBCs at early ring stage (FIG.9b-III, -IV and -V) and mature schizont stage (FIG.9c-III, -IV and -V). In all MIRBCs the PTNB locations coincided with those of the parasite (FIGS.9band9c-II and -III). Identical excitation of the ring and schizont parasite stages returned different signal responses: the lifetime of the PTNB in schizont MIRBCs was ten-fold higher than that in ring MIRBCs (FIGS.9b-IV,9c-IV,10b). These stage-specific differences appear to be a consequence of larger and more abundant Hz crystals in schizont stage parasites (FIGS.7band7c), which greatly facilitates PTNB generation. The lifetimes of PTNB increased with the fluence of laser pulse thus increasing the sensitivity of the detection of parasite (FIG.10b). Like with isolated Hz nanocrystals, we observed much higher efficacy of the PTNB generation with a short, 70 ps pulse compared to a longer, 14 ns pulse. We also found a good correlation between the amplitude of the acoustic trace and the lifetime of the PTNB as measured by optical scattering trace (FIG.10c). This correlation verifies feasibility of acoustic detection of parasites in opaque biological tissue (e.g., through the skin) that would normally compromise optical detection. Unlike MIRBCs, which sustained visible damage after a single laser pulse (FIGS.9b-VI and9c-VI), irradiation of uninfected RBCs under the same conditions did not generate PTNBs detectable by any of the three methods (FIGS.9d-III-9d-V). Even more importantly, no signs of laser-induced damage or significant heating of uninfected RBCs were observed (FIGS.9d-IV,9d-VI). The selective generation of PTNBs in only MIRBC results from the combination of: (1) the five- to seven-fold higher optical absorbance of Hz compared to that of hemoglobin in RBCs and (2) temporally and spatially localized heat release and evaporation of liquid due to the nano-size of the Hz nanocrystals and the short duration of the laser pulse (70 ps) which prevented thermal diffusive losses from the nanocrystal. These experiments demonstrate that the generation ofPlasmodium falciparum-specific PTNBs in individual MIRBCs is similar to the generation of PTNBs around isolated Hz nanocrystals in water and its efficacy is maximal with the picosecond excitation pulses. Hz is found only in blood stage of malaria parasite, therefore laser-induced PTNBs can act as malaria parasite-specific cellular agents even at early ring stages when the Hz crystals are only tens of nanometers in size and difficult to detect in single cells by other known methods. PTNB generation and detection. The duration of each light scattering trace was measured to determine the PTNB lifetime as the metric of the maximal size of the vapor PTNB. We observed steady increases in the PTNB lifetime with increasing fluence of the laser pulse (FIG.10a). Both the threshold for bubble production and its lifetime depended upon laser pulse duration. The shortest, 70 ps, pulse generated the largest PTNBs and required the minimal threshold fluence (about 10 mJ/cm2) whereas the longer 14 ns pulse had a higher threshold (about 40 mJ/cm2) and generated smaller PTNBs (FIG.10a). This pulse duration effect is determined by the size of the optical absorber. Hz nanocrystals are between approximately 50-1000 nm in diameter and generate PTNB more efficiently with a 70 ps pulse rather than with the longer 14 ns pulse. The latter pulse may be too long to prevent thermal losses and de-localization of the photo-heating effect. Absorbance of the 17 ps pulse by a Hz nanocrystal results in rapid evaporation of its surrounding water layers resulting in localized and tunable generation of vapor PTNBs. Identical excitation of the ring and schizont parasite stages returned different signal responses. At low fluence (28 mJ/cm2) only schizont MIRBCs returned PTNB-type responses, whereas the ring MIRBCs did not generate PTNBs (FIG.10b). At higher fluence (40 mJ/cm2), the lifetimes of the PTNBs in schizont MIRBCs was ten-fold greater than those observed in ring MIRBCs (FIGS.9b,9c, and10b). We also studied how the maximal diameter of the PTNB, a parameter that determines diagnostic sensitivity and parasiticidal efficacy, depends upon optical excitation conditions. Using light scattering trace detection (FIG.9-IV), we measured the probability of PTNB generation and its lifetime in individual cells as a function of laser pulse fluence and duration at different parasite stages (FIG.10b). The probability of formation and the lifetime of PTNBs increased with fluence. For the mature schizont stage-infected cells, the PTNB lifetime was more than ten-fold higher than that for early ring stage-infected cells treated with identical fluence of the laser pulse (FIG.10b). These stage-specific differences are likely a consequence of the larger and more abundant Hz crystals in schizont stage parasites (FIGS.7band7c). Increases in size and density of the crystals will likely increase efficacy of PTNB generation. Increased pulse duration from 70 ps to 14 ns under identical laser pulse fluence dramatically reduced the probability and lifetimes of the PTNBs (FIG.10b), likely due to increased thermal diffusive losses during the longer excitation pulse. Similar effects were observed when the longer pulse was used for excitation of isolated Hz nanocrystals (FIG.10a). Thus, short picosecond pulses may be optimal for generating diagnostically reliable vapor PTNBs in MIRBCs. Finally, we compared the acoustic and optical traces of MIRBCs (FIG.10c) and found a good correlation between the acoustic amplitude to the PTNB lifetime as measured by the light scattering signals. This result is important for non-invasive clinical applications. Acoustic detection of parasites can be used with opaque and scattering biological tissues that would normally compromise optical, light scattering detection. These results collectively show that short laser pulses may generate localized PTNB by photothermally exciting Hz nanocrystals in MIRBCs without affecting uninfected RBCs. The maximal diameter of vapor PTNBs is estimated to be 0.5-1 μm for a 100 ns lifetime. This size is sufficient to readily measure optical light scattering (FIGS.9b-III and -IV,9c-III and -IV) and acoustic signals (FIG.9b-V and9c-V column V) due to pressure transients generated during the formation and collapse of the bubble. In addition, the localized explosive effect of PTNB formation is large enough to mechanically burst and destroy the parasite (FIGS.9b-VI,9c-VI,13). PTNB lifetime. Parameters of PTNB were analyzed through the PTNB lifetime (the metric of the maximal diameter of PTNB) as function of laser fluence, pulse duration, and number of laser pulses applied to the same Hz crystals. Dependencies of the PTNB lifetime upon fluence were obtained for two durations of the laser excitation pulse, 500 ps and 70 ps (FIG.11a). We observed good tunability of the PTNB lifetime through the fluence: increase of laser fluence resulted in controllable increase of the lifetime of PTNB. At higher fluence, we observed higher efficacy of PTNB generation for the 500 ps pulse compared to the shorter 70 ps pulse. Based on our previous experience, we estimated that PTNBs with a lifetime above 150 nanoseconds kill the host cell, whereas smaller PTNBs can be generated without disrupting the RBC membrane. Therefore, laser pulse fluence can be used for controlling the therapeutic effect of Hz-generated PTNB. The stability of Hz crystals was studied under pulsed laser exposure, heating, and bubble generation for 500 ps pulses (FIG.11b). The same Hz crystal was exposed to several identical pulses of relatively low fluence with a 5 second interval. We observed a rapid decrease of the PTNB lifetime that was caused by deterioration and destruction of the Hz crystal. Diagnostic properties. The diagnostic properties of laser-induced PTNB were studied in mixtures of MIRBCs and uninfected RBCs with simultaneous scanning of cultures with broad-diameter single laser pulses (532 nm, 70 ps, diameter 210 μm) (FIG.12a). The MIRBCs and their stage (ring or schizont) were identified and counted in each cell field prior to the laser exposure using SYBR green I-specific fluorescence (FIG.12a, inset). We obtained acoustic traces for each laser-exposed field with an acoustic sensor located 2-3 mm from the cells. The ratio of MIRBCs to uninfected RBCs was varied by diluting the infected sample with normal blood. Fields lacking MIRBCs returned no signal (FIG.12b, green trace), whereas fields with even a single, ring stage MIRBC returned PTNB-specific traces at MIRBC to RBC ratios of greater than or equal to 1 to 104(FIG.12b, red trace). The acoustic traces detected for schizont stage MIRBCs had much higher amplitude (FIG.12b, black trace). These differential signals could in principle allow diagnosis of the infection stage with single cell sensitivity. Due to the manual registration and analysis of the signals we limited our counts to between 30 and 40 fields (i.e., between 24 to 32000 cells) and, thus, did not study higher ratios of MIRBCs to RBCs. Nevertheless, these data support the feasibility of detection of MIRBCs with the sensitivity of 1 MIRBC for every 106RBC by automatic counting and analysis of acoustic traces of PTNB during trans-cutaneous delivery of laser pulses into blood vessels just under the skin by externally scanning optical fiber probe with acoustic sensor. This approach has the potential to provide highly sensitive, non-invasive and label- and needle-free in vivo detection of individual MIRBCs within several seconds. Parasiticidal effects of PTNBs were analyzed by comparing the percentage of MIRBCs among all cells as a measure of parasitemia before and after bulk single pulse laser treatment of blood in a flow system (FIG.16). The explosive mechanical action of the intra-parasite PTNB appears to immediately burst and destroy the parasites (FIGS.9b,9cVI; see alsoFIGS.8and13). We applied 70 ps pulses at two fluence levels, 35 mJ/cm2and 130 mJ/cm2, and 14 ns pulses of 70 mJ/cm2fluence that corresponded to 40-60 ns lifetimes of the PTNBs in MIRBCs as was found previously (seeFIG.10b). The flow rate, laser beam diameter and laser pulse repetition rate were synchronized to provide a single laser pulse exposure to each cell flown through the system. The level of parasitemia and the cell concentration were measured for 3000-4000 cells at three time-points: before treatment (0 hours), 24 hours after laser treatment, and 48 hours after laser treatment, using Giemsa bright field and SYBR green 1 fluorescent imaging (FIG.12c). In addition to the bulk PTNB treatment, we applied a standard malaria drug, chloroquine, in a therapeutic dose of 1 μM15. The PTNB mode showed three-fold higher parasiticidal efficacy than chloroquine and rapidly reduced the level of MIRBCs to between 5% and 7% of that in the untreated sample at 24 hours (FIG.12c). The concentration of uninfected RBCs did not show any detectable changes 24 hours or 48 hours after the 70 ps laser treatment. The maximal parasiticidal effect was observed for combinatorial treatment with PTNBs and drugs after 48 hours (FIG.12c). Destruction of malaria parasites. The immediate mechanical destruction caused by rapid expansion of the PTNB around Hz nanocrystals in the parasite food vacuole destroys the parasite but does not immediately cause loss of fluorescence of the SYBR green I dye. DNA, which will also cause SYBR green I fluorescence, is likely still present in the parasite fragments in the original location of the laser-treated cell. Therefore, to quantify remaining viability of infected cells after laser treatment, we quantified the number of the MIRBCs at 24 hours after treatment and 48 hours after treatment (levels of parasitemia). These time intervals are long enough to allow significant multiplication of any viable parasites as was observed for the untreated samples of MIRBCs (FIG.12c). The lack of multiplication and, more importantly, the decrease in the level of MIRBCs after laser treatment (FIG.12c) is most likely due to PTNB-induced lethality of parasites. Generation of PTNB in the MIRBCs under a high fluence of the excitation short laser pulse also often induced the lysis of the host cells (FIG.13) due to mechanical perforation of the RBC membrane. However, even under these more destructive conditions, uninfected RBCs had no detectable damage (FIG.13). This result confirms the localized, malaria parasite-specific nature of the Hz-derived PTNB whose mechanical impact was confined by the MIRBC. It should be noted that increasing the fluence of the short 70 ps pulse beyond 40 mJ/cm2did not enhance the parasiticidal efficacy (FIG.12c), and a longer 14 ns pulse showed lower parasiticidal efficacy (FIG.12c) and, at the same time, lysed roughly 25% of the uninfected RBCs, due to more delocalized photothermal heating. Destruction of malaria parasites: additional data. MIRBCs were modeled by mixing and incubating normal RBC with Hz crystals. Then RBCs containing Hz adsorbed to the cell membranes were mixed with normal RBC (FIG.14a). All cells were treated with single identical laser pulses at the fluence that was previously determined to generate PTNBs around Hz crystals (532 nm, 400 ps, 31 mJ/cm2). Generation of PTNBs was optically monitored through time-resolved optical scattering imaging and through time-responses of individual cells. Cells were imaged before laser treatment (FIG.14a) and immediately after (FIG.14b). We observed selective destruction (lysis) of MIRBC model cells (i.e., Hz adsorbed to the surface), while normal RBCs were not damaged. Such high selectivity of cell destruction correlated very well with the generation of PTNBs: they were observed only in MIRBC models (FIGS.14cand14d), whereas normal RBCs did not produce any PTNBs (FIG.14c,14e). Since the lifetime of Hz-generated PTNB was, as a rule, above 100 ns at the fluence applied, we concluded that MIRBC model cells were destructed with relatively large PTNBs (as shown inFIG.2d). PTNB and hyperthermia. Because Hz crystals were previously reported as the photothermal targets for laser-, radiofrequency- and magnetic-based hyperthermia treatments of malaria, we experimentally compared the efficacy and optical dose in PTNB generation and hyperthermia modes. The heating mode was achieved by using the same optical pulse of low fluence that caused localized transient heating of Hz crystals but without generation of PTNBs (FIG.15). Laser pulses were applied at 10 hertz for 10 seconds and longer. The thermal effect was confirmed with optical responses (FIG.15d) of specific shape that indicated fast heating and gradual cooling. Despite apparent heating of the target and increased optical dose (100 mJ/cm2against 31 mJ/cm2used in the PTNB mode) we did not observe any apparent damage to MIRBC model cells (adsorbed Hz) (FIGS.15band15f), whereas the MIRBC models treated in PTNB mode were destroyed after a single laser pulse. This experiment demonstrated higher efficacy, shorter treatment time, and lower optical dose of the treatment in PTNB mode and a totally different mechanism than that of the hyperthermia mode. Experimental set up for the bulk flow treatment of the blood We designed a closed sterile flow system (FIG.16) that included an optically transparent flow cuvette (μ-Slide VI 0.1, #80666, Ibidi, LLC., Verona, WI) connected to two syringes, one dispensing and one collecting the RBC suspension. Both syringes were synchronously driven with computer-controlled pumps (NE-1000, New Era Pump Systems, Inc., Farmingdale, NY). The excitation laser beam was directed through the cuvette. The geometry of the channel (rectangular cross-section 1 mm wide, 0.1 mm deep and 15 mm length) ensures laminar flow with a two-dimensional monolayer of flowing cells being formed in the middle of the cuvette. The syringes were kept at physiological temperature by the automated heating sleeves. The diameter of the excitation laser beam was increased to 1.8 mm to provide uniform irradiation of all cells in the 1 mm by 1 mm area of the cuvette for each pulse. Flow rate was adjusted to the laser pulse repetition rate (10-40 hertz) to ensure single pulse exposure to each cell flowing through the cuvette. A low flow rate was used to treat 1 mL of the cell suspension in several minutes. The flow rate was limited in our experiments by the energy of the laser pulse and by the pulse repetition rate. Commercial lasers with 200-400 mJ/pulses and 100 hertz repetition rates will allow an increase in the treatment rate to 500 mL/min. This rate would allow the treatment of all the blood cells of a patient in 10 to 20 minutes. We applied the following protocol for the flow treatment of the MIRBCs:The initial level of parasitemia was calculated with the two methods as described above.The cell suspension was adjusted to 3×106cell/mL.Cells were flown through the system and then exposed to a specific pulsed laser fluence.Collected cells were cultured for another 48 hours.Cell concentration and the levels of parasitemia were measured before treatment (0 hours), 24 hours after laser treatment, and 48 hours after laser treatment. In the experiments that included the drug chloroquine, chloroquine (C6628, Sigma-Aldrich LLC, Saint Louis, MO) was added to the cell suspensions immediately prior to the flow treatment. A drug dose of 1 μM was calculated to match the therapeutic level used in most treatment regimens. Each treatment was repeated 3-4 times for different blood samples, each of which was cultured independently.The parasiticidal effect of the bulk flow treatment was analyzed using the following parameters:The absolute level of MIRBCs (parasitemia level) at 24 hours after laser treatment and 48 hours after treatment was measured and compared to that of the initial, untreated samples. This metric was used to estimate the efficacy of the specific treatment mode and to compare different treatments at one time-point.The relative level of MIRBCs was calculated for each time point as the ratio of the absolute levels of MIRBCs in the treated sample to that in the untreated control with intact blood cells. This metric was used to compare the parasiticidal kinetics in the different non-synchronized samples of MIRBCs.The total cell concentration characterized the safety of the treatment to uninfected cells. The concentration of RBCs was measured at each time point and was compared to the initial concentration of the cells in the suspension prior to flow treatment. Devices for malaria diagnostics, therapeutics, and theranostics. Devices for the diagnosis and/or treatment of malaria may include devices similar to those described herein and may include an optically transparent cuvette that allows for blood containing MIRBC to be exposed to short laser pulses (FIG.17a). Diagnostic and treatment devices may be similar to the prototype we constructed, which included a transparent flow cuvette and a syringe pump that flows the cell suspension through the cuvette (FIG.17b). Devices appropriate for in vivo diagnosis and/or treatment may include at least an optical fiber probe, a photodetector, an ultrasound detector, and a computer, or some combination of these components (FIG.18a). Devices appropriate for in vivo applications may allow excitation laser radiation to be directed with a fiber probe into a sub-cutaneous blood vessel or vessels where PTNBs may be generated in MIRBCs (FIG.18b). Certain devices may be similar to the prototype we constructed that include a fiber system for PTNB generation and detection (FIG.18c). The experiments described above demonstrate selective generation of PTNB around Hz crystals, the ability to guide and detect PTNB generation in real time with three different techniques, the therapeutic feasibility of the method for destroying infected RBCs, the high therapeutic selectivity of the method which prevents destruction of uninfected cells, and the possibility combining the diagnosis (based on PTNB detection), guidance of treatment (with PTNB of specific lifetime) and destruction of parasites and/or MIRBCs (based on the parameters of PTNB signals) in one theranostic procedure. Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as illustrated, in part, by the appended claims. | 62,387 |
11859178 | DETAILED DESCRIPTION Sequencing nucleic acid libraries generated from single-cells or spatial array analyses generally biases capture to the 3′ end of captured analytes due to fragmentation and subsequent ligation of sequencing adapters. Strategies are needed to sequence regions more than about 1 kilobase away from the 3′ end of analytes in nucleic acid libraries generated from single-cells or spatial array analyses. Provided herein are methods, compositions, and kits for the manipulation of nucleic acid libraries. Various methods of removing a portion of a sequence from a member of a nucleic acid library or reversing the orientation of the sequence from a member of a nucleic acid library are generally described herein. Some embodiments include double-stranded members of a nucleic acid library. Some embodiments include single-stranded members of a nucleic acid library. Some embodiments of the nucleic acid library methods provided herein remove a portion of a nucleic acid sequence in a nucleic acid library prior to standard sequencing preparation. Some embodiments of the nucleic acid library methods provided herein remove a portion of a captured analyte sequence in a nucleic acid library. Some embodiments of the nucleic acid library methods remove a portion of a constant sequence of a captured analyte. Some embodiments of the nucleic acid library methods reverse the orientation of the nucleic acid, or a portion thereof. Some embodiments of the nucleic acid library methods described herein reverse the orientation of a captured analyte, or a portion thereof. Some embodiments of the nucleic acid library methods described here include the use of nucleic acid libraries prepared from single-cells. Some embodiments of the nucleic acid libraries described herein include the use of nucleic acid libraries from arrays (e.g., a spatial array). Spatial analysis methodologies and compositions described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context. Spatial analysis methods and compositions can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or a nucleic acid) produced by and/or present in a cell. Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte. For example, the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample. Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022, 10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent Application Publication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641, 2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709, 2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322, 2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875, 2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO 2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee et al., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE 14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol. 36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020), both of which are available at the 10× Genomics Support Documentation website, and can be used herein in any combination. Further non-limiting aspects of spatial analysis methodologies and compositions are described herein. Some general terminology that may be used in this disclosure can be found in Section (I)(b) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Typically, a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a capture probe). A barcode can be part of an analyte, or independent of an analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes. For the purpose of this disclosure, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. The term “target” can similarly refer to an analyte of interest. Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, the analyte(s) can be localized to subcellular location(s), including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. Additional examples of analytes can be found in Section (I)(c) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. In some embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein. A “biological sample” is typically obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject. In some embodiments, a biological sample can be a tissue section. In some embodiments, a biological sample can be a fixed and/or stained biological sample (e.g., a fixed and/or stained tissue section). Non-limiting examples of stains include histological stains (e.g., hematoxylin and/or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, a biological sample (e.g., a fixed and/or stained biological sample) can be imaged. Biological samples are also described in Section (I)(d) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. In some embodiments, a biological sample is permeabilized with one or more permeabilization reagents. For example, permeabilization of a biological sample can facilitate analyte capture. Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Array-based spatial analysis methods involve the transfer of one or more analytes from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample. The spatial location of an analyte within the biological sample is determined based on the feature to which the analyte is bound (e.g., directly or indirectly) on the array, and the feature's relative spatial location within the array. A “capture probe” refers to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and/or a unique molecular identifier (UMI)) and a capture domain). In some embodiments, a capture probe can include a cleavage domain and/or a functional domain (e.g., a primer-binding site, such as for next-generation sequencing (NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. In some embodiments, more than one analyte type (e.g., nucleic acids and proteins) from a biological sample can be detected (e.g., simultaneously or sequentially) using any appropriate multiplexing technique, such as those described in Section (IV) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. In some embodiments, detection of one or more analytes (e.g., protein analytes) can be performed using one or more analyte capture agents. As used herein, an “analyte capture agent” refers to an agent that interacts with an analyte (e.g., an analyte in a biological sample) and with a capture probe (e.g., a capture probe attached to a substrate or a feature) to identify the analyte. In some embodiments, the analyte capture agent includes: (i) an analyte binding moiety (e.g., that binds to an analyte), for example, an antibody or antigen-binding fragment thereof (ii) analyte binding moiety barcode; and (iii) an analyte capture sequence. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe. In some cases, an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g., cleaved) from the analyte capture agent. Additional description of analyte capture agents can be found in Section (II)(b)(ix) of WO 2020/176788 and/or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663. There are at least two methods to associate a spatial barcode with one or more neighboring cells, such that the spatial barcode identifies the one or more cells, and/or contents of the one or more cells, as associated with a particular spatial location. One method is to promote analytes or analyte proxies (e.g., intermediate agents) out of a cell and towards a spatially-barcoded array (e.g., including spatially-barcoded capture probes). Another method is to cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and/or into or onto the biological sample. FIG.1is a schematic diagram showing an exemplary capture probe, as described herein. As shown, the capture probe102is optionally coupled to a feature101by a cleavage domain103, such as a disulfide linker. The capture probe can include a functional sequence104that are useful for subsequent processing. The functional sequence104can include all or a part of sequencer specific flow cell attachment sequence (e.g., a P5 or P7 sequence), all or a part of a sequencing primer sequence, (e.g., a R1 primer binding site, a R2 primer binding site), or combinations thereof. The capture probe can also include a spatial barcode105. The capture probe can also include a unique molecular identifier (UMI) sequence106. WhileFIG.1shows the spatial barcode105as being located upstream (5′) of UMI sequence106, it is to be understood that capture probes wherein UMI sequence106is located upstream (5′) of the spatial barcode105is also suitable for use in any of the methods described herein. The capture probe can also include a capture domain107to facilitate capture of a target analyte. In some embodiments, the capture probe comprises one or more additional functional sequences that can be located, for example between the spatial barcode105and the UMI sequence106, between the UMI sequence106and the capture domain107, or following the capture domain107. The capture domain can have a sequence complementary to a sequence of a nucleic acid analyte. The capture domain can have a sequence complementary to a connected probe described herein. The capture domain can have a sequence complementary to a capture handle sequence present in an analyte capture agent. The capture domain can have a sequence complementary to a splint oligonucleotide. Such splint oligonucleotide, in addition to having a sequence complementary to a capture domain of a capture probe, can have a sequence of a nucleic acid analyte, a sequence complementary to a portion of a connected probe described herein, and/or a capture handle sequence described herein. The functional sequences can generally be selected for compatibility with any of a variety of different sequencing systems, e.g., Ion Torrent Proton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore, etc., and the requirements thereof. In some embodiments, functional sequences can be selected for compatibility with non-commercialized sequencing systems. Examples of such sequencing systems and techniques, for which suitable functional sequences can be used, include (but are not limited to) Ion Torrent Proton or PGM sequencing, Illumina sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing. Further, in some embodiments, functional sequences can be selected for compatibility with other sequencing systems, including non-commercialized sequencing systems. In some embodiments, the spatial barcode105and functional sequences104is common to all of the probes attached to a given feature. In some embodiments, the UMI sequence106of a capture probe attached to a given feature is different from the UMI sequence of a different capture probe attached to the given feature. In some cases, capture probes may be configured to prime, replicate, and consequently yield optionally barcoded extension products from a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663 regarding extended capture probes). In some cases, capture probes may be configured to form ligation products with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligation products that serve as proxies for a template. As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to the terminus (e.g., 3′ or 5′ end) of the capture probe thereby extending the overall length of the capture probe. For example, an “extended 3′ end” indicates additional nucleotides were added to the most 3′ nucleotide of the capture probe to extend the length of the capture probe, for example, by polymerization reactions used to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e.g., a DNA polymerase or a reverse transcriptase). In some embodiments, extending the capture probe includes adding to a 3′ end of a capture probe a nucleic acid sequence that is complementary to a nucleic acid sequence of an analyte or intermediate agent specifically bound to the capture domain of the capture probe. In some embodiments, the capture probe is extended using reverse transcription. In some embodiments, the capture probe is extended using one or more DNA polymerases. The extended capture probes include the sequence of the capture probe and the sequence of the spatial barcode of the capture probe. In some embodiments, extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e.g., via DNA sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) act as templates for an amplification reaction (e.g., a polymerase chain reaction). Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Analysis of captured analytes (and/or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes, sequencing (e.g., of a cleaved extended capture probe and/or a cDNA molecule complementary to an extended capture probe), sequencing on the array (e.g., using, for example, in situ hybridization or in situ ligation approaches), temporal analysis, and/or proximity capture, is described in Section (II)(g) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Some quality control measures are described in Section (II)(h) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Spatial information can provide information of biological and/or medical importance. For example, the methods and compositions described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and/or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g., diagnosis) of a subject as having a disease or disorder; identification of stage and/or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and/or selection of a treatment for a subject with a disease or disorder. Spatial information can provide information of biological importance. For example, the methods and compositions described herein can allow for: identification of transcriptome and/or proteome expression profiles (e.g., in healthy and/or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor analysis); determination of up- and/or down-regulated genes and/or proteins in diseased tissue; characterization of tumor microenvironments; characterization of tumor immune responses; characterization of cells types and their co-localization in tissue; and identification of genetic variants within tissues (e.g., based on gene and/or protein expression profiles associated with specific disease or disorder biomarkers). Typically, for spatial array-based methods, a substrate functions as a support for direct or indirect attachment of capture probes to features of the array. A “feature” is an entity that acts as a support or repository for various molecular entities used in spatial analysis. In some embodiments, some or all of the features in an array are functionalized for analyte capture. Exemplary substrates are described in Section (II)(c) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Generally, analytes and/or intermediate agents (or portions thereof) can be captured when contacting a biological sample with a substrate including capture probes (e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or a substrate with features (e.g., beads, wells) comprising capture probes). As used herein, “contact,” “contacted,” and/or “contacting,” a biological sample with a substrate refers to any contact (e.g., direct or indirect) such that capture probes can interact (e.g., bind covalently or non-covalently (e.g., hybridize)) with analytes from the biological sample. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in Section (II)(e) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. In some cases, spatial analysis can be performed by attaching and/or introducing a molecule (e.g., a peptide, a lipid, or a nucleic acid molecule) having a barcode (e.g., a spatial barcode) to a biological sample (e.g., to a cell in a biological sample). In some embodiments, a plurality of molecules (e.g., a plurality of nucleic acid molecules) having a plurality of barcodes (e.g., a plurality of spatial barcodes) are introduced to a biological sample (e.g., to a plurality of cells in a biological sample) for use in spatial analysis. In some embodiments, after attaching and/or introducing a molecule having a barcode to a biological sample, the biological sample can be physically separated (e.g., dissociated) into single cells or cell groups for analysis. Some such methods of spatial analysis are described in Section (III) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. In some cases, spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to an analyte. In some instances, for example, spatial analysis can be performed using RNA-templated ligation (RTL). Methods of RTL have been described previously. See, e.g., Credle et al.,Nucleic Acids Res.2017 Aug. 21; 45(14):e128. Typically, RTL includes hybridization of two oligonucleotides to adjacent sequences on an analyte (e.g., an RNA molecule, such as an mRNA molecule). In some instances, the oligonucleotides are DNA molecules. In some instances, one of the oligonucleotides includes at least two ribonucleic acid bases at the 3′ end and/or the other oligonucleotide includes a phosphorylated nucleotide at the 5′ end. In some instances, one of the two oligonucleotides includes a capture domain (e.g., a poly(A) sequence, a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., SplintR ligase) ligates the two oligonucleotides together, creating a ligation product. In some instances, the two oligonucleotides hybridize to sequences that are not adjacent to one another. For example, hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides. In some instances, a polymerase (e.g., a DNA polymerase) can extend one of the oligonucleotides prior to ligation. After ligation, the ligation product is released from the analyte. In some instances, the ligation product is released using an endonuclease (e.g., RNAse H). The released ligation product can then be captured by capture probes (e.g., instead of direct capture of an analyte) on an array, optionally amplified, and sequenced, thus determining the location and optionally the abundance of the analyte in the biological sample. During analysis of spatial information, sequence information for a spatial barcode associated with an analyte is obtained, and the sequence information can be used to provide information about the spatial distribution of the analyte in the biological sample. Various methods can be used to obtain the spatial information. In some embodiments, specific capture probes and the analytes they capture are associated with specific locations in an array of features on a substrate. For example, specific spatial barcodes can be associated with specific array locations prior to array fabrication, and the sequences of the spatial barcodes can be stored (e.g., in a database) along with specific array location information, so that each spatial barcode uniquely maps to a particular array location. Alternatively, specific spatial barcodes can be deposited at predetermined locations in an array of features during fabrication such that at each location, only one type of spatial barcode is present so that spatial barcodes are uniquely associated with a single feature of the array. Where necessary, the arrays can be decoded using any of the methods described herein so that spatial barcodes are uniquely associated with array feature locations, and this mapping can be stored as described above. When sequence information is obtained for capture probes and/or analytes during analysis of spatial information, the locations of the capture probes and/or analytes can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature location. In this manner, specific capture probes and captured analytes are associated with specific locations in the array of features. Each array feature location represents a position relative to a coordinate reference point (e.g., an array location, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array. Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See, for example, the Exemplary embodiment starting with “In some non-limiting examples of the workflows described herein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020). In some embodiments, spatial analysis can be performed using dedicated hardware and/or software, such as any of the systems described in Sections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, or any of one or more of the devices or methods described in Sections Control Slide for Imaging, Methods of Using Control Slides and Substrates for, Systems of Using Control Slides and Substrates for Imaging, and/or Sample and Array Alignment Devices and Methods, Informational labels of WO 2020/123320. Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or sealable, fluid-tight chamber) for containing a biological sample. The biological sample can be mounted for example, in a biological sample holder. One or more fluid chambers can be connected to the chamber and/or the sample holder via fluid conduits, and fluids can be delivered into the chamber and/or sample holder via fluidic pumps, vacuum sources, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow. One or more valves can also be connected to fluid conduits to regulate the flow of reagents from reservoirs to the chamber and/or sample holder. The systems can optionally include a control unit that includes one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid state storage medium such as, but not limited to, a magnetic, optical, or other solid state, persistent, writeable and/or re-writeable storage medium). The control unit can optionally be connected to one or more remote devices via a network. The control unit (and components thereof) can generally perform any of the steps and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the steps or features described herein. The systems can optionally include one or more detectors (e.g., CCD, CMOS) used to capture images. The systems can also optionally include one or more light sources (e.g., LED-based, diode-based, lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media. The systems can optionally include software instructions encoded and/or implemented in one or more of tangible storage media and hardware components such as application specific integrated circuits. The software instructions, when executed by a control unit (and in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perform any of the method steps or functions described herein. In some cases, the systems described herein can detect (e.g., register an image) the biological sample on the array. Exemplary methods to detect the biological sample on an array are described in PCT Application No. 2020/061064 and/or U.S. patent application Ser. No. 16/951,854. Prior to transferring analytes from the biological sample to the array of features on the substrate, the biological sample can be aligned with the array. Alignment of a biological sample and an array of features including capture probes can facilitate spatial analysis, which can be used to detect differences in analyte presence and/or level within different positions in the biological sample, for example, to generate a three-dimensional map of the analyte presence and/or level. Exemplary methods to generate a two- and/or three-dimensional map of the analyte presence and/or level are described in PCT Application No. 2020/053655 and spatial analysis methods are generally described in WO 2020/061108 and/or U.S. patent application Ser. No. 16/951,864. In some cases, a map of analyte presence and/or level can be aligned to an image of a biological sample using one or more fiducial markers, e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of WO 2020/123320, PCT Application No. 2020/061066, and/or U.S. patent application Ser. No. 16/951,843. Fiducial markers can be used as a point of reference or measurement scale for alignment (e.g., to align a sample and an array, to align two substrates, to determine a location of a sample or array on a substrate relative to a fiducial marker) and/or for quantitative measurements of sizes and/or distances. Methods for Preparing Nucleic Acid Libraries Sequencing nucleic acid libraries generated from single-cell or spatial array analyses generally biases capture to the 3′ end of captured analytes due to fragmentation and ligation of sequencing adapters. Alternative nucleic acid library preparation strategies described herein allow sequencing of regions further away (e.g., 5′) from the 3′ end of analytes in nucleic acid libraries. The 5′ analyte sequence enrichment strategies described herein assist in the identification (e.g., sequencing) of critical sequences (e.g., V(D)J sequences, CDR sequences) important in understanding immune cell receptor clonality in health and disease. For example, nucleic acid libraries (e.g., cDNA libraries) generated in single-cell analysis and arrays, (e.g., spatial arrays described herein), are generally biased to sequences from the 3′ end and as a result sequences more than about 1 kb away from the end of a poly(A) tail are generally not present in the sequencing library, thereby making it difficult to study 5′ coding regions and non-coding regions (e.g., 5′ untranslated region (UTR)) of analytes beyond 1 kb from the end of the poly(A) tail). In some examples described herein, the nucleic acid libraries are generated from single-cell assay systems. In some examples described herein, nucleic acid libraries are generated from array (e.g., spatial array) based assay systems. Provided herein are methods, compositions, and kits for the manipulation of nucleic acid libraries. Various methods of removing a portion of a sequence from a member of a nucleic acid library or reversing the orientation of the sequence from a member of a nucleic acid library are generally described herein. Some embodiments include double-stranded members of a nucleic acid library. Some embodiments include single-stranded members of a nucleic acid library. Some embodiments of the nucleic acid library methods provided herein remove a portion of a nucleic acid sequence in a nucleic acid library prior to standard sequencing preparation. Some embodiments of the nucleic acid library methods provided herein remove a portion of a captured analyte sequence in a nucleic acid library. Some embodiments of the nucleic acid library methods remove a portion of a constant sequence of a captured analyte. Some embodiments of the nucleic acid library methods reverse the orientation of the nucleic acid, or a portion thereof. Some embodiments of the nucleic acid library methods described herein reverse the orientation of a captured analyte, or a portion thereof. Some embodiments of the nucleic acid library methods described here include the use of nucleic acid libraries prepared from single-cells. Some embodiments of the nucleic acid libraries described herein include the use of nucleic acid libraries from arrays (e.g., a spatial array). An example of sequences of interest beyond 1 kb from the end of the poly(A) include, but are not limited to, sequences encoding T-cell receptors (TCRs) and B-cell receptor (BCR) immunoglobulins. Most T-cell receptors are generally composed of a variable alpha chain and a variable beta chain. T-cell receptor genes include multiple V (variable), D (diversity), and J (joining) gene segments in their alpha and beta chains that are rearranged during the development of the lymphocyte to provide the cell with a unique antigen receptor. Similarly, B-cell receptor genes contain multiple V, D, and J gene segments encoding a membrane-bound immunoglobulin molecule of the following isotypes IgD, IgM, IgA, IgG, or IgE. V(D)J sequences from both TCRs and BCRs also include complementarity determining region(s) (CDRs), such as CDR1, CDR 2, and CDR3, which provide specificity to the antigen-binding regions. Generally described herein are preparation methods for nucleic acid libraries. In some embodiments, the nucleic acid library is a DNA library. In some embodiments, the nucleic acid library is a cDNA library. In some embodiments, the nucleic acid library is a double-stranded nucleic acid library. In some embodiments, the nucleic acid library is a single-stranded nucleic acid library. The nucleic acid preparation methods described herein describe various steps, including ligation. In some embodiments ligation includes using a ligase (e.g. any of the ligases described herein). In some embodiments, the ligase is a DNA ligase. In some embodiments, the ligase is T4 ligase. In some embodiments, the ligase is CircLigase. In some embodiments of the nucleic acid preparation methods described herein, a member of a nucleic acid library is circularized. In some embodiments, a member of a nucleic acid library is circularized two times. In some embodiments, a double-stranded member of a nucleic acid library is circularized. In some embodiments, a single-stranded member of a nucleic acid library is circularized. Any suitable method to circularize a member of a nucleic acid library can be used, including the examples described herein. In some embodiments, a member of a nucleic acid library is circularized to bring 5′ sequences of interest closer to domains positioned at the 3′ end of the member of a nucleic acid library. In some embodiments, the 5′ sequences of interest are brought closer to domains (e.g., circularized), such as a unique molecular identifier and a barcode sequence (e.g., a cell barcode, a spatial barcode). In some embodiments, the 5′ sequences of interest are brought closer to domains positioned at the 3′ end by the methods described in Naml, A. S., Somatic mutation and cell identify linked by Genotyping of Transcriptomes,Nature,571, 355-360 (2019), which is incorporated herein by reference in its entirety. In some examples, a single-stranded member of a nucleic acid library is circularized after contacting the member with an enzyme to phosphorylate a 5′ end of a single-stranded member of the nucleic acid library (e.g., polynucleotide kinase). In some embodiments, the phosphorylated single-stranded member of a nucleic acid library can be circularized with CircLigase. In some embodiments, the single-stranded member can be circularized by a templated ligation reaction (e.g., splint ligation). In some embodiments, a splint oligonucleotide can facilitate the ligation reaction where the splint oligonucleotide is complementary to both ends of a linear single stranded member of a nucleic acid library such that hybridization of the splint oligonucleotide to both ends brings the two ends in proximity for a ligation reaction to occur. In some examples, a single-stranded member of a nucleic acid library is amplified with a phosphorylated primer (e.g., a phosphorylated pR1 primer). In some embodiments, the amplicons are denatured to generate single-stranded members of the nucleic acid library. In some embodiments, a splint oligonucleotide can facilitate the ligation reaction as previously described. In some examples, a double-stranded member of a nucleic acid library can be circularized by a Gibson assembly strategy (Gibson, D. G., Enzymatic assembly of DNA molecules up to several hundred kilobases,Nature Methods,6(5): 343-345, doi:10.1038/nmeth.1318 (2009), which is incorporated herein by reference in its entirety). In some embodiments, homologous sequences are designed on either end (e.g., a 3′ end, a 5′ end) of the amplified molecule (e.g., a cDNA molecule). In some embodiments, Gibson assembly of the double stranded product generates a circularized double-stranded member of a nucleic acid library. In some examples, restriction enzyme (e.g., restriction endonucleases) recognition sites can be added to the ends of a member of a nucleic acid, digested with a restriction enzyme, and intramolecularly ligated to generate a circularized nucleic acid product. Any suitable restriction enzyme can be used. In some embodiments, a rare restriction enzyme can be used. As used herein, a “rare restriction enzyme” is a restriction enzyme with a recognition sequence that occurs only rarely in a genome. For example, rare restriction enzymes with a 7-nucleotide recognition site cut once every 47bp (16,384 bp), and those with 8-nucleotide recognition sites cut every 48bp (65,536 bp), respectively. Use of a rare restriction enzyme recognition site in a nucleic acid for subsequence cleavage and circularization could be useful, for example, to help minimize unwanted cleavage within the target nucleic acid which could occur with a restriction enzyme recognition site that is more prevalent within a genome. In some embodiments, a member of a nucleic acid library is circularized by Cre-Lox recombination. In some embodiments, a member (e.g., single-stranded) of a nucleic acid library is circularized by CircLigase™ ligation enzyme. As used herein, a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a capture probe). For example, a barcode can be associated with a location in a biological sample (e.g., a spatial barcode) or a barcode can be associated with one or more cells, or a single-cell (e.g., a cell barcode). In some embodiments of the nucleic acid library preparation methods described herein, the barcode is a spatial barcode. In some embodiments of the nucleic acid library preparation methods described herein, the barcode is a cell barcode. Provided herein are methods for removing all or a portion of a sequence encoding a constant region of an analyte from a double-stranded member of a nucleic acid library, where the double-stranded member of the nucleic acid library includes a first adaptor, a barcode, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor, where the method includes (a) adding to each end of the double-stranded member of the nucleic acid library a first restriction endonuclease recognition sequence, (b) contacting the double-stranded member of the nucleic acid library of step (a) with a first restriction endonuclease that cleaves the first restriction endonuclease recognition sequence at each end of the double-stranded member of the nucleic acid library, (c) ligating the cleaved ends of the double-stranded member of the nucleic acid library of step (b) to generate a first double-stranded circularized nucleic acid, (d) amplifying the first double-stranded circularized nucleic acid using a first and a second primer to generate a first double-stranded nucleic acid product, where the first primer includes (i) a sequence substantially complementary from a 3′ region of the sequence encoding the constant region of the analyte and (ii) a second restriction endonuclease recognition sequence and the second primer includes (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the constant region of the analyte, and (ii) the second restriction endonuclease recognition sequence, (e) contacting the first double-stranded nucleic acid product with a second restriction endonuclease that cleaves the second restriction endonuclease recognition sequence at each end of the first double-stranded nucleic acid product, (f) ligating ends of the first double-stranded nucleic acid product of step (e) to generate a second double-stranded circularized nucleic acid; and (g) amplifying the second double-stranded circularized nucleic acid using a third primer including a sequence that is substantially complementary to the first adapter and a fourth primer including a sequence that is substantially complementary to the second adapter, to generate a version of the double-stranded member of the nucleic acid library lacking all or a portion of the sequence encoding the constant region of the analyte. Also provided herein are methods for removing all or a portion of a sequence encoding a constant region of an analyte from a double-stranded member of a nucleic acid library, where the double-stranded member of the nucleic acid library includes a first adaptor, a barcode, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor, wherein the method includes (a) adding to each end of the double-stranded member of the nucleic acid library a first restriction endonuclease recognition sequence, (b) contacting the double-stranded member of the nucleic acid library of step (a) with a first restriction endonuclease that cleaves the first restriction endonuclease recognition sequence at each end, (c) ligating ends of the double-stranded member of the nucleic acid library of step (b) to generate a first-double-stranded nucleic circularized nucleic acid, and (d) amplifying the double-stranded circularized nucleic acid using a first primer and a second primer to generate a version of the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region of the analyte, wherein: the first primer includes (i) a sequence substantially complementary to a sequence from a 3′ region of the sequence encoding the constant region of the analyte, and (ii) a sequence including a first functional domain; and the second primer includes (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the constant region of the analyte, and (ii) a sequence comprising a second functional domain. In some embodiments of removing all or a portion of a sequence encoding a constant region of an analyte from a double-stranded member of a nucleic acid library, the double-stranded member of the nucleic acid library includes the first adaptor, the barcode, the capture domain, the sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and the second adaptor, in a 5′ to a 3′ direction. In some embodiments, the double-stranded member of the nucleic acid library includes a UMI disposed between the barcode and the capture domain. In some embodiments, the first primer includes (i) the sequence from the 3′ region of the sequence encoding the constant region of the analyte and (ii) the second restriction endonuclease recognition sequence, in a 3′ to a 5′ direction. In some embodiments, the second primer includes (i) the sequence substantially complementary to the sequence from the 5′ region of the sequence encoding the constant region of the analyte, and (ii) the second restriction endonuclease recognition sequence, in a 3′ to a 5′ direction. In some embodiments, ligating in step (c) and/or step (f) is performed using a ligase. In some embodiments, ligating in step (c) and/or step (f) is performed using template-mediated ligation (e.g., a splint oligonucleotide). In some embodiments, the double-stranded member of a nucleic acid library includes a sequence that is complementary to all or a portion of a sequence encoding a variable region of the analyte. In some embodiments, the sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte is positioned 5′ relative to the sequence that is complementary to all or a portion of the sequence encoding the variable region of the analyte. In some embodiments, the sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte is positioned 3′ relative to the sequence that is complementary to all or a portion of the sequence encoding the variable region of the analyte. In some embodiments, the method includes amplifying the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region of the analyte using a third primer and fourth primer, where the third primer is substantially complementary to the first functional domain, and the fourth primer is substantially complementary to the second functional domain. In some embodiments, determining all, or a portion of, the sequence encoding the variable region of the analyte or complement thereof, and all or a portion of the sequence of the barcode or complement thereof. In some embodiments, determining the sequence comprises sequencing (i) all or a portion of the sequence encoding the variable region of the analyte or a complement thereof, and (ii) all or a portion of the sequence of the barcode or a complement thereof. In some embodiments, the first primer includes a sequence substantially complementary to the reverse complement of the first adaptor, and a sequence including the first functional domain, in 3′ to 5′ direction. In some embodiments, the second primer includes a sequence substantially complementary to a sequence of the 5′ region of the sequence encoding the constant region of the analyte, and a sequence including the second functional domain, in a 3′ to 5′ direction. Also provided herein are methods for removing all or a portion of the sequence encoding a constant region of an analyte from a double-stranded member of a nucleic acid library, wherein the double-stranded member of the nucleic acid library includes a ligation sequence, a barcode, a reverse complement of a first adaptor, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor, wherein the method includes ligating ends of the double-stranded member using the ligation sequence as a splint (e.g., splint oligonucleotide) to and splint ligation, to generate a circularized double-stranded nucleic acid, amplifying the circularized double-stranded nucleic acid using a first primer and a second primer to generate a version of the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region, wherein: the first primer includes (i) a sequence substantially complementary to the reverse complement of the first adaptor and (ii) a first functional domain and the second primer includes (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the constant region of the analyte, and (ii) a second functional domain. In some embodiments, the double-stranded member of the nucleic acid library includes the ligation sequence, the barcode, the reverse complement of the first adaptor, the capture domain, the sequence complementary to all or a portion of the sequence encoding the constant region of the analyte, and the second adaptor, in a 5′ to 3′ direction. In some embodiments, the double-stranded member of the nucleic acid library includes a unique molecular identifier (UMI). In some embodiments, the UMI is disposed between the barcode and the reverse complement of the first adaptor. In some embodiments, the first primer includes a sequence substantially complementary to the reverse complement of the first adaptor, and a sequence including the first functional domain, in 3′ to 5′ direction. In some embodiments, the second primer includes a sequence substantially complementary to a sequence of the 5′ region of the sequence encoding the constant region of the analyte, and (ii) the sequence comprising the second functional domain, in a 3′ to 5′ direction. In some embodiments, a third primer is substantially complementary to the first functional domain. In some embodiments, a fourth primer is substantially complementary to the second functional domain. Also provided herein, are methods for removing all or a portion of a sequence encoding an analyte from a double-stranded member of a nucleic acid library, where the double-stranded member of the nucleic acid library includes a first adaptor, a barcode, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the analyte, and a second adaptor, where the method includes (a) adding to each end of the double-stranded member of the nucleic acid library a first restriction endonuclease recognition sequence, (b) contacting the double-stranded member of the nucleic acid library of step (a) with a first restriction endonuclease that cleaves the first restriction endonuclease recognition sequence at each end of the double-stranded member of the nucleic acid library, (c) ligating ends of the double-stranded member of the nucleic acid library of step (b) to generate a first double-stranded circularized nucleic acid, (d) amplifying the first double-stranded circularized nucleic acid using a first and a second primer to generate a first double-stranded nucleic acid product, where the first primer includes (i) a sequence substantially complementary to a 3′ region of the sequence encoding the analyte and (ii) a second restriction endonuclease recognition sequence and the second primer includes (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the analyte, and (ii) the second restriction endonuclease recognition sequence, (e) contacting the first double-stranded nucleic acid product with a second restriction endonuclease that cleaves the second restriction endonuclease recognition sequence at each end of the first double-stranded nucleic acid product, (f) ligating ends of the first double-stranded nucleic acid product of step (e) to generate a second double-stranded circularized nucleic acid, and (g) amplifying the second double-stranded circularized nucleic acid using a third primer including a sequence that is substantially complementary to the first adapter and a fourth primer including a sequence that is substantially complementary to the second adapter, to generate a version of the double-stranded member of the nucleic acid library lacking all or a portion the sequence encoding the analyte. Also provided herein are methods for removing all or a portion of a sequence encoding an analyte from a double-stranded member of a nucleic acid library, where the double-stranded member of the nucleic acid library includes a first adaptor, a barcode, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the analyte, and a second adaptor, where the method includes (a) adding to each end of the double-stranded member of the nucleic acid library a first restriction endonuclease recognition sequence, (b) contacting the double-stranded member of the nucleic acid library of step (a) with a first restriction endonuclease that cleaves the first restriction endonuclease recognition sequence at each end, (c) ligating ends of the double-stranded member of the nucleic acid library of step (b) to generate a first-double-stranded nucleic circularized nucleic acid, and (d) amplifying the double-stranded circularized nucleic acid using a first primer and a second primer to generate a version of the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the analyte, where the first primer includes (i) a sequence substantially complementary to a sequence from a 3′ region of the sequence encoding the analyte, and (ii) a sequence including a first functional domain and the second primer includes (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the analyte, and (ii) a sequence including a second functional domain. Also provided herein are methods for removing all or a portion of the sequence encoding an analyte from a double-stranded member of a nucleic acid library, where the double-stranded member of the nucleic acid library includes a ligation sequence, a barcode, a reverse complement of a first adaptor, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the analyte, and a second adaptor, where the method includes (a) ligating ends of the double-stranded member using the ligation sequence to splint ligation, to generate a circularized double-stranded nucleic acid, (b) amplifying the circularized double-stranded nucleic acid using a first primer and a second primer to generate a version of the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the analyte, where the first primer includes (i) a sequence substantially complementary to the reverse complement of the first adaptor and (ii) a first functional domain and the second primer includes (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the analyte, and (ii) a second functional domain. Also provided herein are methods of reversing the orientation of an analyte sequence of a double-stranded member of a nucleic acid library, wherein the double-stranded member of the nucleic acid library includes a ligation sequence, a barcode, a reverse complement of the first adaptor, an amplification domain, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the analyte, and a second adaptor, wherein the method includes (a) ligating ends of the double-stranded member of the nucleic acid library using the ligation sequence for splint ligation, to generate a circularized double-stranded nucleic acid and (b) amplifying the circularized double-stranded nucleic acid using a first primer and a second primer to generate a double-stranded nucleic acid product, where the first primer includes (i) a sequence substantially complementary to the reverse complement of the first adaptor and (ii) a functional domain; and the second primer includes a sequence substantially complementary to the amplification domain, thereby reversing the orientation of the analyte sequence of the double-stranded member of the nucleic acid library. Some embodiments included herein describe removal of all or a portion of a constant region of an analyte, however, it will be appreciated by one of ordinary skill in the art that any portion of an analyte sequence can be removed by the methods described herein, such as for example, with a pair of primers designed to a 3′ and a 5′ portion of an analyte sequence (e.g., a captured analyte sequence, a complement of an analyte sequence, etc.). In some embodiments, the double-stranded member of the nucleic acid library includes the ligation sequence, the barcode (e.g., a spatial barcode, a cell barcode), the reverse complement of the first adaptor, the amplification domain, the capture domain, the sequence complementary to all or a portion of the sequence encoding an analyte, and the second adaptor, in a 5′ to 3′ direction. In some embodiments, the double-stranded member of the nucleic acid library includes a unique molecular identifier (UMI). In some embodiments, the UMI is disposed between the barcode and the reverse complement of the first adaptor. In some embodiments, the first primer includes a sequence substantially complementary to the reverse complement of the first adaptor (e.g., Read 1), and a sequence comprising the first functional domain, in a 5′ to 3′ direction. In some embodiments, the double-stranded member of the nucleic acid library includes a sequence that is complementary to all, or a portion of, a sequence encoding a 5′ untranslated region of an analyte. In some embodiments, the double-stranded member of the nucleic acid library includes a complementary sequence to all, or a portion of, a sequence encoding a 3′ untranslated region of an analyte. In some embodiments, a complementary sequence to all, or a portion of, the sequence encoding a 5′ untranslated region of the analyte is positioned 5′ relative to the sequence that is complementary to all, or a portion of, the sequence encoding the 3′ untranslated region of the analyte. In some embodiments, the double-stranded member of the nucleic acid library includes one or more exons of the analyte. In some embodiments, the analyte includes a complementary sequence to all, or a portion of, the sequence encoding the 5′ untranslated region of the analyte, the one or more exons, and the sequence that is complementary to all or a portion of the sequence encoding the 3′ untranslated region, in a 5′ to 3′ direction. In some embodiments of the nucleic acid preparation methods described herein, the double-stranded member of the nucleic acid library includes a complementary sequence to all, or a portion of, a sequence encoding a variable region of an analyte. In some embodiments, the sequence encoding the constant region of the analyte is positioned 5′ relative to the sequence that is complementary to all or a portion of the sequence encoding the variable regions of the analyte. In some embodiments, the complementary sequence to all, or a portion of, the sequence encoding the constant region of the analyte is positioned 3′ relative to the sequence that is complementary to all or a portion of the sequence encoding the variable region of the analyte. In some embodiments, circularization of one or more analytes can be performed on single cells, including a general single cell capture configuration and workflow as generally depicted inFIG.6. An exemplary method for capturing analytes from single cells and performing subsequent library preparation including circularization methods as described herein include a support630(e.g., a bead, such as a gel bead) comprising a nucleic acid barcode molecule690that are co-partitioned into a partition amongst a plurality of partitions (e.g., a droplet of a droplet emulsion or a well of a micro/nanowell array). In some embodiments, the partition comprises at most a single cell and a single support630. In some embodiments, nucleic acid barcode molecule690is attached to support630via a releasable linkage640(e.g., comprising a labile bond). Upon release of nucleic acid barcode molecule690from the support630, barcoded molecules may be generated within the partition. In some embodiments, nucleic acid barcode molecule690comprises sequence623complementary to a sequence of an RNA molecule670from a cell. In some instances, sequence623comprises a sequence specific for an RNA molecule. In some instances, sequence623comprises a poly-T sequence. In some instances, sequence623includes a sequence specific for an RNA molecule. In some instances, sequence623includes a sequence complementary to a region of an immune molecule, such as the constant region of a TCR or BCR sequence (as described herein). Sequence623is hybridized to RNA molecule670and a cDNA molecule680is generated in a reverse transcription reaction generating a barcoded nucleic acid molecule including cell (e.g., partition specific) barcode sequence622(or a reverse complement thereof) and a sequence of cDNA (or a portion thereof). Barcoded nucleic acid molecules can then be optionally processed as described elsewhere herein, e.g., to amplify the molecules and/or append sequencing platform specific sequences to the fragments. See, e.g., U.S. Pat. Pub. Nos. 20180105808 and 20190367969 and U.S. Pat. Nos. 10,273,541, 10,480,029, and 10,550,429, each of which is hereby incorporated by reference in its entirety. Barcoded nucleic acid molecules, or derivatives generated therefrom, can then be sequenced on a suitable sequencing platform. The methods described herein for circularization of a nucleic acid library is equally applicable for the libraries generated from a single cell workflow as previously described. Analyte Sequences The analyte sequences present in the nucleic acid library (e.g., nucleic acid library generated from single-cells or from a biological sample on an array) can be captured from a biological sample (e.g., any of the biological samples described herein). In some embodiments, the biological sample is a tissue sample. In some embodiments, the tissue sample is a tissue section. In some embodiments, the tissue section is a fixed tissue section. In some embodiments, the fixed tissue section is formalin-fixed paraffin-embedded tissue section. In some embodiments, the tissue section is a fresh, frozen tissue section. Analyte sequences present in the nucleic acid library (e.g., a nucleic acid library generated from single-cells or from a biological sample on an array) can be obtained from RNA capture (e.g., any of the RNAs described herein). In some embodiments, the RNA is mRNA. In some embodiments, the analyte sequence present in the nucleic acid library are obtained from DNA. In some embodiments, the DNA is genomic DNA. The captured analyte sequences in the nucleic acid library (e.g., nucleic acid library prepared from single-cells or an array) can be any analyte (e.g., mRNA) captured. For example, an analyte of interest can include a sequence of more than about 1 kb away from its 3′ end and can be prepared by any of the methods described herein with analyte specific primers. In some embodiments, analyte sequences in the nucleic acid library include a constant region, such as a constant region present in an analyte encoding immune cell receptors. In some embodiments, analytes encoding immune cell receptors identify clonotypes or receptors from a biological sample, for example V(D)J sequences including CDR sequences (e.g., CDR 1, CDR 2, CDR 3). In some embodiments, the analyte sequence of interest is for an immune cell receptor. In some embodiments, the immune cell receptor is a B cell receptor. In some embodiments, the B cell receptor is an immunoglobulin kappa light chain. In some embodiments, the variable region of the analyte includes a CDR3 of the immunoglobulin kappa light chain. In some embodiments, the variable region of the analyte includes one or both of CDR1 and CDR2 of the immunoglobulin kappa light chain. In some embodiments, the variable region of the analyte includes a full-length variable domain of the immunoglobulin kappa light chain. In some embodiments, the B cell receptor is an immunoglobulin lambda light chain. In some embodiments, the variable region of the analyte includes a CDR3 of the immunoglobulin lambda light chain. In some embodiments, the variable region of the analyte includes one or both of CDR1 and CDR2 of the immunoglobulin lambda light chain. In some embodiments, the variable region of the analyte includes a full-length variable domain of the immunoglobulin lambda light chain. In some embodiments, the B cell receptor is an immunoglobulin heavy chain. In some embodiments, the variable region of the analyte includes a CDR3 of the immunoglobulin heavy chain. In some embodiments, the variable region of the analyte includes one or both of CDR1 and CDR2 of the immunoglobulin heavy chain. In some embodiments, the variable region of the analyte includes a full-length variable domain of the immunoglobulin heavy chain. In some embodiments, the immune cell receptor is a T cell receptor. In some embodiments, the T cell receptor is a T cell receptor alpha chain. In some embodiments, the variable region of the analyte includes a CDR3 of the T cell receptor alpha chain. In some embodiments, the variable region of the analyte includes one or both of CDR1 and CDR2 of the T cell receptor alpha chain. In some embodiments, the variable region of the analyte includes a full-length variable domain of the T cell receptor alpha chain. In some embodiments, the T cell receptor is a T cell receptor beta chain. In some embodiments, the variable region of the analyte includes a CDR3 of the T cell receptor beta chain. In some embodiments, the variable region of the analyte includes one or both of CDR1 and CDR2 of the T cell receptor beta chain. In some embodiments, the variable region of the analyte further includes a full-length variable domain of the T cell receptor beta chain. In some embodiments of the nucleic acid library preparation methods described herein, the methods include determining all or a portion of a sequence encoding the variable region of the analyte or a complement thereof, and all or a portion of the barcode or a complement thereof. In some embodiments, determining a sequence includes sequencing (e.g., any of the sequencing methods described herein) all, or a portion of, the sequence encoding the variable region of the analyte or a complement thereof, and all or a portion of the barcode or a complement thereof. In some embodiments, sequencing is performed using high-throughput sequencing. In some embodiments, sequencing is performed by sequencing-by-synthesis, sequencing-by-ligation, or sequencing-by-hybridization. In some embodiments, the analyte is released from a biological sample. In some embodiments, a location of the analyte in the biological sample is determined using the sequences of a barcode. In some embodiments, the barcode is a spatial barcode. In some embodiments, an analyte is associated with a cell of a biological sample. In some embodiments, the analyte is associated with a cell of a biological sample by the sequence of a cell barcode. In some embodiments of any of the spatial methods described herein, the method includes generating the double-stranded member of the nucleic acid library. In some embodiments, generating the double-stranded member of the nucleic acid library includes contacting the analyte with a capture probe comprising the first adaptor, the barcode (e.g., a spatial barcode, a cell barcode), and the capture domain, where the capture domain binds specifically to a sequence present in the analyte, extending an end of the capture probe using the analyte specifically bound to the capture domain as a template, thereby generating an extended capture probe, and adding the second adaptor to an end of the extended capture probe, thereby generating the double-stranded member of the nucleic acid library. In some embodiments, the capture probe includes the first adapter (e.g., Read 1), the barcode (e.g., a spatial barcode, a cell barcode), and the capture domain in a 5′ to a 3′ direction. In some embodiments, the capture probe is extended by a reverse transcriptase (e.g., any of the reverse transcriptases described herein). In some embodiments, a 3′ end of the capture probe is extended to generate an extended capture probe. In some embodiments, the second adapter (e.g., a template switching oligonucleotide (TSO) sequence) is added to a 5′ end of the extended capture probe. Compositions Provided herein are compositions including a double-stranded member of a nucleic acid library that includes a first adaptor, a barcode, a capture domain, a complementary analyte sequence including a sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor. In some embodiments, a unique molecular identifier is disposed between the barcode and the capture domain. In some embodiments, the barcode is a spatial barcode. In some compositions, the barcode is a cell barcode. In some compositions, the composition includes a first adaptor, a barcode, a UMI, a capture domain, a sequence complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor in a 5′ to 3′ direction. In some compositions, the composition includes a double-stranded member of a nucleic acid library including a first restriction endonuclease recognition sequences added to each end of the double-stranded member of a nucleic acid library. In some compositions, the first restriction endonuclease recognition sequence is digested by a first restriction endonuclease thereby generating sticky ends on the double-stranded member of a nucleic acid library. In some compositions, the sticky ends of the double-stranded member of a nucleic acid library are ligated to each other intramolecularly to generate a first double-stranded circularized nucleic acid. In some compositions, the first double-stranded circularized nucleic acid is amplified with a first primer and second primer to generate a first double-stranded nucleic acid product (e.g., linearized), where a second restriction endonuclease recognition site is added to both ends of the first double-stranded nucleic acid product. In some compositions, the second restriction endonuclease recognition sequence (e.g., site) is digested by a second restriction endonuclease, thereby generating sticky ends on the first double-stranded nucleic acid product. In some compositions, the sticky ends of the first double-stranded nucleic acid product are ligated intramolecularly to generate a second double-stranded circularized nucleic acid. In some compositions, the second double-stranded circularized nucleic acid is amplified with a third primer and fourth primer to generate a version of the double-stranded member (e.g., linearized) of the nucleic acid library lacking all or a portion of the sequence encoding the constant region of the analyte. In some compositions, after the step of generating the first double-stranded circularized nucleic acid, the first double-stranded circularized nucleic acid is amplified with a first primer and a second primer to generate a version of the nucleic acid product lacking all or a portion of the constant region of the analyte. In some compositions, the version of the nucleic acid product lacking all or a portion of the constant region of the analyte includes, in a 5′ to 3′ direction, a first functional domain, a portion of the constant region, a capture domain, a UMI, a barcode, a first adaptor, a second adaptor, the analyte sequence, and a second functional domain. In some compositions, the composition does not include any portion of the constant sequence. Also provided herein are compositions including a double-stranded member of a nucleic acid library that includes a ligation sequence, barcode, a reverse complement of a first adaptor, a capture domain, a complementary analyte sequence including a sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor. In some compositions, a unique molecular identifier is disposed between the barcode and the reverse complement of a first adaptor. In some embodiments, the barcode is a spatial barcode. In some compositions, the barcode is a cell barcode. In some compositions, the composition includes a ligation sequence, a barcode, a UMI, a reverse complement to the first adaptor, a capture domain, a sequence complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor in a 5′ to 3′ direction. In some compositions, the ends of the double-stranded member of the nucleic acid library are ligated intramolecularly to generate a circularized double-stranded nucleic acid product where the ligation sequence splints the ligation. In some compositions, the circularized double-stranded nucleic acid is amplified with a first primer and second primer to generate a version of the double-stranded member (e.g., linearized) of a nucleic acid library lacking all or a portion of the sequence encoding the constant region. In some compositions, the version of the double-stranded member of the nucleic acid library includes, in a 5′ to 3′ direction, a first functional domain (e.g., P5), a first adaptor, a unique molecular identifier, a barcode, a ligation sequence, a second adaptor, and a complementary analyte sequence. Also provided herein are compositions including a double-stranded member of a nucleic acid library that includes a ligation sequence, barcode, a reverse complement of a first adaptor, an amplification domain, a capture domain, a complementary analyte sequence including a sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor. In some compositions, a unique molecular identifier is disposed between the barcode and the reverse complement of a first adaptor. In some embodiments, the barcode is a spatial barcode. In some compositions, the barcode is a cell barcode. In some compositions, the composition includes a ligation sequence, a barcode, a UMI, a reverse complement to the first adaptor, a capture domain, a sequence complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor in a 5′ to 3′ direction. In some compositions, the ends of the double-stranded member of the nucleic acid library are ligated intramolecularly to generate a circularized double-stranded nucleic acid product where the ligation sequence splints the ligation. In some compositions, the circularized double-stranded nucleic acid is amplified with a first primer and second primer to generate a version of the double-stranded member (e.g., linearized) of a nucleic acid library lacking all or a portion of the sequence encoding the constant region. In some compositions the version of the double-stranded member of the nucleic acid library includes, in a 5′ to 3′ direction, a first functional domain (e.g., P5), a first adaptor, a unique molecular identifier, a barcode, a second adaptor, an analyte sequence where the orientation of the analyte sequence is reversed (e.g., the 5′ end of the sequence is located 5′ to the second adaptor), a capture domain, and an amplification domain. Kits Also provided herein are kits including (i) a first restriction endonuclease that cleaves a first restriction endonuclease recognition sequence; (ii) a second restriction endonuclease that cleaves a second restriction endonuclease recognition sequence; (iii) a ligase; and (iv) a first and a second primer, where: the first primer includes (i) a sequence from a 3′ region of a sequence encoding a constant region of an analyte and (ii) the second restriction endonuclease recognition sequence and the second primer includes (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the constant region of the analyte, and (ii) the second restriction endonuclease recognition sequence. In some kits, the kit includes a third primer including a sequence that is substantially complementary to a first adapter and a fourth primer including a sequence that is substantially complementary to a second adapter. In some kits, the first primer includes (i) the sequence from the 3′ region of the sequence encoding the constant region of the analyte and (ii) the second restriction endonuclease recognition sequence, in a 3′ to a 5′ direction. In some kits, the second primer includes (i) the sequence substantially complementary to the sequence from the 5′ region of the sequence encoding the constant region of the analyte, and (ii) the second restriction endonuclease recognition sequence, in a 3′ to a 5′ direction. In some kits, the ligase is a DNA ligase. In some kits, the DNA ligase is T4 ligase. Also provided herein are kits including (i) a first restriction endonuclease that cleaves a first restriction endonuclease recognition sequence, (ii) a ligase, and (iii) a first and a second primer, where the first primer includes: (i) a sequence from a 3′ region of a sequence encoding a constant region of an analyte, and (ii) a sequence including a first functional domain, and the second primer includes (i) a sequence substantially complementary to a sequence from a 5′ region of a sequence encoding the constant region of the analyte, and (ii) a sequence including a second functional domain. In some kits, the kit includes a third primer including a sequence substantially complementary to the first functional domain and a fourth primer including a sequence substantially complementary to the second functional domain. In some kits, the first primer includes (i) the sequence from the 3′ region of sequence encoding a constant region of the analyte, and (ii) the sequence including the first functional domain, in a 3′ to 5′ direction. In some kits, the second primer includes (i) the sequence substantially complementary to the sequence from the 5′ region of the sequence encoding the constant region of the analyte, and (ii) the sequence including the second functional domain, in a 3′ to direction. In some kits, the ligase is a DNA ligase. In some kits, the DNA ligase is T4 ligase. Also provided herein are kits including (i) a first restriction endonuclease that cleaves a first restriction endonuclease recognition sequence, (ii) a ligase, and (iii) a first and a second primer, where the first primer includes (i) a sequence substantially complementary to a reverse complement of a first adaptor, and (ii) a sequence including a first functional domain; and the second primer includes (i) a sequence substantially complementary to a sequence from a 5′ region of a sequence encoding the constant region of the analyte, and (ii) a sequence including a second functional domain. In some kits, the kit includes a third primer including a sequence substantially complementary to the first functional domain and a fourth primer including a sequence substantially complementary to the second functional domain. In some kits, the first primer includes (i) the sequence substantially complementary to the reverse complement of the first adaptor, and (ii) the sequence including the first functional domain, in a 3′ to 5′ direction. In some kits, the second primer includes (i) the sequence substantially complementary to the sequence from the 5′ region of the sequence encoding the constant region of the analyte, and (ii) the sequence including the second functional domain, in a 3′ to 5′ direction. In some kits, the ligase is a DNA ligase. In some kits, the DNA ligase is T4 ligase. Also provided herein are kits including (i) a first restriction endonuclease that cleaves a first restriction endonuclease recognition sequence, (ii) a ligase, and (iii) a first and a second primer, where the first primer includes (i) a sequence substantially complementary to a reverse complement of a first adaptor, and (ii) a functional domain; and the second primer includes a sequence substantially complementary to the amplification domain. In some kits, the kit includes a third primer including a sequence substantially complementary to the functional domain, and a fourth primer including a sequence substantially complementary to a reverse complement of the amplification domain. In some kits, the first primer includes (i) the sequence substantially complementary to the reverse complement of the first adaptor, and (ii) the sequence including the functional domain, in a 3′ to 5′ direction. In some kits, the ligase is a DNA ligase. In some kits, the DNA ligase is T4 ligase. EMBODIMENTS Embodiment 1 is a method for removing all or a portion of a sequence encoding a constant region of an analyte from a double-stranded member of a nucleic acid library, wherein the double-stranded member of the nucleic acid library comprises: a first adaptor, a barcode, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor, wherein the method comprises: (a) adding to each end of the double-stranded member of the nucleic acid library a first restriction endonuclease recognition sequence; (b) contacting the double-stranded member of the nucleic acid library of step (a) with a first restriction endonuclease that cleaves the first restriction endonuclease recognition sequence at each end of the double-stranded member of the nucleic acid library; (c) ligating ends of the double-stranded member of the nucleic acid library of step (b) to generate a first double-stranded circularized nucleic acid; (d) amplifying the first double-stranded circularized nucleic acid using a first and a second primer to generate a first double-stranded nucleic acid product, wherein: the first primer comprises: (i) a sequence substantially complementary to a 3′ region of the sequence encoding the constant region of the analyte and (ii) a second restriction endonuclease recognition sequence; and the second primer comprises: (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the constant region of the analyte, and (ii) the second restriction endonuclease recognition sequence; (e) contacting the first double-stranded nucleic acid product with a second restriction endonuclease that cleaves the second restriction endonuclease recognition sequence at each end of the first double-stranded nucleic acid product; (f) ligating ends of the first double-stranded nucleic acid product of step (e) to generate a second double-stranded circularized nucleic acid; and (g) amplifying the second double-stranded circularized nucleic acid using a third primer comprising a sequence that is substantially complementary to the first adapter and a fourth primer comprising a sequence that is substantially complementary to the second adapter, to generate a version of the double-stranded member of the nucleic acid library lacking all or a portion of the sequence encoding the constant region of the analyte. Embodiment 2 is the method of embodiment 1, wherein the double-stranded member of the nucleic acid library comprises the first adaptor, the barcode, the capture domain, the sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and the second adaptor, in a 5′ to 3′ direction. Embodiment 3 is the method of embodiment 2, wherein the double-stranded member of the nucleic acid library further comprises a UMI disposed between the barcode and the capture domain. Embodiment 4 is the method of any one of embodiments 1-3, wherein the first primer comprises (i) the sequence from the 3′ region of the sequence encoding the constant region of the analyte and (ii) the second restriction endonuclease recognition sequence, in a 3′ to a 5′ direction. Embodiment 5 is the method of any one of embodiments 1-4, wherein the second primer comprises (i) the sequence substantially complementary to the sequence from the 5′ region of the sequence encoding the constant region of the analyte, and (ii) the second restriction endonuclease recognition sequence, in a 3′ to a 5′ direction. Embodiment 6 is the method of any one of embodiments 1-5, wherein the ligating in step (c) and/or step (f) is performed using a ligase or using template mediated ligation. Embodiment 7 is the method of embodiment 6, wherein the ligase is a DNA ligase. Embodiment 8 is the method of embodiment 7, wherein the DNA ligase is a T4 ligase. Embodiment 9 is the method of any one of embodiments 1-8, wherein the barcode is a cell barcode or a spatial barcode. Embodiment 10 is the method of any one of embodiments 1-9, wherein the nucleic acid library is a DNA library. Embodiment 11 is the method of any one of embodiments 1-10, wherein the nucleic acid library is a cDNA library. Embodiment 12 is the method of any one of embodiments 1-11, wherein the double-stranded member of a nucleic acid library further comprises a sequence that is complementary to all or a portion of a sequence encoding a variable region of the analyte. Embodiment 13 is the method of embodiment 12, wherein the sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte is positioned 5′ relative to the sequence that is complementary to all or a portion of the sequence encoding the variable region of the analyte. Embodiment 14 is the method of embodiment 12, wherein the sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte is positioned 3′ relative to the sequence that is complementary to all or a portion of the sequence encoding the variable region of the analyte. Embodiment 15 is the method of any one of embodiments 12-14, wherein the analyte is an immune cell receptor. Embodiment 16 is the method of embodiment 15, wherein the immune cell receptor is a B cell receptor. Embodiment 17 is the method of embodiment 16, wherein the B cell receptor is an immunoglobulin kappa light chain. Embodiment 18 is the method of embodiment 17, wherein the variable region of the analyte comprises a CDR3 of the immunoglobulin kappa light chain. Embodiment 19 is the method of embodiment 18, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the immunoglobulin kappa light chain. Embodiment 20 is the method of embodiment 18, wherein the variable region of the analyte further comprises a full-length variable domain of the immunoglobulin kappa light chain. Embodiment 21. The method of embodiment 16, wherein the B cell receptor is an immunoglobulin lambda light chain. Embodiment 22 is the method of embodiment 21, wherein the variable region of the analyte comprises a CDR3 of the immunoglobulin lambda light chain. Embodiment 23 is the method of embodiment 22, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the immunoglobulin lambda light chain. Embodiment 24 is the method of embodiment 22, wherein the variable region of the analyte further comprises a full-length variable domain of the immunoglobulin lambda light chain. Embodiment 25 is the method of embodiment 16, wherein the B cell receptor is an immunoglobulin heavy chain. Embodiment 26 is the method of embodiment 25, wherein the variable region of the analyte comprises a CDR3 of the immunoglobulin heavy chain. Embodiment 27 is the method of embodiment 26, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the immunoglobulin heavy chain. Embodiment 28 is the method of embodiment 26, wherein the variable region of the analyte further comprises a full-length variable domain of the immunoglobulin heavy chain. Embodiment 29 is the method of embodiment 15, wherein the immune cell receptor is a T cell receptor. Embodiment 30 is the method of embodiment 29, wherein the T cell receptor is a T cell receptor alpha chain. Embodiment 31 is the method of embodiment 30, wherein the variable region of the analyte comprises a CDR3 of the T cell receptor alpha chain. Embodiment 32 is the method of embodiment 31, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the T cell receptor alpha chain. Embodiment 33 is the method of embodiment 31, wherein the variable region of the analyte further comprises a full-length variable domain of the T cell receptor alpha chain. Embodiment 34 is the method of embodiment 29, wherein the T cell receptor is a T cell receptor beta chain. Embodiment 35 is the method of embodiment 34, wherein the variable region of the analyte comprises a CDR3 of the T cell receptor beta chain. Embodiment 36 is the method of embodiment 35, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the T cell receptor beta chain. Embodiment 37 is the method of embodiment 35, wherein the variable region of the analyte further comprises a full-length variable domain of the T cell receptor beta chain. Embodiment 38 is the method of any one of embodiments 12-37, wherein the method further comprises: (h) determining (i) all or a portion of a sequence encoding the variable region of the analyte or a complement thereof, and (ii) all or a portion of the barcode or a complement thereof. Embodiment 39 is the method of embodiment 38, wherein the determining in step (h) comprises sequencing (i) all or a portion of the sequence encoding the variable region of the analyte or a complement thereof, and (ii) all or a portion of the barcode or a complement thereof. Embodiment 40 is the method of embodiment 38 or 39, wherein the analyte was released from a biological sample, and the method further comprises: determining a location of the analyte in the biological sample using the determined sequences of (i) and (ii). Embodiment 41 is the method of any one of embodiments 1-40, further comprising generating the double-stranded member of the nucleic acid library. Embodiment 42 is the method of embodiment 41, wherein the step of generating the double-stranded member of the nucleic acid library comprises: contacting the analyte with a capture probe comprising the first adaptor, the barcode, and the capture domain, wherein the capture domain binds specifically to a sequence present in the analyte; extending an end of the capture probe using the analyte specifically bound to the capture domain as a template, thereby generating an extended capture probe; and adding the second adaptor an end of the extended capture probe, thereby generating the double-stranded member of the nucleic acid library. Embodiment 43 is the method of embodiment 42, wherein the capture probe comprises the first adapter, the barcode, and the capture domain in a 5′ to a 3′ direction. Embodiment 44 is the method of embodiment 42 or 43, wherein a 3′ end of the capture probe is extended. Embodiment 45 is the method of any one of embodiments 42-44, wherein the second adapter is added to a 5′ end of the extended capture probe. Embodiment 46 is the method of any one of embodiments 1-45, wherein the biological sample is a tissue sample, a tissue section or a fixed tissue section. Embodiment 47 is the method of embodiment 46, wherein the fixed tissue section is formalin-fixed paraffin-embedded tissue section or the tissue section is a fresh, frozen tissue section. Embodiment 48 is the method of any one of embodiments 1-47, wherein the analyte is an RNA. Embodiment 49 is the method of embodiment 48, wherein the RNA is an mRNA. Embodiment 50 is the method of any one of embodiments 1-47, wherein the analyte is a DNA. Embodiment 51 is the method of embodiment 50, wherein the DNA is genomic DNA. Embodiment 52 is a kit comprising: (i) a first restriction endonuclease that cleaves a first restriction endonuclease recognition sequence; (ii) a second restriction endonuclease that cleaves a second restriction endonuclease recognition sequence; (iii) a ligase; and (iv) a first and a second primer, wherein: the first primer comprises: (i) a sequence from a 3′ region of a sequence encoding a constant region of an analyte and (ii) the second restriction endonuclease recognition sequence; and the second primer comprises: (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the constant region of the analyte, and (ii) the second restriction endonuclease recognition sequence. Embodiment 53 is the kit of embodiment 53, wherein the kit further comprises: a third primer comprising a sequence that is substantially complementary to a first adapter; and a fourth primer comprising a sequence that is substantially complementary to a second adapter. Embodiment 54 is the kit of embodiment 52 or 53, wherein the first primer comprises (i) the sequence from the 3′ region of the sequence encoding the constant region of the analyte and (ii) the second restriction endonuclease recognition sequence, in a 3′ to a 5′ direction. Embodiment 55 is the kit of any one of embodiments 52-54, wherein the second primer comprises (i) the sequence substantially complementary to the sequence from the 5′ region of the sequence encoding the constant region of the analyte, and (ii) the second restriction endonuclease recognition sequence, in a 3′ to a 5′ direction. Embodiment 56 is the kit of any one of embodiments 52-55, wherein the ligase is a DNA ligase. Embodiment 57 is the kit of embodiment 56, wherein the DNA ligase is T4 ligase. Embodiment 58 is a method for removing all or a portion of a sequence encoding a constant region of an analyte from a double-stranded member of a nucleic acid library, wherein the double-stranded member of the nucleic acid library comprises: a first adaptor, a barcode, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor, wherein the method comprises:(a) adding to each end of the double-stranded member of the nucleic acid library a first restriction endonuclease recognition sequence; (b) contacting the double-stranded member of the nucleic acid library of step (a) with a first restriction endonuclease that cleaves the first restriction endonuclease recognition sequence at each end; (c) ligating ends of the double-stranded member of the nucleic acid library of step (b) to generate a first-double-stranded nucleic circularized nucleic acid; and (d) amplifying the double-stranded circularized nucleic acid using a first primer and a second primer to generate a version of the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region of the analyte, wherein: the first primer comprises: (i) a sequence substantially complementary to a sequence from a 3′ region of the sequence encoding the constant region of the analyte, and (ii) a sequence comprising a first functional domain; and the second primer comprises: (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the constant region of the analyte, and (ii) a sequence comprising a second functional domain. Embodiment 59 is the method of embodiment 58, wherein the double-stranded member of the nucleic acid library comprises the first adaptor, the barcode, the capture domain, the sequence complementary to all or a portion of the sequence encoding the constant region of the analyte, and the second adaptor, in a 5′ to 3′ direction. Embodiment 60 is the method of embodiment 58 or 59, wherein the double-stranded member of the nucleic acid library further comprises a unique molecular identifier (UMI) disposed between the spatial barcode and the capture domain. Embodiment 61 is the method of any one of embodiments 58-60, wherein the first primer comprises (i) the sequence from the 3′ region of the sequence encoding the constant region of the analyte, and (ii) the sequence comprising the first functional domain, in 3′ to 5′ direction; and wherein the second primer comprises (i) the sequence from the 5′ region of the sequence encoding the constant region of the analyte, and (ii) the sequence comprising the second functional domain, in a 3′ to 5′ direction. Embodiment 62 is the method of any one of embodiments 58-61, wherein the barcode is a spatial barcode or a cell barcode. Embodiment 63 is the method of any one of embodiments 58-62, wherein ligating in step (c) is performed using a DNA ligase or using template mediated ligation. Embodiment 64 is the method of embodiment 63, wherein the DNA ligase is T4 ligase. Embodiment 65 is the method of any one of embodiments 58-64, wherein the nucleic acid library is a DNA library. Embodiment 66 is the method of any one of embodiments 58-64, wherein the nucleic acid library is a cDNA library. Embodiment 67 is the method of any one of embodiments 58-66, wherein the double-stranded member of the nucleic acid library further comprises a sequence that is complementary to all or a portion of a sequence encoding a variable region of an analyte. Embodiment 68 is the method of embodiment 67, wherein the sequence complementary to all or a portion of the sequence encoding the constant region of the analyte is positioned 5′ relative to the sequence that is complementary to all or a portion of the sequence encoding the variable regions of the analyte. Embodiment 69 is the method of embodiment 67, wherein the sequence complementary to all or a portion of the sequence encoding the constant region of the analyte is positioned 3′ relative to the sequence that is complementary to all or a portion of the sequence encoding the variable region of the analyte. Embodiment 70 is the method of any one of embodiments 67-69, wherein the analyte is an immune cell receptor. Embodiment 71 is the method of embodiment 70, wherein the immune cell receptor is a B cell receptor. Embodiment 72 is the method of embodiment 71, wherein the B cell receptor is an immunoglobulin kappa light chain. Embodiment 73 is the method of embodiment 72, wherein the variable region of the analyte comprises a CDR3 of the immunoglobulin kappa light chain. Embodiment 74 is the method of embodiment 73, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the immunoglobulin kappa light chain. Embodiment 75 is the method of embodiment 73, wherein the variable region of the analyte further comprises a full-length variable domain of the immunoglobulin kappa light chain. Embodiment 76 is the method of embodiment 71, wherein the B cell receptor is an immunoglobulin lambda light chain. Embodiment 77 is the method of embodiment 76, wherein the variable region of the analyte comprises a CDR3 of the immunoglobulin kappa light chain. Embodiment 78 is the method of embodiment 77, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the immunoglobulin kappa light chain. Embodiment 79 is the method of embodiment 77, wherein the variable region of the analyte further comprises a full-length variable domain of the immunoglobulin lambda light chain. Embodiment 80 is the method of embodiment 71, wherein the B cell receptor is an immunoglobulin heavy chain. Embodiment 81 is the method of embodiment 80, wherein the variable region of the analyte comprises a CDR3 of the immunoglobulin heavy chain. Embodiment 82 is the method of embodiment 81, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the immunoglobulin heavy chain. Embodiment 83 is the method of embodiment 81, wherein the variable region of the analyte further comprises a full-length variable domain of the immunoglobulin heavy chain. Embodiment 84 is the method of embodiment 70, wherein the immune cell receptor is a T cell receptor. Embodiment 85 is the method of embodiment 84, wherein the T cell receptor is a T cell receptor alpha chain. Embodiment 86 is the method of embodiment 85, wherein the variable region of the analyte comprises a CDR3 of the T cell receptor alpha chain. Embodiment 87 is the method of embodiment 86, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the T cell receptor alpha chain. Embodiment 88 is the method of embodiment 86, wherein the variable region of the analyte further comprises a full-length variable domain of the T cell receptor alpha chain. Embodiment 89 is the method of embodiment 84, wherein the T cell receptor is a T cell receptor beta chain. Embodiment 90 is the method of embodiment 89, wherein the variable region of the analyte comprises a CDR3 of the T cell receptor beta chain. Embodiment 91 is the method of embodiment 90, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the T cell receptor beta chain. Embodiment 92 is the method of embodiment 90, wherein the variable region of the analyte further comprises a full-length variable domain of the T cell receptor beta chain. Embodiment 93 is the method of any one of embodiments 58-92, wherein the method further comprises amplifying the version of the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region of the analyte using a third primer and fourth primer, wherein: the third primer is substantially complementary to the first functional domain, and the fourth primer is substantially complementary to the second functional domain. Embodiment 94 is the method of any one of embodiments 58-93, wherein the method further comprises: determining (i) all or a portion of the sequence encoding the variable region of the analyte or complement thereof, and (ii) all or a portion of the sequence of the barcode or complement thereof. Embodiment 95 is the method of embodiment 94, wherein determining the sequence comprises sequencing (i) all or a portion of the sequence encoding the variable region of the analyte or a complement thereof, and (ii) all or a portion of the sequence of the barcode or a complement thereof. Embodiment 96 is the method of embodiment 95, wherein the sequencing is performed by sequence by synthesis, sequence by ligation or sequence by hybridization. Embodiment 97 is the method of any one of embodiments 94-103, wherein the analyte was released from a biological sample, and the method further comprises: determining the location of the analyte in the biological sample using the determined sequence of (i) and (ii). Embodiment 98 is the method of any one of embodiments 58-97, further comprising generating the double-stranded member of the nucleic acid library. Embodiment 99 is the method of embodiment 98, wherein the step of generating the double-stranded member of the nucleic acid library comprises: contacting the analyte with a capture probe comprising the first adaptor, the barcode, and the capture domain, wherein the capture domain binds specifically to a sequence present in the analyte; extending an end of the capture probe using the analyte specifically bound to the capture domain as a template, thereby generating an extended capture probe; and adding the second adaptor to an end of the extended capture probe, thereby generating the double-stranded member of the nucleic acid library. Embodiment 100 is the method of embodiment 99, wherein the capture probe comprises the first adapter, the barcode, and the capture domain in a 5′ to a 3′ direction. Embodiment 101 is the method of embodiment 99 or 100, wherein a 3′ end of the capture probe is extended. Embodiment 102 is the method of any one of embodiments 100-101, wherein the second adapter is added to a 5′ end of the extended capture probe. Embodiment 103 is the method of any one of embodiments 58-102, wherein the biological sample is a tissue sample, a tissue section or a fixed tissue section. Embodiment 104 is the method of embodiment 103, wherein the fixed tissue section is formalin-fixed paraffin-embedded tissue section or a fresh, frozen tissue section. Embodiment 105 is the method of any one of embodiments 58-104, wherein the analyte is an RNA. Embodiment 106 is the method of embodiment 105, wherein the RNA is an mRNA. Embodiment 107 is the method of any one of embodiments 58-104, wherein the analyte is a DNA. Embodiment 108 is the method of embodiment 107, wherein the DNA is genomic DNA. Embodiment 109 is a kit comprising: (i) a first restriction endonuclease that cleaves a first restriction endonuclease recognition sequence; (ii) a ligase; and (iii) a first and a second primer, wherein: the first primer comprises: (i) a sequence from a 3′ region of a sequence encoding a constant region of an analyte, and (ii) a sequence comprising a first functional domain; and the second primer comprises: (i) a sequence substantially complementary to a sequence from a 5′ region of a sequence encoding the constant region of the analyte, and (ii) a sequence comprising a second functional domain. Embodiment 110 is the kit of embodiment 109, wherein the kit further comprises: a third primer comprising a sequence substantially complementary to the first functional domain; and a fourth primer comprising a sequence substantially complementary to the second functional domain. Embodiment 111 is the kit of embodiment 108 or 109, wherein the first primer comprises (i) the sequence from the 3′ region of sequence encoding a constant region of the analyte, and (ii) the sequence comprising the first functional domain, in a 3′ to 5′ direction. Embodiment 112 is the kit of any one of embodiments 108-111, wherein the second primer comprises (i) the sequence substantially complementary to the sequence from the 5′ region of the sequence encoding the constant region of the analyte, and (ii) the sequence comprising the second functional domain, in a 3′ to 5′ direction. Embodiment 113 is the kit of any one of embodiments 108-112, wherein the ligase is a DNA ligase. Embodiment 114 is the kit of embodiment 113, wherein the DNA ligase is T4 ligase. Embodiment 115 is a method for removing all or a portion of the sequence encoding a constant region of an analyte from a double-stranded member of a nucleic acid library, wherein the double-stranded member of the nucleic acid library comprises a ligation sequence, a barcode, a reverse complement of a first adaptor, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor, wherein the method comprises: (a) ligating ends of the double-stranded member using the ligation sequence to splint ligation, to generate a circularized double-stranded nucleic acid; (b) amplifying the circularized double-stranded nucleic acid using a first primer and a second primer to generate a version of the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region, wherein: the first primer comprises: (i) a sequence substantially complementary to the reverse complement of the first adaptor and (ii) a first functional domain; and the second primer comprises: (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the constant region of the analyte, and (ii) a second functional domain. Embodiment 116 is the method of embodiment 115, wherein the double-stranded member of the nucleic acid library comprises the ligation sequence, the barcode, the reverse complement of the first adaptor, the capture domain, the sequence complementary to all or a portion of the sequence encoding the constant region of the analyte, and the second adaptor, in a 5′ to 3′ direction. Embodiment 117 is the method of embodiment 116, wherein the double-stranded member of the nucleic acid library further comprises a unique molecular identifier (UMI) disposed between the barcode and the reverse complement of the first adaptor. Embodiment 118 is the method of any one of embodiments 115-118, wherein the first primer comprises (i) the sequence substantially complementary to the reverse complement of the first adaptor, and (ii) the sequence comprising the first functional domain, in 3′ to 5′ direction; and wherein the second primer comprises (i) the sequence substantially complementary to a sequence of the 5′ region of the sequence encoding the constant region of the analyte, and (ii) the sequence comprising the second functional domain, in a 3′ to 5′ direction. Embodiment 119 is the method of any one of embodiments 115-118, wherein ligating in step (a) is performed using a DNA ligase. Embodiment 120 is the method of embodiment 119, wherein the DNA ligase is T4 ligase. Embodiment 121 is the method of any one of embodiments 115-120, wherein the barcode is a spatial barcode or a cell barcode. Embodiment 122 is the method of any one of embodiments 115-121, wherein the nucleic acid library is a DNA library. Embodiment 123 is the method of any one of embodiments 121-121, wherein the nucleic acid library is a cDNA library. Embodiment 124 is the method of any one of embodiments 115-123, wherein the double-stranded member of the nucleic acid library further comprises a sequence that is complementary to all or a portion of a sequence encoding a variable region of an analyte. Embodiment 125 is the method of embodiment 124, wherein the sequence complementary to all or a portion of the sequence encoding the constant region of the analyte is positioned 5′ relative to the sequence that is complementary to all or a portion of the sequence encoding the variable regions of the analyte. Embodiment 126 is the method of embodiment 124, wherein the sequence complementary to all or a portion of the sequence encoding the constant region of the analyte is positioned 3′ relative to the sequence that is complementary to all or a portion of the sequence encoding the variable region of the analyte. Embodiment 127 is the method of any one of embodiments 124-126, wherein the analyte is an immune cell receptor. Embodiment 128 is the method of embodiment 127, wherein the immune cell receptor is a B cell receptor. Embodiment 129 is the method of embodiment 128, wherein the B cell receptor is an immunoglobulin kappa light chain. Embodiment 130 is the method of embodiment 129, wherein the variable region of the analyte comprises a CDR3 of the immunoglobulin kappa light chain. Embodiment 131 is the method of embodiment 130, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the immunoglobulin kappa light chain. Embodiment 132 is the method of embodiment 130, wherein the variable region of the analyte further comprises a full-length variable domain of the immunoglobulin kappa light chain. Embodiment 133 is the method of embodiment 128, wherein the B cell receptor is an immunoglobulin lambda light chain. Embodiment 134 is the method of embodiment 133, wherein the variable region of the analyte comprises a CDR3 of the immunoglobulin kappa light chain. Embodiment 135 is the method of embodiment 134, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the immunoglobulin kappa light chain. Embodiment 136 is the method of embodiment 134, wherein the variable region of the analyte further comprises a full-length variable domain of the immunoglobulin lambda light chain. Embodiment 137 is the method of embodiment 128, wherein the B cell receptor is an immunoglobulin heavy chain. Embodiment 138 is the method of embodiment 137, wherein the variable region of the analyte comprises a CDR3 of the immunoglobulin heavy chain. Embodiment 139 is the method of embodiment 138, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the immunoglobulin heavy chain. Embodiment 140 is the method of embodiment 138, wherein the variable region of the analyte further comprises a full-length variable domain of the immunoglobulin heavy chain. Embodiment 141 is the method of embodiment 127, wherein the immune cell receptor is a T cell receptor. Embodiment 142 is the method of embodiment 141, wherein the T cell receptor is a T cell receptor alpha chain. Embodiment 143 is the method of embodiment 142, wherein the variable region of the analyte comprises a CDR3 of the T cell receptor alpha chain. Embodiment 144 is the method of embodiment 143, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the T cell receptor alpha chain. Embodiment 145 is the method of embodiment 143, wherein the variable region of the analyte further comprises a full-length variable domain of the T cell receptor alpha chain. Embodiment 146 is the method of embodiment 141, wherein the T cell receptor is a T cell receptor beta chain. Embodiment 147 is the method of embodiment 146, wherein the variable region of the analyte comprises a CDR3 of the T cell receptor beta chain. Embodiment 148 is the method of embodiment 147, wherein the variable region of the analyte further comprises one or both of CDR1 and CDR2 of the T cell receptor beta chain. Embodiment 149 is the method of embodiment 147, wherein the variable region of the analyte further comprises a full-length variable domain of the T cell receptor beta chain. Embodiment 150 is the method of any one of embodiments 115-149, wherein the method further comprises amplifying the version of the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region of the analyte using a third primer and fourth primer, wherein: the third primer is substantially complementary to the first functional domain, and the fourth primer is substantially complementary to the second functional domain. Embodiment 151 is the method of any one of embodiments 115-150, wherein the method further comprises: determining (i) all or a portion of the sequence encoding the variable region of the analyte or complement thereof, and (ii) all or a portion of the sequence of the barcode or complement thereof. Embodiment 152 is the method of embodiment 151, wherein the determining the sequence comprises sequencing (i) all or a portion of the sequence encoding the variable region of the analyte or a complement thereof, and (ii) all or a portion of the sequence of the barcode or a complement thereof. Embodiment 153 is the method of embodiment 152, wherein the sequencing is performed using sequence by synthesis, sequence by ligation or sequence by hybridization. Embodiment 154 is the method of any one of embodiments 151-153, wherein the analyte was released from a biological sample, and the method further comprises: determining the location of the analyte in the biological sample using the determined sequence of (i) and (ii). Embodiment 155 is the method of any one of embodiments 115-154, further comprising generating the double-stranded member of the nucleic acid library. Embodiment 156 is the method of embodiment 155, wherein the step of generating the double-stranded member of the nucleic acid library comprises: contacting the analyte with a capture probe comprising the ligation sequence, the barcode, the reverse complement of the first adaptor, the capture domain a sequence that is complementary to all or a portion of the sequence encoding the constant region of the analyte, and a second adaptor, wherein the capture domain binds specifically to a sequence present in the analyte; extending an end of the capture probe using the analyte specifically bound to the capture domain as a template, thereby generating an extended capture probe; and adding the second adaptor to an end of the extended capture probe, thereby generating the double-stranded member of the nucleic acid library. Embodiment 157 is the method of embodiment 156, wherein the capture probe comprises the ligation sequence, the barcode, the reverse complement of the first adaptor, and the capture domain in a 5′ to a 3′ direction. Embodiment 158 is the method of embodiment 156 or 157, wherein a 3′ end of the capture probe is extended. Embodiment 159 is the method of any one of embodiments 156-158, wherein the second adapter is added to a 5′ end of the extended capture probe. Embodiment 160 is the method of any one of embodiments 115-159, wherein the biological sample is a tissue sample, a tissue section, or a fixed tissue section. Embodiment 161 is the method of embodiment 160, wherein the fixed tissue section is formalin-fixed paraffin-embedded tissue section or the tissue section is a fresh, frozen tissue section. Embodiment 162 is the method of any one of embodiments 115-161, wherein the analyte is an RNA. Embodiment 163 is the method of embodiment 162, wherein the RNA is an mRNA. Embodiment 164 is the method of any one of embodiments 115-161, wherein the analyte is a DNA. Embodiment 165 is the method of embodiment 164, wherein the DNA is genomic DNA. Embodiment 166 is a kit comprising: (i) a first restriction endonuclease that cleaves a first restriction endonuclease recognition sequence; (ii) a ligase; and (iii) a first and a second primer, wherein: the first primer comprises: (i) a sequence substantially complementary to a reverse complement of a first adaptor, and (ii) a sequence comprising a first functional domain; and the second primer comprises: (i) a sequence substantially complementary to a sequence from a 5′ region of a sequence encoding the constant region of the analyte, and (ii) a sequence comprising a second functional domain. Embodiment 167 is the kit of embodiment 166, wherein the kit further comprises: a third primer comprising a sequence substantially complementary to the first functional domain; and a fourth primer comprising a sequence substantially complementary to the second functional domain. Embodiment 168 is the kit of embodiment 166 or 167, wherein the first primer comprises (i) the sequence substantially complementary to the reverse complement of the first adaptor, and (ii) the sequence comprising the first functional domain, in a 3′ to 5′ direction. Embodiment 169 is the kit of any one of embodiments 166-168, wherein the second primer comprises (i) the sequence substantially complementary to the sequence from the 5′ region of the sequence encoding the constant region of the analyte, and (ii) the sequence comprising the second functional domain, in a 3′ to 5′ direction. Embodiment 170 is the kit of any one of embodiments 166-169, wherein the ligase is a DNA ligase. Embodiment 171 is the kit of embodiment 170, wherein the DNA ligase is T4 ligase. Embodiment 172 is a method of reversing the orientation of an analyte sequence of a double-stranded member of a nucleic acid library, wherein the double-stranded member of the nucleic acid library comprises a ligation sequence, a barcode, a reverse complement of the first adaptor, an amplification domain, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the analyte, and a second adaptor, wherein the method comprises: (a) ligating ends of the double-stranded member of the nucleic acid library using the ligation sequence to splint ligation, to generate a circularized double-stranded nucleic acid; and (b) amplifying the circularized double-stranded nucleic acid using a first primer and a second primer to generate a double-stranded nucleic acid product, wherein: the first primer comprises (i) a sequence substantially complementary to the reverse complement of the first adaptor and (ii) a functional domain; and the second primer comprises a sequence substantially complementary to the amplification domain, thereby reversing the orientation of the analyte sequence of the double-stranded member of the nucleic acid library. Embodiment 173 is the method of embodiment 172, wherein the double-stranded member of the nucleic acid library comprises the ligation sequence, the barcode, the reverse complement of the first adaptor, the amplification domain, the capture domain, the sequence complementary to all or a portion of the sequence encoding an analyte, and the second adaptor, in a 5′ to 3′ direction. Embodiment 174 is the method of embodiment 173, wherein the double-stranded member of the nucleic acid library further comprises a unique molecular identifier (UMI) disposed between the barcode and the reverse complement of the first adaptor. Embodiment 175 is the method of any one of embodiments 172-174, wherein the first primer comprises (i) the sequence substantially complementary to the reverse complement of the first adaptor, and (ii) the sequence comprising the first functional domain, in a 5′ to 3′ direction. Embodiment 176 is the method of any one of embodiments 172-175, wherein ligating in step (a) is performed using a ligase. Embodiment 177 is the method of embodiment 176, wherein the ligase is a DNA ligase. Embodiment 178 is the method of embodiment 177, wherein the DNA ligase is T4 ligase. Embodiment 179 is the method of any one of embodiments 172-178, wherein the barcode is a spatial barcode or a cell barcode. Embodiment 180 is the method of any one of embodiments 172-179, wherein the nucleic acid library is a DNA library. Embodiment 181 is the method of any one of embodiments 172-179, wherein the nucleic acid library is a cDNA library. Embodiment 182 is the method of any one of embodiments 172-181, wherein the double-stranded member of the nucleic acid library further comprises a sequence that is complementary to all or a portion of a sequence encoding a 5′ untranslated region of an analyte. Embodiment 183 is the method of any one of embodiments 172-182, wherein the double-stranded member of the nucleic acid library further comprises a sequence that is complementary to all or a portion of a sequence encoding a 3′ untranslated region of an analyte. Embodiment 184 is the method of embodiment 183, wherein the sequence that is complementary to all or a portion of the sequence encoding a 5′ untranslated region of the analyte is positioned 5′ relative to the sequence that is complementary to all or a portion of the sequence encoding the 3′ untranslated region of the analyte. Embodiment 185 is the method of any one of embodiments 172-184, wherein the double-stranded member of the nucleic acid library comprises one or more exons of the analyte. Embodiment 186 is the method of embodiment 185, wherein the analyte comprises the sequence that is complementary to all or a portion of the sequence encoding the 5′ untranslated region of the analyte, the one or more exons, and the sequence that is complementary to all or a portion of the sequence encoding the 3′ untranslated region, in a 5′ to 3′ direction. Embodiment 187 is the method of any one of embodiments 172-186, wherein the method further comprises: (c) determining (i) all or a portion of a sequence encoding the analyte or a complement thereof, and (ii) all or a portion of the barcode, or a complement thereof. Embodiment 188 is the method of embodiment 187, wherein the determining in step (c) comprises sequencing (i) all or a portion of the sequence encoding the analyte or a complement thereof, and (ii) all or a portion of the barcode or a complement thereof. Embodiment 189 is the method of embodiment 188, wherein the sequencing comprises high throughput sequencing. Embodiment 190 is the method of embodiment 188, wherein the sequencing is performed using sequence by synthesis, sequence by ligation or sequence by hybridization. Embodiment 191 is the method of any one of embodiments 188-190, wherein the analyte was released from a biological sample, and the method further comprises: determining a location of the analyte in the biological sample using the determined sequences of (i) and (ii). Embodiment 192 is the method of any one of embodiments 172-191, further comprising generating the double-stranded member of the nucleic acid library. Embodiment 193 is the method of embodiment 192, wherein the step of generating the double-stranded member of the nucleic acid library comprises: contacting the analyte with a capture probe comprising the ligation sequence, the barcode, the reverse complement of the first adaptor, the amplification domain, and the capture domain, wherein the capture domain binds specifically to a sequence present in the analyte; extending an end of the capture probe using the analyte specifically bound to the capture domain as a template, thereby generating an extended capture probe; and adding the second adaptor to an end of the extended capture probe, thereby generating the double-stranded member of the nucleic acid library. Embodiment 194 is the method of embodiment 193, wherein the capture probe comprises the ligation sequence, the barcode, the reverse complement of the first adaptor, the amplification domain, and the capture domain in a 5′ to a 3′ direction. Embodiment 195 is the method of embodiment 193 or 194, wherein a 3′ end of the capture probe is extended. Embodiment 196 is the method of any one of embodiments 193-195, wherein the second adapter is added to a 5′ end of the extended capture domain. Embodiment 197 is the method of any one of embodiments 191-196, wherein the biological sample is a tissue sample, a tissue section or a fixed tissue section. Embodiment 198 is the method of embodiment 197, wherein the fixed tissue section is formalin-fixed paraffin-embedded tissue section or the tissue section is a fresh, frozen tissue section. Embodiment 199 is the method of any one of embodiments 172-198, wherein the analyte is an RNA. Embodiment 200 is the method of embodiment 199, wherein the RNA is an mRNA. Embodiment 201 is the method of any one of embodiments 172-200, wherein the analyte is a DNA. Embodiment 202 is the method of embodiment 201, wherein the DNA is genomic DNA. Embodiment 203 is the method of any one of embodiments 172-202, wherein the analyte is a nucleic acid encoding an immune cell receptor. Embodiment 204 is the method of embodiment 203, wherein the immune cell receptor is a B-cell receptor. Embodiment 205 is the method of embodiment 204, wherein the B cell receptor is one of an immunoglobulin kappa light chain, an immunoglobulin lambda chain, and/or an immunoglobulin heavy chain. Embodiment 206 is the method of embodiment 203, wherein the immune cell receptor is a T cell receptor. Embodiment 207 is the method of embodiment 206, wherein the T cell receptor is one or both of a T cell receptor alpha chain and a T cell receptor beta chain. Embodiment 208 is a kit comprising: (i) a first restriction endonuclease that cleaves a first restriction endonuclease recognition sequence; (ii) a ligase; and (iii) a first and a second primer, wherein: the first primer comprises: (i) a sequence substantially complementary to a reverse complement of a first adaptor, and (ii) a functional domain; and the second primer comprises a sequence substantially complementary to the amplification domain. Embodiment 209 is the kit of embodiment 208, wherein the kit further comprises: a third primer comprising a sequence substantially complementary to the functional domain; and a fourth primer comprising a sequence substantially complementary to a reverse complement of the amplification domain. Embodiment 210 is the kit of embodiment 208 or 209, wherein the first primer comprises (i) the sequence substantially complementary to the reverse complement of the first adaptor, and (ii) the sequence comprising the functional domain, in a 3′ to 5′ direction. Embodiment 211 is the kit of any one of embodiments 208-210, wherein the ligase is a DNA ligase. Embodiment 212 is the kit of embodiment 211, wherein the DNA ligase is T4 ligase. Embodiment 213 is a method for removing all or a portion of a sequence encoding an analyte from a double-stranded member of a nucleic acid library, wherein the double-stranded member of the nucleic acid library comprises: a first adaptor, a barcode, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the analyte, and a second adaptor, wherein the method comprises: (a) adding to each end of the double-stranded member of the nucleic acid library a first restriction endonuclease recognition sequence; (b) contacting the double-stranded member of the nucleic acid library of step (a) with a first restriction endonuclease that cleaves the first restriction endonuclease recognition sequence at each end of the double-stranded member of the nucleic acid library; (c) ligating ends of the double-stranded member of the nucleic acid library of step (b) to generate a first double-stranded circularized nucleic acid; (d) amplifying the first double-stranded circularized nucleic acid using a first and a second primer to generate a first double-stranded nucleic acid product, wherein: the first primer comprises: (i) a sequence substantially complementary to a 3′ region of the sequence encoding the analyte and (ii) a second restriction endonuclease recognition sequence; and the second primer comprises: (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the analyte, and (ii) the second restriction endonuclease recognition sequence; (e) contacting the first double-stranded nucleic acid product with a second restriction endonuclease that cleaves the second restriction endonuclease recognition sequence at each end of the first double-stranded nucleic acid product; (f) ligating ends of the first double-stranded nucleic acid product of step (e) to generate a second double-stranded circularized nucleic acid; and (g) amplifying the second double-stranded circularized nucleic acid using a third primer comprising a sequence that is substantially complementary to the first adapter and a fourth primer comprising a sequence that is substantially complementary to the second adapter, to generate a version of the double-stranded member of the nucleic acid library lacking all or a portion the sequence encoding the analyte. Embodiment 214 is a method for removing all or a portion of a sequence encoding an analyte from a double-stranded member of a nucleic acid library, wherein the double-stranded member of the nucleic acid library comprises: a first adaptor, a barcode, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the analyte, and a second adaptor, wherein the method comprises: (a) adding to each end of the double-stranded member of the nucleic acid library a first restriction endonuclease recognition sequence; (b) contacting the double-stranded member of the nucleic acid library of step (a) with a first restriction endonuclease that cleaves the first restriction endonuclease recognition sequence at each end; (c) ligating ends of the double-stranded member of the nucleic acid library of step (b) to generate a first-double-stranded nucleic circularized nucleic acid; and (d) amplifying the double-stranded circularized nucleic acid using a first primer and a second primer to generate a version of the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the analyte, wherein: the first primer comprises: (i) a sequence substantially complementary to a sequence from a 3′ region of the sequence encoding the analyte, and (ii) a sequence comprising a first functional domain; and the second primer comprises: (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the analyte, and (ii) a sequence comprising a second functional domain. Embodiment 215 is a method for removing all or a portion of the sequence encoding an analyte from a double-stranded member of a nucleic acid library, wherein the double-stranded member of the nucleic acid library comprises a ligation sequence, a barcode, a reverse complement of a first adaptor, a capture domain, a sequence that is complementary to all or a portion of the sequence encoding the analyte, and a second adaptor, wherein the method comprises: ligating ends of the double-stranded member using the ligation sequence to splint ligation, to generate a circularized double-stranded nucleic acid; amplifying the circularized double-stranded nucleic acid using a first primer and a second primer to generate a version of the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the analyte, wherein: the first primer comprises: (i) a sequence substantially complementary to the reverse complement of the first adaptor and (ii) a first functional domain; and the second primer comprises: (i) a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the analyte, and (ii) a second functional domain. EXAMPLES Example 1: Removal of a Portion of a Member of a Nucleic Acid Library Via Circularization FIGS.2A-Ishow an exemplary nucleic acid library preparation method to remove a portion of an analyte sequence via double circularization of a member of a nucleic acid library.FIG.2Ashows an exemplary member of a nucleic acid library including, in a 5′ to 3′ direction, a first adaptor (e.g., primer sequence R1, pR1 (e.g., Read 1)), a barcode (e.g., a spatial barcode or a cell barcode), a unique molecular identifier (UMI), a capture domain (e.g., poly(T) VN sequence), a sequence complementary to an analyte (C, J, D and V), and a second adaptor (e.g., template switching oligonucleotide sequence (TSO)). For purposes of this example an analyte including a constant region (C) and V(D)J sequence are shown, however, the methods described herein can be equally applied to other analyte sequences in a nucleic acid library. FIG.2Bshows the exemplary member of a nucleic acid library where additional sequences can be added to both the 3′ and 5′ ends of the nucleic acid member (shown as a X and Y) via a PCR reaction. The additional sequences added can include a recognition sequence for a restriction enzyme (e.g., restriction endonuclease). The restriction recognition sequence can be for a rare restriction enzyme. The exemplary member of the nucleic acid library shown inFIG.2B, can be digested with a restriction enzyme to generate sticky ends shown inFIG.2C(shown as triangles) and can be intramolecularly circularized by ligation to generate the circularized member of the nucleic acid library shown inFIG.2D. The ligation can be performed with a DNA ligase. The ligase can be T4 ligase. A primer pair can be hybridized to a circularized nucleic acid member, where a first primer hybridizes to a 3′ portion of a sequence encoding the constant region (C) and includes a second restriction enzyme (e.g., restriction endonuclease) sequence that is non-complementary to the analyte sequence, and where a second primer hybridized to a 5′ portion of a sequence encoding the constant region (C), and where the second primer includes a second restriction enzyme sequence (FIG.2E). The first primer and the second primer can generate a linear amplification product (e.g., a first double-stranded nucleic acid product) as shown inFIG.2F, which includes the second restriction enzyme recognition sequences (shown as X and Y end sequences). The linear amplification product (FIG.2F) can be digested with a second restriction enzyme to generate sticky ends and can be intramolecularly ligated with a ligase (e.g., T4 DNA ligase) to generate a second double-stranded circularized nucleic acid product as shown inFIG.2G. The second double-stranded circularized nucleic product (FIG.2G) can be amplified with a third primer, pR1, substantially complementary to the first adaptor (e.g., Read 1) sequence and a fourth primer substantially complementary to the second adapter (e.g., TSO) as shown inFIG.211to generate a version of the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region (C) of the analyte (FIG.21). The resulting double-stranded member of the nucleic acid library lacking all or a portion of the constant region can undergo standard library preparation methods, such as library preparation methods used in single-cell or spatial analyses. For example, the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region of the analyte can be fragmented, followed by end repair, a-tailing, adaptor ligation, and/or additional amplification (e.g., PCR). The fragments can then be sequenced using, for example, paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites or any other sequencing method described herein. As a result of the methods described in this Example, sequences can be determined from regions more than about 1 kb away from the end of an analyte (e.g., 3′ end) and can link such a sequence to a barcode sequence (e.g., a spatial barcode, a cell barcode) in library preparation methods (e.g., sequencing preparation). For purposes of this example an analyte including a constant region (C) and V(D)J sequences are shown, however, the methods described herein can be equally applied to other analyte sequences in a nucleic acid library. Example 2: Removal of a Portion of a Member of a Nucleic Acid Library Via Single Circularization In this Example an exemplary member of a nucleic acid library can be prepared as shown inFIGS.2A-Dto generate a first double-stranded circularized nucleic acid product (FIG.2D) as previously described. A primer pair can be contacted with the double-stranded circularized nucleic acid produce with a first primer that can hybridize to a sequence from a 3′ region of the sequence encoding the constant region of the analyte and a sequence including a first functional domain (e.g., P5). The second primer can hybridize to a sequence from a 5′ region of the sequence encoding the constant region of the analyte, and includes a sequence including a second functional domain (shown as “X”) as shown inFIG.3A. Amplification of the double-stranded circularized nucleic acid product results in a linear product as shown inFIG.3B, where all, or a portion of, the constant region (C) is removed. The first functional domain can include a sequencer specific flow cell attachment sequence (e.g., P5). The second functional domain can include an amplification domain such as a primer sequence to amplify the nucleic acid library prior to further sequencing preparation. The resulting double-stranded member of the nucleic acid library lacking all or a portion of the constant region can undergo standard library preparation methods, such as library preparation methods used in single-cell or spatial analyses. For example, the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region of the analyte can be fragmented, followed by end repair, A-tailing, adaptor ligation, and/or amplification (e.g., PCR) (FIG.3C). The fragments can then be sequenced using, for example, paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites (FIG.3C, arrows), or any other sequencing method described herein. In this Example, after standard library preparation methods described herein, a different sequencing primer for the first adaptor (e.g., Read 1) is used since the orientation of the first adaptor (e.g., Read 1) sequence will be reversed. As a result of the methods described in this Example, sequences can be determined from regions more than about 1 kb away from the end of an analyte (e.g., 3′ end) and can link such a sequence to a barcode sequence (e.g., a spatial barcode, a cell barcode) in further library preparation methods (e.g., sequencing preparation). For purposes of this example an analyte including a constant region (C) and V(D)J sequence are shown, however, the methods described herein can be applied to other analyte sequences in a nucleic acid library as well. Example 3: Removal of a Portion of a Member of a Nucleic Acid Library Via Single Circularization FIGS.4A-Bshow an exemplary nucleic acid library preparation method to remove all or a portion of a constant sequence of an analyte from a member of a nucleic acid library via circularization.FIGS.4A and4Bshows an exemplary member of a nucleic acid library including, in a 5′ to 3′ direction, a ligation sequence, a barcode sequence, a unique molecular identifier, a reverse complement of a first adaptor (e.g., primer sequence pR1 (e.g., Read 1)), a capture domain, a sequence complementary to the captured analyte sequence, and a second adapter (e.g., TSO sequence). The ends of the double-stranded nucleic acid can be ligated together via a ligation reaction where the ligation sequence splints the ligation to generate a circularized double-stranded nucleic acid as shown inFIG.4B. The circularized double-stranded nucleic acid can be amplified with a pair of primers to generate a linear nucleic acid product lacking all or a portion of the constant region of the analyte (FIGS.4B and4C). The first primer can include a sequence substantially complementary to the reverse complement of the first adaptor and a first functional domain. The first functional domain can be a sequencer specific flow cell attachment sequence (e.g., P5). The second primer can include a sequence substantially complementary to a sequence from a 5′ region of the sequence encoding the constant region of the analyte, and a second functional domain. The second functional domain can include an amplification domain such as a primer sequence to amplify the nucleic acid library prior to further sequencing preparation. The resulting double-stranded member of the nucleic acid library lacking all or a portion of the constant region can undergo standard library preparation methods, such as library preparation methods used in single-cell or spatial analyses. For example, the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region of the analyte can be fragmented, followed by end repair, A-tailing, adaptor ligation, and/or amplification (e.g., PCR) (FIG.4C). The fragments can then be sequenced using, for example, paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites, or any other sequencing method described herein (FIG.4D). In this Example, after standard library preparation methods described herein, standard sequencing primers can be used since the orientation of Read 1 will be in the proper orientation for sequencing primer pR1. As a result of the methods described in this Example, sequences can be determined from regions more than about 1 kb away from the end of an analyte (e.g., 3′ end) and can link such a sequence to a barcode sequence (e.g., a spatial barcode, a cell barcode) in further library preparation methods (e.g., sequencing preparation). For purposes of this example an analyte including a constant region (C) and V(D)J sequence are shown, however, the methods described herein can be applied to other analyte sequences in a nucleic acid library as well. Example 4: Reversal of the Orientation of an Analyte Sequence in a Member of a Nucleic Acid Library FIGS.5A-Bshow an exemplary nucleic acid library method to reverse the orientation of an analyte sequence in a member of a nucleic acid library.FIG.5Ashows an exemplary member of a nucleic acid library including, in a 5′ to 3′ direction, a ligation sequence, a barcode (e.g., a spatial barcode or a cell barcode), unique molecular identifier, a reverse complement of a first adaptor, an amplification domain, a capture domain, a sequence complementary to an analyte, and a second adapter. The ends of the double-stranded nucleic acid can be ligated together via a ligation reaction where the ligation sequence splints the ligation to generate a circularized double-stranded nucleic acid also shown inFIG.5A. The circularized double-stranded nucleic acid can be amplified to generate a linearized double-stranded nucleic acid product, where the orientation of the analyte is reversed such that the 5′ sequence (e.g., 5′ UTR) is brought in closer proximity to the barcode (e.g., a spatial barcode or a cell barcode) (FIG.5B). The first primer includes a sequence substantially complementary to the reverse complement of the first adaptor and a functional domain. The functional domain can be a sequencer specific flow cell attachment sequence (e.g., P5). The second primer includes a sequence substantially complementary to the amplification domain. The resulting double-stranded member of the nucleic acid library including a reversed analyte sequence (e.g., the 5′ end of the analyte sequence is brought in closer proximity to the barcode) can undergo standard library preparation methods, such as library preparation methods used in single-cell or spatial analyses. For example, the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region of the analyte can be fragmented, followed by end repair, A-tailing, adaptor ligation, and/or amplification (e.g., PCR) (FIG.5C). The fragments can then be sequenced using, for example, paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites, or any other sequencing method described herein. As a result of the methods described in this Example, sequences from the 5′ end of an analyte will be included in sequencing libraries (e.g., paired end sequencing libraries). Any type of analyte sequence in a nucleic acid library can be prepared by the methods described in this Example (e.g., reversed). | 146,901 |
11859179 | DETAILED DESCRIPTION Through genetic linkage analysis of familial ALS patients, several genes have been identified to be risk factors for ALS. In the first intron of chromosome 9 open reading frame 72 (C9orf72), a large repeat expansion consisting of GGGGCC hexanucleotide has been identified in families of familial ALS patients. These microsatellite expansions can be transcribed in a bidirectional manner, producing both sense and antisense transcripts. The RNA transcripts accumulate in the nucleus of affected regions in the brain as RNA foci; moreover, repeat-associated non-ATG (RAN) translation of the transcripts leads to generation of dipeptide aggregates in the neuronal cytoplasm within the affected region. There is evidence indicating dipeptides and RNA foci may be toxic and may disrupt nucleocytoplasmic transport, autophagy, and immune response. Provided herein are methods and related compositions useful for reducing or removing (e.g., completely removing) GGGGCC (e.g., G4C2) repeat expansions. In some embodiments, methods provided herein reduce the accumulation of RNA foci and dipeptide aggregates in the nucleus and cytoplasm, respectively. To accomplish this, a gene editing approach involving CRISPR/Cas9 nuclease and guide RNAs targeted at different regions of C9orf72 gene were used in some embodiments. In some embodiments, strategies are outlined to excise the GGGGCC repeat in both in vitro and in vivo mice models. Gene Editing Molecules In some aspects, the disclosure provides a recombinant gene editing complex comprising: a recombinant gene editing protein; and, a nucleic acid encoding a guide RNA (gRNA) that specifically hybridizes to a target nucleic acid sequence within the C9ORF72 locus that are useful for excising all or a portion of a GGGGCC repeat expansion. As used herein, “gene editing complex” refers to a biologically active molecule (e.g., a protein, one or more proteins, a nucleic acid, one or more nucleic acids, or any combination of the foregoing) configured for adding, disrupting or changing genomic sequences (e.g., a gene sequence), for example by causing one or more double stranded breaks (DSBs) in a target DNA. Examples of gene editing complexes include but are not limited to Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), engineered meganuclease re-engineered homing endonucleases, the CRISPR/Cas system, and meganucleases (e.g., Meganuclease I-SceI). In some embodiments, a gene editing complex comprises proteins or molecules (e.g., recombinant gene editing proteins) related to the CRISPR/Cas system, including but not limited to Cas9, Cas6, Cpf1, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and variants thereof. In some embodiments, a recombinant gene editing protein is a nuclease. As used herein, the terms “endonuclease” and “nuclease” refer to an enzyme that cleaves a phosphodiester bond or bonds within a polynucleotide chain. Nucleases may be naturally occurring or genetically engineered. Genetically engineered nucleases are particularly useful for genome editing and are generally classified into four families: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases (e.g., engineered meganucleases) and CRISPR-associated proteins (Cas nucleases). In some embodiments, the nuclease is a ZFN. In some embodiments, the ZFN comprises a FokI cleavage domain. In some embodiments, the ZFN comprises Cys2His2fold group. In some embodiments, the nuclease is a TALEN. In some embodiments, the TALEN comprises a FokI cleavage domain. In some embodiments, the nuclease is a meganuclease. Examples of meganucleases include but are not limited to I-SceI, I-CreI, I-DmoI, and combinations thereof (e.g., E-DreI, DmoCre). The term “CRISPR” refers to “clustered regularly interspaced short palindromic repeats”, which are DNA loci containing short repetitions of base sequences. CRISPR loci form a portion of a prokaryotic adaptive immune system that confers resistance to foreign genetic material. Each CRISPR loci is flanked by short segments of “spacer DNA”, which are derived from viral genomic material. In the Type II CRISPR system, spacer DNA hybridizes to transactivating RNA (tracrRNA) and is processed into CRISPR-RNA (crRNA) and subsequently associates with CRISPR-associated nucleases (Cas nucleases) to form complexes that recognize and degrade foreign DNA. In certain embodiments, the nuclease is a CRISPR-associated nuclease (Cas nuclease). Examples of CRISPR nucleases include, but are not limited to Cas9, dCas9, Cas6, Cpf1, and variants thereof. In some embodiments, the nuclease is Cas9. In some embodiments, the Cas9 is derived from the bacteriaStreptococcus pyogenes(e.g., SpCas9) orStaphylococcus aureus(e.g., SaCas9). In some embodiments, a Cas protein or variant thereof does not exceed the packaging capacity of a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector, for example as described by Ran et al. (2015)Nature.520(7546); 186-91. For example, in some embodiments, a nucleic acid encoding a Cas protein is less than about 4.6 kb in length. For the purpose of genome editing, the CRISPR system can be modified to combine the tracrRNA and crRNA in to a single guide RNA (sgRNA) or just (gRNA). As used herein, the terms “guide RNA”, “gRNA”, and “sgRNA” refer to a polynucleotide sequence that is complementary to a target sequence in a cell and associates with a Cas nuclease, thereby directing the Cas nuclease to the target sequence. In some embodiments, a gRNA (e.g., sgRNA) ranges between 1 and 30 nucleotides in length. In some embodiments, a gRNA (e.g., sgRNA) ranges between 5 and 25 nucleotides in length. In some embodiments, a gRNA (e.g., sgRNA) ranges between 10 and 22 nucleotides in length. In some embodiments, a gRNA (e.g., sgRNA) ranges between 14 and 24 nucleotides in length. In some embodiments, a Cas protein and a guide RNA (e.g., sgRNA) are expressed from the same vector. In some embodiments, a Cas protein and a guide RNA (e.g., sgRNA) are expressed from separate vectors (e.g., two or more vectors). Typically, a guide RNA (e.g., a gRNA or sgRNA) hybridizes (e.g., binds specifically to, for example by Watson-Crick base pairing) to a target sequence and thus directs the CRISPR/Cas protein or simple protein to the target sequence. In some embodiments, a guide RNA hybridizes to (e.g., targets) a nucleic acid sequence, e.g., within a C9ORF72 locus. In some embodiments, a guide RNA hybridizes to a target sequence on the sense strand (e.g., 5′-3′ strand) of a gene. In some embodiments, a guide RNA hybridizes to a target sequence on the antisense strand (e.g., 3′-5′ strand) of a gene. In some aspects, the disclosure relates to guide RNAs (gRNAs) that specifically hybridize to a target nucleic acid sequence flanking opposite sides of a G4C2repeat of a C9ORF72 gene. As used herein “flanking opposite sides of a G4C2repeat” refers to a first portion of a target nucleic acid sequence that is upstream (e.g., 5′) with respect to a G4C2repeat and a second portion of a target nucleic acid sequence that is downstream (e.g., 3′) with respect to a G4C2repeat (and also the first portion). For example, gRNA-R9 and gRNA-R1 represent a pair of gRNAs that specifically hybridize to a target nucleic acid sequence flanking opposite sides of a G4C2repeat, as shown inFIG.1A. In some embodiments, a sequence that flanks a G4C2repeat is positioned between 1 nucleotide and 1000 nucleotides (e.g., any integer between 1 and 1000) upstream (e.g., 5′) with respect to a G4C2repeat (e.g., the first GGGGCC unit of the repeat). In some embodiments, a sequence that flanks a G4C2repeat is positioned between 10 nucleotides and 800 nucleotides upstream (e.g., 5′) with respect to a G4C2repeat. In some embodiments, a sequence that flanks a G4C2repeat is positioned between 200 nucleotides and 700 nucleotides upstream (e.g., 5′) with respect to a G4C2repeat. In some embodiments, a sequence that flanks a G4C2repeat is positioned between more than 1000 nucleotides (e.g., 1500, 2000, 2500, 5000, or more) upstream (e.g., 5′) with respect to a G4C2repeat. In some embodiments, a sequence that flanks a G4C2repeat is positioned between 1 nucleotide and 1000 nucleotides (e.g., any integer between 1 and 1000) downstream (e.g., 3′) with respect to a G4C2repeat (e.g., the last GGGGCC unit of the repeat). In some embodiments, a sequence that flanks a G4C2repeat is positioned between 10 nucleotides and 800 nucleotides downstream (e.g., 3′) with respect to a G4C2repeat. In some embodiments, a sequence that flanks a G4C2repeat is positioned between 200 nucleotides and 700 nucleotides downstream (e.g., 3′) with respect to a G4C2repeat. In some embodiments, a sequence that flanks a G4C2repeat is positioned between more than 1000 nucleotides (e.g., 1500, 2000, 2500, 5000, or more) downstream (e.g., 3′) with respect to a G4C2repeat. Methods of Treatment In some aspects, the disclosure provides methods for treating a subject having ALS or at risk of having ALS. A subject can be a human, non-human primate, rat, mouse, cat, dog, or other mammal. As used herein, the terms “treatment”, “treating”, and “therapy” refer to therapeutic treatment and prophylactic or preventative manipulations. The terms further include ameliorating existing symptoms, preventing additional symptoms, ameliorating or preventing the underlying causes of symptoms, preventing or reversing causes of symptoms, for example, symptoms associated with ALS. Thus, the terms denote that a beneficial result has been conferred on a subject having ALS, or with the potential to develop such a disorder. Furthermore, treatment may include the application or administration of an agent (e.g., therapeutic agent or a therapeutic composition) to a subject, or an isolated tissue or cell line from a subject, who may have a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. Therapeutic agents or therapeutic compositions may include a compound, vector, etc. in a pharmaceutically acceptable form that prevents and/or reduces the symptoms of a particular disease (e.g., ALS). For example a therapeutic composition may be a pharmaceutical composition that prevents and/or reduces the symptoms of ALS. In some embodiments, the disclosure provides a composition (e.g., a therapeutic composition) comprising one or more components of, or encoding, a gene editing complex as described by the disclosure, e.g., a vector as described by the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. It is contemplated that the therapeutic composition of the present invention will be provided in any suitable form. The form of the therapeutic composition will depend on a number of factors, including the mode of administration as described herein. The therapeutic composition may contain diluents, adjuvants and excipients, among other ingredients as described herein. Pharmaceutical Compositions In some aspects, the disclosure relates to pharmaceutical compositions comprising a gene editing complex. In some embodiments, the composition comprises gene editing complex and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical compositions can be prepared as described herein. The active ingredients may be admixed or compounded with any conventional, pharmaceutically acceptable carrier or excipient. The compositions may be sterile. Typically, pharmaceutical compositions are formulated for delivering an effective amount of an agent (e.g., gene editing complex). In general, an “effective amount” of an active agent refers to an amount sufficient to elicit the desired biological response. An effective amount of an agent may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated (e.g., ALS), the mode of administration, and the patient. A composition is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known in the art. It will be understood by those skilled in the art that any mode of administration, vehicle or carrier conventionally employed and which is inert with respect to the active agent may be utilized for preparing and administering the pharmaceutical compositions of the present disclosure. An effective amount, also referred to as a therapeutically effective amount, of a compound (for example, a gene editing complex or vector as described by the disclosure) is an amount sufficient to ameliorate at least one adverse effect associated with a condition (e.g., ALS). In the case of viral vectors, an amount of active agent can be included in each dosage form to provide between about 1010, 1011, 1012, 1013, 1014, or 1015genome copies per subject. One of ordinary skill in the art would be able to determine empirically an appropriate therapeutically effective amount. Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The compositions may conveniently be presented in unit dosage form. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. In some embodiments, liquid dose units are vials or ampoules. In some embodiments, solid dose units are tablets, capsules and suppositories. Modes of Administration In some embodiments, a therapeutically effective amount of a gene editing complex or vector as described by the disclosure is delivered to a target tissue or a target cell. The pharmaceutical compositions containing gene editing complex or vector, and/or other compounds can be administered by any suitable route for administering medications. A variety of administration routes are available, including parenterally, intravenously, intrathecally, intracranially, intradermally, intramuscularly or subcutaneously, or transdermally. The methods of this disclosure, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic effect without causing clinically unacceptable adverse effects. Various modes of administration are discussed herein. For use in therapy, an effective amount of the gene editing complex or vector, and/or other therapeutic agent can be administered to a subject by any mode that delivers the agent to the desired tissue, e.g., systemic, intramuscular, etc. In some embodiments, the gene editing complex or vector as described by the disclosure is administered to a subject via intramuscular (IM) injection or intravenously. In some embodiments, a gene editing complex (e.g., a nucleic acid encoding one or more components of a gene editing complex) can be delivered to the cells via an expression vector engineered to express the gene editing complex. An expression vector is one into which a desired sequence may be inserted, e.g., by restriction and ligation, such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. An expression vector typically contains an insert that is a coding sequence for a protein (e.g., gene editing protein, such as a CRISPR/Cas protein) or for a polynucleotide, such as guide RNA (gRNA, sgRNA, etc.). Vectors may further contain one or more marker sequences suitable for use in the identification of cells that have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes that encode enzymes whose activities are detectable by standard assays or fluorescent proteins, etc. As used herein, a coding sequence (e.g., protein coding sequence, miRNA sequence, shRNA sequence) and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. It will be appreciated that a coding sequence may encode an functional RNA. The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5′ non-transcribed and 5′ non-translated sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Such 5′ non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene. However, in some embodiments, a vector does not include a promoter sequence. Regulatory sequences may also include enhancer sequences, upstream activator sequences, internal ribosomal entry sites (IRES), and/or self-processing peptide sequences (e.g., 2A peptide), as desired. The vectors of the disclosure may optionally include 5′ leader or signal sequences. In some embodiments, a virus vector for delivering a nucleic acid molecule is selected from the group consisting of adenoviruses, adeno-associated viruses, lentiviral vectors, etc. In some embodiments, the viral vector is a recombinant adeno-associated virus. The adeno-associated virus is capable of infecting a wide range of cell types and species and can be engineered to be replication-deficient. It further has advantages, such as heat and lipid solvent stability, high transduction frequencies in cells of diverse lineages, including hematopoietic cells, and lack of superinfection inhibition thus allowing multiple series of transductions. The adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression. The adeno-associated virus can also function in an extrachromosomal fashion. In some embodiments, a recombinant AAV vector (rAAV) comprises, at a minimum, a transgene coding sequence (e.g., a nucleic acid sequence encoding a gene editing protein, such as a Cas protein, or a gRNA) and its associated regulatory sequence flanked by two AAV inverted terminal repeat (ITR) sequences. Examples of regulatory sequences include promoters (e.g., constitutive promoters, inducible promoters, tissue-specific promoters), enhancer sequences, etc. In some embodiments, the ITR sequences are AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9 ITR sequences, or variants thereof. In some embodiments, an rAAV vector comprising a nucleic acid encoding all or part of a gene editing complex (e.g., a nucleic acid sequence encoding a gene editing protein, a gRNA, or both) is packaged into a recombinant AAV (rAAV). Typically, an AAV vector is packaged into viral particles comprising one or more AAV capsid proteins. In some embodiments, the AAV capsid is an important element in determining these tissue-specific targeting capabilities. Thus, an rAAV having a capsid appropriate for the tissue being targeted can be selected. In some embodiments, the capsid protein has a serotype selected from AAV2, AAV3, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.39, and AAVrh.43 or suitable variants of any one of them. In some embodiments, the rAAV comprises a capsid protein that targets neuronal cells. In some embodiments, other useful viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include certain retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. In general, the retroviruses are replication-deficient (e.g., capable of directing synthesis of the desired transcripts, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., “Gene Transfer and Expression, A Laboratory Manual,” W.H. Freeman Co., New York (1990) and Murry, E. J. Ed. “Methods in Molecular Biology,” vol. 7, Humana Press, Inc., Clifton, New Jersey (1991). In some embodiments, gene editing complex (e.g., a nucleic acid sequence encoding a gene editing protein, a gRNA, or both) is delivered to a cell (e.g. a cell of a subject) by a lentiviral vector. Various techniques may be employed for introducing nucleic acid molecules of the disclosure into cells, depending on whether the nucleic acid molecules are introduced in vitro or in vivo in a host. Such techniques include transfection of nucleic acid molecule-calcium phosphate precipitates, transfection of nucleic acid molecules associated with DEAE, transfection or infection with the foregoing viruses including the nucleic acid molecule of interest, liposome-mediated transfection, and the like. Other examples include: N-TER™ Nanoparticle Transfection System by Sigma-Aldrich, FectoFly™ transfection reagents for insect cells by Polyplus Transfection, Polyethylenimine “Max” by Polysciences, Inc., Unique, Non-Viral Transfection Tool by Cosmo Bio Co., Ltd., Lipofectamine™ LTX Transfection Reagent by Invitrogen, SatisFection™ Transfection Reagent by Stratagene, Lipofectamine™ Transfection Reagent by Invitrogen, FuGENE® HD Transfection Reagent by Roche Applied Science, GMP compliant in vivo-jetPEI™ transfection reagent by Polyplus Transfection, and Insect GeneJuice® Transfection Reagent by Novagen. EXAMPLES Example 1: Excision of G4C2Expansion Strategy Design and Testing in HEK Cells. This example describes removal of the G4C2expansion repeat in C90rf72 using a CRISPR/Cas9 system. Several guide RNAs targeting the flanking regions of the G4C2expansion were designed. The G4C2expansion and guide RNAs are shown inFIG.1A. Guides determined to be successful in achieving significant editing, as described herein, are shown. In order to test gene editing events, two primers, C9Var1-F and C9In1-R, that span the repeat expansion and the guides were designed (FIGS.1A-1C). These primers can amplify through few repeats, but will generally not amplify through the 45-60 repeats present in the BAC436 mouse model. In order to detect no editing in the BAC436 model, a NoE-F1 primer that can in conjugation with C9In1-R-amplify ˜120 bp band in unedited DNA, was designed (FIGS.1B-1C). Another primer that recognizes the GGGGCC sequence within the repeat was designed (FIGS.1B-1C). This primer was used for the repeat primed PCR (RP-PCR) described herein. Four different guide RNA constructs, two on the 5′ end of the repeat expansion (f1, also referred to as “gRNA1” & r9, also referred to as “gRNA 2”) and two on the 3′ end (r1, also referred to as “gRNA 4”& f11, also referred to as “gRNA 3”) (Table 1), were generated. Then, plasmids expressing two of each guides as follows were generated: gRNA f1-r1, gRNA f1-f11, gRNA r9-f11, gRNA r9-r1. Each of these plasmids was co-transfected into HEK293T cells with another plasmid expressingS. pyogenesCas9. DNA was extracted from these HEK 293T cells and a PCR was performed using C9Var1-F and C9In1-R. The products were run on an agarose gel (FIG.2A). In case no editing occurs, these primers will amplify a 523 bp band. In case editing occurs, gRNA f1-r1 and f1-f11 will produce a ˜250 bp band while r9-f11 and r9-r1 will produce a ˜320 bp band. TABLE 1Guide RNAs generated for“Excision of G4C2expansion.”guide RNA nameguide RNA sequenceSEQ ID NO:gRNA-f11GGGGUUCGGCUGCCGGGAAG1gRNA-r1GGAAGAGGCGCGGGUAGAAG2gRNA-r9GUAGCAAGCUCUGGAACUCA3gRNA-f1UGCUCUCACAGUACUCGCUG4 As seen on the gel (FIG.2A) these four different combination are capable of editing C9 gene in HEK cells, since bands of the anticipated edited size in each of these guide RNA combinations are observed. gRNA f1-r1 and gRNAf1-f11 both have a faint band between 200 and 300 bp, while gRNA r9-f11 and gRNA r9-r1 have a strong band around 320 bp. Both of these bands are absent in the untreated control. However, the combination of r9-f11 and r9-r1 seems to be much more efficient at gene editing, since the edited band is much more intense than f1-r1 and f1-f11 alone. Additionally, the unedited band at 523 bp is almost completely gone from r9-f11 and r9-r1. Bands labeled with arrow heads inFIG.2Awere then extracted and sequenced to ensure that gene editing occurred at the expected locations (FIG.2B). Based on this data an AAV9 virus containing gRNA r9-r1 and r9-f11 was generated to use for the in vivo studies. C9ORf72 Gene Editing in Mice Primary Neurons A mouse model (Bac436) expressing human C9orf72 with 45-65 expanded GGGGCC repeats has been developed. This model contains 6-8 copies of the C9orf72 gene in heterozygous (het) animals and 12-16 copies in homozygous (homo) animals. Additionally, a mouse expressing Cas9 gene, in addition to C9orf72 with the expansion, is observed in this model. In order to determine whether guides will successfully excise the GGGGCC repeat in mice primary neurons, appropriate crosses of the BAC436 mice expressing C9orf72 and Cas9 were set up to produce only heterozygous progeny. Primary neurons were isolated at embryonic day 14 (E14), and cultured appropriately. After 4 days in culture, neurons were either treated with PBS alone, or infected with AAV9 CB-GFP, AAV9 SOD1 guide RNA (control guide), AAV9-CB-GFP-C9gR flank r9-r1, or AAV9-CB-GFP-C9gR flank r9-f11. At 72 hours, 25,000 MOI was recorded and the cells were harvested. The DNA was isolated using QIAGEN™ blood and tissue DNA extraction kit. In order to determine whether editing has occurred in these isolated neuronal cells, a PCR reaction was performed using C9Var1-f and C9In1-R (FIGS.1A and1B). Without gene editing, these primers fail to amplify through the repeat and no band appears on the gel. When gene editing occurs, the repeat is excised out and primers amplify a single band at 321 bp. In both sets of guides a strong band appearing at the right size is observed, while this band is absent in both non-AAV treated neurons and those transfected with CB-GFP, or SOD1-gR (FIG.3). Testing Guide RNA Constructs in Mice Livers In order to determine whether gene editing is also successful in vivo, four groups of Cas9/+, C9/+mice were tail vein injected with PBS alone, AAV9 SOD1 guide RNA, AAV9-CB-GFP-C9gR flank r9-r1, or AAV9-CB-GFP-C9gR flank r9-f11. Two weeks after injection, mice were sacrificed and tissues were harvested. Since tail vein injection is very efficient at transfecting liver cells, DNA isolated from liver was analyzed. A third primer (NoE-F1) that can amplify unedited DNA, in conjugation with C9In1-R, was designed (FIG.1B). To reduce competition between C9Var1-f and NoE-F1, two different PCR reactions were run separately with C9Var1-f and C9In1-R or NoE-F1 and C9In1-R. Products from these two PCRs were mixed and run on the same gel (FIG.4). A 321 bp band appears in samples from mice injected with AAV9-CB-GFP-C9gR flank r9-r1 and AAV9-CB-GFP-C9gR flank r9-f11, but not from mice injected with AAV9 SOD1 guide RNA or PBS alone (FIG.4). Moreover, the 100 bp amplified by NoE-F1 and C9In1-R from unedited DNA was much less intense in r9-r1 and r9-f11 mice in comparison to control mice. The labeled bands were isolated and sequenced to confirm that the correct size gene editing products were made. To further elucidate editing, a Repeat Primed PCR was performed using a FAM-tagged C9Var1-f and c9ccccggLCM13F_MRX-R1b. The latter is a reverse primer that recognizes and binds the GGGGCC repeat. This form of PCR reaction produces different sized fragments based on where in the repeat the reverse primer binds and starts the amplification. These fragments were then analyzed on a fragment analyzer to produce an electropherogram where each peak reflects a different sized fragment and its intensity reflects fragment abundance. As the primer binds deeper into the repeat, it becomes more difficult to amplify and thus the intensity of peaks on the electropherogram decreases with larger fragments. These fragments can only be amplified in unedited DNA, and the shortest most intense fragment is around 330 bp in size. The electropherograms of the Repeat primed PCR products for AAV9 SOD1 guide RNA, AAV9-CB-GFP-C9gR flank r9-r1, AAV9-CB-GFP-C9gR flank r9-f11, and uninjected wild type C57BL mice that don't express human C9 are shown inFIGS.5A-5D, respectively. The results confirm Cas9-mediated C9orf72 G4C2 editing in vivo. Example 2: Induction of Non-Sense Mediated Decay of C9orf72 Transcripts In this example, guide RNAs were designed to target exon 3 after the ATG initiation codon of C9orf72 (Table 2). The strategy was to introduce small indels that will lead to early termination codon, thus inducing non-sense mediated decay of C9orf72 transcripts to reduce RNA foci and dipeptide formation.FIG.6Ashows the human C9orf72 gene sequence of exon 3 with the locations of the non-sense mediated decay (NMD) guide RNA 1r and 2f and the location and sequence of PCR indel analysis primers C9NMD Indel F1 and R1 marked.FIG.6Bshows the results of agarose gel electrophoresis of the PCR products amplified by the C9NMD-Indel F1 and R1 PCR primers. In this example, HEK293T cells were transfected with LV-SpCas9 (Control) or LV-NMDgR-SpCas9 plasmid (2 μg) in triplicate.FIG.6Cshows the results of digital droplet PCT (ddPCR) analysis of the C9orf72 RNA levels fromFIG.6B. TABLE 2Guide RNAs generated for“Non-sense mediated decay.”SEQIDguide RNAguide RNA sequenceNO:NMD gRNA 1rUCGAAAUGCAGAGAGUGGUG5NMD gRNA 2fAAUGGGGAUCGCAGCACAUA6 Example 3: Direct Visualization of C90Rf72 Gene Editing in Primary Neurons A mouse model (BAC111) expressing human C9orf72 with 45-65 expanded GGGGCC repeats has been developed. This model contains 6-8 copies of the C9orf72 gene in heterozygous (het) animals and 12-16 copies in homozygous (homo) animals. Additionally, this mouse model expresses Cas9, in addition to C9orf72 with the expansion. In order to determine whether guides successfully excise the GGGGCC repeat in mice primary neurons, appropriate crosses of the BAC111 mice expressing C9orf72 and Cas9 were set up to produce only heterozygous progeny. Primary neurons were isolated at embryonic day 14 (E14), and cultured appropriately. After 4 days in culture, neurons were either treated with PBS alone, or infected with AAV9 single-stranded-GFP (ss-GFP), AAV9-ROSA-tRFP guide RNA (control guide), AAV9-GFP-C9gR flank gRNA 2 & 3, or AAV9-GFP-C9gR flank gRNA 2 & 4. At 72 hours, 25,000 MOI was recorded and the cells were harvested. The DNA was isolated using QIAGEN™ blood and tissue DNA extraction kit. PCR results are shown inFIG.7. The cultured primary neurons were imaged for GFP or RFP fluorescence to visualize the incorporation of AAV9-gRNA constructs to into primary neurons (FIG.8A). In order to determine whether editing occurred in these isolated neuronal cells, a PCR reaction was performed using C9Var1-F and NoER2 primers (FIG.8B). Without gene editing, these primers fail to amplify through the repeat and no band appears on the gel. When gene editing occurs, the repeat is excised out and primers amplify a single band at about 720 base pairs. In both sets of guides a strong band appearing at the right size is observed, while this band is absent in both non-AAV treated neurons (PBS) and those transfected with ss-GFP, or ROSA-tRFP (FIG.8B). In order to estimate the level of unedited DNA, a PCR reaction was performed using NoE-F1 and NoER2 (FIG.8B). A band of about 500 base pairs appears on a gel when gene editing has not occurred. Control gene editing conditions (PBS, ss-GFP, or ROSA-tRFP) produced an intense band at about 500 base pairs, while both sets of gRNA 2 & 3 and gRNA 2 & 4 guides have less unedited samples. To directly visualize gene editing, cultured primary neurons from BAC111 mice expressing human C9orf72 and Cas9 were isolated and treated with PBS, AAV9-ss-GFP, AAV9-ROSA-tRFP, AAV9-gRNA 2 & 3, AAV9-gRNA 2 & 4 as above. Fluorescence in situ hybridization (FISH) was used to visualize unedited C9orf72 RNA (punctate staining, e.g., foci) and nuclei were stained with DAPI (FIG.9). Almost 55-60% of unedited cells have more than ten foci, while edited cells exhibit significantly less in only 35-40% of cells (FIG.9). Example 4: Exogenous Cas9 Promotes C9ORf72 Gene Editing in Primary Neurons To directly test whether C9orf72 excision of GGGGCC repeats requires endogenous Cas9 expression, BAC111 mouse models expressing C9orf72 and not Cas9 were produced. Primary neurons were isolated at embryonic day 14 (E14), and cultured appropriately. After 4 days in culture, neurons were supplemented with Cas9 and either treated with Cas9 alone, or infected with AAV9-ss-GFP+Cas9, AAV9-ROSA-RFP+Cas9 (control guide), AAV9-GFP-C9gR flank gRNA 2 & 3, or AAV9-GFP-C9gR flank gRNA 2 & 4. At 72 hours, 25,000 MOI was recorded and the cells were harvested. The DNA was isolated using QIAGEN™ blood and tissue DNA extraction kit. The cultured primary neurons were imaged for GFP or RFP fluorescence to visualize the incorporation of AAV9-gRNA constructs to into primary neurons (FIG.10A). PCR amplification of edited DNA from cultured neurons was performed. Briefly, edited DNA was amplified by PCR with C9Var1-F & NoER2 (FIG.10B). Amplification bands occur only in edited cells (e.g., cells treated with AAV9-gRNA 2-3+Cas9, or AAV9-gRNA 2-4+Cas9), as shown inFIG.10B. FIG.11shows direct visualization and quantification of gRNAs bound to unedited DNA from primary cultured neurons isolated from BAC111 mice expressing C9 by FISH. Around 55-60% of cells have foci when unedited (Cas9 only, single stranded GFP, ROSA). Foci in edited cells were reduced to 35-40%. Treatment with both gRNA pairs resulted in a significantly different reduction. Tissue distribution of gene editing constructs (e.g., rAAVs) was examined.FIG.12shows gene editing in vivo in BAC111 mice expressing C9/Cas9 injected with PBS, SOD gRNA (control), R9-r1 (gRNA 2 & 4), or R9-f11 (gRNA 2 & 3). Brain, muscle, and liver tissue samples taken after 8 weeks each demonstrated gene editing with gRNA 2 & 3 and gRNA 2 & 4 guides, but not PBS and control SOD gRNA. FIG.13shows FISH data (sense direction) on frontal sections of CAC111 mice that were facially injected at p1-2. The top panel shows a fluorescence micrograph indicating a reduction in number of foci in edited cells compared to untreated and control cells. The bottom panel shows data indicating the reduction is consistent for heterozygous and homozygous mice. FIGS.14A-14Bshow gene editing through stereotaxic striatal brain injections in Baloh and BAC111 mice.FIG.14Ashows the injection site and the brain slice used for tissue isolation.FIG.14Bshows that injection of PBS+Cas9, ROSA-tRFP+Cas9, gRNA 2 & 3+Cas9, gRNA 2 & 4+Cas9 promotes gene editing in Baloh C9 mice and BAC111 C9/Cas9 mice. While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. | 44,045 |
11859180 | DETAILED DESCRIPTION OF THE INVENTION Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21stedition, 2005; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2ndEdition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety. Unless otherwise indicated, the following terms have the following meanings: As used herein, “aqueous solution” means a solution comprising one or more solutes in water. In certain embodiments, the water is sterile water. In certain embodiments, an aqueous solution is saline. As used herein, “antisense oligonucleotide solution” means an aqueous solution comprising one or more antisense oligonucleotides. In certain embodiments, an antisense oligonucleotide solution comprises an aqueous solution having only one antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide solution comprises an aqueous solution having more than one antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide solution comprises an aqueous solution wherein an antisense oligonucleotide is present at a concentration between 0.1 and 500 mg/mL. As used herein, “excipient” means any compound or composition other than water or an antisense oligonucleotide. As used herein, “excipient that modulates viscosity, turbidity or both viscosity and turbidity” or “excipient that modulates viscosity, turbidity or both” means an excipient, the presence of which increases or decreases the viscosity and/or turbidity of an antisense oligonucleotide solution, compared to the viscosity or turbidity of the antisense oligonucleotide solution at the same concentration and temperature in the absence of the excipient. As used herein, “aromatic compound” refers to a mono- or polycyclic carbocyclic ring system having one or more aromatic rings. Preferred aromatic ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aromatic groups as used herein may optionally include further substituent groups. The term “heterocyclic compound” as used herein, refers to a mono-, or poly-cyclic ring system that includes at least one heteroatom and is unsaturated, partially saturated, or fully saturated, thereby including heteroaryl groups and heterocyclic aromatic compounds. Heterocyclic compound is also meant to include fused ring systems wherein one or more of the fused rings contain at least one heteroatom and the other rings can contain one or more heteroatoms or optionally contain no heteroatoms. A heterocyclic compound typically includes at least one atom selected from sulfur, nitrogen or oxygen. In certain embodiments, a heterocyclic compound may include one or more rings, wherein each ring has one or more heteroatoms. In certain embodiments, a heterocyclic compound includes a monocyclic ring system with one or more heteroatoms. In certain embodiments, a heterocyclic compound includes a monocyclic ring system with two or more heteroatoms. Examples of heterocyclic compounds include, but are not limited to, [1,3]dioxolane, pyrrolidine, pyrazoline, pyrazolidine, imidazoline, imidazolidine, piperidine, piperazine, oxazolidine, isoxazolidine, morpholine, thiazolidine, isothiazolidine, quinoxaline, pyridazinone, tetrahydrofuran and the like. Heterocyclic compounds as used herein may optionally include further substituent groups. As used herein, “heterocyclic aromatic compound” means any compound comprising a mono- or poly-cyclic aromatic ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heterocyclic aromatic compounds also include fused ring systems, including systems where one or more of the fused rings contain no heteroatoms. Heterocyclic aromatic compounds typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heterocyclic aromatic compounds groups include without limitation, pyridine, pyrazine, pyrimidine, pyrrole, pyrazole, imidazole, thiazole, oxazole, isoxazole, thiadiazole, oxadiazole, thiophene, furan, quinoline, isoquinoline, benzimidazole, benzooxazole, quinoxaline and the like. Heterocyclic aromatic compounds can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heterocyclic aromatic compounds as used herein may optionally include further substituent groups. As used herein, “nucleoside” means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety. As used herein, “chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. As used herein, “furanosyl” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom. As used herein, “naturally occurring sugar moiety” means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA. As used herein, “sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside. As used herein, “modified sugar moiety” means a substituted sugar moiety, a bicyclic or tricyclic sugar moiety, or a sugar surrogate. As used herein, “substituted sugar moiety” means a furanosyl comprising at least one substituent group that differs from that of a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyl comprising substituents at the 2′-position, the 3′-position, the 5′-position and/or the 4′-position. As used herein, “2′-substituted sugar moiety” means a furanosyl comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring. As used herein, “MOE” means —OCH2CH2OCH3. As used herein, “bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl. As used herein the term “sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside is capable of (1) incorporation into an oligonucleotide and (2) hybridization to a complementary nucleoside. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholino, modified morpholinos, cyclohexenyls and cyclohexitols. As used herein, “nucleotide” means a nucleoside further comprising a phosphate linking group. As used herein, “linked nucleosides” may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.” As used herein, “linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked). As used herein, “nucleobase” means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified. As used herein, “heterocyclic base” or “heterocyclic nucleobase” means a nucleobase comprising a heterocyclic structure. As used herein the terms, “unmodified nucleobase” or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U). As used herein, “modified nucleobase” means any nucleobase that is not a naturally occurring nucleobase. As used herein, “modified nucleoside” means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase. As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety. As used herein, “constrained ethyl nucleoside” or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)—O-2′ bridge. As used herein, “locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH2—O-2′ bridge. As used herein, “2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted nucleoside is not a bicyclic nucleoside. As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil). As used herein, “oligonucleotide” means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides. As used herein “oligonucleoside” means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage. As used herein “internucleoside linkage” means a covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein “naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage. As used herein, “modified internucleoside linkage” means any internucleoside linkage other than a naturally occurring internucleoside linkage. As used herein, “oligomeric compound” means a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. As used herein, “terminal group” means one or more atom attached to either, or both, the 3′ end or the 5′ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides. As used herein, “conjugate” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties. As used herein, “conjugate linking group” means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound. As used herein, “antisense compound” means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity. As used herein, “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid. As used herein, “detecting” or “measuring” means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed. As used herein, “detectable and/or measureable activity” means a statistically significant activity that is not zero. As used herein, “essentially unchanged” means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero. As used herein, “expression” means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5′-cap), and translation. As used herein, “target nucleic acid” means a nucleic acid molecule to which an antisense compound hybridizes. As used herein, “mRNA” means an RNA molecule that encodes a protein. As used herein, “pre-mRNA” means an RNA transcript that has not been fully processed into mRNA. Pre-RNA includes one or more intron. As used herein, “transcript” means an RNA molecule transcribed from DNA. Transcripts include, but are not limited to mRNA, pre-mRNA, and partially processed RNA. As used herein, “targeting” or “targeted to” means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of a target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions. As used herein, “nucleobase complementarity” or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity. As used herein, “non-complementary” in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another. As used herein, “complementary” in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions. Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary. As used herein, “hybridization” means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. As used herein, “specifically hybridizes” means the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In certain embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site. As used herein, “percent complementarity” means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound. As used herein, “percent identity” means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid. As used herein, “modulation” means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, function, or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include a change in splice site selection of pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice-variant compared to the amount in the absence of modulation. As used herein, “motif” means a pattern of chemical modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound. As used herein, “nucleoside motif” means a pattern of nucleoside modifications in an oligomeric compound or a region thereof. The linkages of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited. As used herein, “sugar motif” means a pattern of sugar modifications in an oligomeric compound or a region thereof. As used herein, “linkage motif” means a pattern of linkage modifications in an oligomeric compound or region thereof. The nucleosides of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited. As used herein, “nucleobase modification motif” means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence. As used herein, “sequence motif” means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications. As used herein, “type of modification” in reference to a nucleoside or a nucleoside of a “type” means the chemical modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside. As used herein, “differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified. As used herein, “the same type of modifications” refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleoside have “the same type of modification,” even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases. As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline. As used herein, “substituent” and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2′-substuent is any atom or group at the 2′-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the present invention have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound. Likewise, as used herein, “substituent” in reference to a chemical functional group means an atom or group of atoms differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)Raa), carboxyl (—C(O)O—Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (—O—Raa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (—N(Rbb)(Rcc)), imino(═NRbb), amido (—C(O)N(Rbb)(Rcc) or —N(Rbb)C(O)Raa), azido (—N3), nitro (—NO2), cyano (—CN), carbamido (—OC(O)N(Rbb)(Rcc) or —N(Rbb)C(O)ORaa), ureido (—N(Rbb)C(O)N(Rbb)(Rcc)), thioureido (—N(Rbb)C(S)N(Rbb)—(Rcc)), guanidinyl (—N(Rbb)C(═NRbb)N(Rbb)(Rcc)), amidinyl (—C(═NRbb)N(Rbb)(Rcc) or —N(Rbb)C(═NRbb)(Raa)), thiol (—SRbb), sulfinyl (—S(O)Rbb), sulfonyl (—S(O)2Rbb) and sulfonimidoyl (—S(O)2N(Rbb)(Rcc) or —N(Rbb)S—(O)2Rbb). Wherein each Raa, Rbband Rccis, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree. As used herein, “alkyl,” as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12alkyl) with from 1 to about 6 carbon atoms being more preferred. As used herein, “alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups. As used herein, “alkynyl,” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups. As used herein, “acyl,” means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups. As used herein, “alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups. As used herein, “aliphatic” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups. As used herein, “alkoxy” means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups. As used herein, “aminoalkyl” means an amino substituted C1-C12alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions. As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that is covalently linked to a C1-C12alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group. As used herein, “aryl” and mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups. As used herein, “halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine. As used herein, “heteroaryl,” mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups. Oligomeric Compounds In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, such oligomeric compounds comprise oligonucleotides optionally comprising one or more conjugate and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. In certain embodiments, oligonucleotides comprise one or more chemical modifications. Such chemical modifications include modifications one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage. Certain Sugar Moieties In certain embodiments, oligomeric compounds of the invention comprise one or more modified nucleosides comprising a modified sugar moiety. Such oligomeric compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to oligomeric compounds comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are substituted sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties. In certain embodiments, modified sugar moieties are substituted sugar moieties comprising one or more substituent, including but not limited to substituents at the 2′ and/or 5′ positions. Examples of sugar substituents suitable for the 2′-position, include, but are not limited to: 2′-F, 2′-OCH3(“OMe” or “O-methyl”), and 2′-O(CH2)2OCH3(“MOE”). In certain embodiments, sugar substituents at the 2′ position is selected from allyl, amino, azido, thio, O-allyl, O—C1-C10alkyl, O—C1-C10substituted alkyl; O—C1-C10alkoxy; O—C1-C10substituted alkoxy, OCF3, O(CH2)2SCH3, O(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10alkyl. Examples of sugar substituents at the 5′-position, include, but are not limited to: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certain embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties (see, e.g., PCT International Application WO 2008/101157, for additional 5′,2′-bis substituted sugar moieties and nucleosides). Nucleosides comprising 2′-substituted sugar moieties are referred to as 2′-substituted nucleosides. In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from halo, allyl, amino, azido, O—C1-C10alkoxy; O—C1-C10substituted alkoxy, SH, CN, OCN, CF3, OCF3, O-alkyl, S-alkyl, N(Rm)-alkyl; O-alkenyl, S-alkenyl, or N(Rm)-alkenyl; O-alkynyl, S-alkynyl, N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rmand Rnis, independently, H, an amino protecting group or substituted or unsubstituted C1-C10alkyl. These 2′-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl. In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from F, NH2, N3, OCF3, O—CH3, O(CH2)3NH2, CH2—CH═CH2, O—CH2—CH═CH2, OCH2CH2OCH3, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O—CH2—C(═O)—N(Rm)(Rn) where each Rmand Rnis, independently, H, an amino protecting group or substituted or unsubstituted C1-C10alkyl. In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, OCF3, O—CH3, OCH2CH2OCH3, O(CH2)2SCH3, O—(CH2)2—O—N(CH3)2, —O(CH2)2O(CH2)2N(CH3)2, and O—CH2—C(═O)—N(H)CH3. In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, O—CH3, and OCH2CH2OCH3. Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ sugar substituents, include, but are not limited to: —[C(Ra)(Rb)]n—, —[C(Ra)(Rb)]n—O—, —C(RaRb—)N(R)—O— or, —C(RaRb)—O—N(R)—; 4′-CH2-2′, 4′-(CH2)2-2′, 4′-(CH2)3-2′, 4′-(CH2)—O-2′ (LNA); 4′-(CH2)—S-2′; 4′-(CH2)2—O-2′ (ENA); 4′-CH(CH3)—O-2′ (cEt) and 4′-CH(CH2OCH3)—O-2′, and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH3)(CH3)—O-2′ and analogs thereof, (see, e.g., WO2009/006478, published Jan. 8, 2009); 4′-CH2—N(OCH3)-2′ and analogs thereof (see, e.g., WO2008/150729, published Dec. 11, 2008); 4′-CH2—O—N(CH3)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004); 4′-CH2—O—N(R)-2′, and 4′-CH2—N(R)—O-2′-, wherein each R is, independently, H, a protecting group, or C1-C12alkyl; 4′-CH2—N(R)—O-2′, wherein R is H, C1-C12alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2—C(H)(CH3)-2′ (see, e.g., Chattopadhyaya, et al.,J. Org. Chem.,2009, 74, 118-134); and 4′-CH2—C(═CH2)-2′ and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008). In certain embodiments, such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from —[C(Ra)(Rb)]n—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —C(═NRa)—, —C(═O)—, —C(═S)—, —O—, —Si(Ra)2—, —S(═O)x—, and —N(Ra)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each Raand Rbis, independently, H, a protecting group, hydroxyl, C1-C12alkyl, substituted C1-C12alkyl, C2-C12alkenyl, substituted C2-C12alkenyl, C2-C12alkynyl, substituted C2-C12alkynyl, C5-C20aryl, substituted C5-C20aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7alicyclic radical, substituted C5-C7alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and each J1and J2is, independently, H, C1-C12alkyl, substituted C1-C12alkyl, C2-C12alkenyl, substituted C2-C12alkenyl, C2-C12alkynyl, substituted C2-C12alkynyl, C5-C20aryl, substituted C5-C20aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12aminoalkyl, substituted C1-C12aminoalkyl, or a protecting group. Nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4′-CH2—O-2′) BNA, (B) β-D-Methyleneoxy (4′-CH2—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4′-(CH2)2—O-2′) BNA, (D) Aminooxy (4′-CH2—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH2—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy) (4′-CH(CH3)—O-2′) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4′-CH2—S-2′) BNA, (H) methylene-amino (4′-CH2—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH2—CH(CH3)-2′) BNA, and (J) propylene carbocyclic (4′-(CH2)3-2′) BNA as depicted below. wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C1-C12alkyl. Additional bicyclic sugar moieties are known in the art, for example: Singh et al.,Chem. Commun.,1998, 4, 455-456; Koshkin et al.,Tetrahedron,1998, 54, 3607-3630; Wahlestedt et al.,Proc. Natl. Acad. Sci. U.S.A.,2000, 97, 5633-5638; Kumar et al.,Bioorg. Med. Chem. Lett.,1998, 8, 2219-2222; Singh et al.,J. Org. Chem.,1998, 63, 10035-10039; Srivastava et al.,J. Am. Chem. Soc.,129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al.,Curr. Opinion Invens. Drugs,2001, 2, 558-561; Braasch et al.,Chem. Biol.,2001, 8, 1-7; Orum et al.,Curr. Opinion Mol. Ther.,2001, 3, 239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Applications Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922. In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH2—O-2′) bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al.,Nucleic Acids Research,2003, 21, 6365-6372). In certain embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinyl group). In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the naturally occurring sugar is substituted, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above. For example, certain sugar surrogates comprise a 4′-sulfur atom and a substitution at the 2′-position (see, e.g., published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005) and/or the 5′ position. By way of additional example, carbocyclic bicyclic nucleosides having a 4′-2′ bridge have been described (see, e.g., Freier et al.,Nucleic Acids Research,1997, 25(22), 4429-4443 and Albaek et al.,J. Org. Chem.,2006, 71, 7731-7740). In certain embodiments, sugar surrogates comprise rings having other than 5-atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J.Bioorg. &Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula VII: wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII: Bx is a nucleobase moiety; T3and T4are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T3and T4is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T3and T4is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group; q1, q2, q3, q4, q5, q6and q7are each, independently, H, C1-C6alkyl, substituted C1-C6alkyl, C2-C6alkenyl, substituted C2-C6alkenyl, C2-C6alkynyl, or substituted C2-C6alkynyl; and each of R1and R2is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2, and CN, wherein X is O, S or NJ1, and each J1, J2, and J3is, independently, H or C1-C6alkyl. In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q1, q2, q3, q4, q5, q6and q7are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6and q7is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6and q7is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R1and R2is F. In certain embodiments, R1is fluoro and R2is H, R1is methoxy and R2is H, and R1is methoxyethoxy and R2is H. Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used to modify nucleosides (see, e.g., review article: Leumann, J. C,Bioorganic&Medicinal Chemistry,2002, 10, 841-854). In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following structure: In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.” Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′, 2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH2—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al.,J. Am. Chem. Soc.2007, 129(26), 8362-8379). Certain Nucleobases In certain embodiments, nucleosides of the present invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise one or more modified nucleobases. In certain embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288. Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety. Certain Internucleoside Linkages In certain embodiments, the present invention provides oligomeric compounds comprising linked nucleosides. In such embodiments, nucleosides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P═S). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (—CH2—N(CH3)—O—CH2—), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2—O—); and N,N′-dimethylhydrazine (—CH2—N(CH3)—N(CH3)—). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art. The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), α or β such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms. Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH2—N(CH3)—O-5′), amide-3 (3′-CH2—C(═O)—N(H)-5′), amide-4 (3′-CH2—N(H)—C(═O)-5′), formacetal (3′-O—CH2—O-5′), and thioformacetal (3′-S—CH2—O-5′). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example:Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2component parts. Certain Motifs In certain embodiments, the present invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modification. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides comprising modified sugars. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides comprising one or more modified nucleobases. In certain embodiments, chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemically modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif. In certain embodiments, the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another. Thus, an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemical modifications to the nucleobases independent of the sequence of nucleobases). Certain Sugar Motifs In certain embodiments, oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications. In certain embodiments, the oligonucleotides comprise or consist of a region having a gapmer sugar modification motif, which comprises two external regions or “wings” and an internal region or “gap.” The three regions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar modification motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar modification motifs of the 5′-wing differs from the sugar modification motif of the 3′-wing (asymmetric gapmer). In certain embodiments, oligonucleotides comprise 2′-MOE modified nucleosides in the wings and 2′-F modified nucleosides in the gap. In certain embodiments, oligonucleotides are fully modified. In certain such embodiments, oligonucleotides are uniformly modified. In certain embodiments, oligonucleotides are uniform 2′-MOE. In certain embodiments, oligonucleotides are uniform 2′-F. In certain embodiments, oligonucleotides are uniform morpholino. In certain embodiments, oligonucleotides are uniform BNA. In certain embodiments, oligonucleotides are uniform LNA. In certain embodiments, oligonucleotides are uniform cEt. In certain embodiments, oligonucleotides comprise a uniformly modified region and additional nucleosides that are unmodified or differently modified. In certain embodiments, the uniformly modified region is at least 5, 10, 15, or 20 nucleosides in length. In certain embodiments, the uniform region is a 2′-MOE region. In certain embodiments, the uniform region is a 2′-F region. In certain embodiments, the uniform region is a morpholino region. In certain embodiments, the uniform region is a BNA region. In certain embodiments, the uniform region is a LNA region. In certain embodiments, the uniform region is a cEt region. In certain embodiments, the oligonucleotide does not comprise more than 4 contiguous unmodified 2′-deoxynucleosides. In certain circumstances, antisesense oligonucleotides comprising more than 4 contiguous 2′-deoxynucleosides activate RNase H, resulting in cleavage of the target RNA. In certain embodiments, such cleavage is avoided by not having more than 4 contiguous 2′-deoxynucleosides, for example, where alteration of splicing and not cleavage of a target RNA is desired. Certain Internucleoside Linkage Motifs In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif, as described above for sugar modification motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The sugar modification motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped sugar modification motif and if it does have a gapped sugar motif, the wing and gap lengths may or may not be the same. In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate. In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide. Certain Nucleobase Modification Motifs In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain such embodiments, nucleobase modifications are arranged in a gapped motif. In certain embodiments, nucleobase modifications are arranged in an alternating motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically modified. In certain embodiments, oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 3′-end of the oligonucleotide. In certain such embodiments, the block is at the 5′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 5′-end of the oligonucleotide. In certain embodiments, nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide. For example, in certain embodiments each purine or each pyrimidine in an oligonucleotide is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each cytosine is modified. In certain embodiments, each uracil is modified. In certain embodiments, some, all, or none of the cytosine moieties in an oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methyl cytosine is not a “modified nucleobase.” Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain embodiments, the methylation state of all or some cytosine nucleobases is specified. Certain Overall Lengths In certain embodiments, the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the invention provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X≤Y. For example, in certain embodiments, the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides of an oligomeric compound or oligonucleotide is limited, whether to a range or to a specific number, the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents. For example, an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents. In certain embodiments, a gapmer oligonucleotide has any of the above lengths. One of skill in the art will appreciate that certain lengths may not be possible for certain motifs. For example: a gapmer having a 5′-wing region consisting of four nucleotides, a gap consisting of at least six nucleotides, and a 3′-wing region consisting of three nucleotides cannot have an overall length less than 13 nucleotides. Thus, one would understand that the lower length limit is 13 and that the limit of 10 in “10-20” has no effect in that embodiment. Further, where an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range. For example, an oligonucleotide consisting of 20-25 linked nucleosides comprising a 5′-wing consisting of 5 linked nucleosides; a 3′-wing consisting of 5 linked nucleosides and a central gap consisting of 10 linked nucleosides (5+5+10=20) may have up to 5 nucleosides that are not part of the 5′-wing, the 3′-wing, or the gap (before reaching the overall length limitation of 25). Such additional nucleosides may be 5′ of the 5′-wing and/or 3′ of the 3′ wing. Certain Oligonucleotides In certain embodiments, oligonucleotides of the present invention are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. Thus, the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region. Likewise, such sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. Herein if a description of an oligonucleotide or oligomeric compound is silent with respect to one or more parameter, such parameter is not limited. Thus, an oligomeric compound described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase modification motif. Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence. Certain Conjugate Groups In certain embodiments, oligomeric compounds are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). In certain embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. In certain embodiments, conjugate groups are directly attached to oligonucleotides in oligomeric compounds. In certain embodiments, conjugate groups are attached to oligonucleotides by a conjugate linking group. In certain such embodiments, conjugate linking groups, including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound. In general a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like. Some nonlimiting examples of conjugate linking moieties include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C10alkyl, substituted or unsubstituted C2-C10alkenyl or substituted or unsubstituted C2-C10alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl. Conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position. In certain embodiments, conjugate groups are at the 3′-end of an oligonucleotide of an oligomeric compound. In certain embodiments, conjugate groups are near the 3′-end. In certain embodiments, conjugates are attached at the 3′ end of an oligomeric compound, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group. In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, oligomeric compounds comprise an oligonucleotide. In certain embodiments, an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups. Such conjugate and/or terminal groups may be added to oligonucleotides having any of the chemical motifs discussed above. Thus, for example, an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group. Antisense Compounds In certain embodiments, oligomeric compounds of the present invention are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid. In certain embodiments, a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid non-specific hybridization to any non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays). In certain embodiments, the present invention provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length. TABLE 1 below provides certain non-limiting examples of antisense compounds and their targets: TABLE 1Antisense CompoundsTargetISIS NoIndicationSequenceChemistrySEQ ID NOFactor XI416858ClottingACGGCATTGGTGCACAGTTT5-10-5 MOE1disorderTTR420915AmyloidesTCTTGGTTACATGAAATCCC5-10-5 MOE2Apo(a)494372CADTGCTCCGTTGGTGCTTGTTC5-10-5 MOE3Alpha1-487660Liver diseaseCCAGCTCAACCCTTCTTTAA5-10-5 MOE4antitrypsinPTP-1B404173DiabetesAATGGTTTATTCCATGGCCA5-10-5 MOE5GCGR449884DiabetesGGTTCCCGAGGTGCCCA3-10-4 MOE6DGAT2501861NASHTCACAGAATTATCAGCAGTA5-10-5 MOE7Factor VII540175Cancer-GGACACCCACGCCCCC3-10-38associatedcEt/MOEthrombosisSMN396443SMATCACTTTCATAATGCTGGUmiform9MOEFGFR4463588ObesityGCACACTCAGCAGGACCCCC5-10-5 MOE10apoB-100301012HighGCCTCAGTCTGCTTCGCACC5-10-5 MOE11CholesterolCRP329993CADAGCATAGTTAACGAGCTCCC5-10-5 MOE12ApoC-III304801HighAGCTTCTTGTCCAGCTTTAT5-10-5 MOE13triglyceridesGCCR426115DiabetesGCAGCCATGGTGATCAGGAG5-10-5 MOE14STAT3481464CancerCTATTTGGATGTCAGC3-10-3 (S)-cEt15eIF-4E183750CancerTGTCATATTCCTGGATCCTT5-10-5 MOE16SOD1333611ALSCCGTCGCCCTTCAGCACGCA5-10-5 MOE17GHR227452AcromegalyTCAGGGCATTCTTTCCATTC5-10-5 MOE18Clusterin112989CancerCAGCAGCAGAGTCTTCATCAT4-13-4 MOE19Hsp27306053CancerGGGACGCGGCGCTCGGTCAT4-12-4 MOE20CMV2922RetinitisGCGTTTGCTCTTCTTCTTGCGUniform deoxy21ICAM-12302UlcerativeGCCCAAGCTGGCATCCGTCAUniform deoxy22colitisVLA-4107248MultipleCTGAGTCTGTTTTCCATTCT3-9-8 MOE23sclerosisCTGF412294FibrosisGTTTGACATGGCACAATGTT2-13-5 MOE24c-raf kinase13650Ocular diseaseTCCCGCCTGTGACATGCATT6-8-6 MOE25 Certain Target Nucleic Acids and Mechanisms In certain embodiments, antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain embodiments, an antisense oligonucleotide modulates splicing of a pre-mRNA. Certain Properties of Antisense Compounds Viscosity In certain embodiments, antisense oligonucleotide solutions possess varying degrees of viscosity, and the viscosity of antisense oligonucleotide solutions depends on many factors. Some factors include, but are not limited to, length, nucleobase sequence, nucleobase modifications, nucleobase modification motif, and/or sugar modification of the antisense oligonucleotide. In certain embodiments, the viscosity of an antisense oligonucleotide solution may be difficult to predict. In certain embodiments an antisense oligonucleotide solution may exhibit a relatively high viscosity, while an antisense oligonucleotide solution comprising a similar antisense oligonucleotide (partially homologous sequence, similar modifications, etc.) may exhibit a relatively low viscosity. In certain embodiments it may be desireable to lower the viscosity of a given antisense oligonucleotide solution, for example, in certain embodiments it may be desirable to to lower the viscosity of a given antisense oligonucleotide solution so that higher amounts of an antisense oligonucleotide may be present in a given volume of solution. In certain embodiments it may be desirable to increase the viscosity of a given antisense oligonucleotide solution. In certain embodiments, viscosity may be increased or decreased depending on a variety of factors, e.g. concentration, volume of solute, temperature, and/or pH. In certain embodiments, the desireable viscosity varies. In certain embodiments it may be desirable to increase or decrease viscosity, depending on the particular route of delivery or application a given antisense oligonucleotide solution. In certain embodiments, viscosity is often a concentration-limiting factor. While not wishing to be bound by theory, in certain embodiments, as the concentration of an antisense oligonucleotide is increased, interactions between antisense oligonucleotide molecules likewise increase. In certain embodiments, certain antisense oligonucleotides may interact to form aggregates which may increase viscosity and/or turbidity. Certain antisense oligonucleotides may interact to form antisense oligonucleotide polymers, which may increase viscosity and/or turbidity. In certain embodiments, as the concentration of an antisense oligonucleotide in solution increases, the viscosity of the antisense oligonucleotide solution also increases. In certain embodiments, it is undesirable to have an antisense oligonucleotide solution having a high viscosity. For example, if the viscosity of an antisense oligonucleotide solution is too high, then it may become difficult to manufacture an antisense oligonucleotide solution including but not limited to an antisense oligonucleotide solution drug product. As another example, if the viscosity of an antisense oligonucleotide solution is too high, filtration time during manufacturing may increase, adding time and cost to the manufacturing process. As another example, if the viscosity of an antisense oligonucleotide solution is too high, it may become more difficult to pre-fill syringes with accurate and precise amounts of the antisense oligonucleotide solution. As another example, if the viscosity of an antisense oligonucleotide solution is too high, it may become more difficult to administer to an animal, human, or patient. As another example, if the viscosity of an antisense oligonucleotide solution is too high, drug clearance from the subcutaneous injection site may be slowed. As another example, if the viscosity of an antisense oligonucleotide solution is too high, a larger gauge needle may be required to effectively administer doses of the antisense oligonucleotide solution, which may exacerbate discomfort upon injection. In certain embodiments, it is therefore desirable to have an antisense oligonucleotide solution having both a high concentration of antisense oligonucleotide and low viscosity. In certain embodiments, if the viscosity of an antisense oligonucleotide solution is too high, then the concentration of the antisense oligonucleotide in solution may be reduced to reduce the viscosity of the antisense oligonucleotide solution to a desirable level. The reduction of the concentration of an antisense oligonucleotide in the antisense oligonucleotide solution may be undesirable for several reasons. For example, as the concentration of an antisense oligonucleotide in an antisense oligonucleotide solution is decreased, animals, humans, and/or patients must receive a larger volume of antisense oligonucleotide solution to receive the desired amount of an antisense oligonucleotide. In certain embodiments, it is therefore desirable to reduce the viscosity of an antisense oligonucleotide solution without reducing the concentration of the antisense oligonucleotide within the antisense oligonucleotide solution. In certain embodiments, it is therefore desirable to have an antisense oligonucleotide solution having both a high concentration of antisense oligonucleotide and low viscosity. In certain embodiments, certain antisense oligonucleotide solutions having desirable concentrations of antisense oligonucleotides have undesirably high viscosities. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both may reduce the viscosity of a certain antisense oligonucleotide solution to a desirable level. In certain embodiments described herein, it is recognized that viscosity of an oligonucleotide solution may vary with temperature. In certain embodiments, the viscosity may be expressed as a range of cP units relative to a concentration of oligonucleotide in solution. For example, in certain embodiments, the viscosity of the antisense oligonucleotide solution is less than 40 cP when the concentration of the antisense oligonucleotide is between 40 to 60 mg/mL. In certain such embodiments, the viscocity of the solution and the concentration of the oligonucleotide represent an approximate measurement of viscocity and approximate concentration of oligonucleotide at a given temperature or range of temperatures. For example, in certain embodiments described herein, the temperature of the solution for which viscocity is measured is about 25 degrees Celsius. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 40 to 60 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 45 to 55 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 80 to 120 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 90 to 110 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 140 to 160 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 165 to 185 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 180 to 220 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 190 to 210 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 210 to 230 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 230 to 260 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 245 to 255 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 260 to 300 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the concentration of the antisense oligonucleotide is between 300 to 400 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the antisense oligonucleotide has one or more modified sugars having 2′-MOE modifications. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the antisense oligonucleotide has one or more modified sugars having 2′-OMe modifications. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the antisense oligonucleotide has one or more modified sugars having 2′-F modifications. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the antisense oligonucleotide has one or more modified sugars having LNA modifications. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the viscosity of the antisense oligonucleotide solution to less than 40 cP when the antisense oligonucleotide has one or more modified sugars having cEt modifications. Turbidity While not wishing to be bound by theory, the presence of turbidity in antisense oligonucleotide solutions is associated with the formation of antisense oligonucleotide strand aggregates. In certain embodiments, the presence of turbidity in antisense oligonucleotide solutions makes the antisense oligonucleotide solution appear white and cloudy. In certain embodiments, the presence of turbidity in antisense oligonucleotide solutions makes the antisense oligonucleotide solution appear to contain small particles. In certain embodiments, it is desirable to have antisense oligonucleotide solutions that have low turbidity. In certain embodiments, it is desirable to have antisense oligonucleotide solutions that appear clear and particle-free when viewed by the naked eye. In certain embodiments, it is desirable to have antisense oligonucleotide solutions that have turbidity below 20 NTU. In certain embodiments, turbidiy may be induced via a freeze-thaw method, wherein an antisense oligonucleotide solution is frozen and then rapidly thawed, as described herein. In certain embodiments, the freese-thaw method induces turbidity wherein a given antisense oligonucleotide solution would not normally demonstrate turbidity, or wherein a given antisense oligonucleotide solution would demonstrate turbidity after a long period of time. In certain embodiments turbidity represents an aesthetic problem, but does not affect the efficacy or safety of a given antisense oligonucleotide solution. In certain embodiments, certain antisense oligonucleotide solutions having desirable concentrations of antisense oligonucleotides have undesirably high turbidity. In certain embodiments, certain antisense oligonucleotide solutions having desirable concentrations of antisense oligonucleotides have undesirably high turbidity wherein the antisense oligonucleotide solution has a cloudy or milky white appearance. In certain embodiments, certain antisense oligonucleotide solutions having desirable concentrations of antisense oligonucleotides have undesirably high turbidity wherein the antisense oligonucleotide solution appears to have small amounts of particulate interspersed throughout the antisense oligonucleotide solution. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both may reduce the turbidity of a certain antisense oligonucleotide solution to a desirable level. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both may reduce the turbidity of a certain antisense oligonucleotide solution from having a cloudy appearance to a having a clear appearance to the naked eye. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both may reduce the turbidity of a certain antisense oligonucleotide solution from having a milky white appearance to a having a clear appearance to the naked eye. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both may reduce the turbidity of a certain antisense oligonucleotide solution from visible particles to a having a clear particle-free appearance to the naked eye. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 40 to 60 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 45 to 55 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 80 to 120 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 90 to 110 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 140 to 160 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 165 to 185 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 180 to 220 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 190 to 210 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 210 to 230 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 230 to 260 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 245 to 255 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 260 to 300 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the concentration of the antisense oligonucleotide is between 300 to 400 mg/mL. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the antisense oligonucleotide has one or more modified sugars having 2′-MOE modifications. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the antisense oligonucleotide has one or more modified sugars having 2′-OMe modifications. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the antisense oligonucleotide has one or more modified sugars having 2′-F modifications. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the antisense oligonucleotide has one or more modified sugars having LNA modifications. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution reduces the turbidity of the antisense oligonucleotide solution to less than 20 NTU when the antisense oligonucleotide has one or more modified sugars having cEt modifications. Osmolarity While not wishing to be bound by theory, antisense oligonucleotide solutions possess varying concentrations of osmolarity, and the osmolarity of antisense oligonucleotide solutions depends on many factors. Some factors include but are not limited to, length, nucleobase sequence, nucleobase modifications, nucleobase modification motif, and/or sugar modification of the antisense oligonucleotide. In certain embodiments, the osmolarity of an antisense oligonucleotide solution may be difficult to predict. In certain embodiments an antisense oligonucleotide solution may exhibit a relatively high concentrations of osmolarity, while a similar antisense oligonucleotide solution may exhibit a relatively low concentrations of osmolarity. In certain embodiments, certain antisense oligonucleotide solutions having desirable concentrations of antisense oligonucleotides have undesirably low osmolarity, and are hypotonic. In certain embodiments, it is undesirable to have an antisense oligonucleotide solution having low osmolarity, and hypotonicity. For example, if the osmoloarity of an antisense oligonucleotide solution is too low, then an animal, human, or patient may experience pain at the injection site. In certain embodiments, it is therefore desirable to increase the osmolarity of an antisense oligonucleotide solution. In certain embodiments, it is therefore desirable to have the antisense oligonucleotide solution become isotonic. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution increases the osmolarity of the antisense oligonucleotide solution. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution increases the osmolarity of the antisense oligonucleotide solution wherein the antisense oligonucleotide solution becomes isotonic. pH While not wishing to be bound by theory, antisense oligonucleotide solutions possess varying pH levels, and the pH levels of antisense oligonucleotide solutions depends on many factors. Some factors include, but are not limited to length, nucleobase sequence, nucleobase modifications, nucleobase modification motif, and/or sugar modification of the antisense oligonucleotide. In certain embodiments, the pH of an antisense oligonucleotide solution may be difficult to predict. In certain embodiments an antisense oligonucleotide solution may exhibit a relatively high pH, while an antisense oligonucleotide solution of a similar antisense oligonucleotide may exhibit a relatively low pH. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution alters the pH of the antisense oligonucleotide solution. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution increases pH of the antisense oligonucleotide solution. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution decreases pH of the antisense oligonucleotide solution. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution buffers pH of the antisense oligonucleotide solution. In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution buffers the pH of the antisense oligonucleotide solution around a pH of 8.0. (e.g., 7.8 to 8.2) In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution buffers the pH of the antisense oligonucleotide solution around a pH of 7.4. (e.g., 7.2-7.6) In certain embodiments, the addition of one or more excipients that modulate viscosity, turbidity or both to the antisense oligonucleotide solution buffers the pH of the antisense oligonucleotide solution around a pH of 7.0. (e.g. 6.8-7.2). Antimicrobial In certain embodiments it is desirable to include an antimicrobial agent to an antisense oligonucleotide solution to facilitate storage or delivery. In certain embodiments it is not necessary to include an antimicrobial agent to an antisense oligonucleotide solution to facilitate storage or delivery of the antisense oligonucleotide solution. In certain embodiments an antimicrobial agent is added to an antisense oligonucleotide solution. In certain embodiments no antimicrobial agents are added to an antisense oligonucleotide solution. In certain embodiments antisense oligonucleotide solutions are prepared without antimicrobial agents. In certain embodiments one or more excipients that modulate viscosity, turbidity or both may also serve as an antimicrobial agent or preservative. In certain embodiments one or more excipients that modulate viscosity, turbidity or both may not have any antimicrobial or preservative properties. Certain Pharmaceutical Compositions In certain embodiments, the present invention provides pharmaceutical compositions comprising one or more antisense compound. In certain embodiments, such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile water. In certain embodiments, the sterile saline is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical grade PBS. In certain embodiments, antisense compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered. Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising antisense compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. A prodrug can include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active antisense oligomeric compound. Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue. In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used. In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody. In certain embodiments, a pharmaceutical composition provided herein comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions. In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration. In certain of such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. In certain embodiments, a pharmaceutical composition provided herein comprises an oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. In certain embodiments, one or more modified oligonucleotide provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form. In certain embodiments, the present invention provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human. In certain embodiments, the present invention provides methods of administering a pharmaceutical composition comprising an oligomeric compound of the present invention to an animal. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the eyes, ears). Certain Excipients As reported in scientific or patent literature, some oligonucleotide compounds form aggregates when in solution and aggregation may causes undesired viscosity, turbidity, or both. In certain embodiments, oligonucleotide solutions having high viscosity are undesirable because it complicates delivery through a syringe. For example, oligonucleotide solutions having high viscosity may only effectively get delivered using a high gauge needle which may cause excess pain or discomfort to a patient. In certain embodiments, oligonucleotide solutions having high turbidity are undesirable because the oligonucleotide solution may appear to have one or more impurities present. In certain embodiments, oligonucleotide solutions having additives can mitigate only turbidity or only viscosity (see US 20110098343). In certain embodiments, this disclosure provides that both turbidity and viscosity can be comitigated by a single excipient, for example, L-tryptophan, pyridoxine, L-Phenylalanine, and nicotinamide. In certain embodiments, additional excipients that have potential to mitigate both turbidity and viscosity include but are not limited to thymine, adenine, riboflavin, thiamine, and tryptamine. In certain embodiments, effective excipients have heterocyclic character and/or an ability to act as a hydrogen bond donor and/or a hydrogen bond acceptor. In certain embodiments, excipients with nonaromatic rings may not be effective for viscosity reduction. In certain embodiments, certain properties of effective excipients, e.g. (i) aromatic homocyclicity or heterocyclicity and (ii) the ability to act as a hydrogen bond donor and/or acceptor, may be consolidated into one excipient or compound. In certain embodiments, two or more excipients may together possess one or more properties of effective excipients. For example, in certain embodiments, a first excipient may be an aromatic homocyclic or heterocyclic compound but may not have the ability to act as a hydrogen bond donor and/or acceptor. In certain such embodiments, a second excipient may have the ability to act as a hydrogen bond donor and/or acceptor, but may not be an aromatic homocyclic or heterocyclic compound. In certain such embodiments, the first excipient may act as a aromatic homocyclic or heterocyclic compound and a second excipient may act as a hydrogen bond donor and/or acceptor and the combination of the first and second excipient may produce an effective excipient for mitigating turbidity, viscocity, or both. In certain embodiments, a mixture of two or more excipients, the sum of which contain (i) aromatic homocyclicity or heterocyclicity and (ii) the ability to act as a hydrogen bond donor and/or acceptor, are effective in mitigating both turbidity and viscosity. In certain embodiments, antisense oligonucleotide compositions provided herein comprise one or more modified antisense oligonucleotides and one or more excipients. Any suitable excipient known to those having skill in the art may be used. For example, suitable excipients may be found in, for example “Handbook of Pharmaceutical Excipients,” American Pharmaceutical Association Publications, Washington D.C., 6thEdition, 2009; which is hereby incorporated herein by reference in its entirety. In certain such embodiments, excipients are selected from salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone. In certain embodiments, excipients are selected from Adenine, Benzyl Alcohol, m-Cresol, Cytidine, Cytidine Monophosphate, Cytosine, Dextran, Guanine Monophosphate, D-Mannitol, Methylparaben, Nicotinamide, Phenol, 2-Phenoxyethanol, L-Phenylalanine, Pyridoxine, Sodium Chloride, Thymine, tryptophan, L-Tryptophan, L-Tyrosine, Ascorbic Acid, Benzamide, o-Benzenediol, Benzenehexol, L-Histidine, Hydroxypyridine, Indole, D-Mannitol+Phenol mixture, D-Mannitol+Pyridine mixture, 2H-Pyran, Pyrazinamide, Pyridine, Pyrimidine, 2-Pyrone, Riboflavin, Thiamine, Tryptamine, ethanol, (2-Hydroxypropyl)-β-cyclodextrin, Niacin, Polyethylene Glycol 600, Polyethylene Glycol 4600, Propylene Glycol, Pyridoxine, Sucrose, Thymidine, Tween 80, Uridine, Thymine, caffeine, acridine orange, ethidium bromide, propidium iodide, cyanine dyes such as PicoGreen®, thiamine hydrochloride, ethylene diamine tetraacetic acid, and 1,2-dihydroxybenzene. In certain embodiments, excipients comprise heterocyclic molecules. In certain embodiments, excipients comprise heterocyclic amines. In certain embodiments, excipients comprise aromatic molecules or molecules having one one or more aromatic ring. In certain embodiments, excipients comprise heterocyclic molecules wherein the heteroatom is oxygen. In certain embodiments, excipients comprise heterocyclic molecules wherein the heteroatom is nitrogen. In certain embodiments, excipients comprise heterocyclic molecules wherein the heteroatom is sulfur. In certain embodiments, the excipient lowers the viscosity of an antisense oligonucleotide composition. In certain embodiments, the excipient lowers the turbidity of an antisense oligonucleotide composition. In certain embodiments, the excipient increases the osmolarity of an antisense oligonucleotide composition. In certain embodiments, the excipient decreases the osmolarity of an antisense oligonucleotide composition. In certain embodiments, the excipient increases the pH of an antisense oligonucleotide composition. In certain embodiments, the excipient decreases the pH of an antisense oligonucleotide composition. In certain embodiments, the excipient buffers the pH of an antisense oligonucleotide composition. In certain embodiments, the excipient lowers the turbidity and the viscosity of an antisense oligonucleotide composition. In certain embodiments, the excipient lowers the turbidity and the viscosity of an antisense oligonucleotide composition and also increases the osmolarity of the antisense composition. In certain embodiments, the excipient lowers the turbidity and the viscosity of an antisense oligonucleotide composition and also decreases the osmolarity of the antisense composition. In certain embodiments, a mixture of two or more excipients lowers the viscosity of an antisense oligonucleotide composition. In certain embodiments, a mixture of two or more excipients lowers the turbidity of an antisense oligonucleotide composition. In certain embodiments, a mixture of two or more excipients increases the osmolarity of an antisense oligonucleotide composition. In certain embodiments, a mixture of two or more excipients decreases the osmolarity of an antisense oligonucleotide composition. In certain embodiments, a mixture of two or more excipients increases the pH of an antisense oligonucleotide composition. In certain embodiments, a mixture of two or more excipients decreases the pH of an antisense oligonucleotide composition. In certain embodiments, a mixture of two or more excipients buffers the pH of an antisense oligonucleotide composition. In certain embodiments, a mixture of two or more excipients lowers the turbidity and the viscosity of an antisense oligonucleotide composition. In certain embodiments, a mixture of two or more excipients lowers the turbidity and the viscosity of an antisense oligonucleotide composition and also increases the osmolarity of the antisense composition. In certain embodiments, a mixture of two or more excipients lowers the turbidity and the viscosity of an antisense oligonucleotide composition and also decreases the osmolarity of the antisense composition. In certain embodiments, one or more excipients may effective reduce the viscosity, turbidity, or both the viscocity and turbidity of a solution of one or more modified antisense oligonucleotides. In certain embodiments, one or more excipients may effective reduce the viscosity, turbidity, or both the viscocity and turbidity of a solution of one or more modified antisense oligonucleotides but this excipient may, for example, increase the osmolality of the solution to an undesirable amount. In certain embodiments, In certain embodiments, salts such as NaCl, KCl, LiCl, MgCl2, or CaCl2may used to modulate the osmolality of an oligonucleotide in solution, but may also induce the turbidity of certain oligonucleotides. In certain such embodiments, it may therefore be desireable to use an excipient selected from among L-Tryptophan, Niacinamide, L-Phenylalanine, and L-Histidine to modulate the osmolality of an oligonucleotide solution and to also modulate the viscosity and/or turbidity of the oligonucleotide solution. In certain such embodiments, it may therefore be desireable to use an excipient selected from among L-Tryptophan, Niacinamide, L-Phenylalanine, and L-Histidine to increase the osmolality of an oligonucleotide solution and to also decrease the viscosity of the oligonucleotide solution. In certain such embodiments, it may therefore be desireable to use an excipient selected from among L-Tryptophan, Niacinamide, L-Phenylalanine, and L-Histidine to increase the osmolality of an oligonucleotide solution and to also decrease the turbidity of the oligonucleotide solution. In certain such embodiments, it may therefore be desireable to use an excipient selected from among L-Tryptophan, Niacinamide, L-Phenylalanine, and L-Histidine to increase the osmolality of an oligonucleotide solution and to also decrease the viscosity and decrease the turbidity of the oligonucleotide solution. Nonlimiting Disclosure and Incorporation by Reference While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety. Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2′-OH for the natural 2′-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA). Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence “ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified or naturally occurring bases, such as “ATmeCGAUCG,” whereinmeC indicates a cytosine base comprising a methyl group at the 5-position. EXAMPLES The following examples illustrate certain embodiments of the present invention and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated. Example 1 General Method for Evaluating Turbidity, and Viscosity The general method for measuring turbidity is as follows. Turbidity qualitative assessment is performed by visually inspecting control and sample vials that have frozen at −20° C. and subsequently thawed at 5° C. Their appearance is noted before the samples equilibrate to room temperature. These results are often documented with photographs. Turbidity quantitative measurement is performed on a Hach 2100 AN Laboratory Turbidimeter and is used to test turbidity of 3.2-3.4 mL of solution filled in a 13-mm glass tube. Typically the solution has received freeze-thaw treatment similar to the one described above to induce turbidity formation prior to measurement. The instrument is standardized with Formazin reference suspensions over a range of Nephelometric Turbidity Units (NTUs) that brackets the turbidity of the samples being tested. Viscosity qualitative assessment is performed by visually inspecting the flow of a sample solution in a container and comparing it to a control. In this case, sample viscosities were noted as increased or decreased only when an obvious difference was noted (e.g., if at 5° C. the control sample appeared to mimic honey in viscosity, the viscosity of excipient samples appearing as free-flowing liquids are noted as “decreased”, while excipient samples appearing as solid gel are noted as “increased”). Quantitative viscosity measurement comprises of two forms, dynamic and kinematic. The form relating to antisense oligonucleotide (ASO) drug product characterization is dynamic viscosity. Two instruments that are currently used in the Pharmaceutical Development (PD) laboratory to measure dynamic viscosity are RheoSense m-VROC viscometer and Malvern Instruments Bohlin CVO 100 rheometer. Further information about the instruments can be obtained from the manufacturer's websites (rheosense.com and malvern.com/CVO). RheoSense m-VROC System The RheoSense m-VROC utilizes a microelectromechanical systems (MEMS) chip which consists of three silicone pressure sensor arrays embedded lengthwise along the center of a rectangular channel. The liquid sample is loaded into a syringe pump, which dispenses the sample into the microfluidic chip at a specified flow rate. The pressure drop over distance of a flowing test liquid is measured, and it is expected to be linear for Newtonian fluids if a fully developed flow is ensured within the channel. The shear stress and viscosity are calculated according to fluid dynamic principles. There are four classifications of chips available for the RheoSense m-VROC, labeled A, B, C, and D. They vary consecutively in size, with the A chip having the smallest inner channel dimensions that enables precise measurements of low viscosity samples, while the D chip is built for high viscosity samples. Within each chip category there are also three channel depths: 20, 50, and 100 μm, indicated as the “02”, “05”, and “10” series. The maximum recommended sample viscosities for use with each chip are shown in Table 2. TABLE 2Recommended upper viscosity limits for each Rheosense m-VROC chipChipMaximum Recommended Viscosity (cP)A100B400C3,000D10,000 Malvern Instruments Bohlin CVO 100 System The Bohlin CVO 100 operates via a bob-and-cup mechanism, whereby the torque required to accelerate a bob in a cup filled with sample fluid to a specified angular speed is measured and converted mathematically to viscosity. A single interface is used for all sample types evaluated in, with the bob and cup being removable for sample loading and cleaning. The instrument specifications are shown in Table 3. TABLE 3Instrument Specifications for Bohlin CVO 100 SystemMethodShear ramp method with a spinning bobDescriptionrheometer and no inertial correctionsCell1 mL Mooney-EwartGap75 micronsTemperatureIsothermal, typically at 25° C., 15° C., or 5° C.Standard and1.2 mLSample VolumeStandards10, 50, and 100 cP polydimethylsiloxaneviscosity reference fluidsModel FitNewtonian Example 1a General Method for Evaluating Osmolality Osmolality is measured using Wescor VAPRO 5600 Vapor Pressure Osmometer. A small paper disc is loaded onto the sampling area, and 10 uL of test solution is dispensed into the disc. Calibration is performed using 100, 290, and 1000 mOsm/kg standards. Further information about this instrument can be obtained from the manufacturer's website (wescor.com/biomedical/osmometer/vapro5600.html). Wescor VAPRO 5600 Vapor Pressure Osmometer VAPRO 5600 osmometer measures a test solution's dew point temperature depression, which is related to its vapor pressure. Vapor pressure is a colligative property of a solution which linearly correlates to the concentration of particles dissolved in the solvent (i.e. osmolality). The particle's size, density, configuration, or electrical charge has no bearing on a colligative property. Increasing osmolality decreases the solution vapor pressure. In the VAPRO system, an internal thermocouple hygrometer joins with the sample holder when the sample has been loaded to form a small chamber enclosing the sample disc. After the chamber temperature has equilibrated, the thermocouple is cooled, thus inducing dew formation on the thermocouple surface. The thermocouple is then heated until it reaches the dew point. The difference between ambient temperature and the dew point temperature is the dew point temperature depression. The instrument processes this result and provides a reading in mmol/kg, equaling mOsm/kg. (Note: mOsm/kg refers to the concentration of dissociated molecules in solution. Therefore, 1 mmol/L NaCl yields 2 mmol/L of dissociated ions; i.e. Na+and Cl−, which equals 2 mOsm/L. Applying the density conversion of 1 kg/L yields 2 mOsm/kg). The instrument specifications are shown in Table 3a. TABLE 3aInstrument Specifications for Wescor VAPRO 5600Sample Volume10μLMeasurement Range20-300mmol/kgMeasurement Time90secondsResolution1mmol/kgCalibrationOpti-mole ™ osmolality standards Example 2 General Method Used to Screen Excipients for the Mitigation of Turbidity and Viscosity Turbidity Screening The screening of the excipients for mitigating turbidity is performed by adding 0.1 to 5% (w/v) of the excipient at pH 7-8 to a solution of ASO in water at a concentration of 200 mg/mL to 250 mg/mL. After the solution is prepared, a 1 mL sample is aliquoted and filled into a 2-mL clean glass vial, stoppered, sealed, frozen at −20° C., and thawed at 5° C. The sample is then evaluated for turbidity with the turbidimeter or more commonly by visual inspection. The results from visual inspection is converted to a score from 0 to 3 with 0 being visually clear (i.e., <20 NTU); 1 being less turbid than a control but not clear; 2 approximately the same turbidity as a control; and 3 being more turbid than a control. A solution of ASO in water is used as the control. The excipients investigated for mitigation of turbidity are independently selected from but are not limited to adenine, benzyl alcohol, m-cresol, cytidine, cytidine monophosphate, cytosine, dextran, guanine monophosphate, D-mannitol, methylparaben, nicotinamide, phenol, 2-phenoxyethanol, L-phenylalanine, pyridoxine, sodium chloride, thymine, L-tryptophan, L-tyrosine, ascorbic acid, benzamide, o-benzenediol, benzenehexol, caffeine, L-histidine, hydroxypyridine, indole, 2H-pyran, pyrazinamide, pyridine, pyrimidine, 2-pyrone, riboflavin, thiamine, thiamine hydrochloride, 1,2-dihydroxybenzene (catechol), ethylene diamine tetraacetic acid (EDTA), tryptamine, calcium folinate, sodium folinate (vitamin B9), D-mannitol and phenol mixture, D-mannitol and pyridine mixture, D-mannitol and niacinamide (nicotinamide) mixture, L-phenylalanine and pyridoxine mixture, pyridoxine and benzyl alcohol mixture, L-phenylalanine and L-histidine mixture; any mixture combination of L-phenylalanine, L-tryptophan, L-histidine, and niacinamide; and dinucleotides or trinucleotides or other shortmer oligonucleotide probes; and nucleic acid stains such as acridine orange, ethidium bromide, propidium iodide, and cyanine dyes such as PicoGreen®. Viscosity Screening A concentrated stock solution of ASO in water is diluted using solid excipient or a concentrated stock solution of excipient. A typical dilution is from a concentration of 250 to 200 mg/mL of ASO at pH 8. Viscosity would either be visually noted, or measured using the RheoSense m-VROC viscometer or Malvern Instruments Bohlin CVO 100 rheometer as described in Example 1. The measured results for viscosity are then obtained and normalized to the results when the diluent is only water with no excipient present. The excipients investigated for mitigation of viscosity are independently selected from but are not limited to benzyl alcohol, m-cresol, cytidine, cytosine, dextran, ethanol, (2-Hydroxypropyl)-β-cyclodextrin, D-mannitol, niacin, nicotinamide, polyethylene glycol 600 (PEG600), polyethylene glycol 4600 (PEG4600), propylene glycol, pyridoxine, sodium chloride, sucrose, thymidine, L-tryptophan, uridine, adenine, thymine, Tween 80, cytidine monophosphate, guanine monophosphate, methylparaben, phenol, 2-phenoxyethanol, L-phenylalanine, L-tyrosine, ascorbic acid, benzamide, o-benzenediol, benzenehexol, caffeine, L-histidine, hydroxypyridine, indole, 2H-pyran, pyrazinamide, pyridine, pyrimidine, 2-pyrone, riboflavin, thiamine, tryptamine, thiamine hydrochloride, 1,2-dihydroxybenzene (catechol), ethylene diamine tetraacetic acid (EDTA), calcium folinate, sodium folinate (vitamin B9), D-mannitol and phenol mixture, D-mannitol and pyridine mixture, D-mannitol and phenol mixture, D-mannitol and pyridine mixture, D-mannitol and niacinamide (nicotinamide) mixture, L-phenylalanine and pyridoxine mixture, pyridoxine and benzyl alcohol mixture, L-phenylalanine and L-histidine mixture; any mixture combination of L-phenylalanine, L-tryptophan, L-histidine, and niacinamide; and dinucleotides or trinucleotides or other shortmer oligonucleotide probes; and nucleic acid stains such as acridine orange, ethidium bromide, propidium iodide, and cyanine dyes such as PicoGreen®. Note: Not all the aforementioned excipients are viable for formulation. Some are used in the screening simply for mechanistic studies. Example 3 Turbidity and Viscosity Evaluation for ISIS NO. 426115 Antisense oligonucleotide Isis No. 426115 was selected for turbidity and viscosity evaluation. The ASO and its motif are described in Table 4. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P═S). Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) modified nucleosides. An “N” indicates a U, T, C,meC, G or A nucleoside. TABLE 4Antisense Oligonucleotide Isis No. 426115Selected for Turbidity and Viscosity EvaluationIsis No.Composition (5′ to 3′)MotifSEQ ID No.426115NeNeNeNeNeNNNNNNNNNNNeNeNeNeNe5-10-514 Turbidity Evaluation for ISIS NO. 426115 Several excipients in Example 2 were selected and screened for their effect in mitigating the turbidity of ISIS NO. 426115. The turbidity experiment was performed in the same manner as described in Example 2. To an aqueous solution of ISIS NO. 426115 at a concentration of 220 mg/mL, the excipient was added at the percentage (%, w/v) as indicated in Table 5. The solution was frozen at −20° C., thawed to 5° C. and subjected to turbidity evaluation. Turbidity was analyzed and compared to a control by visual inspection using a scoring format of 0 to 3 with 0 being visually clear; 1 being less turbid than a control but not clear; 2 approximately the same turbidity as a control; and 3 being more turbid than a control. A solution of ISIS NO. 426115 at a concentration of 220 mg/mL at pH 7-8 in only water was used as the control. The results are presented in Table 5. As illustrated, several excipients when used individually or in combination with other excipients at various concentrations demonstrated desirable reduction in turbidity with a score of 1 or lower as compared to the control. TABLE 5Effect of various excipients on turbidityfor ISIS NO. 426115 at 220 mg/mL at 5° C.Excipient concentrationTurbidityExcipient(%, w/v)(visual inspection)Adenine0.50Ascorbic acid0.52Benzyl alcohol0.521.50Caffeine0.501.50m-Cresol0.33Cytidine0.0120.20.55.00Cytidine Monophosphate0.52Cytosine0.00520.220.501.0Dextran0.521,2-Dihydroxybenzene0.53(catechol)Ethylene diamine0.22tetraacetic acid (EDTA)1.03Guanine Monophosphate0.52L-Histidine1.502.00Hydroxypyridine or 20.51Pyridone1.00D-Mannitol (0.5) +1.0 (total)2Phenol (0.5) mixtureD-Mannitol (0.5) +1.0 (total)1Pyridine (0.5) mixtureD-Mannitol (0.5) +1.0 (total)1Niacinamide (0.5) mixtureD-Mannitol2.025.01015Methylparaben0.230.521.5*0*excipient saturatedand precipitatedNicotinamide or0.52Niacinamide1.011.502.02.53.05.0Phenol0.532-Phenoxyethanol0.53L-Phenylalanine0.511.50Pyrazinamide0.511.00Pyridine0.52Pyridoxine1.021.5Sodium Chloride0.52Thiamine HCl10Thymine0.50L-Tryptophan0.120.20.30.410.501.50L-Tyrosine0.042Calcium Folinate0.52L-Histidine0.521.5120Indole0.122H-Pyran0.52Pyrimidine0.522-Pyrone0.51Riboflavin (Vitamin B2)0.0252D-Mannitol (0.5) +1 (total)1Nicotinamide/Niacinamide(0.5) mixtureL-phenylalanine (1) +1.5 (total)0Pyridoxine (0.5)Pyridoxine (0.5) +1 (total)0Benzyl alcohol (0.5) Viscosity Evaluation for ISIS NO. 426115 Several excipients in Example 2 were selected and screened for their effect in mitigating the viscosity of ISIS NO. 426115. The viscosity experiment was performed in the same manner as described in Example 2. A concentrated stock solution of ASO in water was diluted using solid excipient or a concentrated stock solution of excipient. The dilution was at a concentration of 220 mg/mL of ISIS NO. 426115 at pH 7-8. Viscosity at 25° C. and 5° C. was measured using the RheoSense m-VROC viscometer as described in Example 1. The results for viscosity were obtained and normalized to the results of the control when the diluent was only water. Normalized viscosity was calculated by dividing the viscosity of the excipient present vs the viscosity of the control. The results are presented in Table 6. As illustrated, at 25° C. or 5° C., several excipients when used alone or in combination with other excepients at various concentrations demonstrated desirable reduction in the viscosity with a normalized viscosity below 1.00. TABLE 6Effect of various excipients on viscosity ofISIS NO. 426115 at 220 mg/mL at 25° C. and 5 ° C.ExcipientNormalizedConc.Viscosity (cP)Viscosity*Excipient(%, w/v)25° C.5° C.25° C.5° C.None (Control)07928,5001.001.00L-Tryptophan0.57312,0000.90.41.5647,7580.80.3Nicotinamide1.5503,0000.60.1(Niacinamide)Phenylalanine1.5411,6040.50.1Thiamine HCl19579,0001.22.8Pyridoxine HCl (0.5%) +110619,0001.30.7benzyl alcohol (0.5%)Pyridoxine HCl (0.5%) +1.5462,5390.60.1phenylalanine (1%)Caffeine1.514835,0001.91.2Histidine0.573NT0.9NT1.563NT0.8NT2474,7590.60.2Pyrazinamide159NT0.7NTMethylparaben1.5120NT1.5NT*Normalized to ISIS NO. 426115 at concentration of 220 mg/mL in waterNT = Not Tested As reported in scientific or patent literature, some additives can mitigate only turbidity or only viscosity (see US 20110098343). Unlike the literature, results from Tables 4 and 5 demonstrated that both turbidity and viscosity can be comitigated by a single excipient such as L-tryptophan, benzyl alcohol, L-histidine, L-phenylalanine, and nicotinamide. In certain embodiments, additional excipients that have potential to mitigate both turbidity and viscosity based on our current study include but are not limited to thymine, adenine, riboflavin, thiamine, and tryptamine since, in certain embodiments, several effective excipients have aromatic character (with heterocylicity enhancing their turbidity mitigation property) and an ability to act as a hydrogen bond donor and/or a hydrogen bond acceptor. Osmolality Measurement The osmolality of some samples from the screenings above was measured and compared to the control using the procedure illustrated in Example 1a. The results are presented in Table 6a, below. The molar increase in solution osmolality compared to the control was expected to equal the molar amount of excipient added, since no ionic dissociation was expected. For example, adding 73.4 mmol/kg of L-Tryptophan (which equals to 73.4 mOsm/kg assuming the density is 1 kg/L) to a control solution of 431 mOsm/kg, the expected increase in osmolality of the solution would be 73.4 mOsm/kg (which equals to 504.4 mOsm/kg total solution osmolality). As illustrated in Table 6a, both L-tryptophan and niacinamide showed an increase in osmolality less than expected at 26 mOsm/kg, and 66 mOsm/kg rather than at 73.4 mOsm/kg and 122.8 mOsm/kg, respectively compared to the control. Phenylalanine showed an increase in osmolality as expected, while histidine showed an increase in osmolality more than expected at 119 mOsm/kg. TABLE 6aEffect of various excipients on osmolality for ISIS 426115 at 220 mg/mLExpectedMeasuredExcipientExcipientExpectedMeasuredOsmolalityOsmolalityConc.Conc.OsmolalityOsmolalityIncreaseIncreaseExcipient(%, w/v)(mmol/kg)*(mOsm/kg)(mOsm/kg)(mOsm/kg)(mOsm/kg)Control00.043143100L-Tryptophan1.573.4504.445773.426Niacinamide1.5122.8553.8497122.866L-Phenylalanine1.590.8521.852290.891L-Histidine1.596.7527.755096.7119*assuming density = 1 kg/L Example 4 Evaluation of Properties of Effective Turbidity and Viscosity Comitigator Excipients for ISIS NO. 426115 The properties of excipients which can effectively comitigate turbidity and viscosity of ISIS NO. 426115 were investigated. Several excipients in Example 2 were selected and screened using the same method as described previously. The properties that were examined included, but not limited to aromaticity, homocyclic vs. heterocyclic aromatic rings, and the number of hydrogen bond donor and/or acceptor. The ASO samples were prepared in the same manner as described in Example 3. To an aqueous solution of ISIS NO. 426115 at a concentration of 220 mg/mL, the excipient was added at the percentage (%, w/v) as indicated in Table 7. The solution was frozen at −20° C., thawed to 5° C. and subjected to turbidity and viscosity evaluation. Turbidity and viscosity were analyzed and compared to a control by visual inspection. For turbidity evaluation, a scoring format of 0 to 3 was employed with 0 being visually clear; 1 being less turbid than a control but not clear; 2 approximately the same turbidity as a control; and 3 being more turbid than a control. A solution of ISIS NO. 426115 at a concentration of 220 mg/mL at pH 8 in only water was used as the control. The results are presented in Table 7. As illustrated, ascorbic acid reduced turbidity but not viscosity compared to the control, while L-phenylalanine was able to reduce both turbidity and viscosity. Similarly, pyrazinamide was effective for co-mitigation at 0.5% (w/v). Benzamide was effective for mitigating turbidity but not for viscosity at 2% (w/v), while nicotinamide was able to co-mitigate turbidity and viscosity at the same concentration. Phenol was unable to mitigate turbidity and viscosity, while hydroxypyridine was able to co-mitigate. Thus, in certain embodiments, heterocyclicity with increasing number of non-carbon substituents such as nitrogen and oxygen, seem to improve turbidity reduction. Both phenol and catechol were ineffective in reducing turbidity at 0.5% (w/v). Phenol appeared to increase viscosity while catechol had no effect. Pyridine was ineffective for both turbidity and viscosity reduction, while hydroxypyridine was effective for co-mitigation. These results suggest that excipients with more hydrogen bond donors and/or acceptors can be effective at turbidity and viscosity co-mitigation. In certain embodiments, some excipients effective at decreasing turbidity and/or viscosity have heterocyclic or homocyclic aromatic character. In certain embodiments, some excipients effective at decreasing turbidity and/or viscosity are heterocyclic and nonaromatic, for example, ascorbic acid as shown in Table 7. The results from Table 7 suggest that in certain embodiments, excipients that have heterocyclic aromatic or nonaromatic character and contain hydrogen-bond donor(s) and/or hydrogen-bond acceptor(s) mitigate both turbidity and viscosity. In certain embodiments, the properties given in this example are consistent with single excipients that are effective at mitigating both turbidity and viscosity, for example, compounds that resemble nucleobases, and heterocyclic compounds that possess both hydrogen bond donors and acceptors. In certain embodiments, aromatic ring character appears to provide a benefit based on the planar nature that facilitates: (i) positioning hydrogen bonds at a low energy state and (ii) interference of base stacking. TABLE 7Effect of excipients in mitigating turbidity and viscosity of ISIS NO. 426115ViscosityCompared toTurbidityControlConcentration(visual(visualChemical StructureExcipient(%, w/v)inspection)inspection)Phenol0.53IncreasedCatechol (1,2- Dihydroxybenzene)0.53No notable differenceHydroxypyridine0.51DecreasedBenzamide23DecreasedNicotinamide0.5 22 0No notable difference DecreasedPyrazinamide0.51DecreasedPyridine0.52No notable differenceAscorbic acid0.50No notable differencePhenylalanine0.51Slightly decreased Example 5 Effect of Combining Singly Ineffective Excipients for Co-Mitigation of ISIS NO. 426115 The effect of combining excipients which are ineffective by themselves at mitigating both turbidity and viscosity of ISIS NO. 426115 was investigated. Several excipients in Example 2 were selected and screened using the same method as described previously. The ASO samples were prepared in the same manner as described in Example 3. To an aqueous solution of ISIS NO. 426115 at a concentration of 220 mg/mL at pH 8 the excipient mixture was added at the percentage (%, w/v) indicated in Table 8. The solution was frozen at −20° C., thawed to 5° C. and subjected to turbidity and viscosity evaluation. Turbidity and viscosity were analyzed and compared to a control by visual inspection. For turbidity evaluation, a scoring format of 0 to 3 was employed with 0 being visually clear; 1 being less turbid than a control but not clear; 2 approximately the same turbidity as a control; and 3 being more turbid than a control. A solution of ISIS NO. 426115 at a concentration of 220 mg/mL at pH 8 in only water was used as the control. The results are presented in Table 8. In certain embodiments, the chemical properties listed previously namely (i) aromatic homocyclicity or heterocyclicity and (ii) the ability to act as a hydrogen bond donor and/or acceptor do not have to be consolidated into one excipient or compound. In certain embodiments, it was discovered from our finding that a mixture of two excipients, which satisfies the two properties as a whole, can also be effective in mitigating both turbidity and viscosity. In certain embodiments, this was demonstrated by the mixture of 0.5% (w/v) mannitol and 0.5% (w/v) pyridine. Mannitol is a saturated linear carbon chain with hydroxyl groups, therefore possessing both hydrogen bond donors and acceptors; whereas pyridine is a heterocyclic aromatic compound lacking a hydrogen bond donor but has a hydrogen bond acceptor from its nitrogen atom. This mixture visually reduced both turbidity and viscosity compared to the control. In contrast, mannitol and pyridine by itself are ineffective at mitigating either turbidity or viscosity. In support of our observation for chemical properties of the excipients, it was found that mannitol and phenol mixture lacking a heterocyclic ring showed no significant mitigation in turbidity (Table 5). The results from Table 8 suggest that in certain embodiments, other mixtures may potentially have such synergistic effects for both turbidity and viscosity mitigation as demonstrated by D-mannitol and pyridine mixture. Such mixtures include, but are not limited to, dextran and pyrimidine mixture or ascorbic acid and phenanthroline mixture. TABLE 8Effect of excipient mixtures in mitigating turbidity and viscosity of ISIS NO. 426115ViscosityCompared toTurbidityControlConcentration(visual(visualChemical StructureExcipient(%, w/v)inspection)inspection)Pyridine0.52No notable differenceD-Mannitol2 5 10 152No notable differenceSee boxes aboveD-Mannitol + Pyridine0.5 + 0.51Decreased Example 6 Effect of Temperature and Time Dependency on ISIS NO. 426115 Turbidity The effect of temperature and time dependency on the turbidity profile of freeze-thawed ISIS NO. 426115 at 220 mg/mL as it warmed up from 5° C. to room temperature was evaluated. The experiment was performed in the following manner. Materials The materials used to carry out the experiment included a solution of ISIS NO. 426115 in water at a concentration of 220 mg/mL and 150 mg/mL; a Hach 2100AN Nephelometer; a StablCal Calibration Set for 2100AN Nephelometer, a StablCal Formazin Turbdity Standard at 1000 NTU; a VWR Precision 0.01° thermometer and Gerresheimer 13×100 mm glass culture tubes. Method A 4 mL sample of ISIS NO. 426115 at concentration of 220 mg/mL or 150 mg/mL was pipetted into a sample tube, capped and parafilmed at the cap joint. Both samples were allowed to freeze in a −20° C. storage and then moved to a 2-8° C. storage to thaw. Turbidity Measurement The turbidimeter was calibrated using Formazin turbidity calibration set. The 220 mg/mL sample from 2-8° C. storage was removed and allowed to stand at room temperature. The temperature of the sample was monitored until it reached approximately 13° C. or until condensation does not recur upon wiping of the tube surface and the temperature was recorded. The tube was then inserted in the nephelometer using a tube adaptor and turbidity measurement was taken every 2 minutes until turbidity value does not change by more than 0.1 NTU. Temperature Profile The 150 mg/mL sample from 2-8° C. storage was removed and allowed to stand at room temperature. The sample tube was inserted into the nephelometer (which does not need to be turned on). Condensation was not accounted for since it did not significantly affect the temperature profile. A temperature probe was inserted into the sample tube and the temperature was recorded over time. Note: The temperature profiles of 150 mg/mL and 220 mg/mL samples of ISIS NO. 426115 has been shown to be identical in previous studies. Sample Tube Turbidity Correction Factor (CF) Since the 13-mm sample tubes used differ from the nephelometer's recommended set, a different path length of light applies for taking the sample turbidity. As such a CF was required to convert the readings into accurate NTU (Nephelometric Turbidity Units) values. Formazin turbidity standards were volumetrically prepared by serial dilution of 1000 NTU standard using sterile water for injection. The standard values are 0, 100, 250, 500, 750 or 1000 NTU. The standard solutions were filled into 13-mm sample tubes and turbidity was measured. The standard values were plotted over measured turbidity values and a linear trend over the data points were fitted to obtain the slope. The slope is the CF for 13-mm sample tubes. The CF was used to convert measured values from 220 mg/mL ISIS NO. 426115 sample readings into actual NTU values. Data Analysis and Results The temperature and turbidity measurements over time were tabulated. The temperature profile was plotted by designating time zero to be at the same starting temperature as the turbidity sample. Overlaid the turbidity and temperature profiles over time and matched up the turbidity value with a temperature value along the time profile to generate an approximate turbidity vs temperature profile (FIG.1). The results showed that the CF for the tubes is the slope of the plot which is 1.8148 (FIG.1). A decrease in turbidity appeared to lag behind the increase in temperature as shown inFIGS.2and3. Turbidity remained at approximately 1800 NTU for the first 10 minutes when the temperature had increased by about 5 degrees. The solution temperature reached room temperature after approximately 30 minutes, however visually the turbidity did not completely dissipate until approximately 50 minutes, when it reached 20 NTU.FIG.4shows that 20 NTU is the approximate lower limit of visible turbidity in a solution. In certain embodiments, this observation is indicative of a certain amount of thermal energy being required to break up the turbid species, which has been hypothesized to be self associated oligonucleotides. In addition, in certain embodiments, as shown inFIG.3, there is an apparent melting temperature existing at approximately 19° C. At temperatures below that, the turbid species persist, and at temperatures above that they dissociate. Example 7 Viscosity Evaluation for ISIS NO. 104838 Antisense oligonucleotide Isis No. 104838 was selected for viscosity evaluation. The ASO and its motif are described in Table 9. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P═S). Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) modified nucleosides. An “N” indicates a U, T, C,meC, G or A nucleoside. Several excipients in Example 2 were selected and screened for their effect in mitigating the viscosity of ISIS NO. 104838. The viscosity experiment was performed in the same manner as described in Example 2. A concentrated stock solution of ASO in water was diluted using solid excipient or a concentrated stock solution of excipient. The dilution was at a concentration of 200 mg/mL of ISIS NO. 104838 at pH 8. Viscosity at 25° C. was measured using the Malvern Instruments Bohlin CVO 100 rheometer as described in Example 1. The results for viscosity were obtained and normalized to the results of the control when the diluent was only water. The range of the control viscosity (105.1-113.6 cP) provided in Table 10 was generated from various independent studies and therefore, normalized viscosity was calculated based on the control value obtained from the same study. The results are shown below. As illustrated, several excipients at various concentrations demonstrated a desirable reduction in viscosity with a normalized viscosity of below 1.00 as compared to the control. TABLE 9Antisense Oligonucleotide Isis No. 104838Selected for Viscosity EvaluationIsis No.Composition (5′ to 3′)MotifSEQ ID No.104838NeNeNeNeNeNNNNNNNNNNNeNeNeNeNe5-10-526 TABLE 10Effect of various excipients on viscosityof ISIS NO. 104838 at 200 mg/mL at 25° C.Excipient Conc.ViscosityNormalizedExcipient(%, w/v)(cP)Viscosity*None (control)0105.1-113.61.00PEG46005194.81.85(2-Hydroxypropyl)-5178.21.60β-cyclodextrinPEG6005147.21.40Dextran 15005130.91.18D-Mannitol5115.51.10Sucrose5122.11.10Niacin (Vitamin B3)5123.81.09sodium saltTween 800.5115.11.04Thymidine594.750.86Uridine589.050.81Cytosine181.890.74L-Tryptophan1.182.690.73Benzyl alcohol0.982.090.723.844.080.39Cytidine567.640.61m-Cresol262.400.55Nicotinamide/Niacinamide542.460.38*Normalized to ISIS NO. 104838 concentration of 200 mg/mL in water Example 8 Combining Singly Effective Excipients for Enhanced Turbidity and Viscosity Co-Mitigation of ISIS NO. 426115 The effect of combining effective excipients was evaluated for co-mitigation of turbidity and viscosity of ISIS NO. 426115 beyond what each excipient could perform individually. Tryptophan, niacinamide (nicotinamide), L-phenylalanine and L-histidine were selected for a factorial mixture design-of-experiment (DOE) study, where the total of all excipient mixture combinations equal 60 mM. The ratios are presented in Table 11, below. Stock solutions of 220 mg/mL ISIS NO. 426115 containing 60 mM single excipients were prepared by adding solid ASO and excipient powders, diluting with water, and then pH-adjusting to target pH 7-8 as necessary. Subsequently, the stock solutions were combined in relevant ratios to total 1 mL solutions, and filled into 2-mL clean glass vials which were stoppered and sealed, to produce all 24 samples. Control solution of 220 mg/mL ISIS NO. 426115 without excipient was prepared separately using similar method. These samples were frozen and thawed in two cycles, by first freezing at −20° C. and thawing at 5° C., and then the process was repeated. Turbidity was analyzed after the two freeze-thaw cycles and compared to the control by visual inspection. A scoring format of 0 to 2 was employed with 0 being visually clear; 0.5 being very slightly turbid, 1 being slightly turbid; and 2 being as turbid as the control. Viscosity was measured at 25° C. using the Rheosense m-VROC system as described in Example 1. The results for viscosity were obtained and normalized to the results of the control when the diluent was only water. The results presented in Table 11 were then entered into a DOE software (Design-Expert®8) to determine statistical significance of the data and render a response surface showing the most ideal mixture for viscosity reduction. As illustrated, all combinations of excipients at the ratios tested mitigated turbidity with the score of 1 or lower and also mitigated viscosity to less than 65% of the control. Design-Expert ANOVA analysis determined that the results possess adequate signal-to-noise ratio, and could be used to generate statistically significant models. Additionally, a viscosity response surface generated by the software showed that 50/50 combination of L-phenylalanine and L-histidine proved most effective at lowering viscosity. TABLE 11Effect of excipient mixtures in mitigating turbidity and viscosity of ISIS NO. 426115 at 25° C.L-NicotinamideL-L-TurbiditySampletryptophan(Niacinamide)phenylalaninehistidine(visualViscosityNormalizedType(mM)(mM)(mM)(mM)inspection)(cP)ViscosityControl0000266.841*1.0Test60000038.8550.58Samples06000035.2540.5300600034.2960.5100060134.8630.523030000360.54300300033.3060.50300030038.3780.57030300035.2160.530300300.535.610.530030300.529.1470.442020200035.8630.542020020036.1140.542002020032.2790.4802020200.532.8290.4915151515034.7390.5238888037.6250.5683888034.7780.5288388026.0260.39888380.535.130.5360000041.6620.6206000036.6250.5500600034.0820.5100060135.560.53303000037.4940.56*Measured by taking the average from three independent studies Example 10 Turbidity Evaluation for ISIS 442245 in the Presence of Osmolality Adjusters Another ASO sequence, ISIS 442245 was used for turbidity evaluation. ISIS 442245 and its motif are described in Table 12. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P═S). Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “g” indicate 3′-F-HNA modified nucleosides. An “N” indicates a U, T, C,meC, G or A nucleoside. Osmolality adjusters for ISIS 442245 formulated at low ASO concentration can comprise salts such as NaCl, or sugars such as mannitol, or other substances. The effect of adding 0.6% (w/v) NaCl to 55 mg/mL ISIS 442245 was compared to adding 0.4% (w/v) mannitol. A buffered aqueous solution of ISIS 442245 at a concentration of 55 mg/mL was prepared by adding solid drug substance powder to 5 mM phosphate buffer (pH 7-8). To this stock solution, either 0.4% (w/v) mannitol or 0.6% NaCl (w/v) was added. A control solution was also prepared without osmolality adjusters. The solutions were frozen at −20° C., thawed to 5° C. Turbidity was evaluated by visual inspection and noted as either “clear” or “turbid”. The results are presented in Table 13, below. As illustrated, only ISIS 442245 with NaCl solution became turbid after freeze-thaw. The solution containing mannitol remained clear, which suggests that mannitol or other sugars such as glucose, sucrose, or fructose, may likely be suitable for use as osmolality adjusters replacing NaCl for turbidity mitigation. TABLE 12Antisense Oligonucleotide Isis 442245 Selected for Turbidity EvaluationIsis No.Composition (5′ to 3′)MotifSEQ ID No.442245NgNgNNNNNNNNNNNgNg2-10-227 TABLE 13Turbidity Evaluation of ISIS 442245 in thePresence or Absence of Osmolality AdjustersSamplesTurbidity(all buffered with 5 mMOsmolality(visualphosphate buffer, pH 7-8)(mOsm/kg)inspection)Control: 55 mg/mL 442245274Clear55 mg/mL 442245 + 0.4% mannitol505Clear55 mg/mL 442245 + 0.6% NaCl488Turbid Example 11 Turbidity Evaluation for ISIS 442245 with Excipients Excipients such as tryptophan, niacinamide (nicotinamide) and phenylalanine were selected and screened for their effect in mitigating the turbidity of ISIS 442245 formulated with 5 mM phosphate buffer (pH 7-8) and 0.6% NaCl. The turbidity experiment was performed in the same manner as described in Example 2. To an aqueous solution of ISIS 442245 at a concentration of 55 mg/mL the excipients were added at the percentage (%, w/v) indicated in Table 14. L-Tryptophan and L-Phenylalanine concentrations tested were limited by their solubility. The solutions were frozen at −20° C., thawed to 5° C. and subjected to turbidity evaluation. Turbidity was analyzed and compared to a control by visual inspection using a scoring format of 0 to 3 with 0 being visually clear; 1 being less turbid than a control but not clear; 2 approximately the same turbidity as a control; and 3 being more turbid than a control. A solution of ISIS 442245 at a concentration of 55 mg/mL with 5 mM phosphate buffer (pH 7-8) and 0.6% NaCl is used as the control. The results are presented in Table 14. As illustrated, the control appeared to be a turbid gel at 5° C. L-phenylalanine was shown to be effective in co-mitigating turbidity and viscosity at approximately 200 mM when the solution was stored for several days in 5° C. Upon freeze-thaw, the saturated L-phenylalanine precipitated and therefore caused the solution to become more turbid than the control. L-Tryptophan was also effective at turbidity mitigation at 50 mM after freeze-thaw, and would likely be a successful co-mitigator if it had been more soluble. Further, niacinamide was shown to co-mitigate turbidity and viscosity successfully at 400 mM while remaining stable after freeze-thaw. The results demonstrate that these excipients can be effective at mitigating turbidity and/or viscosity and can be used as co-mitigators for oligonucleotide sequences other than the ones exemplified herein. TABLE 14Effect of various excipients on turbidity and viscosity of ISIS NO. 442245 at 55 mg/mLformulated with 5 mM buffer pH 7-8 and 0.6% NaClTurbidityTurbidityExcipientafter 3 days atafter freeze-Viscosity atViscosityConc.Excipient5° C. (visualthaw (visual5° C. (visualat 5° C.Excipient(% w/v)Conc. (mM)inspection)inspection)inspection)(cP)Control0022N/ASee note**Niacinamide4.940000Decreased5L-Tryptophan1.05021No notableSee note**differenceL-Phenylalanine3.3 *200*03Decreased312.515002DecreasedNT*Due to excipient saturation, some excipients precipitated at this concentration**Viscosity traces were erratic, showing some maximum values above 100 cP and minima around 0 cP. This is likely due to inhomogeneous sample gelling.NT = not tested | 165,245 |
11859181 | DETAILED DESCRIPTION Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended embodiments. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. I. Overview RNA-guided nucleases (RGNs) allow for the targeted manipulation of specific site(s) within a genome and are useful in the context of gene targeting for therapeutic and research applications. In a variety of organisms, including mammals, RNA-guided nucleases have been used for genome engineering by stimulating non-homologous end joining and homologous recombination, for example. The compositions and methods described herein are useful for creating single- or double-stranded breaks in polynucleotides, modifying polynucleotides, detecting a particular site within a polynucleotide, or modifying the expression of a particular gene. The RNA-guided nucleases disclosed herein can alter gene expression by modifying a target sequence. In specific embodiments, the RNA-guided nucleases are directed to the target sequence by a guide RNA (gRNA) as part of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided nuclease system. The RGNs are considered “RNA-guided” because guide RNAs form a complex with the RNA-guided nucleases to direct the RNA-guided nuclease to bind to a target sequence and in some embodiments, introduce a single-stranded or double-stranded break at the target sequence. After the target sequence has been cleaved, the break can be repaired such that the DNA sequence of the target sequence is modified during the repair process. Thus, provided herein are methods for using the RNA-guided nucleases to modify a target sequence in the DNA of host cells. For example, RNA-guided nucleases can be used to modify a target sequence at a genomic locus of eukaryotic cells or prokaryotic cells. II. RNA-Guided Nucleases Provided herein are RNA-guided nucleases. The term RNA-guided nuclease (RGN) refers to a polypeptide that binds to a particular target nucleotide sequence in a sequence-specific manner and is directed to the target nucleotide sequence by a guide RNA molecule that is complexed with the polypeptide and hybridizes with the target sequence. Although an RNA-guided nuclease can be capable of cleaving the target sequence upon binding, the term RNA-guided nuclease also encompasses nuclease-dead RNA-guided nucleases that are capable of binding to, but not cleaving, a target sequence. Cleavage of a target sequence by an RNA-guided nuclease can result in a single- or double-stranded break. RNA-guided nucleases only capable of cleaving a single strand of a double-stranded nucleic acid molecule are referred to herein as nickases. The RNA-guided nucleases disclosed herein include the APG06622, APG02787, APG06248, APG06007, APG02874, APG03850, APG07553, APG03031, APG09208, APG05586, APG08770, APG08167, APG01604, APG03021, APG06015, APG09344, APG07991, APG01868, APG02998, APG09298, APG06251, APG03066, APG01560, APG02777, APG05761, APG02479, APG08385, APG09217, and APG06657 RNA-guided nucleases, the amino acid sequences of which are set forth, respectively, as SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence-specific manner. In some of these embodiments, the active fragment or variant of the APG06622, APG02787, APG06248, APG06007, APG02874, APG03850, APG07553, APG03031, APG09208, APG05586, APG08770, APG08167, APG01604, APG03021, APG06015, APG09344, APG07991, APG01868, APG02998, APG09298, APG06251, APG03066, APG01560, APG02777, APG05761, APG02479, APG08385, APG09217, or APG06657 RGN is capable of cleaving a single- or double-stranded target sequence. In some embodiments, an active variant of the APG06622, APG02787, APG06248, APG06007, APG02874, APG03850, APG07553, APG03031, APG09208, APG05586, APG08770, APG08167, APG01604, APG03021, APG06015, APG09344, APG07991, APG01868, APG02998, APG09298, APG06251, APG03066, APG01560, APG02777, APG05761, APG02479, APG08385, APG09217, or APG06657 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth as SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579. In certain embodiments, an active fragment of the APG06622, APG02787, APG06248, APG06007, APG02874, APG03850, APG07553, APG03031, APG09208, APG05586, APG08770, APG08167, APG01604, APG03021, APG06015, APG09344, APG07991, APG01868, APG02998, APG09298, APG06251, APG03066, APG01560, APG02777, APG05761, APG02479, APG08385, APG09217, or APG06657 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579. RNA-guided nucleases provided herein can comprise at least one nuclease domain (e.g., DNase, RNase domain) and at least one RNA recognition and/or RNA binding domain to interact with guide RNAs. Further domains that can be found in RNA-guided nucleases provided herein include, but are not limited to: DNA binding domains, helicase domains, protein-protein interaction domains, and dimerization domains. In specific embodiments, the RNA-guided nucleases provided herein can comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one or more of a DNA binding domain, helicase domain, protein-protein interaction domain, and dimerization domain. A target nucleotide sequence is bound by an RNA-guided nuclease provided herein and hybridizes with the guide RNA associated with the RNA-guided nuclease. The target sequence can then be subsequently cleaved by the RNA-guided nuclease if the polypeptide possesses nuclease activity. The terms “cleave” or “cleavage” refer to the hydrolysis of at least one phosphodiester bond within the backbone of a target nucleotide sequence that can result in either single-stranded or double-stranded breaks within the target sequence. The presently disclosed RGNs can cleave nucleotides within a polynucleotide, functioning as an endonuclease or can be an exonuclease, removing successive nucleotides from the end (the 5′ and/or the 3′ end) of a polynucleotide. In other embodiments, the disclosed RGNs can cleave nucleotides of a target sequence within any position of a polynucleotide and thus function as both an endonuclease and exonuclease. The cleavage of a target polynucleotide by the presently disclosed RGNs can result in staggered breaks or blunt ends. The presently disclosed RNA-guided nucleases can be wild-type sequences derived from bacterial or archaeal species. Alternatively, the RNA-guided nucleases can be variants or fragments of wild-type polypeptides. The wild-type RGN can be modified to alter nuclease activity or alter PAM specificity, for example. In some embodiments, the RNA-guided nuclease is not naturally-occurring. In certain embodiments, the RNA-guided nuclease functions as a nickase, only cleaving a single strand of the target nucleotide sequence. Such RNA-guided nucleases have a single functioning nuclease domain. In particular embodiments, the nickase is capable of cleaving the positive strand or negative strand. In some of these embodiments, additional nuclease domains have been mutated such that the nuclease activity is reduced or eliminated. In other embodiments, the RNA-guided nuclease lacks nuclease activity altogether and is referred to herein as nuclease-dead or nuclease inactive. Any method known in the art for introducing mutations into an amino acid sequence, such as PCR-mediated mutagenesis and site-directed mutagenesis, can be used for generating nickases or nuclease-dead RGNs. See, e.g., U.S. Publ. No. 2014/0068797 and U.S. Pat. No. 9,790,490; each of which is incorporated by reference in its entirety. RNA-guided nucleases that lack nuclease activity can be used to deliver a fused polypeptide, polynucleotide, or small molecule payload to a particular genomic location. In some of these embodiments, the RGN polypeptide or guide RNA can be fused to a detectable label to allow for detection of a particular sequence. As a non-limiting example, a nuclease-dead RGN can be fused to a detectable label (e.g., fluorescent protein) and targeted to a particular sequence associated with a disease to allow for detection of the disease-associated sequence. Alternatively, nuclease-dead RGNs can be targeted to particular genomic locations to alter the expression of a desired sequence. In some embodiments, the binding of a nuclease-dead RNA-guided nuclease to a target sequence results in the reduction in expression of the target sequence or a gene under transcriptional control by the target sequence by interfering with the binding of RNA polymerase or transcription factors within the targeted genomic region. In other embodiments, the RGN (e.g., a nuclease-dead RGN) or its complexed guide RNA further comprises an expression modulator that, upon binding to a target sequence, serves to either repress or activate the expression of the target sequence or a gene under transcriptional control by the target sequence. In some of these embodiments, the expression modulator modulates the expression of the target sequence or regulated gene through epigenetic mechanisms. In other embodiments, the nuclease-dead RGNs or an RGN with only nickase activity can be targeted to particular genomic locations to modify the sequence of a target polynucleotide through fusion to a base-editing polypeptide, for example a deaminase polypeptide or active variant or fragment thereof, that directly chemically modifies (e.g., deaminates) a nucleobase, resulting in conversion from one nucleotide base to another. The base-editing polypeptide can be fused to the RGN at its N-terminal or C-terminal end. Additionally, the base-editing polypeptide may be fused to the RGN via a peptide linker. A non-limiting example of a deaminase polypeptide that is useful for such compositions and methods includes a cytidine deaminase or an adenosine deaminase (such as the adenine deaminase base editor described in Gaudelli et al. (2017)Nature551:464-471, U.S. Publ. Nos. 2017/0121693 and 2018/0073012, and International Publ. No. WO 2018/027078, or any of the deaminases disclosed in International Publ. No. WO 2020/139873, and U.S. Provisional Appl. Nos. 63/077,089 filed Sep. 11, 2020, 63/146,840 filed Feb. 8, 2021, and 63/164,273 filed Mar. 22, 2021, each of which is herein incorporated by reference in its entirety). Further, it is known in the art that certain fusion proteins between an RGN and a base-editing enzyme may also comprise at least one uracil stabilizing polypeptide that increases the mutation rate of a cytidine, deoxycytidine, or cytosine to a thymidine, deoxythymidine, or thymine in a nucleic acid molecule by a deaminase. Non-limiting examples of uracil stabilizing polypeptides include those disclosed in U.S. Provisional Appl. No. 63/052,175, filed Jul. 15, 2020, including USP2 (SEQ ID NO: 1089), and a uracil glycosylase inhibitor (UGI) domain (SEQ ID NO: 212), which may increase base editing efficiency. Therefore, a fusion protein may comprise an RGN described herein or variant thereof, a deaminase, and optionally at least one uracil stabilizing polypeptide, such as UGI or USP2. In certain embodiments, the RGN that is fused to the base-editing polypeptide is a nickase that cleaves the DNA strand that is not acted upon by the base-editing polypeptide (e.g., deaminase). RNA-guided nucleases that are fused to a polypeptide or domain can be separated or joined by a linker. The term “linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., a binding domain and a cleavage domain of a nuclease. In some embodiments, a linker joins a gRNA binding domain of an RNA guided nuclease and a base-editing polypeptide, such as a deaminase. In some embodiments, a linker joins a nuclease-dead RGN and a deaminase. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. The presently disclosed RNA-guided nucleases can comprise at least one nuclear localization signal (NLS) to enhance transport of the RGN to the nucleus of a cell. Nuclear localization signals are known in the art and generally comprise a stretch of basic amino acids (see, e.g., Lange et al.,J. Biol. Chem. (2007) 282:5101-5105). In particular embodiments, the RGN comprises 2, 3, 4, 5, 6 or more nuclear localization signals. The nuclear localization signal(s) can be a heterologous NLS. Non-limiting examples of nuclear localization signals useful for the presently disclosed RGNs are the nuclear localization signals of SV40 Large T-antigen, nucleoplasmin, and c-Myc (see, e.g., Ray et al. (2015)Bioconjug Chem26(6):1004-7). In particular embodiments, the RGN comprises the NLS sequence set forth as SEQ ID NO: 251 or 253. The RGN can comprise one or more NLS sequences at its N-terminus, C-terminus, or both the N-terminus and C-terminus. For example, the RGN can comprise two NLS sequences at the N-terminal region and four NLS sequences at the C-terminal region. Other localization signal sequences known in the art that localize polypeptides to particular subcellular location(s) can also be used to target the RGNs, including, but not limited to, plastid localization sequences, mitochondrial localization sequences, and dual-targeting signal sequences that target to both the plastid and mitochondria (see, e.g., Nassoury and Morse (2005)Biochim Biophys Acta1743:5-19; Kunze and Berger (2015)Front Physioldx.doi.org/10.3389/fphys.2015.00259; Herrmann and Neupert (2003)IUBMB Life55:219-225; Soll (2002)Curr Opin Plant Biol5:529-535; Carrie and Small (2013)Biochim Biophys Acta1833:253-259; Carrie et al. (2009)FEBS J276:1187-1195; Silva-Filho (2003)Curr Opin Plant Biol6:589-595; Peeters and Small (2001)Biochim Biophys Acta1541:54-63; Murcha et al. (2014)J Exp Bot65:6301-6335; Mackenzie (2005)Trends Cell Biol15:548-554; Glaser et al. (1998)Plant Mol Biol38:311-338). In certain embodiments, the presently disclosed RNA-guided nucleases comprise at least one cell-penetrating domain that facilitates cellular uptake of the RGN. Cell-penetrating domains are known in the art and generally comprise stretches of positively charged amino acid residues (i.e., polycationic cell-penetrating domains), alternating polar amino acid residues and non-polar amino acid residues (i.e., amphipathic cell-penetrating domains), or hydrophobic amino acid residues (i.e., hydrophobic cell-penetrating domains) (see, e.g., Milletti F. (2012)Drug Discov Today17:850-860). A non-limiting example of a cell-penetrating domain is the trans-activating transcriptional activator (TAT) from the human immunodeficiency virus 1. The nuclear localization signal, plastid localization signal, mitochondrial localization signal, dual-targeting localization signal, and/or cell-penetrating domain can be located at the amino-terminus (N-terminus), the carboxyl-terminus (C-terminus), or in an internal location of the RNA-guided nuclease. The presently disclosed RGNs can be fused to an effector domain, such as a cleavage domain, a deaminase domain, or an expression modulator domain, either directly or indirectly via a linker peptide. Such a domain can be located at the N-terminus, the C-terminus, or an internal location of the RNA-guided nuclease. In some of these embodiments, the RGN component of the fusion protein is a nuclease-dead RGN. In some embodiments, the RGN fusion protein comprises a cleavage domain, which is any domain that is capable of cleaving a polynucleotide (i.e., RNA, DNA, or RNA/DNA hybrid) and includes, but is not limited to, restriction endonucleases and homing endonucleases, such as Type IIS endonucleases (e.g., FokI) (see, e.g., Belfort et al. (1997)Nucleic Acids Res.25:3379-3388; Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). In other embodiments, the RGN fusion protein comprises a deaminase domain that deaminates a nucleobase, resulting in conversion from one nucleobase to another, and includes, but is not limited to, a cytidine deaminase or an adenine deaminase base editor (see, e.g., Gaudelli et al. (2017)Nature551:464-471, U.S. Publ. Nos. 2017/0121693 and 2018/0073012, U.S. Pat. No. 9,840,699, and International Publ. No. WO/2018/027078). In some embodiments, the effector domain of the RGN fusion protein can be an expression modulator domain, which is a domain that either serves to upregulate or downregulate transcription. The expression modulator domain can be an epigenetic modification domain, a transcriptional repressor domain or a transcriptional activation domain. In some of these embodiments, the expression modulator of the RGN fusion protein comprises an epigenetic modification domain that covalently modifies DNA or histone proteins to alter histone structure and/or chromosomal structure without altering the DNA sequence, leading to changes in gene expression (i.e., upregulation or downregulation). Non-limiting examples of epigenetic modifications include acetylation or methylation of lysine residues, arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation of histone proteins, and methylation and hydroxymethylation of cytosine residues in DNA. Non-limiting examples of epigenetic modification domains include histone acetyltransferase domains, histone deacetylase domains, histone methyltransferase domains, histone demethylase domains, DNA methyltransferase domains, and DNA demethylase domains. In other embodiments, the expression modulator of the fusion protein comprises a transcriptional repressor domain, which interacts with transcriptional control elements and/or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to reduce or terminate transcription of at least one gene. Transcriptional repressor domains are known in the art and include, but are not limited to, Sp1-like repressors, IκB, and Krüppel associated box (KRAB) domains. In yet other embodiments, the expression modulator of the fusion protein comprises a transcriptional activation domain, which interacts with transcriptional control elements and/or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to increase or activate transcription of at least one gene. Transcriptional activation domains are known in the art and include, but are not limited to, a herpes simplex virus VP16 activation domain and an NFAT activation domain. The presently disclosed RGN polypeptides can comprise a detectable label or a purification tag. The detectable label or purification tag can be located at the N-terminus, the C-terminus, or an internal location of the RNA-guided nuclease, either directly or indirectly via a linker peptide. In some of these embodiments, the RGN component of the fusion protein is a nuclease-dead RGN. In other embodiments, the RGN component of the fusion protein is an RGN with nickase activity. A detectable label is a molecule that can be visualized or otherwise observed. The detectable label may be fused to the RGN as a fusion protein (e.g., fluorescent protein) or may be a small molecule conjugated to the RGN polypeptide that can be detected visually or by other means. Detectable labels that can be fused to the presently disclosed RGNs as a fusion protein include any detectable protein domain, including but not limited to, a fluorescent protein or a protein domain that can be detected with a specific antibody. Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, ZsGreen1) and yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellow1). Non-limiting examples of small molecule detectable labels include radioactive labels, such as3H and35S. RGN polypeptides can also comprise a purification tag, which is any molecule that can be utilized to isolate a protein or fused protein from a mixture (e.g., biological sample, culture medium). Non-limiting examples of purification tags include biotin, myc, maltose binding protein (MBP), glutathione-S-transferase (GST), and 3×FLAG tag. II. Guide RNA The present disclosure provides guide RNAs and polynucleotides encoding the same. The term “guide RNA” refers to a nucleotide sequence having sufficient complementarity with a target nucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of an associated RNA-guided nuclease to the target nucleotide sequence. Thus, an RGN's respective guide RNA is one or more RNA molecules (generally, one or two), that can bind to the RGN and guide the RGN to bind to a particular target nucleotide sequence, and in those embodiments wherein the RGN has nickase or nuclease activity, also cleave the target nucleotide sequence. In general, a guide RNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). Native guide RNAs that comprise both a crRNA and a tracrRNA generally comprise two separate RNA molecules that hybridize to each other through the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA. Native direct repeat sequences within a CRISPR array generally range in length from 28 to 37 base pairs, although the length can vary between about 23 bp to about 55 bp. Spacer sequences within a CRISPR array generally range from about 32 to about 38 bp in length, although the length can be between about 21 bp to about 72 bp. Each CRISPR array generally comprises less than 50 units of the CRISPR repeat-spacer sequence. The CRISPRs are transcribed as part of a long transcript termed the primary CRISPR transcript, which comprises much of the CRISPR array. The primary CRISPR transcript is cleaved by Cas proteins to produce crRNAs or in some cases, to produce pre-crRNAs that are further processed by additional Cas proteins into mature crRNAs. Mature crRNAs comprise a spacer sequence and a CRISPR repeat sequence. In some embodiments in which pre-crRNAs are processed into mature (or processed) crRNAs, maturation involves the removal of about one to about six or more 5′, 3′, or 5′ and 3′ nucleotides. For the purposes of genome editing or targeting a particular target nucleotide sequence of interest, these nucleotides that are removed during maturation of the pre-crRNA molecule are not necessary for generating or designing a guide RNA. A CRISPR RNA (crRNA) comprises a spacer sequence and a CRISPR repeat sequence. The “spacer sequence” is the nucleotide sequence that directly hybridizes with the target nucleotide sequence of interest. The spacer sequence is engineered to be fully or partially complementary with the target sequence of interest. In various embodiments, the spacer sequence can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the spacer sequence can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In some embodiments, the spacer sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In some embodiments, the spacer sequence is about 10 to about 26 nucleotides in length, or about 12 to about 30 nucleotides in length. In particular embodiments, the spacer sequence is about 30 nucleotides in length. In some embodiments, the spacer sequence is 30 nucleotides in length. In some embodiments, the degree of complementarity between a spacer sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is between 50% and 99% or more, including but not limited to about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In particular embodiments, the degree of complementarity between a spacer sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In particular embodiments, the spacer sequence is free of secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (see, e.g., Zuker and Stiegler (1981)Nucleic Acids Res.9:133-148) and RNAfold (see, e.g., Gruber et al. (2008)Cell106(1):23-24). The CRISPR RNA repeat sequence comprises a nucleotide sequence that forms a structure, either on its own or in concert with a hybridized tracrRNA, that is recognized by the RGN molecule. In various embodiments, the CRISPR RNA repeat sequence can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the CRISPR repeat sequence can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In particular embodiments, the CRISPR repeat sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In some embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In particular embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA sequence, when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In particular embodiments, the CRISPR repeat sequence comprises the nucleotide sequence of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, or an active variant or fragment thereof that when comprised within a guide RNA, is capable of directing the sequence-specific binding of an associated RNA-guided nuclease provided herein to a target sequence of interest. In certain embodiments, an active CRISPR repeat sequence variant of a wild-type sequence comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the nucleotide sequence set forth as SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124. In certain embodiments, an active CRISPR repeat sequence fragment of a wild-type sequence comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of the nucleotide sequence set forth as SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124. In certain embodiments, the crRNA is not naturally-occurring. In some of these embodiments, the specific CRISPR repeat sequence is not linked to the engineered spacer sequence in nature and the CRISPR repeat sequence is considered heterologous to the spacer sequence. In certain embodiments, the spacer sequence is an engineered sequence that is not naturally occurring. A trans-activating CRISPR RNA or tracrRNA molecule comprises a nucleotide sequence comprising a region that has sufficient complementarity to hybridize to a CRISPR repeat sequence of a crRNA, which is referred to herein as the anti-repeat region. In some embodiments, the tracrRNA molecule further comprises a region with secondary structure (e.g., stem-loop) or forms secondary structure upon hybridizing with its corresponding crRNA. In particular embodiments, the region of the tracrRNA that is fully or partially complementary to a CRISPR repeat sequence is at the 5′ end of the molecule and the 3′ end of the tracrRNA comprises secondary structure. This region of secondary structure generally comprises several hairpin structures, including the nexus hairpin, which is found adjacent to the anti-repeat sequence. The nexus forms the core of the interactions between the guide RNA and the RGN, and is at the intersection between the guide RNA, the RGN, and the target DNA. The nexus hairpin often has a conserved nucleotide sequence in the base of the hairpin stem, with the motif UNANNC (SEQ ID NO: 132) found in many nexus hairpins in tracrRNAs. Interestingly, several of the RGNS of the invention use tracrRNAs that comprise non-canonical sequences in the base of the hairpin stem of their nexus hairpins, including UNANNA, UNANNU, UNANNG, and CNANNC (SEQ ID NOs: 129, 130, 131, and 133, respectively). There are often terminal hairpins at the 3′ end of the tracrRNA that can vary in structure and number, but often comprise a GC-rich Rho-independent transcriptional terminator hairpin followed by a string of U's at the 3′ end. See, for example, Briner et al. (2014)Molecular Cell56:333-339, Briner and Barrangou (2016)Cold Spring Harb Protoc; doi: 10.1101/pdb.top090902, and U.S. Publication No. 2017/0275648, each of which is herein incorporated by reference in its entirety. In various embodiments, the anti-repeat region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence comprises from about 8 nucleotides to about 30 nucleotides, or more. For example, the region of base pairing between the tracrRNA anti-repeat sequence and the CRISPR repeat sequence can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In particular embodiments, the region of base pairing between the tracrRNA anti-repeat sequence and the CRISPR repeat sequence is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In some embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA anti-repeat sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In particular embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA anti-repeat sequence, when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In various embodiments, the entire tracrRNA can comprise from about 60 nucleotides to more than about 210 nucleotides. For example, the tracrRNA can be about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, or more nucleotides in length. In particular embodiments, the tracrRNA is 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, 190, 200, 210 or more nucleotides in length. In particular embodiments, the tracrRNA is about 80 to about 90 nucleotides in length, including about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, and about 90 nucleotides in length. In particular embodiments, the tracrRNA is 80 to 90 nucleotides in length, including 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90 nucleotides in length. In particular embodiments, the tracrRNA comprises the nucleotide sequence of SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, or an active variant or fragment thereof that when comprised within a guide RNA is capable of directing the sequence-specific binding of an associated RNA-guided nuclease provided herein to a target sequence of interest. In certain embodiments, an active tracrRNA sequence variant of a wild-type sequence comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the nucleotide sequence set forth as SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125. In certain embodiments, an active tracrRNA sequence fragment of a wild-type sequence comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more contiguous nucleotides of the nucleotide sequence set forth as SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125. Two polynucleotide sequences can be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. Likewise, an RGN is considered to bind to a particular target sequence within a sequence-specific manner if the guide RNA bound to the RGN binds to the target sequence under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which the two polynucleotide sequences will hybridize to each other to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short sequences (e.g., 10 to 50 nucleotides) and at least about 60° C. for long sequences (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched sequence. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). The term “sequence specific” can also refer to the binding of a target sequence at a greater frequency than binding to a randomized background sequence. The guide RNA can be a single guide RNA or a dual-guide RNA system. A single guide RNA comprises the crRNA and tracrRNA on a single molecule of RNA, whereas a dual-guide RNA system comprises a crRNA and a tracrRNA present on two distinct RNA molecules, hybridized to one another through at least a portion of the CRISPR repeat sequence of the crRNA and at least a portion of the tracrRNA, which may be fully or partially complementary to the CRISPR repeat sequence of the crRNA. In some of those embodiments wherein the guide RNA is a single guide RNA, the crRNA and tracrRNA are separated by a linker nucleotide sequence. In general, the linker nucleotide sequence is one that does not include complementary bases in order to avoid the formation of secondary structure within or comprising nucleotides of the linker nucleotide sequence. In some embodiments, the linker nucleotide sequence between the crRNA and tracrRNA is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more nucleotides in length. In particular embodiments, the linker nucleotide sequence of a single guide RNA is at least 4 nucleotides in length. In certain embodiments, the linker nucleotide sequence is the nucleotide sequence set forth as SEQ ID NO: 249. The single guide RNA or dual-guide RNA can be synthesized chemically or via in vitro transcription. Assays for determining sequence-specific binding between an RGN and a guide RNA are known in the art and include, but are not limited to, in vitro binding assays between an expressed RGN and the guide RNA, which can be tagged with a detectable label (e.g., biotin) and used in a pull-down detection assay in which the guide RNA:RGN complex is captured via the detectable label (e.g., with streptavidin beads). A control guide RNA with an unrelated sequence or structure to the guide RNA can be used as a negative control for non-specific binding of the RGN to RNA. In certain embodiments, the guide RNA is SEQ ID NO: 4, 11, 18, 25, 32, 39, 46, 53, 59, 66, 73, 79, 86, 92, 99, 106, 113, 120, or 126, wherein the spacer sequence can be any sequence and is indicated as a poly-N sequence. In certain embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as an RNA molecule. The guide RNA can be transcribed in vitro or chemically synthesized. In other embodiments, a nucleotide sequence encoding the guide RNA is introduced into the cell, organelle, or embryo. In some of these embodiments, the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., an RNA polymerase III promoter). The promoter can be a native promoter or heterologous to the guide RNA-encoding nucleotide sequence. In various embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as a ribonucleoprotein complex, as described herein, wherein the guide RNA is bound to an RNA-guided nuclease polypeptide. The guide RNA directs an associated RNA-guided nuclease to a particular target nucleotide sequence of interest through hybridization of the guide RNA to the target nucleotide sequence. A target nucleotide sequence can comprise DNA, RNA, or a combination of both and can be single-stranded or double-stranded. A target nucleotide sequence can be genomic DNA (i.e., chromosomal DNA), plasmid DNA, or an RNA molecule (e.g., messenger RNA, ribosomal RNA, transfer RNA, micro RNA, small interfering RNA). The target nucleotide sequence can be bound (and in some embodiments, cleaved) by an RNA-guided nuclease in vitro or in a cell. The chromosomal sequence targeted by the RGN can be a nuclear, plastid or mitochondrial chromosomal sequence. In some embodiments, the target nucleotide sequence is unique in the target genome. The target nucleotide sequence is adjacent to a protospacer adjacent motif (PAM). A protospacer adjacent motif is generally within about 1 to about 10 nucleotides from the target nucleotide sequence, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides from the target nucleotide sequence. In particular embodiments, a PAM is within 1 to 10 nucleotides from the target nucleotide sequence, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the target nucleotide sequence. The PAM can be 5′ or 3′ of the target sequence. In some embodiments, the PAM is 3′ of the target sequence for the presently disclosed RGNs. Generally, the PAM is a consensus sequence of about 3-4 nucleotides, but in particular embodiments it can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length. In various embodiments, the PAM sequence recognized by the presently disclosed RGNs comprises the consensus sequence set forth as SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 62, 69, 79, 82, 95, 102, 109, or 116. In particular embodiments, an RNA-guided nuclease having SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579 or an active variant or fragment thereof binds a target nucleotide sequence adjacent to a PAM sequence set forth as SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 62, 69, 79, 82, 95, 102, 109, or 116. In some of these embodiments, the RGN binds to a guide sequence comprising a CRISPR repeat sequence set forth in SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, respectively, or an active variant or fragment thereof, and a tracrRNA sequence set forth in SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, respectively, or an active variant or fragment thereof. The RGN systems are described further in Examples 1-3 and Tables 1 and 2 of the present specification. Variants of RGN APG05586 (SEQ ID NO: 63) were produced and have amino acid sequences of SEQ ID NOs: 570-579. RGNs having any one of SEQ ID NOs: 63 and 570-579 can bind a target nucleotide sequence adjacent to a PAM sequence set forth as SEQ ID NO: 79. In some embodiments, the variants of RGN APG05586 bind to a guide sequence comprising a CRISPR repeat sequence set forth in SEQ ID NO: 64 and may also comprise a tracrRNA sequence set forth in SEQ ID NO: 65. These RGN systems are described further in Example 5 of the present specification. It is well-known in the art that PAM sequence specificity for a given nuclease enzyme is affected by enzyme concentration (see, e.g., Karvelis et al. (2015)Genome Biol16:253), which may be modified by altering the promoter used to express the RGN, or the amount of ribonucleoprotein complex delivered to the cell, organelle, or embryo. Upon recognizing its corresponding PAM sequence, the RGN can cleave the target nucleotide sequence at a specific cleavage site. As used herein, a cleavage site is made up of the two particular nucleotides within a target nucleotide sequence between which the nucleotide sequence is cleaved by an RGN. The cleavage site can comprise the 1stand 2nd, 2ndand 3rd, 3rdand 4th4thand 5th5thand 6th, 7thand 8th, or 8thand 9thnucleotides from the PAM in either the 5′ or 3′ direction. In some embodiments, the cleavage site may be over 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the PAM in either the 5′ or 3′ direction. As RGNs can cleave a target nucleotide sequence resulting in staggered ends, in some embodiments, the cleavage site is defined based on the distance of the two nucleotides from the PAM on the positive (+) strand of the polynucleotide and the distance of the two nucleotides from the PAM on the negative (−) strand of the polynucleotide. III. Nucleotides Encoding RNA-Guided Nucleases, CRISPR RNA, and/or tracrRNA The present disclosure provides polynucleotides comprising the presently disclosed CRISPR RNAs, tracrRNAs, and/or sgRNAs and polynucleotides comprising a nucleotide sequence encoding the presently disclosed RNA-guided nucleases, CRISPR RNAs, tracrRNAs, and/or sgRNAs. Presently disclosed polynucleotides include those comprising or encoding a CRISPR repeat sequence comprising the nucleotide sequence of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, or an active variant or fragment thereof that when comprised within a guide RNA is capable of directing the sequence-specific binding of an associated RNA-guided nuclease to a target sequence of interest. Also disclosed are polynucleotides comprising or encoding a tracrRNA comprising the nucleotide sequence of SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, or an active variant or fragment thereof that when comprised within a guide RNA is capable of directing the sequence-specific binding of an associated RNA-guided nuclease to a target sequence of interest. Polynucleotides are also provided that encode an RNA-guided nuclease comprising the amino acid sequence set forth as SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence-specific manner. The use of the term “polynucleotide” or “nucleic acid molecule” is not intended to limit the present disclosure to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides (RNA) and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. These include peptide nucleic acids (PNAs), PNA-DNA chimers, locked nucleic acids (LNAs), and phosphothiorate linked sequences. The polynucleotides disclosed herein also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, DNA-RNA hybrids, triplex structures, stem-and-loop structures, and the like. The nucleic acid molecules encoding RGNs can be codon optimized for expression in an organism of interest. A “codon-optimized” coding sequence is a polynucleotide coding sequence having its frequency of codon usage designed to mimic the frequency of preferred codon usage or transcription conditions of a particular host cell. Expression in the particular host cell or organism is enhanced as a result of the alteration of one or more codons at the nucleic acid level such that the translated amino acid sequence is not changed. Nucleic acid molecules can be codon optimized, either wholly or in part. Codon tables and other references providing preference information for a wide range of organisms are available in the art (see, e.g., Campbell and Gown (1990)Plant Physiol.92:1-11 for a discussion of plant-preferred codon usage). Methods are available in the art for synthesizing plant-preferred genes or mammalian (for example human) codon-optimized coding sequences. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989)Nucleic Acids Res.17:477-498, herein incorporated by reference. Polynucleotides encoding the RGNs, crRNAs, tracrRNAs, and/or sgRNAs provided herein can be provided in expression cassettes for in vitro expression or expression in a cell, organelle, embryo, or organism of interest. The cassette will include 5′ and 3′ regulatory sequences operably linked to a polynucleotide encoding an RGN, crRNA, tracrRNAs, and/or sgRNAs provided herein that allows for expression of the polynucleotide. The cassette may additionally contain at least one additional gene or genetic element to be cotransformed into the organism. Where additional genes or elements are included, the components are operably linked. The term “operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a promoter and a coding region of interest (e.g., region coding for an RGN, crRNA, tracrRNAs, and/or sgRNAs) is a functional link that allows for expression of the coding region of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. Alternatively, the additional gene(s) or element(s) can be provided on multiple expression cassettes. For example, the nucleotide sequence encoding a presently disclosed RGN can be present on one expression cassette, whereas the nucleotide sequence encoding a crRNA, tracrRNA, or complete guide RNA can be on a separate expression cassette. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the polynucleotides to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain a selectable marker gene. The expression cassette will include in the 5′-3′ direction of transcription, a transcriptional (and, in some embodiments, translational) initiation region (i.e., a promoter), an RGN-, crRNA-, tracrRNA- and/or sgRNA-encoding polynucleotide of the invention, and a transcriptional (and in some embodiments, translational) termination region (i.e., termination region) functional in the organism of interest. The promoters of the invention are capable of directing or driving expression of a coding sequence in a host cell. The regulatory regions (e.g., promoters, transcriptional regulatory regions, and translational termination regions) may be endogenous or heterologous to the host cell or to each other. As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Convenient termination regions are available from the Ti-plasmid ofA. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991)Mol. Gen. Genet.262:141-144; Proudfoot (1991)Cell64:671-674; Sanfacon et al. (1991)Genes Dev.5:141-149; Mogen et al. (1990)Plant Cell2:1261-1272; Munroe et al. (1990)Gene91:151-158; Ballas et al. (1989)Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987)Nucleic Acids Res.15:9627-9639. Additional regulatory signals include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter “Sambrook 11”; Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y., and the references cited therein. In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved. A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, inducible, growth stage-specific, cell type-specific, tissue-preferred, tissue-specific, or other promoters for expression in the organism of interest. See, for example, promoters set forth in WO 99/43838 and in U.S. Pat. Nos. 8,575,425; 7,790,846; 8,147,856; 8,586832; 7,772,369; 7,534,939; 6,072,050; 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; herein incorporated by reference. For expression in plants, constitutive promoters also include CaMV 35S promoter (Odell et al. (1985)Nature313:810-812); rice actin (McElroy et al. (1990)Plant Cell2:163-171); ubiquitin (Christensen et al. (1989)Plant Mol. Biol.12:619-632 and Christensen et al. (1992)Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991)Theor. Appl. Genet.81:581-588); and MAS (Velten et al. (1984)EMBO J.3:2723-2730). Examples of inducible promoters are the Adh1 promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, the PPDK promoter and the pepcarboxylase promoter which are both inducible by light. Also useful are promoters which are chemically inducible, such as the In2-2 promoter which is safener induced (U.S. Pat. No. 5,364,780), the Axig 1 promoter which is auxin induced and tapetum specific but also active in callus (PCT US01/22169), the steroid-responsive promoters (see, for example, the ERE promoter which is estrogen induced, and the glucocorticoid-inducible promoter in Schena et al. (1991)Proc. Natl. Acad. Sci. USA88:10421-10425 and McNellis et al. (1998)Plant J.14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991)Mol. Gen. Genet.227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference. Tissue-specific or tissue-preferred promoters can be utilized to target expression of an expression construct within a particular tissue. In certain embodiments, the tissue-specific or tissue-preferred promoters are active in plant tissue. Examples of promoters under developmental control in plants include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers. A “tissue specific” promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of genes, tissue-specific expression is the result of several interacting levels of gene regulation. As such, promoters from homologous or closely related plant species can be preferable to use to achieve efficient and reliable expression of transgenes in particular tissues. In some embodiments, the expression comprises a tissue-preferred promoter. A “tissue preferred” promoter is a promoter that initiates transcription preferentially, but not necessarily entirely or solely in certain tissues. In some embodiments, the nucleic acid molecules encoding an RGN, crRNA, and/or tracrRNA comprise a cell type-specific promoter. A “cell type specific” promoter is a promoter that primarily drives expression in certain cell types in one or more organs. Some examples of plant cells in which cell type specific promoters functional in plants may be primarily active include, for example, BETL cells, vascular cells in roots, leaves, stalk cells, and stem cells. The nucleic acid molecules can also include cell type preferred promoters. A “cell type preferred” promoter is a promoter that primarily drives expression mostly, but not necessarily entirely or solely in certain cell types in one or more organs. Some examples of plant cells in which cell type preferred promoters functional in plants may be preferentially active include, for example, BETL cells, vascular cells in roots, leaves, stalk cells, and stem cells. The nucleic acid sequences encoding the RGNs, crRNAs, tracrRNAs, and/or sgRNAs can be operably linked to a promoter sequence that is recognized by a phage RNA polymerase for example, for in vitro mRNA synthesis. In such embodiments, the in vitro-transcribed RNA can be purified for use in the methods described herein. For example, the promoter sequence can be a T7, T3, or SP6 promoter sequence or a variation of a T7, T3, or SP6 promoter sequence. In such embodiments, the expressed protein and/or RNAs can be purified for use in the methods of genome modification described herein. In certain embodiments, the polynucleotide encoding the RGN, crRNA, tracrRNA, and/or sgRNA also can be linked to a polyadenylation signal (e.g., SV40 polyA signal and other signals functional in plants) and/or at least one transcriptional termination sequence. Additionally, the sequence encoding the RGN also can be linked to sequence(s) encoding at least one nuclear localization signal, at least one cell-penetrating domain, and/or at least one signal peptide capable of trafficking proteins to particular subcellular locations, as described elsewhere herein. The polynucleotide encoding the RGN, crRNA, tracrRNA, and/or sgRNA can be present in a vector or multiple vectors. A “vector” refers to a polynucleotide composition for transferring, delivering, or introducing a nucleic acid into a host cell. Suitable vectors include plasmid vectors, phagemids, cosmids, artificial/mini-chromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, baculoviral vector). The vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like. Additional information can be found in “Current Protocols in Molecular Biology” Ausubel et al., John Wiley & Sons, New York, 2003 or “Molecular Cloning: A Laboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 3rd edition, 2001. The vector can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). In some embodiments, the expression cassette or vector comprising the sequence encoding the RGN polypeptide can further comprise a sequence encoding a crRNA and/or a tracrRNA, or the crRNA and tracrRNA combined to create a guide RNA. The sequence(s) encoding the crRNA and/or tracrRNA can be operably linked to at least one transcriptional control sequence for expression of the crRNA and/or tracrRNA in the organism or host cell of interest. For example, the polynucleotide encoding the crRNA and/or tracrRNA can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III). Examples of suitable Pol III promoters include, but are not limited to, mammalian U6, U3, H1, and 7SL RNA promoters and rice U6 and U3 promoters. As indicated, expression constructs comprising nucleotide sequences encoding the RGNs, crRNA, tracrRNA, and/or sgRNA can be used to transform organisms of interest. Methods for transformation involve introducing a nucleotide construct into an organism of interest. By “introducing” is intended to introduce the nucleotide construct to the host cell in such a manner that the construct gains access to the interior of the host cell. The methods of the invention do not require a particular method for introducing a nucleotide construct to a host organism, only that the nucleotide construct gains access to the interior of at least one cell of the host organism. The host cell can be a eukaryotic or prokaryotic cell. In particular embodiments, the eukaryotic host cell is a plant cell, a mammalian cell, an avian cell, or an insect cell. In some embodiments, the eukaryotic cell that comprises or expresses a presently disclosed RGN or that has been modified by a presently disclosed RGN is a human cell. In some embodiments, the eukaryotic cell that comprises or expresses a presently disclosed RGN or that has been modified by a presently disclosed RGN is a cell of hematopoietic origin, such as an immune cell (i.e., a cell of the innate or adaptive immune system) including but not limited to a B cell, a T cell, a natural killer (NK) cell, a pluripotent stem cell, an induced pluripotent stem cell, a chimeric antigen receptor T (CAR-T) cell, a monocyte, a macrophage, and a dendritic cell. Methods for introducing nucleotide constructs into plants and other host cells are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. The methods result in a transformed organism, such as a plant, including whole plants, as well as plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen). “Transgenic organisms” or “transformed organisms” or “stably transformed” organisms or cells or tissues refers to organisms that have incorporated or integrated a polynucleotide encoding an RGN, crRNA, and/or tracrRNA of the invention. It is recognized that other exogenous or endogenous nucleic acid sequences or DNA fragments may also be incorporated into the host cell.Agrobacterium- and biolistic-mediated transformation remain the two predominantly employed approaches for transformation of plant cells. However, transformation of a host cell may be performed by infection, transfection, microinjection, electroporation, microprojection, biolistics or particle bombardment, electroporation, silica/carbon fibers, ultrasound mediated, PEG mediated, calcium phosphate co-precipitation, polycation DMSO technique, DEAE dextran procedure, and viral mediated, liposome mediated and the like. Viral-mediated introduction of a polynucleotide encoding an RGN, crRNA, and/or tracrRNA includes retroviral, lentiviral, adenoviral, and adeno-associated viral mediated introduction and expression, as well as the use of Caulimoviruses, Geminiviruses, and RNA plant viruses. Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of host cell (e.g., monocot or dicot plant cell) targeted for transformation. Methods for transformation are known in the art and include those set forth in U.S. Pat. Nos. 8,575,425; 7,692,068; 8,802,934; 7,541,517; each of which is herein incorporated by reference. See, also, Rakoczy-Trojanowska, M. (2002)Cell Mol Biol Lett.7:849-858; Jones et al. (2005)Plant Methods1:5; Rivera et al. (2012)Physics of Life Reviews9:308-345; Bartlett et al. (2008)Plant Methods4:1-12; Bates, G. W. (1999)Methods in Molecular Biology111:359-366; Binns and Thomashow (1988)Annual Reviews in Microbiology42:575-606; Christou, P. (1992)The Plant Journal2:275-281; Christou, P. (1995)Euphytica85:13-27; Tzfira et al. (2004)TRENDS in Genetics20:375-383; Yao et al. (2006)Journal of Experimental Botany57:3737-3746; Zupan and Zambryski (1995)Plant Physiology107:1041-1047; Jones et al. (2005)Plant Methods1:5; Transformation may result in stable or transient incorporation of the nucleic acid into the cell. “Stable transformation” is intended to mean that the nucleotide construct introduced into a host cell integrates into the genome of the host cell and is capable of being inherited by the progeny thereof. “Transient transformation” is intended to mean that a polynucleotide is introduced into the host cell and does not integrate into the genome of the host cell. Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990)Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga (1993)Proc. Natl. Acad. Sci. USA90:913-917; Svab and Maliga (1993)EMBO J.12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994)Proc. Natl. Acad. Sci. USA91:7301-7305. The cells that have been transformed may be grown into a transgenic organism, such as a plant, in accordance with conventional ways. See, for example, McCormick et al. (1986)Plant Cell Reports5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as “transgenic seed”) having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome. Alternatively, cells that have been transformed may be introduced into an organism. These cells could have originated from the organism, wherein the cells are transformed in an ex vivo approach. The sequences provided herein may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassicasp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive,papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers. Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genusCurcumissuch as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea,hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, andchrysanthemum. Preferably, plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.). As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides. Further provided is a processed plant product or byproduct that retains the sequences disclosed herein, including for example, soymeal. The polynucleotides encoding the RGNs, crRNAs, and/or tracrRNAs can also be used to transform any prokaryotic species, including but not limited to, archaea and bacteria (e.g.,Bacillussp.,Klebsiellasp.Streptomycessp.,Rhizobiumsp.,Escherichiasp.,Pseudomonassp.,Salmonellasp.,Shigellasp.,Vibriosp.,Yersiniasp.,Mycoplasmasp.,Agrobacterium, Lactobacillussp.). The polynucleotides encoding the RGNs, crRNAs, and/or tracrRNAs can be used to transform any eukaryotic species, including but not limited to animals (e.g., mammals, insects, fish, birds, and reptiles), fungi, amoeba, algae, and yeast. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian, insect, or avian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of an RGN system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256: 808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994). Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787). The use of RNA or DNA viral based systems for the delivery of nucleic acids takes advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Viral. 66:2731-2739 (1992); Johann et al., J. Viral. 66:1635-1640 (1992); Sommnerfelt et al., Viral. 176:58-59 (1990); Wilson et al., J. Viral. 63:2374-2378 (1989); Miller et al., I. Viral. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Katin, Human Gene Therapy 5:793-801 (1994); Muzyczka, I. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., I. Viral. 63:03822-3828 (1989). Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ψJ2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference. In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. In some embodiments, the cell line may be mammalian, insect, or avian cells. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLaS3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CVI, RPTE, AlO, T24, 182, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, lurkat, 145.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4. COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-I cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr−/−, COR-L23, COR-L23/CPR, COR-L235010, CORL23/R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, lurkat, lY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCKII, MDCKII, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of an RGN system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of an RGN system, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds. In some embodiments, one or more vectors described herein are used to produce a non-human transgenic animal or transgenic plant. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, hamster, rabbit, cow, or pig. In some embodiments, the transgenic animal is a bird, such as a chicken or a duck. In some embodiments, the transgenic animal is an insect, such as a mosquito or a tick. IV. Variants and Fragments of Polypeptides and Polynucleotides The present disclosure provides active variants and fragments of a naturally-occurring (i.e., wild-type) RNA-guided nuclease, the amino acid sequence of which is set forth as SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579, as well as active variants and fragments of naturally-occurring CRISPR repeats, such as the sequence set forth as SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, and active variant and fragments of naturally-occurring tracrRNAs, such as the sequence set forth as SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, and polynucleotides encoding the same. While the activity of a variant or fragment may be altered compared to the polynucleotide or polypeptide of interest, the variant and fragment should retain the functionality of the polynucleotide or polypeptide of interest. For example, a variant or fragment may have increased activity, decreased activity, different spectrum of activity or any other alteration in activity when compared to the polynucleotide or polypeptide of interest. Fragments and variants of naturally-occurring RGN polypeptides, such as those disclosed herein, will retain sequence-specific, RNA-guided DNA-binding activity. In particular embodiments, fragments and variants of naturally-occurring RGN polypeptides, such as those disclosed herein, will retain nuclease activity (single-stranded or double-stranded). Fragments and variants of naturally-occurring CRISPR repeats, such as those disclosed herein, will retain the ability, when part of a guide RNA (comprising a tracrRNA), to bind to and guide an RNA-guided nuclease (complexed with the guide RNA) to a target nucleotide sequence in a sequence-specific manner. Fragments and variants of naturally-occurring tracrRNAs, such as those disclosed herein, will retain the ability, when part of a guide RNA (comprising a CRISPR RNA), to guide an RNA-guided nuclease (complexed with the guide RNA) to a target nucleotide sequence in a sequence-specific manner. The term “fragment” refers to a portion of a polynucleotide or polypeptide sequence of the invention. “Fragments” or “biologically active portions” include polynucleotides comprising a sufficient number of contiguous nucleotides to retain the biological activity (i.e., binding to and directing an RGN in a sequence-specific manner to a target nucleotide sequence when comprised within a guideRNA). “Fragments” or “biologically active portions” include polypeptides comprising a sufficient number of contiguous amino acid residues to retain the biological activity (i.e., binding to a target nucleotide sequence in a sequence-specific manner when complexed with a guide RNA). Fragments of the RGN proteins include those that are shorter than the full-length sequences due to the use of an alternate downstream start site. A biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700 or more contiguous amino acid residues of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579. Such biologically active portions can be prepared by recombinant techniques and evaluated for sequence-specific, RNA-guided DNA-binding activity. A biologically active fragment of a CRISPR repeat sequence can comprise at least 8 contiguous amino acids of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124. A biologically active portion of a CRISPR repeat sequence can be a polynucleotide that comprises, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124. A biologically active portion of a tracrRNA can be a polynucleotide that comprises, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110 or more contiguous nucleotides of SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125. In general, “variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” or “wild type” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the native amino acid sequence of the gene of interest. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode the polypeptide or the polynucleotide of interest. Generally, variants of a particular polynucleotide disclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein. Variants of a particular polynucleotide disclosed herein (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides disclosed herein is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In particular embodiments, the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to an amino acid sequence of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579. In some embodiments, variants of SEQ ID NO: 63 maintain the isoleucine at an amino acid position corresponding to 305, the valine at an amino acid position corresponding to 328, the leucine at an amino acid position corresponding to 366, the threonine at an amino acid position corresponding to 368, and the valine at an amino acid position corresponding to 405 of SEQ ID NO: 63. An amino acid position of a first amino acid sequence “corresponding to” a particular position of a second amino acid sequence refers to the position in the first amino acid sequence when the first and second amino acid sequences are optimally aligned that lines up with the specified amino acid residue position in the second sequence. In particular embodiments, variants of SEQ ID NO: 63 have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 63 outside of these amino acid residues that are maintained from SEQ ID NO: 63 (i.e., I305, V328, L366, T368, and V405). A biologically active variant of an RGN polypeptide of the invention may differ by as few as about 1-15 amino acid residues, as few as about 1-10, such as about 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue. In specific embodiments, the polypeptides can comprise an N-terminal or a C-terminal truncation, which can comprise at least a deletion of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700 amino acids or more from either the N or C terminus of the polypeptide. In certain embodiments, the presently disclosed polynucleotides comprise or encode a CRISPR repeat comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the nucleotide sequence set forth as SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124. The presently disclosed polynucleotides can comprise or encode a tracrRNA comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the nucleotide sequence set forth as SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125. Biologically active variants of a CRISPR repeat or tracrRNA of the invention may differ by as few as about 1-15 nucleotides, as few as about 1-10, such as about 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 nucleotide. In specific embodiments, the polynucleotides can comprise a 5′ or 3′ truncation, which can comprise at least a deletion of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110 nucleotides or more from either the 5′ or 3′ end of the polynucleotide. It is recognized that modifications may be made to the RGN polypeptides, CRISPR repeats, and tracrRNAs provided herein creating variant proteins and polynucleotides. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques. Alternatively, native, as yet-unknown or as yet unidentified polynucleotides and/or polypeptides structurally and/or functionally-related to the sequences disclosed herein may also be identified that fall within the scope of the present invention. Conservative amino acid substitutions may be made in nonconserved regions that do not alter the function of the RGN proteins. Alternatively, modifications may be made that improve the activity of the RGN. Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different RGN proteins disclosed herein (e.g., SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579) is manipulated to create a new RGN protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the RGN sequences provided herein and other known RGN genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased Kmin the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994)Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994)Nature370:389-391; Crameri et al. (1997)Nature Biotech.15:436-438; Moore et al. (1997)J. Mol. Biol.272:336-347; Zhang et al. (1997)Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998)Nature391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458. A “shuffled” nucleic acid is a nucleic acid produced by a shuffling procedure such as any shuffling procedure set forth herein. Shuffled nucleic acids are produced by recombining (physically or virtually) two or more nucleic acids (or character strings), for example in an artificial, and optionally recursive, fashion. Generally, one or more screening steps are used in shuffling processes to identify nucleic acids of interest; this screening step can be performed before or after any recombination step. In some (but not all) shuffling embodiments, it is desirable to perform multiple rounds of recombination prior to selection to increase the diversity of the pool to be screened. The overall process of recombination and selection are optionally repeated recursively. Depending on context, shuffling can refer to an overall process of recombination and selection, or, alternately, can simply refer to the recombinational portions of the overall process. As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California). As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10. Two sequences are “optimally aligned” when they are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences. Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art and described, e.g., in Dayhoff et al. (1978) “A model of evolutionary change in proteins.” In “Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3 (ed. M. O. Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. and Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919. The BLOSUM62 matrix is often used as a default scoring substitution matrix in sequence alignment protocols. The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The alignment is defined by the amino acids positions of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences, so as to arrive at the highest possible score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer-implemented alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul et al. (1997)Nucleic Acids Res.25:3389-3402, and made available to the public at the National Center for Biotechnology Information Website (www.ncbi.nlm.nih.gov). Optimal alignments, including multiple alignments, can be prepared using, e.g., PSI-BLAST, available through www.ncbi.nlm.nih.gov and described by Altschul et al. (1997)Nucleic Acids Res.25:3389-3402. With respect to an amino acid sequence that is optimally aligned with a reference sequence, an amino acid residue “corresponds to” the position in the reference sequence with which the residue is paired in the alignment. The “position” is denoted by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus. Owing to deletions, insertion, truncations, fusions, etc., that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence as determined by simply counting from the N-terminal will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where there is a deletion in an aligned test sequence, there will be no amino acid that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to any amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence. V. Antibodies Antibodies to the RGN polypeptides or ribonucleoproteins comprising the RGN polypeptides of the present invention, including those having the amino acid sequence set forth as SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579 or active variants or fragments thereof, are also encompassed. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; and U.S. Pat. No. 4,196,265). These antibodies can be used in kits for the detection and isolation of RGN polypeptides or ribonucleoproteins. Thus, this disclosure provides kits comprising antibodies that specifically bind to the polypeptides or ribonucleoproteins described herein, including, for example, polypeptides having the sequence of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579. VI. Systems and Ribonucleoprotein Complexes for Binding a Target Sequence of Interest and Methods of Making the Same The present disclosure provides a system for binding a target sequence of interest, wherein the system comprises at least one guide RNA or a nucleotide sequence encoding the same, and at least one RNA-guided nuclease or a nucleotide sequence encoding the same. The guide RNA hybridizes to the target sequence of interest and also forms a complex with the RGN polypeptide, thereby directing the RGN polypeptide to bind to the target sequence. In some of these embodiments, the RGN comprises an amino acid sequence of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence comprising the nucleotide sequence of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, or an active variant or fragment thereof. In particular embodiments, the guide RNA comprises a tracrRNA comprising a nucleotide sequence of SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, or an active variant or fragment thereof. The guide RNA of the system can be a single guide RNA or a dual-guide RNA. In particular embodiments, the system comprises an RNA-guided nuclease that is heterologous to the guideRNA, wherein the RGN and guideRNA are not found complexed to one another (i.e., bound to one another) in nature. The system for binding a target sequence of interest provided herein can be a ribonucleoprotein complex, which is at least one molecule of an RNA bound to at least one protein. The ribonucleoprotein complexes provided herein comprise at least one guide RNA as the RNA component and an RNA-guided nuclease as the protein component. Such ribonucleoprotein complexes can be purified from a cell or organism that naturally expresses an RGN polypeptide and has been engineered to express a particular guide RNA that is specific for a target sequence of interest. Alternatively, the ribonucleoprotein complex can be purified from a cell or organism that has been transformed with polynucleotides that encode an RGN polypeptide and a guide RNA and cultured under conditions to allow for the expression of the RGN polypeptide and guide RNA. Thus, methods are provided for making an RGN polypeptide or an RGN ribonucleoprotein complex. Such methods comprise culturing a cell comprising a nucleotide sequence encoding an RGN polypeptide, and in some embodiments a nucleotide sequence encoding a guide RNA, under conditions in which the RGN polypeptide (and in some embodiments, the guide RNA) is expressed. The RGN polypeptide or RGN ribonucleoprotein can then be purified from a lysate of the cultured cells. Methods for purifying an RGN polypeptide or RGN ribonucleoprotein complex from a lysate of a biological sample are known in the art (e.g., size exclusion and/or affinity chromatography, 2D-PAGE, HPLC, reversed-phase chromatography, immunoprecipitation). In particular methods, the RGN polypeptide is recombinantly produced and comprises a purification tag to aid in its purification, including but not limited to, glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6×His, 10×His, biotin carboxyl carrier protein (BCCP), and calmodulin. Generally, the tagged RGN polypeptide or RGN ribonucleoprotein complex is purified using immobilized metal affinity chromatography. It will be appreciated that other similar methods known in the art may be used, including other forms of chromatography or for example immunoprecipitation, either alone or in combination. An “isolated” or “purified” polypeptide, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polypeptide as found in its naturally occurring environment. Thus, an isolated or purified polypeptide is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals. Particular methods provided herein for binding and/or cleaving a target sequence of interest involve the use of an in vitro assembled RGN ribonucleoprotein complex. In vitro assembly of an RGN ribonucleoprotein complex can be performed using any method known in the art in which an RGN polypeptide is contacted with a guide RNA under conditions to allow for binding of the RGN polypeptide to the guide RNA. As used herein, “contact”, contacting”, “contacted,” refer to placing the components of a desired reaction together under conditions suitable for carrying out the desired reaction. The RGN polypeptide can be purified from a biological sample, cell lysate, or culture medium, produced via in vitro translation, or chemically synthesized. The guide RNA can be purified from a biological sample, cell lysate, or culture medium, transcribed in vitro, or chemically synthesized. The RGN polypeptide and guide RNA can be brought into contact in solution (e.g., buffered saline solution) to allow for in vitro assembly of the RGN ribonucleoprotein complex. VII. Methods of Binding, Cleaving, or Modifying a Target Sequence The present disclosure provides methods for binding, cleaving, and/or modifying a target nucleotide sequence of interest. The methods include delivering a system comprising at least one guide RNA or a polynucleotide encoding the same, and at least one RGN polypeptide or a polynucleotide encoding the same to the target sequence or a cell, organelle, or embryo comprising the target sequence. In some of these embodiments, the RGN comprises the amino acid sequence of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence comprising the nucleotide sequence of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, or an active variant or fragment thereof. In particular embodiments, the guide RNA comprises a tracrRNA comprising the nucleotide sequence of SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, or an active variant or fragment thereof. The guide RNA of the system can be a single guide RNA or a dual-guide RNA. The RGN of the system may be nuclease dead RGN, have nickase activity, or may be a fusion polypeptide. In some embodiments, the fusion polypeptide comprises a base-editing polypeptide, for example a cytidine deaminase or an adenosine deaminase. In other embodiments, the RGN fusion protein comprises a reverse transcriptase. In other embodiments, the RGN fusion protein comprises a polypeptide that recruits members of a functional nucleic acid repair complex, such as a member of the nucleotide excision repair (NER) or transcription coupled-nucleotide excision repair (TC-NER) pathway (Wei et al., 2015, PNAS USA112(27):E3495-504; Troelstra et al., 1992, Cell71:939-953; Marnef et al., 2017, J Mol Biol429(9):1277-1288), as described in U.S. Provisional Application No. 62/966,203, which was filed on Jan. 27, 2020, and is incorporated by reference in its entirety. In some embodiments, the RGN fusion protein comprises CSB (van den Boom et al., 2004, J Cell Biol166(1):27-36; van Gool et al., 1997, EMBO J16(19):5955-65; an example of which is set forth as SEQ ID NO: 608), which is a member of the TC-NER (nucleotide excision repair) pathway and functions in the recruitment of other members. In further embodiments, the RGN fusion protein comprises an active domain of CSB, such as the acidic domain of CSB which comprises amino acid residues 356-394 of SEQ ID NO: 608 (Teng et al., 2018, Nat Commun9(1):4115). In particular embodiments, the RGN and/or guide RNA is heterologous to the cell, organelle, or embryo to which the RGN and/or guide RNA (or polynucleotide(s) encoding at least one of the RGN and guide RNA) are introduced. In those embodiments wherein the method comprises delivering a polynucleotide encoding a guide RNA and/or an RGN polypeptide, the cell or embryo can then be cultured under conditions in which the guide RNA and/or RGN polypeptide are expressed. In various embodiments, the method comprises contacting a target sequence with an RGN ribonucleoprotein complex. The RGN ribonucleoprotein complex may comprise an RGN that is nuclease dead or has nickase activity. In some embodiments, the RGN of the ribonucleoprotein complex is a fusion polypeptide comprising a base-editing polypeptide. In certain embodiments, the method comprises introducing into a cell, organelle, or embryo comprising a target sequence an RGN ribonucleoprotein complex. The RGN ribonucleoprotein complex can be one that has been purified from a biological sample, recombinantly produced and subsequently purified, or in vitro-assembled as described herein. In those embodiments wherein the RGN ribonucleoprotein complex that is contacted with the target sequence or a cell organelle, or embryo has been assembled in vitro, the method can further comprise the in vitro assembly of the complex prior to contact with the target sequence, cell, organelle, or embryo. A purified or in vitro assembled RGN ribonucleoprotein complex can be introduced into a cell, organelle, or embryo using any method known in the art, including, but not limited to electroporation. Alternatively, an RGN polypeptide and/or polynucleotide encoding or comprising the guide RNA can be introduced into a cell, organelle, or embryo using any method known in the art (e.g., electroporation). Upon delivery to or contact with the target sequence or cell, organelle, or embryo comprising the target sequence, the guide RNA directs the RGN to bind to the target sequence in a sequence-specific manner. In those embodiments wherein the RGN has nuclease activity, the RGN polypeptide cleaves the target sequence of interest upon binding. The target sequence can subsequently be modified via endogenous repair mechanisms, such as non-homologous end joining, or homology-directed repair with a provided donor polynucleotide. Methods to measure binding of an RGN polypeptide to a target sequence are known in the art and include chromatin immunoprecipitation assays, gel mobility shift assays, DNA pull-down assays, reporter assays, microplate capture and detection assays. Likewise, methods to measure cleavage or modification of a target sequence are known in the art and include in vitro or in vivo cleavage assays wherein cleavage is confirmed using PCR, sequencing, or gel electrophoresis, with or without the attachment of an appropriate label (e.g., radioisotope, fluorescent substance) to the target sequence to facilitate detection of degradation products. Alternatively, the nicking triggered exponential amplification reaction (NTEXPAR) assay can be used (see, e.g., Zhang et al. (2016)Chem. Sci.7:4951-4957). In vivo cleavage can be evaluated using the Surveyor assay (Guschin et al. (2010)Methods Mol Biol649:247-256). In some embodiments, the methods involve the use of a single type of RGN complexed with more than one guide RNA. The more than one guide RNA can target different regions of a single gene or can target multiple genes. In those embodiments wherein a donor polynucleotide is not provided, a double-stranded break introduced by an RGN polypeptide can be repaired by a non-homologous end-joining (NHEJ) repair process. Due to the error-prone nature of NHEJ, repair of the double-stranded break can result in a modification to the target sequence. As used herein, a “modification” in reference to a nucleic acid molecule refers to a change in the nucleotide sequence of the nucleic acid molecule, which can be a deletion, insertion, or substitution of one or more nucleotides, or a combination thereof. Modification of the target sequence can result in the expression of an altered protein product or inactivation of a coding sequence. In those embodiments wherein a donor polynucleotide is present, the donor sequence in the donor polynucleotide can be integrated into or exchanged with the target nucleotide sequence during the course of repair of the introduced double-stranded break, resulting in the introduction of the exogenous donor sequence. A donor polynucleotide thus comprises a donor sequence that is desired to be introduced into a target sequence of interest. In some embodiments, the donor sequence alters the original target nucleotide sequence such that the newly integrated donor sequence will not be recognized and cleaved by the RGN. Integration of the donor sequence can be enhanced by the inclusion within the donor polynucleotide of flanking sequences, referred to herein as “homology arms” that have substantial sequence identity with the sequences flanking the target nucleotide sequence, allowing for a homology-directed repair process. In some embodiments, homology arms have a length of at least 50 base pairs, at least 100 base pairs, and up to 2000 base pairs or more, and have at least 90%, at least 95%, or more, sequence homology to their corresponding sequence within the target nucleotide sequence. In those embodiments wherein the RGN polypeptide introduces double-stranded staggered breaks, the donor polynucleotide can comprise a donor sequence flanked by compatible overhangs, allowing for direct ligation of the donor sequence to the cleaved target nucleotide sequence comprising overhangs by a non-homologous repair process during repair of the double-stranded break. In those embodiments wherein the method involves the use of an RGN that is a nickase (i.e., is only able to cleave a single strand of a double-stranded polynucleotide), the method can comprise introducing two RGN nickases that target identical or overlapping target sequences and cleave different strands of the polynucleotide. For example, an RGN nickase that only cleaves the positive (+) strand of a double-stranded polynucleotide can be introduced along with a second RGN nickase that only cleaves the negative (−) strand of a double-stranded polynucleotide. In various embodiments, a method is provided for binding a target nucleotide sequence and detecting the target sequence, wherein the method comprises introducing into a cell, organelle, or embryo at least one guide RNA or a polynucleotide encoding the same, and at least one RGN polypeptide or a polynucleotide encoding the same, expressing the guide RNA and/or RGN polypeptide (if coding sequences are introduced), wherein the RGN polypeptide is a nuclease-dead RGN and further comprises a detectable label, and the method further comprises detecting the detectable label. The detectable label may be fused to the RGN as a fusion protein (e.g., fluorescent protein) or may be a small molecule conjugated to or incorporated within the RGN polypeptide that can be detected visually or by other means. Also provided herein are methods for modulating the expression of a target sequence or a gene of interest under the regulation of a target sequence. The methods comprise introducing into a cell, organelle, or embryo at least one guide RNA or a polynucleotide encoding the same, and at least one RGN polypeptide or a polynucleotide encoding the same, expressing the guide RNA and/or RGN polypeptide (if coding sequences are introduced), wherein the RGN polypeptide is a nuclease-dead RGN. In some of these embodiments, the nuclease-dead RGN is a fusion protein comprising an expression modulator domain (i.e., epigenetic modification domain, transcriptional activation domain or a transcriptional repressor domain) as described herein. The present disclosure also provides methods for binding and/or modifying a target nucleotide sequence of interest. The methods include delivering a system comprising at least one guide RNA or a polynucleotide encoding the same, and at least one fusion polypeptide comprises an RGN of the invention and a base-editing polypeptide, for example a cytidine deaminase or an adenosine deaminase, or a polynucleotide encoding the fusion polypeptide, to the target sequence or a cell, organelle, or embryo comprising the target sequence. One of ordinary skill in the art will appreciate that any of the presently disclosed methods can be used to target a single target sequence or multiple target sequences. Thus, methods comprise the use of a single RGN polypeptide in combination with multiple, distinct guide RNAs, which can target multiple, distinct sequences within a single gene and/or multiple genes. Also encompassed herein are methods wherein multiple, distinct guide RNAs are introduced in combination with multiple, distinct RGN polypeptides. These guide RNAs and guide RNA/RGN polypeptide systems can target multiple, distinct sequences within a single gene and/or multiple genes. In one aspect, the invention provides kits containing any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably linked to a DNA sequence encoding the crRNA sequence and one or more insertion sites for inserting a guide sequence upstream of the encoded crRNA sequence, wherein when expressed, the guide sequence directs sequence-specific binding of an RGN complex to a target sequence in a eukaryotic cell, wherein the RGN complex comprises an RGN enzyme complexed with the guide RNA polynucleotide; and/or (b) a second regulatory element operably linked to an enzyme coding sequence encoding said RGN enzyme comprising a nuclear localization sequence. Elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube. In some embodiments, the kit includes instructions in one or more languages. In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element. In some embodiments, the kit comprises a homologous recombination template polynucleotide. In one aspect, the invention provides methods for using one or more elements of an RGN system. The RGN system of the invention provides an effective means for modifying a target polynucleotide. The RGN system of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating, base editing) a target polynucleotide in a multiplicity of cell types. As such the RGN system of the invention has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary RGN system, or RGN complex, comprises an RGN enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide. VIII. Target Polynucleotides In one aspect, the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including microalgae) and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant (including microalgae). Using natural variability, plant breeders combine most useful genes for desirable qualities, such as yield, quality, uniformity, hardiness, and resistance against pests. These desirable qualities also include growth, day length preferences, temperature requirements, initiation date of floral or reproductive development, fatty acid content, insect resistance, disease resistance, nematode resistance, fungal resistance, herbicide resistance, tolerance to various environmental factors including drought, heat, wet, cold, wind, and adverse soil conditions including high salinity The sources of these useful genes include native or foreign varieties, heirloom varieties, wild plant relatives, and induced mutations, e.g., treating plant material with mutagenic agents. Using the present invention, plant breeders are provided with a new tool to induce mutations. Accordingly, one skilled in the art can analyze the genome for sources of useful genes, and in varieties having desired characteristics or traits employ the present invention to induce the rise of useful genes, with more precision than previous mutagenic agents and hence accelerate and improve plant breeding programs. The target polynucleotide of an RGN system can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell. The target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA). Without wishing to be bound by theory, the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized by the RGN system. The precise sequence and length requirements for the PAM differ depending on the RGN used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). The target polynucleotide of an RGN system may include a number of disease-associated genes and polynucleotides as well as signaling biochemical pathway-associated genes and polynucleotides. Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A “disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease (e.g., a causal mutation). The transcribed or translated products may be known or unknown, and further may be at a normal or abnormal level. In some embodiments, the disease may be an animal disease. In some embodiments, the disease may be an avian disease. In other embodiments, the disease may be a mammalian disease. In further embodiments, the disease may be a human disease. Examples of disease-associated genes and polynucleotides in humans are available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web. Although RGN systems are particularly useful for their relative ease in targeting to genomic sequences of interest, there still remains an issue of what the RGN can do to address a causal mutation. One approach is to produce a fusion protein between an RGN (preferably an inactive or nickase variant of the RGN) and a base-editing enzyme or the active domain of a base editing enzyme, such as a cytidine deaminase or an adenosine deaminase base editor (U.S. Pat. No. 9,840,699, herein incorporated by reference). In some embodiments, the methods comprise contacting a DNA molecule with (a) a fusion protein comprising an RGN of the invention and a base-editing polypeptide such as a deaminase; and (b) a gRNA targeting the fusion protein of (a) to a target nucleotide sequence of the DNA strand; wherein the DNA molecule is contacted with the fusion protein and the gRNA in an amount effective and under conditions suitable for the deamination of a nucleobase. In some embodiments, the target DNA sequence comprises a sequence associated with a disease or disorder, and wherein the deamination of the nucleobase results in a sequence that is not associated with a disease or disorder. In some embodiments, the target DNA sequence resides in an allele of a crop plant, wherein the particular allele of the trait of interest results in a plant of lesser agronomic value. The deamination of the nucleobase results in an allele that improves the trait and increases the agronomic value of the plant. In some embodiments, the DNA sequence comprises a T→C or A→G point mutation associated with a disease or disorder, and wherein the deamination of the mutant C or G base results in a sequence that is not associated with a disease or disorder. In some embodiments, the deamination corrects a point mutation in the sequence associated with the disease or disorder. In some embodiments, the sequence associated with the disease or disorder encodes a protein, and wherein the deamination introduces a stop codon into the sequence associated with the disease or disorder, resulting in a truncation of the encoded protein. In some embodiments, the contacting is performed in vivo in a subject susceptible to having, having, or diagnosed with the disease or disorder. In some embodiments, the disease or disorder is a disease associated with a point mutation, or a single-base mutation, in the genome. In some embodiments, the disease is a genetic disease, a cancer, a metabolic disease, or a lysosomal storage disease. IX. Pharmaceutical Compositions and Methods of Treatment Pharmaceutical compositions comprising the presently disclosed RGN polypeptides and active variants and fragments thereof, as well as polynucleotides encoding the same, the presently disclosed gRNAs or polynucleotides encoding the same, the presently disclosed systems, or cells comprising any of the RGN polypeptides or RGN-encoding polynucleotides, gRNA or gRNA-encoding polynucleotides, or the RGN systems, and a pharmaceutically acceptable carrier are provided. A pharmaceutical composition is a composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition or disease that comprises an active ingredient (i.e., RGN polypeptides, RGN-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RGN systems, or cells comprising any one of these) and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” refers to a material that does not cause significant irritation to an organism and does not abrogate the activity and properties of the active ingredient (i.e., RGN polypeptides, RGN-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RGN systems, or cells comprising any one of these). Carriers must be of sufficiently high purity and of sufficiently low toxicity to render them suitable for administration to a subject being treated. The carrier can be inert, or it can possess pharmaceutical benefits. In some embodiments, a pharmaceutically acceptable carrier comprises one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. In some embodiments, the pharmaceutically acceptable carrier is not naturally-occurring. In some embodiments, the pharmaceutically acceptable carrier and the active ingredient are not found together in nature. Pharmaceutical compositions used in the presently disclosed methods can be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. A multitude of appropriate formulations are known to those skilled in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21st ed. 2005). Suitable formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN vesicles), lipid nanoparticles, DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Pharmaceutical compositions for oral or parenteral use may be prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. In some embodiments wherein cells comprising or modified with the presently disclosed RGN, gRNAs, RGN systems or polynucleotides encoding the same are administered to a subject, the cells are administered as a suspension with a pharmaceutically acceptable carrier. One of skill in the art will recognize that a pharmaceutically acceptable carrier to be used in a cell composition will not include buffers, compounds, cryopreservation agents, preservatives, or other agents in amounts that substantially interfere with the viability of the cells to be delivered to the subject. A formulation comprising cells can include e.g., osmotic buffers that permit cell membrane integrity to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those of skill in the art and/or can be adapted for use with the cells described herein using routine experimentation. A cell composition can also be emulsified or presented as a liposome composition, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, and in amounts suitable for use in the therapeutic methods described herein. Additional agents included in a cell composition can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable and pharmaceutically acceptable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active compound used in the cell compositions that is effective in the treatment of a particular disorder or condition can depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The presently disclosed RGN polypeptides, guide RNAs, RGN systems or polynucleotides encoding the same can be formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending upon the particular mode of administration and dosage form. In some embodiments, these pharmaceutical compositions are formulated to achieve a physiologically compatible pH, and range from a pH of about 3 to a pH of about 11, about pH 3 to about pH 7, depending on the formulation and route of administration. In some embodiments, the pH can be adjusted to a range from about pH 5.0 to about pH 8. In some embodiments, the compositions can comprise a therapeutically effective amount of at least one compound as described herein, together with one or more pharmaceutically acceptable excipients. In some embodiments, the compositions comprise a combination of the compounds described herein, or include a second active ingredient useful in the treatment or prevention of bacterial growth (for example and without limitation, anti-bacterial or anti-microbial agents), or include a combination of reagents of the present disclosure. Suitable excipients include, for example, carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients can include antioxidants (for example and without limitation, ascorbic acid), chelating agents (for example and without limitation, EDTA), carbohydrates (for example and without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example and without limitation, oils, water, saline, glycerol and ethanol), wetting or emulsifying agents, pH buffering substances, and the like. In some embodiments, the formulations are provided in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring the addition of the sterile liquid carrier, for example, saline, water-for-injection, a semi-liquid foam, or gel, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. In some embodiments, the active ingredient is dissolved in a buffered liquid solution that is frozen in a unit-dose or multi-dose container and later thawed for injection or kept/stabilized under refrigeration until use. The therapeutic agent(s) may be contained in controlled release systems. In order to prolong the effect of a drug, it often is desirable to slow the absorption of the drug from subcutaneous, intrathecal, or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. In some embodiments, the use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term sustained release implants are well-known to those of ordinary skill in the art. Methods of treating a disease in a subject in need thereof are provided herein. The methods comprise administering to a subject in need thereof an effective amount of a presently disclosed RGN polypeptide or active variant or fragment thereof or a polynucleotide encoding the same, a presently disclosed gRNA or a polynucleotide encoding the same, a presently disclosed RGN system, or a cell modified by or comprising any one of these compositions. In some embodiments, the treatment comprises in vivo gene editing by administering a presently disclosed RGN polypeptide, gRNA, or RGN system or polynucleotide(s) encoding the same. In some embodiments, the treatment comprises ex vivo gene editing wherein cells are genetically modified ex vivo with a presently disclosed RGN polypeptide, gRNA, or RGN system or polynucleotide(s) encoding the same and then the modified cells are administered to a subject. In some embodiments, the genetically modified cells originate from the subject that is then administered the modified cells, and the transplanted cells are referred to herein as autologous. In some embodiments, the genetically modified cells originate from a different subject (i.e., donor) within the same species as the subject that is administered the modified cells (i.e., recipient), and the transplanted cells are referred to herein as allogeneic. In some examples described herein, the cells can be expanded in culture prior to administration to a subject in need thereof. In some embodiments, the disease to be treated with the presently disclosed compositions is one that can be treated with immunotherapy, such as with a chimeric antigen receptor (CAR) T cell. Such diseases include but are not limited to cancer. In some embodiments, the disease to be treated with the presently disclosed compositions is associated with a causal mutation. As used herein, a “causal mutation” refers to a particular nucleotide, nucleotides, or nucleotide sequence in the genome that contributes to the severity or presence of a disease or disorder in a subject. The correction of the causal mutation leads to the improvement of at least one symptom resulting from a disease or disorder. In some embodiments, the causal mutation is adjacent to a PAM site recognized by an RGN disclosed herein. The causal mutation can be corrected with a presently disclosed RGN or a fusion polypeptide comprising a presently disclosed RGN and a base-editing polypeptide (i.e., a base editor). Non-limiting examples of diseases associated with a causal mutation include cystic fibrosis, Hurler syndrome, Friedreich's Ataxia, Huntington's Disease, and sickle cell disease. In some embodiments, the disease to be treated with the presently disclosed RGNs is a disease listed in Table 11. Additional non-limiting examples of disease-associated genes and mutations are available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web. As used herein, “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested. The term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, and the delivery system in which it is carried. The term “administering” refers to the placement of an active ingredient into a subject, by a method or route that results in at least partial localization of the introduced active ingredient at a desired site, such as a site of injury or repair, such that a desired effect(s) is produced. In those embodiments wherein cells are administered, the cells can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the life time of the patient, i.e., long-term engraftment. For example, in some aspects described herein, an effective amount of photoreceptor cells or retinal progenitor cells is administered via a systemic route of administration, such as an intraperitoneal or intravenous route. In some embodiments, the administering comprises administering by viral delivery. In some embodiments, the administering comprises administering by electroporation. In some embodiments, the administering comprises administering by nanoparticle delivery. In some embodiments, the administering comprises administering by liposome delivery. Any effective route of administration can be used to administer an effective amount of a pharmaceutical composition described herein. In some embodiments, the administering comprises administering by a method selected from the group consisting of: intravenously, subcutaneously, intramuscularly, orally, rectally, by aerosol, parenterally, ophthalmicly, pulmonarily, transdermally, vaginally, otically, nasally, and by topical administration, or any combination thereof. In some embodiments, for the delivery of cells, administration by injection or infusion is used. As used herein, the term “subject” refers to any individual for whom diagnosis, treatment or therapy is desired. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human being. The efficacy of a treatment can be determined by the skilled clinician. However, a treatment is considered an “effective treatment,” if any one or all of the signs or symptoms of a disease or disorder are altered in a beneficial manner (e.g., decreased by at least 10%), or other clinically accepted symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art. Treatment includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms. A. Modifying Causal Mutations Using Base-Editing An example of a genetically inherited disease which could be corrected using an approach that relies on an RGN-base editor fusion protein of the invention is Hurler Syndrome. Hurler Syndrome, also known as MPS-1, is the result of a deficiency of α-L-iduronidase (IDUA) resulting in a lysosomal storage disease characterized at the molecular level by the accumulation of dermatan sulfate and heparan sulfate in lysosomes. This disease is generally an inherited genetic disorder caused by mutations in the IDUA gene encoding α-L-iduronidase. Common IDUA mutations are W402X and Q70X, both nonsense mutations resulting in premature termination of translation. Such mutations are well addressed by precise genome editing (PGE) approaches, since reversion of a single nucleotide, for example by a base-editing approach, would restore the wild-type coding sequence and result in protein expression controlled by the endogenous regulatory mechanisms of the genetic locus. Additionally, since heterozygotes are known to be asymptomatic, a PGE therapy that targets one of these mutations would be useful to a large proportion of patients with this disease, as only one of the mutated alleles needs to be corrected (Bunge et al. (1994) Hum. Mol. Genet. 3(6): 861-866, herein incorporated by reference). Current treatments for Hurler Syndrome include enzyme replacement therapy and bone marrow transplants (Vellodi et al. (1997) Arch. Dis. Child. 76(2): 92-99; Peters et al. (1998) Blood 91(7): 2601-2608, herein incorporated by reference). While enzyme replacement therapy has had a dramatic effect on the survival and quality of life of Hurler Syndrome patients, this approach requires costly and time-consuming weekly infusions. Additional approaches include the delivery of the IDUA gene on an expression vector or the insertion of the gene into a highly expressed locus such as that of serum albumin (U.S. Pat. No. 9,956,247, herein incorporated by reference). However, these approaches do not restore the original IDUA locus to the correct coding sequence. A genome-editing strategy would have a number of advantages, most notably that regulation of gene expression would be controlled by the natural mechanisms present in healthy individuals. Additionally, using base editing does not necessitate causing a double stranded DNA breaks, which could lead to large chromosomal rearrangements, cell death, or oncogenecity by the disruption of tumor suppression mechanisms. A general strategy may be directed toward using RGN-base editor fusion proteins of the invention to target and correct certain disease-causing mutations in the human genome. It will be appreciated that similar approaches to target diseases that can be corrected by base-editing may also be pursued. It will be further appreciated that similar approaches to target disease-causing mutations in other species, particularly common household pets or livestock, can also be deployed using the RGNs of the invention. Common household pets and livestock include dogs, cats, horses, pigs, cows, sheep, chickens, donkeys, snakes, ferrets, and fish including salmon and shrimp. B. Modifying Causal Mutations by Targeted Deletion RGNs of the invention could also be useful in human therapeutic approaches where the causal mutation is more complicated. For example, some diseases such as Friedreich's Ataxia and Huntington's Disease are the result of a significant increase in repeats of a three nucleotide motif at a particular region of a gene, which affects the ability of the expressed protein to function or to be expressed. Friedreich's Ataxia (FRDA) is an autosomal recessive disease resulting in progressive degeneration of nervous tissue in the spinal cord. Reduced levels of the frataxin (FXN) protein in the mitochondria cause oxidative damages and iron deficiencies at the cellular level. The reduced FXN expression has been linked to a GAA triplet expansion within the intron 1 of the somatic and germline FXN gene. In FRDA patients, the GAA repeat frequently consists of more than 70, sometimes even more than 1000 (most commonly 600-900) triplets, whereas unaffected individuals have about 40 repeats or less (Pandolfo et al. (2012) Handbook of Clinical Neurology 103: 275-294; Campuzano et al. (1996) Science 271: 1423-1427; Pandolfo (2002) Adv. Exp. Med. Biol. 516: 99-118; all herein incorporated by reference). The expansion of the trinucleotide repeat sequence causing Friedreich's Ataxia (FRDA) occurs in a defined genetic locus within the FXN gene, referred to as the FRDA instability region. RNA guided nucleases (RGNs) may be used for excising the instability region in FRDA patient cells. This approach requires 1) an RGN and guide RNA sequence that can be programmed to target the allele in the human genome; and 2) a delivery approach for the RGN and guide sequence. Many nucleases used for genome editing, such as the commonly used Cas9 nuclease fromS. pyogenes(SpCas9), are too large to be packaged into adeno-associated viral (AAV) vectors, especially when considering the length of the SpCas9 gene and the guide RNA in addition to other genetic elements required for functional expression cassettes. This makes an approach using SpCas9 more difficult. Certain RNA guided nucleases of the invention are well suited for packaging into an AAV vector along with a guide RNA. Packing two guide RNAs would likely require a second vector, but this approach still compares favorably to what would be required of a larger nuclease such as SpCas9, which may require splitting the protein sequence between two vectors. The present invention encompasses a strategy using RGNs of the invention in which a region of genomic instability is removed. Such a strategy is applicable to other diseases and disorders which have a similar genetic basis, such as Huntington's Disease. Similar strategies using RGNs of the invention may also be applicable to similar diseases and disorders in non-human animals of agronomic or economic importance, including dogs, cats, horses, pigs, cows, sheep, chickens, donkeys, snakes, ferrets, and fish including salmon and shrimp. C. Modifying Causal Mutations by Targeted Mutagenesis RGNs of the invention could also be to introduce disruptive mutations that may result in a beneficial effect. Genetic defects in the genes encoding hemoglobin, particularly the beta globin chain (the HBB gene), can be responsible for a number of diseases known as hemoglobinopathies, including sickle cell anemia and thalassemias. In adult humans, hemoglobin is a heterotetramer comprising two alpha (α)-like globin chains and two beta (β)-like globin chains and 4 heme groups. In adults the α2β2 tetramer is referred to as Hemoglobin A (HbA) or adult hemoglobin. Typically, the alpha and beta globin chains are synthesized in an approximate 1:1 ratio and this ratio seems to be critical in terms of hemoglobin and red blood cell (RBC) stabilization. In a developing fetus, a different form of hemoglobin, fetal hemoglobin (HbF), is produced which has a higher binding affinity for oxygen than Hemoglobin A such that oxygen can be delivered to the baby's system via the mother's blood stream. Fetal hemoglobin also contains two α globin chains, but in place of the adult β-globin chains, it has two fetal gamma (γ)-globin chains (i.e., fetal hemoglobin is α2γ2). The regulation of the switch from production of gamma- to beta-globin is quite complex, and primarily involves a down-regulation of gamma globin transcription with a simultaneous up-regulation of beta globin transcription. At approximately 30 weeks of gestation, the synthesis of gamma globin in the fetus starts to drop while the production of beta globin increases. By approximately 10 months of age, the newborn's hemoglobin is nearly all α2β2 although some HbF persists into adulthood (approximately 1-3% of total hemoglobin). In the majority of patients with hemoglobinopathies, the genes encoding gamma globin remain present, but expression is relatively low due to normal gene repression occurring around parturition as described above. Sickle cell disease is caused by a V6E mutation in the β globin gene (HBB) (a GAG to GTG at the DNA level), where the resultant hemoglobin is referred to as “hemoglobinS” or “HbS.” Under lower oxygen conditions, HbS molecules aggregate and form fibrous precipitates. These aggregates cause the abnormality or ‘sickling’ of the RBCs, resulting in a loss of flexibility of the cells. The sickling RBCs are no longer able to squeeze into the capillary beds and can result in vaso-occlusive crisis in sickle cell patients. In addition, sickled RBCs are more fragile than normal RBCs, and tend towards hemolysis, eventually leading to anemia in the patient. Treatment and management of sickle cell patients is a life-long proposition involving antibiotic treatment, pain management and transfusions during acute episodes. One approach is the use of hydroxyurea, which exerts its effects in part by increasing the production of gamma globin. Long term side effects of chronic hydroxyurea therapy are still unknown, however, and treatment gives unwanted side effects and can have variable efficacy from patient to patient. Despite an increase in the efficacy of sickle cell treatments, the life expectancy of patients is still only in the mid to late 50's and the associated morbidities of the disease have a profound impact on a patient's quality of life. Thalassemias (alpha thalassemias and beta thalassemia) are also diseases relating to hemoglobin and typically involve a reduced expression of globin chains. This can occur through mutations in the regulatory regions of the genes or from a mutation in a globin coding sequence that results in reduced expression or reduced levels or functional globin protein. Treatment of thalassemias usually involves blood transfusions and iron chelation therapy. Bone marrow transplants are also being used for treatment of people with severe thalassemias if an appropriate donor can be identified, but this procedure can have significant risks. One approach that has been proposed for the treatment of both sickle cell disease (SCD) and beta thalassemias is to increase the expression of gamma globin so that HbF functionally replaces the aberrant adult hemoglobin. As mentioned above, treatment of SCD patients with hydroxyurea is thought to be successful in part due to its effect on increasing gamma globin expression (DeSimone (1982) Proc Nat'l Acad Sci USA 79(14):4428-31; Ley, et al., (1982) N. Engl. J. Medicine, 307: 1469-1475; Ley, et al., (1983) Blood 62: 370-380; Constantoulakis et al., (1988) Blood 72(6):1961-1967, all herein incorporated by reference). Increasing the expression of HbF involves identification of genes whose products play a role in the regulation of gamma globin expression. One such gene is BCL11A. BCL11A encodes a zinc finger protein that expressed in adult erythroid precursor cells, and down-regulation of its expression leads to an increase in gamma globin expression (Sankaran et at (2008) Science 322: 1839, herein incorporated by reference). Use of an inhibitory RNA targeted to the BCL11A gene has been proposed (e.g., U.S. Patent Publication 2011/0182867, herein incorporated by reference) but this technology has several potential drawbacks, including that complete knock down may not be achieved, delivery of such RNAs may be problematic, and the RNAs must be present continuously, requiring multiple treatments for life. RGNs of the invention may be used to target the BCL11A enhancer region to disrupt expression of BCL11A, thereby increasing gamma globin expression. This targeted disruption can be achieved by non-homologous end joining (NHEJ), whereby an RGN of the invention targets to a particular sequence within the BCL11A enhancer region, makes a double-stranded break, and the cell's machinery repairs the break, typically simultaneously introducing deleterious mutations. Similar to what is described for other disease targets, RGNs of the invention may have advantages over other known RGNs due to their relatively small size, which enables packaging expression cassettes for the RGN and its guide RNA into a single AAV vector for in vivo delivery. Similar strategies using RGNs of the invention may also be applicable to similar diseases and disorders in both humans and in non-human animals of agronomic or economic importance. X. Cells Comprising a Polynucleotide Genetic Modification Provided herein are cells and organisms comprising a target sequence of interest that has been modified using a process mediated by an RGN, crRNA, and/or tracrRNA as described herein. In some of these embodiments, the RGN comprises the amino acid sequence of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence comprising the nucleotide sequence of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, or an active variant or fragment thereof. In particular embodiments, the guide RNA comprises a tracrRNA comprising the nucleotide sequence of SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, or an active variant or fragment thereof. The guide RNA of the system can be a single guide RNA or a dual-guide RNA. The modified cells can be eukaryotic (e.g., mammalian, plant, insect cell) or prokaryotic. Also provided are organelles and embryos comprising at least one nucleotide sequence that has been modified by a process utilizing an RGN, crRNA, and/or tracrRNA as described herein. The genetically modified cells, organisms, organelles, and embryos can be heterozygous or homozygous for the modified nucleotide sequence. The chromosomal modification of the cell, organism, organelle, or embryo can result in altered expression (up-regulation or down-regulation), inactivation, or the expression of an altered protein product or an integrated sequence. In those embodiments wherein the chromosomal modification results in either the inactivation of a gene or the expression of a non-functional protein product, the genetically modified cell, organism, organelle, or embryo is referred to as a “knock out”. The knock out phenotype can be the result of a deletion mutation (i.e., deletion of at least one nucleotide), an insertion mutation (i.e., insertion of at least one nucleotide), or a nonsense mutation (i.e., substitution of at least one nucleotide such that a stop codon is introduced). Alternatively, the chromosomal modification of a cell, organism, organelle, or embryo can produce a “knock in”, which results from the chromosomal integration of a nucleotide sequence that encodes a protein. In some of these embodiments, the coding sequence is integrated into the chromosome such that the chromosomal sequence encoding the wild-type protein is inactivated, but the exogenously introduced protein is expressed. In other embodiments, the chromosomal modification results in the production of a variant protein product. The expressed variant protein product can have at least one amino acid substitution and/or the addition or deletion of at least one amino acid. The variant protein product encoded by the altered chromosomal sequence can exhibit modified characteristics or activities when compared to the wild-type protein, including but not limited to altered enzymatic activity or substrate specificity. In yet other embodiments, the chromosomal modification can result in an altered expression pattern of a protein. As a non-limiting example, chromosomal alterations in the regulatory regions controlling the expression of a protein product can result in the overexpression or downregulation of the protein product or an altered tissue or temporal expression pattern. The cells that have been modified can be grown into an organism, such as a plant, in accordance with conventional ways. See, for example, McCormick et al. (1986)Plant Cell Reports5:81-84. These plants may then be grown, and either pollinated with the same modified strain or different strains, and the resulting hybrid having the genetic modification. The present invention provides genetically modified seed. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the genetic modification. Further provided is a processed plant product or byproduct that retains the genetic modification, including for example, soymeal. The methods provided herein may be used for modification of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassicasp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive,papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers. Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genusCurcumissuch as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea,hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, andchrysanthemum. Preferably, plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.). The methods provided herein can also be used to genetically modify any prokaryotic species, including but not limited to, archaea and bacteria (e.g.,Bacillussp.,Klebsiellasp.Streptomycessp.,Rhizobiumsp.,Escherichiasp.,Pseudomonassp.,Salmonellasp.,Shigellasp.,Vibriosp.,Yersiniasp.,Mycoplasmasp.,Agrobacterium, Lactobacillussp.). The methods provided herein can be used to genetically modify any eukaryotic species or cells therefrom, including but not limited to animals (e.g., mammals, insects, fish, birds, and reptiles), fungi, amoeba, algae, and yeast. In some embodiments, the cell that is modified by the presently disclosed methods include cells of hematopoietic origin, such as cells of the immune system including but not limited to B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells. Cells that have been modified may be introduced into an organism. These cells could have originated from the same organism (e.g., person) in the case of autologous cellular transplants, wherein the cells are modified in an ex vivo approach. Alternatively, the cells originated from another organism within the same species (e.g., another person) in the case of allogeneic cellular transplants. The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a polypeptide” means one or more polypeptides. All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended embodiments. Non-limiting embodiments include: 1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein said polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579;wherein said RGN polypeptide is capable of binding a target DNA sequence in an RNA-guided sequence specific manner when bound to a guide RNA (gRNA) capable of hybridizing to said target DNA sequence, andwherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide.2. The nucleic acid molecule of embodiment 1, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.3. The nucleic acid molecule of embodiment 1, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.4. The nucleic acid molecule of embodiment 1, wherein said RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 63 and has an isoleucine at an amino acid position corresponding to 305, a valine at an amino acid position corresponding to 328, a leucine at an amino acid position corresponding to 366, a threonine at an amino acid position corresponding to 368, and a valine at an amino acid position corresponding to 405 of SEQ ID NO: 63.5. The nucleic acid molecule of any one of embodiments 1-4, wherein said RGN polypeptide is capable of cleaving said target DNA sequence upon binding.6. The nucleic acid molecule of embodiment 5, wherein said RGN polypeptide is capable of generating a double-stranded break.7. The nucleic acid molecule of embodiment 5, wherein said RGN polypeptide is capable of generating a single-stranded break.8. The nucleic acid molecule of any one of embodiments 1-4, wherein said RGN polypeptide is nuclease inactive or is a nickase.9. The nucleic acid molecule of any one of embodiments 1-8, wherein the RGN polypeptide is operably fused to a base-editing polypeptide.10. The nucleic acid molecule of embodiment 9, wherein the base-editing polypeptide is a deaminase.11. The nucleic acid molecule of embodiment 10, wherein the deaminase is a cytidine deaminase or an adenine deaminase.12. The nucleic acid molecule of any one of embodiments 1-11, wherein the RGN polypeptide comprises one or more nuclear localization signals.13. The nucleic acid molecule of any one of embodiments 1-12, wherein the RGN polypeptide is codon optimized for expression in a eukaryotic cell.14. The nucleic acid molecule of any one of embodiments 1-13, wherein said target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).15. A vector comprising the nucleic acid molecule of any one of embodiments 1-14.16. The vector of embodiment 15, further comprising at least one nucleotide sequence encoding said gRNA capable of hybridizing to said target DNA sequence.17. The vector of embodiment 16, wherein the guide RNA is selected from the group consisting of:a) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 2; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 3;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1;b) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 9; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 10;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8;c) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 17;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15;d) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 24;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22;e) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 30; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29;f) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 37; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 38;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 36;g) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 44; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 45;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 43;h) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 51; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 52;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 50;i) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 57; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 58;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 56;j) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 64; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 65;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 71; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 72;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 70;l) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 77; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 78;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 76;m) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 84; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 85;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83;n) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 90; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 91;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 89;o) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 97; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 98;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 96;p) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 104; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 105;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 103;q) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 111; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 112;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 110;r) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 118; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 119;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 117;s) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 124; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 125;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 123; andt) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 84; andii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 78;wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83.18. The vector of embodiment 16, wherein the guide RNA is selected from the group consisting of:a) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 2; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 3;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1;b) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 9; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 10;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8;c) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 16; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 17;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 15;d) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 23; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 24;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 22;e) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 30; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 31;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 29;f) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 37; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 38;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 36;g) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 44; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 45;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 43;h) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 51; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 52;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 50;i) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 57; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 58;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 56;j) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 64; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 65;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 71; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 72;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 70;l) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 77; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 78;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 76;m) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 84; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 85;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 83;n) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 90; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 91;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 89;o) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 97; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 98;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 96;p) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 104; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 105;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 103;q) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 111; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 112;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 110;r) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 118; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 119;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117;s) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 124; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 125;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 123; andt) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 84; andii) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 78;wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 83.19. The vector of embodiment 16, wherein the guide RNA is selected from the group consisting of:a) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 2; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 3;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 1;b) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 9; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 10;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 8;c) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 16; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 17;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 15;d) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 23; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 24;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 22;e) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 30; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 31;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 29;f) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 37; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 38;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 36;g) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 44; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 45;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 43;h) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 51; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 52;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 50;i) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 57; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 58;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 56;j) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 64; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 65;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 71; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 72;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 70;l) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 77; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 78;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 76;m) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 84; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 85;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 83;n) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 90; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 91;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 89;o) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 97; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 98;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 96;p) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 104; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 105;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 103;q) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 111; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 112;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 110;r) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 118; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 119;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 117;s) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 124; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 125;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 123; andt) a guide RNA comprising:i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 84; andii) a tracrRNA having 100% sequence identity to SEQ ID NO: 78;wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 83.20. The vector of any one of embodiments 16-19, where said gRNA is a single guide RNA.21. The vector of any one of embodiments 16-19, wherein said gRNA is a dual-guide RNA.22. A cell comprising the nucleic acid molecule of any one of embodiments 1-14 or the vector of any one of embodiments 15-21.23. A method for making an RGN polypeptide comprising culturing the cell of embodiment 22 under conditions in which the RGN polypeptide is expressed.24. A method for making an RGN polypeptide comprising introducing into a cell a heterologous nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579;wherein said RGN polypeptide is capable of binding a target DNA sequence in an RNA-guided sequence specific manner when bound to a guide RNA (gRNA) capable of hybridizing to said target DNA sequence;and culturing said cell under conditions in which the RGN polypeptide is expressed.25. The method of embodiment 24, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.26. The method of embodiment 24, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.27. The method of embodiment 24, wherein said RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 63 and has an isoleucine at an amino acid position corresponding to 305, a valine at an amino acid position corresponding to 328, a leucine at an amino acid position corresponding to 366, a threonine at an amino acid position corresponding to 368, and a valine at an amino acid position corresponding to 405 of SEQ ID NO: 63.28. The method of any one of embodiments 23-27, further comprising purifying said RGN polypeptide.29. The method of any one of embodiments 23-27, wherein said cell further expresses one or more guide RNAs capable of binding to said RGN polypeptide to form an RGN ribonucleoprotein complex.30. The method of embodiment 29, further comprising purifying said RGN ribonucleoprotein complex.31. An isolated RNA-guided nuclease (RGN) polypeptide, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579; andwherein said RGN polypeptide is capable of binding a target DNA sequence of a DNA molecule in an RNA-guided sequence specific manner when bound to a guide RNA (gRNA) capable of hybridizing to said target DNA sequence.32. The isolated RGN polypeptide of embodiment 31, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.33. The isolated RGN polypeptide of embodiment 31, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.34. The isolated RGN polypeptide of embodiment 31, wherein said RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 63 and has an isoleucine at an amino acid position corresponding to 305, a valine at an amino acid position corresponding to 328, a leucine at an amino acid position corresponding to 366, a threonine at an amino acid position corresponding to 368, and a valine at an amino acid position corresponding to 405 of SEQ ID NO: 63.35. The isolated RGN polypeptide of any one of embodiments 31-34, wherein said RGN polypeptide is capable of cleaving said target DNA sequence upon binding.36. The isolated RGN polypeptide of embodiment 35, wherein cleavage by said RGN polypeptide generates a double-stranded break.37. The isolated RGN polypeptide of embodiment 35, wherein cleavage by said RGN polypeptide generates a single-stranded break.38. The isolated RGN polypeptide of any one of embodiments 31-34, wherein said RGN polypeptide is nuclease inactive or a nickase.39. The isolated RGN polypeptide of any one of embodiments 31-38, wherein the RGN polypeptide is operably fused to a base-editing polypeptide.40. The isolated RGN polypeptide of embodiment 39, wherein the base-editing polypeptide is a deaminase.41. The isolated RGN polypeptide of any one of embodiments 31-40, wherein said target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).42. The isolated RGN polypeptide of any one of embodiments 31-41, wherein the RGN polypeptide comprises one or more nuclear localization signals.43. A nucleic acid molecule comprising a polynucleotide encoding a CRISPR RNA (crRNA), wherein said crRNA comprises a spacer sequence and a CRISPR repeat sequence, wherein said CRISPR repeat sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, and 124;wherein a guide RNA comprising:a) said crRNA; andb) a trans-activating CRISPR RNA (tracrRNA) hybridized to said CRISPR repeat sequence of said crRNA;is capable of hybridizing to a target DNA sequence in a sequence specific manner through the spacer sequence of said crRNA when said guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide, andwherein said polynucleotide encoding a crRNA is operably linked to a promoter heterologous to said polynucleotide.44. The nucleic acid molecule of embodiment 43, wherein said CRISPR repeat sequence comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, and 124.45. The nucleic acid molecule of embodiment 43, wherein said CRISPR repeat sequence comprises a nucleotide sequence having 100% sequence identity to any one of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, and 124.46. A vector comprising the nucleic acid molecule of any one of embodiments 43-45.47. The vector of embodiment 46, wherein said vector further comprises a polynucleotide encoding said tracrRNA.48. The vector of embodiment 47, wherein said tracrRNA is selected from the group consisting of:a) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 3, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 2;b) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 10, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 9;c) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 17, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 16;d) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 24, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 23;e) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 30;f) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 38, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 37;g) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 45, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 44;h) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 52, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 51;i) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 58, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 57;j) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 65, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 64;k) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 72, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 71;l) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 78, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 77;m) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 85, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 84;n) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 91, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 90;o) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 98, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 97;p) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 105, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 104;q) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 112, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 111;r) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 119, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 118; ands) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 125, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 124.49. The vector of embodiment 47, wherein said tracrRNA is selected from the group consisting of:a) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 3, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 2;b) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 10, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 9;c) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 17, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 16;d) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 24, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 23;e) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 31, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 30;f) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 38, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 37;g) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 45, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 44;h) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 52, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 51;i) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 58, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 57;j) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 65, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 64;k) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 72, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 71;l) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 78, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 77;m) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 85, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 84;n) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 91, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 90;o) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 98, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 97;p) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 105, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 104;q) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 112, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 111;r) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 119, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 118; ands) a tracrRNA having at least 95% sequence identity to SEQ ID NO: 125, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 124.50. The vector of embodiment 47, wherein said tracrRNA is selected from the group consisting of:a) a tracrRNA having 100% sequence identity to SEQ ID NO: 3, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 2;b) a tracrRNA having 100% sequence identity to SEQ ID NO: 10, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 9;c) a tracrRNA having 100% sequence identity to SEQ ID NO: 17, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 16;d) a tracrRNA having 100% sequence identity to SEQ ID NO: 24, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 23;e) a tracrRNA having 100% sequence identity to SEQ ID NO: 31, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 30;f) a tracrRNA having 100% sequence identity to SEQ ID NO: 38, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 37;g) a tracrRNA having 100% sequence identity to SEQ ID NO: 45, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 44;h) a tracrRNA having 100% sequence identity to SEQ ID NO: 52, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 51;i) a tracrRNA having 100% sequence identity to SEQ ID NO: 58, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 57;j) a tracrRNA having 100% sequence identity to SEQ ID NO: 65, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 64;k) a tracrRNA having 100% sequence identity to SEQ ID NO: 72, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 71;l) a tracrRNA having 100% sequence identity to SEQ ID NO: 78, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 77;m) a tracrRNA having 100% sequence identity to SEQ ID NO: 85, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 84;n) a tracrRNA having 100% sequence identity to SEQ ID NO: 91, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 90;o) a tracrRNA having 100% sequence identity to SEQ ID NO: 98, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 97;p) a tracrRNA having 100% sequence identity to SEQ ID NO: 105, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 104;q) a tracrRNA having 100% sequence identity to SEQ ID NO: 112, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 111;r) a tracrRNA having 100% sequence identity to SEQ ID NO: 119, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 118; ands) a tracrRNA having 100% sequence identity to SEQ ID NO: 125, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 124.51. The vector of any one of embodiments 47-50, wherein said polynucleotide encoding said crRNA and said polynucleotide encoding said tracrRNA are operably linked to the same promoter and are encoded as a single guide RNA.52. The vector of any one of embodiments 47-50, wherein said polynucleotide encoding said crRNA and said polynucleotide encoding said tracrRNA are operably linked to separate promoters.53. The vector of any one of embodiments 46-52, wherein said vector further comprises a polynucleotide encoding said RGN polypeptide.54. The vector of embodiment 53, wherein said RGN polypeptide is selected from the group consisting of:a) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 1, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 2 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 3;b) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 8, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 9 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 10;c) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 15, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 16 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 17;d) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 22, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 23 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 24;e) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 29, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 30 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 31;f) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 36, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 37 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 38;g) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 43, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 44 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 45;h) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 50, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 51 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 52;i) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 56, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 57 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 58;j) a RGN polypeptide having at least 90% sequence identity to any one of SEQ ID NO: 63 and 570-579, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 64 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 65;k) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 70, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 71 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 72;l) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 76, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 77 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 78;m) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 83, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 84 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 85;n) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 89, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 90 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 91;o) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 96, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 97 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 98;p) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 103, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 104 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 105;q) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 110, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 111 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 112;r) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 117, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 118 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 119;s) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 123, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 124 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 125; andt) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 83, wherein said CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 84 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 78.55. The vector of embodiment 53, wherein said RGN polypeptide is selected from the group consisting of:a) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 1, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 2 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 3;b) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 8, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 9 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 10;c) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 15, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 16 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 17;d) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 22, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 23 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 24;e) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 29, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 30 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 31;f) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 36, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 37 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 38;g) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 43, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 44 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 45;h) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 50, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 51 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 52;i) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 56, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 57 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 58;j) a RGN polypeptide having at least 95% sequence identity to any one of SEQ ID NO: 63 and 570-579, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 64 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 65;k) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 70, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 71 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 72;l) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 76, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 77 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 78;m) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 83, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 84 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 85;n) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 89, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 90 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 91;o) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 96, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 97 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 98;p) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 103, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 104 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 105;q) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 110, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 111 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 112;r) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 117, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 118 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 119;s) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 123, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 124 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 125; andt) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 83, wherein said CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 84 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 78.56. The vector of embodiment 53, wherein said RGN polypeptide is selected from the group consisting of:a) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 1, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 2 and said tracrRNA has 100% sequence identity to SEQ ID NO: 3;b) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 8, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 9 and said tracrRNA has 100% sequence identity to SEQ ID NO: 10;c) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 15, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 16 and said tracrRNA has 100% sequence identity to SEQ ID NO: 17;d) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 22, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 23 and said tracrRNA has 100% sequence identity to SEQ ID NO: 24;e) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 29, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 30 and said tracrRNA has 100% sequence identity to SEQ ID NO: 31;f) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 36, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 37 and said tracrRNA has 100% sequence identity to SEQ ID NO: 38;g) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 43, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 44 and said tracrRNA has 100% sequence identity to SEQ ID NO: 45;h) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 50, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 51 and said tracrRNA has 100% sequence identity to SEQ ID NO: 52;i) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 56, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 57 and said tracrRNA has 100% sequence identity to SEQ ID NO: 58;j) a RGN polypeptide having 100% sequence identity to any one of SEQ ID NO: 63 and 570-579, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 64 and said tracrRNA has 100% sequence identity to SEQ ID NO: 65;k) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 70, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 71 and said tracrRNA has 100% sequence identity to SEQ ID NO: 72;l) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 76, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 77 and said tracrRNA has 100% sequence identity to SEQ ID NO: 78;m) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 83, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 84 and said tracrRNA has 100% sequence identity to SEQ ID NO: 85;n) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 89, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 90 and said tracrRNA has 100% sequence identity to SEQ ID NO: 91;o) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 96, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 97 and said tracrRNA has 100% sequence identity to SEQ ID NO: 98;p) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 103, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 104 and said tracrRNA has 100% sequence identity to SEQ ID NO: 105;q) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 110, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 111 and said tracrRNA has 100% sequence identity to SEQ ID NO: 112;r) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 117, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 118 and said tracrRNA has 100% sequence identity to SEQ ID NO: 119;s) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 123, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 124 and said tracrRNA has 100% sequence identity to SEQ ID NO: 125; andt) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 83, wherein said CRISPR repeat sequence has 100% sequence identity to SEQ ID NO: 84 and said tracrRNA has 100% sequence identity to SEQ ID NO: 78.57. A nucleic acid molecule comprising a polynucleotide encoding a trans-activating CRISPR RNA (tracrRNA) comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125;wherein a guide RNA comprising:a) said tracrRNA; andb) a crRNA comprising a spacer sequence and a CRISPR repeat sequence, wherein said tracrRNA hybridizes with said CRISPR repeat sequence of said crRNA;is capable of hybridizing to a target DNA sequence in a sequence specific manner through the spacer sequence of said crRNA when said guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide, andwherein said polynucleotide encoding a tracrRNA is operably linked to a promoter heterologous to said polynucleotide.58. The nucleic acid molecule of embodiment 57, wherein said tracrRNA comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125.59. The nucleic acid molecule of embodiment 57, wherein said tracrRNA comprises a nucleotide sequence having 100% sequence identity to SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125.60. A vector comprising the nucleic acid molecule of any one of embodiments 57-59.61. The vector of embodiment 60, wherein said vector further comprises a polynucleotide encoding said crRNA.62. The vector of embodiment 61, wherein said crRNA comprises a CRISPR repeat sequence selected from the group consisting of:a) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 2, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 3;b) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 9, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 10;c) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 17;d) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 24;e) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 30, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 31;f) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 37, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 38;g) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 44, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 45;h) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 51, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 52;i) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 57, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 58;j) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 64, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 65;k) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 71, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 72;l) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 77, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 78;m) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 84, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 85;n) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 90, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 91;o) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 97, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 98;p) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 104, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 105;q) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 111, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 112;r) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 118, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 119; ands) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 124, wherein said tracrRNA has at least 90% sequence identity to SEQ ID NO: 125.63. The vector of embodiment 61, wherein said crRNA comprises a CRISPR repeat sequence selected from the group consisting of:a) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 2, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 3;b) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 9, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 10;c) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 16, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 17;d) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 23, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 24;e) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 30, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 31;f) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 37, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 38;g) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 44, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 45;h) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 51, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 52;i) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 57, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 58;j) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 64, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 65;k) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 71, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 72;l) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 77, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 78;m) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 84, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 85;n) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 90, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 91;o) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 97, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 98;p) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 104, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 105;q) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 111, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 112;r) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 118, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 119; ands) a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 124, wherein said tracrRNA has at least 95% sequence identity to SEQ ID NO: 125.64. The vector of embodiment 61, wherein said crRNA comprises a CRISPR repeat sequence selected from the group consisting of:a) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 2, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 3;b) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 9, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 10;c) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 16, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 17;d) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 23, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 24;e) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 30, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 31;f) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 37, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 38;g) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 44, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 45;h) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 51, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 52;i) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 57, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 58;j) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 64, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 65;k) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 71, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 72;l) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 77, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 78;m) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 84, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 85;n) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 90, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 91;o) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 97, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 98;p) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 104, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 105;q) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 111, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 112;r) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 118, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 119; ands) a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 124, wherein said tracrRNA has 100% sequence identity to SEQ ID NO: 125.65. The vector of any one of embodiments 61-64, wherein said polynucleotide encoding said crRNA and said polynucleotide encoding said tracrRNA are operably linked to the same promoter and are encoded as a single guide RNA.66. The vector of any one of embodiments 61-64, wherein said polynucleotide encoding said crRNA and said polynucleotide encoding said tracrRNA are operably linked to separate promoters.67. The vector of any one of embodiments 60-66, wherein said vector further comprises a polynucleotide encoding said RGN polypeptide.68. The vector of embodiment 67, wherein said RGN polypeptide is selected from the group consisting of:a) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 1, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 2 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 3;b) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 8, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 9 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 10;c) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 15, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 17;d) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 22, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 24;e) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 29, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 30 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 31;f) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 36, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 37 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 38;g) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 43, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 44 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 45;h) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 50, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 51 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 52;i) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 56, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 57 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 58;j) a RGN polypeptide having at least 90% sequence identity to any one of SEQ ID NO: 63 and 570-579, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 64 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 65;k) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 70, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 71 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 72;l) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 76, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 77 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 78;m) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 83, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 84 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 85;n) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 89, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 90 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 91;o) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 96, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 97 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 98;p) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 103, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 104 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 105;q) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 110, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 111 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 112;r) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 117, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 118 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 119;s) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 123, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 124 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 125; andt) a RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 83, wherein said crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 84 and said tracrRNA has at least 90% sequence identity to SEQ ID NO: 78.69. The vector of embodiment 67, wherein said RGN polypeptide is selected from the group consisting of:a) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 1, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 2 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 3;b) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 8, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 9 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 10;c) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 15, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 16 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 17;d) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 22, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 23 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 24;e) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 29, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 30 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 31;f) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 36, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 37 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 38;g) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 43, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 44 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 45;h) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 50, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 51 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 52;i) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 56, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 57 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 58;j) a RGN polypeptide having at least 95% sequence identity to any one of SEQ ID NO: 63 and 570-579, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 64 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 65;k) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 70, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 71 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 72;l) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 76, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 77 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 78;m) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 83, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 84 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 85;n) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 89, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 90 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 91;o) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 96, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 97 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 98;p) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 103, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 104 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 105;q) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 110, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 111 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 112;r) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 117, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 118 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 119;s) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 123, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 124 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 125; andt) a RGN polypeptide having at least 95% sequence identity to SEQ ID NO: 83, wherein said crRNA comprises a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 84 and said tracrRNA has at least 95% sequence identity to SEQ ID NO: 78.70. The vector of embodiment 67, wherein said RGN polypeptide is selected from the group consisting of:a) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 1, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 2 and said tracrRNA has 100% sequence identity to SEQ ID NO: 3;b) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 8, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 9 and said tracrRNA has 100% sequence identity to SEQ ID NO: 10;c) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 15, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 16 and said tracrRNA has 100% sequence identity to SEQ ID NO: 17;d) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 22, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 23 and said tracrRNA has 100% sequence identity to SEQ ID NO: 24;e) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 29, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 30 and said tracrRNA has 100% sequence identity to SEQ ID NO: 31;f) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 36, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 37 and said tracrRNA has 100% sequence identity to SEQ ID NO: 38;g) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 43, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 44 and said tracrRNA has 100% sequence identity to SEQ ID NO: 45;h) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 50, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 51 and said tracrRNA has 100% sequence identity to SEQ ID NO: 52;i) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 56, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 57 and said tracrRNA has 100% sequence identity to SEQ ID NO: 58;j) a RGN polypeptide having 100% sequence identity to any one of SEQ ID NO: 63 and 570-579, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 64 and said tracrRNA has 100% sequence identity to SEQ ID NO: 65;k) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 70, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 71 and said tracrRNA has 100% sequence identity to SEQ ID NO: 72;l) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 76, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 77 and said tracrRNA has 100% sequence identity to SEQ ID NO: 78;m) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 83, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 84 and said tracrRNA has 100% sequence identity to SEQ ID NO: 85;n) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 89, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 90 and said tracrRNA has 100% sequence identity to SEQ ID NO: 91;o) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 96, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 97 and said tracrRNA has 100% sequence identity to SEQ ID NO: 98;p) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 103, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 104 and said tracrRNA has 100% sequence identity to SEQ ID NO: 105;q) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 110, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 111 and said tracrRNA has 100% sequence identity to SEQ ID NO: 112;r) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 117, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 118 and said tracrRNA has 100% sequence identity to SEQ ID NO: 119;s) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 123, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 124 and said tracrRNA has 100% sequence identity to SEQ ID NO: 125; andt) a RGN polypeptide having 100% sequence identity to SEQ ID NO: 83, wherein said crRNA comprises a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 84 and said tracrRNA has 100% sequence identity to SEQ ID NO: 78.71. A system for binding a target DNA sequence of a DNA molecule, said system comprising:a) one or more guide RNAs capable of hybridizing to said target DNA sequence or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more guide RNAs (gRNAs); andb) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579 or a polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide;wherein at least one of said nucleotide sequences encoding the one or more guide RNAs and said nucleotide sequence encoding the RGN polypeptide is operably linked to a promoter heterologous to said nucleotide sequence;wherein the one or more guide RNAs are capable of hybridizing to the target DNA sequence, andwherein the one or more guide RNAs are capable of forming a complex with the RGN polypeptide in order to direct said RGN polypeptide to bind to said target DNA sequence of the DNA molecule.72. A system for binding a target DNA sequence of a DNA molecule, said system comprising:a) one or more guide RNAs capable of hybridizing to said target DNA sequence or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more guide RNAs (gRNAs); andb) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579;wherein the one or more guide RNAs are capable of hybridizing to the target DNA sequence, andwherein the one or more guide RNAs are capable of forming a complex with the RGN polypeptide in order to direct said RGN polypeptide to bind to said target DNA sequence of the DNA molecule.73. The system of embodiment 71 or 72, wherein at least one of said nucleotides sequences encoding the one or more guide RNAs is operably linked to a promoter heterologous to said nucleotide sequence.74. The system of any one of embodiments 71-73, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579. 75. The system of any one of embodiments 71-73, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.76. The system of any one of embodiments 71-73, wherein said RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 63 and has an isoleucine at an amino acid position corresponding to 305, a valine at an amino acid position corresponding to 328, a leucine at an amino acid position corresponding to 366, a threonine at an amino acid position corresponding to 368, and a valine at an amino acid position corresponding to 405 of SEQ ID NO: 63.77. The system of any one of embodiments 71-76, wherein said RGN polypeptide and said one or more guide RNAs are not found complexed to one another in nature.78. The system of any one of embodiments 71-77, wherein said target DNA sequence is a eukaryotic target DNA sequence.79. The system of any one of embodiments 71-78, wherein said gRNA is a single guide RNA (sgRNA).80. The system of any one of embodiments 71-78, wherein said gRNA is a dual-guide RNA.81. The system of any one of embodiments 71-80, wherein said gRNA is selected from the group consisting of:a) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 2 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 3, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1;b) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 9 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 10, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8;c) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 17, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15;d) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 24, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22;e) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 30 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29;f) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 38, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 36;g) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 44 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 45, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 43;h) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 51 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 52, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 50;i) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 57 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 58, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 56;j) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 64 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 65, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 71 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 72, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 70;l) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 77 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 76;m) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 85, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83;n) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 90 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 91, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 89;o) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 97 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 98, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 96;p) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 104 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 105, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 103;q) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 111 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 112, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 110;r) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 118 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 119, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 117;s) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 124 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 125, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 123; andt) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83.82. The system of any one of embodiments 71-80, wherein said gRNA is selected from the group consisting of:a) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 2 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 3, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1;b) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 9 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 10, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8;c) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 17, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 15;d) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 23 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 24, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 22;e) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 30 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 31, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 29;f) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 38, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 36;g) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 44 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 45, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 43;h) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 51 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 52, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 50;i) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 57 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 58, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 56;j) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 64 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 65, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 71 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 72, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 70;l) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 77 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 76;m) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 85, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 83;n) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 90 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 91, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 89;o) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 97 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 98, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 96;p) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 104 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 105, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 103;q) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 111 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 112, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 110;r) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 118 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 119, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117;s) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 124 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 125, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 123; andt) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 83.83. The system of any one of embodiments 71-80, wherein said gRNA is selected from the group consisting of:a) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 2 and a tracrRNA having 100% sequence identity to SEQ ID NO: 3, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 1;b) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 9 and a tracrRNA having 100% sequence identity to SEQ ID NO: 10, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 8;c) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 16 and a tracrRNA having 100% sequence identity to SEQ ID NO: 17, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 15;d) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 23 and a tracrRNA having 100% sequence identity to SEQ ID NO: 24, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 22;e) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 30 and a tracrRNA having 100% sequence identity to SEQ ID NO: 31, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 29;f) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 37 and a tracrRNA having 100% sequence identity to SEQ ID NO: 38, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 36;g) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 44 and a tracrRNA having 100% sequence identity to SEQ ID NO: 45, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 43;h) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 51 and a tracrRNA having 100% sequence identity to SEQ ID NO: 52, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 50;i) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 57 and a tracrRNA having 100% sequence identity to SEQ ID NO: 58, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 56;j) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 64 and a tracrRNA having 100% sequence identity to SEQ ID NO: 65, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 71 and a tracrRNA having 100% sequence identity to SEQ ID NO: 72, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 70;l) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 77 and a tracrRNA having 100% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 76;m) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 84 and a tracrRNA having 100% sequence identity to SEQ ID NO: 85, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 83;n) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 90 and a tracrRNA having 100% sequence identity to SEQ ID NO: 91, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 89;o) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 97 and a tracrRNA having 100% sequence identity to SEQ ID NO: 98, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 96;p) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 104 and a tracrRNA having 100% sequence identity to SEQ ID NO: 105, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 103;q) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 111 and a tracrRNA having 100% sequence identity to SEQ ID NO: 112, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 110;r) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 118 and a tracrRNA having 100% sequence identity to SEQ ID NO: 119, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 117;s) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 124 and a tracrRNA having 100% sequence identity to SEQ ID NO: 125, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 123; andt) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 84 and a tracrRNA having 100% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 83.84. The system of any one of embodiments 71-83, wherein said target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).85. The system of any one of embodiments 71-84, wherein the target DNA sequence is within a cell.86. The system of embodiment 85, wherein the cell is a eukaryotic cell.87. The system of embodiment 86, wherein the eukaryotic cell is a plant cell.88. The system of embodiment 86, wherein the eukaryotic cell is a mammalian cell.89. The system of embodiment 88, wherein said mammalian cell is a human cell.90. The system of embodiment 89, wherein said human cell is an immune cell.91. The system of embodiment 90, wherein said immune cell is a stem cell.92. The system of embodiment 91, wherein the stem cell is an induced pluripotent stem cell.93. The system of embodiment 86, wherein the eukaryotic cell is an insect cell.94. The system of embodiment 85, wherein the cell is a prokaryotic cell.95. The system of any one of embodiments 71-94, wherein when transcribed the one or more guide RNAs is capable of hybridizing to the target DNA sequence and the guide RNA is capable of forming a complex with the RGN polypeptide to direct cleavage of the target DNA sequence.96. The system of embodiment 95, wherein the cleavage generates a double-stranded break.97. The system of embodiment 95, wherein the cleavage generates a single-stranded break.98. The system of any one of embodiments 71-94, wherein said RGN polypeptide is nuclease inactive or is a nickase.99. The system of any one of embodiments 71-98, wherein the RGN polypeptide is operably linked to a base-editing polypeptide.100. The system of embodiment 99, wherein the base-editing polypeptide is a deaminase.101. The system of embodiment 100, wherein the deaminase is a cytidine deaminase or an adenine deaminase.102. The system of any one of embodiments 71-101, wherein the RGN polypeptide comprises one or more nuclear localization signals.103. The system of any one of embodiments 71-102, wherein the RGN polypeptide is codon optimized for expression in a eukaryotic cell.104. The system of any one of embodiments 71-103, wherein nucleotide sequences encoding the one or more guide RNAs and the nucleotide sequence encoding an RGN polypeptide are located on one vector.105. The system of any one of embodiments 71-104, wherein said system further comprises one or more donor polynucleotides or one or more nucleotide sequences encoding the one or more donor polynucleotides.106. A pharmaceutical composition comprising the nucleic acid molecule of any one of embodiments 1-14, 43-45, and 57-59, the vector of any one of embodiments 15-21, 46-56, and 60-70, the cell of embodiment 22, the isolated RGN polypeptide of any one of embodiments 31-42, or the system of any one of embodiments 71-105, and a pharmaceutically acceptable carrier.107. A method for binding a target DNA sequence of a DNA molecule comprising delivering a system according to any one of embodiments 71-105, to said target DNA sequence or a cell comprising the target DNA sequence.108. The method of embodiment 107, wherein said RGN polypeptide or said guide RNA further comprises a detectable label, thereby allowing for detection of said target DNA sequence.109. The method of embodiment 107, wherein said guide RNA or said RGN polypeptide further comprises an expression modulator, thereby modulating expression of said target DNA sequence or a gene under transcriptional control by said target DNA sequence.110. A method for cleaving or modifying a target DNA sequence of a DNA molecule comprising delivering a system according to any one of embodiments 71-105 to said target DNA sequence or a cell comprising the DNA molecule, and cleavage or modification of said target DNA sequence occurs.111. The method of embodiment 110, wherein said modified target DNA sequence comprises insertion of heterologous DNA into the target DNA sequence.112. The method of embodiment 110, wherein said modified target DNA sequence comprises deletion of at least one nucleotide from the target DNA sequence.113. The method of embodiment 110, wherein said modified target DNA sequence comprises mutation of at least one nucleotide in the target DNA sequence.114. A method for binding a target DNA sequence of a DNA molecule comprising:a) assembling an RNA-guided nuclease (RGN) ribonucleotide complex in vitro by combining:i) one or more guide RNAs capable of hybridizing to the target DNA sequence; andii) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579;under conditions suitable for formation of the RGN ribonucleotide complex; andb) contacting said target DNA sequence or a cell comprising said target DNA sequence with the in vitro-assembled RGN ribonucleotide complex;wherein the one or more guide RNAs hybridize to the target DNA sequence, thereby directing said RGN polypeptide to bind to said target DNA sequence.115. The method of embodiment 114, wherein said RGN polypeptide or said guide RNA further comprises a detectable label, thereby allowing for detection of said target DNA sequence.116. The method of embodiment 114, wherein said guide RNA or said RGN polypeptide further comprises an expression modulator, thereby allowing for the modulation of expression of said target DNA sequence.117. A method for cleaving and/or modifying a target DNA sequence of a DNA molecule, comprising contacting the DNA molecule with:a) an RNA-guided nuclease (RGN) polypeptide, wherein said RGN comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579; andb) one or more guide RNAs capable of targeting the RGN of (a) to the target DNA sequence;wherein the one or more guide RNAs hybridize to the target DNA sequence, thereby directing said RGN polypeptide to bind to said target DNA sequence and cleavage and/or modification of said target DNA sequence occurs.118. The method of embodiment 117, wherein cleavage by said RGN polypeptide generates a double-stranded break.119. The method of embodiment 117, wherein cleavage by said RGN polypeptide generates a single-stranded break.120. The method of embodiment 117, wherein said RGN polypeptide is nuclease inactive or a nickase and is operably fused to a base-editing polypeptide.121. The method of embodiment 120, wherein the base-editing polypeptide is a deaminase.122. The method of embodiment 121, wherein the deaminase is a cytidine deaminase or an adenine deaminase.123. The method of embodiment 117, wherein said modified target DNA sequence comprises insertion of heterologous DNA into the target DNA sequence.124. The method of embodiment 117, wherein said modified target DNA sequence comprises deletion of at least one nucleotide from the target DNA sequence.125. The method of embodiment 117, wherein said modified target DNA sequence comprises mutation of at least one nucleotide in the target DNA sequence.126. The method of any one of embodiments 114-125, wherein said target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).127. The method of any one of embodiments 114-126, wherein said target DNA sequence is a eukaryotic target DNA sequence.128. The method of any one of embodiments 114-127, wherein said gRNA is a single guide RNA (sgRNA).129. The method of any one of embodiments 114-127, wherein said gRNA is a dual-guide RNA.130. The method of any one of embodiments 114-129, wherein said RGN comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.131. The method of any one of embodiments 114-129, wherein said RGN comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.132. The method of any one of embodiments 114-129, wherein said RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 63 and has an isoleucine at an amino acid position corresponding to 305, a valine at an amino acid position corresponding to 328, a leucine at an amino acid position corresponding to 366, a threonine at an amino acid position corresponding to 368, and a valine at an amino acid position corresponding to 405 of SEQ ID NO: 63.133. The method of any one of embodiments 114-129, wherein:a) said RGN has at least 90% sequence identity to SEQ ID NO: 1, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 2 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 3;b) said RGN has at least 90% sequence identity to SEQ ID NO: 8, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 9 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 10;c) said RGN has at least 90% sequence identity to SEQ ID NO: 15, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 17;d) said RGN has at least 90% sequence identity to SEQ ID NO: 22, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 23 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 24;e) said RGN has at least 90% sequence identity to SEQ ID NO: 29, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 30 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31;f) said RGN has at least 90% sequence identity to SEQ ID NO: 36, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 38;g) said RGN has at least 90% sequence identity to SEQ ID NO: 43, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 44 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 45;h) said RGN has at least 90% sequence identity to SEQ ID NO: 50, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 51 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 52;i) said RGN has at least 90% sequence identity to SEQ ID NO: 56, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 57 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 58;j) said RGN has at least 90% sequence identity to any one of SEQ ID NOs: 63 and 570-579, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 64 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 65;k) said RGN has at least 90% sequence identity to SEQ ID NO: 70, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 71 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 72;l) said RGN has at least 90% sequence identity to SEQ ID NO: 76, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 77 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 78;m) said RGN has at least 90% sequence identity to SEQ ID NO: 83, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 85;n) said RGN has at least 90% sequence identity to SEQ ID NO: 89, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 90 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 91;o) said RGN has at least 90% sequence identity to SEQ ID NO: 96, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 97 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 98;p) said RGN has at least 90% sequence identity to SEQ ID NO: 103, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 104 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 105;q) said RGN has at least 90% sequence identity to SEQ ID NO: 110, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 111 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 112;r) said RGN has at least 90% sequence identity to SEQ ID NO: 117, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 118 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 119;s) said RGN has at least 90% sequence identity to SEQ ID NO: 123, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 124 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 125; ort) said RGN has at least 90% sequence identity to SEQ ID NO: 83, said guideRNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 78.134. The method of any one of embodiments 114-129, wherein:a) said RGN has at least 95% sequence identity to SEQ ID NO: 1, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 2 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 3;b) said RGN has at least 95% sequence identity to SEQ ID NO: 8, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 9 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 10;c) said RGN has at least 95% sequence identity to SEQ ID NO: 15, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 17;d) said RGN has at least 95% sequence identity to SEQ ID NO: 22, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 23 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 24;e) said RGN has at least 95% sequence identity to SEQ ID NO: 29, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 30 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 31;f) said RGN has at least 95% sequence identity to SEQ ID NO: 36, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 38;g) said RGN has at least 95% sequence identity to SEQ ID NO: 43, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 44 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 45;h) said RGN has at least 95% sequence identity to SEQ ID NO: 50, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 51 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 52;i) said RGN has at least 95% sequence identity to SEQ ID NO: 56, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 57 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 58;j) said RGN has at least 95% sequence identity to any one of SEQ ID NOs: 63 and 570-579, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 64 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 65;k) said RGN has at least 95% sequence identity to SEQ ID NO: 70, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 71 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 72;l) said RGN has at least 95% sequence identity to SEQ ID NO: 76, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 77 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 78;m) said RGN has at least 95% sequence identity to SEQ ID NO: 83, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 85;n) said RGN has at least 95% sequence identity to SEQ ID NO: 89, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 90 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 91;o) said RGN has at least 95% sequence identity to SEQ ID NO: 96, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 97 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 98;p) said RGN has at least 95% sequence identity to SEQ ID NO: 103, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 104 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 105;q) said RGN has at least 95% sequence identity to SEQ ID NO: 110, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 111 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 112;r) said RGN has at least 95% sequence identity to SEQ ID NO: 117, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 118 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 119;s) said RGN has at least 95% sequence identity to SEQ ID NO: 123, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 124 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 125; ort) said RGN has at least 95% sequence identity to SEQ ID NO: 83, said guideRNA comprises a crRNA repeat sequence having at least 95% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 78.135. The method of any one of embodiments 114-129, wherein:a) said RGN has 100% sequence identity to SEQ ID NO: 1, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 2 and a tracrRNA having 100% sequence identity to SEQ ID NO: 3;b) said RGN has 100% sequence identity to SEQ ID NO: 8, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 9 and a tracrRNA having 100% sequence identity to SEQ ID NO: 10;c) said RGN has 100% sequence identity to SEQ ID NO: 15, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 16 and a tracrRNA having 100% sequence identity to SEQ ID NO: 17;d) said RGN has 100% sequence identity to SEQ ID NO: 22, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 23 and a tracrRNA having 100% sequence identity to SEQ ID NO: 24;e) said RGN has 100% sequence identity to SEQ ID NO: 29, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 30 and a tracrRNA having 100% sequence identity to SEQ ID NO: 31;f) said RGN has 100% sequence identity to SEQ ID NO: 36, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 37 and a tracrRNA having 100% sequence identity to SEQ ID NO: 38;g) said RGN has 100% sequence identity to SEQ ID NO: 43, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 44 and a tracrRNA having 100% sequence identity to SEQ ID NO: 45;h) said RGN has 100% sequence identity to SEQ ID NO: 50, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 51 and a tracrRNA having 100% sequence identity to SEQ ID NO: 52;i) said RGN has 100% sequence identity to SEQ ID NO: 56, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 57 and a tracrRNA having 100% sequence identity to SEQ ID NO: 58;j) said RGN has 100% sequence identity to any one of SEQ ID NOs: 63 and 570-579, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 64 and a tracrRNA having 100% sequence identity to SEQ ID NO: 65;k) said RGN has 100% sequence identity to SEQ ID NO: 70, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 71 and a tracrRNA having 100% sequence identity to SEQ ID NO: 72;l) said RGN has 100% sequence identity to SEQ ID NO: 76, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 77 and a tracrRNA having 100% sequence identity to SEQ ID NO: 78;m) said RGN has 100% sequence identity to SEQ ID NO: 83, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 84 and a tracrRNA having 100% sequence identity to SEQ ID NO: 85;n) said RGN has 100% sequence identity to SEQ ID NO: 89, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 90 and a tracrRNA having 100% sequence identity to SEQ ID NO: 91;o) said RGN has 100% sequence identity to SEQ ID NO: 96, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 97 and a tracrRNA having 100% sequence identity to SEQ ID NO: 98;p) said RGN has 100% sequence identity to SEQ ID NO: 103, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 104 and a tracrRNA having 100% sequence identity to SEQ ID NO: 105;q) said RGN has 100% sequence identity to SEQ ID NO: 110, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 111 and a tracrRNA having 100% sequence identity to SEQ ID NO: 112;r) said RGN has 100% sequence identity to SEQ ID NO: 117, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 118 and a tracrRNA having 100% sequence identity to SEQ ID NO: 119;s) said RGN has 100% sequence identity to SEQ ID NO: 123, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 124 and a tracrRNA having 100% sequence identity to SEQ ID NO: 125; ort) said RGN has 100% sequence identity to SEQ ID NO: 83, said guideRNA comprises a crRNA repeat sequence having 100% sequence identity to SEQ ID NO: 84 and a tracrRNA having 100% sequence identity to SEQ ID NO: 78.136. The method of any one of embodiments 107-135, wherein the target DNA sequence is within a cell.137. The method of embodiment 136, wherein the cell is a eukaryotic cell.138. The method of embodiment 137, wherein the eukaryotic cell is a plant cell.139. The method of embodiment 137, wherein the eukaryotic cell is a mammalian cell.140. The method of embodiment 139, wherein said mammalian cell is a human cell.141. The method of embodiment 140, wherein said human cell is an immune cell.142. The method of embodiment 141, wherein said immune cell is a stem cell.143. The method of embodiment 142, wherein said stem cell is an induced pluripotent stem cell.144. The method of embodiment 137, wherein the eukaryotic cell is an insect cell.145. The method of embodiment 136, wherein the cell is a prokaryotic cell.146. The method of any one of embodiments 136-145, further comprising culturing the cell under conditions in which the RGN polypeptide is expressed and cleaves the target DNA sequence to produce a DNA molecule comprising a modified DNA sequence; and selecting a cell comprising said modified target DNA sequence.147. A cell comprising a modified target DNA sequence according to the method of embodiment 146.148. The cell of embodiment 147, wherein the cell is a eukaryotic cell.149. The cell of embodiment 148, wherein the eukaryotic cell is a plant cell.150. A plant comprising the cell of embodiment 149.151. A seed comprising the cell of embodiment 149.152. The cell of embodiment 148, wherein the eukaryotic cell is a mammalian cell.153. The cell of embodiment 152, wherein said mammalian cell is a human cell.154. The cell of embodiment 153, wherein said human cell is an immune cell.155. The cell of embodiment 154, wherein said immune cell is a stem cell.156. The cell of embodiment 155, wherein said stem cell is an induced pluripotent stem cell.157. The cell of embodiment 148, wherein the eukaryotic cell is an insect cell.158. The cell of embodiment 147, wherein the cell is a prokaryotic cell.159. A pharmaceutical composition comprising the cell of any one of embodiments 148 and 152-156 and a pharmaceutically acceptable carrier.160. A method for producing a genetically modified cell with a correction in a causal mutation for a genetically inherited disease, the method comprising introducing into the cell:a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579, or a polynucleotide encoding said RGN polypeptide, wherein said polynucleotide encoding the RGN polypeptide is operably linked to a promoter to enable expression of the RGN polypeptide in the cell; andb) a guide RNA (gRNA) or a polynucleotide encoding said gRNA, wherein said polynucleotide encoding the gRNA is operably linked to a promoter to enable expression of the gRNA in the cellwhereby the RGN and gRNA target to the genomic location of the causal mutation and modify the genomic sequence to remove the causal mutation.161. The method of embodiment 160, wherein the RGN is nuclease inactive or a nickase and is fused to a polypeptide which has base-editing activity.162. The method of embodiment 161, wherein the base-editing polypeptide is a deaminase.163. The method of embodiment 162, wherein the polypeptide with base-editing activity is a cytidine deaminase or an adenine deaminase.164. The method of any one of embodiments 160-163, wherein the genetically inherited disease is caused by a single nucleotide polymorphism.165. The method of any one of embodiments 160-163, wherein the genetically inherited disease is Hurler Syndrome.166. The method of any one of embodiments 160-163, wherein the gRNA further comprises a spacer sequence that targets a region proximal to the causal single nucleotide polymorphism.167. A method for producing a genetically modified cell with a deletion in a disease-causing genomic region of instability, the method comprising introducing into the cell:a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579, or a polynucleotide encoding said RGN polypeptide, wherein said polynucleotide encoding the RGN polypeptide is operably linked to a promoter to enable expression of the RGN polypeptide in the cell; andb) a first guide RNA (gRNA) or a polynucleotide encoding said gRNA, wherein said polynucleotide encoding the gRNA is operably linked to a promoter to enable expression of the gRNA in the cell, and further wherein the gRNA comprises a spacer sequence that targets the 5′ flank of the genomic region of instability; andc) a second guide RNA (gRNA) or a polynucleotide encoding said gRNA, wherein said polynucleotide encoding the gRNA is operably linked to a promoter to enable expression of the gRNA in the cell, and further wherein said second gRNA comprises a spacer sequence that targets the 3′ flank of the genomic region of instability;whereby the RGN and the two gRNAs target to the genomic region of instability and at least a portion of the genomic region of instability is removed.168. The method of embodiment 167, wherein the genetically inherited disease is Friedrich's Ataxia or Huntington's Disease.169. The method of embodiment 167, wherein the first gRNA further comprises a spacer sequence that targets a region within or proximal to the genomic region of instability.170. The method of embodiment 169, wherein the second gRNA further comprises a spacer sequence that targets a region within or proximal to the genomic region of instability.171. The method of any one of embodiments 160-170, wherein said RGN polypeptide has at least 95% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579.172. The method of any one of embodiments 160-170, wherein said RGN polypeptide has 100% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579.173. The method of any one of embodiments 160-170, wherein said RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 63 and has an isoleucine at an amino acid position corresponding to 305, a valine at an amino acid position corresponding to 328, a leucine at an amino acid position corresponding to 366, a threonine at an amino acid position corresponding to 368, and a valine at an amino acid position corresponding to 405 of SEQ ID NO: 63.174. The method of any one of embodiments 160-170, wherein said gRNA, said first gRNA, said second gRNA, or said first gRNA and said second gRNA is selected from a gRNA selected from the group consisting of:a) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 2 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 3, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1;b) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 9 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 10, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8;c) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 17, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15;d) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 24, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22;e) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 30 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29;f) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 38, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 36;g) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 44 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 45, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 43;h) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 51 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 52, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 50;i) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 57 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 58, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 56;j) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 64 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 65, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 71 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 72, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 70;l) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 77 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 76;m) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 85, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83;n) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 90 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 91, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 89;o) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 97 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 98, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 96;p) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 104 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 105, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 103;q) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 111 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 112, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 110;r) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 118 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 119, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 117;s) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 124 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 125, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 123; andt) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83.175. The method of any one of embodiments 160-170, wherein said gRNA, said first gRNA, said second gRNA, or said first gRNA and said second gRNA is selected from a gRNA selected from the group consisting of:a) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 2 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 3, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1;b) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 9 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 10, wherein NO: 8;c) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 17, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 15;d) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 23 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 24, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 22;e) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 30 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 31, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 29;f) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 38, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 36;g) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 44 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 45, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 43;h) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 51 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 52, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 50;i) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 57 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 58, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 56;j) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 64 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 65, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 71 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 72, wherein NO: 70;l) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 77 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 76;m) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 85, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 83;n) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 90 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 91, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 89;o) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 97 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 98, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 96;p) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 104 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 105, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 103;q) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 111 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 112, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 110;r) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 118 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 119, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117;s) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 124 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 125, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 123; andt) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 78, wherein NO: 83.176. The method of any one of embodiments 160-170, wherein said gRNA, said first gRNA, said second gRNA, or said first gRNA and said second gRNA is selected from a gRNA selected from the group consisting of:a) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 2 and a tracrRNA having 100% sequence identity to SEQ ID NO: 3, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 1;b) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 9 and a tracrRNA having 100% sequence identity to SEQ ID NO: 10, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 8;c) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 16 and a tracrRNA having 100% sequence identity to SEQ ID NO: 17, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 15;d) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 23 and a tracrRNA having 100% sequence identity to SEQ ID NO: 24, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 22;e) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 30 and a tracrRNA having 100% sequence identity to SEQ ID NO: 31, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 29;f) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 37 and a tracrRNA having 100% sequence identity to SEQ ID NO: 38, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 36;g) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 44 and a tracrRNA having 100% sequence identity to SEQ ID NO: 45, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 43;h) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 51 and a tracrRNA having 100% sequence identity to SEQ ID NO: 52, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 50;i) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 57 and a tracrRNA having 100% sequence identity to SEQ ID NO: 58, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 56;j) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 64 and a tracrRNA having 100% sequence identity to SEQ ID NO: 65, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 71 and a tracrRNA having 100% sequence identity to SEQ ID NO: 72, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 70;l) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 77 and a tracrRNA having 100% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 76;m) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 84 and a tracrRNA having 100% sequence identity to SEQ ID NO: 85, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 83;n) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 90 and a tracrRNA having 100% sequence identity to SEQ ID NO: 91, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 89;o) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 97 and a tracrRNA having 100% sequence identity to SEQ ID NO: 98, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 96;p) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 104 and a tracrRNA having 100% sequence identity to SEQ ID NO: 105, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 103;q) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 111 and a tracrRNA having 100% sequence identity to SEQ ID NO: 112, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 110;r) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 118 and a tracrRNA having 100% sequence identity to SEQ ID NO: 119, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 117;s) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 124 and a tracrRNA having 100% sequence identity to SEQ ID NO: 125, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 123; andt) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 84 and a tracrRNA having 100% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 83.177. The method of any one of embodiments 160-176, wherein the cell is an animal cell.178. The method of embodiment 177, wherein the animal cell is a mammalian cell.179. The method of embodiment 177, wherein the cell is derived from a dog, cat, mouse, rat, rabbit, horse, cow, pig, or human.180. A method for producing a genetically modified mammalian hematopoietic progenitor cell having decreased BCL11A mRNA and protein expression, the method comprising introducing into an isolated human hematopoietic progenitor cell:a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579, or a polynucleotide encoding said RGN polypeptide, wherein said polynucleotide encoding the RGN polypeptide is operably linked to a promoter to enable expression of the RGN polypeptide in the cell; andb) a guide RNA (gRNA) or a polynucleotide encoding said gRNA, wherein said polynucleotide encoding the gRNA is operably linked to a promoter to enable expression of the gRNA in the cell,whereby the RGN and gRNA are expressed in the cell and cleave at the BCL11A enhancer region, resulting in genetic modification of the human hematopoietic progenitor cell and reducing the mRNA and/or protein expression of BCL11A.181. The method of embodiment 180, wherein said RGN polypeptide has at least 95% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579.182. The method of embodiment 180, wherein said RGN polypeptide has 100% sequence identity to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579.183. The method of embodiment 180, wherein said RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 63 and has an isoleucine at an amino acid position corresponding to 305, a valine at an amino acid position corresponding to 328, a leucine at an amino acid position corresponding to 366, a threonine at an amino acid position corresponding to 368, and a valine at an amino acid position corresponding to 405 of SEQ ID NO: 63.184. The method of embodiment 180, wherein said gRNA is selected from the group consisting of:a) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 2 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 3, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1;b) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 9 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 10, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8;c) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 17, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15;d) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 24, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22;e) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 30 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29;f) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 38, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 36;g) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 44 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 45, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 43;h) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 51 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 52, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 50;i) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 57 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 58, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 56;j) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 64 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 65, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 71 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 72, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 70;l) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 77 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 76;m) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 85, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83;n) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 90 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 91, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 89;o) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 97 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 98, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 96;p) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 104 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 105, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 103;q) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 111 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 112, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 110;r) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 118 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 119, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 117;s) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 124 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 125, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 123; andt) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83.185. The method of embodiment 180, wherein said gRNA is selected from the group consisting of:a) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 2 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 3, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1;b) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 9 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 10, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8;c) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 17, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 15;d) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 23 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 24, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 22;e) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 30 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 31, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 29;f) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 38, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 36;g) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 44 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 45, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 43;h) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 51 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 52, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 50;i) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 57 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 58, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 56;j) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 64 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 65, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 71 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 72, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 70;l) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 77 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 76;m) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 85, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 83;n) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 90 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 91, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 89;o) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 97 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 98, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 96;p) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 104 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 105, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 103;q) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 111 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 112, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 110;r) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 118 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 119, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117;s) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 124 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 125, wherein NO: 123; andt) a gRNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 84 and a tracrRNA having at least 95% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 83.186. The method of embodiment 180, wherein said gRNA is selected from the group consisting of:a) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 2 and a tracrRNA having 100% sequence identity to SEQ ID NO: 3, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 1;b) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 9 and a tracrRNA having 100% sequence identity to SEQ ID NO: 10, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 8;c) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 16 and a tracrRNA having 100% sequence identity to SEQ ID NO: 17, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 15;d) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 23 and a tracrRNA having 100% sequence identity to SEQ ID NO: 24, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 22;e) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 30 and a tracrRNA having 100% sequence identity to SEQ ID NO: 31, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 29;f) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 37 and a tracrRNA having 100% sequence identity to SEQ ID NO: 38, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 36;g) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 44 and a tracrRNA having 100% sequence identity to SEQ ID NO: 45, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 43;h) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 51 and a tracrRNA having 100% sequence identity to SEQ ID NO: 52, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 50;i) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 57 and a tracrRNA having 100% sequence identity to SEQ ID NO: 58, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 56;j) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 64 and a tracrRNA having 100% sequence identity to SEQ ID NO: 65, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 63 and 570-579;k) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 71 and a tracrRNA having 100% sequence identity to SEQ ID NO: 72, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 70;l) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 77 and a tracrRNA having 100% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 76;m) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 84 and a tracrRNA having 100% sequence identity to SEQ ID NO: 85, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 83;n) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 90 and a tracrRNA having 100% sequence identity to SEQ ID NO: 91, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 89;o) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 97 and a tracrRNA having 100% sequence identity to SEQ ID NO: 98, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 96;p) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 104 and a tracrRNA having 100% sequence identity to SEQ ID NO: 105, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 103;q) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 111 and a tracrRNA having 100% sequence identity to SEQ ID NO: 112, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 110;r) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 118 and a tracrRNA having 100% sequence identity to SEQ ID NO: 119, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 117;s) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 124 and a tracrRNA having 100% sequence identity to SEQ ID NO: 125, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 123; andt) a gRNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 84 and a tracrRNA having 100% sequence identity to SEQ ID NO: 78, wherein said RGN polypeptide has an amino acid sequence having 100% sequence identity to SEQ ID NO: 83.187. The method of any one of embodiments 180-186, wherein the gRNA further comprises a spacer sequence that targets a region within or proximal to the BCL11A enhancer region.188. A method of treating a disease, said method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of embodiment 106 or 159.189. The method of embodiment 188, wherein said disease is associated with a causal mutation and said effective amount of said pharmaceutical composition corrects said causal mutation.190. Use of the nucleic acid molecule of any one of embodiments 1-14, 43-45, and 57-59, the vector of any one of embodiments 15-21, 46-56, and 60-70, the cell of any one of embodiments 22, 147, 148, and 152-156, the isolated RGN polypeptide of any one of embodiments 31-42, or the system of any one of embodiments 71-105 for the treatment of a disease in a subject.191. The use of embodiment 190, wherein said disease is associated with a causal mutation and said treating comprises correcting said causal mutation.192. Use of the nucleic acid molecule of any one of embodiments 1-14, 43-45, and 57-59, the vector of any one of embodiments 15-21, 46-56, and 60-70, the cell of any one of embodiments 22, 147, 148, and 152-156, the isolated RGN polypeptide of any one of embodiments 31-42, or the system of any one of embodiments 71-105 for the manufacture of a medicament useful for treating a disease.193. The use of embodiment 192, wherein said disease is associated with a causal mutation and an effective amount of said medicament corrects said causal mutation. The following examples are offered by way of illustration and not by way of limitation. EXAMPLES Example 1. Identification of RNA-Guided Nuclease Nineteen distinct CRISPR-associated RNA-guided nucleases (RGNs) were identified and are described in Table 1 below. Table 1 provides the name of each RGN, its amino acid sequence, the source from which it was derived, and processed crRNA and tracrRNA sequences (see Example 2 for methods of identification). Table 1 further provides a generic single guide RNA (sgRNA) sequence, where the poly-N indicates the location of the spacer sequence which determines the nucleic acid target sequence of the sgRNA. For RGN systems APG06622, APG02787, and APG06248, the conserved sequence in the base of the hairpin stem of the tracrRNA is UNANNA (SEQ ID NO: 129). For APG06007, APG09344, and APG07991, the sequence in the same location is UNANNU (SEQ ID NO: 130). For APG02874, APG03850, and APG07553, the sequence in the same location is UNANNG (SEQ ID NO: 131). For RGN systems APG03031, APG09208, APG05586, APG08770, APG03021, APG06015, APG01868, and APG02998, the conserved sequence in the base of the hairpin stem of the tracrRNA is UNANNC (SEQ ID NO: 132). For APG08167 and APG01604, the sequence in the same location is CNANNC (SEQ ID NO: 133). TABLE 1Summary of SEQ IDs and CRISPR associated systemscrRNArepeatsgRNASEQseqtracrRNAbackboneID(SEQ(SEQ(SEQRGN IDNO.SourceID NO.)ID NO.)ID NO)APG066221Pedobactersp.234APG027878Chitinophagasp.91011APG0624815Mucilaginibactersp.161718APG0600722Acidovoraxsp.232425APG0287429Bacillussp.303132APG0385036Bacillussp.373839APG0755343Bacillussp.444546APG0303150Chryseobacteriumsp.515253APG0920856Bacillussp.575859APG0558663Enterococcussp.646566APG0877070Enterococcussp.717273APG0816776Staphylococcussp.777879APG0160483Staphylococcussp.848586APG0302189Streptococcussp.909192APG0601596Pediococcussp.979899APG09344103Weissellasp.104105106APG07991110Enterococcussp.111112113APG01868117Enterococcussp.118119120APG02998123Enterococcussp.124125126 Example 2: Guide RNA Identification and sgRNA Construction Cultures of bacteria that natively express the RNA-guided nuclease system under investigation were grown to mid-log phase (OD600 of ˜0.600), pelleted, and flash frozen. RNA was isolated from the pellets using a mirVANA miRNA Isolation Kit (Life Technologies, Carlsbad, CA), and sequencing libraries were prepared from the isolated RNA using an NEBNext Small RNA Library Prep kit (NEB, Beverly, MA). The library prep was fractionated on a 6% polyacrylamide gel to capture the RNA species less than 200 nt to detect crRNAs and tracrRNAs, respectively. Deep sequencing (75 bp paired-end) was performed on a Next Seq 500 (High Output kit) by a service provider (MoGene, St. Louis, MO). Reads were quality trimmed using Cutadapt and mapped to reference genomes using Bowtie2. A custom RNAseq pipeline was written in python to detect the crRNA and tracrRNA transcripts. Processed crRNA boundaries were determined by sequence coverage of the native repeat spacer array. The anti-repeat portion of the tracrRNA was identified using permissive BLASTn parameters. RNA sequencing depth confirmed the boundaries of the processed tracrRNA by identifying the transcript containing the anti-repeat. Manual curation of RNAs was performed using secondary structure prediction by NUPACK, an RNA folding software. sgRNA cassettes were prepared by DNA synthesis and were generally designed as follows (5′→3′): 20-30 bp spacer sequence, operably linked at its 3′ end to the processed repeat portion of the crRNA, operably linked to a 4 bp noncomplementary linker (AAAG; SEQ ID NO: 249), operably linked at its 3′ end to the processed tracrRNA. Other 4 bp noncomplementary linkers may also be used. For in vitro assays, sgRNAs were synthesized by in vitro transcription of the sgRNA cassettes with a GeneArt™ Precision gRNA Synthesis Kit (ThermoFisher). Processed crRNA and tracrRNA sequences for each of the RGN polypeptides are identified and are set forth in Table 1. See below for the sgRNAs constructed for PAM libraries 1 and 2. Example 3: Determination of PAM Requirements for Each RGN PAM requirements for each RGN were determined using a PAM depletion assay essentially adapted from Kleinstiver et al. (2015)Nature523:481-485 and Zetsche et al. (2015)Cell163:759-771. Briefly, two plasmid libraries (L1 and L2) were generated in a pUC18 backbone (ampR), with each containing a distinct 30 bp protospacer (target) sequence flanked by 8 random nucleotides (i.e., the PAM region). The target sequence and flanking PAM region of library 1 and library 2 for each RGN are set forth in Table 2. The libraries were separately electroporated intoE. coliBL21(DE3) cells harboring pRSF-1b expression vectors containing an RGN of the invention (codon optimized forE. coli) along with a cognate sgRNA containing a spacer sequence corresponding to the protospacer in L1 or L2. Sufficient library plasmid was used in the transformation reaction to obtain >106CFU. Both the RGN and sgRNA in the pRSF-1b backbone were under the control of T7 promoters. The transformation reaction was allowed to recover for 1 hr after which it was diluted into LB media containing carbenicillin and kanamycin and grown overnight. The following day, the mixture was diluted into self-inducing Overnight Express™ Instant TB Medium (Millipore Sigma) to allow expression of the RGN and sgRNA, and grown for an additional 4 h or 20 h after which the cells were spun down and plasmid DNA was isolated with a Mini-prep kit (Qiagen, Germantown, MD). In the presence of the appropriate sgRNA, plasmids containing a PAM that is recognizable by the RGN will be cleaved resulting in their removal from the population. Plasmids containing PAMs that are not recognizable by the RGN, or that are transformed into bacteria not containing an appropriate sgRNA, will survive and replicate. The PAM and protospacer regions of uncleaved plasmids were PCR-amplified and prepared for sequencing following published protocols (16s-metagenomic library prep guide 15044223B, Illumina, San Diego, CA). Deep sequencing (75 bp single end reads) was performed on a MiSeq (Illumina) by a service provider (MoGene, St. Louis, MO). Typically, 1-4M reads were obtained per amplicon. PAM regions were extracted, counted, and normalized to total reads for each sample. PAMs that lead to plasmid cleavage were identified by being underrepresented when compared to controls (i.e., when the library is transformed intoE. colicontaining the RGN but lacking an appropriate sgRNA). To represent PAM requirements for a novel RGN, the depletion ratios (frequency in sample/frequency in control) for all sequences in the region in question were converted to enrichment values with a −log base 2 transformation. Sufficient PAMs were defined as those with enrichment values >2.3 (which corresponds to depletion ratios <˜0.2). PAMs above this threshold in both libraries were collected and used to generate web logos, which for example can be generated using a web-based service on the internet known as “weblogo”. PAM sequences were identified and reported when there was a consistent pattern in the top enriched PAMs. A consensus PAM (having an enrichment factor (EF)>2.3) for each RGN is provided in Table 2. The PAM orientation is also indicated in Table 2. TABLE 2PAM or PAM-like determinationsgRNA L1sgRNA L2PAM(SEQ(SEQ(SEQPAMRGN IDID NO.)ID NO.)ID NO.)orientationAPG066225675′-target-PAM-3′APG027871213145′-target-PAM-3′APG062481920215′-target-PAM-3′APG060072627285′-target-PAM-3′APG028743334355′-target-PAM-3′APG038504041425′-target-PAM-3′APG075534748495′-target-PAM-3′APG030315455355′-target-PAM-3′APG092086061625′-target-PAM-3′APG055866768695′-target-PAM-3′APG087707475695′-target-PAM-3′APG081678081825′-target-PAM-3′APG016048788825′-target-PAM-3′APG030219394955′-target-PAM-3′APG060151001011025′-target-PAM-3′APG093441071081095′-target-PAM-3′APG079911141151165′-target-PAM-3′APG018681211221165′-target-PAM-3′APG029981271281165′-target-PAM-3′ Example 4: Demonstration of Gene Editing Activity in Mammalian Cells RGN expression cassettes were produced and introduced into vectors for mammalian expression. Each RGN was codon-optimized for human expression (SEQ ID NOs: 134-152), and operably fused at the 5′ end to an SV40 nuclear localization sequence (NLS; SEQ ID NO: 251) and to 3×FLAG tags (SEQ ID NO: 252), and operably fused at the 3′ end to nucleoplasmin NLS sequences (SEQ ID NO: 253). Two copies of the NLS sequence were used, operably fused in tandem. Each expression cassette was under control of a cytomegalovirus (CMV) promoter (SEQ ID NO: 258). It is known in the art that the CMB transcription enhancer (SEQ ID NO: 259) may also be included in constructs comprising the CMV promoter. Guide RNA expression constructs encoding a single gRNA each under the control of a human RNA polymerase III U6 promoter (SEQ ID NO: 260) were produced and introduced into the pTwist High Copy Amp vector. Sequences for the target sequences for each guide are in Table 3. Several of the constructs described above were introduced into mammalian cells. One day prior to transfection, 1×105HEK293T cells (Sigma) were plated in 24-well dishes in Dulbecco's modified Eagle medium (DMEM) plus 10% (vol/vol) fetal bovine serum (Gibco) and 1% Penicillin-Streptomycin (Gibco). The next day when the cells were at 50-60% confluency, 500 ng of an RGN expression plasmid plus 500 ng of a single gRNA expression plasmid were co-transfected using 1.5 μL of Lipofectamine 3000 (Thermo Scientific) per well, following the manufacturer's instructions. After 48 hours of growth, total genomic DNA was harvested using a genomic DNA isolation kit (Machery-Nagel) according to the manufacturer's instructions. The total genomic DNA was then analyzed to determine the rate of editing for each RGN for each genomic target. First, oligonucleotides were produced to be used for PCR amplification and subsequent analysis of the amplified genomic target site. Oligonucleotide sequences used are listed in Table 4. All PCR reactions were performed using 10 μL of 2× Master Mix Phusion High-Fidelity DNA polymerase (Thermo Scientific) in a 20 μL reaction including 0.5 μM of each primer. Large genomic regions encompassing each target gene were first amplified using PCR #1 primers, using a program of: 98° C., 1 min; 30 cycles of [98° C., 10 sec; 62° C., 15 sec; 72° C., 5 min]; 72° C., 5 min; 12° C., forever. One microliter of this PCR reaction was then further amplified using primers specific for each guide (PCR #2 primers), using a program of: 98° C., 1 min; 35 cycles of [98° C., 10 sec; 67° C., 15 sec; 72° C., 30 sec]; 72° C., 5 min; 12° C., forever. Primers for PCR #2 include Nextera Read 1 and Read 2 Transposase Adapter overhang sequences for Illumina sequencing. For RGNs APG02874, APG03850, and APG09208, methods were carried out as described above. A number of different genes in the human genome were targeted for RNA-guided cleavage. These loci are included in Table 3 below, along with the reference to the SEQ ID NO of the sgRNA. The indel percentage, which is an indication of RGN activity, is also shown. TABLE 3Target and sgRNA sequences for guide RNAs used totest gene editing activity in mammalian cellsTarget SequencesgRNARGN IDGeneGuide ID(SEQ ID NO.)(SEQ ID NO.)APG02874, APG09208RelASGN000973,153188, 206SGN000778APG02874RelASGN000974154189APG02874RelASGN000975,155190, 208SGN000780APG02874, APG09208AurkBSGN000976,156191, 204SGN000775APG02874, APG09208AurkBSGN000977,157192, 205SGN000776APG02874AurkBSGN000978158193APG02874VEGFASGN000979159194APG02874VEGFASGN000981160195APG03850RelASGN000982161196APG03850RelASGN000983162197APG03850RelASGN000984163198APG03850AurkBSGN000985164199APG03850AurkBSGN000986165200APG03850AurkBSGN000987166201APG03850VEGFASGN000988167202APG03850VEGFASGN000990168203APG09208RelASGN000779169207APG09208AurkBSGN000793170209APG09208AurkBSGN000794171210APG05586TRASGN001163553557APG05586TRASGN001164554558APG05586VEGFASGN001165555559APG05586VEGFASGN001166556560APG09208RelASGN000778153206APG09208AurkBSGN000793609811APG05586, APG08770,EMX1SGN001159610812APG09298APG05586, APG08770,TRASGN001162611813APG09298APG05586, APG08770,TRASGN001163612814APG09298APG05586, APG08770,TRASGN001164613815APG09298APG05586, APG08770,VEGFASGN001165614816APG09298APG05586, APG08770,VEGFASGN001166615817APG09298APG05586, APG09298VEGFASGN001167616818APG09208RelASGN001213617819APG08167, APG01604VEGFASGN001245618820APG08167, APG01604VEGFASGN001246619821APG08167, APG01604VEGFASGN001247620822APG08167, APG01604RelASGN001248621823APG08167, APG01604RelASGN001249622824APG08167, APG01604RelASGN001250623825APG08167, APG01604AurkBSGN001251624826APG08167, APG01604AurkBSGN001252625827APG08167, APG01604AurkBSGN001253626828APG07991RelASGN001312627829APG07991RelASGN001313628830APG07991RelASGN001314629831APG01868RelASGN001315630832APG01868RelASGN001316631833APG01868RelASGN001317632834APG02998RelASGN001318633835APG02998RelASGN001319634836APG02998RelASGN001320635837APG09344RelASGN001321636838APG06015RelASGN001322637839APG03021RelASGN001323638840APG09344RelASGN001324639841APG06015RelASGN001325640842APG03021RelASGN001326641843APG06015RelASGN001327642844APG03021RelASGN001328643845APG09344RelASGN001329644846APG03021TRASGN001330645847APG03021TRASGN001331646848APG03021TRASGN001332647849APG06015TRASGN001333648850APG06015TRASGN001334649851APG06015TRASGN001335650852APG09344TRASGN001336651853APG09344TRASGN001337652854APG09344TRASGN001338653855APG07991TRASGN001339654856APG07991TRASGN001340655857APG07991TRASGN001341656858APG01868TRASGN001342657859APG01868TRASGN001343658860APG01868TRASGN001344659861APG01868TRASGN001692660862APG02998TRASGN001345661863APG02998TRASGN001346662864APG02998TRASGN001347663865APG03021HA01SGN001348664866APG03021HAO1SGN001349665867APG03021HAO1SGN001350666868APG06015HAO1SGN001351667869APG06015HAO1SGN001352668870APG06015HAO1SGN001353669871APG09344HAO1SGN001354670872APG09344HAO1SGN001355671873APG09344HAO1SGN001356672874APG07991HAO1SGN001357673875APG07991HAO1SGN001358674876APG07991HAO1SGN001359675877APG01868HAO1SGN001360676878APG01868HAO1SGN001361677879APG01868HAO1SGN001362678880APG02998HAO1SGN001363679881APG02998HAO1SGN001364680882APG02998HAO1SGN001365681883APG05586, APG09298TRASGN001371682884APG05586, APG09298TRASGN001372683885APG05586, APG09298TRASGN001373684886APG05586, APG09298TRASGN001374685887APG05586, APG09298TRASGN001375686888APG05586, APG09298TRASGN001376687889APG05586, APG09298TRASGN001377688890APG05586, APG09298TRASGN001378689891APG05586, APG09298TRASGN001379690892APG05586, APG09298TRASGN001380691893APG05586, APG09298TRASGN001381692894APG05586, APG09298TRASGN001382693895APG05586, APG09298B2MSGN001383694896APG05586, APG09298B2MSGN001384695897APG05586, APG09298B2MSGN001385696898APG05586, APG09298B2MSGN001386697899APG05586, APG09298B2MSGN001387698900APG05586, APG09298B2MSGN001388699901APG05586, APG09298B2MSGN001389700902APG05586, APG09298B2MSGN001390701903APG05586, APG09298B2MSGN001391702904APG05586, APG09298B2MSGN001392703905APG05586, APG09298B2MSGN001393704906APG05586, APG09298B2MSGN001394705907APG05586, APG09298LDHASGN001395706908APG05586, APG09298LDHASGN001396707909APG05586, APG09298LDHASGN001397708910APG05586, APG09298LDHASGN001399709911APG05586, APG09298LDHASGN001400710912APG05586, APG09298LDHASGN001401711913APG05586, APG09298LDHASGN001402712914APG05586, APG09298LDHASGN001403713915APG05586, APG09298LDHASGN001404714916APG05586, APG09298LDHASGN001405715917APG05586, APG09298HAO1SGN001406716918APG05586, APG09298HAO1SGN001407717919APG05586, APG09298HAO1SGN001408718920APG05586, APG09298HAO1SGN001409719921APG05586, APG09298HAO1SGN001410720922APG05586, APG09298HAO1SGN001411721923APG05586, APG09298HAO1SGN001412722924APG05586, APG09298HAO1SGN001413723925APG05586, APG09298HAO1SGN001414724926APG05586, APG09298HAO1SGN001415725927APG05586, 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 TABLE 4Oligonucleotides for detection of gene editing activity in mammalian cellsSEQDescriptionPrimer SequenceID NOSGN000977 FWDCTTGTAGCTGGAGGTCCATC172SGN000977 REVTGTTGGCAAATCTAGTCTCG173SGN000978 FWDACATTTGACGAGCAGCGAA174SGN000978 REVGGCCCCTGGAGAGGTTTTAA175SGN000979, SGN000982, SGN000988,ACACAGCTTCCCGTTCTCAG176SGN000990 FWDSGN000979, SGN000982, SGN000988,ATTCACCCAGCTTCCCTGTG177SGN000990 REVSGN000981 FWDGGCGTCGCACTGAAACTTTT178SGN000981 REVAGTTCATGGTTTCGGAGGCC179SGN000983 FWDCGACCAAACAAGTGCAAAGG180SGN000983 REVGGGTTGTTGTTGGTCTGGAT181SGN000775, SGN000776, SGN000793,ACTGCCATGGGAAGAAGGTG182SGN000794, SGN000976, SGN000985,SGN000986, SGN000987 FWDSGN000775, SGN000776, SGN000793,ACAATTCTCCTGCCTCAGCC183SGN000794, SGN000976, SGN000985,SGN000986, SGN000987 REVSGN000778, SGN000973, SGN000984 FWDTGGCCCCTATGTGGAGATCA184SGN000778, SGN000973, SGN000984 REVGGCAGAGCTCAGCCTCATAG185SGN000779, SGN000780, SGN000974,ATATCCCCACTTCCCCTGCT186SGN000975 FWDSGN000779, SGN000780, SGN000974,CACCTCAAGGACAGCTCTGG187SGN000975 REVSGN001163, SGN001164 FWDTTGATAGCTTGTGCCTGTCC561SGN01163, SGN001164REVAGAGTCTCTCAGCTGGTACA562SGN001165, SGN001166 FWDGCGACAGGGGCAAAGTGAGT563SGN001165, SGN001166REVCTAGCACTTCTCGCGGCTCC564SGN001382 FWDAACTCATGCCTGCTGCTCTT1023SGN001382 REVCAGTCTCACGCAGTCACTCA1024SGN001371, SGN001372, SGN001373, SGN001689,AACTGAGGCGGCTGAAATGA1025SGN001690 FWDSGN001371, SGN001372, SGN001373, SGN001689,TGGGACATGCAAGCCCATAA1026SGN001690 REVSGN001730 FWDAAGATGTTGACATGCTCTTCC1027SGN001730 REVTATGCAGTCAAAAGCCTCA1028SGN001353, SGN001356, SGN001359, SGN001362,AAGTCATTTGCTTGTTTGGA1029SGN001365, SGN001406, SGN001407, SGN001412,SGN001413, SGN001414, SGN001622, SGN001623,SGN001624, SGN001625, SGN001626, SGN001627FWDSGN001353, SGN001356, SGN001359, SGN001362,TGGTGCATTCAGAGAAGGAG1030SGN001365, SGN001406, SGN001407, SGN001412,SGN001413, SGN001414, SGN001622, SGN001623,SGN001624, SGN001625, SGN001626, SGN001627REVSGN001375, SGN001376, SGN001377 FWDAATGAAGCCAGGCAAGAGCA1031SGN001375, SGN001376, SGN001377 REVCTGTGCAAACCCAGGCTAGA1032SGN001251, SGN001252 FWDACATTTGACGAGCAGCGAA1033SGN001251, SGN001252 REVGGCCCCTGGAGAGGTTTTAA1034SGN001380 FWDACCCGGCCTGCTTTTCTTAA1035SGN001380 REVGGCAGCGAGGCATACATAGT1036SGN001731, SGN001732, SGN001733, SGN001734,ACCCTGCTTTTTCTGCCTTT1037SGN001735 FWDSGN001731, SGN001732, SGN001733, SGN001734,CAGGCCTAATGGACATTAATC1038SGN001735 REVCTSGN001374, SGN001688 FWDACTCACTAAGGGGCCCATCT1039SGN001374, SGN001688 REVCAGGAGGAGGATTCGGAACC1040SGN001726, SGN001727, SGN001728, SGN001729AGGAAAATGAATCACAATTAC1041FWDTSGN001726, SGN001727, SGN001728, SGN001729GTGCGAAAGGGCAAGATTCT1042REVSGN001397 FWDAGGCCTTTCAACTCTCTTTTGG1043CASGN001397 REVGGATGGGGTCAAGGTATGGGC1044SGN001213, SGN001248, SGN001249, SGN001321,ATGACATTCAGGCCACAGTG1045SGN001322, SGN001323 FWDSGN001213, SGN001248, SGN001249, SGN001321,CTTCCTCCTATTCAGGCCCA1046SGN001322, SGN001323 REVSGN001725 FWDCAGCTTTTGAAATGGGGTGC1047SGN001725 REVCAACAAATGGAGACCATCTGG1048ASGN001381 FWDCAGTATTCTAAGGACGCCAGA1049AASGN001381 REVGCACTTTGGGAGGCTGAA1050SGN001390, SGN001391, SGN001392, SGN001393,CGGGCATTCCTGAAGCTG1051SGN001394, SGN001592, SGN001593, SGN001594,SGN001595, SGN001596, SGN001597, SGN001598,SGN001599, SGN001600, SGN001601, SGN001602,SGN001603 FWDSGN001390, SGN001391, SGN001392, SGN001393,GTAGGCCAAAGGTCTCCCC1052SGN001394, SGN001592, SGN001593, SGN001594,SGN001595, SGN001596, SGN001597, SGN001598,SGN001599, SGN001600, SGN001601, SGN001602,SGN001603 REVSGN001383, SGN001384, SGN001385, SGN001386,CTTGACACCAAGTTAGCCCC1053SGN001387, SGN001388, SGN001389 FWDSGN001383, SGN001384, SGN001385, SGN001386,TCATACACAACTTTCAGCAGC1054SGN001387, SGN001388, SGN001389 REVSGN000793, SGN001253 FWDCTTGTAGCTGGAGGTCCATC1055SGN000793, SGN001253 REVTGTTGGCAAATCTAGTCTCG1056SGN001379 FWDGAGCAGCTGAGTCAATGATAG1057TSGN001379 REVGGAGAGATCTGGAGGGAACTT1058ASGN001165, SGN001166, SGN001245 FWDGCAAAGTGAGTGACCTGCTT1059SGN001165, SGN001166, SGN001245 REVGAGCTAGCACTTCTCGCG1060SGN001738 FWDGCTGTTTGGGAGGTCAGAAA1061SGN001738 REVGAATATTGAAGGGGGCAGGG1062SGN001167, SGN001246, SGN001247, SGN001785GGACACTTCCCAAAGGACC1063FWDSGNOO1167, SGN001246, SGN001247, SGN001785CACGTCCTCACTCTCGAAGA1064REVSGN001399, SGN001402, SGN001403, SGN001646,GGCCTTCACTCTTCACAGACCC1065SGN001647, SGN001648, SGN001649, SGN001650,SGN001651 FWDSGN001399, SGN001402, SGN001403, SGN001646,GGATGGGGTCAAGGTATGGGC1066SGN001647, SGN001648, SGN001649, SGN001650,SGN001651 REVSGN001162, SGN001163, SGN001164, SGN001330,GGGCAAAGAGGGAAATGAGA1067SGN001331, SGN001332, SGN001333, SGN001334,SGN001335, SGN001336, SGN001337, SGN001338,SGN001339, SGN001340, SGN001341, SGN001342,SGN001343, SGN001344, SGN001345, SGN001346,SGN001347, SGN001664, SGN001665, SGN001666,SGN001667, SGN001668, SGN001669, SGN001670,SGN001671, SGN001672, SGN001673, SGN001674,SGN001675, SGN001684, SGN001685, SGN001686,SGN001687, SGN001691, SGN001692 FWDSGN001162, SGN001163, SGN001164, SGN001330,GAACCTGGCCATTCCTGAAG1068SGN001331, SGN001332, SGN001333, SGN001334,SGN001335, SGN001336, SGN001337, SGN001338,SGN001339, SGN001340, SGN001341, SGN001342,SGN001343, SGN001344, SGN001345, SGN001346,SGN001347, SGN001664, SGN001665, SGN001666,SGN001667, SGN001668, SGN001669, SGN001670,SGN001671, SGN001672, SGN001673, SGN001674,SGN001675, SGN001684, SGN001685, SGN001686,SGN001687, SGN001691, SGN001692 REVSGN001714, SGN001722 FWDGGTTTTTGGAGGTGGAGTTGA1069SGN001714, SGN001722 REVCCCCCTAACCAAGTGAAAAGA1070SGN001716, SGN001717, SGN001721 FWDTAGATAAATGAGCAGTGAACA1071GCCSGN001716, SGN001717, SGN001721 REVTCCACAAAGGATCACAAAGTC1072ASGN001313, SGN001316, SGN001319 FWDTGAGAGACAGTGGGACAGAC1073SGN001313, SGN001316, SGN001319 REVAGTCCTAGAGGAGGCAGAAC1074SGN001702, SGN001703, SGN001707, SGN001709,TGAGTCCGAGCAGAAGAAGA1075SGN001710, SGN001711 FWDSGN001702, SGN001703, SGN001707, SGN001709,GGAGATTGGAGACACGGAGA1076SGN001710, SGN001711 REVSGN001723 FWDTGGAGGTGGAGTTGAATAACA1077SGN001723 REVCTTTCTCCCCCTAACCAAGTG1078SGN001395, SGN001396 FWDTGGATCTCCAACATGGCAGCC1079SGN001395, SGN001396 REVCGGAAGGCTAAGGAGGGAGG1080ASGN000778, SGN001250, SGN001312, SGN001314,TGGCCCCTATGTGGAGATCA1081SGN001315, SGN001317, SGN001318, SGN001320,SGN001324, SGN001325, SGN001326, SGN001327,SGN001328, SGN001329 FWDSGN000778, SGN001250, SGN001312, SGN001314,GGCAGAGCTCAGCCTCATAG1082SGN001315, SGN001317, SGN001318, SGN001320,SGN001324, SGN001325, SGN001326, SGN001327,SGN001328, SGN001329 REVSGN001159, SGN001697, SGN001698, SGN001699,TGTTAGACCCATGGGAGCAG1083SGNOO1700, SGN001701, SGN001704, SGN001705,SGN001706, SGN001708 FWDSGN001159, SGN001697, SGN001698, SGN001699,GTTGCCCACCCTAGTCATT1084SGN001700, SGN001701, SGN001704, SGN001705,SGN001706, SGN001708 REVSGN001400, SGN001401, SGN001404, SGN001405,TTCCACGCTAAGGTATGGGCC1085SGN001640, SGN001641, SGN001642, SGN001643,SGN001644, SGN001645 FWDSGN001400, SGN001401, SGN001404, SGN001405,GCCAACAGCACCAACCCCAA1086SGN001640, SGN001641, SGN001642, SGN001643,SGN001644, SGN001645 REVSGN001348, SGN001349, SGN001350, SGN001351,TTGCTCACTTGATGTAAGCAA1087SGN001352, SGN001354, SGN001355, SGN001357,SGN001358, SGN001360, SGN001361, SGN001363,SGN001364, SGN001408, SGN001409, SGN001410,SGN001411, SGN001415, SGN001416, SGN001616,SGN001617, SGN001618, SGN001619, SGN001620,SGN001621, SGN001713, SGN001715, SGN001718,SGN001719, SGN001724 FWDSGN001348, SGN001349, SGN001350, SGN001351,TTTTGGTACGGTCTTTGTGT1088SGN001352, SGN001354, SGN001355, SGN001357,SGN001358, SGN001360, SGN001361, SGN001363,SGN001364, SGN001408, SGN001409, SGN001410,SGN001411, SGN001415, SGN001416, SGN001616,SGN001617, SGN001618, SGN001619, SGN001620,SGN001621, SGN001713, SGN001715, SGN001718,SGN001719, SGN001724 REV Purified genomic DNA was subjected to PCR #1 and PCR #2 as above. Following the second PCR amplification, DNA was cleaned using a PCR cleanup kit (Zymo) according to the manufacturer's instructions and eluted in water. 200-500 ng of purified PCR #2 product was combined with 2 μL of 10×NEB Buffer 2 and water in a 20 μL reaction and annealed to form heteroduplex DNA using a program of: 95° C., 5 min; 95-85° C., cooled at a rate of 2° C./sec; 85-25° C., cooled at a rate of 0.1° C./sec.; 12° C., forever. Following annealing 5 μL of DNA was removed as a no enzyme control, and 1 μL of T7 Endonuclease I (NEB) was added and the reaction incubated at 37° C. for 1 hr. After incubation 5× FlashGel loading dye (Lonza) was added and 5 μL of each reaction and controls were analyzed by a 2.2% agarose FlashGel (Lonza) using gel electrophoresis. Following visualization of the gel, the percentage of non-homologous end joining (NHEJ) was determined using the following equation: % NHEJ events=100×[1−(1−fraction cleaved)(½)], where (fraction cleaved) is defined as: (density of digested products)/(density of digested products+undigested parental band). For some samples, SURVEYOR® was used to analyze the results following expression in mammalian cells. Cells were incubated at 37° C. for 72 h post-transfection before genomic DNA extraction. Genomic DNA was extracted using the QuickExtract DNA Extraction Solution (Epicentre) following the manufacturer's protocol. The genomic region flanking the RGN target site was PCR amplified, and products were purified using QiaQuick Spin Column (Qiagen) following the manufacturer's protocol. 200-500 ng total of the purified PCR products were mixed with 1 μl 10× Taq DNA Polymerase PCR buffer (Enzymatics) and ultrapure water to a final volume of 10 μl, and subjected to a re-annealing process to enable heteroduplex formation: 95° C. for 10 min, 95° C. to 85° C. ramping at −2° C./s, 85° C. to 25° C. at −0.25° C./s, and 25° C. hold for 1 min. After reannealing, products were treated with SURVEYOR® nuclease and SURVEYOR® enhancer S (Integrated DNA Technologies) following the manufacturer's recommended protocol and analyzed on 4-20% Novex TBE polyacrylamide gels (Life Technologies). Gels were stained with SYBR Gold DNA stain (Life Technologies) for 10 min and imaged with a Gel Doc gel imaging system (Bio-rad). Quantification was based on relative band intensities. Indel percentage was determined by the formula, 100×(1−(1−(b+c)/(a+b+c))½), where a is the integrated intensity of the undigested PCR product, and b and c are the integrated intensities of each cleavage product. Additionally, products from PCR #2 containing Illumina overhang sequences underwent library preparation following the Illumina 16S Metagenomic Sequencing Library protocol. Deep sequencing was performed on an Illumina Mi-Seq platform by a service provider (MOGene). Typically, 200,000 of 250 bp paired-end reads (2×100,000 reads) are generated per amplicon. The reads were analyzed using CRISPResso (Pinello, et al. 2016 Nature Biotech, 34:695-697) to calculate the rates of editing. Output alignments were hand-curated to confirm insertion and deletion sites as well as identify microhomology sites at the recombination sites. The rates of editing are shown in Table 5. All experiments were performed in human cells. The “target sequence” is the targeted sequence within the gene target. For each target sequence, the guide RNA comprised the complementary RNA target sequence and the appropriate sgRNA depending on the RGN used. A selected breakdown of experiments by guide RNA is shown in Tables 6.1-6.3. TABLE 5Activity of RGNs in mammalian cellsOverallDeletionInsertionGeneEditingRate inRate inRGNGuide IDtargetRateSampleSampleAPG02874SGN000973RelAN.D.APG02874SGN000974RelA0.10%37.23%62.77%APG02874SGN000975RelAN.D.APG02874SGN000976AurkB0.56%35.17%64.84%APG02874SGN000977AurkB0.30%76.34%23.65%APG02874SGN000978AurkBN.D.APG02874SGN000979VEGFA0.17%13.95%86.05%APG02874SGN000981VEGFAN.D.APG03850SGN000982RelA0.19%40.55%59.45%APG03850SGN000983RelAN.D.APG03850SGN000984RelA0.05%72.17%27.84%APG03850SGN000985AurkB0.37%66.62%33.37%APG03850SGN000986AurkBN.D.APG03850SGN000987AurkBN.D.APG03850SGN000988VEGFAN.D.APG03850SGN000990VEGFAN.D.APG09208SGN000775AurkBN.D.APG09208SGN000776AurkBN.D.APG09208SGN000778RelAN.D.APG09208SGN000779RelAN.D.APG09208SGN000780RelAN.D.APG09208SGN000793AurkB1.18%59.97%40.02%APG09208SGN000794AurkBN.D.APG05586SGN001163TRA23.49%95.03%5.41%APG05586SGN001164TRA1.37%95.65%4.36%APG05586SGN001165VEGFA65.59%98.58%2.03%APG05586SGN001166VEGFA10.48%94.98%5.02%APG06015SGN001322RelAN.D.APG06015SGN001325RelAN.D.APG06015SGN001327RelAN.D.APG06015SGN001333IRAN.D.APG06015SGN001334TRA0.03100%0%APG06015SGN001335TRAN.D.APG06015SGN001351HAO1N.D.APG06015SGN001352HAO1N.D.APG06015SGN001353HAO1N.D.APG09344SGN001321RelA0.020%100%APG09344SGN001324RelAN.D.APG09344SGN001329RelAN.D.APG09344SGN001336TRAN.D.APG09344SGN001337TRAN.D.APG09344SGN001338TRAN.D.APG09344SGN001354HAO1N.D.APG09344SGN001355HAO1N.D.APG09344SGN001356HAO1N.D.APG07991SGN001312RelA2.1656.61%43.39%APG07991SGN001313RelA2.6480.6%19.39%APG07991SGN001314RelA2.4932.48%67.51%APG07991SGN001339TRA6.7576.47%27.42%APG07991SGN001340TRA6.6669.19%33.89%APG07991SGN001341TRA2.8460.63%39.38%APG07991SGN001357HAO113.3479.66%21.31%APG07991SGN001358HAO10.0560.78%39.22%APG07991SGN001359HAO10.2180.53%19.48%APG01868SGN001315RelA10.7864.07%36.36%APG01868SGN001316RelA6.2687.09%13.73%APG01868SGN001317RelA19.4262.57%38.11%APG01868SGN001342TRA4.6639.32%60.68%APG01868SGN001343TRA41.3960.13%41.39%APG01868SGN001344TRA5.4260.46%39.53%APG01868SGN001360HAO10.3869.28%30.72%APG01868SGN001361HAO19.4884.92%17.07%APG01868SGN001362HAO10.3228.97%71.03%APG02998SGN001318RelA1.1372.78%27.22%APG02998SGN001319RelA2.5591.76%8.22%APG02998SGN001320RelA3.4843.97%56.04%APG02998SGN001345TRA2.0179.92%26.06%APG02998SGN001346TRA5.8740.14%61.02%APG02998SGN001347TRA2.2659.08%40.89%APG02998SGN001363HAO10.416.34%83.66%APG02998SGN001364HAO1N.D.APG02998SGN001365HAO11.1465.98%34.02%APG09208SGN000778RelA0.260%100.00%APG09208SGN000793AurkB1.6450.94%49.06%APG09208SGN001213RelA0.05100%0.00%APG05586SGN001159EMX140.1383.86%16.13%APG05586SGN001162TRA46.1286.07%14.45%APG05586SGN001163TRA43.7793.38%7.58%APG05586SGN001164TRA895.23%4.80%APG05586SGN001165VEGFA65.5998.58%2.03%APG05586SGN001166VEGFA10.4894.98%5.02%APG05586SGN001167VEGFA43.892.97%7.35%APG08770SGN001159EMX111.980.98%20.13%APG08770SGN001162TRA14.5790.2%10.24%APG08770SGN001163TRA12.7895.47%5.49%APG08770SGN001164TRA3.0992.84%9.00%APG08770SGN001165VEGFA23.1292.75%8.00%APG08770SGN001166VEGFA10.5293.45%6.92%APG08167SGN001245VEGFAN.D.APG08167SGN001246VEGFAN.D.APG08167SGN001247VEGFAN.D.APG08167SGN001248RelAN.D.APG08167SGN001249RelAN.D.APG08167SGN001250RelAN.D.APG08167SGN001251AurkBN.D.APG08167SGN001252AurkBN.D.APG08167SGN001253AurkBN.D.APG01604SGN001245VEGFA69.1396.82%7.23%APG01604SGN001246VEGFA4.5779.07%24.78%APG01604SGN001247VEGFA18.4996.17%5.09%APG01604SGN001248RelA17.0494.78%5.61%APG01604SGN001249RelA5.5387.88%14.96%APG01604SGN001250RelA21.1881.7%19.19%APG01604SGN001251AurkB8.3884.67%15.34%APG01604SGN001252AurkB24.7490.49%10.22%APG01604SGN001253AurkB0.3286.44%13.56%APG03021SGN001323RelA13.7387.67%12.31%APG03021SGN001326RelA1.0378.9%21.11%APG03021SGN001328RelA7.1292.18%9.68%APG03021SGN001330IRAN.D.APG03021SGN001331TRA0.4523.15%76.85%APG03021SGN001332TRA0.2549.44%50.57%APG03021SGN001348HAO10.0830.16%69.84%APG03021SGN001349HAO10.0358.33%41.67%APG03021SGN001350HAO10.3670.53%29.48% Specific insertions and deletions for respective guides are shown in Tables 6.1-6.3. In these tables, the target sequence is identified by bold upper case letters. The 8mer PAM regions are double underlined, with the main recognized nucleotides in bold. Insertions are identified by lowercase letters. Deletions are indicated with dashes ( - - - ). The INDEL location is calculated from the PAM proximal edge of the target sequence, with the edge being location 0. The location is positive (+) if the location is on the target side of the edge; the location is negative (−) if the location is on the PAM side of the edge. TABLE 6.1Specific insertions and deletions for Guide 977 using RGN APG02874#%% ofINDELGuide SGN000977 (SEQ ID NO: 157)ReadsReadsINDELsTypeLocationSizeGGAGAGGTTTTAATGGCCCAGCCTGG21865199.7GGAGAGGTTTTAATGGCCCAG---GG1610.0724.88Deletion-14GGAGAGGTTTTAATGGCCCAGtacCCT1530.0723.65Insertion+33AGGGGAGAGGTTTTAATGGCCCAG----G1370.0621.17Deletion-912GGAGAGGTTTTAATGGCCCAGC--GG1300.0620.09Deletion-35GGAGAGGTTT-AATGGCCCAGCCTGG330.025.1Deletion+131GGAGAGGTTTTAATGGCC-AGCCTGG330.025.1Deletion+51 TABLE 6.2Specific insertions and deletions for Guide 985 using RGN APG03850#%% ofINDELSizeGuide SGN000985 (SEQ ID NO: 164)ReadsReadsINDELSTypeLocationAGGCTGGGCCATTAAAACCTCTCCAGG21836799.63AGGCTGGGCCATTAAAACCTCTCCaAGG2680.1233.37Insertion31AGGCTGGGCCATTAAAACCTCT-CAGG2040.0925.4Deletion41AGGCTGGGCCATTAAAACCTCT-----1400.0617.43Deletion-27AGGCTGGGCCATTAAAACCTCTCC-GG1370.0617.06Deletion21AGGCT-GGCCATTAAAACCTCTCCAGG280.013.49Deletion211C---------------------------------260.013.24Deletion-3988-------------------------------------------------------T TABLE 6.3Specific insertions and deletions for Guide 793 using RGN APG09208#%% ofINDELGuide SGN000793 (SEQ ID NO: 170)ReadsReadsINDELsTypeLocationSizeAGGTTTTAATGGCCCAGCCTCACACCC16957898.82C---------------------------------4710.2723.25Deletion-1780-----------------------------------------------GTGGAGAGGTTTTAATGGCCCAGCCTCACAaCCCA3980.2319.64Insertion+31CCTCCCCTGGAGAGGTTTTAATGGCCCAGCCTCACA----1900.119.38Deletion-1518--------------GCTGGAGAGGTTTTAATGGCCCAGCCTCACAC---1330.086.56Deletion-1012---------TCCCCTGGAGAGGTTTTAATGGCCCAGCCTCACACC-1100.065.43Deletion01CCTCCCCTGGAGAGGTTTTAATGGCCCAG-----------1060.065.23Deletion-212CCTCCCCTGGAGAGGTTTTAATGGCCCAGCCTCACAggCC1020.065.03Insertion+32CCCTCCCCTGGAGAGGTTTTAATGGCCCAGCC-------920.054.54Deletion+17CAGGTCTGGCCTCCCCTGGAGAGGTTTTAATGGCCCAGCCTCACAcCCC820.054.05Insertion01CCTCCCCTGGAGAGGTTTTAATGGCCCAGCCTCACACtgt610.043.01Insertion+338ttgacctggagccactctctgcaccccgctgaccCCCCCTCCCC---------------------------------500.032.47Deletion-1044-----------TCCCCTGGAGAGGTTTTAATGGCCCAGCCTCACAacta480.032.37Insertion+329ggtgtattataagaatcttataaacCCCGGCCTCCCCTGGAGAGGTTTTAATGGCCCAGCCTCACAccag390.021.92Insertion+343ctttcgttcgcaactcgagtggaagattggacttgcctgCCCCCTCCCCTGGAGAGGTTTTAATGGCCCAGCCTC-------360.021.78Deletion-1218-----------CCCTGGAGAGGTTTTAATGTCCCAGCCTCACAgcac340.021.68Insertion+346tgttcacgtggctgatcatacactgatcacgtgattgatcatCCCCCTCCCCTGGAGAGGTTTTAATGGCCCA------------270.021.33Deletion-2334----------------------TCTGGAGAGGTTTTAATGGCCCAGCCTCACAgatg240.011.18Insertion+355cgacgctgcgcgtcttatactcccacatatgccagattcagcaacggatacCCCCCTCCCCTGGAGAGGTTTTAATGGCCCagcctcacaggta230.011.14Insertion+381gctggactatgcatgtgatggctggtgctcaagcagccatcttgccctaagaagtgagagccaggagccaaggatagCCCCCTCCC TABLE 6.4Specific insertions and deletions for Guide 1166 using RGN APG005586%% ofINDELGuide SGN001166 (SEQ ID NO: 556)#ReadsReadsINDELSTypeLocationSizeCGCGCGGACCACGGCTCCTCCGAAGCGAGAACA19714889.52GCCCAGAAGCGCGCGGACCACGGCTCCTCCGA-----GAACA38291.7416.59Deletion50GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAAG----AACA19850.98.6Deletion4-1GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAA-------CA13000.595.63Deletion7-3GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAA-CGAGAACA12230.565.3Deletion13GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAAG-AGAACA12210.555.29Deletion21GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAA--------9360.434.06Deletion117-113-------------------------------------------------------------------------------------------------------------CCGCGCGGACCACGGCTCCTCCGAAGC------8450.383.66Deletion9-7---CCAGAAGCGCGCGGACCACGGCTCCTCC-----------7150.323.1Deletion64-57-----------------------------------------------------TCGCGCGGACCACGGCTCCTCCGAAG-------6150.282.66Deletion140-137---------------------------------------------------------------------GCGCGCGGACCACGG------------------6100.282.64Deletion132-118----------------------------------------------------------------------------------TCGCGCGGACCACGGCTCCTCC----CGAGAACA5510.252.39Deletion43GCCCAGAAGCGCGCGGACCACGGCTCCTCCGA-CGAGAACA4970.232.15Deletion23GCCCAGAAGGCGCGGACcACGGCTCCTCCGAAGTCGAGAACA4380.21.9Insertion13GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAAG-GAGAACA3810.171.65Deletion12GCCCAGAAGG-------------------gaGGCgg------3340.151.45Deletion49-13------------------------GCGCGCGGACCACGGCTCCTCCGAA---------3280.151.42Deletion27-23------------------ACGCGGACCACGGCTCCTCCGAAGGagGAGAACA3260.151.41Insertion22GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAAGC-------3210.151.39Deletion11-9----AGAAGCGCGCGGACCACGGCTCCTCCGAAGAG------3010.141.3Deletion18-17------------GCGCGCGGACCACGGCTCCTCCGAAG--------2900.131.26Deletion14-11------AAGCGCGCGGACCACGGCTCC--------GAGAACA2660.121.15Deletion82GCCCAGAAGCGCGCGGACCACGGCTCCTC------GAGAACA2650.121.15Deletion62GCCCAGAAGCGCGCGGACCACGGCTC---------GAGAACA2320.111.01Deletion92GCCCAGAAGGCGCGGACCACGGCTCCTCCGAAGcCGAGAACA2170.10.94Insertion12GCCCAGAAGCGCGCGGACCACGGCTCC-------CGAGAACA1990.090.86Deletion73GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAAG--------1920.090.83Deletion17-14---------TCGCGCGGACCACGGCTCCTCC------------1770.080.77Deletion15-8---CAGAAGCGCGCGGACCACGGC-----------GAGAACA1580.070.68Deletion112GCCCAGAAGCGCGCGGACCACGGCTCCTCC------------1490.070.65Deletion97-90-------------------------------------------------------------------------------------TCGCGCGGACCACGGC----------CGAGAACA1470.070.64Deletion103GCCCAGAAGCGCGCGGACCACGGCTCC---------------1310.060.57Deletion18-8---CAGAAGCGCGCGGACCACGGCTCCTCCGAAGC-------1270.060.55Deletion10-8---CAGAAGCGCGCGGACCACGGCTCCTCCGAAG--------1240.060.54Deletion53-50---------------------------------------------GCGCGCGGACCACGGCCCCTCCGA-----GAACA1130.050.49Deletion50GCCCAGAAGCGCGCGGACCACGGCTCCTCC------------1120.050.49Deletion16-9----AGAAGCGCGCGGACCACGGCTCC--------------A1040.050.45Deletion14-4GCCCAGAAGCGCGCGGA--------------------GAACA1020.050.44Deletion200GCCCAGAAGCGCGCGGACCACGGC------------------1010.050.44Deletion20-7--CCAGAAGCGCGCG---------------------AGAACA980.040.42Deletion211GCCCAGAAGCGCGCGGACCACGGCTCCTCCG---CGAGAACA970.040.42Deletion33GCCCAGAAGCGCGCGGACCACGGCTCCTCCG-----------940.040.41Deletion78-72-------------------------------------------------------------------GCGCGCGGACCACGGCTCCTCCGAAG---GAACA910.040.39Deletion30GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAAG--------900.040.39Deletion12-9----AGAAGCGCGC----------------------------800.040.35Deletion30-7--CCAGAAGGCGCGGACCACGGCTCCTCCGAAGcCCAGAACA740.030.32Insertion11GCCCAGAAGC--------------------------------730.030.32Deletion56-9------------------------AGAAGCGCGCGGACCACGGCTCCTCCGAAG--------670.030.29Deletion47-44---------------------------------------ACGCGCGGACCACGGCTCCTCCGAA-CGAGAACA660.030.29Deletion13GCCCAGACGCGCGCGGACCACGGCTC----------------630.030.27Deletion20-9----AGAAGCGCGCGGACC----------------------630.030.27Deletion26-8----CAGAAGCGCGCGGACCACGGCTCCTCCG-----------620.030.27Deletion179-173------------------------------------------------------------------------------------------------------------------------------------------------------------------------TC--------------------------------600.030.26Deletion51-9-------------------AGAAGCGCGCGGACCACGGCTCCTCC-----------A590.030.26Deletion11-4GCCCAGAAGCGCGCGGACCAC---------------------560.030.24Deletion25-9----AGAAGCGCGCGGAC-----------------GAGAACA550.020.24Deletion172GCCCAGAAGCGCGCGGACCACGGCTCCTCT----CGAGAACA540.020.23Deletion43GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAAG--------540.020.23Deletion8-5GCCCAGAAGG--------------------------------530.020.23Deletion391-------AGAACAGCCCAGAAGCGCGCGGACCA----------------------520.020.23Deletion27-10-----GAAGCGCGCGGACCACGGCTCC---------------520.020.23Deletion19-9----AGAAGCGCGCGGACCACGGCTCCTCCG----GAGAACA500.020.22Deletion42GCCCAGAAGCGCGCGGACCACGGCTCC---------------480.020.21Deletion16-6-CCCAGAAGCGCGCGGACCACGGCTCCTCCGA----------480.020.21Deletion15-10-----GAAGCGCGCGGA----------------------ACA470.020.2Deletion222GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAAG-----ACA460.020.2Deletion5-2GCCCAGAAGCGCGCGGACCACGGCTCCT-----GCGAGAACA450.020.19Deletion54GCCCAGAAGCGCGCGGACC---------------CGAGAACA450.020.19Deletion153GCCCAGAAGCGCG-----------------------AGAACA440.020.19Deletion231GCCCAGAAGCGCGCGGACCA-----------------GAACA440.020.19Deletion170GCCCAGAAGCG------------------------------A440.020.19Deletion30-4GCCCAGAAGCGCGCGGACCACGGCTCCTCCGA-----GAACA440.020.19Deletion50GCCCAGACGCGCGCGGACCACGGCTCCTCGGA-----GAACA420.020.18Deletion50GCCCAGAAGCGCGCGGACCACGGCTCCTCCTC-CGAGAACA420.020.18Deletion23GCCCAGAAGCGCGCGG-----------------GGAAGAACA410.020.18Deletion174GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAAG--------390.020.17Deletion13-10-----GAAGCGCGCGGACC----------------GAGAACA390.020.17Deletion162GCCCAGAAGCGCGCGGACCACGGCTCCTCCGAAG--------390.020.17Deletion17-16-----GA----GGCGGACCACGGCTCCTCCGAAGaagCGAGAACA380.020.16Insertion33GCCCAGAAGGGACCACGGCTCCTCCGAAGGAGGagGAGAACA380.020.16Insertion52GCCCAGAAGCGCGCGGACCAC---------------------380.020.16Deletion27-11------AAGCGCGCGGACCACGGCTCCTCCGAAGC------A380.020.16Deletion6-4GCCCAGAAGT--------------------------------370.020.16Deletion47-15---------------TCGCGCGGACCACGGCTGTTCTGA-----GAACA370.020.16Deletion50GCCCAGAAGC-------------------------------360.020.16Deletion313CGAGAACAGCCCAGAAGCGCGCGGACCACGGC-----------------360.020.16Deletion23-10------GAAGCGCGCGGA-------------------------350.020.15Deletion32-12-------AGCGCGCGGACCACGG---------------AACA340.020.15Deletion15-1GCCCAGAAGCGCGCGGACCACGGCTC------------AACA340.020.15Deletion12-1GCCCAGAAGCGCGCGGACCACGGCCCCTCC----CGAGAACA330.010.14Deletion43GCCCAGAAGG--------------------------------330.010.14Deletion57-21-------------------------ACGCGCGGACCACGGCTCCTCCGA-GCGAGAACA320.010.14Deletion14GCCCAGAAGCGCGCGGACCACGTT----------CGAGAACA320.010.14Deletion103GCCCAGAAGCGCGA----------------------AGAACA310.010.13Deletion221GCCCAGAAGCGCGCGGACCACGG-------------------310.010.13Deletion25-11------AAGCGCG-------------------------AACA300.010.13Deletion36-20GCCC-----------GCGCGCGGACCACGGCTCCTCCGAAG----AACA300.010.13Deletion4-1GCCCAGCAGCGCGCGGACCACGGCTCCT---------GAACA300.010.13Deletion90GCCCAGAAGCGCGGACCACGGCTCCTCCGAAGcGCGAGAACA300.010.13Insertion21GCCCAGAAGCG-------------------------290.010.13Deletion251AGAACAGCCCAGAAGCGCGCGGACCACGGCCCCTCCGAAG----290.010.13Deletion4-1AACAGCCCAGAAGCGCGCGGACCAC--------------------290.010.13Deletion20-4AGCCCAGAAGCGCGCGGACCACGG----------------290.010.13Deletion16-2ACAGCCCAGAAGCGCGCGGACCACGGCTCCTC------------280.010.12Deletion17-9-----AGAAG The robustness of several nucleases was tested by assaying their ability to edit at many different target sites across several genes. Over 40 targets were tested in the expanded guide panel. All proteins tested showed robust editing at the diverse sites. Results are shown in Table 7. TABLE 7Robustness of select RNA-guided nucleasesOverallSGNEditingRGNGeneNumberRate (%)APG01868TRASGN00168442.62SGN0016851.43SGN0016868.19SGN0016878.83SGN0016880.21SGN0016890SGN0016900SGN00169120.86EMX1SGN0016970.93SGN0016980SGN00169918.84SGN00170021.8SGN00170139.94SGN0017022.16SGN0017030.79SGN0017043.49SGN00170514.49SGN0017061.04SGN0017072.51SGN0017081.75SGN0017093.62SGN0017100SGN00171113.39HAO1SGN00171322.29SGN0017140.67SGN0017150.23SGN0017167.1SGN0017170.68SGN0017180.21SGN0017190SGN0017210.17SGN0017221.36SGN00172324.16SGN0017240.2LDHASGN0017254.92SGN0017260SGN0017271.32SGN0017280.28SGN0017291.94SGN0017305.2SGN00173111.28SGN0017326.04SGN0017331.12SGN0017342.11SGN0017357APG05586TRASGN00137130.52SGN00137226.01SGN00137326.67SGN00137422.94SGN00137521.17SGN0013760SGN0013770SGN00137830.04SGN00137939.13SGN0013804.93SGN00138117.14SGN0013825.6B2MSGN00138336.23SGN00138444.28SGN0013856.33SGN0013865.55SGN00138748.71SGN00138831.78SGN00138941SGN00139048.15SGN00139146.19SGN00139237.22SGN00139331.62SGN00139429.72LDHASGN00139632.88SGN00139741.45SGN00139943.28SGN0014002.52SGN00140137.52SGN0014020.37SGN00140353.13SGN00140444.06SGN0014051.46HAO1SGN00140621.98SGN0014079.13SGN00140825.06SGN00140943.81SGN00141037.6SGN00141140.75SGN00141218.2SGN00141328.44SGN00141429.39SGN0014150.39SGN00141643.59APG01604B2MSGN00159223.77SGN00159322.7SGN00159423.6SGN00159533.23SGN00159620.88SGN0015971.26SGN00159826.7SGN0015999.41SGN00160028.88SGN0016017.39SGN00160227.8SGN0016036.93HAO1SGN0016160SGN0016172.55SGN0016180.6SGN0016196.29SGN0016207.92SGN00162113.03SGN0016225.32SGN00162318.58SGN00162420.03SGN0016251.65SGN0016260.18SGN0016270.73LDHASGN0016403.75SGN0016411.13SGN00164214.2SGN00164312.16SGN0016444.9SGN0016454.78SGN0016460.74SGN0016472.89SGN0016480SGN0016490.15SGN0016504.4SGN0016517.26TRASGN0016640.44SGN0016653.4SGN00166617.24SGN0016672.33SGN0016680.3SGN00166913.68SGN0016700.54SGN0016715.09SGN0016720.22SGN00167312.22SGN00167417.33SGN00167511.69APG09298B2MSGN00138322.25SGN0013849.5SGN0013852.37SGN0013860.4SGN00138738.03SGN00138817.66SGN00138942.03SGN00139042.88SGN00139116.31SGN00139216.5SGN0013936.27SGN00139417.15HAO1SGN0014063.46SGN0014071.87SGN00140810.71SGN00140918.79SGN00141015.09SGN00141114.81SGN0014120.63SGN0014135.92SGN0014145.24SGN0014150.03SGN00141615.41LDHASGN0013953.15SGN00139628.53SGN00139711.69SGN00139923.92SGN0014000.89SGN00140133.97SGN0014020SGN00140325.62SGN00140416.96SGN0014050.11TRASGN00116244.26SGN00116342.06SGN0011648.1SGN00137121.05SGN0013723.95SGN0013737.8SGN0013749.04SGN0013752.94SGN0013760SGN0013770SGN00137822.14SGN00137927.43SGN0013800.16SGN00138114.18SGN0013822.78 Example 5: Protein Engineering of APG05586 Conserved and variable residues of APG05586 were identified by comparison of APG05586 to closely related homologs, including APG08770 (SEQ ID NO: 70), APG09882 (set forth as SEQ ID NO: 568 and described in International Appl. No. PCT/US2020/045759, which is incorporated by reference in its entirety), and APG01658 (set forth as SEQ ID NO: 569 and described in International Appl. No. PCT/US2020/045759). Several variants were generated containing mutations at the nonconserved locations to identify critical residues in the APG05586 protein. In total, 132 residues were altered in ten APG05586 engineered variant RGNs (SEQ ID NOs: 570-579). These ten APG05586 variants and wild type APG05586 were then assayed for activity in mammalian cells. Using the guide RNA backbone for APG05586 (SEQ ID NO: 66), the RGNs were tested for activity at six targeted genomic locations (Table 8) following the methods described in Example 4. Mammalian codon-optimized coding sequences for each variant are provided as SEQ ID NOs: 580-589. 5′ and 3′ primer nucleotide sequences useful for detection of gene editing activity are also provided in Table 8. Editing rates are shown in Table 9. TABLE 8Target sequencesTargetSequencesgRNA5′3′(SEQ(SEQPrimer forPrimer forGuide IDGeneID NO.)ID NO.)amplificationamplificationSGN001159EMX1590591592593SGN001162TRA594595596597SGN001163TRA598599596597SGN001164TRA600601596597SGN001165VEGFA602603604605SGN001166VEGFA606607604605 TABLE 9Editing rates for APG05586 and variants thereofRGNSEQRGN IDID NO.SGN001159SGN001162SGN001163SGN001164SGN001165SGN001166APG0929857033.40%44.26%42.06%8.10%33.94%46.19%APG062515710%43.59%48.82%5.90%25.07%35.08%APG030665720%0%0%0%0%0%APG0156057315.12%16.38%17.59%2.52%44.99%20.68%APG027775740%0.08%0%0%0%0%APG0576157524.25%21.95%21.86%10.45%23.72%20.17%APG024795765.61%9.25%11.00%1.45%4.53%6.34%APG0838557731.16%39.95%35.42%2.37%31.41%26.65%APG0921757829.43%41.12%44.71%4.79%38.16%31.76%APG0665757936.81%44.20%41.54%4.44%16.29%21.86%APG055866340.13%46.12%43.77%8.00%96.76%77.38% The relative activity of the variants suggests which locations in the protein tolerate mutations. Table 10 shown below is a summary of the activity of the variant RGN with the number of mutations introduced compared to APG005586. “−” is no activity; “+” is 1-15% editing in at least 4 out of 6 targets; “++” is 10-25% editing in at least 4 out of 6 targets; and “+++” is 20-50% editing in at least 4 out of 6 targets. TABLE 10Summary of protein engineering changes and editing ratesActivityNumber ofProteinOutcomemutationsAPG09298+++10APG06251+++12APG03066−87APG01560++10APG02777−37APG05761++10APG02479+12APG08385+++12APG09217+++14APG06657+++14APG05586+++0 Variants APG03066 and APG02777 contained too many mutations to identify specific residues important for function, however, the low activity of these variants indicates that extensive changes to the bridge helix and recognition domain are not tolerated in this protein. All other variants contained 14 or fewer mutations, which enabled identification of specific residues important for activity. Based on these results, several residues were identified as important for function of the protein. I305L, V328A, L366I, T368S, and V405A mutations resulted in decreased activity in the assayed variants. All of these mutations are predicted to be in the recognition domain of the protein. The decrease in activity for APG01560 is a result of multiple changes that are not localized to a specific region within the protein. Example 6: Identification of Disease Targets A database of clinical variants was obtained from NCBI ClinVar database, which is available through the world wide web at the NCBI ClinVar website. Pathogenic Single Nucleotide Polymorphisms (SNPs) were identified from this list. Using the genomic locus information, CRIgvSPR targets in the region overlapping and surrounding each SNP were identified. A selection of SNPs that can be corrected using base editing in combination with the RGNs of the invention to target the causal mutation (“Casl Mut.”) is listed in Table 11. In Table 11, only one alias of each disease is listed. The “RS #” corresponds to the RS accession number through the SNP database at the NCBI website. The AlleleID corresponds to a causal allele accession number, and the Chromosome Accession number also provides accession reference information found through the NCBI website. Table 11 also provides genomic target sequence information suitable for the RGN listed for each disease. The target sequence information also provides protospacer sequence for the production of the necessary sgRNA for the corresponding RGN of the invention. TABLE 11Disease Targets for RGNsTarget(SEQCaslAlleleGeneIDDiseaseRS#RGNMut.IDChromosome AccessionSymbolNO.)Stargardt disease 11800553APG06622C > T22927NC_000001.10, NC_000001.11ABCA4261Stargardt disease 11800728APG06622A > G98777NC_000001.10, NC_000001.11ABCA4262Stargardt disease 161751374APG06622G > A22933NC_000001.10, NC_000001.11ABCA4263Stargardt disease 161750641APG02874,G > A105317NC_000001.10, NC_000001.11ABCA4264APG03031,APG09208Stargardt disease 11800553APG02787G > A22927NC_000001.10, NC_000001.11ABCA4265Stargardt disease 11800553APG06007G > A22927NC_000001.10, NC_000001.11ABCA4266Stargardt disease 11800553APG03850G > A22927NC_000001.10, NC_000001.11ABCA4267Stargardt disease 11800553APG05586G > A22927NC_000001.10, NC_000001.11ABCA4268Stargardt disease 11800553APG08167,C > T22927NC_000001.10, NC_000001.11ABCA4269APG01604Familial hyperinsulinism1.51E+08APG02874,G > A24127NC_000011.9, NC_000011.10ABCC8270APG03031,APG09208Very long chain acyl-CoA1.14E+08APG06622T > C33877NC_000017.10, NC_000017.11ACADVL271dehydrogenase deficiencyVery long chain acyl-CoA1.14E+08APG06248T > C33877NC_000017.10, NC_000017.11ACADVL272dehydrogenase deficiencyVery long chain acyl-CoA3.7E+08APG02874,G > A98197NC_000017.10, NC_000017.11ACADVL273dehydrogenase deficiencyAPG03031,APG09208Baraitser-Winter syndrome 12.82E+08APG02874,G > A38553NC_000007.13, NC_000007.14ACTB274APG03031,APG09208Severe immunodeficiency1.22E+08APG02874,G > A16996NC_000020.10, NC_000020.11ADA275due to ADA deficiencyAPG03031,APG09208Severe immunodeficiency1.22E+08APG02874,T > C17004NC_000020.10, NC_000020.11ADA276due to ADA deficiencyAPG03031,APG09208Primary hyperoxaluria1.22E+08APG06622G > A38436NC_000002.11, NC_000002.12AGXT277Congenital disorder of28939378APG06622C > T19763NC_000016.9, NC_000016.10ALG1278glycosylationHypophosphatasia1.22E+08APG06622G > A28709NC_000001.10, NC_000001.11ALPL279Hypophosphatasia1.22E+08APG02874,G > A28709NC_000001.10, NC_000001.11ALPL280APG03031,APG09208Colorectal cancer1.38E+08APG06622C > T15837NC_000005.9, NC_000005.10APC281Metachromatic80338815APG06622C > T18090NC_000022.10, NC_000022.11ARSA282leukodystrophyWilson disease1.94E+08APG02874,G > A44393NC_000013.10, NC_000013.11ATP7B283APG03031,APG09208Cardio-facio-cutaneous1.8E+08APG06622T > C29012NC_000007.13, NC_000007.14BRAF284syndromeCardio-facio-cutaneous1.8E+08APG06248T > C29012NC_000007.13, NC_000007.14BRAF285syndromeBreast and/or ovarian41293455APG06622G > A32714NC_000017.10, NC_000017.11BRCA1286cancerBreast and/or ovarian41293465APG06622G > A70268NC_000017.10, NC_000017.11BRCA1287cancerBreast and colorectal cancer55770810APG06622G > A70063NC_000017.10, NC_000017.11BRCA1288Breast and/or ovarian62625307APG06622G > A69596NC_000017.10, NC_000017.11BRCA1289cancerBreast and/or ovarian62625308APG06622G > A32710NC_000017.10, NC_000017.11BRCA1290cancerBreast and/or ovarian80356962APG06622C > T70247NC_000017.10, NC_000017.11BRCA1291cancerBreast and/or ovarian41293455APG02787C > T32714NC_000017.10, NC_000017.11BRCA1292cancerBreast and/or ovarian41293455APG06007C > T32714NC_000017.10, NC_000017.11BRCA1293cancerBreast and/or ovarian41293455APG03850C > T32714NC_000017.10, NC_000017.11BRCA1294cancerBreast and/or ovarian41293455APG05586C > T32714NC_000017.10, NC_000017.11BRCA1295cancerBreast and/or ovarian41293455APG08167,G > A32714NC_000017.10, NC_000017.11BRCA1296cancerAPG01604Breast and/or ovarian80356962APG08167,C > T70247NC_000017.10, NC_000017.11BRCA1297cancerAPG01604Breast and/or ovarian80358163APG08167,T > C46006NC_000017.10, NC_000017.11BRCA1298cancerAPG01604Breast and/or ovarian45580035APG06622C > T67431NC_000013.10, NC_000013.11BRCA2299cancerBreast and/or ovarian80359212APG06622C > T67494NC_000013.10, NC_000013.11BRCA2300cancerBreast and/or ovarian80359003APG02874,G > A67069NC_000013.10, NC_000013.11BRCA2301cancerAPG03031,APG09208Breast and/or ovarian80359004APG02874,G > A46672NC_000013.10, NC_000013.11BRCA2302cancerAPG03031,APG09208Breast and/or ovarian80359212APG02787C > T67494NC_000013.10, NC_000013.11BRCA2303cancerBreast and/or ovarian80359212APG06007C > T67494NC_000013.10, NC_000013.11BRCA2304cancerBreast and/or ovarian80359212APG03850C > T67494NC_000013.10, NC_000013.11BRCA2305cancerBreast and/or ovarian80359212APG05586C > T67494NC_000013.10, NC_000013.11BRCA2306cancerBreast and/or ovarian80359212APG08167,C > T67494NC_000013.10, NC_000013.11BRCA2307cancerAPG01604Breast and/or ovarian80359071APG08167,G > A67203NC_000013.10, NC_000013.11BRCA2308cancerAPG01604CAPN3-Related Disorders1.21E+08APG02874,G > A32661NC_000015.9, NC_000015.10CAPN3;309APG03031,POMT1APG09208CBS-deficiency5742905APG06622A > G15159NC_000021.8, NC_000021.9CBS310CBS-deficiency5742905APG02874,T > C15159NC_000021.8, NC_000021.9CBS311APG03031,APG09208CBS-deficiency1.22E+08APG02874,G > A15156NC_000021.8, NC_000021.9CBS312APG03031,APG09208Cystic fibrosis77010898APG06622G > A22168NC_000007.13, NC_000007.14CFTR313Cystic fibrosis75096551APG02874,G > A33858NC_000007.13, NC_000007.14CFTR314APG03031,APG09208Cystic fibrosis75527207APG02787G > A22159NC_000007.13, NC_000007.14CFTR315Cystic fibrosis75527207APG06007G > A22159NC_000007.13, NC_000007.14CFTR316Cystic fibrosis75527207APG03850G > A22159NC_000007.13, NC_000007.14CFTR317Cystic fibrosis75527207APG05586G > A22159NC_000007.13, NC_000007.14CFTR318Cystic fibrosis78655421APG02787G > A22148NC_000007.13, NC_000007.14CFTR319Cystic fibrosis78655421APG06007G > A22148NC_000007.13, NC_000007.14CFTR320Cystic fibrosis78655421APG03850G > A22148NC_000007.13, NC_000007.14CFTR321Cystic fibrosis78655421APG05586G > A22148NC_000007.13, NC_000007.14CFTR322Cystic fibrosis75527207APG08167,G > A22159NC_000007.13, NC_000007.14CFTR323APG01604Congenital myotonia80356701APG02874,T > C33902NC_000007.13, NC_000007.14CLCN1324APG03031,APG09208Osteogenesis imperfecta72645321APG02874,G > A414022NC_000017.10, NC_000017.11COL1A1325type 1APG03031,APG09208Osteogenesis imperfecta72645321APG02874,G > A414022NC_000017.10, NC_000017.11COL1A1326type 1APG03031,APG09208Alport syndrome 1, X-1.05E+08APG02874,G > A35796NC_000023.10, NC_000023.11COL4A5327linked recessiveAPG03031,APG09208Carnitine74315294APG06622C > T23992NC_000001.10, NC_000001.11CPT2328palmitoyltransferase IIdeficiencyCarnitine74315294APG06248C > T23992NC_000001.10, NC_000001.11CPT2329palmitoyltransferase IIdeficiencyCarnitine74315294APG06007C > T23992NC_000001.10, NC_000001.11CPT2330palmitoyltransferase IIdeficiencyCarnitine74315294APG03850C > T23992NC_000001.10, NC_000001.11CPT2331palmitoyltransferase IIdeficiencyCarnitine74315294APG05586C > T23992NC_000001.10, NC_000001.11CPT2332palmitoyltransferase IIdeficiencyCarnitine74315294APG08167,C > T23992NC_000001.10, NC_000001.11CPT2333palmitoyltransferase IIAPG01604deficiencyDopamine beta hydroxylase74853476APG02874,T > C16789NC_000009.11, NC_000009.12DBH334deficiencyAPG03031,APG09208Congenital microcephaly11555217APG06622C > T34125NC_000011.9, NC_000011.10DHCR7335Smith-Lemli-Opitz80338853APG06622G > A21822NC_000011.9, NC_000011.10DHCR7336syndromeSmith-Lemli-Opitz80338857APG02874,G > A34128NC_000011.9, NC_000011.10DHCR7337syndromeAPG03031,APG09208Smith-Lemli-Opitz11555217APG06007G > A34125NC_000011.9, NC_000011.10DHCR7338syndromeSmith-Lemli-Opitz11555217APG03850G > A34125NC_000011.9, NC_000011.10DHCR7339syndromeSmith-Lemli-Opitz11555217APG05586G > A34125NC_000011.9, NC_000011.10DHCR7340syndromeFamilial dysautonomia1.11E+08APG06248A > G21124NC_000009.11, NC_000009.12ELP1341Hypertyrosinemia80338901APG06622G > A26909NC_000015.9, NC_000015.10FAH342Hypertyrosinemia80338901APG06248G > A26909NC_000015.9, NC_000015.10FAH343Fanconi anemia1.05E+08APG06622G > A27086NC_000009.11, NC_000009.12FANCC344Marfan Syndrome3.98E+08APG06622C > T51454NC_000015.9, NC_000015.10FBN1345Marfan Syndrome7.28E+08APG06622A > G175979NC_000015.9, NC_000015.10FBN1346Marfan Syndrome1.38E+08APG02874,G > A31496NC_000015.9, NC_000015.10FBN1347APG03031,APG09208Marfan Syndrome1.38E+08APG02874,G > A31496NC_000015.9, NC_000015.10FBN1348APG03031,APG09208Marfan Syndrome3.88E+08APG02874,G > A38652NC_000015.9, NC_000015.10FBN1349APG03031,APG09208FGFR3-Related Disorders1.22E+08APG06622C > T31371NC_000004.11, NC_000004.12FGFR3350Glycogen storage disease1801175APG08167,C > T27037NC_000017.10, NC_000017.11G6PC351type 1AAPG01604Glycogen storage disease,3.98E+08APG02874,G > A415590NC_000017.10, NC_000017.11GAA352type IIAPG03031,APG09208Deficiency of UDPglucose-75391579APG06622A > G18653NC_000009.11, NC_000009.12GALT353hexose-1-phosphateuridylyltransferaseGlutaric aciduria, type 11.21E+08APG02874,G > A17127NC_000019.9, NC_000019.10GCDH354APG03031,APG09208Deafness, X-linked76434661APG06622C > T53916NC_000013.10, NC_000013.11GJB2355Deafness, X-linked80338945APG06622A > G32055NC_000013.10, NC_000013.11GJB2356Deafness, X-linked80338945APG02874,T > C32055NC_000013.10, NC_000013.11GJB2357APG03031,APG09208Deafness, X-linked80338945APG02874,T > C32055NC_000013.10, NC_000013.11GJB2358APG03031,APG09208Deafness, X-linked1.11E+08APG02874,G > A53902NC_000013.10, NC_000013.11GJB2359APG03031,APG09208Deafness, X-linked1.05E+08APG06007G > A32041NC_000013.10, NC_000013.11GJB2360Deafness, X-linked1.05E+08APG03850G > A32041NC_000013.10, NC_000013.11GJB2361Deafness, X-linked1.05E+08APG08167,C > T32041NC_000013.10, NC_000013.11GJB2362APG01604Deafness, X-linked80338945APG08167,A > G32055NC_000013.10, NC_000013.11GJB2363APG01604Inclusion body myopathy 228937594APG06622A > G21064NC_000009.11, NC_000009.12GNE364Inclusion body myopathy 228937594APG06248A > G21064NC_000009.11, NC_000009.12GNE365beta Thalassemia33930165APG02874,G > A30165NC_000011.9, NC_000011.10HBB366APG03031,APG09208Mucopolysaccharidosis type1.22E+08APG06622G > A26947NC_000004.11, NC_000004.12IDUA367IMucopolysaccharidosis type1.22E+08APG06622C > T26948NC_000004.11, NC_000004.12IDUA368IMucopolysaccharidosis type1.22E+08APG02874,G > A26947NC_000004.11, NC_000004.12IDUA369IAPG03031,APG09208Congenital long QT1.99E+08APG02874,G > A67758NC_000011.9, NC_000011.10KCNQ1370syndromeAPG03031,APG09208Congenital long QT1.99E+08APG02874,T > C67776NC_000011.9, NC_000011.10KCNQ1371syndromeAPG03031,APG09208Familial28942080APG06622G > A18735NC_000019.9, NC_000019.10LDLR372hypercholesterolemiaFamilial1.22E+08APG06622C > T18725NC_000019.9, NC_000019.10LDLR373hypercholesterolemiaFamilial1.38E+08APG06622G > A171217NC_000019.9, NC_000019.10LDLR374hypercholesterolemiaFamilial7.46E+08APG06622C > T228192NC_000019.9, NC_000019.10LDLR375hypercholesterolemiaFamilial7.66E+08APG06622G > A228162NC_000019.10, NC_000019.9LDLR376hypercholesterolemiaFamilial7.69E+08APG06622G > A228176NC_000019.10, NC_000019.9LDLR377hypercholesterolemiaFamilial7.46E+08APG06248C > T228192NC_000019.9, NC_000019.10LDLR378hypercholesterolemiaFamilial7.69E+08APG06248G > A228176NC_000019.10, NC_000019.9LDLR379hypercholesterolemiaFamilial3.76E+08APG02874,G > A198012NC_000019.10, NC_000019.9LDLR380hypercholesterolemiaAPG03031,APG09208Familial7.69E+08APG02874,G > A228176NC_000019.10, NC_000019.9LDLR381hypercholesterolemiaAPG03031,APG09208Familial7.75E+08APG02874,T > C228197NC_000019.9, NC_000019.10LDLR382hypercholesterolemiaAPG03031,APG09208Familial7.76E+08APG02874,G > A246116NC_000019.9, NC_000019.10LDLR383hypercholesterolemiaAPG03031,APG09208Familial8.79E+08APG02874,T > C246008NC_000019.10, NC_000019.9LDLR384hypercholesterolemiaAPG03031,APG09208Familial1.38E+08APG02787G > A171217NC_000019.9, NC_000019.10LDLR385hypercholesterolemiaFamilial1.38E+08APG06007G > A171217NC_000019.9, NC_000019.10LDLR386hypercholesterolemiaFamilial1.38E+08APG03850G > A171217NC_000019.9, NC_000019.10LDLR387hypercholesterolemiaFamilial1.38E+08APG05586G > A171217NC_000019.9, NC_000019.10LDLR388hypercholesterolemiaFamilial1.38E+08APG08167,G > A171217NC_000019.9, NC_000019.10LDLR389hypercholesterolemiaAPG01604Familial1.22E+08APG08167,C > T18725NC_000019.9, NC_000019.10LDLR390hypercholesterolemiaAPG01604Cardio-facio-cutaneous1.22E+08APG06622A > G28390NC_000015.9, NC_000015.10MAP2K1391syndromeMECP2-Related Disorders28934906APG06622G > A26850NC_000023.10, NC_000023.11MECP2392MECP2-Related Disorders28935468APG06622G > A26863NC_000023.10, NC_000023.11MECP2393MECP2-Related Disorders61749721APG06622G > A26868NC_000023.10, NC_000023.11MECP2394MECP2-Related Disorders61750240APG06622G > A26854NC_000023.10, NC_000023.11MECP2395MECP2-Related Disorders28935468APG06248G > A26863NC_000023.10, NC_000023.11MECP2396MECP2-Related Disorders28934906APG06007C > T26850NC_000023.10, NC_000023.11MECP2397MECP2-Related Disorders28934906APG03850C > T26850NC_000023.10, NC_000023.11MECP2398MECP2-Related Disorders61750240APG02787C > T26854NC_000023.10, NC_000023.11MECP2399MECP2-Related Disorders61750240APG06007C > T26854NC_000023.10, NC_000023.11MECP2400MECP2-Related Disorders61750240APG03850C > T26854NC_000023.10, NC_000023.11MECP2401MECP2-Related Disorders61750240APG05586C > T26854NC_000023.10, NC_000023.11MECP2402Angelman syndrome61751362APG02787C > T26858NC_000023.10, NC_000023.11MECP2403Angelman syndrome61751362APG06007C > T26858NC_000023.10, NC_000023.11MECP2404Angelman syndrome61751362APG03850C > T26858NC_000023.10, NC_000023.11MECP2405Angelman syndrome61751362APG05586C > T26858NC_000023.10, NC_000023.11MECP2406Angelman syndrome28934906APG08167,G > A26850NC_000023.10, NC_000023.11MECP2407APG01604Familial Mediterranean28940579APG06622A > G17579NC_000016.9, NC_000016.10MEFV408feverFamilial Mediterranean61752717APG06622T > C17577NC_000016.9, NC_000016.10MEFV409feverFamilial Mediterranean1.05E+08APG06622C > T17588NC_000016.9, NC_000016.10MEFV410feverFamilial Mediterranean61752717APG06248T > C17577NC_000016.9, NC_000016.10MEFV411feverFamilial Mediterranean1.05E+08APG06248C > T17588NC_000016.9, NC_000016.10MEFV412feverFamilial Mediterranean1.05E+08APG02874,G > A17588NC_000016.9, NC_000016.10MEFV413feverAPG03031,APG09208Familial Mediterranean28940579APG08167,A > G17579NC_000016.9, NC_000016.10MEFV414feverAPG01604Charcot-Marie-Tooth28940293APG02874,T > C17309NC_000001.10, NC_000001.11MFN2415disease, type 2APG03031,APG09208Hereditary nonpolyposis63751657APG02874,G > A95331NC_000003.11, NC_000003.12MLH1416colon cancerAPG03031,APG09208Hereditary nonpolyposis63751711APG02874,G > A95792NC_000003.11, NC_000003.12MLH1417colon cancerAPG03031,APG09208Methylmalonic acidemia1.22E+08APG06622C > T16462NC_000001.10, NC_000001.11MMACHC418Methylmalonic acidemia1.22E+08APG06248C > T16462NC_000001.10, NC_000001.11MMACHC419Methylmalonic acidemia1.22E+08APG02874,G > A16464NC_000001.10, NC_000001.11MMACHC420APG03031,APG09208Hereditary cancer-63750636APG06622C > T96378NC_000002.11, NC_000002.12MSH2421predisposing syndromeHereditary cancer-63749843APG06622C > T94826NC_000002.11, NC_000002.12MSH6422predisposing syndromeHereditary cancer-7.86E+08APG06622C > T181998NC_000002.12, NC_000002.11MSH6423predisposing syndromeHereditary cancer-63750741APG02874,T > C94663NC_000002.11, NC_000002.12MSH6424predisposing syndromeAPG03031,APG09208Hereditary cancer-63749843APG02787C > T94826NC_000002.11, NC_000002.12MSH6425predisposing syndromeHereditary cancer-63749843APG06007C > T94826NC_000002.11, NC_000002.12MSH6426predisposing syndromeHereditary cancer-63749843APG03850C > T94826NC_000002.11, NC_000002.12MSH6427predisposing syndromeHereditary cancer-63749843APG05586C > T94826NC_000002.11, NC_000002.12MSH6428predisposing syndromeHereditary cancer-63751017APG02787C > T94786NC_000002.11, NC_000002.12MSH6429predisposing syndromeHereditary cancer-63751017APG06007C > T94786NC_000002.11, NC_000002.12MSH6430predisposing syndromeHereditary cancer-63751017APG03850C > T94786NC_000002.11, NC_000002.12MSH6431predisposing syndromeHereditary cancer-63751017APG05586C > T94786NC_000002.11, NC_000002.12MSH6432predisposing syndromeHereditary cancer-63751017APG08167,C > T94786NC_000002.11, NC_000002.12MSH6433predisposing syndromeAPG01604MUTYH-associated34612342APG06622T > C20332NC_000001.10, NC_000001.11MUTYH434polyposisMUTYH-associated36053993APG06622C > T20333NC_000001.10, NC_000001.11MUTYH435polyposisMUTYH-associated36053993APG02787G > A20333NC_000001.10, NC_000001.11MUTYH436polyposisMUTYH-associated36053993APG06007G > A20333NC_000001.10, NC_000001.11MUTYH437polyposisMUTYH-associated36053993APG03850G > A20333NC_000001.10, NC_000001.11MUTYH438polyposisMUTYH-associated36053993APG05586G > A20333NC_000001.10, NC_000001.11MUTYH439polyposisMUTYH-associated36053993APG08167,C > T20333NC_000001.10, NC_000001.11MUTYH440polyposisAPG01604MUTYH-associated34612342APG08167,T > C20332NC_000001.10, NC_000001.11MUTYH441polyposisAPG01604Hyperimmunoglobulin D28934897APG06622G > A26968NC_000012.11, NC_000012.12MVK442with periodic feverMYBPC3-Related2E+08APG06622C > T174776NC_000011.9, NC_000011.10MYBPC3443DisordersMYBPC3-Related3.88E+08APG06622G > A45725NC_000011.9, NC_000011.10MYBPC3444DisordersMYBPC3-Related3.98E+08APG06622C > T51962NC_000011.9, NC_000011.10MYBPC3445DisordersMYBPC3-Related2E+08APG06248C > T174776NC_000011.9, NC_000011.10MYBPC3446DisordersMYBPC3-Related1.88E+08APG02874,T > C45267NC_000011.9, NC_000011.10MYBPC3447DisordersAPG03031,APG09208MYBPC3-Related2E+08APG02874,G > A174776NC_000011.9, NC_000011.10MYBPC3448DisordersAPG03031,APG09208MYBPC3-Related3.98E+08APG02874,G > A51820NC_000011.9, NC_000011.10MYBPC3449DisordersAPG03031,APG09208MYBPC3-Related3.98E+08APG02874,G > A51962NC_000011.9, NC_000011.10MYBPC3450DisordersAPG03031,APG09208Cardiomyopathy3218716APG06622C > T52071NC_000014.8, NC_000014.9MYH7451Cardiomyopathy36211715APG06622C > T29159NC_000014.8, NC_000014.9MYH7452Cardiomyopathy1.22E+08APG06622G > A29128NC_000014.8, NC_000014.9MYH7453Cardiomyopathy3.72E+08APG06622C > T52045NC_000014.8, NC_000014.9MYH7454Cardiomyopathy3.98E+08APG02874,T > C52276NC_000014.8, NC_000014.9MYH7455APG03031,APG09208Cardiomyopathy3.72E+08APG06007G > A52045NC_000014.8, NC_000014.9MYH7456Cardiomyopathy3.72E+08APG03850G > A52045NC_000014.8, NC_000014.9MYH7457Deafness, autosomal1.11E+08APG02874,G > A52388NC_000011.9, NC_000011.10MYO7A458recessiveAPG03031,APG09208Inborn genetic diseases80358259APG06622A > G18006NC_000018.9, NC_000018.10NPC1459Inborn genetic diseases80358259APG02874,T > C18006NC_000018.9, NC_000018.10NPC1460APG03031,APG09208Inborn genetic diseases1.2E+08APG02874,G > A18010NC_000018.9, NC_000018.10NPC1461APG03031,APG09208Phenylketonuria5030851APG06622G > A15628NC_000012.11, NC_000012.12PAH462Phenylketonuria5030858APG06622G > A15616NC_000012.11, NC_000012.12PAH463Hyperphenylalaninemia5030860APG06622T > C15632NC_000012.11, NC_000012.12PAH464Phenylketonuria62516101APG02874,G > A15658NC_000012.11, NC_000012.12PAH465APG03031,APG09208Hyperphenylalaninemia,62644499APG02874,G > A15656NC_000012.11, NC_000012.12PAH466non-pkuAPG03031,APG09208Phenylketonuria5030858APG02787C > T15616NC_000012.11, NC_000012.12PAH467Phenylketonuria5030858APG06007C > T15616NC_000012.11, NC_000012.12PAH468Phenylketonuria5030858APG03850C > T15616NC_000012.11, NC_000012.12PAH469Phenylketonuria5030858APG05586C > T15616NC_000012.11, NC_000012.12PAH470Hyperphenylalaninemia,5030860APG08167,T > C15632NC_000012.11, NC_000012.12PAH471non-pkuAPG01604Hyperphenylalaninemia,62642937APG08167,G > A15667NC_000012.11, NC_000012.12PAH472non-pkuAPG01604Familial cancer of breast1.8E+08APG06622G > A132139NC_000016.10, NC_000016.9PALB2473Familial cancer of breast1.8E+08APG02874,G > A132185NC_000016.10, NC_000016.9PALB2474APG03031,APG09208Peroxisome biogenesis61750420APG08167,C > T22555NC_000007.13, NC_000007.14PEX1475disorder 1BAPG01604Immunodeficiency 143.98E+08APG06622G > A94255NC_000001.10, NC_000001.11PIK3CD476Polycystic kidney dysplasia1.38E+08APG06622G > A19147NC_000006.11, NC_000006.12PKHD1477Carbohydrate-deficient28936415APG02787G > A22745NC_000016.9, NC_000016.10PMM2478glycoprotein syndrome typeICarbohydrate-deficient28936415APG06007G > A22745NC_000016.9, NC_000016.10PMM2479glycoprotein syndrome typeICarbohydrate-deficient28936415APG03850G > A22745NC_000016.9, NC_000016.10PMM2480glycoprotein syndrome typeICarbohydrate-deficient28936415APG05586G > A22745NC_000016.9, NC_000016.10PMM2481glycoprotein syndrome typeICarbohydrate-deficient28936415APG08167,G > A22745NC_000016.9, NC_000016.10PMM2482glycoprotein syndrome typeAPG01604IPOLG-related condition1.14E+08APG06622C > T28535NC_000015.9, NC_000015.10POLG483POLG-related condition1.14E+08APG06622C > T28541NC_000015.9, NC_000015.10POLG484POLG-related condition1.14E+08APG02874,G > A28535NC_000015.9, NC_000015.10POLG485APG03031,APG09208POLG-related condition1.14E+08APG06007G > A28535NC_000015.9, NC_000015.10POLG486POLG-related condition1.14E+08APG03850G > A28535NC_000015.9, NC_000015.10POLG487POLG-related condition1.14E+08APG08167,C > T28541NC_000015.9, NC_000015.10POLG488APG01604Ceroid lipofuscinosis1.38E+08APG06622G > A23943NC_000001.10, NC_000001.11PPT1489neuronal 1Ceroid lipofuscinosis1.38E+08APG02787C > T23943NC_000001.10, NC_000001.11PPT1490neuronal 1Ceroid lipofuscinosis1.38E+08APG06007C > T23943NC_000001.10, NC_000001.11PPT1491neuronal 1Ceroid lipofuscinosis1.38E+08APG03850C > T23943NC_000001.10, NC_000001.11PPT1492neuronal 1Ceroid lipofuscinosis1.38E+08APG05586C > T23943NC_000001.10, NC_000001.11PPT1493neuronal 1Ceroid lipofuscinosis1.38E+08APG08167,G > A23943NC_000001.10, NC_000001.11PPT1494neuronal 1APG01604Cardiomyopathy1.22E+08APG06622C > T21885NC_000007.13, NC_000007.14PRKAG2495Cowden syndrome1.22E+08APG06622C > T22852NC_000010.10, NC_000010.11PTEN496PTPN11-related disorder1.22E+08APG06622C > T28370NC_000012.11, NC_000012.12PTPN11497B lymphoblastic leukemia1.22E+08APG06622A > G28372NC_000012.11, NC_000012.12PTPN11498lymphoma, no ICD-OsubtypePTPN11-related disorder3.98E+08APG06622A > G49032NC_000012.11, NC_000012.12PTPN11499PTPN11-related disorder1.22E+08APG06248C > T28370NC_000012.11, NC_000012.12PTPN11500PTPN11-related disorder1.22E+08APG06248A > G28379NC_000012.11, NC_000012.12PTPN11501PTPN11-related disorder28933386APG02787A > G28365NC_000012.11, NC_000012.12PTPN11502PTPN11-related disorder28933386APG06007A > G28365NC_000012.11, NC_000012.12PTPN11503PTPN11-related disorder28933386APG03850A > G28365NC_000012.11, NC_000012.12PTPN11504PTPN11-related disorder28933386APG05586A > G28365NC_000012.11, NC_000012.12PTPN11505PTPN11-related disorder1.22E+08APG02787A > G28372NC_000012.11, NC_000012.12PTPN11506PTPN11-related disorder1.22E+08APG06007A > G28372NC_000012.11, NC_000012.12PTPN11507PTPN11-related disorder1.22E+08APG03850A > G28372NC_000012.11, NC_000012.12PTPN11508PTPN11-related disorder1.22E+08APG05586A > G28372NC_000012.11, NC_000012.12PTPN11509PTPN11-related disorder28933386APG08167,A > G28365NC_000012.11, NC_000012.12PTPN11510APG01604Glycogen storage disease1.17E+08APG06622G > A17337NC_000011.9, NC_000011.10PYGM511Glycogen storage disease1.17E+08APG06248G > A17337NC_000011.9, NC_000011.10PYGM512Breast-ovarian cancer,3.88E+08APG06622G > A39241NC_000017.10, NC_000017.11RAD51D513familial 4Dilated cardiomyopathy2.68E+08APG02874,G > A15310NC_000010.10, NC_000010.11RBM205141DDAPG03031,APG09208RET-Related Disorders74799832APG06622T > C28958NC_000010.10, NC_000010.11RET515RET-Related Disorders74799832APG02874,T > C28958NC_000010.10, NC_000010.11RET516APG03031,APG09208RYR1-Related Disorders1.18E+08APG06622C > T28003NC_000019.9, NC_000019.10RYR1517RYR1-Related Disorders2.01E+08APG06622C > T169564NC_000019.9, NC_000019.10RYR1518RYR1-Related Disorders1.18E+08APG02874,G > A76888NC_000019.9, NC_000019.10RYR1519APG03031,APG09208RYR1-Related Disorders1.18E+08APG02874,G > A76888NC_000019.9, NC_000019.10RYR1520APG03031,APG09208RYR1-Related Disorders1.18E+08APG02874,G > A76835NC_000019.9, NC_000019.10RYR1521APG03031,APG09208RYR1-Related Disorders1.18E+08APG02874,T > C28014NC_000019.9, NC_000019.10RYR1522APG03031,APG09208Shwachman syndrome1.14E+08APG06622A > G18235NC_000007.13, NC_000007.14SBDS523Brugada syndrome1.38E+08APG06622C > T24416NC_000003.11, NC_000003.12SCN5A524Brugada syndrome28937316APG02874,G > A24408NC_000003.11, NC_000003.12SCN5A525APG03031,APG09208Brugada syndrome45546039APG02874,G > A48043NC_000003.11, NC_000003.12SCN5A526APG03031,APG09208Brugada syndrome72549410APG02874,G > A78547NC_000003.11, NC_000003.12SCN5A527APG03031,APG09208Cowden syndrome 380338844APG06622C > T21935NC_000011.9, NC_000011.10SDHD528Cowden syndrome 380338844APG06248C > T21935NC_000011.9, NC_000011.10SDHD529Alpha-1-antitrypsin28929474APG06622C > T33006NC_000014.8, NC_000014.9SERPINA1530deficiencyAlpha-1-antitrypsin28929474APG02787G > A33006NC_000014.8, NC_000014.9SERPINA1531deficiencyAlpha-1-antitrypsin28929474APG03850G > A33006NC_000014.8, NC_000014.9SERPINA1532deficiencyAlpha-1-antitrypsin28929474APG05586G > A33006NC_000014.8, NC_000014.9SERPINA1533deficiencyAlpha-1-antitrypsin28929474APG08167,C > T33006NC_000014.8, NC_000014.9SERPINA1534deficiencyAPG01604Limb-girdle muscular28933693APG06622C > T24476NC_000017.10, NC_000017.11SGCA535dystrophy, type 2DMucopolysaccharidosis,1.05E+08APG06622C > T20146NC_000017.10, NC_000017.11SGSH536MPS-III-AMucopolysaccharidosis,1.05E+08APG02874,G > A20146NC_000017.10, NC_000017.11SGSH537MPS-III-AAPG03031,APG09208Noonan syndrome2.68E+08APG02787A > G21860NC_000010.10, NC_000010.11SHOC2538Noonan syndrome2.68E+08APG06007A > G21860NC_000010.10, NC_000010.11SHOC2539Noonan syndrome2.68E+08APG03850A > G21860NC_000010.10, NC_000010.11SHOC2540Noonan syndrome2.68E+08APG05586A > G21860NC_000010.10, NC_000010.11SHOC2541Noonan syndrome2.68E+08APG08167,A > G21860NC_000010.10, NC_000010.11SHOC2542APG01604SLC26A2-Related1.05E+08APG06622C > T19128NC_000005.9, NC_000005.10SLC26A2543DisordersSLC26A2-Related1.11E+08APG02874,G > A52666NC_000007.13, NC_000007.14SLC26A4544DisordersAPG03031,APG09208Familial hypertrophic7.28E+08APG02874,G > A172354NC_000001.10, NC_000001.11TNNT2545cardiomyopathy 2APG03031,APG09208Charcot-Marie-Tooth3.98E+08APG02874,G > A48018NC_000012.11, NC_000012.12TRPV4546disease type 2CAPG03031,APG09208Focal cortical dysplasia type28934872APG06622G > A27436NC_000016.9, NC_000016.10TSC2547IIAmyloidogenic76992529APG06622G > A28465NC_000018.9, NC_000018.10TTR548transthyretin amyloidosisAmyloidogenic76992529APG06248G > A28465NC_000018.9, NC_000018.10TTR549transthyretin amyloidosisOculocutaneous albinism1.05E+08APG06622C > T18816NC_000011.9, NC_000011.10TYR550Oculocutaneous albinism1.22E+08APG02874,G > A18814NC_000011.9, NC_000011.10TYR551APG03031,APG09208Von Willebrand disease41276738APG06622C > T15335NC_000012.11, NC_000012.12VWF552 Example 7: Targeting Mutations Responsible for Hurler Syndrome The following describes a potential treatment for Hurler Syndrome, also referred to as MPS-1, using an RNA directed base editing system that corrects a mutation responsible for Hurler syndrome in a large proportion of patients with the disease. This approach utilizes a base editing fusion protein that is RNA guided and that can be packaged into a single AAV vector for delivery to a wide range of tissue types. Depending on the exact regulatory elements and base editor domain used, it may also be possible to engineer a single vector that encodes for both the base editing fusion protein and a single guide RNA to target the diseased locus. Example 7.1: Identifying RGN with Ideal PAM The genetic disease MPS-1 is a lysosomal storage disease characterized at the molecular level by the accumulation of dermatan sulfate and heparan sulfate in lysosomes. This disease is generally an inherited genetic disorder caused by mutations in the IDUA gene (NCBI Reference sequence NG_008103.1), which encodes α-L-iduronidase. The disease is a result of a deficiency of α-L-iduronidase. The most common IDUA mutations found in studies of individuals of Northern European background are W402X and Q70X, both nonsense mutations resulting in premature termination of translation (Bunge et al. (1994), Hum. Mol. Genet, 3(6): 861-866, herein incorporated by reference). Reversion of a single nucleotide would restore the wild-type coding sequence and result in protein expression controlled by the endogenous regulatory mechanisms of the genetic locus. The W402X mutation of the human Idua gene accounts for a high proportion of MPS-1H cases. Base editors can target a narrow sequence window relative to the binding site of the protospacer component of the guide RNA and thus the presence of a PAM sequence a specific distance from the target locus is essential for the success of the strategy. Given the constraints that the target mutation must be on the exposed non-target strand (NTS) during the interaction of the base editing protein and that the footprint of the RGN domain will block access to the region near the PAM, an accessible locus is thought to be 10-30 bp from the PAM. To avoid editing and mutagenesis of other nearby adenosine bases in this window, different linkers are screened. The ideal window is 12-16 bp from the PAM. A PAM sequence compatible with APG02874, APG09208, and APG05586 is readily apparent at the genetic locus. These nucleases have a PAM sequence of 5′-nnnnCC-3′ (SEQ ID NO: 35), 5′-nnnnC-3′ (SEQ ID NO: 62) and 5′-nnRYA-3′ (SEQ ID NO: 69), respectively, and are compact in size—potentially allowing delivery via a single AAV vector. This delivery approach bestows multiple advantages relative to others, such as access to a wide range of tissues (liver, muscle, CNS) and well established safety profile and manufacturing techniques. Cas9 fromS. pyogenes(SpyCas9) requires a PAM sequence of NGG (SEQ ID NO: 256), which is present near the W402X locus, but the size of SpyCas9 prevents packaging into a single AAV vector, and thus forgoes the aforementioned advantages of this approach. While a dual delivery strategy may be employed (for example, Ryu et al, (2018), Nat. Biotechnol., 36(6): 536-539, herein incorporated by reference), it would add significant manufacturing complexity and cost. Additionally, dual viral vector delivery significantly decreases the efficiency of gene correction, since a successful edit in a given cell requires infection with both vectors and assembly of the fusion protein in the cell. A commonly used Cas9 ortholog fromS. aureus(SauCas9) is considerably smaller in size relative to SpyCas9 but has a more complex PAM requirement—NGRRT (SEQ ID NO: 257). This sequence is not within a range expected to be useful for base editing of the causative locus. Example 7.2: RGN Fusion Constructs and sgRNA Sequences A DNA sequence encoding a fusion protein with the following domains is produced using standard molecular biology techniques: 1) an RGN domain with mutations that inactivate the DNA cleavage activity (“dead” or “nickase”); 2) an adenosine deaminase useful for base editing. All constructs described in the table below comprise a fusion protein with the base editing active domain, in this example ADAT (SEQ ID NO: 211), operably fused to the N-terminal end of the a dead RGN APG02874 (SEQ ID NO: 214), APG09208 (SEQ ID NO: 216), and APG005586 (SEQ ID NO: 567). Other adenosine deaminases useful for base editing DNA may also be used (see for example PCT application PCT/US2019/068079). It is known in the art that a fusion protein could also be made with the base-editing enzyme at the C-terminal end of the RGN. Additionally, the RGN and the base editor of the fusion protein are typically separated by a linker amino sequence. It is known in the art that lengths of standard linkers range from 15-30 amino acids. Further, it is known in the art that certain fusion proteins between an RGN and a base-editing enzyme may also comprise at least one uracil glycosylase inhibitor (UGI) domain (SEQ ID NO: 212), which may increase base editing efficiency (U.S. Pat. No. 10,167,457, herein incorporated by reference). Therefore, a fusion protein may comprise RGN APG02874, APG09208, or a variant thereof, an adenosine deaminase, and optionally at least one UGI. TABLE 12Constructs for RNA-targeted base editingSEQ IDDead (D)orBaseNO.ConstructRGNNickase (N)editor213Nuc-ADAT-Linker-dAPG02874DADATAPG02874 -Linker-SV40215Nuc-ADAT-Linker-dAPG09208DADATAPG09208-Linker-SV40565Nuc-ADAT-Linker-dAPG05586DADATAPG05586-Linker-SV40 The accessible editing sites of an RGN are determined by the PAM sequence. When combining an RGN with a base editing domain, the target residue for editing must reside on the non-target strand (NTS), since the NTS is single stranded while the RGN is associated with the locus. Evaluating a number of nucleases and corresponding guide RNAs enables the selection of the most appropriate gene editing tool for this particular locus. Several potential PAM sequences that can be targeted by the constructs described above in the human Idua gene are in the proximity of the mutant nucleotide responsible for the W402X mutation. A sequence encoding a guide RNA transcript containing 1) a “spacer” that is complementary to the non-coding DNA strand at the disease locus; and 2) RNA sequence required for association of the guide RNA with the RGN is also produced. Such a sgRNA may be encoded by, for example, SEQ ID NO: 217 for the APG02874 RGN system, SEQ ID NO: 218 for the APG09208 RGN system, or SEQ ID NO: 566 for the APG005586 RGN system. These sgRNA molecules, and similar sgRNAs that may be devised by one of skill in the art, can be evaluated for their efficiency in directing the base editors above to the locus of interest. Example 7.3: Assay for Activity in Cells from Hurler Disease Patients To verify the genotype strategy and evaluate the constructs described above, fibroblasts from Hurler disease patients are used. A vector is designed containing appropriate promoters upstream of the fusion protein coding sequence and the sgRNA encoding sequence for expression of these in human cells, similar to those vectors described in Example 4. It is recognized that promoters and other DNA elements (for example enhancers, or terminators) which either are known for high levels of expression in human cells or may specifically express well in fibroblast cells may also be used. The vector is transfected into the fibroblasts using standard techniques, for example transfection similar to what is described in Example 4. Alternatively, electroporation may be used. The cells are cultured for 1-3 days. Genomic DNA (gDNA) is isolated using standard techniques. The editing efficiency is determined by performing a qPCR genotyping assay and/or next generation sequencing on the purified gDNA, as described further below. Taqman™ qPCR analysis utilizes probes specific for the wild-type and mutant allele. These probes bear fluorophores which are resolved by their spectral excitation and/or emission properties using a qPCR instrument. A genotyping kit containing PCR primers and probes can be obtained commercially (i.e. Thermo Fisher Taqman™ SNP genotyping assayID C_27862753_10 for SNP ID rs121965019) or designed. An example of a designed primer and probe set is shown in Table 13. TABLE 13RT-PCR primers and probesDescriptionSequenceSEQ ID NO.Forward Amplification Primer5′-GACTCCTTCACCAAG-3′219Reverse Amplification Primer5′-GTAGATCAGCACCG-3′220Wild Type Probe5′-CTCTGGGCCGAAGT-3′221W402X Probe5′-CTCTAGGCCGAAGT-3′222 Following the editing experiment, the gDNA is subjected to qPCR analysis using standard methods and the primers and probes described above. Expected results are shown in Table 14. This in vitro system can be used to expediently evaluate constructs and choose one with high editing efficiency for further studies. The systems will be evaluated in comparison with cells with and without the W402X mutation, and preferably with some that are heterozygous for this mutation. The Ct values will be compared to either a reference gene or the total amplification of the locus using a dye such as Sybr green. TABLE 14Expected qPCR resultsTransfected withGenotypebase editorExpected PCR resultIduaWT/WTNoHomozygous WTIduaWT/W402XNoHeterozygous: 50% WT,50% W402XIduaW402X/W402XNoHomozygous W402XIduaW402X/W402XYesVariable The tissues can also be analyzed by next generation sequencing. Primer binding sites such as the ones shown below (Table 15), or other suitable primer binding sites that can be identified by a person of skill in the art, can be used. Following PCR amplification, products containing Illumina Nextera XT overhang sequences undergo library preparation following the Illumina 16S Metagenomic Sequencing Library protocol. Deep sequencing is performed on an Illumina Mi-Seq platform. Typically, 200,000 of 250 bp paired-end reads (2×100,000 reads) are generated per amplicon. The reads are analyzed using CRISPResso (Pinello et al., 2016) to calculate the rates of editing. Output alignments are hand-curated to confirm insertion and deletion sites as well as identify microhomology sites at the recombination sites. TABLE 15NGS primer binding sitesDirectionSequenceSEQ ID NO.Forward5'-ACTTCCTCCAGCC-3'223Reverse5'-GAACCCCGGCTTA-3'224 Western blotting of cell lysate of transfected cells and control cells using an anti-IDUA antibody is performed to verify expression of the full-length protein and an enzyme activity assay on the cell lysate using substrate 4-methylumbelliferyl a-L-iduronide verifies that the enzyme is catalytically active (Hopwood et al., Clin. Chim. ACta (1979), 92(2): 257-265, incorporated by reference herein). These experiments are performed in comparison with the original IduaW402X/W402Xcell line (without transfection), the IduaW402X/W402Xcell line transfected with the base editing construct and a random guide sequence, and a cell line expressing wild-type IDUA. Example 7.4: Disease Treatment Validation in a Murine Model To verify the efficacy of this therapeutic approach, a mouse model with a nonsense mutation in the analogous amino acid is used. The mouse strain bears a W392X mutation in its Idua gene (Gene ID: 15932) which corresponds to the homologous mutation in Hurler syndrome patients (Bunge et al., (1994), Hum. Mol. Genet. 3(6): 861-866, incorporated by reference herein). This locus comprises a distinct nucleotide sequence relative to that in humans, which lacks the PAM sequence necessary for correction with the base editors described in the previous examples, and thus necessitates design of a distinct fusion protein to perform the nucleotide correction. Amelioration of the disease in this animal can validate the therapeutic approach of correcting the mutation in tissues accessible by a gene delivery vector. Mice homozygous for this mutation display a number of phenotypic characteristics similar to Hurler syndrome patients. A base editing-RGN fusion protein as described above (Table 12) along with an RNA guide sequence are incorporated into an expression vector that allows protein expression and RNA transcription in mice. A study design is shown below in Table 16. The study includes groups that are treated with a high dose of the expression vector comprising the base-editing fusion protein and RNA guide sequence, a low dose of same expression vector, control which is the model mouse treated with an expression vector that does not comprise the base editing fusion protein or the guide RNA, and a second control which is a wild type mouse treated with the same empty vector. TABLE 16Genome editing experiment in murine modelGroupMouse strainNTreatment1Idua-W392X1≥5Low dose of vector2Idua-W392X≥5High dose of vector3Idua-W392X≥5Vehicle4129/Sv (WT)5Vehicle Endpoints to evaluate include body weight, urine GAG excretion, serum IDUA enzymatic activity, IDUA activity in tissues of interest, tissue pathology, genotyping of tissues of interest to verify correction of the SNP, and behavioral and neurological evaluation. Since some endpoints are terminal, additional groups may be added for evaluation of, for example, tissue pathology and tissue IDUA activities before the end of the study. Additional examples of endpoints can be found in published papers establishing Hurler syndrome animal models (Shull et al. (1994), Proc. Natl. Acad. Sci. U.S.A., 91(26): 12937-12941; Wang et al. (2010), Mol. Genet. Metab., 99(1): 62-71; Hartung et al. (2004), Mol. Ther., 9(6): 866-875; Liu et al. (2005), Mol. Ther., 11(1): 35-47; Clarke et al. (1997), Hum. Mol. Genet. 6(4): 503-511; all herein incorporated by reference). One possible delivery vector utilizes the adeno associated virus (AAV). A vector is produced to include a base editor-dRGN fusion protein coding sequence (for example, Nuc-ADAT-Linker-dAPG19748-Linker-SV40, as described above) preceded by a CMV enhancer (SEQ ID NO: 259) and promoter (SEQ ID NO: 258), or other suitable enhancer and promoter combination), optionally a Kozak sequence, and operably fused at the 3′ end to a terminator sequence and a poly-adenylation sequence such as the minimal sequence described in Levitt, N.; Briggs, D.; Gil, A.; Proudfoot, N. J. Definition of an Efficient Synthetic Poly(A) Site. Genes Dev. 1989, 3 (7), 1019-1025. The vector may further comprise an expression cassette encoding for a single guide RNA operably linked at its 5′ end to a human U6 promoter (SEQ ID NO: 260) or another promoter suitable for production of small non-coding RNAs, and further comprising inverted terminal repeat (ITR) sequences necessary and well-known in the art for packaging into the AAV capsid. Production and viral packaging is performed by standard methods, such as those described in U.S. Pat. No. 9,587,250, herein incorporated by reference. Other possible viral vectors include adenovirus and lentivirus vectors, which are commonly used and would contain similar elements, with different packaging capabilities and requirements. Non-viral delivery methods also be used, such as mRNA and sgRNA encapsulated by lipid nanoparticles (Cullis, P. R. and Allen, T. M. (2013), Adv. Drug Deliv. Rev. 65(1): 36-48; Finn et al. (2018), Cell Rep. 22(9): 2227-2235, both incorporated by reference) hydrodynamic injection of plasmid DNA (Suda T and Liu D,)2007) Mol. Ther. 15(12): 2063-2069, herein incorporated by reference), or ribonucleoprotein complexes of sgRNA and associated with gold nanoparticles (Lee, K.; Conboy, M.; Park, H. M.; Jiang, F.; Kim, H. J.; Dewitt, M. A.; Mackley, V. A.; Chang, K.; Rao, A.; Skinner, C.; et al. Nanoparticle Delivery of Cas9 Ribonucleoprotein and Donor DNA in Vivo Induces Homology-Directed DNA Repair. Nat. Biomed. Eng. 2017, 1 (11), 889-90). Example 7.5: Disease Correction in a Murine Model with a Humanized Locus To evaluate the efficacy of an identical base editor construct as would be used for human therapy, a mouse model in which the nucleotides near W392 are altered to match the sequence in humans around W402 is needed. This can be accomplished by a variety of techniques, including use of an RGN and an HDR template to cut and replace the locus in mouse embryos. Due to the high degree of amino acid conservation, most nucleotides in the mouse locus can be altered to those of the human sequence with silent mutations as shown in Table 17. The only base changes resulting in altered coding sequence in the resulting engineered mouse genome occur after the introduced stop codon. TABLE 17Nucleotide mutations to generate a humanized mouse locusHuman (W402X)Mouse (W392X)Humanized MouseNucleotideEn-NucleotideEn-NucleotideEn-(SEQ IDcoded(SEQ IDcoded(SEQ IDcodedFeatureNO: 225)AANO: 226)AANO: 227)AAProto-GEAGGGspacerGEGEGEAAAGAGCQCQCQAAAGAGCLCLCLTTTCCCTSTOPTSTOPTSTOPAAAGGGGAGAGACCCCACGEGEGEAAAAGAGVGVGVTTTGCGTSTSTSCCCGAGPAM,CQAKCQnon-AAAcriticalGGGGAGAGAPAM,CCCcriticalCTC Upon engineering of this mouse strain, similar experiments will be performed as described in Example 7.4. Example 8: Targeting Mutations Responsible for Friedreich Ataxia The expansion of the trinucleotide repeat sequence causing Friedreich's Ataxia (FRDA) occurs in a defined genetic locus within the FXN gene, referred to as the FRDA instability region. RNA guided nucleases (RGNs) may be used for excising the instability region in FRDA patient cells. This approach requires 1) an RGN and guide RNA sequence that can be programmed to target the allele in the human genome; and 2) a delivery approach for the RGN and guide sequence. Many nucleases used for genome editing, such as the commonly used Cas9 nuclease fromS. pyogenes(SpCas9), are too large to be packaged into adeno-associated viral (AAV) vectors, especially when considering the length of the SpCas9 gene and the guide RNA in addition to other genetic elements required for functional expression cassettes. This makes a viable approach using SpCas9 unlikely. Compact RNA guided nucleases of the invention, for example APG03850, are uniquely well suited for the excision of the FRDA instability region. APG03850 has a PAM requirement that is in the vicinity of the FRDA instability region. Additionally, APG03850 can be packaged into an AAV vector along with a guide RNA. Packing two guide RNAs would likely require a second vector, but this approach still compares favorably to what would be required of a larger nuclease such as SpCas9, which would require splitting the protein sequence between two vectors. Table 18 shows the location of genomic target sequences suitable for targeting APG03850 to the 5′ and 3′ flanks of the FRDA instability region, as well as the sequence of the sgRNAs for the genomic targets. Once at the locus, the RGN would excise the FA instability region. Excision of the region can be verified with Illumina sequencing of the locus. TABLE 18Genomic target sequences for RGN systemsGenome targetGuideLocation relative to FRDAsequencesgRNANo.instability region(SEQ ID NO.)(SEQ ID NO.)15′22822925′23023133′23223343′234235 Example 9: Targeting Mutations Responsible for Sickle Cell Diseases Targeting sequences within the BCL11A enhancer region (SEQ ID NO: 236) may provide a mechanism for increasing fetal hemoglobulin (HbF) to either cure or alleviate the symptoms of sickle cell diseases. For example, genome wide association studies have identified a set of genetic variations at BCL11A that are associated with increased HbF levels. These variations are a collection of SNPs found in non-coding regions of BCL11A that function as a stage-specific, lineage-restricted enhancer region. Further investigation revealed that this BCL11A enhancer is required in erythroid cells for BCL11A expression (Bauer et al, (2013) Science 343:253-257, incorporated by reference herein). The enhancer region was found within intron 2 of the BCL11A gene, and three areas of DNaseI hypersensitivity (often indicative of a chromatin state that is associated with regulatory potential) in intron 2 were identified. These three areas were identified as “+62”, “+58” and “+55” in accordance with the distance in kilobases from the transcription start site of BCL11A. These enhancer regions are roughly 350 (+55); 550 (+58); and 350 (+62) nucleotides in length (Bauer et al., 2013). Example 9.1: Identifying Preferred RGN Systems Here we describe a potential treatment for beta-hemoglobinopathies using an RGN system that disrupts BCL11A binding to its binding site within the HBB locus, which is the gene responsible for making beta-globin in adult hemoglobin. This approach uses NHEJ which is more efficient in mammalian cells. In addition, this approach uses a nuclease of sufficiently small size that can be packaged into a single AAV vector for in vivo delivery. The GATA1 enhancer motif in the human BCL11A enhancer region (SEQ ID NO: 236) is an ideal target for disruption using RNA guided nucleases (RGNs) to reduce BCL11A expression with concurrent re-expression of HbF in adult human erythrocytes (Wu et al. (2019) Nat Med 387:2554). Several PAM sequences compatible with APG03850 or APG09208 are readily apparent at the genetic locus surrounding this GATA1 site. These nucleases have a PAM sequence of 5′-nnnnG-3′ (SEQ ID NO: 42) and 5′-nnnnC-3′ (SEQ ID NO: 62), respectively, and are compact in size, potentially allowing their delivery along with an appropriate guide RNA in a single AAV or adenoviral vector. This delivery approach bestows multiple advantages relative to others, such as access to hematopoietic stem cells and a well-established safety profile and manufacturing techniques. The commonly used Cas9 nuclease fromS. pyogenes(SpyCas9) requires a PAM sequence of 5′-NGG-3′, (SEQ ID NO: 256) several of which are present near the GATA1 motif. However, the size of SpyCas9 prevents packaging into a single AAV or adenoviral vector and thus forgoes the aforementioned advantages of this approach. While a dual delivery strategy may be employed, it would add significant manufacturing complexity and cost. Additionally, dual viral vector delivery significantly decreases the efficiency of gene correction, since a successful edit in a given cell requires infection with both vectors. An expression cassette encoding a human codon optimized APG03850 or APG09208 is produced, similar to those described in Example 4. Expression cassettes which express guide RNAs for RGNs APG03850 or APG09208 are also produced. These guide RNAs comprise: 1) a protospacer sequence that is complementary to either the non-coding or coding DNA strand within the BCL11A enhancer locus (the target sequence) and 2) an RNA sequence required for association of the guide RNA with the RGN. Because several potential PAM sequences for targeting by APG03850 or APG09208 surround the BCL11A GATA1 enhancer motif, several potential guide RNA constructs are produced to determine the best protospacer sequence that produces robust cleavage and NHEJ mediated disruption of the BCL11A GATA1 enhancer sequence. The target genomic sequences in Table 19 are evaluated to direct the RGN to this locus using the sgRNA provided in Table 19. TABLE 19Target Sequences for BCL11A GATA1 enhancerlocus using APG03850 or APG09208Target genomic sequencesgRNAGuideRGN(SEQ ID NO.)(SEQ ID NO.)1APG038502372382APG038502392403APG038502412424APG092082432445APG092082452466APG09208247248 To evaluate the efficiency with which APG03850 or APG09208 generates insertions or deletions that disrupt the BCL11A enhancer region, human cell lines such as human embryonic kidney cells (HEK cells) are used. A DNA vector comprising an RGN expression cassette (for example, as described in Example 4) is produced. A separate vector comprising an expression cassette comprising a coding sequence for a guide RNA sequence of Table 16 is also produced. Such an expression cassette may further comprise a human RNA polymerase III U6 promoter (SEQ ID NO: 260), as described in Example 4. Alternatively, a single vector comprising expression cassettes of both the RGN and guide RNA may be used. The vector is introduced into HEK cells using standard techniques such as those described in Example 4, and the cells are cultured for 1-3 days. Following this culture period, genomic DNA is isolated and the frequency of insertions or deletions is determined by using T7 Endonuclease I digestion and/or direct DNA sequencing, as described in Example 4. A region of DNA encompassing the target BCL11A region is amplified by PCR with primers containing Illumina Nextera XT overhang sequences. These PCR amplicons are either examined for NHEJ formation using T7 Endonuclease I digestion, or undergo library preparation following the Illumina 16S Metagenomic Sequencing Library protocol or a similar Next Generation Sequencing (NGS) library preparation. Following deep sequencing, the reads generated are analyzed by CRISPResso to calculate rates of editing. Output alignments are hand-curated to confirm insertion and deletion sites. This analysis identifies the preferred RGN and the corresponding preferred guide RNA (sgRNA). The analysis may result in both APG03850 and APG09208 being equally preferred. Additionally, the analysis may determine there is more than one preferred guide RNA, or that all target genomic sequences in Table 16 are equally preferred. Example 9.2: Assay for Expression of Fetal Hemoglobin In this example, APG03850 or APG09208 generated insertions or deletions disrupting the BCL11A enhancer region are assayed for expression of fetal hemoglobin. Healthy human donor CD34+hematopoietic stem cells (HSCs) are used. These HSCs are cultured and vector(s) comprising expression cassettes comprising the coding regions of the preferred RGN and the preferred sgRNA are introduced using methods similar to those described in Example 8.1. Following electroporation, these cells are differentiated in vitro into erythrocytes using established protocols (for example, Giarratana et al. (2004) Nat Biotechnology 23:69-74, herein incorporated by reference). The expression of HbF is then measured using western blotting with an anti-human HbF antibody, or quantified via High Performance Liquid Chromatography (HPLC). It is expected that successful disruption of the BCL11A enhancer locus will lead to an increase in HbF production when compared to HSCs electroporated with only the RGN but no guide. Example 9.3: Assay for Decreased Sickle Cell Formation In this example, APG03850- or APG09208-generated insertions or deletions disrupting the BCL11A enhancer region are assayed for decreased sickle-cell formation. Donor CD34+hematopoietic stem cells (HSCs) from patients afflicted with sickle cell disease are used. These HSCs are cultured and vector(s) comprising expression cassettes comprising the coding regions of preferred RGN and the preferred sgRNA are introduced using methods similar to those described in Example 8.1. Following electroporation, these cells are differentiated in vitro into erythrocytes using established protocols (Giarratana et al. (2004) Nat Biotechnology 23:69-74). The expression of HbF is then measured using western blotting with an anti-human HbF antibody, or quantified via High Performance Liquid Chromatography (HPLC). It is expected that successful disruption of the BCL11A enhancer locus will lead to an increase in HbF production when compared to HSCs electroporated with only the RGN but no guide. Sickle cell formation is induced in these differentiated erythrocytes by the addition of metabisulfite. The numbers of sickled vs normal erythrocytes are counted using a microscope. It is expected that the numbers of sickled cells are less in cells treated with APG03850 or APG09208 plus sgRNAs than with cells untreated, or treated with RGNs alone. Example 9.4: Disease Treatment Validation in a Murine Model To evaluate the efficacy of using APG03850 or APG09208 for disruption of the BCL11A locus, suitable humanized mouse models of sickle cell anemia are used. Expression cassettes encoding for the preferred RGN and for the preferred sgRNA are packaged into AAV vectors or adenovirus vectors. In particular, adenovirus type Ad5/35 is effective at targeting HSCs. A suitable mouse model containing a humanized HBB locus with sickle cell alleles is chosen, such as B6; FVB-Tg(LCR-HBA2,LCR-HBB*E26K)53Hhb/J or B6.Cg-Hbatm1Paz Hbbtm1Tow Tg(HBA-HBBs)41Paz/HhbJ. These mice are treated with granulocyte colony-stimulating factor alone or in combination with plerixafor to mobilize HSCs into circulation. AAVs or adenoviruses carrying the RGN and guide plasmid are then injected intravenously, and the mice are allowed to recover for a week. Blood obtained from these mice is tested in an in vitro sickling assay using metabisulfite, and the mice are followed longitudinally to monitor mortality rates and hematopoietic function. It is expected that treatment with AAVs or adenoviruses carrying an RGN and guide RNA will reduce sickling, mortality, and improve hematopoietic function when compared to mice treated with viruses lacking both expression cassettes, or with viruses carrying the RGN expression cassette alone. Example 10: Testing Different Delivery Formats To determine if the RGNs are capable of delivery in different formats, mRNA and RNP nucleofection delivery was tested with primary T-cells. Purified CD3+ T-cells or peripheral blood mononuclear cells (PBMCs) were thawed, activated using CD3/CD28 beads (ThermoFisher) for 3 days, then nucleofected using the Lonza 4D-Nucleofector X unit and Nucleocuvette strips. The P3 Primary Cell kit was used for both mRNA and RNP delivery. Cells were transfected using the EO-115 and EH-115 programs for mRNA and RNP delivery respectively. Cells were cultured in CTS OpTimizer T cell expansion medium (ThermoFisher) containing IL-2, IL-7, and IL-15 (Miltenyi Biotec) for 4 days post nucleofection before being harvested using a Nucleospin Tissue genomic DNA isolation kit (Machery Nagel). Amplicons surrounding the editing sites were generated by PCR using primers identified in Table 4 and subjected to NGS sequencing using the Illumina Nextera platform using 2×250 bp paired end sequencing following the method in Example 4. TABLE 20mRNA and RNP delivery of RGNs in primary T-cellsRGNDelivery MethodSGN% EditingAPG01604mRNA, NucleofectionSGN0016711.18APG01604mRNA, NucleofectionSGN00167322.7APG01604RNP, NucleofectionSGN0016730.56APG01604mRNA, NucleofectionSGN0016740.63APG01604RNP, NucleofectionSGN0016740.77APG01868mRNA, NucleofectionSGN00168477.85APG01868RNP, NucleofectionSGN00168483.73APG01868mRNA, NucleofectionSGN00169182.53APG01868RNP, NucleofectionSGN00169180.7APG01868mRNA, NucleofectionSGN00169276.84APG01868RNP, NucleofectionSGN00169286.06APG01868mRNA, NucleofectionSGN0017850.22APG01868RNP, NucleofectionSGN0017850.43 Both mRNA and RNP delivery showed successful editing with APG01868. APG01868 showed successful editing with RNPs at several genomic targets. RNP delivery was limited with APG01604, but showed equal editing rates with mRNA delivery. Example 11: Base Editor Testing To determine if APG09298 and APG01604 could perform cytosine base editing in mammalian cells, a cytosine deaminase was operably fused to the nickase version of each RGN to produce a fusion protein. Residues predicted to deactivate the RuvC domain of the RGNs APG09298 and APG01604 were identified and the RGNs were modified to nickase variants. A nickase variant of an RGN is referred to herein as “nRGN”. It should be understood that any nickase variant of an RGN may be used to produce a fusion protein of the invention. Deaminase and nRGN nucleotide sequences codon optimized for mammalian expression were synthesized as fusion proteins with an N-terminal nuclear localization tag and cloned into the pTwist CMV (Twist Biosciences) expression plasmid. Each fusion protein comprises, starting at the amino terminus, the SV40 NLS (SEQ ID NO: 251) operably linked at the C-terminal end to 3×FLAG Tag (SEQ ID NO: 252), operably linked at the C-terminal end to a deaminase (APG05840), operably linked at the C-terminal end to a peptide linker, operably linked at the C-terminal end to the nRGN (nAPG09298 or nAPG01604), operably linked at the C-terminal end to a peptide linker, operably linked at the C-terminal end to a uracil stabilizing protein (USP2 set forth as SEQ ID NO: 1089), finally operably linked at the C-terminal end to the nucleoplasmin NLS (SEQ ID NO: 253). The amino acid sequence of the APG05840-nAPG09298-USP2 and APG05840-nAPG01604-USP2 fusion proteins are set forth as SEQ ID NOs: 1090 and 1091, respectively. Expression plasmids comprising an expression cassette encoding a sgRNA were also produced. Human genomic target sequences and the sgRNA sequences for guiding the fusion proteins to the genomic targets are indicated in Table 3 and the primers for amplification of the genomic region are listed in Table 4. The same methods in Example 4 for plasmid delivery to mammalian cells and amplicon sequencing were used to test the base editing capabilities of these RGNs when tethered to a cytosine deaminase. TABLE 21Estimated Base Editing Rates for each RGN testedConstructTarget% Mutated ReadsAPG05840-SGN0011596.96nAPG09298-USP2APG05840-SGN0011599.91nAPG09298-USP2APG05840-SGN00116221.59nAPG09298-USP2APG05840-SGN00116227.05nAPG09298-USP2APG05840-SGN00116317.85nAPG09298-USP2APG05840-SGN00116320.74nAPG09298-USP2APG05840-SGN0011644.74nAPG09298-USP2APG05840-SGN0011647.89nAPG09298-USP2APG05840-SGN00116531.99nAPG09298-USP2APG05840-SGN00116562.47nAPG09298-USP2APG05840-SGN00116623.49nAPG09298-USP2APG05840-SGN00116629.56nAPG09298-USP2APG05840-SGN0012459.67nAPG01604-USP2APG05840-SGN0012461.4nAPG01604-USP2APG05840-SGN0012491.72nAPG01604-USP2APG05840-SGN00125013.41nAPG01604-USP2APG05840-SGN0012510.87nAPG01604-USP2APG05840-SGN0012524.28nAPG01604-USP2 Example 12: Off Target Analysis To assess the specificity of the nucleases, off target editing was determined at potential sites identified via bioinformatics. Potential off target sites for APG01604 were identified by targets with less than five mismatches in the target sequence and at least one residue match in the PAM sequence. The same methods as those described in Example 4 for plasmid delivery to mammalian cells and amplicon sequencing were used to test the specificity and off target editing of APG01604. From the same experiment where the SGNs in Table 22 were tested for on target editing, the potential off target locations in Table 23 were assayed for potential editing. The primers in Table 24 were used to amplify potential off target sites with sequence similarity to the on target site to look for off target editing. TABLE 22SGNs used to look for off target editingOn Target Forward PrimerOn Target Reverse PrimerSGNGene(SEQ ID NO)(SEQ ID NO)SGN001675TRACCCTTGTCCATCACTGGCATACCAAAGCTGCCCTTACCTG(1096)(1097)SGN001594B2MCCTTAATGTGCCTCCAGCCTAGGAGAGACTCACGCTGGAT(1092)(1093)SGN001674TRACCCTTGTCCATCACTGGCATACCAAAGCTGCCCTTACCTG(1094)(1095) TABLE 23Off target sequences assayedOffSEQTargetOff targetIDSGNNumberlocus sequenceNOSGN0015941594-2AGCACAGCTAAGGCCTAAAGTTGAA1098SGN0015941594-3AGCACAGCTAAGGCACCGGATTGGA1099SGN0015941594-4AGCACAGCCAAGGCCAAGGGCTGAC1100SGN0015941594-5AGCAGAGCTAAGGCCAAGGCAGGTG1101SGN0015941594-6GGCACAGCTAAGGCCAGCAGTGGCC1102SGN0016741674-2GTCTCTGAGCTGGTACATGGCAGAG1103SGN0016741674-3GTCTTTTAGCTGGTACACGTGTGTC1104SGN0016751675-2AGAAGATTTGTCACTGGATTCTGAG1105SGN0016751675-3AGGACACTTGTCACTGGATTTAGGA1106SGN0016751675-4AGGACACTTGTCACTGGATTTAGGA1107SGN0016751675-5TGAGGACTTGTCACTGGATTCAGGG1108SGN0016751675-6AGGAGACTTTTCACTGGATTTAGGG1109SGN0016751675-7TGGACACTTGTCACTGGATTTAGGG1110SGN0016751675-8CACAGACTTGTCACTGGATGTGGGG1111SGN0016751675-9TACAGACATGTCACTGGATCTGGAA1112SGN0016751675-10AACACACTTGTCATTGGATTTAGGG1113SGN0016751675-11TACAGACTGGTCACTGGATGCTGGT1114SGN0016751675-12AGGACACTTGTCACTGGATTTAGCA1115SGN0016751675-13CTAAGACTTGTTACTGGATTGTGTG1116SGN0016751675-14CTCAGACTGGTCACTGGATAATGTA1117 TABLE 24Primers used to amplify off target regionsDescriptionPrimer sequenceSEQ ID NO1594-2 FWDCCAGAAGCCAGCAGATGACA11181594-2 REVGAGTGGTGGGCTCTCAATCC11191594-3 FWDAGCAACACCATCCAAAGGTT11201594-3 REVGGGAGTGATGATAATGCGGG11211594-4 FWDTGGACTAGAGAGGGTTGGGG11221594-4 REVTCTCTTTCCACGAGCAGCAG11231594-5 FWDGCCTTTGACCTTCCCAGATT11241594-5 REVACCATTGGAAAGGTGGATGC11251594-6 FWDGGCTTCAGGCTTTCCTCTGT11261594-6 REVAGCATGCTGGCCTAAAGTGA11271674-2 FWDACCATTGGTCTGCTCAGGTG11281674-2 REVCCAAAACCTGCAGTGGCTTC11291674-3 FWDGGAGAGGAACTGGGCATGAG11301674-3 REVTCCGTCTCTCCTAGGTCTGC11311675-2 FWDCCATGACTGGCCCTTCTGTT11321675-2 REVGGGTAGAGTACATGGCGACG11331675-3 FWDCCCTCCCACCAGAAGCTCTA11341675-3 REVGATAAGAGGCCCAAGGACCG11351675-4 FWDCACTTGACACGTGAGCCTCT11361675-4 REVCCTCTTTCAGCCTCTGGTGG11371675-5 FWDGACAGACTTGGTTCTGCCCT11381675-5 REVCCTCCCCTCCTTCCTAGCTT11391675-6 FWDTGCTGTAGTGGGTCGAACAG11401675-6 REVTTTCACCATGCTGGCTAGGC11411675-7 FWDCCCAGCCACATGGAACTAAG11421675-7 REVGGACCCTAGGAGTTCCTTGT11431675-8 FWDGCAGGTTGATAGGGAAGAGC11441675-8 REVTCATCTCCCAGCTGATGACA11451675-9 FWDCCACCTGTTGCACAAATCCG11461675-9 REVTCCTCCCCTGGGAACATGAT11471675-10 FWDGAGGTACTGGGAGTGGGGAT11481675-10 REVCTTCCTGCCTCTTCCAGCTC11491675-11 FWDGCACAGCTTTTGTCATGGGG11501675-11 REVGGGGATGAGAAAACAGAGCCA11511675-12 FWDCCATGCCCCATTCTGAAGGT11521675-12 REVTTCCCCTCCTCTAGACTGCC11531675-13 FWDCTTGAGCCCAGGAGTTTGAG11541675-13 REVTGCATTCTTGGGATGACCTC11551675-14 FWDACTGCTTTTCCCTGGACACA11561675-14 REVGGTCACAAGTCCCACTGGTC1157 Using plasmid delivery, there was no detectable off target editing at two of the three guides tested. One off target site for SGN001675, 1675-8, showed 2% editing at the off target locus. There was duplication of 132 bp of the genomic region at the cut site in the NGS reads. Two off target sites, 1675-7 and 1675-13, were unable to be sequenced due to poor primer amplification. TABLE 25Off Target Editing for SGN001594 and APG08167TotalTotalTotalOff targetMismatchesMismatchesMatches inNumberin Targetin SeedPAM% EditingSGN00159400342.791594-222301594-32230.011594-421201594-532201594-62110 TABLE 26Off Target Editing for SGN001675 and APG08167TotalTotalTotalOfftargetMismatchesMismatchesMatches inNumberin Targetin SeedPAM% EditingSGN00167500333.1151675-82132.021675-54030.061675-103130.031675-124020.021675-640301675-240301675-340301675-440301675-931301675-1120201675-1441201675-13402Issuesequencing1675-7403Issuesequencing TABLE 27Off Target Editing for SGN001674 and APG08167TotalTotalTotalOfftargetMismatchesMismatchesMatches inNumberin Targetin SeedPAM% EditingSGN00167400352.171674-221301674-32010.03 Example 13: Guide RNA Backbone Variant Testing APG08167 and APG01604 are approximately 62.72% identical, but recognize the same PAM, share the same crRNA, and have a closely related tracrRNA sequence. Because of the similarity of the guide RNA sequences, all data previously generated for APG01604 used the APG08167 tracrRNA backbone. These studies were performed to confirm that the APG01604 protein is more active with a tracrRNA encoded in the APG08167 genome (the native APG08167 tracrRNA). Different spacer lengths in the crRNA were also tested to determine if the APG01604 protein prefers a 20 or 25 base pair spacer sequence. To do this, synthetic crRNAs with six different target sequences that contained a 20 or 25 base pair target were generated. The different crRNAs were combinatorially combined with a synthetic tracrRNA from the APG08167 or APG01604 genome. RNPs were formed and the RNP complexes were nucleofected into HEK293T cells. The standard methods in Example 4 were used for determining the editing rate in cells. TABLE 28crRNA SequencesGeneSpacerSpacer LengthSEQ ID NOB2MA201158B201159C201160D201161A251162B251163C251164D251165TRAE201166F201167E251168F251169 TABLE 29tracrRNA SequencestracrRNASEQ ID NOAPG08167 tracrRNA1170APG01604 tracrRNA1171 TABLE 30Sequencing Primer SequencesPrimerSEQ ID NOTRAC Left Primer1170TRAC Right Primer1171B2M Left Primer1172B2M Right Primer1173 TABLE 31Editing results for APG01604 backbone and target length testingSpacerEditingGeneSpacerLengthBackboneefficiency (%)B2MA20APG01604 tr0.21B20APG01604 tr0.08C20APG01604 tr0.27D20APG01604 tr0.17A25APG01604 tr0.15B25APG01604 tr0C25APG01604 tr1.33D25APG01604 tr0.31A20APG08167 tr6.58B20APG08167 tr1.75C20APG08167 tr6.99D20APG08167 tr64.6A25APG08167 tr22.7B25APG08167 tr2.36C25APG08167 tr52.51D25APG08167 tr82.58TRAE20APG01604 tr0.05F20APG01604 tr0.01E25APG01604 tr0.02F25APG01604 tr0.01 | 452,618 |
11859182 | It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features. Definitions All of the functionalities described in connection with one embodiment of the compositions and/or methods described herein are intended to be applicable to the additional embodiments of the compositions and/or methods except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment. Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “a system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Additionally, it is to be understood that terms such as “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,” “outer” that may be used herein merely describe points of reference and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention. Conventional methods are used for the procedures described herein, such as those provided in the art, and demonstrated in the Examples and various general references. Unless otherwise stated, nucleic acid sequences described herein are given, when read from left to right, in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNA, as RNA, or a combination of DNA and RNA (e.g., a chimeric nucleic acid). Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. The term “and/or” where used herein is to be taken as specific disclosure of each of the multiple specified features or components with or without another. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art. As used herein, the term “about,” as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). As used herein, the terms “binding affinity” or “dissociation constant” or “Kd” refer to the tendency of a molecule to bind (covalently or non-covalently) to a different molecule. A high Kd(which in the context of the present disclosure refers to blocked nucleic acid molecules or blocked primer molecules binding to RNP2) indicates the presence of more unbound molecules, and a low IQ (which in the context of the present disclosure refers to unblocked nucleic acid molecules or unblocked primer molecules binding to RNP2) indicates the presence of more bound molecules. In the context of the present disclosure and the binding of blocked or unblocked nucleic acid molecules or blocked or unblocked primer molecules to RNP2, low IQ values are in a range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high Kdvalues are in the range of 100 nM-100 μM (10 mM) and thus are about 105-to 1010-fold or higher as compared to low Kdvalues. As used herein, the terms “binding domain” or “binding site” refer to a region on a protein, DNA, or RNA, to which specific molecules and/or ions (ligands) may form a covalent or non-covalent bond. By way of example, a polynucleotide sequence present on a nucleic acid molecule (e.g., a primer binding domain) may serve as a binding domain for a different nucleic acid molecule (e.g., an unblocked primer nucleic acid molecule). Characteristics of binding sites are chemical specificity, a measure of the types of ligands that will bond, and affinity, which is a measure of the strength of the chemical bond. As used herein, the term “blocked nucleic acid molecule” refers to nucleic acid molecules that cannot bind to the first or second RNP complex (i.e., RNP1 or RNP2) to activate cis- or trans-cleavage. “Unblocked nucleic acid molecule” refers to a formerly blocked nucleic acid molecule that can bind to the second RNP complex (RNP2) to activate trans-cleavage of additional blocked nucleic acid molecules. A “blocked nucleic acid molecule” may be a “blocked primer molecule” in some embodiments of the cascade assay. The terms “Cos RNA-guided endonuclease” or “CRISPR nuclease” or “nucleic acid-guided nuclease” refer to a CRISPR-associated protein that is an RNA-guided endonuclease suitable for assembly with a sequence-specific gRNA to form a ribonucleoprotein (RNP) complex. As used herein, the terms “cis-cleavage”, “cis-endonuclease activity”, “cis-mediated endonuclease activity”, “cis-nuclease activity”, “cis-mediated nuclease activity”, and variations thereof refer to sequence-specific cleavage of a target nucleic acid of interest, including an unblocked nucleic acid molecule or synthesized activating molecule, by a nucleic acid-guided nuclease in an RNP complex. Cis-cleavage is a single turn-over cleavage event in that only one substrate molecule is cleaved per event. The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen-bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10, or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementary to the nucleotide sequence 5′-AGCT-3; and the nucleotide sequence 3′-ATCGAT-5′ is 100% complementary to a region of the nucleotide sequence 5′-GCTAGCTAG-3′. As used herein, the term “contacting” refers to placement of two moieties in direct physical association, including in solid or liquid form. Contacting can occur in vitro with isolated cells (for example in a tissue culture dish or other vessel) or in samples or in vivo by administering an agent to a subject. A “control” is a reference standard of a known value or range of values. The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) a crRNA region or guide sequence capable of hybridizing to the target strand of a target nucleic acid of interest, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease. The crRNA region of the gRNA is a customizable component that enables specificity in every nucleic acid-guided nuclease reaction. A gRNA can include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest to hybridize with the target nucleic acid of interest and to direct sequence-specific binding of a ribonucleoprotein (RNP) complex containing the gRNA and nucleic acid-guided nuclease to the target nucleic acid. Target nucleic acids of interest may include a protospacer adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region on the target nucleic acid of interest, including on an unblocked nucleic acid molecule or synthesized activating molecule. A gRNA may contain a spacer sequence including a plurality of bases complementary to a protospacer sequence in the target nucleic acid. For example, a spacer can contain about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more bases. The gRNA spacer may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its corresponding target nucleic acid of interest. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences. A guide RNA may be from about 20 nucleotides to about 300 nucleotides long. Guide RNAs may be produced synthetically or generated from a DNA template. “Modified” refers to a changed state or structure of a molecule. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, a nucleic acid molecule (for example, a blocked nucleic acid molecule) may be modified by the introduction of non-natural nucleosides, nucleotides, and/or internucleoside linkages. In another embodiment, a modified protein (e.g., a nucleic acid-guided nuclease) may refer to any polypeptide sequence alteration which is different from the wildtype. The terms “percent sequence identity”, “percent identity”, or “sequence identity” refer to percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence following alignment by standard techniques. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST, or MEGALIGN™ software. In some embodiments, the software is MUSCLE (Edgar, Nucleic Acids Res., 32(5):1792 1797 (2004)). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, in embodiments, percent sequence identity values are generated using the sequence comparison computer program BLAST (Altschul, et al., J. Mol. Biol., 215:403-410 (1990)). As used herein, the terms “preassembled ribonucleoprotein complex”, “ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complex containing a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to a target nucleic acid of interest, guides the RNP to the target nucleic acid of interest and hybridizes to it. The hybridized target nucleic acid-gRNA units are cleaved by the nucleic acid-guided nuclease. In the cascade assays described herein, a first ribonucleoprotein complex (RNP1) includes a first guide RNA (gRNA) specific to a nucleic acid target nucleic acid of interest, and a first nucleic acid-guided nuclease, such as, for example, cas 12a or cas 14a for a DNA target nucleic acid, or cas 13a for an RNA target nucleic acid. A second ribonucleoprotein complex (RNP2) for signal amplification includes a second guide RNA specific to an unblocked nucleic acid or synthesized activating molecule, and a second nucleic acid-guided nuclease, which may be different from or the same as the first nucleic acid-guided nuclease. As used herein, the terms “protein” and “polypeptide” are used interchangeably. Proteins may or may not be made up entirely of amino acids. As used herein, the term “sample” refers to tissues; cells or component parts; body fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. “Sample” may also refer to specimen or aliquots from food; agricultural products; pharmaceuticals; cosmetics, nutraceuticals; personal care products; environmental substances such as soil, water, air, or sewer sample; industrial sites and products; and chemicals and compounds. A sample further may include a homogenate, lysate or extract. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules. The terms “target DNA sequence”, “target sequence”, “target nucleic acid of interest”, “target molecule of interest”, “target nucleic acid”, or “target of interest” refer to any locus that is recognized by a gRNA sequence in vitro or in vivo. The “target strand” of a target nucleic acid of interest is the strand of the double-stranded target nucleic acid that is complementary to a gRNA. The spacer sequence of a gRNA may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or more complementary to the target nucleic acid of interest. Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences. Full complementarity is not necessarily required provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of an RNP complex. A target nucleic acid of interest can include any polynucleotide, such as DNA (ssDNA or dsDNA) or RNA polynucleotides. A target nucleic acid of interest may be located in the nucleus or cytoplasm of a cell such as, for example, within an organelle of a eukaryotic cell, such as a mitochondrion or a chloroplast, or it can be exogenous to a host cell, such as a eukaryotic cell or a prokaryotic cell. The target nucleic acid of interest may be present in a sample, such as a biological or environmental sample, and it can be a viral nucleic acid molecule, a bacterial nucleic acid molecule, a fungal nucleic acid molecule, or a polynucleotide of another organism, such as a coding or a non-coding sequence, and it may include single-stranded or double-stranded DNA molecules, such as a cDNA or genomic DNA, or RNA molecules, such as pre-mRNA, mRNA, tRNA, and rRNA. The target nucleic acid of interest may be associated with a protospacer adjacent motif (PAM) sequence, which may include a 2-5 base pair sequence adjacent to the protospacer. In some embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target nucleic acids can be detected by the disclosed method. As used herein, the terms “trans-cleavage”, “trans-endonuclease activity”, “trans-mediated endonuclease activity”, “trans-nuclease activity”, “trans-mediated nuclease activity” and variations thereof refer to indiscriminate, non-sequence-specific cleavage of a nucleic acid molecule by an endonuclease (such as by a Cas12, Cas13, and Cas14) which is triggered by cis-(sequence-specific) cleavage. Trans-cleavage is a “multiple turn-over” event, in that more than one substrate molecule is cleaved after initiation by a single turn over cis-cleavage event. Type V CRISPR/Cas nucleic acid-guided nucleases are a subtype of Class 2 CRISPR/Cas effector nucleases such as, but not limited to, engineered Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), CasY (Cas12d), Cas13a nucleases or naturally-occurring proteins, such as a Cas12a isolated from, for example,Francisella tularensissubsp.novicida(Gene ID: 60806594),Candidatus Methanoplasma termitum(Gene ID: 24818655),Candidatus Methanomethylophilus alvus(Gene ID: 15139718), andEubacteriumeligens ATCC 27750 (Gene ID: 41356122), and an artificial polypeptide, such as a chimeric protein. The term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many if not most regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A variant of a polypeptide may be a conservatively modified variant. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code (e.g., a non-natural amino acid). A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Variants include modifications-including chemical modifications—to one or more amino acids that do not involve amino acid substitutions, additions or deletions. A “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like. DETAILED DESCRIPTION The present disclosure provides compositions of matter and cascade assay methods for detecting nucleic acids where the compositions of matter allow for the reaction kinetics of the cascade assay to be adjusted or “tuned.” The compositions and methods provide for massive multiplexing, high accuracy, low cost, minimum workflow, with results in some embodiments virtually instantaneously, even at ambient temperatures of 16-25° C. or less, or, if desired, with slower reaction times but with the ability to quantify the target nucleic acids of interest with exquisite accuracy. The cascade assays described herein comprise first and second ribonucleoprotein complexes and either blocked nucleic acid molecules or blocked primer molecules. The blocked nucleic acid molecules or blocked primer molecules keep the second ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex, and the molecular design or configuration of the blocked nucleic acid molecules (or blocked primer molecules) confers the “tunability” to the cascade assay. Again, by “locked” it is meant that the blocked nucleic acid molecules or blocked primer molecules are designed in such a way that they are largely blocked from interacting with the ribonucleoprotein complexes; therefore, the ribonucleoprotein complexes remain largely inactive (i.e., “locked”) unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex The methods comprise the steps of providing cascade assay components, contacting the cascade assay components with a sample, and detecting a signal that is generated only when a target nucleic acid of interest is present in the sample. Early and accurate identification of, e.g., infectious agents, microbe contamination, variant nucleic acid sequences that indicate the presence of such diseases such as cancer or contamination by heterologous sources is important in order to select correct treatment; identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment. However, currently available state-of-the-art nucleic acid detection such as quantitative PCR (also known as real time PCR or qPCR) relies on DNA amplification, which requires time and may lead to changes to the relative proportion of nucleic acids, particularly in multiplexed nucleic acid assays. The lack of rapidity for qPCR assays is due to the fact that there is a significant lag phase early in the amplification process where fluorescence above background cannot be detected. That is, there is a lag until the cycle threshold or Ct value, which is the number of amplification cycles required for the fluorescent signal to exceed the background level of fluorescence, is achieved and can be quantified. The present disclosure describes a signal boost cascade assay and improvements thereto that can detect one or more target nucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex amplification, such as primer-dimerization. In addition, the cascade assay is tunable, such that in some embodiments detection of target nucleic acids of interest can happen virtually instantaneously, or, alternatively, over a longer period of time. Additionally, the cascade assay can be tuned—via varying the molecular configuration of the blocked nucleic acid molecules or blocked primer molecules—to quantify the target nucleic acids of interest over a desired range of concentration; thus providing flexibility for virtually any application. As described in detail below, the cascade assays utilize signal amplification mechanisms comprising various components including nucleic acid-guided nucleases, guide RNAs (gRNAs) incorporated into ribonucleoprotein complexes (RNP complexes), blocked nucleic acid molecules or blocked primer molecules, reporter moieties, and, in some embodiments, polymerases and template molecules where the polymerases copy but do not amplify the template molecules. A particularly advantageous feature of the cascade assay is that, with the exception of the gRNA (gRNA1) in RNP1, the cascade assay components can be essentially identical no matter what target nucleic acid(s) of interest are being detected, and gRNA1 is easily programmable. Further, in the context of tunability, the cascade assay is tunable by use of different blocked nucleic acid molecules (or blocked primer molecules) used to activate RNP2 (described in detail below). The improvement to the signal amplification or signal boost cascade assay described herein is drawn to being able to “tune” the cascade assay by employing differently configured blocked nucleic acid molecules (or blocked primer molecules) that activate RNP2. The present disclosure demonstrates that by altering the Gibbs free energy (i.e., molecular configuration and composition) of the blocked nucleic acid molecules (or blocked primer molecules) employed in the cascade assay, the kinetics of the cascade assay can be “tuned.” FIG.1Aprovides a simplified diagram demonstrating a prior art method for quantifying target nucleic acids of interest in a sample; namely, the quantitative polymerase chain reaction or qPCR, which to date may be considered the gold standard for quantitative detection assays. The difference between PCR and qPCR is that PCR is a qualitative technique that indicates the presence or absence of a target nucleic acid of interest in a sample, where qPCR allows for quantification of target nucleic acids of interest in a sample. qPCR involves selective amplification and quantitative detection of specific regions of DNA or cDNA (i.e., the target nucleic acid of interest) using oligonucleotide primers that flank the specific region(s) in the target nucleic acid(s) of interest. The primers are used to amplify the specific regions using a polymerase. Like PCR, repeated cycling of the amplification process leads to an exponential increase in the number of copies of the region(s) of interest; however, unlike traditional PCR, the increase is tracked using an intercalating dye or, as shown inFIG.1A, a sequence-specific probe (e.g., a “Taq-man probe”) the fluorescence of which is detected in real time. RT-qPCR differs from qPCR in that a reverse transcriptase is used to first copy RNA molecules to produce cDNA before the qPCR process commences. FIG.1Ais an overview of a qPCR assay where target nucleic acids of interest from a sample are amplified before detection.FIG.1Ashows the qPCR method (10), comprising a double-stranded DNA template (12) and a sequence-specific Taq-man probe (14) comprising a region complementary to the target nucleic acid of interest (20), a quencher (16), a quenched fluorophore (18) where (22) denotes quenching between the quencher (18) and quenched fluorophore (16). Upon denaturation, the two strands of the double-stranded DNA template (12) separate into complementary single strands (26) and (28). In the next step, primers (24) and (24′), anneal to complementary single strands (26) and (28), as does the sequence-specific Taq-man probe (14) via the region complementary (20) to complementary strand (26). Initially the Taq-man probe is annealed to complementary strand (26) of the target region of interest intact; however, primers (24) and (24′) are extended by polymerase (30) forming a complement (32) of complementary strand (26); however, the Taq-man probe is not, due to the absence of a 3′ hydroxy group. Instead, the exonuclease activity of the polymerase “chews up” the Taq-man probe, thereby separating the quencher (16) from the quenched fluorophore (18) resulting in an unquenched or excited-state fluorophore (34). The fluorescence quenching ensures that fluorescence occurs only when target nucleic acids of interest are present and being copied, where the fluorescent signal is proportional to the number of single strand target nucleic acids being amplified. As noted above, one downside to currently available detection assays is that they rely on DNA amplification, which, in addition to issues with multiplexing, significantly hinders the ability to perform rapid testing, e.g., in the field, where the present cascade assay works at ambient temperatures, including room temperature and below. Assays that require amplification of the target nucleic acids of interest do not work well at lower temperatures—even those assays utilizing isothermal amplification—due to non-specific binding of the primers and low polymerase activity. Further, primer design is far more challenging. As for the lack of rapidity of qPCR, a significant lag phase occurs early in the amplification process where fluorescence above background cannot be detected, particularly in samples with very low copy numbers of the target nucleic acid of interest. And, again, amplification, particularly multiplex amplification, may cause changes to the relative proportion of nucleic acids in samples that, in turn, lead to artifacts or inaccurate results. A second downside to PCR is that reaction kinetics are defined by primer binding efficiency and the rate of primer extension by the polymerase. The reaction temperature for a PCR reaction is typically equal to the Tmof the primer plus 5° C. This temperature cannot be altered significantly without either decreasing the amount of primer that binds the target nucleic acids of interest or increasing non-specific or mis-priming events. Thus, essentially qPCR cannot be tuned to vary reaction time or to quantify target nucleic acids of interest within a specific window. Another downside to PCR includes complex temperature cycling (e.g., 95° C. for denaturing, at least 5° C. below Tmfor annealing, and at least 5° C. above Tmfor extension), which in turn is dependent on the PCR reagents (e.g., primer concentration, primer length, polymerase half-life, and the polymerase's rate of polymerization). FIG.1Bprovides a simplified diagram demonstrating a prior art method (51) of a CRISPR-based nucleic acid-guided nuclease detection assay where target nucleic acids of interest from a sample must be amplified in order to be detected, which, like qPCR is not tunable kinetically except via reaction temperature. First, assuming the presence of a target nucleic acid of interest in a sample, the target nucleic acid of interest (52) is amplified to produce many copies of the target nucleic acid of interest (54). The detection assay is initiated (step2) when the target nucleic acid of interest (54) is combined with and binds to a pre-assembled ribonucleoprotein complex (56), which is part of a reaction mixture. The ribonucleoprotein complex (56) comprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to the target nucleic acid of interest, guides the RNP complex to the target nucleic acid of interest and hybridizes to it, thereby activating the ribonucleoprotein complex (58). The nucleic acid-guided nuclease exhibits (i.e., possesses) both cis- and trans-cleavage activity, where trans-cleavage activity is initiated after cis-cleavage activity, or at least upon specific binding of N nucleotide bases of a target nucleic acid molecule to the ribonucleoprotein complex. Cis-cleavage activity occurs as the target nucleic acid of interest binds to the gRNA and is cleaved by the nucleic acid-guided nuclease (i.e., activation). Once an initial cis-cleavage of the target nucleic acid of interest is completed, trans-cleavage activity is triggered, where trans-cleavage activity is an indiscriminate, non-sequence-specific, and multi-turnover cleavage event of nucleic acid molecules in the sample. In step3, the trans-cleavage activity triggers activation of reporter moieties (62) that are present in the reaction mixture. The reporter moieties (62) may be a synthetic molecule linked or conjugated to a quencher (64) and a fluorophore (66) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The quencher (64) and fluorophore (66) typically are about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Reporter moieties (62) are described in greater detail below. As more ribonucleoprotein complexes (56) are activated (56→58), more trans-cleavage activity of the nucleic acid-guided nuclease in the ribonucleoprotein complex is activated and more reporter moieties (68) are activated (where here, “activated” means unquenched); thus, the binding of the target nucleic acid of interest (54). The signal change (70) increases as more reporter moieties (68) are activated. As noted above, the downside to currently available nucleic acid-guided nuclease detection assays is that they rely on DNA amplification, which, in addition to issues with multiplexing, significantly hinders the ability to perform rapid point-of-care testing. The lack of rapidity is, at least in-part, due to cis-cleavage of a target nucleic acid of interest being a single turnover event in which the number of activated enzyme complexes is, at most, equal to the number of copies of the target nucleic acids of interest in the sample; thus, PCR amplification affects the rapidity of currently available nucleic acid-guided nuclease detection systems. Once the ribonucleoprotein complex is activated after completion of cis-cleavage, trans-cleavage activity of the reporter moieties that are initially quenched is generated. However, the turnover (K) of, e.g., activated Cas12a complex is 17/sec and 3/sec for dsDNA and ssDNA targets, respectively. Therefore, for less than 10,000 target copies, the number of reporters cleaved is not sufficient to generate a signal in less than 30-60 minutes. Thus, like qPCR, a typical CRISPR-based nucleic acid-guided nuclease detection assay cannot be tuned to vary reaction times or to quantify target nucleic acids of interest over a specific concentration window. In contrast, the cascade assay described herein which utilizes two ribonucleoprotein (RNP) complexes can be tuned, allowing for maximum flexibility.FIG.1Cprovides a simplified diagram demonstrating a method (100) of a cascade assay. The cascade assay is initiated when the target nucleic acid of interest (104) binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1) (102). A ribonucleoprotein complex comprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to the target nucleic acid of interest, guides an RNP complex to the target nucleic acid of interest and hybridizes to it. Typically, preassembled RNP complexes are employed in the reaction mixture—as opposed to separate nucleic acid-guided nucleases and gRNAs—to facilitate rapid (virtually instantaneous) detection of the target nucleic acid(s) of interest, if desired. “Activation” of RNP1 (106) in the context of the cascade assay refers to activating trans-cleavage activity of the nucleic acid-guided nuclease in RNP1 (106) by first initiating cis-cleavage where the target nucleic acid of interest is cleaved by the nucleic acid-guided nuclease, or at least upon specific binding of N nucleotide bases of a target nucleic acid molecule to the ribonucleoprotein complex. This cis-cleavage activity then initiates trans-cleavage activity (i.e., multi-turnover activity) of the nucleic acid-guided nuclease, where trans-cleavage is indiscriminate, leading to non-sequence-specific cutting of nucleic acid molecules by the nucleic acid-guided nuclease of RNP1 (102). This trans-cleavage activity triggers activation of blocked ribonucleoprotein complexes (RNP2s) (108) via blocked nucleic acid molecules (or in an alternative embodiment, blocked primer molecules), which are described in detail below. Each newly activated RNP2 (110) activates more RNP2 (108→110), which in turn cleave reporter moieties (112). The reporter moieties (112) may be a synthetic molecule linked or conjugated to a quencher (114) and a fluorophore (116) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The quencher (114) and fluorophore (116) can be about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Reporter moieties may also be incorporated into blocked nucleic acid molecules or blocked primer molecules—which also affects the kinetics of the cascade assay reaction—and are described in greater detail below. As more RNP2s are activated (108→110), more trans-cleavage activity is activated and more reporter moieties (118) are unquenched; thus, the binding of the target nucleic acid of interest (104) to RNP1 (102) initiates what becomes a cascade of signal production (120), which increases exponentially, hence, the terms signal amplification or signal boost. The cascade assay thus comprises a single turnover event that triggers a multi-turnover event that then triggers another multi-turnover event. As described below in relation toFIG.4, the reporter moieties (112) may be provided as molecules that are separate from the other components of the nucleic acid-guided nuclease cascade assay, or the reporter moieties may be covalently or non-covalently linked to the blocked nucleic acid molecules or synthesized activating molecules (i.e., the target molecules for the RNP2). As described in detail below, the present description presents blocked nucleic acid molecules, which can be “tuned” to provide varying reaction kinetics for the cascade assay. Target Nucleic Acids of Interest The target nucleic acid of interest may be a DNA, RNA, or cDNA molecule. Target nucleic acids of interest may be isolated from a sample or organism by standard laboratory techniques or may be synthesized by standard laboratory techniques (e.g., RT-PCR). The target nucleic acids of interest are identified in a sample, such as a biological sample from a subject (including non-human animals or plants), items of manufacture, or an environmental sample (e.g., water or soil). Non-limiting examples of biological samples include blood, serum, plasma, saliva, mucus, a nasal swab, a buccal swab, a cell, a cell culture, and tissue. The source of the sample could be any mammal, such as, but not limited to, a human, primate, monkey, cat, dog, mouse, pig, cow, horse, sheep (and other livestock), and bat. Samples may also be obtained from any other source, such as air, water, soil, surfaces, food, beverages, nutraceuticals, clinical sites or products, industrial sites and products, plants and grains, cosmetics, personal care products, pharmaceuticals, medical devices, agricultural equipment and sites, and commercial samples. In some embodiments, the target nucleic acid of interest is from an infectious agent (e.g., a bacteria, protozoan, insect, worm, virus, or fungus) that affects mammals. As a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from bacteria, such asBordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Acinetobacter calcoaceticus-baumanniicomplex,Bacteroides fragilis, Enterobacter cloacaecomplex,Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniaegroup,Moraxella catarrhalis, Proteusspp.,Salmonella enterica, Serratia marcescens, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydia tracomatis, Neisseria gonorrhoeae, Syphilis (Treponema pallidum),Ureaplasma urealyticum, Mycoplasma genitalium, and/orGardnerella vaginalis. As a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a virus, such as adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus 0C43, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human metapneumovirus, human rhinovirus, enterovirus, influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus, herpes simplex virus 1, herpes simplex virus 2, human immunodeficiency virus (HIV), human papillomavirus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and/or human parvovirus B19 (B19V). Also, as a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a fungus, such asCandida albicans, Candida auris, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and/orCryptococcus gattii. As another non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a protozoan, such asTrichomonas vaginalis, Bonamia exitiosa, Bonamia ostreae, Leishmania amazonensis, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania major, Leishmania mexicana, Leishmania tropica, Marteilia refringens, Perkinsus marinus, Perkinsus olseni, Theileria annulata, Theileria equi, Theileria parva, Tritrichomonas foetus, Trypanosoma brucei, Trypanosoma congolense, Trypanosoma equiperdum, Trypanosoma evansiand,Trypanosoma vivax. Additionally, the target nucleic acid of interest may originate in an organism such as a bacterium, virus, fungus or other pest that infects livestock or agricultural crops. Such organisms include avian influenza viruses,mycoplasmaand other bovine mastitis pathogens,Clostridium perfringens, Campylobactersp.,Salmonellasp., Pospirivoidae, Avsunvirodiae,Panteoea stewartii, Mycoplasma genitalium, Sprioplasmasp.,Pseudomonas solanacearum, Erwinia amylovora, Erwinia carotovora, Pseudomonas syringae, Xanthomonas campestris, Agrobacterium tumefaciens, Spiroplasma citri, Phytophthora infestans, Endothia parasitica, Ceratocysis ulmi, Puccinia graminis, Hemilea vastatrix, Ustilage maydis, Ustilage nuda, Guignardia bidwellii, Uncinula necator, Botrytis cincerea, Plasmopara viticola, orBotryotinis fuckleina. In some embodiments, other target nucleic acids of interest may be for non infectious conditions, e.g., to be used for genotyping, including non-invasive prenatal diagnosis of, e.g, trisomies, other chromosomal abnormalities, and known genetic diseases such as Tay Sachs disease and sickle cell anemia. Other target nucleic acids of interest and samples are described herein. Target nucleic acids of interest may include engineered biologics, including cells such as chimeric antigen receptor T (CAR-T) cells, or target nucleic acids of interest from very small or rare samples, where only small volumes are available for testing. The cascade assays described herein are particularly well-suited for simultaneous testing of multiple targets. Pools of two to 10,000 target nucleic acids of interest may be employed, e.g., 2-1000, 2-100, 2-50, or 2-10. Further testing may be used to identify the specific member of the pool, if warranted. While the methods described herein do not require the target nucleic acid of interest to be DNA (and in fact it is specifically contemplated that the target nucleic acid of interest may be RNA), it is understood by those in the field that a reverse transcription step to convert target RNA to cDNA may be performed prior to or while contacting the biological sample with the composition. Alternatively, RNA target nucleic acids of interest can be detected directly via RNA-specific nucleic acid nucleases such as Cas13a or Cas12g. Nucleic Acid-Guided Nucleases The cascade assays comprise nucleic acid-guided nucleases in the reaction mixture, either provided as a protein, a coding sequence for the protein, or, in many embodiments, in a pre-assembled ribonucleoprotein (RNP) complex. In some embodiments, the one or more nucleic acid-guided nucleases in the reaction mixture may be, for example, a Cas endonuclease. Any nucleic acid-guided nuclease having both cis- and trans-endonuclease activity may be employed, and the same nucleic acid-guided nuclease may be used for both RNP complexes or different nucleic acid-guided nucleases may be used in RNP1 and RNP2. Note that trans-cleavage activity is not triggered unless and until cis-cleavage activity (i.e., sequence-specific activity) is initiated. Nucleic acid-guided nucleases include Type V and Type VI nucleic acid-guided nucleases, as well as nucleic acid-guided nucleases that comprise a RuvC nuclease domain or a RuvC-like nuclease domain but lack an HNH nuclease domain. Nucleic acid-guided nucleases with these properties are reviewed in Makarova and Koonin, Methods Mol. Biol., 1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology, 37:67-78 (2020) and updated databases of nucleic acid-guided nucleases and nuclease systems that include newly-discovered systems include BioGRID ORCS (orcs:thebiogrid.org); GenomeCRISPR (genomecrispr.org); Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder (crispercas.i2bc.paris-saclay.fr). The type of nucleic acid-guided nuclease utilized in the method of detection depends on the type of target nucleic acid of interest to be detected. For example, a DNA nucleic acid-guided nuclease (e.g., a Cas12a, Cas14a, or Cas3) should be utilized if the target nucleic acid of interest is a DNA molecule, and an RNA nucleic acid-guided nuclease (e.g., Cas13a or Cas12g) should be utilized if the target nucleic acid of interest is an RNA molecule. Exemplary nucleic acid-guided nucleases include, but are not limited to, Cas RNA-guided DNA endonucleases, such as Cas3, Cas12a (e.g., AsCas12a, LbCas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, and Cas12j; Cas RNA-guided RNA endonucleases, such as Cas13a (LbaCas13, LbuCas13, LwaCas13), Cas13b (e.g., CccaCas13b, PsmCas13b), and Cas12g; and any other nucleic acid (DNA, RNA, or cDNA) targeting nucleic acid-guided nuclease with cis-cleavage activity and collateral trans-cleavage activity. In some embodiments, the nucleic acid-guided nuclease is a Type V CRISPR-Cas nuclease, such as a Cas12a, Cas13a, or Cas14a. In some embodiments, the nucleic acid-guided nuclease is a Type I CRISPR-Cas nuclease, such as Cas3. Type II and Type VI nucleic acid-guided nucleases may also be employed. Guide RNA (gRNA) The present disclosure detects a target nucleic acid of interest via a reaction mixture containing at least two guide RNAs (gRNAs) each incorporated into an RNP complex (i.e., RNP1 or RNP2). Suitable gRNAs include at least one crRNA region to enable specificity in every reaction. The gRNA of RNP1 is specific to a target nucleic acid of interest and the gRNA of RNP2 is specific to an unblocked nucleic acid or a synthesized activating molecule (both described in detail below). As will be clear given the description below, an advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (i.e., the gRNA specific to the target nucleic acid of interest), the cascade assay components can stay the same (i.e., are identical or substantially identical) no matter what target nucleic acid(s) of interest are being detected, and the gRNA in RNP1 is easily reprogrammable. In the context of tunability, the cascade assay is tunable by use of blocked nucleic acid molecules or blocked primer molecules having various molecular configurations (i.e., free energies). Once desired reaction kinetics and/or a target quantification window is identified, this particular version of the cascade assay can be reprogrammed by changing the gRNA in RNP1. Like the nucleic acid-guided nuclease, the gRNA may be provided in the cascade assay reaction mixture in a preassembled RNP, as an RNA molecule, or may also be provided as a DNA sequence to be transcribed, in, e.g., a vector backbone. Providing the gRNA in a pre-assembled RNP complex (i.e., RNP1 or RNP2) is preferred if rapid assay kinetics are preferred. If provided as a gRNA molecule, the gRNA sequence may include multiple endoribonuclease recognition sites (e.g., Csy4) for multiplex processing. Alternatively, if provided as a DNA sequence to be transcribed, an endoribonuclease recognition site is encoded between neighboring gRNA sequences and more than one gRNA can be transcribed in a single expression cassette. Direct repeats can also serve as endoribonuclease recognition sites for multiplex processing. Guide RNAs are generally about 20 nucleotides to about 300 nucleotides in length and may contain a spacer sequence containing a plurality of bases and complementarity to a protospacer sequence in the target sequence. The gRNA spacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its intended target nucleic acid of interest. The gRNA of RNP1 is capable of complexing with the nucleic acid-guided nuclease of RNP1 to perform cis-cleavage of a target nucleic acid of interest (i.e., a DNA or RNA), which triggers non-sequence-specific trans-cleavage of other molecules in the reaction mixture. Guide RNAs include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest (or target sequences generated by unblocking blocked nucleic acid molecules or target sequences generated by synthesizing activating molecules as described below). Target nucleic acids of interest may include a protospacer-adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region of the target nucleic acid of interest. In some embodiments, the gRNA (e.g., of RNP1) is an exo-resistant circular molecule that can include several DNA bases between the 5′ end and the 3′ end of a natural guide RNA and is capable of binding a target sequence. The length of the circularized guide for RNP1 can be such that the circular form of guide can be complexed with a nucleic acid-guided nuclease to form a modified RNP1 which can still retain its cis-cleavage i.e., (specific) and trans-cleavage (i.e., non-specific) nuclease activity. In any of the foregoing embodiments, the gRNA may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the gRNAs of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). By way of further example, a modified nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described herein. Ribonucleoprotein (RNP) Complex As described above, although the assay “reaction mixture” may comprise separate nucleic acid-guided nucleases and gRNAs (or coding sequences therefor), the cascade assays preferably comprise preassembled ribonucleoprotein complexes (RNPs) in the reaction mixture, allowing for faster detection kinetics. The present cascade assay employs at least two types of RNP complexes, RNP1 and RNP2, each type containing a nucleic acid-guided nuclease and a gRNA. RNP1 and RNP2 may comprise the same nucleic acid-guided nuclease or may comprise different nucleic acid-guided nucleases; however, the gRNAs in RNP1 and RNP2 are different and are configured to detect different nucleic acids. In some embodiments, the reaction mixture contains about 1 fM to about 10 μM of a given RNP1, or about 1 pM to about 1 μM of a given RNP1, or about 10 pM to about 500 pM of a given RNP1. In some embodiments the reaction mixture contains about 6×104to about 6×1012complexes per microliter (μl) of a given RNP1, or about 6×106to about 6×1010complexes per microliter (μl) of a given RNP1. In some embodiments, the reaction mixture contains about 1 fM to about 500 μM of a given RNP2, or about 1 pM to about 250 μM of a given RNP2, or about 10 pM to about 100 μM of a given RNP2. In some embodiments the reaction mixture contains about 6×104to about 6×1012complexes per microliter (μl) of a given RNP2 or about 6×106to about 6×1012complexes per microliter (μl) of a given RNP2. In any of the embodiments of the disclosure, the reaction mixture includes 1 to about 1,000 different RNP1s (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,0000 RNP1s), where different RNP1s comprise a different gRNA (or crRNA thereof) polynucleotide sequence. For example, a reaction mixture designed for environmental or oncology testing comprises more than one unique RNP1-gRNA (or RNP1-crRNA) ribonucleoprotein complex for the purpose of detecting more than one target nucleic acid of interest. That is, more than one RNP1 may be present for the purpose of targeting one target nucleic acid of interest from many sources or more than one RNP1 may be present for targeting more than one target nucleic acid of interest from a single organism or condition. In any of the foregoing embodiments, the gRNA of RNP1 may be homologous or heterologous, relative to the gRNA of other RNP1(s) present in the reaction mixture. A homologous mixture of RNP1 gRNAs has a number of gRNAs with the same nucleotide sequence, whereas a heterologous mixture of RNP1 gRNAs has multiple gRNAs with different nucleotide sequences (e.g., gRNAs targeting different loci, genes, variants, and/or microbial species). Therefore, the disclosed methods of identifying one or more target nucleic acids of interest may include a reaction mixture containing more than two heterologous gRNAs, more than three heterologous gRNAs, more than four heterologous gRNAs, more than five heterologous gRNAs, more than six heterologous gRNAs, more than seven heterologous gRNAs, more than eight heterologous gRNAs, more than nine heterologous gRNAs, more than ten heterologous gRNAs, more than eleven heterologous gRNAs, more than twelve heterologous gRNAs, more than thirteen heterologous gRNAs, more than fourteen heterologous gRNAs, more than fifteen heterologous gRNAs, more than sixteen heterologous gRNAs, more than seventeen heterologous gRNAs, more than eighteen heterologous gRNAs, more than nineteen heterologous gRNAs, more than twenty heterologous gRNAs, more than twenty-one heterologous gRNAs, more than twenty-three heterologous gRNAs, more than twenty-four heterologous gRNAs, or more than twenty-five heterologous gRNAs. Such a heterologous mixture of RNP1 gRNAs in a single reaction enables multiplex testing. As a first non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNP1s having a gRNA targeting parainfluenza virus 1; a number of RNP1 s having a gRNA targeting human metapneumovirus; a number of RNP1s having a gRNA targeting human rhinovirus; a number of RNP1 s having a gRNA targeting human enterovirus; a number of RNP1 having a gRNA targeting respiratory syncytial virus; and a number of RNP1s having a gRNA targeting coronavirus HKU1. As a second non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNP1 s containing a gRNA targeting two or more SARS-Co-V-2 variants, e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 and subvariants thereof. As another non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain RNP1 s targeting two or more target nucleic acids of interest from, e.g., organisms that infect vineyards, such asGuignardia bidwellii, Uncinula necator, Botrytis cincerea, Plasmopara viticola, andBotryotinis fuckleina. Reporter Moieties The cascade assay detects a target nucleic acid of interest via detection of a signal generated in the reaction mixture by a reporter moiety. In some embodiments the detection of the target nucleic acid of interest occurs virtually instantaneously at 3M or 30 copies and within 1 minute or less at 3 copies (see, e.g.,FIGS.6B-6H). Depending on the type of reporter moiety used, trans- and/or cis-cleavage by the nucleic acid-guided nuclease in RNP2 releases a signal. In some embodiments, trans-cleavage of stand-alone (e.g., not bound to any blocked nucleic acid molecules) reporter moieties may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time (shown at bottom inFIGS.2A,3A and3B). Trans-cleavage by either an activated RNP1 or an activated RNP2 may release a signal; thus, when the reporter moiety is a separate molecule, the reporter moieties are activated quickly by the trans-cleavage activity. The reporter moiety can comprise DNA, RNA, a chimera of DNA and RNA, or an oligonucleotide with modified nucleic acids. The reporter moiety also can comprise both single- and double-stranded portions. In alternative embodiments and preferably, the reporter moiety may be bound to the blocked nucleic acid molecule, where trans-cleavage of the blocked nucleic acid molecule and conversion to an unblocked nucleic acid molecule may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time, thus allowing for real time reporting of results (shown atFIG.4, center). In this embodiment, the reaction kinetics of signal generation match that of the cascade assay reaction rate. The signal is generated as the blocked nucleic acid molecule is unblocked, whether quickly or slowly. In yet another embodiment, the reporter moiety may be bound to a blocked nucleic acid molecule such that cis-cleavage following the binding of the RNP2 to an unblocked nucleic acid molecule releases a PAM distal sequence, which in turn generates a signal at rates that are proportional to the cleavage rate (shown atFIG.4, bottom). In this case, activation of RNP2 by cis-(target specific) cleavage of the unblocked nucleic acid molecule directly produces a signal, rather than producing a signal via indiscriminate trans-cleavage activity. Alternatively or in addition, the reporter moiety may be bound to the gRNA. The reporter moiety may be a synthetic molecule linked or conjugated to a reporter and quencher such as, for example, a TAQMAN® probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The reporter and quencher may be about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Alternatively, signal generation may occur through different mechanisms. Other detectable moieties, labels, or reporters can also be used to detect a target nucleic acid of interest as described herein. Reporter moieties can be labeled in a variety of ways, including direct or indirect attachment of a detectable moiety such as a fluorescent moiety, hapten, or colorimetric moiety. Examples of detectable moieties include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, and protein-protein binding pairs, e.g., protein-antibody binding pairs. Examples of fluorescent moieties include, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansyl chloride, phycocyanin, and phycoerythrin. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, and aequorin. Examples of enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucorinidases, phosphatases, peroxidases, and cholinesterases. Identifiable markers also include radioactive elements such as125I,35S,14C, or3H. Reporters can also include a change in pH or charge of the cascade assay reaction mixture. The methods used to detect the generated signal will depend on the reporter moiety or moieties used. For example, a radioactive label can be detected using a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging. Fluorescent labels can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Simple colorimetric labels can be detected by observing the color associated with the label. When pairs of fluorophores are used in an assay, fluorophores are chosen that have distinct emission patterns (wavelengths) so that they can be easily distinguished. In some embodiments, the signal can be detected by lateral flow assays (LFAs). Lateral flow tests are simple devices intended to detect the presence or absence of a target nucleic acid of interest in a sample. LFAs can use nucleic acid molecules conjugated nanoparticles (often gold, e.g., RNA-AuNPs or DNA-AuNPs) as a detection probe, which hybridizes to a complementary target sequence. (SeeFIG.5and the description thereof below.) The classic example of an LFA is the home pregnancy test. Single-stranded nucleic acid reporter moieties such as ssDNA reporter moieties or RNA molecules can be introduced to show a signal change proportional to the cleavage rate, which increases with every new activated RNP2 complex over time. In some embodiments and as described in detail below, single-stranded nucleic acid reporter moieties can also be embedded into the blocked nucleic acid molecules for real time reporting of results. For example, the method of detecting a target nucleic acid molecule in a sample using a cascade assay as described herein can involve contacting the reaction mixture with a labeled detection ssDNA containing a fluorescent resonance energy transfer (FRET) pair, a quencher/phosphor pair, or both. A FRET pair consists of a donor chromophore and an acceptor chromophore, where the acceptor chromophore may be a quencher molecule. FRET pairs (donor/acceptor) suitable for use include, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL, fluorescein/QSY™ (succinimidyl ester)-7, fluorescein/LC Red 640, fluorescein/Cy 5.5, Texas Red/DABCYL, BODIPY™ (4,4-difluoro-4-bora-3A,4A-diaza-s-indacene)/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL, and fluorescein/LC Red 705. In addition, a fluorophore/quantum dot donor/acceptor pair can be used. EDANS (5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is 5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid); DABCYL is 4-(4-dimethylaminophenyl) diazenylbenzoic acid. Useful quenchers include, but are not limited to, DABCYL, QSY™ (succinimidyl ester) 7 and QSY™ (succinimidyl ester) 33. In any of the foregoing embodiments, the reporter moiety may comprise one or more modified nucleic acid molecules, containing a modified nucleoside or nucleotide. In some embodiments the modified nucleoside or nucleotide is chosen from 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described below. Nucleic Acid Modifications For any of the nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, template molecules, synthesized activating molecules, and reporter moieties), the nucleic acid molecules may be used in a wholly or partially modified form. Typically, modifications to the blocked nucleic acids, gRNAs, template molecules, reporter moieties, and blocked primer molecules described herein are introduced to optimize the molecule's biophysical properties (e.g., increasing endonuclease resistance and/or increasing thermal stability). Modifications typically are achieved by the incorporation of, for example, one or more alternative nucleosides, alternative sugar moieties, and/or alternative internucleoside linkages. For example, one or more of the cascade assay components may include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and/or 3-deazaguanine and 3-deazaadenine. The nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, reporter molecules, synthesized activating molecules, and template molecules) may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and/or 2-pyridone. Further modification of the nucleic acid molecules described herein may include nucleobases disclosed in U.S. Pat. No. 3,687,808; Kroschwitz, ed.,The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch, et al., Angewandte Chemie, 30:613 (1991); and Sanghvi, Chapter 16, Antisense Research and Applications, CRC Press, Gait, ed., 1993, pp. 289-302. In addition to or as an alternative to nucleoside modifications, the cascade assay components may comprise 2′ sugar modifications, including 2′-O-methyl (2′-O-Me), 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE), 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2group, also known as 2′-DMAOE, and/or 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH2OCH2N(CH3)2. Other possible 2′-modifications that can modify the nucleic acid molecules described herein (i.e., blocked nucleic acids, gRNAs, synthesized activating molecules, reporter molecules, and blocked primer molecules) may include all possible orientations of OH; F; O-, S-, or N-alkyl (mono- or di-); O-, S-, or N-alkenyl (mono- or di-); O-, S- or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (—OCH2CH2CH2NH2), ally (—CH2—CH═CH2), —O-ally (—O—CH2—CH═CH2) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the interfering RNA molecule, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Finally, modifications to the cascade assay components may comprise internucleoside modifications such as phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. The Signal Boosting Cascade Assay Employing Blocked Nucleic Acids Before getting to the details relating to tuning the kinetics of the cascade assay via the blocked nucleic acid molecules (or blocked primer molecules), understanding the cascade assay itself is key.FIG.1C, described above, depicts the cascade assay generally. A specific embodiment of the cascade assay utilizing blocked nucleic acids is depicted inFIG.2Aand described in detail below. In this embodiment, a blocked nucleic acid is used to prevent the activation of RNP2 in the absence of a target nucleic acid of interest. The method (200) inFIG.2Abegins with providing the cascade assay components RNP1 (201), RNP2 (202) and blocked nucleic acid molecules (203). RNP1 (201) comprises a gRNA specific for a target nucleic acid of interest and a nucleic acid-guided nuclease (e.g., Cas12a or Cas14 for a DNA target nucleic acid of interest or a Cas 13a for an RNA target nucleic acid of interest) and RNP2 (202) comprises a gRNA specific for an unblocked nucleic acid molecule and a nucleic acid-guided nuclease (again, Cas12a or Cas14 for a DNA unblocked nucleic acid molecule or a Cas13a for an RNA unblocked nucleic acid molecule). As described above, the nucleic acid-guided nucleases in RNP1 (201) and RNP2 (202) can be the same or different depending on the type of target nucleic acid of interest and unblocked nucleic acid molecule. What is key, however, is that the nucleic acid-guided nucleases in RNP1 and RNP2 may be activated to have trans-cleavage activity following initiation of cis-cleavage activity. In a first step, a sample comprising a target nucleic acid of interest (204) is added to the cascade assay reaction mixture. The target nucleic acid of interest (204) combines with and activates RNP1 (205) but does not interact with or activate RNP2 (202). Once activated, RNP1 cuts the target nucleic acid of interest (204) via sequence-specific cis-cleavage, which then activates non-specific trans-cleavage of other nucleic acids present in the reaction mixture, including the blocked nucleic acid molecules (203). At least one of the blocked nucleic acid molecules (203) becomes an unblocked nucleic acid molecule (206) when the blocking moiety (207) is removed. As described below, “blocking moiety” may refer to nucleoside modifications, topographical configurations such as secondary structures, and/or structural modifications. Once at least one of the blocked nucleic acid molecules (203) is unblocked, the unblocked nucleic acid molecule (206) can then interact with and activate an RNP2 (208). Because the nucleic acid-guided nucleases in the RNP1s (205) and RNP2s (208) have both cis- and trans-cleavage activity, more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering activation of more RNP2s (208) and more trans-cleavage activity in a cascade.FIG.2Aat bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (209) comprise a quencher (210) and a fluorophore (211) linked by a nucleic acid sequence. As described above in relation toFIG.1C, the reporter moieties are also subject to trans-cleavage by activated RNP1 (205) and RNP2 (208). The intact reporter moieties (209) become activated reporter moieties (212) when the quencher (210) is separated from the fluorophore (211), emitting a fluorescent signal (213). Signal strength increases rapidly as more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering cis-cleavage activation of more RNP2s (208) and thus more trans-cleavage activity of the reporter moieties (209). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation toFIG.4. One particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (gRNA1), the cascade assay components are modular in the sense that the components stay the same no matter what target nucleic acid(s) of interest are being detected. Further, as described below, the cascade assay is tunable by use of blocked nucleic acid molecules or blocked primer molecules having different configurations and free energies. FIG.2Bis a diagram showing an exemplary blocked nucleic acid molecule (220) and an exemplary technique for unblocking the blocked nucleic acid molecules described herein. A blocked single-stranded or double-stranded, circular or linear, DNA or RNA molecule (220) comprising a target strand (222) may contain a partial hybridization with a complementary non-target strand nucleic acid molecule (224) containing unhybridized and cleavable secondary loop structures (226) (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Trans-cleavage of the loops by, e.g., activated RNP1s or RNP2s, generates short strand nucleotide sequences (228) which, because of the short length and low melting temperature Tm, can dehybridize at room temperature (e.g., 15°−25° C.), thereby unblocking the blocked nucleic acid molecule (220) to create an unblocked nucleic acid molecule (230), enabling the internalization of the unblocked nucleic acid molecule (230) (target strand) into an RNP2, leading to RNP2 activation. A blocked nucleic acid molecule may be single-stranded or double-stranded, circular or linear, and may further contain a partially hybridized nucleic acid sequence containing cleavable secondary loop structures, as exemplified by “L” inFIGS.2C-2E. Such blocked nucleic acids typically have a low binding affinity, or high dissociation constant (Kd) in relation to binding to RNP2 and may be referred to herein as a high Kdnucleic acid molecule. In the context of the present disclosure, the binding of blocked or unblocked nucleic acid molecules or blocked primer molecules or synthesized activating molecules to RNP2 have low Kdvalues ranging from about 100 fM to about 1 aM or lower (e.g., 100 zM). High Kdvalues range from 100 nM to about 10-100 10 mM; thus, high Kdvalues are about 105-, 106-,107-, 108-, 109-to 1010-fold or higher as compared to low Kdvalues. Of course, the ideal blocked nucleic acid molecule would have an “infinite Kd.” The blocked nucleic acid molecules (high Kdmolecules) described herein can be converted into unblocked nucleic acid molecules (low Kdmolecules—also in relation to binding to RNP2) via cleavage of nuclease-cleavable regions (e.g., via active RNP1s and RNP2s). The unblocked nucleic acid molecule has a higher binding affinity for the gRNA in the RNP2 than does the blocked nucleic acid molecule, although, as described below, there may be some “leakiness” where some blocked nucleic acid molecules are able to interact with the gRNA in the RNP2. Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2 activation triggers trans-cleavage activity, which in turn leads to more RNP2 activation by further cleaving blocked nucleic acid molecules to produce more unblocked nucleic acid molecules, resulting in a positive feedback loop. In embodiments where blocked nucleic acid molecules are linear and/or form a secondary structure, the blocked nucleic acid molecules may be single-stranded (ss) or double-stranded (ds) and contain a first nucleotide sequence and a second nucleotide sequence. The first nucleotide sequence has sufficient complementarity to hybridize to a gRNA of RNP2, and the second nucleotide sequence does not. The first and second nucleotide sequences of a blocked nucleic acid molecule may be on the same nucleic acid molecule (e.g., for single-strand embodiments) or on separate nucleic acid molecules (e.g., for double strand embodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the second nucleotide sequence converts the blocked nucleic acid molecule to a single strand unblocked nucleic acid molecule. The unblocked nucleic acid molecule contains only the first nucleotide sequence, which has sufficient complementarity to hybridize to the gRNA of RNP2, thereby activating the trans-endonuclease activity of RNP2. In some embodiments, the second nucleotide sequence at least partially hybridizes to the first nucleotide sequence, resulting in a secondary structure containing at least one loop (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Such loops block the nucleic acid molecule from binding or incorporating into an RNP complex thereby initiating cis- or trans-cleavage (see, e.g., the exemplary structures inFIGS.2C-2E). In some embodiments, the blocked nucleic acid molecule may contain a protospacer adjacent motif (PAM) sequence, or partial PAM sequence, positioned between the first and second nucleotide sequences, where the first sequence is 5′ to the PAM sequence, or partial PAM sequence. Inclusion of a PAM sequence may increase the reaction kinetics internalizing the unblocked nucleic acid molecule into RNP2 and thus decrease the time to detection. In other embodiments, the blocked nucleic acid molecule does not contain a PAM sequence. In some embodiments, the blocked nucleic acid molecules (i.e., high IQ nucleic acid molecules—in relation to binding to RNP2) of the disclosure may include a structure represented by Formula I (e.g.,FIG.2C), Formula II (e.g.,FIG.2D), Formula III (e.g.,FIG.2E), or Formula N (e.g.,FIG.2F) wherein Formulas I-N are in the 5′-to-3′ direction: A-(B-L)J-C-M-T-D (Formula I);wherein A is 0-15 nucleotides in length;B is 4-12 nucleotides in length;L is 3-25 nucleotides in length;J is an integer between 1 and 10;C is 4-15 nucleotides in length;M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)J-C and T-D are separate nucleic acid strands;T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or 17-25) and comprises a sequence complementary to B and C; andD is 0-10 nucleotides in length and comprises a sequence complementary to A; D-T-T′-C-(L-B)J-A (Formula II);wherein D is 0-10 nucleotides in length;T-T′ is 17-135 nucleotides in length (e.g., 17-100, 17-50, or 17-25);T′ is 1-10 nucleotides in length and does not hybridize with T;C is 4-15 nucleotides in length and comprises a sequence complementary to T;L is 3-25 nucleotides in length and does not hybridize with T;B is 4-12 nucleotides in length and comprises a sequence complementary to T;J is an integer between 1 and 10;A is 0-15 nucleotides in length and comprises a sequence complementary to D; T-D-M-A-(B-L)J-C (Formula III);wherein T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or 17-25);D is 0-10 nucleotides in length;M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L)J-C are separate nucleic acid strands;A is 0-15 nucleotides in length and comprises a sequence complementary to D;B is 4-12 nucleotides in length and comprises a sequence complementary to T;L is 3-25 nucleotides in length;J is an integer between 1 and 10; andC is 4-15 nucleotides in length; T-D-M-A-Lp-C (Formula N);wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25);D is 0-15 nucleotides in length;M is 1-25 nucleotides in length;A is 0-15 nucleotides in length and comprises a sequence complementary to D; andL is 3-25 nucleotides in length;p is 0 or 1;C is 4-15 nucleotides in length and comprises a sequence complementary to T. In alternative embodiments of any of these molecules, T (or T-T′) can have a maximum length of 1000 nucleotides, e.g., at most 750, at most 500, at most 250, at most 200, at most 135, at most 75, at most 50, or at most 25. Nucleotide mismatches can be introduced in any of the above structures containing double strand segments (for example, where M is absent in Formula I or Formula III) to reduce the melting temperature (Tm) of the segment such that once the loop (L) is cleaved, the double strand segment is unstable and dehybridizes rapidly. The percentage of nucleotide mismatches of a given segment may vary between 0% and 50%; however, the maximum number of nucleotide mismatches is limited to a number where the secondary loop structure still forms. “Segments” in the above statement refers to A, B, and C. In other words, the number of hybridized bases can be less than or equal to the length of each double strand segment and vary based on number of mismatches introduced. In any blocked nucleic acid molecule having the structure of Formula I, III, or N, T will have sequence complementarity to a nucleotide sequence (e.g., a spacer sequence) within a gRNA of RNP2. The nucleotide sequence of T is to be designed such that hybridization of T to the gRNA of RNP2 activates the trans-nuclease activity of RNP2. In any blocked nucleic acid molecule having structure of Formula II, T-T′ will have sequence complementarity to a sequence (e.g., a spacer sequence) within the gRNA of RNP2. The nucleotide sequence of T-T′ is to be designed such that hybridization of T-T′ τo the gRNA of RNP2 activates the trans-cleavage activity of RNP2. For T or T-T′, full complementarity to the gRNA is not necessarily required, provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of RNP2. In any of the foregoing embodiments, the blocked nucleic acid molecules of the disclosure may and preferably do further contain a reporter moiety attached thereto such that cleavage of the blocked nucleic acid releases a signal from the reporter moiety. (SeeFIG.4, mechanisms depicted at center and bottom.) Also, in any of the foregoing embodiments, the blocked nucleic acid molecule may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the blocked nucleic acid molecules of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). The blocked nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, any other nucleic acid molecule modifications described above, and any combination thereof. The Signal Boosting Cascade Assay Employing Blocked Primer Molecules The blocked nucleic acids described above may also, in an alternative embodiment, be blocked primer molecules. Blocked primer molecules include a sequence complementary to a primer binding domain (PBD) on a template molecule (see description below in reference toFIGS.3A and3B) and can have the same general structures as the blocked nucleic acid molecules described above. A PBD serves as a nucleotide sequence for primer hybridization followed by primer extension by a polymerase. In any of Formulas I, II, or III described above, the blocked primer nucleic acid molecule may include a sequence complementary to the PBD on the 5′ end of T. The unblocked primer nucleic acid molecule can bind to a template molecule at the PBD and copy the template molecule via polymerization by a polymerase. Other specific embodiments of the cascade assay that utilize blocked primer molecules are depicted inFIGS.3A and3B. In the embodiments using blocked nucleic acid molecules described above, activation of RNP1 and trans-cleavage of the blocked nucleic acid molecules were used to activate RNP2—that is, the unblocked nucleic acid molecules are a target sequence for the gRNA in RNP2. In contrast in the embodiments using blocked primers, activation of RNP1 and trans-cleavage unblocks a blocked primer molecule that is then used to prime a template molecule for extension by a polymerase, thereby synthesizing activating molecules that are the target sequence for the gRNA in RNP2. FIG.3Ais a diagram showing the sequence of steps in an exemplary cascade assay (300) involving circular blocked primer molecules and linear template molecules. At left ofFIG.3Ais a cascade assay reaction mixture comprising 1) RNP1 s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) linear template molecules (330) (which is the non-target strand); 4) a circular blocked primer molecule (334) (i.e., a high Kdmolecule); and 5) a polymerase (338), such as a Phi29 (029) polymerase. The linear template molecule (330) (non-target strand) comprises a PAM sequence (331), a primer binding domain (PBD) (332) and, optionally, a nucleoside modification (333) to protect the linear template molecule (330) from 3′→5′ exonuclease activity. Blocked primer molecule (334) comprises a cleavable region (335) and a complementary region (336) to the PBD (332) on the linear template molecule (330). Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) combines with and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence-specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mixture, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334) (i.e., a high Kdmolecule, where high Kdrelates to binding to RNP2) upon cleavage becomes an unblocked linear primer molecule (344) (a low Kdmolecule, where low Kdrelates to binding to RNP2), which has a region (336) complementary to the PBD (332) on the linear template molecule (330) and can bind to the linear template molecule (330). Once the unblocked linear primer molecule (344) and the linear template molecule (330) are hybridized (i.e., hybridized at the PBD (332) of the linear template molecule (330) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′→5′ exonuclease activity of the polymerase (338) removes any unhybridized single-stranded DNA at the end of the unblocked primer molecule (344) and the polymerase (338) can copy the linear template molecule (330) to produce a synthesized activating molecule (346) which is a complement of the non-target strand, which is a target strand. The synthesized activating molecule (346) is capable of binding to the gRNA (306) of RNP2 and activating RNP2 (302→308). As described above, because the nucleic acid-guided nuclease in the RNP2 (308) complex exhibits (that is, possesses) both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade. As stated above in relation to blocked and unblocked nucleic acid molecules (both linear and circular), the unblocked primer molecule has a higher binding affinity for the gRNA in RNP2 than does the synthesized activating molecule, although there may be some “leakiness” where some blocked primer molecules are able to interact with the gRNA in RNP2. However, an unblocked primer molecule has a substantially higher likelihood than a blocked primer molecule to hybridize with the gRNA of RNP2. FIG.3Aat bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation toFIG.1C, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorophore emits a fluorescent signal (313). Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating molecules (346) and triggering activation of more RNP2 (308) complexes and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation toFIG.4. Also, as with the cascade assay embodiment utilizing blocked nucleic acid molecules that are not blocked primers, with the exception of the gRNA in RNP1, the cascade assay components may stay the same no matter what target nucleic acid(s) of interest are being detected. Further, the cascade assay is tunable by employing blocked primer molecules having different configurations (i.e., loop sizes, clamp sizes, GC content) and thus different free energies. FIG.3Bis a diagram showing the sequence of steps in an exemplary cascade assay (350) involving blocked primer molecules and circular template molecules. The cascade assay ofFIG.3Bdiffers from that depicted inFIG.3Aby the configuration of the template molecule. Where the template molecule inFIG.3Awas linear, inFIG.3Bthe template molecule is circular. At left inFIG.3Bis a cascade assay reaction mixture comprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) a circular template molecule (352) (non-target strand); 4) a circular blocked primer molecule (334); and 5) a polymerase (338), such as a X29 polymerase. The circular template molecule (352) (non-target strand) comprises a PAM sequence (331) and a primer binding domain (PBD) (332). Blocked primer molecule (334) comprises a cleavable region (335) and a complementary region (336) to the PBD (332) on the circular template molecule (352). Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) combines with and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence-specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mixture, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334), upon cleavage, becomes an unblocked linear primer molecule (344), which has a region (336) complementary to the PBD (332) on the circular template molecule (352) and can hybridize with the circular template molecule (352). Once the unblocked linear primer molecule (344) and the circular template molecule (352) are hybridized (i.e., hybridized at the PBD (332) of the circular template molecule (352) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′→5′ exonuclease activity of the polymerase (338) removes any unhybridized single-stranded DNA at the 3′ end of the unblocked primer molecule (344). The polymerase (338) can now use the circular template molecule (352) (non-target strand) to produce concatenated activating nucleic acid molecules (360) (which are concatenated target strands), which will be cleaved by the trans-cleavage activity of activated RNP1. The cleaved regions of the concatenated synthesized activating molecules (360) (target strand) are capable of binding to the gRNA (306) of RNP2 and activating the RNP2 (302308) complex. As described above, because the nucleic acid-guided nuclease in RNP2 (308) comprises both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade.FIG.3Bat bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation toFIG.1C, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorescent signal (313) is unquenched and can be detected. Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating nucleic acid molecules and triggering activation of more RNP2s (308) and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation toFIG.4. Also note that as with the other embodiments of the cascade assay, in this embodiment, with the exception of the gRNA in RNP1, the cascade assay components can stay the same no matter what target nucleic acid(s) of interest are being detected. The polymerases used in the “blocked primer molecule” embodiments serve to polymerize a reverse complement strand of the template molecule (non-target strand) to generate a synthesized activating molecule (target strand) as described above. In some embodiments, the polymerase is a DNA polymerase, such as a BST, T4, or Therminator polymerase (New England BioLabs Inc., Ipswich MA., USA). In some embodiments, the polymerase is a Klenow fragment of a DNA polymerase. In some embodiments the polymerase is a DNA polymerase with 5′→3′ DNA polymerase activity and 3‘-’ 5′ exonuclease activity, such as a Type I, Type II, or Type III DNA polymerase. In some embodiments, the DNA polymerase, including the X29, T7, Q5®, Q5U®, Phusion®, OneTaq®, LongAmp®, Vent®, or Deep Vent® DNA polymerases (New England BioLabs Inc., Ipswich MA., USA), or any active portion or variant thereof. Also, a 3′ to 5′ exonuclease can be separately used if the polymerase lacks this activity. FIG.4depicts three mechanisms in which a cascade assay reaction can release a signal from a reporter moiety.FIG.4at top shows the mechanism discussed in relation toFIGS.2A,3A and3B. In this embodiment, a reporter moiety (409) is a separate molecule from the blocked nucleic acid molecules present in the reaction mixture. Reporter moiety (409) comprises a quencher (410) and a fluorophore (411). An activated reporter moiety (412) emits a signal from the fluorophore (411) once it has been physically separated from the quencher (410). Again, if the reporter moiety is a separate molecule that is not activated as part of the blocked nucleic acid molecule (or blocked primer molecule), then activation kinetics of the reporter will be more rapid; however, if activation of the reporter moiety is coupled to unblocking of the blocked nucleic acid molecules (or blocked primer molecules), activation kinetics will be slower. FIG.4at center shows a blocked nucleic acid molecule (403), which is also a reporter moiety. In addition to quencher (410) and fluorophore (411), a blocking moiety (407) can be seen (see also blocked nucleic acid molecules203inFIG.2A). Blocked nucleic acid molecule/reporter moiety (403) comprises a quencher (410) and a fluorophore (411). In this embodiment of the cascade assay, when the blocked nucleic acid molecule (403) is unblocked due to trans-cleavage initiated by the target nucleic acid of interest binding to RNP1, the unblocked nucleic acid molecule (406) also becomes an activated reporter moiety with fluorophore (411) separated from quencher (410). Note both the blocking moiety (407) and the quencher (410) are removed. In this embodiment, reporter signal is directly generated as the blocked nucleic acid molecules become unblocked. FIG.4at the bottom shows that cis-cleavage of an unblocked nucleic acid or a synthesized activation molecule at a PAM distal sequence by RNP2 generates a signal. Shown are activated RNP2 (408), unblocked nucleic acid molecule (461), quencher (410), and fluorophore (411) forming an activated RNP2 with the unblocked nucleic acid/reporter moiety intact (460). Cis-cleavage of the unblocked nucleic acid/reporter moiety (461) results in an activated RNP2 with the reporter moiety activated (462), comprising the activated RNP2 (408), the unblocked nucleic acid molecule with the reporter moiety activated (463), quencher (410) and fluorophore (411). Tuning the Cascade Assay Using Blocked Nucleic Acid Molecules or Blocked Primer Molecules The present disclosure improves upon the signal cascade assay described in U.S. Ser. Nos. 17/861,207; 17/861,208; and 17/861,209 by configuring the blocked nucleic acid molecules or blocked primer molecules to increase reaction kinetics, decrease reaction kinetics, provide detection over a large range of concentrations of the target nucleic acids of interest or provide accurate quantification of target nucleic acids of interest within a narrow range of concentrations. As described above in detail in relation toFIGS.1C,2A,2B,3A,3B, and4, the cascade assay is initiated when a target nucleic acid of interest binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1). The guide nucleic acid of RNP1 (i.e., gRNA1), comprising a sequence complementary to the target nucleic acid of interest, guides RNP1 to the target nucleic acid of interest. Upon binding of N nucleotide bases of the target nucleic acid of interest to RNP1, RNP1 becomes activated, cleaving the target nucleic acid of interest in a sequence-specific manner (i.e., cis-cleavage) leading to non-sequence-specific, indiscriminate trans-cleavage activity which unblocks the blocked nucleic acid molecules in the reaction mixture. The unblocked nucleic acid molecules can then activate a second pre-assembled ribonucleoprotein complex (RNP2), where RNP2 comprises a second gRNA (gRNA2) comprising a sequence complementary to the unblocked nucleic acid molecules, and at least one of the unblocked nucleic acid molecules is cleaved in a sequence-specific manner. Cis-cleavage of the unblocked nucleic acid molecule then leads to non-sequence-specific, indiscriminate trans-cleavage activity by RNP2, which in turn unblocks more blocked nucleic acid molecules (and reporter moieties) in the reaction mixture activating more RNP2s. Each newly activated RNP2 activates more RNP2s, which in turn cleave more blocked nucleic acid molecules and reporter moieties in a reaction cascade. The improvement to the signal cascade or signal boost cascade assay described herein is drawn to being able to “tune” the cascade assay by employing differently configured blocked nucleic acid molecules (or blocked primer molecules) that activate RNP2. “Tuning” relates to controlling kinetics of the assay by two orders of magnitude, from under one minute of target nucleic acids of interest to detection over 100 minutes or more. The present disclosure demonstrates that by altering the Gibbs free energy (i.e., molecular configuration and composition) via varying loop numbers, “clamp” lengths, and GC content of the blocked nucleic acid molecules employed in the cascade assay, the kinetics of the cascade assay can be “tuned” regardless of RNP1 target concentrations. There are various methods to calculate free energy (i.e., Gibbs free energy). In one method, Gibbs free energy changes can be calculated using enthalpy and entropy values according to: ΔG°(T)=(ΔH°−TΔS°)cal mol−1 where T is the temperature at which Gibbs free energy is assessed. Hybridization enthalpy (ΔH°) was calculated as a difference of the oligonucleotides' total energy in the double-stranded (ds) states (Edstot) and in the single-stranded (ss) states (Ess1totand Ess2tot): ΔH°≈Edstot−(Ess1+Ess2) There is a linear dependence of the hybridization entropy on the enthalpy of the complex formation with a very high correlation coefficient (R2=0.995). This dependence is described by the equation: ΔS°=2.678ΔH°/1000−6.0 cal mol−1K−1. (See, e.g., Lomzov, et al., J. Phys. Chem., 119(49):15221-234 (°15). In another method, Gibbs free energy is calculated using enthalpy and entropy values. The following formula is used to calculate the free energy for each base pair: ΔG° (T)=(ΔH°−TΔS°)cal mol−1 The total ΔG° is given by: ΔG° (total)=ΣiniΔG° (i)+ΔG° (init with termG·C)+ΔG° (init with termA·T)+ΔG° (sym) Where ΔG° (i) are the standard free energy changes for the 10 possible Watson-Crick NNs (e.g., ΔG° (1)=ΔG°37(AA/TT), ΔG° (2)=ΔG°37(TA/AT), . . . etc.), n; is the number of occurrences of each nearest neighbor, i, and ΔG° (sym) equals +0.43 kcal/mol (1 cal=4.184 J) if the duplex is self-complementary and zero if it is non-self-complementary. An example of total Gibbs free energy is shown on CGTTGA·TCAACG hybridized DNA: ↓ ↓ ↓5′ C-G-T-T-G-A 3′* * * * * *3′ G-C-A-A-C-T 5′↑ ↑ ΔG°37(pred)=ΔG°37(CG/GC)+ΔG°37(GT/CA)+ΔG°37(TT/AA)+ΔG°37(TG/AC)+ΔG°37(GA/CT)+ΔG°(init.)=−2.17-1.44-1.00-1.45-1.30+0.98+1.03 ΔG°37(pred.)=−5.35 kcal/mol ΔG°37(obs.)=−5.20 kcal/mol The ΔH° and ΔS° parameters are analogously calculated from the parameters in Table 1. TABLE 1Unified oligonucleotides ΔHº andΔSº NN parameters in 1 M NaClSequenceΔHº kcal/molΔSº kcal/molAA/TT-7.9-22.2AT/TA-7.2-20.4TA/TA-7.2-21.3CA/GT-8.5-22.7GT/CA-8.4-22.4CT/GA-7.8-21.0GA/CT-8.2-22.2CG/GC-10.6-27.2GC/CG-9.8-24.4GG/CC-8.0-19.9Init. w/ term. G-C0.1-2.8Init. w/ term. A-T2.34.1Symmetry correction0-1.4 See, e.g., SantaLucia, et al., PNAS, 95(4):1460-65 (1998). Long regions of hybridization (or self-hybridization), i.e., the “clamps” or “clamp regions” of the blocked nucleic acid molecules, lead to higher Tmand thus slower kinetics. Further, the more loops present that need to be cleaved to unblock the blocked nucleic acid molecules or blocked primer molecules, the slower the reaction kinetics will be. Also, if the reporter moiety is incorporated into the blocked nucleic acid molecule design, the detection kinetics of the reporter moiety will be slow compared to when reporter moieties are present in the cascade assay reaction mixture as separate molecules. Finally, with the blocked nucleic acid molecules and the blocked primer molecules, the reaction rate increases as GC content increases and the reaction rate decreases as GC content decreases, particularly in relation to the GC content of a clamp region. Reaction kinetics of course are also affected by temperature. The higher the temperature, the more rapid the reaction. Like Gibbs free energy, melting temperature (Tm) can be calculated using one of several calculations known in the art. For example, for sequences less than 14 nucleotides, the formula is: Tm=(wA+xT)*2+(yG+zC)*4where w, x, y, z are the number of bases A, T, G, C in the sequence respectively. For sequences longer than 13 nucleotides, the equation used is: Tm=64.9+41*(yG+zC−16.4)/(wA+xT+yG+zC) Both equations assume that annealing occurs under the standard conditions of 50 nM primer, 50 nM Na+, and pH 7.0. See, e.g., Mamur and Doty, JMB, 5(1):109-18 (1962) and Wallace, et al., NAR, 6:3543-57 (1979). In distinguishing blocked nucleic acid molecules, the Gibbs free energy of the blocked nucleic acid molecules (or blocked primer molecules) has to be negative enough to be stable in the cascade assay reaction mixture under the desired assay conditions. If the blocked nucleic acid molecules are not stable, unblocking will not be specific; that is, unblocking the blocked nucleic acid molecules may take place without activation of RNP1 by the target nucleic acid of interest, resulting in false positive. For example, note that the clamp regions of molecule U29 (FIG.6A) are 5 and 6 basepairs in length, resulting in a blocked nucleic acid molecule with a Gibbs free energy of −5.85 kcal/mol. Shorter clamps of, e.g., 4 basepairs in length may result in an unstable blocked nucleic acid molecule at the reaction temperature of 25° C. (see the assay results shown inFIGS.6B-6Hand the descriptions thereof below) and thus would be unsuitable for the cascade assay reaction even if instantaneous detection is desired. Thus, at a reaction temperature of 25° C., the Gibbs free energy of the blocked nucleic acid molecule will be about −5.5 kcal/mol to about −20.0 kcal/mol, or about −6.0 kcal/mol to about −18.0 kcal/mol, or about −8.0 kcal/mol to about −16.0 kcal/mol. If faster kinetics are desired, at a reaction temperature of 25° C., the Gibbs free energy of the blocked nucleic acid molecule will be about −5.0 kcal/mol to about −12.0 kcal/mol, or about −6.0 kcal/mol to about 10.0 kcal/mol. If faster kinetics are desired, at a reaction temperature of 25° C., the Gibbs free energy of the blocked nucleic acid molecule will be about −12.0 kcal/mol to about −20.0 kcal/mol, or about −14.0 kcal/mol to about −18.0 kcal/mol. In addition, the tunable blocked nucleic acid molecules can comprise a PAM sequence or lack a PAM sequence, but if a PAM sequence is present, it is present in a loop sequence. In addition to slowing down the cascade assay reaction kinetics in a customizable manner, increasing reaction kinetics (i.e., slower=more quantifiable) allows for quantification of small differences in the number of target nucleic acids of interest, in an almost digital manner. For example, the configuration of the blocked nucleic acid molecule can be chosen so as to, e.g., distinguish between one copy of a target nucleic acid of interest from two copies of the target nucleic acid of interest, or, e.g., two copies of a target nucleic acid of interest from three copies of the target nucleic acid of interest, or e.g., three copies of a target nucleic acid of interest from five copies of the target nucleic acid of interest, or e.g., ten copies of a target nucleic acid of interest from fifteen copies of the target nucleic acid of interest. Again, the higher the Tm of a blocked nucleic acid molecule or blocked primer molecule, the more loops that need to be cleaved, the longer the clamp regions, the higher the GC content of the claim regions, the slower the reaction kinetics leading to more distinction between small differences in copy number. Once putative blocked nucleic acid molecules or blocked primer molecules are designed, Gibbs free energy is calculated and then a selection of blocked nucleic acid molecules or blocked primer molecules are tested under various reaction conditions. The choice of a final blocked nucleic acid molecule or blocked primer molecule candidate is thus determined empirically. Selecting the optimal design of the blocked nucleic acid molecule (or blocked primer molecule) for detection of a specific target nucleic acid of interest in a desired range of copy numbers requires selection of a desired reaction temperature and experimentation as described below to establish detection curves as shown inFIGS.6B-6H,7B,8B,9B,10B and11b. Once a “sweet spot” for copy number detection (and reaction rate) is achieved, the cascade assay can be programmed to detect virtually any target nucleic acid of interest (or combinations thereof) by changing the guide nucleic acid(s) in RNP1. The cascade assay reaction may proceed to completion with measurement continuously or at specific timepoints, and/or the cascade assay reaction may be arrested or quenched at a desired timepoint by, e.g., addition of EDTA. Applications of the Cascade Assay The present disclosure describes cascade assays for detecting one or more target nucleic acids of interest in a sample. The cascade assays allow for massive multiplexing and minimum workflow yet provide accurate results at low cost. In embodiments, the cascade assay can be tuned to detect target nucleic acids of interest instantaneously or nearly so, even at ambient temperatures above 16° C.; detect target nucleic acids of interest over a longer period of time; detect target nucleic acids of interest over large copy number concentrations; or detect copies of target nucleic acids of interest quantitatively over a small range in an almost digital manner. That is, the present disclosure describes methods to “tune” the assay such that reaction kinetics can be controlled over multiple orders of magnitude. Moreover, the various embodiments of the cascade assay are notable in that, with the exception of the gRNA in RNP1, the cascade assay components can stay the same no matter what target nucleic acid(s) of interest are being detected and RNP1 is easily reprogrammed. Further, this remains true in the context of tunability, as the cascade assay is tunable by use of different blocked nucleic acid molecules (or blocked primer molecules) used to activate RNP2 once desired reaction kinetics have been chosen and is independent of the RNP1 target nucleic acid concentration. Note this is not true of, e.g., PCR, where the Ct value depends on the concentration of the target nucleic acid. If single copy differences in the number of target nucleic acids of interest are required, such as, e.g., in oncology applications, one can design the best blocked nucleic acid molecule for this purpose. Such a blocked nucleic acid molecule might comprise a Gibbs free energy of −15 to −20 kcal/mol, such as molecules T135, T134, or T119 seen inFIGS.9A,10A and11A, respectively. If, in contrast, determining the presence of a target nucleic acid of interest virtually instantaneously is desired, again the best blocked nucleic acid for this purpose can be designed, and may comprise a Gibbs free energy of approximately −5 kcal/mol and a molecular structure similar to, e.g., that of U29 seen inFIG.6A. Target nucleic acids of interest are derived from samples as described in more detail above. Suitable samples for testing include, but are not limited to, any environmental sample, such as air, water, soil, surface, food, clinical sites and products, industrial sites and products, pharmaceuticals, medical devices, nutraceuticals, cosmetics, personal care products, agricultural equipment and sites, and commercial samples, and any biological sample obtained from an organism or a part thereof, such as a plant, animal, or microbe. In some embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample is any solid or fluid sample obtained from, excreted by or secreted by any living organism, including, without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms including plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as an infection with a pathogenic microorganism, such as a pathogenic bacteria or virus. For example, a biological sample can be a biological fluid obtained from a human or non-human (e.g., livestock, pets, wildlife) animal, and may include but is not limited to blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or a swab of skin or mucosal membrane surface (e.g., a nasal or buccal swab). In some embodiments, the sample can be a viral or bacterial sample or a biological sample that has been minimally processed, e.g., only treated with a brief lysis step prior to detection. In other embodiments, minimal processing can include thermal lysis at an elevated temperature to release nucleic acids. Suitable methods are contemplated in U.S. Pat. No. 9,493,736, among other references. Common methods for cell lysis involve thermal, chemical, enzymatic, or mechanical treatment of the sample or a combination of those (see, e.g., Example I below). In some embodiments, minimal processing can include treating the sample with chaotropic salts such as guanidine isothiocyanate or guanidine HCl. Suitable methods are contemplated in U.S. Pat. Nos. 8,809,519 and 7,893,251, among other references. In some embodiments, minimal processing may include contacting the sample with reducing agents such as DTT or TCEP and EDTA to inactivate inhibitors and/or other nucleases present in the crude samples. In other embodiments, minimal processing for biofluids may include centrifuging the samples to obtain cell-debris free supernatant before applying the reagents. Suitable methods are contemplated in U.S. Pat. No. 8,809,519, among other references. In still other embodiments, minimal processing may include performing DNA/RNA extraction to get purified nucleic acids before applying CRISPR Cascade reagents. Table 2 below lists exemplary commercial sample processing kits, and Table 3 below lists point of care processing techniques. TABLE 2Exemplary Commercial Sample and Nucleic Acid Processing KitsManufacturerKitSample TypeOutputLysing and extraction methodsQiagen ®DNeasy ™ Bloodsmall volumesgenomicIsolation of Genomic DNA from& Tissue Kitsof bloodDNASmall Volumes of Blooddried blood1. Uses Chemical andspotsBiological/Enzymatic lysis methodsurine2. Uses solid phase extraction (SPE)tissueswith Column Purificationlaser-Isolation of Genomic DNA frommicrodissectedTissuestissues1. Uses Chemical andBiological/Enzymatic lysis methods2. Used to dissolve and lyse tissuesections completely, highertemperature and longer timeincubations up to 24 hours are usedQiagen ®QIAamp ® UCPwhole bloodmicrobialSpecific pretreatment protocols arePathogenswabsDNAsuggested depending on sample typeMini Handbookcultures -with or without the use of kits formicrobial DNApelletedMechanical Lysis Method beforepurificationmicrobial cellsdownstream applications.body fluidsDownstream applications contain:1. Chemical andBiological/Enzymatic lysis methods2. SPE with Column PurificationQiagen ®QIAamp ® Viralplasma andviral DNA1. Uses Chemical lysis methodsRNA Kitsserum2. Uses SPE with ColumnCSFPurificationurineother cell-freebody fluidscell-culturesupernatantsswabsZymoQuick-whole bloodgenomic1. Uses chemical lysis methodsResearch ™DNA ™MicroprepplasmaDNA2. Uses SPE with columnKitserumpurificationbody fluidsbuffy coatlymphocytesswabscultured cellsZymoQuick-DNA ™A. fumigatusMicrobialUses Bead lysis and pretreatmentResearch ™Fungal/BacterialC. albicansDNAwith:Miniprep KitN. crassa1. Chemical lysis methods withS. cerevisiaechaotropic saltsS. pombe2. Nucleic acid extraction (NAE)myceliumwith SPE with silica matricesGram positivebacteriaGram negativebacteria TABLE 3Point of Care Sample Processing TechniquesStepsProtocol Example 1Protocol Example 2Protocol Example 3Field-deployable viralStreamlinedLucira Health ™diagnostics usinginactivation,CRISPR-Cas13amplification, andScience,Cas13-based detection27; 360(6387): 444-448of SARS-CoV-2(2018)Nat Commun, 11: 5921(2020)1. Cell disruptionSamples were thermallyA nasopharyngeal (NP)Lucira Health uses a(lysis) andtreated at ~40° C. for ~15swab or saliva samplesingle buffer that lysesinactivation ofminutes for nucleasewas lysed andand inactivatesnucleasesdeactivation, thereafterinactivated for 10nucleases and/orIn point-of-care setting,at 90° C. for 5 minutesminutes with thermalinhibitors.cell disruption andfor viral deactivation.treatment. TheseA nasal swab is directlyinactivation ofSample Types:samples were incubatedadded to a singlenucleases is doneUrinefor 5 min at 40° C.,lysing/reaction buffercommonly throughSalivafollowed by 5 min atand vigorously stirredthermal lysis.Diluted blood70° C. (or 5 min at 95° C.,to release the viral(1:3 with PBS)if saliva)particulates from theTargets: Virusesswab.Target: SARS-Cov-22. Assay on crudeThermally treatedThermally treatedProcessed biologicalsamplebiological samplesbiological samplessample is used in anThis is usually a direct(above) were used(above) were usedisothermal reaction forassay on the crudedirectly fordirectly forpathogenic nucleic acidsample post cellamplification andamplification anddetection.disruption anddetection of pathogenicdetection of pathogenicinactivation ofnucleic acid.nucleic acid.nucleases. Noextraction is usuallyperformed. FIG.5shows a lateral flow assay (LFA) device that can be used to detect the cleavage and separation of a signal from a reporter moiety. For example, the reporter moiety may be a single-stranded or double-stranded oligonucleotide with terminal biotin and fluorescein amidite (FAM) modifications; and, as described above, the reporter moiety may also be part of a blocked nucleic acid. The LFA device may include a pad with binding particles, such as gold nanoparticles functionalized with anti-FAM antibodies; a control line with a first binding moiety attached, such as avidin or streptavidin; a test line with a second binding moiety attached, such as antibodies; and an absorption pad. After completion of a cascade assay (seeFIGS.2A,3A, and3B), the assay reaction mixture is added to the pad containing the binding particles, (e.g., antibody labeled gold nanoparticles). When the target nucleic acid of interest is present, a reporter moiety is cleaved, and when the target nucleic acid of interest is absent, the reporter is not cleaved. A moiety on the reporter binds to the binding particles and is transported to the control line. When the target nucleic acid of interest is absent, the reporter moiety is not cleaved, and the first binding moiety binds to the reporter moiety, with the binding particles attached. When the target nucleic acid of interest is present, one portion of the cleaved reporter moiety binds to the first binding moiety, and another portion of the cleaved reporter moiety bound to the binding particles via the moiety binds to the second binding moiety. In one example, anti-FAM gold nanoparticles bind to a FAM terminus of a reporter moiety and flow sequentially toward the control line and then to the test line. For reporters that are not trans-cleaved, gold nanoparticles attach to the control line via biotin-streptavidin and result in a dark control line. In a negative test, since the reporter has not been cleaved, all gold conjugates are trapped on control line due to attachment via biotin-streptavidin. A negative test will result in a dark control line with a blank test line. In a positive test, reporter moieties have been trans-cleaved by the cascade assay, thereby separating the biotin terminus from the FAM terminus. For cleaved reporter moieties, nanoparticles are captured at the test line due to anti-FAM antibodies. This positive test results in a dark test line in addition to a dark control line. The components of the cascade assay may be provided in various kits for testing at, e.g., point of care facilities, in the field, pandemic testing sites, and the like. In one aspect, the kit for detecting a target nucleic acid of interest in a sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), blocked nucleic acid molecules, and reporter moieties. The first complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid(s) of interest. Binding of the first complex (RNP1) to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The blocked nucleic acid molecule comprises a sequence complementary to the second gRNA, where trans-cleavage of the blocked nucleic acid molecule results in an unblocked nucleic acid molecule and the unblocked nucleic acid molecule can bind to the second complex (RNP2), thereby activating the trans-cleavage activity of the second nucleic acid-guided nuclease. Activating trans-cleavage activity in RNP2 results in an exponential increase in unblocked nucleic acid molecules and in active reporter moieties, where reporter moieties are nucleic acid molecules and/or are operably linked to the blocked nucleic acid molecules and produce a detectable signal upon cleavage by RNP2. In a second aspect, the kit for detecting a target nucleic acid molecule in sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), template molecules, blocked primer molecules, a polymerase, nucleotide triphosphates (NTPs), and reporter moieties. The first ribonucleoprotein complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid of interest and where binding of RNP1 to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The template molecules comprise a primer binding domain (PBD) sequence as well as a sequence corresponding to a spacer sequence of the second gRNA. The blocked primer molecules comprise a sequence that is complementary to the PBD on the template nucleic acid molecule and a blocking moiety. Upon binding to the target nucleic acid of interest, RNP1 becomes active triggering trans-cleavage activity that cuts at least one of the blocked primer molecules to produce at least one unblocked primer molecule. The unblocked primer molecule hybridizes to the PBD of one of the template nucleic acid molecules, is trimmed of any excess nucleotides by the 3′-to-5′ exonuclease activity of the polymerase and is then extended by the polymerase with NTPs to form a synthesized activating molecule with a sequence that is complementary to the second gRNA of RNP2. Upon activating RNP2, additional trans-cleavage activity is initiated, cleaving at least one additional blocked primer molecule. Continued cleavage of blocked primer molecules and subsequent activation of more RNP2s proceeds at an exponential rate. A signal is generated upon cleavage of a reporter molecule by active RNP2 complexes; therefore, a change in signal production indicates the presence of the target nucleic acid molecule. Any of the kits described herein may further include a sample collection device, e.g., a syringe, lancet, nasal swab, or buccal swab for collecting a biological sample from a subject, and/or a sample preparation reagent, e.g., a lysis reagent. Each component of the kit may be in separate container or two or more components may be in the same container. The kit may further include a lateral flow device used for contacting the biological sample with the reaction mixture, where a signal is generated to indicate the presence or absence of the target nucleic acid molecule of interest. In addition, the kit may further include instructions for use and other information. EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive. Example I: Preparation of Nucleic Acids of Interest Mechanical lysis: Nucleic acids of interest may be isolated by various methods depending on the cell type and source (e.g., tissue, blood, saliva, environmental sample, etc.). Mechanical lysis is a widely-used cell lysis method and may be used to extract nucleic acids from bacterial, yeast, plant and mammalian cells. Cells are disrupted by agitating a cell suspension with “beads” at high speeds (beads for disrupting various types of cells can be sourced from, e.g., OPS Diagnostics (Lebanon NJ, US) and MP Biomedicals (Irvine, CA, USA)). Mechanical lysis via beads begins with harvesting cells in a tissue or liquid, where the cells are first centrifuged and pelleted. The supernatant is removed and replaced with a buffer containing detergents as well as lysozyme and protease. The cell suspension is mixed to promote breakdown of the proteins in the cells and the cell suspension then is combined with small beads (e.g., glass, steel, or ceramic beads) that are mixed (e.g., vortexed) with the cell suspension at high speeds. The beads collide with the cells, breaking open the cell membrane with shear forces. After “bead beating”, the cell suspension is centrifuged to pellet the cellular debris and beads, and the supernatant may be purified via a nucleic acid binding column (such as the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit from ThermoFisher (Waltham, MA, USA) and others from Qiagen (Hilden, Germany), TakaraBio (San Jose, CA, USA), and Biocomma (Shenzen, China)) to collect the nucleic acids (see the discussion of solid phase extraction below). Solid phase extraction (SPE): Another method for capturing nucleic acids is through solid phase extraction. SPE involves a liquid and stationary phase, which selectively separate the target analyte (here, nucleic acids) from the liquid in which the cells are suspended based on specific hydrophobic, polar, and/or ionic properties of the target analyte in the liquid and the stationary solid matrix. Silica binding columns and their derivatives are the most commonly used SPE techniques, having a high binding affinity for DNA under alkaline conditions and increased salt concentration; thus, a highly alkaline and concentrated salt buffer is used. The nucleic acid sample is centrifuged through a column with a highly porous and high surface area silica matrix, where binding occurs via the affinity between negatively charged nucleic acids and positively charged silica material. The nucleic acids bind to the silica matrices, while the other cell components and chemicals pass through the matrix without binding. One or more wash steps typically are performed after the initial sample binding (i.e., the nucleic acids to the matrix), to further purify the bound nucleic acids, removing excess chemicals and cellular components non-specifically bound to the silica matrix. Alternative versions of SPE include reverse SPE and ion exchange SPE, and use of glass particles, cellulose matrices, and magnetic beads. Thermal lysis: Thermal lysis involves heating a sample of mammalian cells, virions, or bacterial cells at high temperatures thereby damaging the cellular membranes by denaturizing the membrane proteins. Denaturizing the membrane proteins results in the release of intracellular DNA. Cells are generally heated above 90° C., however time and temperature may vary depending on sample volume and sample type. Once lysed, typically one or more downstream methods, such as use of nucleic acid binding columns for solid phase extraction as described above, are required to further purify the nucleic acids. Physical lysis: Common physical lysis methods include sonication and osmotic shock. Sonication involves creating and rupturing of cavities or bubbles to release shockwaves, thereby disintegrating the cellular membranes of the cells. In the sonication process, cells are added into lysis buffer, often containing phenylmethylsulfonyl fluoride, to inhibit proteases. The cell samples are then placed in a water bath and a sonication wand is placed directly into the sample solution. Sonication typically occurs between 20-50 kHz, causing cavities to be formed throughout the solution as a result of the ultrasonic vibrations; subsequent reduction of pressure then causes the collapse of the cavity or bubble resulting in a large amount of mechanical energy being released in the form of a shockwave that propagates through the solution and disintegrates the cellular membrane. The duration of the sonication pulses and number of pulses performed varies depending on cell type and the downstream application. After sonication, the cell suspension typically is centrifuged to pellet the cellular debris and the supernatant containing the nucleic acids may be further purified by solid phase extraction as described above. Another form of physical lysis is osmotic shock, which is most typically used with mammalian cells. Osmotic shock involves placing cells in DI/distilled water with no salt added. Because the salt concentration is lower in the solution than in the cells, water is forced into the cell causing the cell to burst, thereby rupturing the cellular membrane. The sample is typically purified and extracted by techniques such as e.g., solid phase extraction or other techniques known to those of skill in the art. Chemical lysis: Chemical lysis involves rupturing cellular and nuclear membranes by disrupting the hydrophobic-hydrophilic interactions in the membrane bilayers via detergents. Salts and buffers (such as, e.g., Tris-HCl pH8) are used to stabilize pH during extraction, and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)) and inhibitors (e.g., Proteinase K) are also added to preserve the integrity of the nucleic acids and protect against degradation. Often, chemical lysis is used with enzymatic disruption methods (see below) for lysing bacterial cell walls. In addition, detergents are used to lyse and break down cellular membranes by solubilizing the lipids and membrane proteins on the surface of cells. The contents of the cells include, in addition to the desired nucleic acids, inner cellular proteins and cellular debris. Enzymes and other inhibitors are added after lysis to inactivate nucleases that may degrade the nucleic acids. Proteinase K is commonly added after lysis, destroying DNase and RNase enzymes capable of degrading the nucleic acids. After treatment with enzymes, the sample is centrifuged, pelleting cellular debris, while the nucleic acids remain in the solution. The nucleic acids may be further purified as described above. Another form of chemical lysis is the widely-used procedure of phenol-chloroform extraction. Phenol-chloroform extraction involves the ability for nucleic acids to remain soluble in an aqueous solution in an acidic environment, while the proteins and cellular debris can be pelleted down via centrifugation. Phenol and chloroform ensure a clear separation of the aqueous and organic (debris) phases. For DNA, a pH of 7-8 is used, and for RNA, a more acidic pH of 4.5 is used. Enzymatic lysis: Enzymatic disruption methods are commonly combined with other lysis methods such as those described above to disrupt cellular walls (bacteria and plants) and membranes. Enzymes such as lysozyme, lysostaphin, zymolase, and protease are often used in combination with other techniques such as physical and chemical lysis. For example, one can use cellulase to disrupt plant cell walls, lysosomes to disrupt bacterial cell walls and zymolase to disrupt yeast cell walls. Example II: RNP Formation For RNP complex formation, 250 nM of LbCas12a nuclease protein was incubated with 375 nM of a target specific gRNA in 1× Buffer (10 mM Tris-HCl, 100 μg/mL BSA) with 2-15 mM MgCl2at 25° C. for 20 minutes. The total reaction volume was 2 μL. Other ratios of LbCas12a nuclease to gRNAs were tested, including 1:1, 1:2 and 1:5. The incubation temperature can range from 20° C.-37° C., and the incubation time can range from 10 minutes to 4 hours. Example III: Blocked Nucleic Acid Molecule Formation Ramp cooling: For formation of the secondary structure of blocked nucleic acids, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-N) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl2for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to 37° C. at 0.015° C./second to form the desired secondary structure. Snap cooling: For formation of the secondary structure of blocked nucleic acids, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-N) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl2for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by removing the heat source to form the desired secondary structure. Snap cooling on ice: For formation of the secondary structure of blocked nucleic acids, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-N) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl2for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by placing the reaction tube on ice to form the desired secondary structure. Example IV: Reporter Moiety Formation The reporter moieties used in the reactions herein were single-stranded DNA oligonucleotides 5-10 bases in length (e.g., with sequences of TTATT, TTTATTT, ATTAT, ATTTATTTA, AAAAA, or AAAAAAAAA) with a fluorophore and a quencher attached on the 5′ and 3′ ends, respectively. In one example using a Cas12a cascade, the fluorophore was FAM-6, and the quencher was IOWA BLACK® (Integrated DNA Technologies, Coralville, IA). In another example using a Cas13 cascade, the reporter moieties were single stranded RNA oligonucleotides 5-10 bases in length (e.g., r(U)n, r(UUAUU)n, r(A)n). Example V: Cascade Assay First Format (final reaction mixture components added at the same time): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the Methicillin resistantStaphylococcus aureus(MRSA) DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VIII). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-N) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl2at 25° C. for 20-40 minutes. Following incubation, RNPIs were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. Thereafter, the final reaction was carried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl2, 4 mM NaCl, 15 nM LbCas12a: 22.5 nM gRNA RNP1, 20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I-N) in a total volume of 9 μL. 1 μL of MRSA DNA target (with samples having as low as three copies and as many as 30000 copies—seeFIGS.6-11) was added to make a final volume of 10 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute. Second Format (RNP1 and MRSA target pre-incubated before addition to final reaction mixture): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to RNP formation protocol described in Example II (for this sequence, see Example VIII). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl2at 25° C. for 20-40 minutes. Following incubation, RNPIs were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1 μL of MRSA DNA target and incubated at 20° C.-37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl2, 4 mM NaCl, the pre-incubated and activated RNP1, 20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I-N) in a total volume of 9 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute. Third Format (RNP1 and MRSA target pre-incubated before addition to final reaction mixture and blocked nucleic acid molecule added to final reaction mixture last): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VIII). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl2at 25° C. for 20-40 minutes. Following incubation, RNPIs were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1 μL of MRSA DNA target and incubated at 20° C.-37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl2, 4 mM NaCl, the pre-incubated and activated RNP1, and 20 nM LbCas12a: 35 nM gRNA RNP2 in a total volume of 9 μL. Once the reaction mixture was made, 1 μL (50 nM) blocked nucleic acid molecule (any one of Formula I-N) was added for a total volume of 10 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute. Example VI: Detection of MRSA and Test Reaction Conditions To detect the presence of Methicillin resistantStaphylococcus aureus(MRSA) and determine the sensitivity of detection with the cascade assay, titration experiments with a MRSA DNA target nucleic acid of interest were performed. The MRSA mecA gene DNA sequence (NCBI Reference Sequence NC: 007793.1) is as follows. SEQ ID NO: 1:ATGAAAAAGATAAAAATTGTTCCACTTATTTTAATAGTTGTAGTTGTCGGGTTTGGTATATATTTTTATGCTTCAAAAGATAAAGAAATTAATAATACTATTGATGCAATTGAAGATAAAAATTTCAAACAAGTTTATAAAGATAGCAGTTATATTTCTAAAAGCGATAATGGTGAAGTAGAAATGACTGAACGTCCGATAAAAATATATAATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAATAAAAAACGAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGTTAAAGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTATTCCAGGAATGCAGAAAGACCAAAGCATACATATTGAAAATTTAAAATCAGAACGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCAATACAGGAACAGCATATGAGATAGGCATCGTTCCAAAGAATGTATCTAAAAAAGATTATAAAGCAATCGCTAAAGAACTAAGTATTTCTGAAGACTATATCAAACAACAAATGGATCAAAATTGGGTACAAGATGATACCTTCGTTCCACTTAAAACCGTTAAAAAAATGGATGAATATTTAAGTGATTTCGCAAAAAAATTTCATCTTACAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGGAAAAGCGACTTCACATCTATTAGGTTATGTTGGTCCCATTAACTCTGAAGAATTAAAACAAAAAGAATATAAAGGCTATAAAGATGATGCAGTTATTGGTAAAAAGGGACTCGAAAAACTTTACGATAAAAAGCTCCAACATGAAGATGGCTATCGTGTCACAATCGTTGACGATAATAGCAATACAATCGCACATACATTAATAGAGAAAAAGAAAAAAGATGGCAAAGATATTCAACTAACTATTGATGCTAAAGTTCAAAAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTATCCACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCATATGACGTCTATCCATTTATGTATGGCATGAGTAACGAAGAATATAATAAATTAACCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGATTACAACTTCACCAGGTTCAACTCAAAAAATATTAACAGCAATGATTGGGTTAAATAACAAAACATTAGACGATAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAAAAAGATAAATCTTGGGGTGGTTACAACGTTACAAGATATGAAGTGGTAAATGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCTTTGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATGAAAAAACTAGGTGTTGGTGAAGATATACCAAGTGATTATCCATTTTATAATGCTCAAATTTCAAACAAAAATTTAGATAATGAAATATTATTAGCTGATTCAGGTTACGGACAAGGTGAAATACTGATTAACCCAGTACAGATCCTTTCAATCTATAGCGCATTAGAAAATAATGGCAATATTAACGCACCTCACTTATTAAAAGACACGAAAAACAAAGTTTGGAAGAAAAATATTATTTCCAAAGAAAATATCAATCTATTAACTGATGGTATGCAACAAGTCGTAAATAAAACACATAAAGAAGATATTTATAGATCTTATGCAAACTTAATTGGCAAATCCGGTACTGCAGAACTCAAAATGAAACAAGGAGAAACTGGCAGACAAATTGGGTGGTTTATATCATATGATAAAGATAATCCAAACATGATGATGGCTATTAATGTTAAAGATGTACAAGATAAAGGAATGGCTAGCTACAATGCCAAAATCTCAGGTAAAGTGTATGATGAGCTATATGAGAACGGTAATAAAAAATACGATATAGATGAATAA Briefly, an RNP1 was preassembled with a gRNA sequence designed to target MRSA DNA. Specifically, RNP1 was designed to target a 20 bp region of the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 2). An RNP2 was preassembled with a gRNA sequence designed to target the unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) blocked nucleic acid molecule U29 (FIG.6A). The reaction mixture contained the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl2and 101 mM NaCl and the reaction was performed at 25° C. As stated above, the present disclosure describes controlling reaction kinetics in a cascade assay via molecular design of one of the assay components, the blocked nucleic acid molecule or the blocked primer molecule that serves as the target molecule of RNP2. As shown below, stronger regions of hybridization (or self-hybridization) via both length and GC content—leads to slower kinetics; that is, the more negative the Gibbs free energy of the blocked nucleic acid molecule or blocked primer molecule, the slower the reaction kinetics. FIG.6Ashows the structure and segment parameters of molecule U29. Note molecule U29 has a secondary structure Gibbs free energy value of −5.85 kcal/mol and relatively short self-hybridizing, double stranded regions (“clamps”) of 5 bases and 6 bases.FIGS.6B-6Hshow the results achieved for detection of 3E4 copies, 30 copies, 3 copies and 0 copies of the mecA gene of MRSA (n=3) at 25° C. with varying concentrations of blocked nucleic acid, RNP2 and reporter moiety.FIG.6Bshows the results achieved when 100 nM blocked nucleic acid molecules, 10 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 10:1. Note first that with 3M copies, nearly 100% of the reporters are cleaved at t=1 with a signal-to-noise ratio of 28.06 at 0 minutes, a signal-to-noise ratio of 24.23 at 5 minutes, and a signal-to-noise ratio of 21.01 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 12.45 at 0 minutes, 14.07 at 5 minutes and 16.16 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.79 at 0 minutes, 1.64 at 5 minutes and is 2.04 at 10 minutes. Note the measured fluorescence for 0 copies of MRSA target increases only slightly over the 10- and 30-minutes intervals, resulting in a flat negative. A flat negative signal (the results obtained over the time period for 0 copies) demonstrates that there is very little undesired signal generation in the system. Note that the negative signal when the ratio of blocked nucleic acid molecules to RNP2s is 10:1 is flatter than those inFIGS.6C through6H. FIG.6Cshows the results achieved when 50 nM blocked nucleic acid molecules, 10 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. Note first that with 3E4 copies, again nearly 100% of the reporters are cleaved at t=1 with a signal-to-noise ratio of 12.85, a signal-to-noise ratio of 10.51 at 5 minutes, and a signal-to-noise ratio of 8.18 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.85 at 0 minutes, 6.44 at 5 minutes and 6.48 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.54 at 0 minutes, 1.61 at 5 minutes and is 1.71 at 10 minutes. Note the measured fluorescence at 0 copies of MRSA target increases, resulting a less flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2. FIG.6Dshows the results achieved when 50 nM blocked nucleic acid molecules, 10 nM RNP2s and 2500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=1 with a signal-to-noise ratio of 34.92, a signal-to-noise ratio of 30.62 at 5 minutes, and a signal-to-noise ratio of 25.81 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 7.97 at 0 minutes, 1.73 at 5 minutes and 10.50 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.65 at 0 minutes, 1.73 at 5 minutes and is 1.82 at 10 minutes. Note the measured fluorescence at 0 copies of MRSA target increases, resulting in a less flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s, but possibly due to the 5× increase in the concentration of reporter moieties; however, note also that a higher concentration of reporter moieties allows for a higher signal-to-noise ratio for 3E4 and 30 copies of MRSA target. FIG.6Eshows the results achieved when 100 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and 4 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1 but double the concentration of both of these molecules than that shown inFIGS.6C and6D. With 3M copies, again nearly 100% of the reporters are cleaved at t=1 with a signal-to-noise ratio of 11.89, a signal-to-noise ratio of 8.97 at 5 minutes, and a signal-to-noise ratio of 6.53 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.46 at 0 minutes, 5.85 at 5 minutes and 5.43 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.58 at 0 minutes, 1.65 at 5 minutes and is 1.80 at 10 minutes. Note the measured fluorescence at 0 copies of MRSA increases, resulting in a less flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown inFIG.6B. Note also that the ratio of blocked nucleic acid molecules to RNP2s (5:1) appears to be more important than the ultimate concentration (100 nM/20 nM) by comparison toFIG.6Dwhere the ratio of blocked nucleic acid molecules to RNP2s was also 5:1; however, the concentration of blocked nucleic acid molecules was 50 nM and the concentration of RNP2 was 10 nM. FIG.6Fshows the results achieved when 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 4 mM NaCl. In this experiment the ratio of blocked nucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=1 with a signal-to-noise ratio of 25.85, a signal-to-noise ratio of 21.36 at 5 minutes, and a signal-to-noise ratio of 16.24 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.28 at 0 minutes, 6.19 at 5 minutes and 7.02 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is very low at 0 minutes, 1.53 at 5 minutes and is 1.73 at 10 minutes. Note the measured fluorescence at 0 copies of MRSA target increases, resulting in a less flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown inFIG.6B. Note also that the signal-to-noise ratio for all concentrations was reduced at the 2.5:1 ratio of blocked nucleic acid molecules to RNP2s. FIG.6Gshows the results achieved when 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 10 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=1 with a signal-to-noise ratio of 12.75, a signal-to-noise ratio of 7.78 at 5 minutes, and a signal-to-noise ratio of 3.66 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 6.09 at 0 minutes, 6.23 at 5 minutes and 3.58 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is very low at 0 minutes, 1.40 at 5 minutes and is 1.62 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown inFIG.6B. Note also that the signal-to-noise ratio for all concentrations was reduced substantially at the 2.5:1 ratio of blocked nucleic acid molecules to RNP2s and that the NaCl concentration at 10 mM vs. 4 mM (FIG.6F) did not make much of a difference. FIG.6Hshows the results achieved when 100 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 10 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=1 with a signal-to-noise ratio of 77.38, a signal-to-noise ratio of 74.18 at 5 minutes, and a signal-to-noise ratio of 67.90 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.94 at 0 minutes, 7,45 at 5 minutes and 9.73 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.66 at 0 minutes, 2.13 at 5 minutes and is 2.38 at 10 minutes. Note the measured fluorescence at 0 copies of MRSA target increases slightly, resulting in a less flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown inFIG.6B. Note also that the signal-to-noise ratio for all concentrations was increased substantially at the 5:1 ratio of blocked nucleic acid molecules to RNP2s as compared to the 2.5:1 ratio of blocked nucleic acid molecules to RNP2s. In summary, the results shown inFIGS.6B-6Hindicate that a 5:1 ratio of blocked nucleic acid molecules to RNP2s or greater leads to higher signal-to-noise ratios for all concentrations of MRSA target. FIG.7Ashows the structure and segment parameters of molecule F375. Note molecule F375 has a secondary structure Gibbs free energy value of −14.50 kcal/mol with longer clamps (7 bases and 7 bases) relative to molecule U29 (5 bases and 6 bases).FIG.7Bshows the results achieved for detection of 3E4 copies, 30 copies, 3 copies and 0 copies of the mecA gene of MRSA (n=3) at 25° C. at 10 or 30 minutes as indicated, where 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. For 3E4 copies of MRSA target, a signal-to-noise ratio of 21.82 is achieved at 0 minutes, a signal-to-noise ratio of 43.81 is achieved at 5 minutes, and a signal-to-noise ratio of 56.19 is achieved at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA is 3.99 at 0 minutes 7.44 at 5 minutes and 11.55 at 10 minutes. The signal-to-noise ratio for detection with 3 copies of MRSA is nearly 1 at 0 minutes, 1.95 at 10 minutes and is 2.43 at 30 minutes. Note that the reaction kinetics for molecule F375 vs. U29 are much slower. (Note thatFIG.6Cshows the results for U29 with comparable conditions.) For U29 at t=1 and 3e4 copies of MRSA target, almost 100% of the reporter molecules are cleaved but not so with F375. Also, for both 30 copies and 3 copies of MRSA target, the U29 blocked nucleic acid molecule has much higher fluorescence at t=1 than F375. FIG.8Ashows the structure and segment parameters of molecule U250. Note molecule U250 has a secondary structure Gibbs free energy value of −9.01 kcal/mol with longer clamp regions (7 bases and 7 bases) than U29 (5 bases and 6 bases) but equal-sized clamp regions of F375 (7 bases and 7 bases), but that U250 has a larger loop region (11 bases vs. 7 bases).FIG.8Bshows the results achieved for detection of 3M copies, 30 copies, 3 copies and 0 copies of the mecA gene of MRSA (n=3) at 25° C. at 10 or 30 minutes as indicated, where 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. For 3M copies, a signal-to-noise ratio of 10.37 is achieved at 0 minutes, a signal-to-noise ratio of 17.70 is achieved at 5 minutes, and a signal-to-noise ratio of 28.62 is achieved at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 6.49 at 0 minutes 7.99 at 5 minutes and 10.33 at 10 minutes. The signal-to-noise ratio for detection with 3 copies of MRSA target is nearly 1 at 0 minutes, 3.45 at 10 minutes and is 3.46 at 30 minutes. Note that the reaction kinetics for U250 are similar to those of molecule F375, and far slower than those for U29. (Note thatFIG.6Cshows the results for U29 with comparable conditions.) For U29 at t=1 and 3M copies of MRSA target, almost 100% of the reporter molecules are cleaved but not so with F375. For both 30 copies and 3 copies of MRSA target, the U29 blocked nucleic acid molecule has much higher fluorescence at t=1 than F375. FIG.9Ashows the structure and segment parameters of molecule T135. Note molecule T135 has a secondary structure Gibbs free energy value of −17.60 kcal/mol with longer clamp regions (10 bases and 12 bases) than all of U29, F375 and U250.FIG.9Bshows the results achieved for detection of 3M copies, 30 copies, 3 copies and 0 copies of the mecA gene of MRSA (n=3) at 25° C. at 10 or 30 minutes as indicated, where 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment as in the others where the results are shown inFIGS.6B,7B and8B, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. For 3M copies, a signal-to-noise ratio of 42.74 is achieved at 0 minutes, a signal-to-noise ratio of 106.61 is achieved at 5 minutes, and a signal-to-noise ratio of 91.04 is achieved at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 9.75 at 0 minutes, 15.77 at 5 minutes and 20.04 at 10 minutes. The signal-to-noise ratio for detection with 3 copies of MRSA target is nearly 1 at 0 minutes, 3.18 at 5 minutes, 3.07 at 10 minutes and is 3.64 at 30 minutes. Note that the reaction kinetics for T135 is similar to those for F375 and U250, but much slower than the reaction kinetics for U29. Note also that the signal-to-noise ratio for 3M and 30 copies of MRSA target was very high. FIG.10Ashows the structure and segment parameters of molecule T134. Note molecule T134 has a secondary structure Gibbs free energy value of −16.13 kcal/mol with clamp regions of 9 bases and 12 bases, on par with those of molecule T135.FIG.10Bshows the results achieved for detection of 3E4 copies, 30 copies, 3 copies and 0 copies of the mecA gene of MRSA (n=3) at 25° C. at 10 or 30 minutes as indicated, where 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment as in the others where the results are shown inFIGS.6B,7B,8B, and10B the ratio of blocked nucleic acid molecules to RNP2s is 5:1. For 3E4 copies, a signal-to-noise ratio of 63.06 is achieved at 0 minutes, a signal-to-noise ratio of 194 is achieved at 5 minutes, and a signal-to-noise ratio of 194 is achieved at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 13.99 at 0 minutes, 32.23 at 5 minutes and 26.82 at 10 minutes. The signal-to-noise ratio for detection with 3 copies of MRSA target is nearly 1 at 0 minutes, 2.34 at 10 minutes and is 2.44 at 30 minutes. The reaction kinetics for molecule T134 is roughly comparable to the reaction kinetics for molecule T135; also with exceptional signal-to-noise ratios at 10 minutes, and even at t=1 and t=5. FIG.11Ashows the structure and segment parameters of molecule T119. Note molecule T119 has a secondary structure Gibbs free energy value of −17.53 kcal/mol with longer clamp regions (10 bases and 12 bases) than all of U29, F375, U250, T135 and T134.FIG.11Bshows the results achieved for detection of 3E4 copies, 30 copies, 3 copies and 0 copies of the mecA gene of MRSA (n=3) at 25° C. at 10 or 30 minutes as indicated, where 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment as in the others where the results are shown inFIGS.6B,7B,8B,9B, and10Bthe ratio of blocked nucleic acid molecules to RNP2s is 5:1. For 3M copies, a signal-to-noise ratio of 21.27 is achieved at 0 minutes, a signal-to-noise ratio of 69.55 is achieved at 5 minutes, and a signal-to-noise ratio of 231 is achieved at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA is 4.56 at 0 minutes, 10.88 at 5 minutes and 25.66 at 10 minutes. The signal-to-noise ratio for detection with 3 copies of MRSA is nearly 1 at 0 minutes, 7.09 at 10 minutes and is 3.36 at 30 minutes. The kinetics for molecule T119 are similar to those of T134, also with exceptional signal-to-noise ratios at t=1, t=5 and t=10. In summary, different designs with different clamps allow for different detection kinetics; that is, the kinetics of the detection reaction can be controlled by the design of the blocked nucleic acid molecule; thus, the blocked nucleic acid molecules are quantitative or semi-quantitative by design. Very long clamps lead to longer detection times but also allow for higher resolution for quantifying target nucleic acids of interest over the longer time period. Note again that the cascade assay reactions were carried out at 25° C. While certain embodiments have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the present disclosures. Indeed, the novel methods, apparatuses, modules, instruments and systems described herein can be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the methods, apparatuses, modules, instruments and systems described herein can be made without departing from the spirit of the present disclosures. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the present disclosures. PARTIES TO A JOINT RESEARCH AGREEMENT The presently claimed invention was made by or on behalf of the below-listed parties to a joint research agreement. The joint research agreement was in effect on or before the date the claimed invention was made, and the claimed invention was part of the joint research agreement and made as a result of activities undertaken within the scope of the joint research agreement. The parties to the joint research agreement are The Board of Trustees of the University of Illinois and LabSimply, Inc. (now VedaBio, Inc.). | 159,775 |
11859183 | DESCRIPTION OF EMBODIMENTS In the present disclosure, computational and TCGA analyses was performed to identify novel miRNAs that can target CDK4/6 and that can be used for therapeutic treatment of colorectal cancer (CRC). The 3′-UTR of CDK4/6 mRNA is shown here to be novel targets of a previously uncharacterized family of miRNAs encompassing miR-6883-5p, miR-149*, miR-6785-5p, and miR-4728-5p. The data presented herein of miRs 6883-5p and 149* revealed that both miRNAs downregulate CDK4 and CDK6 protein and mRNA expression when ectopically expressed in human CRC cell lines. RNA-seq data indicated an inverse relationship between the expression of CDK4/6 and miR-149* and intronic miRNA-6883-5p encoding gene PER1 in CRC patient samples. Restoring expression of miRs 6883-5p and 149* had significant anti-proliferative effects, G0/G1-arrest, and apoptosis in CRC cell lines. Targeting of CDK4/6 by miR-6883-5p and miR-149* can only, in part, explain the anti-proliferative effects of these miRNAs as seen on silencing CDK4/6 in CRC cell lines. Lastly, both miRNAs synergized with frontline CRC chemotherapy irinotecan and sensitized mutant p53 cell lines to 5-FU. Thus, the miRNA-based therapeutic strategy to target CDK4/6 can be used as both a single agent and combinatorial therapy, and to identify biomarkers of response. In particular, in the present disclosure, a combination of in silico prediction algorithms and TCGA analysis was used to identify tumor suppressor miRNAs that can translate to single agent and/or combinatorial therapies for CRC. There have been previous studies describing miRNAs regulating of CDK4 expression in melanoma and NSCLC (Georgantas et al., Pigment Cell Melanoma Res., 2014, 27, 275-86; Lin et al., Cancer Res., 2010, 70, 9473-82; Shao et al., Oncotarget, 2016, 7, 34011-21; and Deng et al., J. Neurooncol., 2013, 114, 263-74). However, there are no such tumor suppressor miRs identified in CRC and/or studies evaluating their use as therapeutics. In the present disclosure, a new family of miRNAs, whose expression is lost in CRC, has be identified. The primary gene network (i.e., G1-S phase of cell cycle, which is regulated by two of the four miRNAs in the family) was characterized herein. Within the CDK4/6-Rb pathway, the present disclosure presents data that gene targets mediating the anti-proliferative effects of miR-6883-5p and miR-149* include CDK4, CDK6 and FOXM1, which has recently been identified to be phosphorylated by CDK4/6 (Anders et al., Cancer Cell, 2011, 20, 620-34). FOXM1 is a Forkhead Box family transcription factor that has also been also linked to resistance to different chemotherapies like cisplatin and 5-FU (Wang et al., Lung Cancer, 2013, 79, 173-9) and is an important oncogenic target in preclinical development. Thus, the inhibition of CDK4/6 and its downstream effector FOXM1 by miRNAs has an advantage of targeting the CDK4/6-Rb pathway at different levels. The combination experiments with FDA-approved chemotherapies Irinotecan and 5-FU, as demonstrated herein, show that both the miRNAs can sensitize CRC cell lines to each of the drugs and indicate that the use of combinations of miRNAs as adjuvant therapeutics for the treatment of colorectal cancer is a viable clinical strategy. In addition to cell cycle, the pro-apoptotic functions of miR-6883-5p and miR-149* has been demonstrated herein. Both miRNAs, by downregulating anti-apoptotic proteins BCLxL and XIAP, lead to apoptosis both as single and combination in CRC cell lines. In summary, new miRNAs, including miR-6883-5p and miR-149*, have been identified herein as direct negative regulators of CDK4 and CDK6. Restoring the expression of miR-6883-5p and miR-149* in cancer cells showed anti-proliferative effects and apoptosis as single agents and in combination, respectively. Thus, miRNA mimics as adjuvant therapy for cancer is an alternate to small molecule inhibitors. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the arts to which the claimed subject matter. As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. As used herein, “colon cancer” means malignancy of the colon, either a primary cancer or metastasized cancer. As used herein, “subject” means a human or non-human animal selected for treatment or therapy. As used herein, “in need thereof” means a subject identified as in need of a therapy or treatment. In some embodiments, a subject has a tumor or cancer, such as colon cancer. In such embodiments, a subject has one or more clinical indications of a tumor or cancer, such as colon cancer, or is at risk for developing a tumor or cancer, such as colon cancer. As used herein, “administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering. As used herein, “parenteral administration,” means administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration. As used herein, “subcutaneous administration” means administration just below the skin. As used herein, “intravenous administration” means administration into a vein. As used herein, “intratumoral administration” means administration within a tumor. As used herein, “intraperitoneal administration” means administration into the peritoneum (i.e., body cavity). As used herein, “chemoembolization” means a procedure in which the blood supply to a tumor is blocked surgically, mechanically, or chemically and chemotherapeutic agents are administered directly into the tumor. As used herein, “duration” means the period of time during which an activity or event continues. In some embodiments, the duration of treatment is the period of time during which one or more doses of a pharmaceutical agent or pharmaceutical composition are administered. As used herein, “therapy” means a disease treatment method. In some embodiments, therapy includes, but is not limited to, chemotherapy, surgical resection, and/or chemoembolization. As used herein, “treatment” means the application of one or more specific procedures used for the cure or amelioration of a disease. In some embodiments, the specific procedure is the administration of one or more pharmaceutical agents. As used herein, “amelioration” means a lessening of severity of at least one indicator of a condition or disease. In some embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art. As used herein, “prevention” refers to delaying or forestalling the onset or development or progression of a condition or disease for a period of time, including weeks, months, or years. As used herein, “therapeutic agent” means a pharmaceutical agent used for the cure, amelioration or prevention of a disease. As used herein, “chemotherapeutic agent” means a pharmaceutical agent used to treat cancer. As used herein, “chemotherapy” means treatment of a subject with one or more pharmaceutical agents that kills cancer cells and/or slows the growth of cancer cells. As used herein, “dose” means a specific quantity of a pharmaceutical agent provided in a single administration. A dose may be administered in two or more boluses, tablets, or injections. In some embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual. As used herein, “dosage unit” means a form in which a pharmaceutical agent is provided. In some embodiments, a dosage unit is a vial containing lyophilized oligonucleotide. In some embodiments, a dosage unit is a vial containing reconstituted oligonucleotide. As used herein, “therapeutically effective amount” refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to an animal. As used herein, “pharmaceutical composition” means a mixture of substances suitable for administering to a subject that includes a pharmaceutical agent. For example, a pharmaceutical composition may comprise a modified oligonucleotide and a sterile aqueous solution. As used herein, “pharmaceutical agent” means a substance that provides a therapeutic effect when administered to a subject. As used herein, “active pharmaceutical ingredient” means the substance in a pharmaceutical composition that provides a desired effect. As used herein, “metastasis” means the process by which cancer spreads from the place at which it first arose as a primary tumor to other locations in the body. The metastatic progression of a primary tumor reflects multiple stages, including dissociation from neighboring primary tumor cells, survival in the circulation, and growth in a secondary location. As used herein, “overall survival time” means the time period for which a subject survives after diagnosis of or treatment for a disease. As used herein, “progression-free survival” means the time period for which a subject having a disease survives, without the disease getting worse. In some embodiments, progression-free survival is assessed by staging or scoring the disease. In some embodiments, progression-free survival of a subject having colon cancer is assessed by evaluating tumor size, tumor number, and/or metastasis. As used herein, “improved colon function” means the change in colon function toward normal limits. As used herein, “acceptable safety profile” means a pattern of side effects that is within clinically acceptable limits. As used herein, “side effect” means a physiological response attributable to a treatment other than desired effects. In some embodiments, side effects include, without limitation, injection site reactions, colon function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies. Such side effects may be detected directly or indirectly. As used herein, “injection site reaction” means inflammation or abnormal redness of skin at a site of injection in an individual. As used herein, “subject compliance” means adherence to a recommended or prescribed therapy by a subject. As used herein, “comply” means the adherence with a recommended therapy by a subject. As used herein, “recommended therapy” means a treatment recommended by a medical professional for the treatment, amelioration, or prevention of a disease. As used herein, “targeting” means the process of design and selection of nucleobase sequence that will hybridize to a target nucleic acid and induce a desired effect. As used herein, “targeted to” means having a nucleobase sequence that will allow hybridization to a target nucleic acid to induce a desired effect. In some embodiments, a desired effect is reduction of a target nucleic acid. As used herein, “modulation” means to a perturbation of function or activity. In some embodiments, modulation means an increase in gene expression. In some embodiments, modulation means a decrease in gene expression. As used herein, “expression” means any functions and steps by which a gene's coded information is converted into structures present and operating in a cell. As used herein, “nucleobase sequence” means the order of contiguous nucleobases, in a 5′ to 3′ orientation, independent of any sugar, linkage, and/or nucleobase modification. As used herein, “contiguous nucleobases” means nucleobases immediately adjacent to each other in a nucleic acid. As used herein, “percent identity” means the number of nucleobases in first nucleic acid that are identical to nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid. Percent identity (or percent complementarity) between particular stretches of nucleotide sequences within nucleic acid molecules or amino acid sequences within polypeptides can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Herein, if reference is made to percent sequence identity, the higher percentages of sequence identity are preferred over the lower ones. As used herein, “substantially identical” used herein may mean that a first and second nucleobase sequence are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% at least 99%, or 100%, identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, or 40 or more nucleobases. As used herein, “hybridize” means the annealing of complementary nucleic acids that occurs through nucleobase complementarity. As used herein, “mismatch” means a nucleobase of a first nucleic acid that is not capable of pairing with a nucleobase at a corresponding position of a second nucleic acid. As used herein, “identical” means having the same nucleobase sequence. As used herein, “hsa-miR-6883-5p” means the modified oligonucleotide having the nucleobase sequence set forth in SEQ ID NO: 1. As used herein, “hsa-miR-149-3p” means the modified oligonucleotide having the nucleobase sequence set forth in SEQ ID NO: 2. As used herein, “hsa-miR-6785-5p” means the modified oligonucleotide having the nucleobase sequence set forth in SEQ ID NO: 3. As used herein, “hsa-miR-4728-5p” means the modified oligonucleotide having the nucleobase sequence set forth in SEQ ID NO: 4. As used herein, “miRNA” or “miR” means a non-coding RNA from about 18 to about nucleobases in length. As used herein, “oligomeric compound” means a compound comprising a polymer of linked monomeric subunits. As used herein, “oligonucleotide” means a polymer of linked nucleosides, each of which can be modified or unmodified, independent from one another. As used herein, “naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage between nucleosides. As used herein, “natural sugar” means a sugar found in DNA (2′-H) or RNA (2′-OH). As used herein, “natural nucleobase” means a nucleobase that is unmodified relative to its naturally occurring “internucleoside linkage” means a covalent linkage between adjacent nucleosides. As used herein, “linked nucleosides” means nucleosides joined by a covalent linkage. As used herein, “nucleobase” means a heterocyclic moiety capable of non-covalently pairing with another nucleobase. As used herein, “nucleoside” means a nucleobase linked to a sugar. As used herein, “nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of a nucleoside. As used herein, “modified oligonucleotide” means an oligonucleotide having one or more modifications relative to a naturally occurring terminus, sugar, nucleobase, and/or internucleoside linkage. As used herein, “modified internucleoside linkage” means any change from a naturally occurring internucleoside linkage. As used herein, “phosphorothioate internucleoside linkage” means a linkage between nucleosides where one of the non-bridging atoms is a sulfur atom. As used herein, “modified sugar” means substitution and/or any change from a natural sugar. As used herein, “modified nucleobase” means any substitution and/or change from a natural nucleobase. As used herein, “5-methylcytosine” means a cytosine modified with a methyl group attached to the 5′ position. As used herein, “2′-O-methyl sugar” or “2′-O-Me sugar” means a sugar having an O-methyl modification at the 2′ position. As used herein, “2′-O-methoxyethyl sugar” or “2′-MOE sugar” means a sugar having a 0-methoxyethyl modification at the 2′ position. As used herein, “2′-O-fluoro” or “2-F” means a sugar having a fluoro modification of the 2′ position. As used herein, “bicyclic sugar moiety” means a sugar modified by the bridging of two non-geminal ring atoms. As used herein, “2′-O-methoxyethyl nucleoside” means a 2′-modified nucleoside having a 2′-O-methoxyethyl sugar modification. As used herein, “2′-fluoro nucleoside” means a 2′-modified nucleoside having a 2′-fluoro sugar modification. As used herein, “2′-O-methyl” nucleoside means a 2′-modified nucleoside having a 2′-O-methyl sugar modification. As used herein, “bicyclic nucleoside” means a 2′-modified nucleoside having a bicyclic sugar moiety. As used herein, “motif” means a pattern of modified and/or unmodified nucleobases, sugars, and/or internucleoside linkages in an oligonucleotide. As used herein, a “fully modified oligonucleotide” means each nucleobase, each sugar, and/or each internucleoside linkage is modified. As used herein, a “uniformly modified oligonucleotide” means each nucleobase, each sugar, and/or each internucleoside linkage has the same modification throughout the modified oligonucleotide. As used herein, a “stabilizing modification” means a modification to a nucleoside that provides enhanced stability to a modified oligonucleotide, in the presence of nucleases, relative to that provided by 2′-deoxynucleosides linked by phosphodiester internucleoside linkages. For example, in some embodiments, a stabilizing modification is a stabilizing nucleoside modification. In some embodiments, a stabilizing modification is a internucleoside linkage modification. As used herein, a “stabilizing nucleoside” means a nucleoside modified to provide enhanced nuclease stability to an oligonucleotide, relative to that provided by a 2′-deoxynucleoside. In one embodiment, a stabilizing nucleoside is a 2′-modified nucleoside. As used herein, a “stabilizing internucleoside linkage” means an internucleoside linkage that provides enhanced nuclease stability to an oligonucleotide relative to that provided by a phosphodiester internucleoside linkage. In one embodiment, a stabilizing internucleoside linkage is a phosphorothioate internucleoside linkage. The present disclosure provides oligonucleotides, such as modified oligonucleotides, consisting of 15 to 40 linked nucleobases, or a salt thereof, wherein the oligonucleotide comprises a nucleobase sequence that is at least 80% identical to a nucleobase sequence of hsa-miR-6883-5p, hsa-miR-149-3p, hsa-miR-6785-5p, or hsa-miR-4728-5p. In some embodiments, hsa-miR-6883-5p comprises the nucleobase sequence agggagggugugguauggaugu (SEQ ID NO:1). In some embodiments, hsa-miR-149-3p comprises the nucleobase sequence agggagggacgggggcugugc (SEQ ID NO:2). In some embodiments, hsa-miR-6785-5p comprises the nucleobase sequence ugggagggcguggaugauggug (SEQ ID NO:3). In some embodiments, hsa-miR-4728-5p comprises the nucleobase sequence ugggaggggagaggcagcaagca (SEQ ID NO:4). In some embodiments, the oligonucleotide comprises a nucleobase sequence that is at least 85% identical to a nucleobase sequence of hsa-miR-6883-5p, hsa-miR-149-3p, hsa-miR-6785-5p, or hsa-miR-4728-5p. In some embodiments, the oligonucleotide comprises a nucleobase sequence that is at least 90% identical to a nucleobase sequence of hsa-miR-6883-5p, hsa-miR-149-3p, hsa-miR-6785-5p, or hsa-miR-4728-5p. In some embodiments, the oligonucleotide comprises a nucleobase sequence that is at least 95% identical to a nucleobase sequence of hsa-miR-6883-5p, hsa-miR-149-3p, hsa-miR-6785-5p, or hsa-miR-4728-5p. In some embodiments, an oligonucleotide consists of 15 to 30 linked nucleobases. In some embodiments, an oligonucleotide consists of 19 to 24 linked nucleobases. In some embodiments, an oligonucleotide consists of 21 to 24 linked nucleobases. In some embodiments, the oligonucleotide consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 linked nucleobases. In some embodiments, the oligonucleotide consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 linked nucleobases. In some embodiments, the oligonucleotide consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 linked nucleobases. In some embodiments, the oligonucleotide comprises a nucleobase sequence comprising at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of a nucleobase sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In each of these embodiments, the oligonucleotide can be a modified oligonucleotide. In some embodiments, the nucleobase sequence of the oligonucleotide has no more than two mismatches compared to a nucleobase sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the nucleobase sequence of the oligonucleotide has no more than one mismatch compared to a nucleobase sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the nucleobase sequence of the oligonucleotide has one mismatch compared to a nucleobase sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the nucleobase sequence of the oligonucleotide has no mismatches compared to a nucleobase sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In each of these embodiments, the oligonucleotide can be a modified oligonucleotide. Suitable nucleic acids include, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), modified DNA or RNA, peptide nucleic acid (PNA), morpholino, locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), DNA containing phosphorothioate residues (S-oligos) and derivatives thereof, or any combination thereof. In some embodiments, one or more additional nucleobases may be added to either or both of the 3′ terminus and 5′ terminus of an oligonucleotide in comparison to the nucleobases sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the one or more additional linked nucleobases are at the 3′ terminus. In some embodiments, the one or more additional linked nucleosides are at the 5′ terminus. In some embodiments, two additional linked nucleosides are linked to a terminus. In some embodiments, one additional nucleoside is linked to a terminus. In each of these embodiments, the oligonucleotide can be a modified oligonucleotide. In some embodiments, the oligonucleotide comprises one or more modified internucleoside linkages, modified sugars, or modified nucleobases, or any combination thereof. The nucleobase sequences set forth herein, including but not limited to those found in the Examples and in the sequence listing, are independent of any modification to the nucleic acid. As such, nucleic acids defined by a SEQ ID NO: may comprise, independently, one or more modifications to one or more sugar moieties, to one or more internucleoside linkages, and/or to one or more nucleobases. A modified nucleobase, sugar, and/or internucleoside linkage may be selected over an unmodified form because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets and increased stability in the presence of nucleases. In some embodiments, at least one internucleoside linkage is a modified internucleoside linkage. In some embodiments, each internucleoside linkage is a modified internucleoside linkage. In some embodiments, a modified internucleoside linkage comprises a phosphorus atom. In some embodiments, a modified oligonucleotide comprises at least one phosphorothioate internucleoside linkage. In some embodiments, each internucleoside linkage of a modified oligonucleotide is a phosphorothioate internucleoside linkage. In some embodiments, a modified internucleoside linkage does not comprise a phosphorus atom. In some such embodiments, an internucleoside linkage is formed by a short chain alkyl internucleoside linkage. In some such embodiments, an internucleoside linkage is formed by a cycloalkyl internucleoside linkages. In some such embodiments, an internucleoside linkage is formed by a mixed heteroatom and alkyl internucleoside linkage. In some such embodiments, an internucleoside linkage is formed by a mixed heteroatom and cycloalkyl internucleoside linkages. In some such embodiments, an internucleoside linkage is formed by one or more short chain heteroatomic internucleoside linkages. In some such embodiments, an internucleoside linkage is formed by one or more heterocyclic internucleoside linkages. In some such embodiments, an internucleoside linkage has an amide backbone. In some such embodiments, an internucleoside linkage has mixed N, O, S and CH2component parts. In some embodiments, at least one nucleobase of the modified oligonucleotide comprises a modified sugar. In some embodiments, each of a plurality of nucleosides comprises a modified sugar. In some embodiments, each nucleoside of the modified oligonucleotide comprises a modified sugar. In each of these embodiments, the modified sugar may be a 2′-O-methoxyethyl sugar, a 2′-fluoro sugar, a 2′-O-methyl sugar, or a bicyclic sugar moiety. In some embodiments, each of a plurality of nucleosides comprises a 2′-O-methoxyethyl sugar and each of a plurality of nucleosides comprises a 2′-fluoro sugar. In some embodiments, the sugar-modified nucleosides can further comprise a natural or modified heterocyclic base moiety and/or a natural or modified internucleoside linkage and may include further modifications independent from the sugar modification. In some embodiments, a sugar modified nucleoside is a 2′-modified nucleoside, wherein the sugar ring is modified at the 2′ carbon from natural ribose or 2′-deoxyribose. In some embodiments, a 2′-modified nucleoside has a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety is a D sugar in the alpha configuration. In some such embodiments, the bicyclic sugar moiety is a D sugar in the beta configuration. In some such embodiments, the bicyclic sugar moiety is an L sugar in the alpha configuration. In some such embodiments, the bicyclic sugar moiety is an L sugar in the beta configuration. In some embodiments, the bicyclic sugar moiety comprises a bridge group between the 2′ and the 4′-carbon atoms. In some such embodiments, the bridge group comprises from 1 to 8 linked biradical groups. In some embodiments, the bicyclic sugar moiety comprises from 1 to 4 linked biradical groups. In some embodiments, the bicyclic sugar moiety comprises 2 or 3 linked biradical groups. In some embodiments, the bicyclic sugar moiety comprises 2 linked biradical groups. Biradical groups are well known in the art. In some embodiments, the modified oligonucleotide comprises at least one modified nucleobase. In some embodiments, the modified nucleobase is selected from 5-hydroxymethyl cytosine, 7-deazaguanine and 7-deazaadenine. In some embodiments, the modified nucleobase is selected from 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. In some embodiments, the modified nucleobase is selected from 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. In some embodiments, the modified nucleobase is a 5-methylcytosine. In some embodiments, at least one nucleoside comprises a cytosine, wherein the cytosine is a 5-methylcytosine. In some embodiments, each cytosine is a 5-methylcytosine. In some embodiments, a 2′-modified nucleoside comprises a 2′-substituent group selected from halo, allyl, amino, azido, —SH, —CN, —OCN, —CF3, —OCF3, —O—, —S—, or —N(Rm)-alkyl; —O—, —S—, or —N(Rm)-alkenyl; —O—, —S— or —N(Rm)-alkynyl; —O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, —O-alkaryl, —O-aralkyl, —O(CH2)2SCH3, —O—(CH2)2—O—N(Rm)(Rn) or —O—CH2—C(═O)—N(Rm)(Rn), where each Rmand Rnis, independently, H, an amino protecting group or substituted or unsubstituted C1-10alkyl. These 2′-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl. In some embodiments, a 2′-modified nucleoside comprises a 2′-substituent group selected from F, NH2, N3, OCF3, O—CH3, O(CH2)3NH2, CH2—CH═CH2, O—CH2—CH═CH2, OCH2CH2OCH3, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), —O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O—CH2—C(═O)—N(Rm)(Rn) where each Rmand Rnis, independently, H, an amino protecting group or substituted or unsubstituted C1-10alkyl. In some embodiments, a 2′-modified nucleoside comprises a 2′-substituent group selected from F, OCF3, O—CH3, OCH2CH2OCH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(CH3)2, —O(CH2)2O(CH2)2N(CH3)2, and O—CH2—C(═O)—N(H)CH3. In some embodiments, a 2-modified nucleoside comprises a 2′-substituent group selected from F, O—CH3, and OCH2CH2OCH3. In some embodiments, a sugar-modified nucleoside is a 4′-thio modified nucleoside. In some embodiments, a sugar-modified nucleoside is a 4′-thio-2′-modified nucleoside. A 4′-thio modified nucleoside has a B-D-ribonucleoside where the 4′-0 replaced with 4′-S. A 4′-thio-2′-modified nucleoside is a 4′-thio modified nucleoside having the 2′-OH replaced with a 2′-substituent group. Suitable 2′-substituent groups include 2′-OCH3, 2′-O—(CH2)2—OCH3, and 2′-F. In some embodiments, a modified nucleobase comprises a polycyclic heterocycle. In some embodiments, a modified nucleobase comprises a tricyclic heterocycle. In some embodiments, a modified nucleobase comprises a phenoxazine derivative. In some embodiments, the phenoxazine can be further modified to form a nucleobase known in the art as a G-clamp. In some embodiments, the oligonucleotide compound comprises a modified oligonucleotide conjugated to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. In some such embodiments, the moiety is a cholesterol moiety or a lipid moiety. Additional moieties for conjugation include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. In some embodiments, a conjugate group is attached directly to a modified oligonucleotide. In some embodiments, a conjugate group is attached to a modified oligonucleotide by a linking moiety selected from amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted C1-10alkyl, substituted or unsubstituted C2-10alkenyl, and substituted or unsubstituted C2-10alkynyl. In some such embodiments, a substituent group is selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl. In some such embodiments, the oligonucleotide compound comprises a modified oligonucleotide having one or more stabilizing groups that are attached to one or both termini of a modified oligonucleotide to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect a modified oligonucleotide from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures include, for example, inverted deoxy abasic caps. Additional cap structures include, but are not limited to, a 4′,5′-methylene nucleotide, a 1-(beta-D-erythrofuranosyl) nucleotide, a 4′-thio nucleotide, a carbocyclic nucleotide, a 1,5-anhydrohexitol nucleotide, an L-nucleotide, an alpha-nucleotide, a modified base nucleotide, a phosphorodithioate linkage, a threopentofuranosyl nucleotide, an acyclic 3′,4′-seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide, an acyclic 3,5-dihydroxypentyl nucleotide, a 3′-3′-inverted nucleotide moiety, a 3′-3′-inverted abasic moiety, a 3′-2′-inverted nucleotide moiety, a 3′-2′-inverted abasic moiety, a 1,4-butanediol phosphate, a 3′-phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a 3′-phosphate, a 3′-phosphorothioate, a phosphorodithioate, a bridging methylphosphonate moiety, and a non-bridging methylphosphonate moiety 5′-amino-alkyl phosphate, a 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2-aminododecyl phosphate, a hydroxypropyl phosphate, a 5′-5′-inverted nucleotide moiety, a 5′-5′-inverted abasic moiety, a 5′-phosphoramidate, a 5′-phosphorothioate, a 5′-amino, a bridging and/or non-bridging 5′-phosphoramidate, a phosphorothioate, and a 5′-mercapto moiety. The present disclosure also provides pharmaceutical compositions comprising one or more of the oligonucleotides described herein. In some embodiments, the oligonucleotide consists of 15 to 30 linked nucleosides, or a salt thereof, wherein the modified oligonucleotide comprises a nucleobase sequence that is at least 80% identical to a nucleobase sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, and a pharmaceutically acceptable carrier or diluent. In each of these embodiments, the oligonucleotide can be a modified oligonucleotide. In some embodiments, the compositions may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation. In some embodiments, pharmaceutical compositions comprise one or more modified oligonucleotides and one or more excipients. In some such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone. In some embodiments, a pharmaceutical composition is prepared using known techniques, including, but not limited to mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tab letting processes. In some embodiments, a pharmaceutical composition is a liquid (e.g., a suspension, elixir and/or solution). In some such embodiments, a liquid pharmaceutical composition is prepared using ingredients known in the art, including, but not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. In some embodiments, a pharmaceutical composition is a solid (e.g., a powder, tablet, and/or capsule). In some such embodiments, a solid pharmaceutical composition comprising one or more oligonucleotides is prepared using ingredients known in the art, including, but not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents. In some embodiments, a pharmaceutical composition is formulated as a depot preparation. Some such depot preparations are typically longer acting than non-depot preparations. In some embodiments, such preparations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. In some embodiments, depot preparations are prepared using suitable polymeric or hydrophobic materials (for example an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. In some embodiments, a pharmaceutical composition comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Delivery systems are useful for preparing pharmaceutical compositions including those comprising hydrophobic compounds. In some embodiments, some organic solvents such as dimethylsulfoxide are used. In some embodiments, presently available RNAi packaging technology can be used to packing the miRNA in lipid complexes and to deliver the miRNA. The delivery system can also comprise nanoparticles or nano-complexes. The delivery system can also comprise bacterial mini-cells comprising RNA duplexes. In some embodiments, a pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents to specific tissues or cell types. For example, in some embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody. In some embodiments, a pharmaceutical composition comprises a cosolvent system. Some such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In some embodiments, such cosolvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. In some embodiments, a pharmaceutical composition comprises a sustained-release system. A non-limiting example of such a sustained-release system is a semi-permeable matrix of solid hydrophobic polymers. In some embodiments, sustained-release systems may, depending on their chemical nature, release pharmaceutical agents over a period of hours, days, weeks or months. In some embodiments, a pharmaceutical composition is prepared for oral administration. In some such embodiments, a pharmaceutical composition is formulated by combining one or more compounds comprising any one or more of the oligonucleotides described herein with one or more pharmaceutically acceptable carriers. Some such carriers enable pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject. In some embodiments, pharmaceutical compositions for oral use are obtained by mixing oligonucleotide and one or more solid excipient. Suitable excipients include, but are not limited to, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In some embodiments, such a mixture is optionally ground and auxiliaries are optionally added. In some embodiments, pharmaceutical compositions are formed to obtain tablets or dragee cores. In some embodiments, disintegrating agents (e.g., cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate) are added. In some embodiments, dragee cores are provided with coatings. In some such embodiments, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to tablets or dragee coatings. In some embodiments, pharmaceutical compositions for oral administration are push-fit capsules made of gelatin. Some such push-fit capsules comprise one or more of the oligonucleotides described herein in admixture with one or more filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In some embodiments, pharmaceutical compositions for oral administration are soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In some soft capsules, one or more of the oligonucleotides described herein are be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. In some embodiments, pharmaceutical compositions are prepared for buccal administration. Some such pharmaceutical compositions are tablets or lozenges formulated in conventional manner. In some embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, etc.). In some such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In some embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In some embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Some pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Some pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Some solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the oligonucleotides described herein to allow for the preparation of highly concentrated solutions. In some embodiments, a pharmaceutical composition is prepared for transmucosal administration. In some such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. In some embodiments, a pharmaceutical composition is prepared for administration by inhalation. Some such pharmaceutical compositions for inhalation are prepared in the form of an aerosol spray in a pressurized pack or a nebulizer. Some such pharmaceutical compositions comprise a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In some embodiments using a pressurized aerosol, the dosage unit may be determined with a valve that delivers a metered amount. In some embodiments, capsules and cartridges for use in an inhaler or insufflator may be formulated. Some such formulations comprise a powder mixture of one or more of the oligonucleotides described herein and a suitable powder base such as lactose or starch. In some embodiments, a pharmaceutical composition is prepared for rectal administration, such as a suppositories or retention enema. Some such pharmaceutical compositions comprise known ingredients, such as cocoa butter and/or other glycerides. In some embodiments, a pharmaceutical composition is prepared for topical administration. Some such pharmaceutical compositions comprise bland moisturizing bases, such as ointments or creams. Exemplary suitable ointment bases include, but are not limited to, petrolatum, petrolatum plus volatile silicones, and lanolin and water in oil emulsions. Exemplary suitable cream bases include, but are not limited to, cold cream and hydrophilic ointment. In some embodiments, a pharmaceutical composition comprises a modified oligonucleotide in a therapeutically effective amount. In some embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. In some embodiments, the pharmaceutical composition may further comprise at least one additional therapeutic agent. The additional therapeutic agent may be a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a platinum-based chemotherapeutic agent such as, for example, cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin. In some embodiments, the chemotherapeutic agent is a taxane such as, for example, paclitaxel, docetaxel, or cabazitaxel. In some embodiments, the chemotherapeutic agent is a type I topoisomerase inhibitor such as, for example, irinotecan, topotecan, camptothecin, or lamellarin D. In some embodiments, the chemotherapeutic agent is a type II topoisomerase inhibitor such as, for example, etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, or HU-331. In some embodiments, the chemotherapeutic agent is a combination of chemotherapeutic agents such as, for example, CHOP (cyclophosphamide, doxorubicin (hydroxydaunomycin), vincristine (Oncovin®), and prednisolone). In some embodiments, the chemotherapeutic agent may be selected from 5-fluorouracil, cisplatin, gemcitabine, doxorubicine, mitomycin c, sorafenib, etoposide, carboplatin, epirubicin, irinotecan and oxaliplatin. In some embodiments, the chemotherapeutic is 5-fluorouracil or irinotecan. The pharmaceutical composition may comprise one or more of the oligonucleotide compounds described herein in combination with one or more of the additional therapeutic agents. For example, the pharmaceutical composition may comprise an one of more oligonucleotides consisting of 15 to 40 linked nucleobases, or a salt thereof, wherein the oligonucleotide comprises a nucleobase sequence that is at least 80% identical to a nucleobase sequence of hsa-miR-6883-5p, hsa-miR-149-3p, hsa-miR-6785-5p, or hsa-miR-4728-5p, in combination with any one or more of 5-fluorouracil, cisplatin, gemcitabine, doxorubicine, mitomycin c, sorafenib, etoposide, carboplatin, epirubicin, irinotecan and oxaliplatin. In some embodiments, the pharmaceutical composition comprises an one of more oligonucleotides consisting of 15 to 40 linked nucleobases, or a salt thereof, wherein the oligonucleotide comprises a nucleobase sequence that is at least 80% identical to a nucleobase sequence of hsa-miR-6883-5p, hsa-miR-149-3p, hsa-miR-6785-5p, or hsa-miR-4728-5p, in combination with 5-fluorouracil and/or irinotecan. In some embodiments, an additional therapy may be a pharmaceutical agent that enhances the body's immune system, including low-dose cyclophosphamide, thymostimulin, vitamins and nutritional supplements (e.g., antioxidants, including vitamins A, C, E, beta-carotene, zinc, selenium, glutathione, coenzyme Q-10 and echinacea), and vaccines, e.g., the immunostimulating complex (ISCOM), which comprises a vaccine formulation that combines a multimeric presentation of antigen and an adjuvant. In some embodiments, a pharmaceutical agent that induces the expression of the miRNAs disclosed herein, or induces the expression of PER1 or regulates the expression of PER1, such as atypical psychotics including, but not limited to, quetiapine and haloperidol can be used. In some embodiments, the pharmaceutical agent is melatonin. In some embodiments, the pharmaceutical agent for inducing the expression of the miRNAs or PER1 is forskolin, interleukin-6, or Sp-5,6-DCI-cBiMPS. These pharmaceutical agents may be present in a pharmaceutical composition. The present disclosure also provides methods for treating a tumor or cancer, comprising administering to a subject in need thereof one or more of the oligonucleotides described herein, and/or a pharmaceutical agent that induces the production of the one or more oligonucleotides and/or induces PER1 expression. In some embodiments, the oligonucleotide consists of 15 to 30 linked nucleosides, wherein the oligonucleotide comprises a nucleobase sequence that is at least 80% identical to a nucleobase sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the oligonucleotide is a modified oligonucleotide as described herein. The present disclosure also provides methods for treating a tumor or cancer, comprising administering to a subject in need thereof a pharmaceutical composition comprising one or more of the oligonucleotides described herein, or a pharmaceutical agent that induces the production of the one or more oligonucleotides and/or induces PER1 expression. In some embodiments, the oligonucleotide consists of 15 to 30 linked nucleosides, wherein the oligonucleotide comprises a nucleobase sequence that is at least 80% identical to a nucleobase sequence selected from SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the oligonucleotide is a modified oligonucleotide as described herein. In some embodiments, the cancer being treated is a tumor or solid tumor. In some embodiments, the cancer cell or tumor overexpresses a CDK, such as CDK4 and/or CDK6. In some embodiments, the cancer cell or tumor exhibits a loss of p16. In some embodiments, the cancer cell or tumor exhibits a loss of Rb. In some embodiments, the cancer cell or tumor exhibits overexpression of cyclin D1. In some embodiments, the cancer cell or tumor is in need of inhibition of a CDK, such as CDK4 and/or CDK6. In some embodiments, the tumor or cancer is pancreatic, melanoma, colorectal, colon, lung, breast, or leukemia. In some embodiments, the cancer is colon cancer, colorectal cancer, lung cancer, or melanoma. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the tumors may be less well-oxygenated than the normal tissues from which they arose (i.e., “tumor hypoxia”) which, in some cases, may lead to resistance to radiotherapy and anticancer chemotherapy as well as predisposing for increased tumor metastases. In some embodiments, the methods described herein use one or more oligonucleotides or modified oligonucleotides that is/are targeted to CDK4 and/or CDK6. The oligonucleotides or modified oligonucleotides can be administered with or without being integrated into a vector. The oligonucleotides or modified oligonucleotides can also be used in the form of double stranded entities, whereby the appropriate strand is produced inside a cell. In some embodiments, administration of a compound comprises intravenous administration, subcutaneous administration, intratumoral administration, intraperitoneal administration, or chemoembolization. In some embodiments, the methods further comprise administering at least one additional therapy. The additional therapy may be a chemotherapeutic agent. The chemotherapeutic agent may be selected from 5-fluorouracil, cisplatin, gemcitabine, doxorubicine, mitomycin c, sorafenib, etoposide, carboplatin, epirubicin, irinotecan and oxaliplatin. In some embodiments, the chemotherapeutic is 5-fluorouracil or irinotecan. The additional therapy may be administered at the same time, less frequently, or more frequently than a compound or pharmaceutical composition described herein. In some embodiments, the additional therapy is surgical resection and/or chemoembolization. In some embodiments, any one or more of the oligonucleotides described herein is administered at a dose selected from 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mg. The oligonucleotide may be administered one per day, once per week, once per two weeks, once per three weeks, or once per four weeks. In some embodiments, the administration of a compound results in reduction of tumor size and/or tumor number. In some embodiments, the administration of a compound prevents an increase in tumor size and/or tumor number. In some embodiments, the administration of a compound prevents, slows, and/or stops metastatic progression. In some embodiments, the administration of a compound extends the overall survival time of the subject. In some embodiments, the administration of a compound extends the progression-free survival of the subject. In some embodiments, administration of a compound prevents the recurrence of tumors. In some embodiments, administration of a compound prevents recurrence of tumor metastasis. A subject may be diagnosed with a tumor or cancer, such as colon cancer, following the administration of medical tests well known to those in the medical profession. The diagnosis of a tumor or cancer, such as colon cancer, can be made by imaging tests such as ultrasound, helical computed tomography (CT) scan, triple phase CT scan, or magnetic resonance imaging (MRI). The imaging tests allow the assessment of the tumor size, number, location, metastasis, patency and/or invasion of adjacent tissue by the tumor. This assessment aids the decision as to the mode of therapeutic or palliative intervention that is appropriate. The final diagnosis is typically confirmed by needle biopsy and histopathological examination. Administration of a pharmaceutical composition to a subject having a tumor can result in one or more clinically desirable outcomes. Such clinically desirable outcomes include reduction of tumor number or reduction of tumor size. Additional clinically desirable outcomes include the extension of overall survival time of the subject, and/or extension of progression-free survival time of the subject. In some embodiments, administration of a pharmaceutical composition prevents an increase in tumor size and/or tumor number. In some embodiments, administration of a pharmaceutical composition prevents metastatic progression. In some embodiments, administration of a pharmaceutical composition slows or stops metastatic progression. In some embodiments, administration of a pharmaceutical composition prevents the recurrence of tumors. In some embodiments, administration of a pharmaceutical composition prevents recurrence of tumor metastasis. In some embodiments, administration of a pharmaceutical composition prevents the recurrence of tumors. Administration of a pharmaceutical composition to tumor cells may result in desirable phenotypic effects. In some embodiments, an oligonucleotide may stop, slow or reduce the uncontrolled proliferation of tumor cells. In some embodiments, an oligonucleotide may induce apoptosis in tumor cells. In some embodiments, an oligonucleotide may reduce tumor cell survival. In some embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are administered at the same time. In some embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are administered at different times. In some embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are prepared together in a single formulation. In some embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are prepared separately. In some embodiments, a pharmaceutical composition is administered in the form of a dosage unit (e.g., tablet, capsule, bolus, etc.). In some embodiments, such pharmaceutical compositions comprise any one or more of the oligonucleotides or modified oligonucleotides described herein in a dose selected from 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg, 445 mg, 450 mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg, 490 mg, 495 mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg, 530 mg, 535 mg, 540 mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg, 575 mg, 580 mg, 585 mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg, 620 mg, 625 mg, 630 mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg, 665 mg, 670 mg, 675 mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg, 710 mg, 715 mg, 720 mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg, 750 mg, 755 mg, 760 mg, 765 mg, 770 mg, 775 mg, 780 mg, 785 mg, 790 mg, 795 mg, and 800 mg. In some such embodiments, a pharmaceutical composition comprises a dose of modified oligonucleotide selected from 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, and 800 mg. In some embodiments, a pharmaceutical agent is sterile lyophilized oligonucleotide that is reconstituted with a suitable diluent, e.g., sterile water for injection or sterile saline for injection. The reconstituted product is administered as a subcutaneous injection or as an intravenous infusion after dilution into saline. The lyophilized drug product consists of any one or more of the oligonucleotides or modified oligonucleotides described herein which has been prepared in water for injection, or in saline for injection, adjusted to pH 7.0-9.0 with acid or base during preparation, and then lyophilized. The lyophilized modified oligonucleotide may be 25-800 mg of any one or more of the oligonucleotides or modified oligonucleotides described herein. It is understood that this encompasses 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mg of modified lyophilized oligonucleotide. The lyophilized drug product may be packaged in a 2 mL Type I, clear glass vial (ammonium sulfate-treated), stoppered with a bromobutyl rubber closure and sealed with an aluminum FLIP-OFF® overseal. The present disclosure also provides methods of detecting and/or determining the level of any one or more of the miRNA described herein. The detection and/or level determination can be carried out by conventional means known in the art. The level of particular miRNAs can be used as disease progress markers for any of the cancers disclosed herein. The miRNAs can also be used to predict and/or monitor a therapeutic response. The present disclosure also provides any one or more of the oligonucleotide compounds described herein, or compositions comprising the same, for use in treating or preventing cancer or tumors. The present disclosure also provides any one or more of the oligonucleotide compounds described herein, or compositions comprising the same, for use in the manufacture of a medicament for treating or preventing cancer or tumors. The present disclosure also provides uses of any one or more of the oligonucleotide compounds described herein, or compositions comprising the same, for treating or preventing cancer or tumors. The present disclosure also provides uses of any one or more of the oligonucleotide compounds described herein, or compositions comprising the same, in the manufacture of a medicament for treating or preventing cancer or tumors. The present disclosure also provides any one or more of the oligonucleotide compounds described herein, or compositions comprising the same, or methods of preparing the same, or methods of using the same, or uses any one or more of the oligonucleotide compounds described herein, or compositions comprising the same, substantially as described with reference to the accompanying examples and/or figures. In order that the subject matter disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the claimed subject matter in any manner. Throughout these examples, molecular cloning reactions, and other standard recombinant DNA techniques, were carried out according to methods described in Maniatis et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, except where otherwise noted. EXAMPLES Example 1: General Materials and Methods Cell culture and reagents: All colorectal, pancreatic and melanoma cell lines were obtained from American Type Culture Collection and maintained in the recommended media. miRNA mimics for hsa-miR-6883-5p, hsa-miR-149*, and hsa-miR-206 (HMI2616, HMI0241 and HMI0364) were purchased from Sigma-Aldrich. siRNA for CDK4 and CDK6 (sc-29261 and sc-29264) were purchased from Santa Cruz Biotechnology. The CDK4-Luciferase construct was obtained from Origene Technologies. Transfection of miRNA mimics, siRNA and plasmid constructs: All miRNA mimics and siRNA transfections were performed by reverse transfection using Lipofectamine RNAiMAX (Life technologies, Grand Island, NY). 80 nM siRNA was used in all experiments for HT-29, RKO and SW-480 cell lines. 40 nM siRNA was used for HCT-116 cells. miRNA mimics were transfected at concentrations of either 25 nM, 50 nM, or 100 nM, as indicated in respective assays. CDK4-Luciferase vector was transfected in HCT-116 cells using Lipofectamine 3000 (Life technologies, Grand Island, NY) and stable cells were selected using G418 antibiotic (500 μg/mL). Luciferase assays: CDK4-Luciferase containing stable HCT-116 cells were reverse-transfected with either scramble duplex or 50 nM miRNA mimics. Luciferase signal was detected 48 hours post-transfection and Relative Luciferase Units (RLU) were calculated by normalizing luciferase signal per μg of protein per assay well. All transfections were performed in triplicates and reported as RLU units±SEM. Cell Proliferation assays: Five thousand to ten thousand cells were reverse transfected with either scramble duplex or miRNA to a net concentration of 50 nM and plated in 96-well plate. Cell viability was measured 72 hours post-transfection using CellTiter-Glo® Luminescent Cell Viability Assay (Promega). Percent cell viability was calculated by normalizing the luminescence signal to scramble duplex wells. All transfections were performed in triplicates and reported as % Viability±SEM. Cell Cycle Analysis: All cell lines were reverse transfected with either scramble duplex or miRNA mimic. At 72 hours post-transfection, both floating and adherent cells were collected and fixed in 70% ethanol, followed by RNase A treatment and PI staining. Cell death (sub-G1) was quantified by propidium iodide (PI) staining and fluorescence-activated cell sorting (FACS). Flo-Jo analysis was performed to quantify the distribution of cells in G1, S, and G2-M phases of cell cycle under different transfection conditions. Colony formation assays: A total of 0.1×106cells were reverse transfected with either scramble duplex or miRNA mimics to net concentration of 50 nM (HT-29 and HCT-116) or 100 nM (RKO and SW-480) for 72 hours. At 72 hours, transfected cells were harvested and 500 cells per treatment group were plated in triplicate in 6-well plates for colony formation. Colonies were crystal violet stained on Day 14, imaged, counted and reported as the number of colonies±SEM. Quantitative RT-PCR (qRT-PCR): Total RNA, which includes miRNA, was isolated using the Quick-RNA™ MiniPrep kit (Zymo Research, Irvine, CA). One μg of total RNA from each sample was subjected to cDNA synthesis using SuperScript® III Reverse Transcriptase kit (Life Technologies, Grand Island, NY), for detection of CDK4, CDK6, and housekeeping genes. For detection of miRNAs, 0.5 μg of total RNA was reverse transcribed using TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies, Grand Island, NY). The relative expression of the reported genes and miRNAs was determined using real-time PCR performed on Applied Biosystems 7900HT Fast Real-Time PCR system. GAPDH and RNU6B were used as the endogenous controls for mRNA and miRNA samples respectively. Each cDNA sample was amplified using Power SYBR Green (Applied Biosystems, CA) and miRNA components were quantified using TaqMan® Universal Master Mix II, no UNG (Applied Biosystems, CA). TaqMan miRNA assays were purchased from Applied Biosystems and used as per manufacturer's instructions. Western blot: Western blotting was performed by routine and well known procedures. The following antibodies were used: CDK4 (Santa Cruz Biotechnology, sc-260), CDK6 (CST, D4S8S), CDK1 (Santa Cruz Biotechnology, sc-54), Cyclin-D1 (CST, 92G2), p-Rb (S795) (CST, 9301S), Total Rb, BCLxL (CST, 2764S), PARP (CST, 9542), p53-DO1 (Santa Cruz Biotechnology, sc-126), p21 (Calbiochem, OP64), and R-actin (Sigma, A5441). Statistical analysis: Data are presented as the mean±standard error of mean from at least three replicates. The Student's two-tailed t-test in GraphPad Prism was used for pairwise analysis. Statistically significant changes (*p≤0.05, **p≤0.01 and ***≤0.001) are indicated. Example 2: CDK4 and CDK6 are Important Therapeutic Targets and can be Regulated with miRNAs in CRC Events of overexpression of cell cycle oncogenes (CDKs, Cyclins) and suppression of tumor suppressors (p16, Rb) in the CDK4/6-Rb pathway are mutually exclusive and tumor-type specific. To assess the status of these events in CRC, RNA-sequencing data from The Cancer Genome Atlas (TCGA) for normal and tumor samples (T/N) was analyzed. Of the 50 T/N samples analyzed as shown inFIG.1A, both CDK4 (left panel) and CDK6 (right panel) expression were significantly high in the tumor samples (p=2.2 e-16 and p=1.594 e-08 respectively). There was no significant change in the RNA levels of tumor suppressors p16 and Rb. These findings are in consensus with previous reports which have shown overexpression of CDK4 and CDK6 in IHC samples with differences on staging (Zhao et al., World J. Gastroenterol., 2003, 9, 2202-2206; and Zhao et al., World J. Gastroenterol., 2006, 12, 6391-6396). To determine whether CDK4 and CDK6 can be negatively regulated by miRNAs, an in silico approach using TargetScan, an online computational algorithm, was taken to find miRNAs that could target the 3′UTR of both CDK4 and CDK6. There have been prior reports of conserved miRNAs, including miR-206, miR-124-3p, and miR-15-5p, that examine CDK4 targeting alone in melanoma and other tumor types. One goal was to focus on novel and uncharacterized miRNAs that also had relevance in CRC and could target both CDK4 and CDK6. Based on a combination of TCGA analysis and TargetScan, a new family of miRNAs encompassing miRs 6883-5p, 149*, 6785-5p and 4728-5p was developed. Each of these miRNAs were predicted to target both CDK4 and CDK6 3′UTR with 8-mer and 7-mer-lA binding sites (Table 1 and 2, respectively, ofFIG.1B). miRs 6883-5p and 149* were further examined for their novelty and relevance to CRC. As seen inFIG.1C, expression of miR-149* was significantly lost in 11 patient CRC tumors as compared to normal tissue (p=0.0049) as assayed by RNA-seq using TCGA analysis. Loss of miR-149* was also correlative to the staging of the tumor (data not shown). Each of the 11 patients also had significant increase in CDK4 expression with little to no change in the other markers of the CDK4/6-Rb pathway (see,FIG.1C). As for miR-6883-5p, no data was located in TCGA. The TCGA analysis showed that RNA expression of PER1 is significantly lost in the same 50 T/N samples assayed prior (see,FIG.1D). Thus, the in silico analysis suggests that restoring expression of miRs 6883-5p and 149* can be used as therapies in treating CRC. Referring in particular toFIGS.1A,1B,1C, and1D, data is presented showing that CDK4 and CDK6 are important therapeutic targets in CRC.FIG.1Ashows RNA expression data from TCGA CRC patient samples showing expression of CDK4 and CDK6 in 50 tumor and normal samples. Box plots indicate the log10RNA expression of normal samples compared to tumor samples for every gene of interest. p-values were obtained from the Wilcoxon test for unpaired samples and are indicated in the figures.FIG.1Bshows Tables 1 and 2 indicating TargetScan analysis of putative binding site(s) of the family of four miRNAs in the 3′UTR regions of CDK4 and CDK6, respectively.FIG.1C(left panel) shows a scatter plot of expression of miR-149* in 11 CRC patient tumors compared to matched normal tissue. Corresponding p-values obtained from the Wilcoxon test for paired samples is indicated.FIG.1C(right panel) shows a histogram of CDK4/6, p16 and Rb status in the same 11 patients.FIG.1Dshows box plots showing the log10RNA expression of PER1 genes in the same 50 normal samples asFIG.1Acompared to tumor samples. p-values were obtained from the Wilcoxon test for unpaired samples and are indicated. Example 3: miR-6883-5p and miR-149* Repress Expression of CDK4 and CDK6 Whether the in silico predictions regarding miR-6883-5p and miR-149* regulation of CDK4/6 translated to in vitro results was examined. A panel of CRC cell lines was used: HCT-116 (p53+/+), RKO (p53+/+), HT-29 (p53 R273H), and SW-480 (p53 R273H/P309S). All the CRC cell lines used are proficient for Rb and p16 except SW-480, which is p16−/− (seeFIG.6). Each of the cell lines was reverse transfected with both miR-6883-5p and miR-149* and examined for protein and RNA expression of CDK4 and CDK6. As shown inFIGS.2A and2B, miR-6883-5p targeted both CDK4 and CDK6 at both the protein and RNA level. miR-149*, on the contrary, was more potent in reducing levels of CDK6 and had no impact on CDK4. This data for potency and specificity of targeting CDKs was compared with miR-206, which has previously been reported to target CDK4 (Georgantas et al., Pigment Cell Melanoma Res., 2014, 27, 275-86). It was further confirmed using the pMirtarget-CDK4-Luciferase vector which has the CDK4 3′UTR cloned with the luciferase gene that both miR-6883-5p and miR-149* directly bind to the 3′UTR region of CDK4. As indicated inFIG.2C, miR-6883-5p significantly reduced luciferase signal compared to miR-206 and miR-149*, respectively. Referring in particular toFIGS.2A,2B, and2C, data is presented showing that miR-6883-5p and miR-149* negatively regulate expression of CD4 and CDK6 in CRC cell lines. Referring toFIG.2A, CDK4 and CDK6 protein levels were detected in a panel of CRC cell lines reverse transfected with 50 nM of miRNA mimics or scrambled duplex (SCR) for 72 hours. Referring toFIG.2B, qRT-PCR was performed for CDK4 and CDK6 in SCR or miRNA mimics transfected in CRC cell lines (50 nM, 72 hours, n=3). *, ** and *** indicate p-value relative to SCR expression. Referring toFIG.2C, HCT-116 cells stably selected with CDK4-Luciferase construct were reverse transfected with SCR or 50 nM of the indicated miRNA mimics for 48 hours. Measured luciferase activities were normalized per μg of protein for indicated samples and reported as RLU±SEM (n=3). * indicates p-value relative SCR RLU. Example 4: Restoring Expression of miR-6883-5p and miR-149* Results in Gr-Arrest and Cell Death Given the ability to target CDK4/6, the functional consequences of restoring the expression of miRs-6883-5p and 149* as compared to miR-206 in the panel of CRC cell lines was determined. Since the miRNAs target proteins in the cell cycle, it was believed that they could affect cell proliferation, both short-term and long-term. As shown inFIG.3A, all three miRNAs had comparable and moderate short-term anti-proliferative effect on the panel of CRC cell lines as measured by cell viability, 72 hours post-transfection. There was greater than 50-70% inhibition on the long-term proliferation of these cells as seen from colony formation assay in all four cell lines (see,FIG.3B). The effects of the miRNAs on the cell cycle markers were also examined to determine whether the inhibition of CDK4/6 would lead to G1-arrest in all the cell lines. As shown inFIG.3C, in all four cell lines, miR-6883-5p and miR-149* reduced levels of phosphorylated Rb (S795), indicating G1-arrest and inhibition of CDK4/6 activity. These findings were further confirmed by PI-staining and looking at the cell cycle profiles of each of these cell lines. As shown inFIG.3E, both RKO and HT-29 cells showed G1-arrest on transfection with each of the miRNAs and a small fraction of cells underwent cell death. However, in HCT-116 alone, expression of all miRNAs led to apoptosis, with between 30-50% cells in sub-G1 phase. This result further replicated with the PARP-cleavage data seen inFIG.3D. While in HCT-116 cell lines, all three miRNAs induced increased apoptosis; in RKO and SW-480 cells, 6883-5p was the most potent in inducing apoptosis. No change in the p53 levels indicated that apoptosis was not p53-dependent in p53+/+ cells. Interestingly, miR-6883-5p downregulated XIAP and BCLXL, which in part could explain the induction of apoptosis by miR-6883-5p. While XIAP is a predicted target of the family of miRNAs, BCLXLis not. However, both BCLXLand XIAP contribute to the pro-apoptotic effects of the miRs. Referring in particular toFIGS.3A,3B,3C,3D, and3E, data is presented showing that restoring expression of miR-6883-5p and miR-149* results in G1-arrest and cell death in CRC cell lines. Referring toFIG.3A, a panel of CRC cell lines was reverse transfected with 50 nM SCR or 50 nM of indicated miRNA mimics. The effects on cell viability were measured 72 hours post-transfection using CellTiter-Glo assay. Referring toFIG.3B, the effects of the miRNA mimics on long-term cell proliferation of CRC cell lines was assessed by colony formation assays performed in 6-well plates. Cells were reverse transfected with 100 nM of SCR or indicated miRNA mimic. After 72 hours, 500 cells were seeded per well in triplicate for each condition and stained with crystal violet on Day 14. Representative images of cells stained with crystal violet are shown (left panel) and relative colony number (n=3) is represented graphically (right panel). All four CRC cell lines were reverse transfected with 100 nM SCR or indicated miRNA mimics. The effects on cell cycle markers (see,FIG.3C) and markers of apoptosis (see,FIG.3D) were evaluated by western blot 72 hours post-transfection. Representative western blots are shown (n=3). Referring toFIG.3E, cell cycle profiles and apoptotic cells were assessed in three CRC cell lines by reverse transfecting with SCR or 50 nM (HCT-116 and HT-29) or 100 nM (RKO) miRNA mimics. 72 hours post-transfection, cells were fixed, stained with PI, and analyzed by FACS. Representative results of changes G1 and sub-G1 phases of cell cycle are graphically represented (n=3). Example 5: Silencing of CDK4 and CDK6 Phenocopies the Effects of miR-6883-5p and miR-149* Mimics in CRC Cell Lines To determine whether the biological effects of miR-6883-5p and miR-149* could be attributed to the direct targeting of CDK4/6, the expression of CDK4/6 was silenced by siRNA and the associated functional consequences were detected. As shown inFIG.4A, knockdown of CDK4 and CDK6 had similar effects on short-term proliferation of CRC cell lines as with overexpression of miRNAs. However, silencing the expression of CDK4/6 was less potent in preventing long-term proliferation of CRC cell lines, especially RKO and HT-29, as seen inFIG.4B. This indicates that targeting of CDK4/6 by miR-6883-5p and miR-149* can only, in part, explain the anti-proliferative effects of these miRNAs. Knockdown of CDK4 and CDK6 siRNAs arrested cells in G1phase of cell cycle (see,FIGS.4C and4D). However, unlike the miRNAs, knockdown of CDK4 alone lead to cell death HCT-116 and SW-480 cells. Given the leakiness of the CDK4 siRNA, knockdown of both CDK4 and CDK6 is more potent and needed to induce apoptosis in CRC cell lines compared to either gene alone. Thus, the dual targeting of CDK4 and CDK6 by miRNAs has therapeutic benefits in CRC cell lines. Referring in particular toFIGS.4A,4B,4C, and4D, the effect of silencing of CDK4 and CDK6 phenocopies on miRNA mimics is shown. Referring toFIG.4A, all four CRC cell lines were reverse transfected with SCR or 80 nM of CDK4 or CDK6 siRNA. The effects of short-term cell proliferation were measured 72 hours post-transfection using CellTiter-Glo assay. Referring toFIG.4B, the long-term effects on cell proliferation by silencing CDK4 or CDK6 were assessed by a colony formation assay performed in 6-well plates. All four cell lines were reverse transfected with 80 nM CDK4 or CDK6. At 72 hours post-transfection, 500 cells were seeded per well in triplicate and stained with crystal violet on Day 14. Representative images of cells stained with crystal violet are shown (left panel) and relative colony number (n=3) is represented graphically (right panel). Referring toFIG.4C, all four CRC cell lines were reverse transfected with 80 nM CDK4 or CDK6 siRNA. The effects on markers of cell cycle and apoptosis were evaluated by western blot 72 hours post-transfection. Representative western blots are shown (n=3). Referring toFIG.4D, cell cycle profiles and apoptotic cells were assessed in three CRC cell lines by reverse transfecting with SCR or 40 nM (HCT-116) or 80 nM (HT-29 and RKO) siRNA of CDK4 and CDK6. 72 hours post-transfection, cells were fixed, stained with PI, and analyzed by FACS. Representative results of changes G1 and sub-G1 phases of cell cycle are graphically represented (n=3). Example 6: miR-6883-5p and miR-149* Synergize with FDA-Approved Therapeutics for CRC The combinatorial effect of miR-6883-5p and miR-149* with frontline therapeutics Irinotecan and 5-FU was evaluated. As shown inFIG.5A, both miR-6883-5p and miR-149* synergized with Irinotecan in all four cell lines. Further, both the miRNAs also increased the sensitivity of p53 mutant cell lines HT-29 and SW-480 to 5-FU (see,FIG.5B). The synergistic combinations led to cell death in all four cell lines as measured by PARP cleavage (see,FIGS.5C and5D). The Irinotecan-miRNA combination engaged the intrinsic pathway of cell death as measured by Cleaved Caspase-9, BCLXLand XIAP (see,FIG.5C). In cells treated with 5-FU, single agent miR-149* and 5-FU caused cell cycle arrest in, as seen by p21 levels. The combination however, led to apoptosis (seeFIG.5D). Thus, both miR-6883-5p and miR-149* are combination agents in CRC. Referring in particular toFIGS.5A,5B,5C, and5D, data is presented that demonstrates that miR-6883-5p and miR-149* synergize with Irinotecan and 5-FU in CRC cell lines. A panel of CRC cell lines was reverse transfected with 25 nM (HCT-116) or 50 nM of SCR or indicated miRNA mimics. At 16 hours post-transfection, Irinotecan (see,FIG.5A) or 5-FU (see,FIG.5B) at indicated doses were added. Synergy of miRNA-drug was measured by cell viability 72 hours post-transfection using CellTiter-Glo assay. The effect on apoptosis and cell cycle markers with miRNA alone or combination were assessed using 50 nM of miRNA mimic and 5M (HT-29 and SW-480) or 2.5 μM Irinotecan (HCT-116 and RKO) by western blot. For 5-FU, 384 μM (HT-29 and SW-480) and 25 μM (HCT-116 and RKO) were used. Representative western blots are shown inFIG.5CandFIG.5D, respectively. Example 7: Efficacy in Cell Lines RNA expression data for CDK4, CDK6 and PER1 was obtained from TCGA CRC patient samples in 50 matched tumor and normal samples (see,FIG.8). The scatter plots indicate the log10RNA expression of normal samples compared to tumor samples for the indicated gene of interest. p-values were obtained from the Wilcoxon test for paired samples and are indicated. Cell viability, cell proliferation, and Western blot analysis of pancreatic cancer cell lines treated with SCR or the indicated miRNA was examined (see,FIG.9). Cell viability, cell proliferation, and Western blot analysis of melanoma cancer cell lines treated with SCR or the indicated miRNA was also examined (see,FIG.10). In addition, cell viability and Western blot analysis of the induction of G1-cell cycle arrest by targeting CDK4 and CDK6, independent of p53 status, was also examined (see,FIG.11). Various modifications of the described subject matter, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, and the like) cited in the present application is incorporated herein by reference in its entirety. The subject matter described herein was made with government support under Grant Nos. R01 CA 176289 and P30 CA 006927 awarded by The National Institutes of Health (NIH). | 82,939 |
11859184 | EXAMPLES Mode for Invention Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention. Example 1: Preparation of Double-Stranded siRNA Via Hydrogen Bond of Sense Strand siRNA and Antisense Strand siRNA Having Substitution with Same Functional Group at their Ends and Preparation of Multi-Conjugate of siRNA Using Cross-Linking Agent 100 nmol of sense or antisense strand siRNA having substitution with sulfhydryl group at 3′ end was dissolved in 260 μl of 1×PBS, which stood at 37° C. for 1 hour, resulting in double-stranded siRNA. To reduce sulfhydryl group at both ends of the prepared double-stranded siRNA, 22 μl of 25×PBS, 260 μl of 2M DTT (dithiothreitol) solution and 4 μl of 5N NaOH solution (to adjust pH) were added thereto, followed by reaction for 12 hours. Upon completion of the reaction, remaining DTT was eliminated by dialysis and the solution was concentrated to 1 nmol/μl. 25×PBS was added to adjust the final concentration to 5×PBS. The cross-linking agent DTME or BM(PEG)2was added at the concentration of half the concentration of thiol group, followed by reaction at room temperature for 24 hours. Upon completion of the reaction, remaining foreign materials such as cross-linking agent, etc, were eliminated by dialysis and the solution was concentrated to make the final concentration to 1˜2 μg/μl to prepare a siRNA multi-conjugate (seeFIG.1A). The prepared siRNA was confirmed by electrophoresis. (seeFIG.2) A multi-conjugate was prepared by direct covalent bonding of double-stranded siRNA mediated by oxidation without using a cross-linking agent. Double-stranded siRNA having substitution with thiol group at 3′ ends of sense and antisense strands was treated with DTT by the same manner as described above, followed by dialysis and concentration to make the final concentration of the solution to 1 nmol/μl. DMSO and diamide were added to the above solution to oxidize sulfhydryl, resulting in the formation of disulfide bond. The prepared double-stranded siRNA multi-conjugate was confirmed by electrophoresis (seeFIG.3A). Example 2: Preparation of Dimer of Each Sense Strand siRNA and Antisense Strand siRNA Having the Substitution with Same Functional Group at the End Using Cross-Linking Agent and Preparation of siRNA Multi-Conjugate Via Hydrogen Bond 100 nmol of sense or antisense strand siRNA having the substitution with sulfhydryl group at 3′ end was dissolved in 260 μl of DEPC (Diethyl pyrocarbonate) treated deionized water, to which 22 μl of 25×PBS was added. 260 μl of 2M DTT (dithiothreitol) was added thereto and then 4 μl of 5N NaOH was added to adjust pH, followed by reaction for 12 hours. Upon completion of the reaction, remaining DTT was eliminated by dialysis and the solution was concentrated. As a result, sense or antisense strand siRNA having the final concentration of 1 nmol/μl was prepared. 25×PBS was added to adjust the final concentration to 5×PBS. The cross-linking agent DIME or BM(PEG)2was added thereto at the concentration of half the concentration of thiol group, followed by reaction at room temperature for hours. Upon completion of the reaction, foreign materials such as cross-linking agent, etc, were eliminated by dialysis, and the solution was concentrated to prepare the dimer form of sense or antisense siRNA having the final concentration of 1-2 μg/μl (seeFIG.1B). The dimer prepared by cleavable disulfide bond (seeFIG.3C) or non-cleavable covalent bond (seeFIG.3D) were confirmed by electrophoresis. Equal amount of sense and antisense dimers stood in PBS at 37° C. for 1 hour to induce hydrogen bond. As a result, a siRNA multi-conjugate was prepared and confirmed by electrophoresis (seeFIG.3B). Example 3: Preparation of Double-Stranded siRNA by Hydrogen Bonding of Sense Strand siRNA and Antisense Strand siRNA Having Different Functional Groups at their Ends and Preparation of siRNA Conjugate Using Cross-Linking Agent Sense strand and antisense strand siRNA having respectively amine group and sulfhydryl group at 3′end were prepared. 100 nmol of each sense and antisense siRNA was dissolved in 260 μl of PBS, which stood at 37° C. for 1 hour, resulting in the preparation of double-stranded siRNA. DTT was treated thereto in order to prepare single-stranded siRNA having sulfhydryl group substituted at the end, followed by dialysis and concentration to make the final concentration of 1 nmol/μl. The cross-linking agent sulfo-SMCC (sulfosuccinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxylate) was added to the prepared double stranded siRNA, followed by reaction for 24 hours to prepare a multi-conjugate of siRNA. Upon completion of the reaction, remaining foreign materials such as cross-linking agent, etc, were eliminated by dialysis and the solution was concentrated to make the final concentration to 1˜2 μg/μl (seeFIG.1C). Example 4: Preparation of Dimer of Each Sense Strand siRNA and Antisense Strand siRNA Having the Substitution with Different Functional Groups at their Ends Using Cross-Linking Agent and Preparation of siRNA Multi-Conjugate Via Hydrogen Bond Sense and antisense siRNA each having amine group and sulfhydryl group at 3′ end were linked to make double-stranded siRNA using the cross-linking agent sulfo-SMCC. Single-stranded siRNA having the substitution with sulfhydryl group at the end was treated with DTT, followed by dialysis and concentration until the final concentration reached 1 nmol/μl. Single-stranded siRNA having the substitution with amine group at the end was dissolved in DEPC treated distilled water at the concentration of 1 nmol/μl. Each solution containing sense and antisense having respectively amine group and sulfhydryl group was treated with sulfo-SMCC to prepare sense or antisense dimer. The prepared sense or antisense dimer was mixed in PBS, which stood at 37° C. for 1 hour, resulting in the preparation of a double-stranded siRNA multi-conjugate (seeFIG.1D). Experimental Example 1: Measurement of GFP Expression An ionic complex was prepared from the siRNA multi-conjugate (prepared by the method ofFIG.1A) linked by cleavable disulfide bond or non-cleavable covalent bond using the siRNA inhibiting GFP gene, the conventional siRNA, a cross linking agent and linear PEI (polyethyleneimine). The prepared ionic complex was treated to the cancer cell line MDA-MB-435 expressing GFP stably for 5 hours. 48 hours later, GFP expression was quantified with a fluorophotometer. As a result, the siRNA multi-conjugate of the present invention demonstrated excellent gene delivery efficiency using a cationic gene carrier and excellent target gene inhibition activity, compared with the conventional siRNA (seeFIG.4). Experimental Example 2: Measurement of Binding Strength with Cationic Gene Carrier and Stability To confirm whether the siRNA multi-conjugate (prepared by the method ofFIG.1B) had excellent binding strength with a cationic gene carrier and could form a stable ionic complex, compared with the conventional siRNA, the present inventors mixed the representative gene carrier Linear PEI and each siRNA to produce each ionic complex. And then, shape and size of each complex were observed by AFM. As a result, the siRNA multi-conjugate of the present invention demonstrated excellent binding strength with a cationic polymer and was capable of forming small but even nano particles, compared with the conventional siRNA (seeFIG.5). To investigate the amount of the cationic polymer binding to each siRNA, gel retardation assay was performed. As a result, the siRNA multi-conjugate of the present invention had higher charge density than the conventional siRNA, suggesting that the siRNA multi-conjugate of the present invention can form an ionic complex by binding with a cationic polymer even at a low concentration (seeFIG.6). Experimental Example 3: Investigation of Gene Inhibition Efficiency Using VEGF An ionic complex was prepared from the siRNA multi-conjugate (prepared by the method ofFIG.1B) linked by cleavable disulfide bond or non-cleavable covalent bond using the siRNA inhibiting VEGF gene, the conventional siRNA, and linear PEI (polyethyleneimine) The prepared ionic complex was treated to PC3 cancer cells for 5 hours. Then VEGF secreted for 21 hours was quantified by ELISA. The experiment was performed over the siRNA concentration (0, 18, 45, and 90 nM) and over the NP ratio (0, 10, 15, and 20), which was the ratio of amine in the cationic carrier to phosphate of nucleotide. To investigate weather intracellular mRNA was reduced selectively, each ionic complex was treated to cancer cells for 5 hours. 18 hours later, RNA was extracted, followed by PCR to measure the level of intracellular VEGF mRNA. As a result, the siRNA multi-conjugate of the present invention could form a stable and even ionic complex with a cationic polymer, compared with the conventional siRNA, and demonstrated excellent gene delivery efficiency and target gene inhibition activity (seeFIG.7). Experimental Example 4: Investigation of VEGF Inhibition Efficiency Electrophoresis was performed with the VEGF siRNA multi-conjugate prepared by the method ofFIG.1B, and siRNA was sorted over the size by gel separation method, followed by electrophoresis to confirm thereof. Each siRNA was mixed with Linear PEI to form a complex. 90 nM of the siRNA conjugate was treated to PC3 cell, and then VEGF gene inhibition effect was measured. As a result, as molecular weight of the siRNA multi-conjugate increased, charge density was increased, suggesting the improvement of gene delivery efficiency using a cationic polymer (seeFIG.8). Experimental Example 5: Induction of Immune Response by siRNA Multi-Conjugate To investigate immune response induction capacity of the siRNA multi-conjugate prepared by the method ofFIG.1B, peripheral blood mononuclear cells (PBMC) were separated from human blood by using Fisher lymphocyte separation medium. An ionic complex was prepared from the conventional VEGF siRNA or the siRNA multi-conjugate linked by cleavable disulfide bond or non-cleavable covalent bond by using the cationic gene carrier linear PEI, jet PEI or DOTAP. The siRNA complex was treated to PBMC at the final concentration of 360 nM for 24 hours. INF-alpha level in the supernatant was measured by ELISA. As a result, the siRNA multi-conjugate of the present invention did not induce INF-alpha significantly, compared with the conventional siRNA. In particular, the siRNA multi-conjugate prepared by disulfide bond demonstrated similar INF-alpha induction to the conventional siRNA (seeFIG.9A). To confirm whether the prepared siRNA multi-conjugate could induce INF-alpha secretion in mouse, 40 μl of the conventional siRNA or the siRNA multi-conjugate prepared by cleavable disulfide bond or non-cleavable covalent bond was mixed with the cationic gene carrier linear PEI to form an ionic complex, which was injected intravenously into ICR mouse at 7 weeks. After 6 hours of the treatment, blood was taken from the heart of the mouse, followed by ELISA to quantify blood siRNA. As a result, compared with the conventional siRNA, the siRNA multi-conjugate of the present invention did not induce INF-alpha secretion significantly in the animal model (seeFIG.9B). INDUSTRIAL APPLICABILITY The siRNA multi-conjugate of the present invention can be applied in medicinal field including gene therapy owing to the improved gene delivery efficiency and thereby further contributes to the advancement of national industry by realizing diverse applications thereof in related fields. Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims. | 12,278 |
11859185 | DETAILED DESCRIPTION OF THE INVENTION The present invention provides iRNA compositions, which affect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a glucokinase (GCK) gene. The GCK gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of a glucokinase (GCK) gene and/or for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a GCK gene, such as a glycogen storage disease (GSD), e.g., type Ia GSD, and one or more of the signs or symptoms associated therewith. The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a GCK gene. In certain embodiments, the iRNAs of the invention include an RNA strand (the antisense strand) which can include longer lengths, for example up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of a GCK gene. These iRNAs with the longer length antisense strands include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides. The use of these iRNAs enables the targeted degradation of mRNAs of a GCK gene in mammals. Very low dosages of GCK iRNAs, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of a GCK gene. Using cell-based assays, the present inventors have demonstrated that iRNAs targeting GCK can mediate RNAi, resulting in significant inhibition of expression of a GCK gene. Thus, methods and compositions including these iRNAs are useful for treating a subject who would benefit by a reduction in the levels and/or activity of a GCK protein, such as a subject having a glycogen storage disease (GSD), e.g., type Ia GSD. The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of a GCK gene, as well as compositions and methods for treating subjects having diseases and disorders that would benefit from inhibition and/or reduction of the expression of this gene. I. Definitions In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements. The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”. The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise. The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as within about 2 standard deviations from the mean. In certain embodiments, about means+10%. In certain embodiments, about means+5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range. The terms “GCK,” “glucokinase,” “hexokinase D,” and “hexokinase 4” refer to an enzyme that facilitates phosphorylation of glucose to glucose-6-phosphate having an amino acid sequence from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise. The term also refers to fragments and variants of native GCK that maintain at least one in vivo or in vitro activity of a native GCK. The term encompasses full-length unprocessed precursor forms of GCK as well as mature forms resulting from, e.g., post-translational processing. The sequence of a human GCK mRNA transcript (transcript variant 2) can be found at, for example, GenBank Accession No. GI: 15967158 (NM_033507; NCBI GeneID: 2645; SEQ ID NO:1). The sequence of another human GCK mRNA transcript (transcript variant 1) can be found at, for example, GenBank Accession No. GI: 167621407 (NM_000162); SEQ ID NO:2). The sequence of yet another human GCK mRNA transcript (transcript variant 3) can be found at, for example, GenBank Accession No. GI: 15967160 (NM_033508); SEQ ID NO:3). The predicted sequence of a rhesus GCK mRNA transcript (transcript variant X1) can be found at, for example, GenBank Accession No. GI: 544420246 (XM_005549685; SEQ ID NO:4). The predicted sequence of another rhesus GCK mRNA transcript (transcript variant X2) can be found at, for example, GenBank Accession No. GI: 544420248 (XM_005549686; SEQ ID NO:5). The predicted sequence of yet another rhesus GCK mRNA transcript (transcript variant X3) can be found at, for example, GenBank Accession No. GI: 544420250 (XM_005549687; SEQ ID NO:6). The predicted sequence of another rhesus GCK mRNA transcript (transcript variant X4) can be found at, for example, GenBank Accession No. GI: 544420252 (XM_005549688; SEQ ID NO:7). The sequence of a mouse GCK mRNA transcript (transcript variant 1)can be found at, for example, GenBank Accession No. GI: 565671706 (NM_010292; SEQ ID NO:8). The sequence of another mouse GCK mRNA transcript (transcript variant 2)can be found at, for example, GenBank Accession No. GI: 565671714 (NM_001287386; SEQ ID NO:9). The sequence of a rat GCK mRNA transcript (transcript variant 2) can be found at, for example, GenBank Accession No. GI: 399220372 (NM_012565; SEQ ID NO:10). The sequence of another rat GCK mRNA transcript (transcript variant 1) can be found at, for example, GenBank Accession No. GI: 399220370 (NM_001270849; SEQ ID NO:11). The sequence of yet another rat GCK mRNA transcript (transcript variant 3) can be found at, for example, GenBank Accession No. GI: 399220373 (NM_001270850; SEQ ID NO:12). Additional examples of GCK mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM. As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a GCK gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a GCK gene. The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention. As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature. “G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention. The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of GCK in a cell, e.g., a cell within a subject, such as a mammalian subject. In one embodiment, an RNAi agent of the invention includes a single stranded RNAi that interacts with a target RNA sequence, e.g., a GCK target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into double stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer (Sharp et al. (2001)Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001)Nature409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001)Cell107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001)Genes Dev.15:188). Thus, in one aspect the invention relates to a single stranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., a GCK gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above. In another embodiment, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012)Cell150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012)Cell150:883-894. In another embodiment, an “iRNA” for use in the compositions and methods of the invention is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., a GCK gene. In some embodiments of the invention, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi. The majority of nucleotides of each strand of a dsRNA molecule may be ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides. As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, and/or modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims. The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs. In one embodiment, an RNAi agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, e.g., a GCK gene, without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001)Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001)Nature409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001)Cell107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001)Genes Dev.15:188). In one embodiment, an RNAi agent of the invention is a dsRNA of 24-30 nucleotides that interacts with a target RNA sequence, e.g., a prolyl hydroxylase domain-containing gene, i.e., a PHD1 target mRNA sequence, a PHD2 target mRNA sequence, or a PHD3 target mRNA sequence, to direct the cleavage of the target RNA. As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA. In one embodiment of the dsRNA, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide. In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′ end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′ end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′ end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′ end of the antisense strand of the duplex. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double stranded over its entire length. The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a GCK mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a GCK nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA. The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein. As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13. As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides. Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein. “Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing. The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use. As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding GCK). For example, a polynucleotide is complementary to at least a part of a GCK mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding GCK. Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target GCK sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target GCK sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:1, or a fragment of SEQ ID NO:1, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target GCK sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2, 3, 6, and 7, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2, 3, 6, and 7, such as at least 85%, 90%, 95% complementary, or 100% complementary. In one embodiment, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target GCK sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:5, or a fragment of any one of SEQ ID NO:5, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary. The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition. The phrase “inhibiting expression of a GCK,” as used herein, includes inhibition of expression of any GCK gene (such as, e.g., a mouse GCK gene, a rat GCK gene, a monkey GCK gene, or a human GCK gene) as well as variants or mutants of a GCK gene that encode a GCK protein. “Inhibiting expression of a GCK gene” includes any level of inhibition of a GCK gene, e.g., at least partial suppression of the expression of a GCK gene, such as an inhibition by at least about 20%. In certain embodiments, inhibition is by at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. The expression of a GCK gene may be assessed based on the level of any variable associated with GCK gene expression, e.g., GCK mRNA level or GCK protein level. The expression of a GCK may also be assessed indirectly based on, e.g., blood glucose levels, serum ketone body levels, and/or serum fatty acid levels. Inhibition may be assessed by a change in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control). In one embodiment, at least partial suppression of the expression of a GCK gene, is assessed by a reduction of the amount of GCK mRNA which can be isolated from or detected in a first cell or group of cells in which a GCK gene is transcribed and which has or have been treated such that the expression of a GCK gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of: (mRNAincontrolcells)-(mRNAintreatedcells)(mRNAincontrolcells)·100% The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell. Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain and/or be coupled to a ligand, e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject. In one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art. The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference. As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). It is understood that the sequence of the GCK gene must be sufficiently complementary to the antisense strand of the iRNA agent for the agent to be used in the indicated species. In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in GCK expression; a human at risk for a disease, disorder or condition that would benefit from reduction in GCK expression; a human having a disease, disorder or condition that would benefit from reduction in GCK expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in GCK expression as described herein. As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, such as increasing blood glucose levels in a subject. The terms “treating” or “treatment” also include, but are not limited to, alleviation or amelioration of one or more symptoms of a glycogen storage disease (GSD), e.g., type Ia GSD, or at least one sign or symptom associated with GSD, such as hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia, hepatomegaly, kidney disease as a result of glycogen accumulation, hunger, jitteriness, lethargy, apnea, seizures, diaphoresis, confusion, headaches, dizziness, unusual mood or behavior changes, loss of consciousness, coma, muscle cramps, bleeding diathesis, short stature, osteoporosis, delayed puberty, gout, renal disease, systemic hypertension, pulmonary hypertension, hepatic adenomas, pancreatitis, anemia, vitamin D deficiency, polycystic ovaries, irregular menstrual cycles, menorrhagia, and eruptive xanthomata. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment. By “lower” in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, down to a level accepted as within the range of normal for an individual without such disorder, or to below the level of detection of the assay. In certain embodiments, the decrease is down to a level accepted as within the range of normal for an individual without such disorder which can also be referred to as a normalization of a level. For example, lowering cholesterol to 180 mg/dl or lower would be considered to be within the range of normal for a subject. A subject having a cholesterol level of 230 mg/dl with a cholesterol level decreased to 210 mg/dl would have a cholesterol level that was decreased by 40% towards normal (230−210/230−180=20/50=40% reduction). In certain embodiments, the reduction is the normalization of the level of a sign or symptom of a disease, a reduction in the difference between the subject level of a sign of the disease and the normal level of the sign for the disease (e.g., to the upper level of normal when the value for the subject must be decreased to reach a normal value, and to the lower level of normal when the value for the subject must be increased to reach a normal level). In certain embodiments, the methods include a clinically relevant inhibition of expression of a GCK gene, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of a GCK gene. As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of a GCK gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such disease, disorder, or condition, e.g., a glycogen storage disease (GSD), e.g., type Ia GSD. The likelihood of developing type Ia GSD is reduced, for example, when an individual having one or more risk factors for type Ia GSD, e.g., a genetic disorder, either fails to develop type Ia GSD, or signs or symptoms thereof, or develops type Ia GSD, or signs or symptoms thereof, with less severity relative to a population having the same risk factors and not receiving treatment as described herein. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months, or years) is considered effective prevention. Prevention can require administration of more than one dose of an agent described herein. As used herein, the term “blood glucose level” refers to the level of glucose present in blood as determined by any routine method known in the art. It is understood that the glucose level in a subject sample is dependent on, for example, whether the subject has had a meal, whether the subject has fasted, the time of day and the level of activity of the subject. Therefore, the glucose level must be compared to an appropriate control to determine if the glucose level is, in fact, altered from a normal level or from a level obtained from the subject at an earlier time point, e.g., prior to treatment. In general, blood glucose levels, e.g., fasting blood glucose levels, in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein is about 70 to about 120 mg/dL. As used herein, the term “plasma lactate level” refers to the level of lactate present in blood as determined by any routine method known in the art. It is understood that the lactate level in a subject sample is dependent on, for example, whether the subject has fasted, the time of day and the level of activity of the subject. Therefore, the lactate level must be compared to an appropriate control to determine if the lactate level is, in fact, altered from a normal level or from a level obtained from the subject at an earlier time point, e.g., prior to treatment. In general, plasma lactate levels in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein is about 0.5 to about 2.2 mmol/L. As used herein, the term “plasma uric acid level” refers to the level of uric acid present in blood as determined by any routine method known in the art. It is understood that the lactate level in a subject sample is dependent on, for example, whether the subject has fasted, the time of day and the level of activity of the subject. Therefore, the uric acid level must be compared to an appropriate control to determine if the uric acid level is, in fact, altered from a normal level or from a level obtained from the subject at an earlier time point, e.g., prior to treatment. In general, plasma uric acid levels in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein is about 2.0 to about 5.0 mg/dL. As used herein, the term “plasma triglyceride level” refers to the level of triglycerides present in blood as determined by any routine method known in the art. It is understood that the triglyceride level in a subject sample is dependent on, for example, whether the subject has fasted, the time of day and the level of activity of the subject. Therefore, the triglyceride level must be compared to an appropriate control to determine if the triglyceride level is, in fact, altered from a normal level or from a level obtained from the subject at an earlier time point, e.g., prior to treatment. In general, plasma triglyceride levels in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein is about 150 to about 200 mg/dL. As used herein, the term “total cholesterol level” refers to the level of high density lipoprotein (HDL) plus low density lipoprotein (LDL) plus 20% of the triglyceride level as determined by any routine method known in the art. It is understood that the total cholesterol level in a subject sample is dependent on, for example, whether the subject has fasted, the time of day and the level of activity of the subject. Therefore, the total cholesterol level must be compared to an appropriate control to determine if the total cholesterol level is, in fact, altered from a normal level or from a level obtained from the subject at an earlier time point, e.g., prior to treatment. In general, plasma cholesterol levels in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein is about 100 to about 200 mg/dL. As used herein, the term “hepatomegaly” refers to swelling of the liver beyond its normal size as determined by any routine method known in the art. It is understood that the size and weight of the liver in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein increases with age and body weight. Sex and body shape also influence the size of the liver, e.g., by percussion, the mean liver size is about 7.5 centimeters in adult women and about 10.5 centimeters in adult men; it may be about 3 centimeters larger or smaller and still be normal. As used herein, a “disease or disorder that would benefit from reduction in GCK expression” is a disease or disorder associated with or caused by a clinically relevant hypoglycemia. For example, this term includes any disorder, disease or condition resulting in one or more signs or symptoms of a glycogen storage disease (GSD), e.g., type Ia GSD, including, but not limited to, hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia, hepatomegaly, kidney disease as a result of glycogen accumulation, hunger, jitteriness, lethargy, apnea, seizures, diaphoresis, confusion, headaches, dizziness, unusual mood or behavior changes, loss of consciousness, coma, muscle cramps, bleeding diathesis, short stature, osteoporosis, delayed puberty, gout, renal disease, systemic hypertension, pulmonary hypertension, hepatic adenomas, pancreatitis, anemia, vitamin D deficiency, polycystic ovaries, irregular menstrual cycles, menorrhagia, and eruptive xanthomata. In certain embodiments, a “disease or disorder that would benefit from reduction in GCK expression” meets the diagnostic requirements of a type Ia GSD. “Glycogen storage disease” or “GSD” is an inherited genetic disorder due to an absence or deficiency of one of the enzymes responsible for making or breaking down glycogen in the body. This enzyme deficiency causes either abnormal tissue concentrations of glycogen or incorrectly or abnormally formed glycogen. There are about 11 known types of GSD, which are classified based on the missing or defective enzymes. For example, Type Ia GSD, the most common form of GSD, is caused by a genetic defect in the enzyme glucose-6-phosphatase. Hepatomegaly due to inappropriate glycogen accumulation and various metabolic disarrangements from inappropriate glucose-6-phosphate metabolism are predominant features of many of the various glycogen storage diseases, such as Types Ia, Ib, III, IV, VI and IX. Symptoms of GSD vary based on the enzyme that is missing and usually result from the buildup of glycogen or from the inability to produce glucose when needed. Glycogen storage disease is usually diagnosed in infancy or childhood, e.g., at about age 3-4. Affected children typically present with hepatomegaly, lactic acidosis, hyperuricemia, hyperlipidemia, hypertriglyceridemia and/or hypoglycemic seizures. Further, affected children often have doll-like faces with fat cheeks, relatively thin extremities, short stature, and protuberant abdomen. Xanthoma and diarrhea may be present. Impaired platelet function can lead to a bleeding tendency with frequent epistaxis. The diagnostic criteria for GSD, e.g., type Ia GSD, include, for example, fasting blood glucose concentration lower than 60 mg/dL; plasma lactate higher than 2.5 mmol/L; plasma uric acid higher than 5.0 mg/dL; triglycerides higher than 250 mg/dL; total plasma cholesterol higher than 200 mg/dL; administration of glucagon or epinephrine (i.e., a glucagon or epinephrine challenge test) causes little or no increase in blood glucose concentration, but both increase serum lactate concentrations significantly; histopathologic liver findings which include distention of the liver cells by glycogen and fat; PAS positive and diastase sensitive glycogen that is uniformly distributed within the cytoplasm; normal or only modestly increased glycogen; and large and numerous lipid vacuoles; biallelic mutations in either G6PC (GSDIa) or SLC37A4 (GSDIb) (Veiga-da-Cunha et al (1998)Am J Hum Genet.63:976-83; Chou et al (2002)Hum Mutat.29:921-30; Matern et al (2002)Eur J Pediatr.161 Suppl 1:S10-9; Rake et al (2002)Eur J Pediatr.161 Suppl 1:S20-34; and Ekstein et al (2004)Am J Med Genet.129A:162-4); deficient, e.g., (lower than 10% of normal) glucose-6-phosphatase (G6Pase) catalytic activity (the normal G6Pase enzyme activity level in liver is 3.50±0.8 μmol/min/g tissue, although in rare individuals with milder clinical manifestations, the G6Pase enzyme activity can be higher, e.g., >1.0 μmol/min/g tissue and <2.0 μmol/min/g tissue. “Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a glycogen storage disease (GSD), e.g., type Ia GSD, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated. “Prophylactically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a subject having a glycogen storage disease (GSD), e.g., type Ia GSD, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the iRNA, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated. A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. iRNA employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a “sample derived from a subject” refers to blood drawn from the subject or plasma isolated therefrom, saliva, or urine, typically a 24 hour urine sample. II. iRNAs of the Invention Described herein are iRNAs which inhibit the expression of a GCK gene. In one embodiment, the iRNA agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a GCK gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having a glycogen storage disease (GSD), e.g., type Ia GSD. The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a GCK gene. The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the GCK gene, the iRNA inhibits the expression of the GCK gene (e.g., a human, a primate, a non-primate, or a bird GCK gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting, or flowcytometric techniques. In preferred embodiments, inhibition of expression is determined by the qPCR method provided in the examples. For in vitro assessment of activity, percent inhibition is determined using the methods provided herein at a single dose at, for example, a 10 nM duplex final concentration. For in vivo studies, the level after treatment can be compared to, for example, an appropriate historical control or a pooled population sample control to determine the level of reduction, e.g., when a baseline value is no available for the subject. A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of a GCK gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides. Generally, the duplex structure is between 15 and 30 base pairs in length, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention. Similarly, the region of complementarity to the target sequence is between 15 and 30 nucleotides in length, e.g., between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention. In some embodiments, the dsRNA is between about 15 and about 23 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target GCK expression is not generated in the target cell by cleavage of a larger dsRNA. A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end, or both ends of either an antisense or sense strand of a dsRNA. In certain embodiments, longer, extended overhangs are possible. A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch™, Applied Biosystems™, Inc. iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both. In certain aspects, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand sequence is selected from the group of sequences provided in any of Tables 2, 3, 6, and 7 and the corresponding nucleotide sequence of the antisense strand of the sense strand is selected from the group of sequences provided in any of Tables 2, 3, 6, and 7. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of a GCK gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any of Tables 2, 3, 6, and 7, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any of Tables 2, 3, 6, and 7. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide. It will be understood that, although the sequences in Tables 3 and 7 are described as modified and/or conjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNA of the invention, may comprise any one of the sequences set forth in any one of Tables 3 and 7 that is un-modified, un-conjugated, and/or modified and/or conjugated differently than described therein. In another aspect, a double stranded ribonucleic acid (dsRNA) of the invention for inhibiting expression of GCK comprises, consists essentially of, or consists of a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of a sense strand in any of Tables 2, 3, 6, and 7 and the antisense strand comprises the nucleotide sequence of the corresponding antisense strand in any of Tables 2, 3, 6, and 7. The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001)EMBO J.,20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005)Nat Biotech23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided herein, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein, and differing in their ability to inhibit the expression of a GCK gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention. In addition, the RNAs described herein identify a site(s) in a GCK transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within this site(s). As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a GCK gene. While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified herein represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics. Further, it is contemplated that for any sequence identified herein, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor. An iRNA agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of a GCK gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a GCK gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of a GCK gene is important, especially if the particular region of complementarity in a GCK gene is known to have polymorphic sequence variation within the population. III. Modified iRNAs of the Invention In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA, is un-modified, and does not comprise, e.g., chemical modifications and/or conjugations known in the art and described herein. In another embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified. iRNAs of the invention in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides. The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone. Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference. Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2component parts. Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference. In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al.,Science,1991, 254, 1497-1500. Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2-[known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2-4 wherein the native phosphodiester backbone is represented as —O—P—O—CH2-] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506. Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1to C10alkyl or C2to C10alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C1to C10lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al.,Helv. Chim. Acta,1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2. Further exemplary modifications include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide). Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference. An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991)Angewandte Chemie, International Edition,30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications. Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference. The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther6(3):833-843; Grunweller, A. et al., (2003)Nucleic Acids Research31(12):3185-3193). In some embodiments, the iRNA of the invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (seeNuc. Acids Symp. Series,52, 133-134 (2008) and Fluiter et al.,Mol. Biosyst.,2009, 10, 1039 hereby incorporated by reference). Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference. The RNA of an iRNA can also be modified to include one or more bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A“bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther6(3):833-843; Grunweller, A. et al., (2003)Nucleic Acids Research31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al.,J. Org. Chem.,2009, 74, 118-134); and 4′-CH2C(═CH2)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference. Additional representative U.S. Patents and US Patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference. Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226). The RNA of an iRNA can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)-0-2′ bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.” An iRNA of the invention may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering. Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US Patent Publication No. 2013/0190383; and PCT publication WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference. Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861. Other modifications of the nucleotides of an iRNA of the invention include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of an RNAi agent. Suitable phosphate mimics are disclosed in, for example US Patent Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference. IV. iRNAs Conjugated to Ligands Another modification of the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., (1989)Proc. Natl. Acid. Sci. USA,86: 6553-6556), cholic acid (Manoharan et al., (1994)Biorg. Med. Chem. Let.,4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., (1992)Ann. N.Y. Acad. Sci.,660:306-309; Manoharan et al., (1993)Biorg. Med. Chem. Let.,3:2765-2770), a thiocholesterol (Oberhauser et al., (1992)Nucl. Acids Res.,20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991)EMBO J,10:1111-1118; Kabanov et al., (1990)FEBS Lett.,259:327-330; Svinarchuk et al., (1993)Biochimie,75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995)Tetrahedron Lett.,36:3651-3654; Shea et al., (1990)Nucl. Acids Res.,18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., (1995)Nucleosides&Nucleotides,14:969-973), or adamantane acetic acid (Manoharan et al., (1995)Tetrahedron Lett.,36:3651-3654), a palmityl moiety (Mishra et al., (1995)Biochim. Biophys. Acta,1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996)J. Pharmacol. Exp. Ther.,277:923-937). In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid. Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide. Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, monovalent or multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, ligands include monovalent or multivalent galactose. In certain embodiments, ligands include cholesterol. Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP. Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein. Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto. The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems™ (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives. In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks. When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis. A. Lipid Conjugates In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA. A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney. In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed. In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand. In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL). B. Cell Permeation Agents In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase. The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 25). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 26) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 27) and theDrosophilaAntennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 28) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized. An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glyciosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF. A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003). C. Carbohydrate Conjugates In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8). In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of: In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to, when one of X or Y is an oligonucleotide, the other is a hydrogen. In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In one embodiment, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In another embodiment, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers. In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex. In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide. Additional carbohydrate conjugates (and linkers) suitable for use in the present invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference. D. Linkers In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non cleavable. The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms. A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum). Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases. A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell. A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis. Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes. In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions). i. Redox Cleavable Linking Groups In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media. ii. Phosphate-Based Cleavable Linking Groups In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above. iii. Acid Cleavable Linking Groups In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above. iv. Ester-Based Linking Groups In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above. v. Peptide-Based Cleaving Groups In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to, when one of X or Y is an oligonucleotide, the other is a hydrogen. In certain embodiments of the compositions and methods of the invention, a ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker. In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXII)-(XXXV): wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5Care each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5Care independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R′)═C(R″), C≡C or C(O); R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5Care each independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), —C(O)—CH(Ra)—NH—, CO, L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5Band L5Crepresent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Rais H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXVI): wherein L5A, L5Band L5Crepresent a monosaccharide, such as GalNAc derivative. Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII. Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference. It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds. “Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an amino linker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate. IV. Delivery of an iRNA of the Invention The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a glycogen storage disease (GSD), e.g., type Ia GSD) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below. In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L., (1992)Trends Cell. Biol.2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J. et al., (2004)Retina24:132-138) and subretinal injections in mice (Reich, S J. et al. (2003)Mol. Vis.9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J. et al. (2005)Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. et al., (2006)Mol. Ther.14:343-350; Li, S. et al., (2007)Mol. Ther.15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004)Nucleic Acids32:e49; Tan, P H. et al. (2005)Gene Ther.12:59-66; Makimura, H. et a.l (2002)BMC Neurosci.3:18; Shishkina, G T., et al. (2004)Neuroscience129:521-528; Thakker, E R., et al. (2004)Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al. (2005)J Neurophysiol.93:594-602) and to the lungs by intranasal administration (Howard, K A. et al., (2006)Mol. Ther.14:476-484; Zhang, X. et al., (2004)J. Biol. Chem.279:10677-10684; Bitko, V. et al., (2005)Nat. Med.11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004)Nature432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O. et al., (2006)Nat. Biotechnol.24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H. et al., (2008)Journal of Controlled Release129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003)J. Mol. Biol327:761-766; Verma, U N. et al., (2003)Clin. Cancer Res.9:1291-1300; Arnold, A S et al., (2007)J. Hypertens.25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S. et al., (2006)Nature441:111-114), cardiolipin (Chien, P Y. et al., (2005)Cancer Gene Ther.12:321-328; Pal, A. et al., (2005)Intl Oncol.26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008)Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006)J. Biomed. Biotechnol.71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006)Mol. Pharm.3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007)Biochem. Soc. Trans.35:61-67; Yoo, H. et al., (1999)Pharm. Res.16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety. A. Vector Encoded iRNAs of the Invention iRNA targeting the GCK gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995)Proc. Natl. Acad. Sci. USA92:1292). The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure. iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell. Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (0 polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are known in the art. V. Pharmaceutical Compositions of the Invention The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of a GCK gene, such as, a glycogen storage disease (GSD), e.g., type Ia GSD, or one or more signs or symptoms of GSD, such as hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia, hepatomegaly, kidney disease as a result of glycogen accumulation, hunger, jitteriness, lethargy, apnea, seizures, diaphoresis, confusion, headaches, dizziness, unusual mood or behavior changes, loss of consciousness, coma, muscle cramps, bleeding diathesis, short stature, osteoporosis, delayed puberty, gout, renal disease, systemic hypertension, pulmonary hypertension, hepatic adenomas, pancreatitis, anemia, vitamin D deficiency, polycystic ovaries, irregular menstrual cycles, menorrhagia, and eruptive xanthomata. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) or for subcutaneous delivery. Another example is compositions that are formulated for direct delivery into the liver, e.g., by infusion into the liver, such as by continuous pump infusion. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a GCK gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. Typically, a suitable dose of an iRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg. A repeat-dose regimine may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day or once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months). After an initial treatment regimen, the treatments can be administered on a less frequent basis. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein. Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as disorders of excess glucose that would benefit from reduction in the expression of GCK. The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration. The iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver). Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline), negative (e.g., dimyristoylphosphatidyl glycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride or diglyceride; or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference. A. iRNA Formulations Comprising Membranous Molecular Assemblies An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types. A liposome containing a RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent. If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation. Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., (1987)Proc. Natl. Acad. Sci. USA8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham et al., (1965)M Mol. Biol.23:238; Olson et al., (1979)Biochim. Biophys. Acta557:9; Szoka et al., (1978)Proc. Natl. Acad. Sci.75: 4194; Mayhew et al., (1984)Biochim. Biophys. Acta775:169; Kim et al., (1983)Biochim. Biophys. Acta728:339; and Fukunaga et al., (1984)Endocrinol.115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986)Biochim. Biophys. Acta858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984)Biochim. Biophys. Acta775:169. These methods are readily adapted to packaging RNAi agent preparations into liposomes. Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987)Biochem. Biophys. Res. Commun.,147:980-985). Liposomes, which are pH-sensitive or negatively charged, entrap nucleic acids rather than complex with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992)Journal of Controlled Release,19:269-274). One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol. Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, (1994)J Biol. Chem.269:2550; Nabel, (1993)Proc. Natl. Acad. Sci.90:11307; Nabel, (1992)Human Gene Ther.3:649; Gershon, (1993)Biochem.32:7143; and Strauss, (1992)EMBO J.11:417. Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994)S.T.P.Pharma. Sci.,4(6):466). Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987)FEBS Letters,223:42; Wu et al., (1993)Cancer Research,53:3765). Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. USA., (1988), 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al). In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages. Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes. A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., (1987)Proc. Natl. Acad. Sci. USA8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA). A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim™, Indianapolis, Indiana) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages. Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega™, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678). Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991)Biochim. Biophys. Res. Commun.179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991)Biochim. Biophys. Acta1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical™, La Jolla, California) and Lipofectamine™ (DOSPA) (Life Technology™, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194. Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992)Journal of Drug Targeting, vol. 2, 405-410 and du Plessis et al., (1992)Antiviral Research,18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998)Biotechniques6:682-690; Itani, T. et al., (1987)Gene56:267-276; Nicolau, C. et al. (1987)Meth. Enzymol.149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983)Meth. Enzymol.101:512-527; Wang, C. Y. and Huang, L., (1987)Proc. Natl. Acad. Sci. USA84:7851-7855). Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder. Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin. Other formulations amenable to the present invention are described in, for example, PCT Publication No. WO 2008/042973. Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin. Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285). If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class. If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps. If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class. If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides. The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285). The iRNA for use in the methods of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic. A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C8to C22alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles. In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing. Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition. For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray. Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used. The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract. B. Lipid Particles iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle. As used herein, the term “LNP” refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964. In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention. The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyOdidodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle. In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference. In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio. The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol; or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle. The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle. In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle. In one embodiment, the lipidoid ND98-4HC1 (MW 1487) (see US20090023673, filed Mar. 26, 2008, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich™), and PEG-Ceramide C16 (Avanti™ Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference. Additional exemplary lipid-dsRNA formulations are described in the table below. cationic lipid/non-cationiclipid/cholesterol/PEG-lipidconjugateIonizable/Cationic LipidLipid:siRNA ratioSNALP-1l,2-Dilinolenyloxy-N,N-DLinDMA/DPPC/Cholesterol/PEG-cDMAdimethylaminopropane (DLinDMA)(57.1/7.1/34.4/1.4)lipid:siRNA ~7:12-XTC2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DPPC/Cholesterol/PEG-cDMAdioxolane (XTC)57.1/7.1/34.4/1.4lipid:siRNA ~7:1LNP052,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC)57.5/7.5/31.5/3.5lipid:siRNA ~6:1LNP062,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC)57.5/7.5/31.5/3.5lipid:siRNA ~11:1LNP072,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC)60/7.5/31/1.5,lipid:siRNA ~6:1LNP082,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSC/Cholesterol/PEG-DMGdioxolane (XTC)60/7.5/31/1.5,lipid:siRNA ~11:1LNP092,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC)50/10/38.5/1.5Lipid:siRNA 10:1LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-ALN100/DSPC/Cholesterol/PEG-DMGoctadeca-9,12-dienyl)tetrahydro-3aH-50/10/38.5/1.5cyclopenta[d][1,3]dioxol-5-amine (ALN100)Lipid:siRNA 10:1LNP11(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-MC-3/DSPC/Cholesterol/PEG-DMGtetraen-19-yl 4-(dimethylamino)butanoate50/10/38.5/1.5(MC3)Lipid:siRNA 10:1LNP121,1′-(2-(4-(2-((2-(bis(2-Tech G1/DSPC/Cholesterol/PEG-DMGhydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5hydroxydodecyl)amino)ethyl)piperazin-1-Lipid:siRNA 10:1yl)ethylazanediyl)didodecan-2-ol (Tech G1)LNP13XTCXTC/DSPC/Chol/PEG-DMG50/10/38.5/1.5Lipid:siRNA: 33:1LNP14MC3MC3/DSPC/Chol/PEG-DMG40/15/40/5Lipid:siRNA: 11:1LNP15MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG50/10/35/4.5/0.5Lipid:siRNA: 11:1LNP16MC3MC3/DSPC/Chol/PEG-DMG50/10/38.5/1.5Lipid:siRNA: 7:1LNP17MC3MC3/DSPC/Chol/PEG-DSG50/10/38.5/1.5Lipid:siRNA: 10:1LNP18MC3MC3/DSPC/Chol/PEG-DMG50/10/38.5/1.5Lipid:siRNA: 12:1LNP19MC3MC3/DSPC/Chol/PEG-DMG50/10/35/5Lipid:siRNA: 8:1LNP20MC3MC3/DSPC/Chol/PEG-DPG50/10/38.5/1.5Lipid:siRNA: 10:1LNP21C12-200C12-200/DSPC/Chol/PEG-DSG50/10/38.5/1.5Lipid:siRNA: 7:1LNP22XTCXTC/DSPC/Chol/PEG-DSG50/10/38.5/1.5Lipid:siRNA: 10:1DSPC: distearoylphosphatidylcholineDPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)SNALP (l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15. 2009. which is hereby incorporated by reference.XTC comprising formulations are described in, for example, PCT Publication No. WO 2010/088537, the entire contents of which are hereby incorporated herein by reference.MC3 comprising formulations are described, e.g., in U.S. Publication No. 2010/0324120, filed Jun. 10. 2010, the entire contents of which are hereby incorporated by reference.ALNY-100 comprising formulations are described in, for example, PCT Publication No. WO 2010/054406, the entire contents of which are hereby incorporated herein by reference.C12-200 comprising formulations are described in, for example, PCT Publication No. WO 2010/129709, the entire contents of which are hereby incorporated herein by reference. Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions, or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride or a diglyceride; or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include polyamino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference. Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular, or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients. Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma. The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers. C. Additional Formulations i. Emulsions The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion. Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285). Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and nonpolar solids such as carbon or glyceryl tristearate. A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase. Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols, and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin. The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins, and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions. ii. Microemulsions In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271). The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously. Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij™ 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, or 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex™ 300, Captex™ 355, Capmul™ MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oil. Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids. Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories-surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above. iii. Microparticles an RNAi agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques. iv. Penetration Enhancers In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Such compounds are well known in the art. v. Carriers Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183. vi. Excipients In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc). Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone; and the like. Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like. vii. Other Components The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents, and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings, and/or aromatic substances; and the like which do not deleteriously interact with the nucleic acid(s) of the formulation. Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. The suspension can also contain stabilizers. In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating a GCK-associated disorder. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50(the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50(i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by GCK expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein. VI. Methods of the Invention The present invention also provides methods of using an iRNA of the invention and/or a composition containing an iRNA of the invention to reduce and/or inhibit glucokinase (GCK) expression in a cell. The methods include contacting the cell with a dsRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of a GCK gene, thereby inhibiting expression of the GCK gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of GCK may be determined by determining the mRNA expression level of GCK using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of GCK using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques. A reduction in the expression of GCK may also be assessed indirectly by measuring a decrease in biological activity of GCK. In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject. A cell suitable for treatment using the methods of the invention may be any cell that expresses a GCK gene. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell. GCK expression is inhibited in the cell by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%. In preferred embodiments, GCK expression is inhibited by at least 20%. The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the GCK gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of GCK, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection. In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver. The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting. In one aspect, the present invention also provides methods for inhibiting the expression of a GCK gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets a GCK gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the GCK gene, thereby inhibiting expression of the GCK gene in the cell. Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in GCK gene and/or protein expression. The present invention further provides methods of treatment of a subject in need thereof. The treatment methods of the invention include administering an iRNA of the invention to a subject, e.g., a subject that would benefit from a reduction and/or inhibition of GCK expression, in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene. In addition, the present invention provides methods of increasing the blood glucose levels, e.g., fasting blood glucose levels, in a subject having a disease or disorder that would benefit from a reduction in GCK expression. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene. Further, the present invention provides methods of decreasing plasma lactate levels in a subject having a disease or disorder that would benefit from a reduction in GCK expression, e.g., a GSD, e.g., type Ia GSD. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene. The present invention also provides methods of decreasing plasma uric acid levels in a subject having a disease or disorder that would benefit from a reduction in GCK expression, e.g., a GSD, e.g., type Ia GSD. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene. The present invention provides methods of decreasing plasma triglyceride levels in a subject having a disease or disorder that would benefit from a reduction in GCK expression, e.g., a GSD, e.g., type Ia GSD. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene. The present invention further provides methods of decreasing total plasma cholesterol levels in a subject having a disease or disorder that would benefit from a reduction in GCK expression, e.g., a GSD, e.g., type Ia GSD. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene. The present invention also provides methods of decreasing hepatomegaly in a subject having a disease or disorder that would benefit from a reduction in GCK expression, e.g., a GSD, e.g., type Ia GSD. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene. An iRNA of the invention may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject. Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation. Subjects that would benefit from a reduction and/or inhibition of GCK gene expression include those having a glycogen storage disease (GSD), e.g., type Ia GSD. In one embodiment, subjects that would benefit from a reduction and/or inhibition of GCK gene expression are those having one or more signs or symptoms associated with a glycogen storage disease (GSD), e.g., type Ia GSD, including, but not limited to, hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia, hepatomegaly, kidney disease as a result of glycogen accumulation, hunger, jitteriness, lethargy, apnea, seizures, diaphoresis, confusion, headaches, dizziness, unusual mood or behavior changes, loss of consciousness, coma, muscle cramps, bleeding diathesis, short stature, osteoporosis, delayed puberty, gout, renal disease, systemic hypertension, pulmonary hypertension, hepatic adenomas, pancreatitis, anemia, vitamin D deficiency, polycystic ovaries, irregular menstrual cycles, menorrhagia, and eruptive xanthomata. Treatment of a subject that would benefit from a reduction and/or inhibition of GCK gene expression and normalization of blood glucose levels includes therapeutic treatment (e.g., of a subject suffering from a glycogen storage disease (GSD)) and prophylactic treatment (e.g., of a subject that does not meet the diagnostic criteria of a glycogen storage disease (GSD), or a subject who may be at risk of developing a glycogen storage disease (GSD)). The invention further provides methods for the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction and/or inhibition of GCK expression, e.g., a subject having a glycogen storage disease (GSD), e.g., type Ia GSD, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an iRNA targeting GCK is administered to a subject having a GSD in combination with, e.g., antihypertensive agents, such as Diazoxide, somatostatin analogues, such as Octreotide, calcium channel blockers, such as, Nifedipine, Thiazide diuretics, such as chlorothiazide (e.g., in combination with Diazoxide), intravenous infusion of glucagon, parenterally administered dextrose; and/or partial pancreatectomy. In some embodiments, an iRNA targeting GCK is administered to a subject having type Ia GSD in combination with, e.g., a sodium-glucose co-transporter 2 (SGLT2) inhibitor, e.g., Dapagliflozin, Canagliflozin, Ipragliflozin (ASP-1941), Tofogliflozin, Empagliflozin, Sergliflozin etabonate, Remogliflozin etabonate (BHV091009), and Ertugliflozin (PF-04971729/MK-8835). The iRNA and additional therapeutic agents may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein. In one embodiment, the method includes administering a composition featured herein such that expression of the target GCK gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target GCK gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer. Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target GCK gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein. Administration of the dsRNA according to the methods of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a glycogen storage disease (GSD), e.g., type Ia GSD. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker, or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a glycogen storage disease (GSD) may be assessed, for example, by periodic monitoring of, e.g., blood glucose levels. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting GCK or pharmaceutical composition thereof, “effective against” a glycogen storage disease (GSD) indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating a glycogen storage disease (GSD) and the related causes and effects. A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed. Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale such as those provided above. Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an iRNA or iRNA formulation as described herein. Subjects can be administered a therapeutic amount of dsRNA, such as about 0.01 mg/kg to about 200 mg/kg. The iRNA can be administered by intravenous infusion over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Administration of the iRNA can reduce GCK levels, e.g., in a cell, tissue, blood, urine, or other compartment of the patient by at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more. Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels. Alternatively, the iRNA can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day or to once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. EXAMPLES Example 1. iRNA Design, Synthesis, Selection, and In Vitro Evaluation This Example describes methods for the design, synthesis, selection, and in vitro evaluation of GCK iRNA agents. Source of Reagents Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology. Bioinformatics A set of siRNAs targeting the human GCK, “glucokinase (hexokinase 4)”, (human: NCBI refseqID NM_033507; NCBI GeneID: 2645), as well as toxicology-species GCK orthologs (cynomolgus monkey: XM_005549685; mouse: NM_010292; rat, NM_012565) were designed using custom R and Python scripts. The human NM_033507 REFSEQ mRNA, version 1, has a length of 2442 bases. The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer iRNA from position 1 through position 2442 (the coding region and 3′ UTR) was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct iRNA designs targeting a large number of vertebrate genes. Subsets of the GCK iRNAs were designed with perfect or near-perfect matches between human, cynomolgus and rhesus monkey. A further subset was designed with perfect or near-perfect matches to mouse and rat GCK orthologs. For each strand of the iRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the iRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the iRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8; 1.2:1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by assuming the value of each weighted mismatch. Preference was given to iRNAs whose antisense score in human and cynomolgus monkey was >=2.0 and predicted efficacy was >=50% knockdown of the GCK transcript. Synthesis of GCK Sequences Synthesis of GCK Single Strands and Duplexes GCK siRNA sequences were synthesized at 1 umol scale on Mermade 192 synthesizer (BioAutomation) using the solid support mediated phosphoramidite chemistry. The solid support was controlled pore glass (500° A) loaded with custom GalNAc ligand or universal solid support (AM biochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA and deoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee, WI) and Hongene (China). 2′F, 2′-O-Methyl, RNA, DNA and other modified nucleosides were introduced in the sequences using the corresponding phosphoramidites. Synthesis of 3′ GalNAc conjugated single strands was performed on a GalNAc modified CPG support. Custom CPG universal solid support was used for the synthesis of antisense single strands. Coupling time for all phosphoramidites (100 mM in acetonitrile) was 5 min employing 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 M in acetonitrile). Phosphorothioate linkages were generated using a 50 mM solution of 3-((Dimethylamino-methylidene) amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, MA, USA) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time was 3 minutes. All sequences were synthesized with final removal of the DMT group (“DMT off”). Upon completion of the solid phase synthesis, single strands were cleaved from the solid support and deprotected in sealed 96 deep well plates using 200 μL Aqueous Methylamine reagent at 60° C. for 20 minutes. For sequences containing 2′ ribo residues (2′-OH) that are protected with tert-butyl dimethyl silyl (TBDMS) group, a second step deprotection was performed using TEA.3HF (triethylamine trihydro fluoride) reagent. To the methylamine deprotection solution, 200 μL of dimethyl sulfoxide (DMSO) and 300 μl TEA.3HF reagent was added and the solution was incubated for additional 20 min at 60° C. At the end of cleavage and deprotection step, the synthesis plate was allowed to come to room temperature and was precipitated by addition of 1 mL of acetontile: ethanol mixture (9:1). The plates were cooled at −80° C. for 2 hrs and the supernatant decanted carefully with the aid of a multi-channel pipette. The oligonucleotide pellet was re-suspended in 20 mM NaOAc buffer and were desalted using a 5 mL HiTrap size exclusion column (GE Healthcare) on an AKTA Purifier System equipped with an A905 autosampler and a Frac 950 fraction collector. Desalted samples were collected in 96 well plates. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV (260 nm) for quantification and a selected set of samples by IEX chromatography to determine purity. Annealing of GCK single strands was performed on a Tecan liquid handling robot. Equimolar mixture of sense and antisense single strands were combined and annealed in 96 well plates. After combining the complementary single strands, the 96 well plate was sealed tightly and heated in an oven at 100° C. for 10 minutes and allowed to come slowly to room temperature over a period 2-3 hours. The concentration of each duplex was normalized to 10 uM in 1×PBS and then submitted for in vitro screening assays. Cell Culture and Transfections for Single Dose and Dose Response Studies Primary mouse hepatocytes (PMH) (GIBCO) or Primary Cynomolgus monkey hepatocytes (PCH) (Celsis) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad CA cat #13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Forty μl of William's E Medium (Life Tech) containing about 5×103cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration. Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12) Total RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl of Lysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150 μl Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured and supernatant removed. cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat #4368813) Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 125× dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H2O per reaction was added to the RNA isolated as described above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 h 37° C. Plates were then incubated at 80° C. for 8 minutes. Real Time PCR Two μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), or 0.5 μl of Custom made Cyno GAPDH Taqman Probe, 0.5 μl GCK mouse probe (Mm00439129 ml) or 0.5 μl cyno probe (Mf02827184 ml) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plate (Roche cat #04887301001). Real time PCR is done in an Roche Lightcycler Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections. To calculate relative fold change, real time data are analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with a non-targeting control siRNA, AD-1955 The sense and antisense sequences of AD-1955 are: SENSE:(SEQ ID NO: 29)cuuAcGcuGAGuAcuucGAdTsdTANTISENSE:(SEQ ID NO: 30)UCGAAGuACUcAGCGuAAGdTsdT. TABLE 1Abbreviations of nucleotide monomers usedin nucleic acid sequence representation.It will be understood that these monomers, when present in anoligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds.AbbreviationNucleotide(s)AAdenosine-3′-phosphateAf2′-fluoroadenosine-3′-phosphateAfs2′-fluoroadenosine-3′-phosphorothioateAsadenosine-3′-phosphorothioateCcytidine-3′-phosphateCf2′-fluorocytidine-3′-phosphateCfs2′-fluorocytidine-3′-phosphorothioateCscytidine-3′-phosphorothioateGguanosine-3′-phosphateGf2′-fluoroguanosine-3′-phosphateGfs2′-fluoroguanosine-3′-phosphorothioateGsguanosine-3′-phosphorothioateT5′-methyluridine-3′-phosphateTf2′-fluoro-5-methyluridine-3′-phosphateTfs2′-fluoro-5-methyluridine-3′-phosphorothioateTs5-methyluridine-3′-phosphorothioateUUridine-3′-phosphateUf2′-fluorouridine-3′-phosphateUfs2′-fluorouridine-3′-phosphorothioateUsuridine-3′-phosphorothioateNany nucleotide (G, A, C, T or U)a2′-O-methyladenosine-3′-phosphateas2′-O-methyladenosine-3′-phosphorothioatec2′-O-methylcytidine-3′-phosphatecs2′-O-methylcytidine-3′-phosphorothioateg2′-O-methylguanosine-3′-phosphategs2′-O-methylguanosine-3′-phosphorothioatet2′-O-methyl-5-methyluridine-3′-phosphatets2′-O-methyl-5-methyluridine-3′-phosphorothioateu2′-O-methyluridine-3′-phosphateus2′-O-methyluridine-3′-phosphorothioatesphosphorothioate linkageL96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinolHyp-(GalNAc-alkyl)3dT2′-deoxythymidine-3′-phosphatedC2′-deoxycytidine-3′-phosphate A detailed list of the unmodified GCK sense and antisense strand sequences is shown in Table 2 and a detailed list of the modified GCK sense and antisense strand sequences is shown in Table 3. Table 4 shows the results of a single dose screen in primary mouse hepatocytes transfected with the indicated modified siRNAs. Data are expressed as percent of message remaining relative to cells treated with a non-targeting control siRNA. Table 5 shows the results of a single dose screen in primaryCynomologoushepatocytes transfected with the indicated modified siRNAs. Data are expressed as percent of message remaining relative to cells treated with a non-targeting control siRNA. TABLE 2GCK Unmodified SequencesSenseSEQAnti-AntisenseSEQDuplexSenseSequenceIDPostion insenseSequenceIDPostion inIDID(5′-3′)NO:NM_033507.1ID(5′-3′)NO:NM_033507.1cross_ spAD-69366A-139669GAGGACCUGAA31_253-273_sA-139670UAUCACCUUCUU120_251-273_ashcmrGAAGGUGAUACAGGUCCUCCUAD-69368A-139673GGACCUGAAGA32_255-275_sA-139674UUCAUCACCUUC121_253-275_ashcmrAGGUGAUGAAUUCAGCUCCUCAD-69411A-139758GGACCUGAAGA33_257-277_sA-139759UAUCACCUUCUU122_255-277_ashcmrAGGUGAUACAGGUCCAD-69413A-139762ACCUGAAGAAG34_259-279_sA-139763UUCAUCACCUUC123_257-279_ashcmrGUGAUGAAUUCAGGUAD-69367A-139671GGUGAUGAGAC35_267-287_sA-139672UUCUGCAUCCGU124_265-287_ashcmrGGAUGCAGAACUCAUCACCUUAD-69369A-139675UGAUGAGACGG36_269-289_sA-139676UCUUCUGCAUCC125_267-289_ashcmrAUGCAGAAGAGUCUCAUCACCAD-69412A-139760UGAUGAGACGG37_271-291_sA-139761UUCUGCAUCCGU126_269-291_ashcmrAUGCAGAACUCAUCAAD-69414A-139764AUGAGACGGAU38_273-293_sA-139765UCUUCUGCAUCC127_271-293_ashcmrGCAGAAGAGUCUCAUAD-69371A-139679ACGGAUGCAGA39_276-296_sA-139680UCCAUCUCCUUC128_274-296_ashcmrAGGAGAUGGAUGCAUCCGUCUAD-69416A-139768GGAUGCAGAAG40_280-300_sA-139769UCCAUCUCCUUC129_278-300_ashcmrGAGAUGGAUGCAUCCAD-69370A-139677ACCCAUGAAGA41_316-336_sA-139678UACACUGGCCUC130_314-336_ashcmrGGCCAGUGUAUUCAUGGGUCUAD-69415A-139766CCAUGAAGAGG42_320-340_sA-139767UACACUGGCCUC131_318-340_ashcmrCCAGUGUAUUCAUGGAD-69372A-139681CAGUGUGAAGA43_330-350_sA-139682UUGGGCAGCAUC132_328-350_ashcUGCUGCCCAAUUCACACUGGCAD-69417A-139770GUGUGAAGAUG44_334-354_sA-139771UUGGGCAGCAUC133_332-354_ashcCUGCCCAAUUCACACAD-69373A-139683GUGAAGGUGGG45_436-456_sA-139684UUCACCUUCUCC134_434-456_ashcAGAAGGUGAACACCUUCACCAAD-69418A-139772GAAGGUGGGAG46_440-460_sA-139773UUCACCUUCUCC135_438-460_ashcAAGGUGAACACCUUCAD-69374A-139685GAGCGUGAAGA47_168-488_sA-139686UGGUGUUUGGUC136_466-488_ashcCCAAACACCAUUCACGCUCCAAD-69419A-139774GCGUGAAGACC48_472-492_sA-139775UGGUGUUUGGUC137_470-492_ashcAAACACCAUUCACGCAD-69375A-139687GAAGACCAAAC49_474-494_sA-139688UACAUCUGGUGU138_472-494_ashcmrACCAGAUGUAUUGGUCUUCACAD-69420A-139776AGACCAAACAC50_478-498_sA-139777UACAUCUGGUGU139_476-498_ashcmrCAGAUGUAUUGGUCUAD-69376A-139689GACUUCCUGGA51_565-585_sA-139690UUGAUGCUUGUC140_563-585_ashcmrCAAGCAUCAACAGGAAGUCGGAD-69377A-139691CUUCCUGGACA52_567-587_sA-139692AUCUGAUGCUUG141_565-587_ashcmrAGCAUCAGAUUCCAGGAAGUCAD-69421A-139778CUUCCUGGACA53_569-589_sA-139779UUGAUGCUUGUC142_567-589_ashcmrAGCAUCAACAGGAAGAD-69422A-139780UCCUGGACAAG54_571-591_sA-139781AUCUGAUGCUUG143_569-591_ashcmrCAUCAGAUUCCAGGAAD-69378A-139693CCUGGACAAGC55_570-590_sA-139694UUCAUCUGAUGC144_568-590_ashcmrAUCAGAUGAAUUGUCCAGGAAAD-69379A-139695CUGGACAAGCA56_571-591_sA-139696UUUCAUCUGAUG145_569-591_ashcmrUCAGAUGAAACUUGUCCAGGAAD-69423A-139782UGGACAAGCAU57_574-594_sA-139783UUCAUCUGAUGC146_572-594_ashcmrCAGAUGAAUUGUCCAAD-69424A-139784GGACAAGCAUC58_575-595_sA-139785UUUCAUCUGAUG147_573-595_ashcmrAGAUGAAACUUGUCCAD-69381A-139699AGGCACGAAGA59_634-654_sA-139700UUUAUCGAUGUC148_632-654_ashcCAUCGAUAAAUUCGUGCCUCAAD-69426A-139788GCACGAAGACA60_638-658_sA-139789UUUAUCGAUGUC149_636-658_ashcUCGAUAAAUUCGUGCAD-69382A-139701ACAUCGAUAAG61_644-664_sA-139702UAAGGAUGCCCU150_642-664_ashcGGCAUCCUUAUAUCGAUGUCUAD-69427A-139790AUCGAUAAGGG62_648-668_sA-139791UAAGGAUGCCCU151_646-668_ashcCAUCCUUAUAUCGAUAD-69383A-139703CUGGACCAAGG63_669-689_sA-139704UCCUUGAAGCCC152_667-6X9_ashcmrGCUUCAAGGAUUGGUCCAGUUAD-69428A-139792GGACCAAGGGC64_673-693_sA-139793UCCUUGAAGCCC153_671-693_ashcmrUUCAAGGAUUGGUCCAD-69384A-139705GGGCUUCAAGG65_678-698_sA-139706UCUCCUGAGGCC154_676-698_ashcCCUCACGAGAUUGAACCCCUUAD-69429A-139794GCUUCAAGGCC66_682-702_sA-139795UCUCCUGAGGCC155_680-702_ashcUCAGGAGAUUGAAGCAD-69385A-139707CAGGAGCAGAA67_692-712_sA-139708UAUUGUUCCCUU156_690-712_ashcGGGAACAAUACUGCUCCUGAGAD-69386A-139709GGAGCAGAAGG68_694-714_sA-139710UACAUUGUUCCC157_692-714_ashcGAACAAUGUAUUCUGCUCCUGAD-69430A-139796GGAGCAGAAGG69_696-716_sA-139797UAUUGUUCCCUU158_694-716_ashcGAACAAUACUGCUCCAD-69387A-139711GAGCAGAAGGG70_695-715_sA-139712UGACAUUGUUCC159_693-715_ashcAACAAUGUCACUUCUGCUCCUAD-69431A-139798AGCAGAAGGGA71_698-718_sA-139799UACAUUGUUCCC160_696-718_ashcACAAUGUAUUCUGCUAD-69432A-139800GCAGAAGGGAA72_699-719_sA-139801UGACAUUGUUCC161_697-719_ashcCAAUGUCACUUCUGCAD-69388A-139713GACUUUGAAAU73_751-771_sA-139714UACCACAUCCAU162_749-771_ashcmrGGAUGUGGUAUUCAAAGUCCCAD-69389A-139715CUUUGAAAUGG74_753-773_sA-139716UCCACCACAUCC163_751-773_ashcmrAUGUGGUGGAAUUUCAAAGUCAD-69433A-139802CUUUGAAAUGG75_755-775_sA-139803UACCACAUCCAU164_753-775_ashcmrAUGUGGUAUUCAAAGAD-69434A-139804UUGAAAUGGAU76_757-777_sA-139805UCCACCACAUCC165_755-777_ashcmrGUGGUGGAAUUUCAAAD-69391A-139719UGAAAUGGAUG77_756-776_sA-139720AUUGCCACCACA166_754-776_ashcmrUGGUGGCAAUUCCAUUUCAAAAD-69436A-139808AAAUGGAUGUG78_760-780_sA-139809AUUGCCACCACA167_758-780_ashcmrGUGGCAAUUCCAUUUAD-69392A-139721AUGGAUGUGGU79_760-780_sA-139722UACCAUUGCCAC168_758-780_ashcmrGGCAAUGGUACACAUCCAUUUAD-69402A-139721AUGGAUGUGGU80_760-780_sA-139741UACCAUUGCCAC169_758-780_asmrGGCAAUGGUACACAUCCAUCUAD-69393A-139723GGAUGUGGUGG81_762-782_sA-139724UUCACCAUUGCC170_760-782_ashcmrCAAUGGUGAAACCACAUCCAUAD-69437A-139810GGAUGUGGUGG82_764-784_sA-1398IIUACCAUUGCCAC171_762-784_ashcmrCAAUGGUACACAUCCAD-69438A-139812AUGUGGUGGCA83_766-786_sA-139813UUCACCAUUGCC172_764-786_ashcmrAUGGUGAAACCACAUAD-69395A-139727GCAAUGGUGAA84_772-792_sA-139728UACCGUGUCAUU173_770-792_ashcUGACACGGUACACCAUUGCCAAD-69440A-139816AAUGGUGAAUG85_776-796_sA-139817UACCGUGUCAUU174_774-796_ashcACACGGUACACCAUUAD-69396A-139729CAGUGCGAGGU86_826-846_sA-139730UAUCAUGCCGAC175_824-846_ashcmrCGGCAUGAUACUCGCACUGAUAD-69441A-139818GUGCGAGGUCG87_830-850_sA-139819UAUCAUGCCGAC176_828-850_ashcmrGCAUGAUACUCGCACAD-69397A-139731ACAUGGAGGAG88_872-892_sA-139732UAUUCUGCAUCU177_870-892_ashcAUGCAGAAUACCUCCAUGUAGAD-69442A-139820AUGGAGGAGAU89_876-896_sA-139821UAUUCUGCAUCU178_874-896_ashcGCAGAAUACCUCCAUAD-69394A-139725GAUGCAGAAUG90_882-902_sA-139726ACCAGCUCCACA179_880-902_ashcmrUGGAGCUGGUUUCUGCAUCUCAD-69439A-139814UGCAGAAUGUG91_886-906_sA-139815ACCAGCUCCACA180_884-906_ashcmrGAGCUGGUUUCUGCAAD-69398A-139733GUGGACGAGAG92_1000-1020_sA-139734UUUUGCAGAGCU181_998-1020_ashcCUCUGCAAAACUCGUCCACCAAD-69443A-139822GGACGAGAGCU93_1004-1024_sA-139823UUUUGCAGAGCU182_1002-1024_ashcCUGCAAAACUCGUCCAD-69380A-139697AAGUACAUGGG94_1057-1077_sA-139698UACCAGCUCGCC183_1055-1077_ashcmrCGAGCUGGUACAUGUACUUGCAD-69425A-139786GUACAUGGGCG95_1061-1081_sA-139787UACCAGCUCGCC184_1059-1081_ashcmrAGCUGGUACAUGUACAD-69403A-139742AGGCUCGUGGA96_1093-1113_sA-139743UAGGUUUUCGUC185_1091-1113_ashcCGAAAACCUACACGAGCCUGAAD-69447A-139830GCUCGUGGACG97_1097-1117_sA-139831UAGGUUUUCGUC186_1095-1117_ashcAAAACCUACACGAGCAD-69404A-139744CGUGGACGAAA98_1098-1118_sA-139745AAGAGCAGGUUU187_1096-1118_ashcACCUGCUCUUUCGUCCACGAGAD-69405A-139746GUGGACGAAAA99_1099-1119_sA-139747UAAGAGCAGGUU188_1097-1119_ashcCCUGCUCUUAUUCGUCCACGAAD-69448A-139832UGGACGAAAAC100_1102-1122_sA-139833AAGAGCAGGUUU189_1100-1122_ashcCUGCUCUUUCGUCCAAD-69449A-139834GGACCAAAACC101_1103-1123_sA-139835UAAGAGCAGGUU190_1101-1123_ashcUGCUCUUAUUCGUCCAD-69406A-139748CGCAAGCAGAU102_1204-1224_sA-139749UAUGUUGUAGAU191_1202-1224_ashcCUACAACAUACUGCUUGCGGUAD-69450A-139836CAAGCAGAUCU103_1208-1228_sA-139837UAUGUUGUAGAU192_1206-1228_ashcACAACAUACUGCUUGAD-69390A-139717AGCUGCGAGAU104_1468-1488_sA-139718UAUGAAGGUGAU193_1466-1488_ashcmrCACCUUCAUACUCGCAGCUGGAD-69435A-139806CUGCGAGAUCA105_1472-1492_sA-139807UAUGAAGGUGAU194_1470-1492_ashcmrCCUUCAUACUCGCAGAD-69408A-139752CCAGUCCUGGC106_2049-2069_sA-139753UAAGAAAAUGGC195_2047-2069_ashcCAUUUUCUUACAGGACUGGGUAD-69452A-139840AGUCCUGGCCA107_2053-2073_sA-139841UAAGAAAAUGGC196_2051-2073_ashcUUUUCUUACAGGACUAD-69409A-139754CACUGAGUGGC108_2170-2190_sA-139755AGAAUCACAAGC197_2168-2190_ashcUUGUGAUUCUCACUCAGUGAUAD-69453A-139842CUGAGUGGCUU109_2174-2194_sA-139843AGAAUCACAAGC198_2172-2194_ashcGUGAUUCUCACUCAGAD-69410A-139756AAUGUUAAAAG110_2413-2433_sA-139757AUGUUUAAAACU199_2411-2433_ashcUUUUAAACAUUUUAACAUUUUAD-69454A-139844UGUUAAAAGUU111_2417-2437_sA-139845AUGUUUAAAACU200_2415-2437_ashcUUAAACAUUUUAACAAD-69444A-139824AGCAGAAGGGA112_698-718_sA-139825UAUGUUGUUCCC201_696-718_asmrACAACAUAUUCUGCUAD-69399A-139735GGAGCAGAAGG113_694-714_sA-139736UAUGUUGUUCCC202_692-714_asmrGAACAACAUAUUCUGCUCCGGAD-69445A-139826UCUCCGAGAUG114_725-745_sA-139827UUUGAUAGCAUC203_723-745_asmrCUAUCAAAUCGGAGAAD-69400A-139737CUUCUCCGAGA115_721-741_sA-139738UUUGAUAGCAUC204_719-741_asmrUGCUAUCAAAUCGGAGAAGUCAD-69446A-139828AGAUGGAUGUG116_760-780_sA-139829AUUGCCACCACA205_758-780_asmrGUGGCAAUUCCAUCUAD-69401A-139739UGAGAUGGAUG117_756-776_sA-139740AUUGCCACCACA206_754-776_asmrUGGUGGCAAUUCCAUCUCAAAAD-69451A-139838CUGCGAAAUCA118_1472-1492_sA-139839AAUGAAGGUGAU207_1470-1492_asmrCCUUCAUUUUCGCAGAD-69407A-139750AACUGCGAAAU119_1468-1488_sA-139751AAUGAAGGUGAU208_1466-1488_asmrCACCUUCAUUUUCGCAGUUGG TABLE 3GCK Modified SequencesSenseSEQAnti-AntisenseSEQSEQDuplexSensesequenceIDsensesequenceIDmRNAIDIDID(5′-3′)NO:ID(5′-3′)NO:sequenceNO:AD-69366A-139669gsasggacCfuGfAf209A-139670usAfsucaCfcUfUfcu298AGGAGGACCUGA387AfgaaggugauaL96ucAfgGfuccucscsuAGAAGGUGAUAAD-69368A-139673gsgsaccuGfaAfGf210A-139674usUfscauCfaCfCfuu299GAGGACCUGAAG388AfaggugaugaaL96cuUfcAfgguccsuscAAGGUGAUGAAAD-69411A-139758GGACCUGAAGAAGGU211A-139759UAUCACCUUCUUCAGGU300GGACCUGAAGAA389GAUAdTdTCCdTdTGGUGAUAAD-69413A-139762ACCUGAAGAAGGUGA212A-139763UUCAUCACCUUCUUCAG301ACCUGAAGAAGG390UGAAdTdTGUdTdTUGAUGAAAD-69367A-139671gsgsugauGfaGfAf213A-139672usUfscugCfaUfCfcg302AAGGUGAUGAGA391CfggaugcagaaL96ucUfcAfucaccsusuCGGAUGCAGAAAD-69369A-139675usgsaugaGfaCfGf214A-139676usCfsuucUfgCfAfuc303GGUGAUGAGACG392GfaugcagaagaL96cgUfcUfcaucascscGAUGCAGAAGAAD-69412A-139760UGAUGAGACGGAUGC215A-139761UUCUGCAUCCGUCUCAU304UGAUGAGACGGA393AGAAdTdTCAdTdTUGCAGAAAD-69414A-139764AUGAGACGGAUGCAG216A-139765UCUUCUGCAUCCGUCUC305AUGAGACGGAUG394AAGAdTdTAUdTdTCAGAAGAAD-69371A-139679ascsggauGfcAfGf217A-139680usCfscauCfuCrCfuu306AGACGGAUGCAG395AfaggagauggaL96cuGfcAfuccguscsuAAGGAGAUGGAAD-69416A-139768GGAUGCAGAAGGAGA218A-139769UCCAUCUCCUUCUGCAU307GGAUGCAGAAGG396UGGAdTdTCCdTdTAGAUGGAAD-69370A-139677ascsccauGfaAfGf219A-139678usAfscacUfgGfCfcu308AGACCCAUGAAG397ATggccaguguaL96cuUfcAfuggguscsuAGGCCAGUGUAAD-69415A-139766CCAUGAAGAGGCCAG220A-139767UACACUGGCCUCUUCAU309CCAUGAAGAGGC398UGUAdTdTGGdTdTCAGUGUAAD-69372A-139681csasguguGfaAfGf221A-139682usUfsgggCfaGfCfau310GCCAGUGUGAAG399AfugcugcccaaL96cuUfcAfcacugsgscAUGCUGCCCAAAD-69417A-139770GUGUGAAGAUGCUGC222A-139771UUGGGCAGCAUCUUCAC311GUGUGAAGAUGC400CCAAdTdTACdTdTUGCCCAAAD-69373A-139683gsusgaagGfuGfGf223A-139684usUfscacCfuUfCfuc312UGGUGAAGGUGG401GfagaaggugaaL96ccAfcCfuucacscsaGAGAAGGUGAAAD-69418A-139772GAAGGUGGGAGAAGG224A-139773UUCACCUUCUCCCACCU313GAAGGUGGGAGA402UGAAdTdTUCdTdTAGGUGAAAD-69374A-139685gsasgcguGfaAfGf225A-139686usGfsgugUfuUfGfgu314UGGAGCGUGAAG403AfccaaacaccaL96cuUfcAfcgcucscsaACCAAACACCAAD-69419A-139774GCGUGAAGACCAAAC226A-139775UGGUGUUUGGLCUUCAC315GCGUGAAGACCA404ACCAdTdTGCdTdTAACACCAAD-69375A-139687gsasagacCfaAfAf227A-139688usAfscauCfuGfGfug316GUGAAGACCAAA405CfaccagauguaL96uuUfgGfucuucsascCACCAGAUGUAAD-69420A-139776AGACCAAACACCAGA228A-139777UACAUCUGGUGUUUGGU317AGACCAAACACC406UGUAdTdTCUdTdTAGAUGUAAD-69376A-139689gsascuucCTuGfGf229A-139690usUfsgauGfcUfUfgu318CCGACUUCCUGG407AfcaagcaucaaL96ccAfgGfaagucsgsgACAAGCAUCAAAD-69377A-139691csusuccuGfgAfCf230A-139692asUfscugAfuGfCfuu319GACUUCCUGGAC408AfagcaucagauL96guCfcAfggaagsuscAAGCAUCAGAUAD-69421A-139778CUUCCUGGACAAGCA231A-139779UCGAUGCUUGUCCAGGA320CUUCCUGGACAA409UCAAdTdTAGdTdTGCAUCAAAD-69422A-139780UCCUGGACAAGCAUC232A-139781AUCUGAUGCCUGUCCAG321UCCUGGACAAGC410AGAUdTdTGAdTdTAUCAGAUAD-69378A-139693cscsuggaCfaAfGf233A-139694usUfscauCfuGfAfug322UUCCUGGACAAG411CfaucagaugaaL96cuUfgUfccaggsasaCAUCAGAUGAAAD-69379A-139695csusggacAfaGfCf234A-13%%usUfsucaUfcUfGfau323UCCUGGACAAGC412AfucagaugaaaL96gcUfuGfuccagsgsaAUCAGAUGAAAAD-69423A-139782UGGACAAGCAUCAGA235A-139783UUCAUCUGAUGCUUGUC324UGGACAAGCAUC413UGAAdTdTCAdTdTAGAUGAAAD-69424A-139784GGACAAGCAUCAGAC236A-139785UUUCAUCUGAUGCUUGU325GGACAAGCAUCA414GAAAdTdTCCdTdTGAUGAAAAD-69381A-139699asgsgcacGfaAfGf237A-139700usUfsuauCfgAfUfgu326UGAGGCACGAAG415AfcaucgauaaaL96cuUfcGfugccuscsaACAUCGAUAAAAD-69426A-139788GCACGAAGACAUCGA238A-139789UUUAUCGAUGUCUUCGU327GCACGAAGACAU416UAAAdTdTGCdTdTCGAUAAAAD-69382A-139701ascsaucgAfuAfAf239A-139702usAfsaggAfuGfCfcc328AGACAUCGAUAA417GfggcauccuuaL96uuAfuCfgauguscsuGGGCAUCCUUAAD-69427A-139790AUCGAUAAGGGCAUC240A-139791UAAGGAUGCCCUUAUCG329AUCGAUAAGGGC418CUUAdTdTAUdTdTAUCCUUAAD-69383A-139703csusggacCfaAfGf241A-139704usCfscuuGfaAfGfcc330AACUGGACCAAG419GfgcuucaaggaL96cuUfgGfuccagsusuGGCUUCAAGGAAD-69428A-139792GGACCAAGGGCLUCA242A-139793UCCUUGAAGCCCUUGGU331GGACCAAGGGCU420AGGAdTdTCCdTdTUCAAGGAAD-69384A-139705gsgsgcuuCfaAfGf243A-139706usCfsuccUfgAfGfgc332AAGGGCUUCAAG421GfccucaggagaL96cuUfgAfagcccsusuGCCUCAGGAGAAD-69429A-139794GCUUCAAGGCCUCAG244A-139795UCUCCUGAGGCCUUGAA333GCUUCAAGGCCU422GAGAdTdTGCdTdTCAGGAGAAD-69385A-139707csasggagCfaGfAf245A-139708usAfsuugUfuCfCfcu334CUCAGGAGCAGA423AfgggaacaauaL96ucUfgCfuccugsasgAGGGAACAAUAAD-69386A-139709gsgsagcaGfaAfGf246A-139710usAfscauUfgUfUfcc335CAGGAGCAGAAG424GfgaacaauguaL96cuUfcUfgcuccsusgGGAACAAUGUAAD-69430A-139796GGAGCAGAAGGGAAC247A-139797UAUUGUUCCCUUCUGCU336GGAGCAGAAGGG425AAUAdTdTCCdTdTAACAAUAAD-69387A-139711gsasgcagAfaGfGf248A-139712usGfsacaUfuGfUfuc337AGGAGCAGAAGG426GfaacaaugucaL96ccUfuCfugcucscsuGAACAAUGUCAAD-69431A-139798AGCAGAAGGGAACAA249A-139799UACAUUGUUCCCUUCUG338AGCAGAAGGGAA427UGUAdTdTCUdTdTCAAUGUAAD-69432A-139800GCAGAAGGGAACAAU250A-139801UGACAUUGUUCCCUUCU339GCAGAAGGGAAC428GUCAdTdTGCdTdTAAUGUCAAD-69388A-139713gsascuuuGfaAfAf251A-139714usAfsccaCfaUfCfca340GGGACUUUGAAA429UfggaugugguaL96uuUfcAfaagucscscUGGAUGUGGUAAD-69389A-139715csusuugaAfaUfGf252A-139716usCfscacCfaCfAfuc341GACUUUGAAAUG430GfaugugguggaL96caUfuUfcaaagsuscGAUGUGGUGGAAD-69433A-139802CUUUGAAAUGGAUGU253A-139803UACCACAUCCAUUUCAA342CUUUGAAAUGGA431GGUAdTdTAGdTdTUGUGGUAAD-69434A-139804UUGAAAUGGAUGUGG254A-139805UCCACCACAUCCAUUUC343UUGAAAUGGAUG432UGGAdTdTAAdTdTUGGUGGAAD-69391A-139719usgsaaauGfgAfUf255A-139720asUfsugcCfaCfCfac344UUUGAAAUGGAU433GfugguggcaauL96auCfcAfuuucasasaGUGGUGGCAAUAD-69436A-139808AAAUGGAUGUGGUGG256A-139809AUUGCCACCACAUCCAU345AAAUGGAUGUGG434CAAUdTdTUUdTdTUGGCAAUAD-69392A-139721asusggauGfuGfCf257A-139722usAfsccaUfuGfCfca346AAAUGGAUGUGG435UfggcaaugguaL96ccAfcAfuccaususuUGGCAAUGGUAAD-69402A-139721asusggauGfuGfGf258A-139741usAfsccaUfuGfCfca347AGAUGGAUGUGG436UfggcaaugguaL96ccAfcAfuccauscsuUGGCAAUGGUAAD-69393A-139723gsgsauguGfgUfGf259A-139724usUfscacCfaUfUfgc348AUGGAUGUGGUG437CfcaauggugaaL96caCfcAfcauccsasuGCAAUGGUGAAAD-69437A-139810GGAUGUGGUGGCAAU260A-139811UACCAUUGCCACCACAU349GGAUGUGGUGGC438GGUAdTdTCCdTdTAAUGGUAAD-69438A-139812AUGUGGUGGCAAUGG261A-139813UUCACCAUUGCCACCAC350AUGUGGUGGCAA439UGAAdTdTAUdTdTUGGUGAAAD-69395A-139727gscsaaugGfuGfAf262A-139728usAfsccgUfgUfCfau351UGGCAAUGGUGA440AfugacacgguaL96ucAfcCfauugcscsaAUGACACGGUAAD-69440A-139816AAUGGUGAAUGACAC263A-139817UACCGUGLCAUUCACCA352AAUGGUGAAUGA441GGUAdTdTUUdTdTCACGGUAAD-69396A-139729csasgugcGfaGfGf264A-139730usAfsucaUfgCfCfga353AUCAGUGCGAGG442UfcggcaugauaL96ccUfcGfcacugsasuUCGGCAUGAUAAD-69441A-139818GUGCGAGGUCGGCAU265A-139819UAUCALGCCGACCUCGC354GUGCGAGGUCGG443GAUAdTdTACdTdTCAUGALAAD-69397A-139731ascsauggAfgGfAf266A-139732usAfsuucUfgCfAfuc355CUACAUGGAGGA444GfaugcagaauaL96ucCfuCfcaugusasgGAUGCAGAAUAAD-69442A-139820AUGGAGGAGAUGCAG267A-139821UAUUCUGCAUCUCCUCC356AUGGAGGAGAUG445AAUAdTdTAUdTdTCAGAAUAAD-69394A-139725gsasugcaGfaAfUf268A-139726asCfscagCfuCfCfac357GAGAUGCAGAAU446GfuggagcugguL96auUfcUfgcaucsuscGUGGAGCUGGUAD-69439A-139814UGCAGAAUGUGGAGC269A-139815ACCAGCUCCACAUUCUG358UGCAGAAUGUGG447UGGUdTdTCAdTdTAGCUGGUAD-69398A-139733gsusggacGfaGfAf270A-139734usUfsuugCfaGfAfgc359UGGUGGACGAGA448GfcucugcaaaaL96ucUfcGfuccacscsaGCUCUGCAAAAAD-69443A-139822GGACGAGAGCUCUGC271A-139823UUUUGCAGAGCUCUCGU360GGACGAGAGCUC449AAAAdTdTCCdTdTUGCAAAAAD-69380A-139697asasguacAfuGfGf272A-139698usAfsccaGfcUfCfgc361GCAAGUACAUGG450GfcgagcugguaL96ccAfuGfuacuusgscGCGAGCUGGUAAD-69425A-139786GUACAUGGGCGAGCU273A-139787UACCAGCUCGCCCAUGU362GUACAUGGGCGA451GGUAdTdTACdTdTGCUGGUAAD-69403A-139742asgsgcucGfuGfGf274A-139743usAfsgguUfuUfCfgu363UCAGGCUCGUGG452AfcgaaaaccuaL96ccAfcGfagccusgsaACGAAAACCUAAD-69447A-139830GCUCGUGGACGAAAA275A-139831UAGGUUUUCGUCCACGA364GCUCGUGGACGA453CCUAdTdTGCdTdTAAACCUAAD-69404A-139744csgsuggaCfgAfAf276A-139745asAfsgagCfaGfGfuu365CUCGUGGACGAA454AfaccugcucuuL96uuCfgUfccacgsasgAACCUGCUCUUAD-69405A-139746gsusggacGfaAfAf277A-139747usAfsagaGfcAfGfgu366UCGUGGACGAAA455AfccugcucuuaL96uuUfcGfuccacsgsaACCUGCUCUUAAD-69448A-139832UGGACGAAAACCUGC278A-139833AAGAGCAGGUUUUCGUC367UGGACGAAAACC456UCUUdTdTCAdTdTUGCUCUUAD-69449A-139834GGACGAAAACCUGCU279A-139835UAAGAGGAGGUUUUCGU368GGACGAAAACCU457CUUAdTdTCCdTdTGCUCUUAAD-69406A-139748csgscaagCfaGfAf280A-139749usAfsuguUfgUfAfga369ACCGCAAGCAGA458UfcuacaacauaL96ucUfgCfuugcgsgsuUCUACAACAUAAD-69450A-139836CAAGCAGAUCUACAA281A-139837UAUGUUGUAGAUCUGCU370CAAGCAGAUCUA459CAUAdTdTUGdTdTCAACAUAAD-69390A-139717asgscugcGfaGfAf282A-139718usAfsugaAfgGftfga371CCAGCUGCGAGA460UfcaccuucauaL96ncUfcGfcagcusgsgUCACCUUCAUAAD-69435A-139806CtGCGAGAUCACCtU283A-139807UAUGAAGGUGAUCUCGC372CUGCGAGAUCAC461CAUAdTdTAGdTdTCUUCAUAAD-69408A-139752cscsagucCfuGfGf284A-139753usAfsagaAfaAfUfgg373ACCCAGUCCUGG462CfcauuuuucuuaL96ccAfgGfacuggsgsuCCAUUUUCUUAAD-69452A-139840AGUCCUGGCCAUUUUC285A-139841UAAGAAAAUGGCCAGGA374AGUCCUGGCCAU463UUAdTdTCUdTdTUUUCUUAAD-69409A-139754csascugaGfuGfGfC286A-139755asGfsaauCfaCfAfag375AUCACUGAGUGG464fuugugauucuL96ccAfcUfcagugsasuCUUGUGAUUCUAD-69453A-139842CUGAGUGGCUUGUGAU287A-139843AGAAUCACAAGCCACUC376CUGAGUGGCUUG465UCUdTdTAGdTdTUGAUUCUAD-69410A-139756asasuguuAfaAfAfG288A-139757asUfsguuUfaAfAfac377AAAAUGUUAAAA466fuuuuaaacauL96uuUfuAfacauususuGUUUUAAACAUAD-69454A-139844UGUUAAAAGUUUUAAA289A-139845AUGUUUAAAACUUUUAA378UGUUAAAAGUUU467CAUdTdTCAdTdTUAAACAUAD-69444A-139824AGCAGAAGGGAACAAC290A-139825UAUGUUGUUCCCUUCUG379AGCAGAAGGGAA468AUAdTdTCUdTdTCAACAUAAD-69399A-139735gsgsagcaGfaAfGfG291A-139736usAfsuguUfgUfUfcc380CCGGAGCAGAAG469fgaacaacauaL96cuUfcUfgcuccsgsgGGAACAACAUAAD-69445A-139826UCUCCGAGAUGCUAUC292A-139827UUUGAUAGCAUCUCGGA381UCUCCGAGAUGC470AAAdTdTGAdTdTUAUCAAAAD-69400A-139737csusucucCfgAfCfA293A-139738usUfsugaUfaGfCfau382GACUUCUCCGAG471fugcuaucaaaL96cuCfgGfagaagsuscAUGCUAUCAAAAD-69446A-139828AGAUGGAUGUGGUGGC294A-139829AUUGCCACCACAUCCAU383AGAUGGAUGUGG472AAUdTdTCUdTdTUGGCAAUAD-69401A-139739usgsagauGfgAfUfG295A-139740asUfsugcCfaCfCfac384UUUGAGAUGGAU473fugguggcaauL96auCfcAfucucasasaGUGGUGGCAAUAD-69451A-139838CUGCGAAAUCACCUUC296A-139839AAUGAAGGUGAUUUCGC385CUGCGAAAUCAC474AUUdTdTAGdTdTCUUCAUUAD-69407A-139750asascugcGfaAfAfU297A-139751asAfsuguAfgGfUfga386CCAACUGCGAAA475fcaccuucauuL96uuUfcGfcaguusgsgUCACCUUCAUU TABLE 4GCK Single Dose Screen in Primary Mouse HepatocytesPrimary Mouse HepatocytesDuplex Name10 nM Avg10 nM SD0.1 nM Avg0.1 nM SDAD-6936682.254.170.212.8AD-6936847.85.391.73.6AD-6941117.64.827.92.8AD-6941315.95.143.74.8AD-6936758.211.092.010.5AD-6936960.310.688.06.5AD-6941224.15.371.715.6AD-6941452.57.782.77.2AD-6937177.59.789.112.3AD-6941611.82.425.14.0AD-6937099.510.196.95.1AD-6941590.39.391.315.2AD-6937227.92.249.810.2AD-6941729.92.163.612.6AD-6937396.717.1104.748.8AD-6941886.234.062.09.9AD-6937433.74.871.77.8AD-6941917.13.244.84.4AD-6937572.08.3104.55.3AD-6942020.32.046.67.2AD-6937622.12.359.09.7AD-6937723.02.059.26.6AD-6942116.12.129.93.5AD-6942215.21.238.92.4AD-6937824.82.170.14.2AD-6937911.20.918.03.2AD-6942311.71.341.13.6AD-694249.51.613.93.5AD-6938173.613.399.936.4AD-6942684.56.376.56.1AD-6938291.95.691.63.3AD-6942797.62.588.84.5AD-6938395.93.2100.59.3AD-6942832.74.366.15.0AD-6938488.39.897.98.6AD-6942990.710.695.95.7AD-6938529.24.245.14.4AD-6938692.59.097.89.8AD-6943021.21.431.82.1AD-6938769.79.077.87.1AD-6943269.53.279.613.6AD-6938866.911.384.711.9AD-6938976.318.386.96.0AD-6943370.02.676.45.0AD-6943497.66.671.69.5AD-6939121.75.159.85.6AD-6943619.91.954.314.6AD-6939239.24.881.512.7AD-6940232.25.162.912.4AD-6939334.95.478.513.0AD-6943714.02.527.34.6AD-6943811.70.728.42.5AD-6939528.26.855.412.2AD-6944019.42.834.77.3AD-6939629.73.057.29.8AD-6944123.84.144.67.4AD-6939739.511.867.911.6AD-6944229.32.440.18.4AD-6939439.44.279.53.2AD-6943937.63.265.93.0AD-6939892.29.6111.316.2AD-69443115.114.597.45.1AD-6938023.63.265.011.2AD-6942511.91.229.46.5AD-6940364.213.077.013.9AD-6944787.47.984.311.7AD-6940467.210.683.916.6AD-6940569.010.986.123.6AD-69448107.86.4120.436.9AD-69449129.637.4120.26.4AD-6940663.84.791.710.8AD-6945084.512.4109.55.3AD-6939016.94.238.911.6AD-694359.01.520.93.5AD-6940867.34.783.616.3AD-6945292.610.4111.85.1AD-6940974.84.879.712.3AD-6945387.21.493.73.0AD-6941071.24.990.29.4AD-6945490.010.986.07.8AD-6939924.42.545.86.1AD-6944516.00.644.44.4AD-6940012.21.434.82.3AD-6944614.91.330.42.9AD-6940115.41.629.44.9AD-694516.91.319.23.6AD-694074.10.411.82.8AD-1955101.28316.1184 TABLE 5GCK Single Dose Screen in Primary Cynomologous HepatocytesPrimary Cyno HepatocytesduplexName10 nM Avg10 nM SD0.1 nM Avg0.1 nM SDAD-6936629.814.337.49.3AD-6936812.24.628.14.9AD-6941114.24.522.610.8AD-6941312.13.230.014.0AD-6936714.92.639.97.1AD-6936936.614.254.29.5AD-6941219.36.531.24.6AD-6941426.411.963.68.7AD-6937150.220.591.317.3AD-6941624.58.321.83.6AD-6937043.410.369.411.6AD-6941515.46.234.213.2AD-6937219.87.923.78.7AD-6941723.810.531.416.1AD-6937387.722.166.810.6AD-6941820.44.925.76.4AD-6937424.715.041.86.2AD-6941921.96.732.812.9AD-6937520.65.549.516.0AD-6942015.32.941.821.6AD-6937617.75.322.412.1AD-6937721.65.626.46.2AD-6942122.14.722.18.9AD-6942222.75.738.211.8AD-6937814.76.631.926.2AD-6937910.52.714.42.9AD-6942320.38.242.210.8AD-6942420.05.117.510.3AD-6938146.612.856.920.6AD-6942649.38.476.729.0AD-6938274.022.754.614.2AD-6942733.95.966.313.8AD-6938363.418.681.422.7AD-6942839.416.069.710.0AD-6938458.117.489.122.8AD-6942930.97.347.917.9AD-6938527.39.237.07.7AD-6938641.416.661.318.6AD-6943024.28.829.813.7AD-6938728.23.030.911.1AD-6943223.57.824.45.3AD-6938863.711.070.87.3AD-6938959.713.877.815.6AD-6943337.03.362.231.0AD-6943476.327.994.18.0AD-6939122.011.225.210.0AD-6943624.54.841.32.7AD-6939223.310.152.911.4AD-6940221.97.659.129.2AD-6939322.92.337.32.9AD-6943721.86.630.47.7AD-6943821.55.932.49.6AD-6939517.45.732.34.6AD-6944025.715.035.113.5AD-6939628.89.316.65.1AD-6944126.38.439.28.9AD-6939762.433.348.626.1AD-6944241.012.350.412.5AD-6939438.012.965.713.1AD-6943930.29.051.610.8AD-6939821.13.823.16.5AD-6944320.07.629.34.2AD-6938043.815.364.726.0AD-6942535.59.838.58.5AD-6940334.512.035.17.0AD-6944723.57.455.021.7AD-6940441.223.930.05.5AD-6940520.27.735.014.4AD-6944853.116.442.36.3AD-6944937.814.742.64.0AD-6940621.613.723.47.1AD-6945019.64.523.95.1AD-6939018.88.021.04.2AD-6943520.017.323.99.7AD-6940840.017.934.118.5AD-6945228.99.645.513.6AD-6940940.010.852.731.5AD-6945339.26.557.620.9AD-6941062.08.778.610.5AD-6945486.56.9102.427.8AD-6939988.817.581.621.4AD-6944568.919.5118.326.5AD-6940032.711.931.110.1AD-6944623.510.838.34.1AD-6940128.418.324.75.7AD-6945179.623.5102.941.6AD-6940716.87.729.212.6AD-1955102.91229.2078 Example 2. iRNA Design, Synthesis, Selection, and In Vitro Evaluation This Example describes methods for the design, synthesis, selection, and in vitro evaluation of additional GCK iRNA agents. Bioinformatics A set of siRNAs targeting human glucokinase (GCK) (human NCBI refseqID: NM_033507; NCBI GeneID: 2645) were designed using custom R and Python scripts. The human GCK REFSEQ mRNA has a length of 2442 bases. The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 10 through position 2442 was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. The custom Python script built the set of siRNAs by systematically selecting an siRNA every 11 bases along the target mRNA nucleotide sequence starting at position 10. At each of the positions, the neighboring siRNA (one position to the 5′ end of the mRNA, one position to the 3′ end of the mRNA) was swapped into the design set if the predicted efficacy was better than the efficacy at the exact every-eleventh siRNA. Low complexity siRNAs, e.g., those with Shannon Entropy measures below 1.35, were excluded from the set. Cell Culture and Transfections Primary Cyno Hepatocytes (PCH) cells were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad CA cat #13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Forty μl of EMEM containing about 5×103cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 20 nM final duplex concentration. Total RNA Isolation Using DYNABEADS mRNA Isolation Kit RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl of Lysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed two times with 150 μl Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured, and the supernatant was removed. cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat #4368813) Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 125× dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H2O per reaction was added to RNA isolated as described above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hours at 37° C. Real Time PCR Two μl of cDNA were added to a master mix containing 0.5 μl of Custom Cyno GAPDH TaqMan Probe, 0.5 μl cyno GCK probe (Mf02827184 ml) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was performed in a LightCycler480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. Each duplex was tested in four independent transfections. To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 20 nM AD-1955, or mock transfected cells. A detailed list of the unmodified GCK sense and antisense strand sequences is shown in Table 6 and a detailed list of the modified GCK sense and antisense strand sequences is shown in Table 7. Table 8 shows the results of a single dose screen in primary Cynomolgus hepatocytes (PCH) transfected with the indicated modified siRNAs. Data are expressed as percent of message remaining relative to cells treated with a non-targeting control siRNA, AD-1955. TABLE 6GCK Unmodified SequencesAnti-SenseSEQssenseSEQDuplexOligoSense SequenceIDOligoSense SequenceIDPosition inNameName(5′ to 3′)NO:Name(5′ to 3′)NO:NM_033507.1AD-71009A-142377CUGCCAGCCUCAGGCAGCU476A-142378AGCUGCCUGAGGCUGGCAG68424-42AD-71010A-142379UCAGGCAGCUCUCCAUCCA477A-142380UGGAUGGAGAGCUGCCUGA68533-51AD-71011A-142381CCAUCCAAGCAGCCGUUGA478A-142382UCAACGGCUGCUUGGAUGG68645-63AD-71012A-142383AGCCGUUGCUGCCACAGGA479A-142384UCCUGUGGCAGCAACGGCU68755-73AD-71013A-142385ACAGGCGGGCCUUACGCUA480A-142386UAGCGUAAGGCCCGCCUGU68868-86AD-71014A-142387UUACGCUCCAAGGCUACAA481A-142388UUGUAGCCUUGGAGCGUAA68979-97AD-71015A-142389AAGGCUACAGCAUGUGCUA482A-142390UAGCACAUGCUGUAGCCUU69088-106AD-71016A-142391UGUGCUAGGCCUCAGCAGA483A-142392UCUGCUGAGGCCUAGCACA691100-118AD-71017A-142393UCAGCAGGCAGGAGCAUCU484A-142394AGAUGCUCCUGCCUGCUGA692111-129AD-71018A-142395AGCAUCUCUGCCUCCCAAA485A-142396UUUGGGAGGCAGAGAUGCU693123-141AD-71019A-142397CCUCCCAAAGCAUCUACCU486A-142398AGGUAGAUGCUUUGGGAGG694133-151AD-71020A-142401UAGCCCCUCGGAGAGAUGA487A-142402UCAUCUCUCCGAGGGGCUA695154-172AD-71021A-142403AGAGAUGGCGAUGGAUGUA488A-142404UACAUCCAUCGCCAUCUCU696165-183AD-71022A-142405UGGAUGUCACAAGGAGCCA489A-142406UGGCUCCUUGUGACAUCCA697176-194AD-71023A-142407AGGAGCCAGGCCCAGACAA490A-142408UUGUCUGGGCCUGGCUCCU698187-205AD-71024A-142411ACUCUGGUAGAGCAGAUCA491A-142412UGAUCUGCUCUACCAGAGU699211-229AD-71025A-142413AGCAGAUCCUGGCAGAGUU492A-142414AACUCUGCCAGGAUCUGCU700221-239AD-71026A-142415CAGAGUUCCAGCUGCAGGA493A-142416UCCUGCAGCUGGAACUCUG701233-251AD-71027A-142417AGCUGCAGGAGGAGGACCU494A-142418AGGUCCUCCUCCUGCAGCU702242-260AD-71028A-142419AGGACCUGAAGAAGGUGAU495A-142420AUCACCUUCUUCAGGUCCU703254-272AD-71029A-142421AAGGUGAUGAGACGGAUGA496A-142422UCAUCCGUCUCAUCACCUU704265-283AD-71030A-142423CGGAUGCAGAAGGAGAUGA497A-142424UCAUCUCCUUCUGCAUCCG705277-295AD-71031A-142425AAGGAGAUGGACCGCGGCA498A-142426UGCCGCGGUCCAUCUCCUU706286-304AD-71032A-142427CGCGGCCUGAGGCUGGAGA499A-142428UCUCCAGCCUCAGGCCGCG707298-316AD-71033A-142429CUGGAGACCCAUGAAGAGA500A-142430UCUCUUCAUGGGUCUCCAG708310-328AD-71034A-142431CAUGAAGAGGCCAGUGUGA501A-142432UCACACUGGCCUCUUCAUG709319-337AD-71035A-142433CAGUGUGAAGAUGCUGCCA502A-142434UGGCAGCAUCUUCACACUG710330-348AD-71036A-142435UGCUGCCCACCUACGUGCA503A-142436UGCACGUAGGUGGGCAGCA711341-359AD-71037A-142437UACGUGCGCUCCACCCCAA504A-142438UUGGGGUGGAGCGCACGUA712352-370AD-71038A-142439ACCCCAGAAGGCUCAGAAA505A-142440UUUCUGAGCCUUCUGGGGU713364-382AD-71039A-142441UCAGAAGUCGGGGACUUCA506A-142442UGAAGUCCCCGACUUCUGA714376-394AD-71040A-142443GGGGACUUCCUCUCCCUGA507A-142444UCAGGGAGAGGAAGUCCCC715385-403AD-71041A-142445UCCCUGGACCUGGGUGGCA508A-142446UGCCACCCAGGUCCAGGGA716397-415AD-71042A-142447UGGGUGGCACUAACUUCAA509A-142448UUGAAGUUAGUGCCACCCA717407-425AD-71043A-142449ACUUCAGGGUGAUGCUGGU510A-142450ACCAGCAUCACCCUGAAGU718419-437AD-71044A-142453AGGUGGGAGAAGGUGAGGA511A-142454UCCUCACCUUCUCCCACCU719440-458AD-71045A-142457CAGUGGAGCGUGAAGACCA512A-142458UGGUCUUCACGCUCCACUG720463-481AD-71046A-142461CCAGAUGUACUCCAUCCCA513A-142462UGGGAUGGAGUACAUCUGG721486-504AD-71047A-142467ACCGGCACUGCUGAGAUGA514A-142468UCAUCUCAGCAGUGCCGGU722517-535AD-71048A-142469AGAUGCUCUUCGACUACAU515A-142470AUGUAGUCGAAGAGCAUCU723530-548AD-71049A-142471UCGACUACAUCUCUGAGUA516A-142472UACUCAGAGAUGUAGUCGA724539-557AD-71050A-142473UCUGAGUGCAUCUCCGACU517A-142474AGUCGGAGAUGCACUCAGA725550-568AD-71051A-142475UCCGACUUCCUGGACAAGA518A-142476UCUUGUCCAGGAAGUCGGA726562-580AD-71052A-142477GACAAGCAUCAGAUGAAAC519A-142478GUUUCAUCUGAUGCUUGUC727574-592AD-71053A-142479AGAUGAAACACAAGAAGCU520A-142480AGCUUCUUGUGUUUCAUCU728584-602AD-71054A-142481AGAAGCUGCCCCUGGGCUU521A-142482AAGCCCAGGGGCAGCUUCU729596-614AD-71055A-142483CCUGGGCUUCACCUUCUCA522A-142484UGAGAAGGUGAAGCCCAGG730606-624AD-71056A-142485ACCUUCUCCUUUCCUGUGA523A-142486UCACAGGAAAGGAGAAGGU731616-634AD-71057A-142487CUGUGAGGCACGAAGACAU524A-142488AUGUCUUCGUGCCUCACAG732629-647AD-71058A-142489GAAGACAUCGAUAAGGGCA525A-142490UGCCCUUAUCGAUGUCUUC733640-658AD-71059A-142491GAUAAGGGCAUCCUUCUCA526A-142492UGAGAAGGAUGCCCUUAUC734649-667AD-71060A-142493UUCUCAACUGGACCAAGGA527A-142494UCCUUGGUCCAGUUGAGAA735662-680AD-71061A-142495ACCAAGGGCUUCAAGGCCU528A-142496AGGCCUUGAAGCCCUUGGU736673-691AD-71062A-142497CAAGGCCUCAGGAGCAGAA529A-142498UUCUGCUCCUGAGGCCUUG737684-702AD-71063A-142499AGGAGCAGAAGGGAACAAU530A-142500AUUGUUCCCUUCUGCUCCU738693-711AD-71064A-142501AACAAUGUCGUGGGGCUUA531A-142502UAAGCCCCACGACAUUGUU739706-724AD-71065A-142503UGGGGCUUCUGCGAGACGA532A-142504UCGUCUCGCAGAAGCCCCA740716-734AD-71066A-142505CGAGACGCUAUCAAACGGA533A-142506UCCGUUUGAUAGCGUCUCG741727-745AD-71067A-142507AAACGGAGAGGGGACUUUA534A-142508UAAAGUCCCCUCUCCGUUU742739-757AD-71068A-142509GGGACUUUGAAAUGGAUGU535A-142510ACAUCCAUUUCAAAGUCCC743749-767AD-71069A-142513GCAAUGGUGAAUGACACGA536A-142514UCGUGUCAUUCACCAUUGC744772-790AD-71070A-142515AAUGACACGGUGGCCACGA537A-142516UCGUGGCCACCGUGUCAUU745781-799AD-71071A-142517GCCACGAUGAUCUCCUGCU538A-142518AGCAGGAGAUCAUCGUGGC746793-811AD-71072A-142519UCCUGCUACUACGAAGACA539A-142520UGUCUUCGUAGUAGCAGGA747805-823AD-71073A-142523AGUGCGAGGUCGGCAUGAU540A-142524AUCAUGCCGACCUCGCACU748827-845AD-71074A-142525GGCAUGAUCGUGGGCACGA541A-142526UCGUGCCCACGAUCAUGCC749838-856AD-71075A-142527GUGGGCACGGGCUGCAAUA542A-142528UAUUGCAGCCCGUGCCCAC750847-865AD-71076A-142529UGCAAUGCCUGCUACAUGA543A-142530UCAUGUAGCAGGCAUUGCA751859-877AD-71077A-142531UACAUGGAGGAGAUGCAGA544A-142532UCUGCAUCUCCUCCAUGUA752871-889AD-71078A-142533AGAUGCAGAAUGUGGAGCU545A-142534AGCUCCACAUUCUGCAUCU753881-899AD-71079A-142535UGUGGAGCUGGUGGAGGGA546A-142536UCCCUCCACCAGCUCCACA754891-909AD-71080A-142537UGGAGGGGGACGAGGGCCA547A-142538UGGCCCUCGUCCCCCUCCA755902-920AD-71081A-142539GAGGGCCGCAUGUGCGUCA548A-142540UGACGCACAUGCGGCCCUC756913-931AD-71082A-142541UGCGUCAAUACCGAGUGGA549A-142542UCCACUCGGUAUUGACGCA757925-943AD-71083A-142543CGAGUGGGGCGCCUUCGGA550A-142544UCCGAAGGCGCCCCACUCG758936-954AD-71084A-142545GCCUUCGGGGACUCCGGCA551A-142546UGCCGGAGUCCCCGAAGGC759946-964AD-71085A-142547UCCGGCGAGCUGGACGAGU552A-142548ACUCGUCCAGCUCGCCGGA760958-976AD-71086A-142549GACGAGUUCCUGCUGGAGU553A-142550ACUCCAGCAGGAACUCGUC761970-988AD-71087A-142551UGCUGGAGUAUGACCGCCU554A-142552AGGCGGUCAUACUCCAGCA762980-998AD-71088A-142553GACCGCCUGGUGGACGAGA555A-142554UCUCGUCCACCAGGCGGUC763991-1009AD-71089A-142555GGACGAGAGCUCUGCAAAC556A-142556GUUUGCAGAGCUCUCGUCC7641002-1020AD-71090A-142557UCUGCAAACCCCGGUCAGA557A-142558UCUGACCGGGGUUUGCAGA7651012-1030AD-71091A-142559GGUCAGCAGCUGUAUGAGA558A-142560UCUCAUACAGCUGCUGACC7661024-1042AD-71092A-142561UAUGAGAAGCUCAUAGGUA559A-142562UACCUAUGAGCUUCUCAUA7671036-1054AD-71093A-142563UCAUAGGUGGCAAGUACAU560A-142564AUGUACUUGCCACCUAUGA7681046-1064AD-71094A-142565AAGUACAUGGGCGAGCUGA561A-142566UCAGCUCGCCCAUGUACUU7691057-1075AD-71095A-142567GCGAGCUGGUGCGGCUUGU562A-142568ACAAGCCGCACCAGCUCGC7701067-1085AD-71096A-142569GGCUUGUGCUGCUCAGGCU563A-142570AGCCUGAGCAGCACAAGCC7711079-1097AD-71097A-142571UCAGGCUCGUGGACGAAAA564A-142572UUUUCGUCCACGAGCCUGA7721091-1109AD-69448A-139832UGGACGAAAACCUGCUCUU565A-139833AAGAGCAGGUUUUCGUCCA7731100-1118AD-71098A-142573UGCUCUUCCACGGGGAGGA566A-142574UCCUCCCCGUGGAAGAGCA7741112-1130AD-71099A-142575GGGAGGCCUCCGAGCAGCU567A-142576AGCUGCUCGGAGGCCUCCC7751124-1142AD-71100A-142577CGAGCAGCUGCGCACACGA568A-142578UCGUGUGCGCAGCUGCUCG7761134-1152AD-71101A-142581AGCCUUCGAGACGCGCUUA569A-142582UAAGCGCGUCUCGAAGGCU7771155-1173AD-71102A-142583CGCUUCGUGUCGCAGGUGA570A-142584UCACCUGCGACACGAAGCG7781168-1186AD-71103A-142585UCGCAGGUGGAGAGCGACA571A-142586UGUCGCUCUCCACCUGCGA7791177-1195AD-71104A-142587AGCGACACGGGCGACCGCA572A-142588UGCGGUCGCCCGUGUCGCU7801189-1207AD-71105A-142589CGACCGCAAGCAGAUCUAA573A-142590UUAGAUCUGCUUGCGGUCG7811200-1218AD-71106A-142591CAGAUCUACAACAUCCUGA574A-142592UCAGGAUGUUGUAGAUCUG7821210-1228AD-71107A-142593UCCUGAGCACGCUGGGGCU575A-142594AGCCCCAGCGUGCUCAGGA7831223-1241AD-71108A-142595CUGGGGCUGCGACCCUCGA576A-142596UCGAGGGUCGCAGCCCCAG7841234-1252AD-71109A-142597CGACCCUCGACCACCGACU577A-142598AGUCGGUGGUCGAGGGUCG7851243-1261AD-71110A-142599CACCGACUGCGACAUCGUA578A-142600UACGAUGUCGCAGUCGGUG7861254-1272AD-71111A-142601CAUCGUGCGCCGCGCCUGA579A-142602UCAGGCGCGGCGCACGAUG7871266-1284AD-71112A-142603CGCGCCUGCGAGAGCGUGU580A-142604ACACGCUCUCGCAGGCGCG7881276-1294AD-71113A-142605AGCGUGUCUACGCGCGCUA581A-142606UAGCGCGCGUAGACACGCU7891288-1306AD-71114A-142607CGCGCUGCGCACAUGUGCU582A-142608AGCACAUGUGCGCAGCGCG7901300-1318AD-71115A-142609ACAUGUGCUCGGCGGGGCU583A-142610AGCCCCGCCGAGCACAUGU7911310-1328AD-71116A-142611CGGGGCUGGCGGGCGUCAU584A-142612AUGACGCCCGCCAGCCCCG7921322-1340AD-71117A-142613CGGGCGUCAUCAACCGCAU585A-142614AUGCGGUUGAUGACGCCCG7931331-1349AD-71118A-142615AACCGCAUGCGCGAGAGCA586A-142616UGCUCUCGCGCAUGCGGUU7941342-1360AD-71119A-142617AGAGCCGCAGCGAGGACGU587A-142618ACGUCCUCGCUGCGGCUCU7951355-1373AD-71120A-142619CGAGGACGUAAUGCGCAUA588A-142620UAUGCGCAUUACGUCCUCG7961365-1383AD-71121A-142621UGCGCAUCACUGUGGGCGU589A-142622ACGCCCACAGUGAUGCGCA7971376-1394AD-71122A-142623UGGGCGUGGAUGGCUCCGU590A-142624ACGGAGCCAUCCACGCCCA7981388-1406AD-71123A-142625UGGCUCCGUGUACAAGCUA591A-142626UAGCUUGUACACGGAGCCA7991398-1416AD-71124A-142627UACAAGCUGCACCCCAGCU592A-142628AGCUGGGGUGCAGCUUGUA8001408-1426AD-71125A-142629CCAGCUUCAAGGAGCGGUU593A-142630AACCGCUCCUUGAAGCUGG8011421-1439AD-71126A-142631AGGAGCGGUUCCAUGCCAA594A-142632UUGGCAUGGAACCGCUCCU8021430-1448AD-71127A-142633AUGCCAGCGUGCGCAGGCU595A-142634AGCCUGCGCACGCUGGCAU8031442-1460AD-71128A-142635CGCAGGCUGACGCCCAGCU596A-142636AGCUGGGCGUCAGCCUGCG8041453-1471AD-71129A-142637CCCAGCUGCGAGAUCACCU597A-142638AGGUGAUCUCGCAGCUGGG8051465-1483AD-71130A-142639GAGAUCACCUUCAUCGAGU598A-142640ACUCGAUGAAGGUGAUCUC8061474-1492AD-71131A-142641AUCGAGUCGGAGGAGGGCA599A-142642UGCCCUCCUCCGACUCGAU8071486-1504AD-71132A-142643AGGAGGGCAGUGGCCGGGA600A-142644UCCCGGCCACUGCCCUCCU8081496-1514AD-71133A-142645CCGGGGCGCGGCCCUGGUA601A-142646UACCAGGGCCGCGCCCCGG8091509-1527AD-71134A-142647CCCUGGUCUCGGCGGUGGA602A-142648UCCACCGCCGAGACCAGGG8101520-1538AD-71135A-142649GCGGUGGCCUGUAAGAAGA603A-142650UCUUCUUACAGGCCACCGC8111531-1549AD-71136A-142651UAAGAAGGCCUGUAUGCUA604A-142652UAGCAUACAGGCCUUCUUA8121542-1560AD-71137A-142653CUGUAUGCUGGGCCAGUGA605A-142654UCACUGGCCCAGCAUACAG8131551-1569AD-71138A-142655CAGUGAGAGCAGUGGCCGA606A-142656UCGGCCACUGCUCUCACUG8141564-1582AD-71139A-142657CAGUGGCCGCAAGCGCAGA607A-142658UCUGCGCUUGCGGCCACUG8151573-1591AD-71140A-142659AGCGCAGGGAGGAUGCCAA608A-142660UUGGCAUCCUCCCUGCGCU8161584-1602AD-71141A-142661UGCCACAGCCCCACAGCAA609A-142662UUGCUGUGGGGCUGUGGCA8171597-1615AD-71142A-142663CACAGCACCCAGGCUCCAU610A-142664AUGGAGCCUGGGUGCUGUG8181608-1626AD-71143A-142665AGGCUCCAUGGGGAAGUGA611A-142666UCACUUCCCCAUGGAGCCU8191618-1636AD-71144A-142667GGAAGUGCUCCCCACACGU612A-142668ACGUGUGGGGAGCACUUCC8201629-1647AD-71145A-142669CCACACGUGCUCGCAGCCU613A-142670AGGCUGCGAGCACGUGUGG8211640-1658AD-71146A-142671UCGCAGCCUGGCGGGGCAA614A-142672UUGCCCCGCCAGGCUGCGA8221650-1668AD-71147A-142673CGGGGCAGGAGGCCUGGCA615A-142674UGCCAGGCCUCCUGCCCCG8231661-1679AD-71148A-142675CCUGGCCUUGUCAGGACCA616A-142676UGGUCCUGACAAGGCCAGG8241673-1691AD-71149A-142677CAGGACCCAGGCCGCCUGA617A-142678UCAGGCGGCCUGGGUCCUG8251684-1702AD-71150A-142679CCGCCUGCCAUACCGCUGA618A-142680UCAGCGGUAUGGCAGGCGG8261695-1713AD-71151A-142681UACCGCUGGGGAACAGAGA619A-142682UCUCUGUUCCCCAGCGGUA8271705-1723AD-71152A-142683AACAGAGCGGGCCUCUUCA620A-142684UGAAGAGGCCCGCUCUGUU8281716-1734AD-71153A-142685CUCUUCCCUCAGUUUUUCA621A-142686UGAAAAACUGAGGGAAGAG8291728-1746AD-71154A-142687UUUUUCGGUGGGACAGCCA622A-142688UGGCUGUCCCACCGAAAAA8301740-1758AD-71155A-142689GGGACAGCCCCAGGGCCCU623A-142690AGGGCCCUGGGGCUGUCCC8311749-1767AD-71156A-142691AGGGCCCUAACGGGGGUGA624A-142692UCACCCCCGUUAGGGCCCU8321760-1778AD-71157A-142693GGGUGCGGCAGGAGCAGGA625A-142694UCCUGCUCCUGCCGCACCC8331773-1791AD-71158A-142695AGGAGCAGGAACAGAGACU626A-142696AGUCUCUGUUCCUGCUCCU8341782-1800AD-71159A-142697AGAGACUCUGGAAGCCCCA627A-142698UGGGGCUUCCAGAGUCUCU8351794-1812AD-71160A-142699AAGCCCCCCACCUUUCUCA628A-142700UGAGAAAGGUGGGGGGCUU8361805-1823AD-71161A-142701UUUCUCGCUGGAAUCAAUU629A-142702AAUUGAUUCCAGCGAGAAA8371817-1835AD-71162A-142703AAUCAAUUUCCCAGAAGGA630A-142704UCCUUCUGGGAAAUUGAUU8381828-1846AD-71163A-142705CCCAGAAGGGAGUUGCUCA631A-142706UGAGCAACUCCCUUCUGGG8391837-1855AD-71164A-142707UUGCUCACUCAGGACUUUA632A-142708UAAAGUCCUGAGUGAGCAA8401849-1867AD-71165A-142709AGGACUUUGAUGCAUUUCA633A-142710UGAAAUGCAUCAAAGUCCU8411859-1877AD-71166A-142711AUUUCCACACUGUCAGAGA634A-142712UCUCUGACAGUGUGGAAAU8421872-1890AD-71167A-142713UGUCAGAGCUGUUGGCCUA635A-142714UAGGCCAACAGCUCUGACA8431882-1900AD-71168A-142715UUGGCCUCGCCUGGGCCCA636A-142716UGGGCCCAGGCGAGGCCAA8441893-1911AD-71169A-142717CUGGGCCCAGGCUCUGGGA637A-142718UCCCAGAGCCUGGGCCCAG8451903-1921AD-71170A-142719CUCUGGGAAGGGGUGCCCU638A-142720AGGGCACCCCUUCCCAGAG8461914-1932AD-71171A-142721UGCCCUCUGGAUCCUGCUA639A-142722UAGCAGGAUCCAGAGGGCA8471927-1945AD-71172A-142723UCCUGCUGUGGCCUCACUU640A-142724AAGUGAGGCCACAGCAGGA8481938-1956AD-71173A-142725CCUCACUUCCCUGGGAACU641A-142726AGUUCCCAGGGAAGUGAGG8491949-1967AD-71174A-142727CUGGGAACUCAUCCUGUGU642A-142728ACACAGGAUGAGUUCCCAG8501959-1977AD-71175A-142729CCUGUGUGGGGAGGCAGCU643A-142730AGCUGCCUCCCCACACAGG8511971-1989AD-71176A-142731GGAGGCAGCUCCAACAGCU644A-142732AGCUGUUGGAGCUGCCUCC8521980-1998AD-71177A-142733CAACAGCUUGACCAGACCU645A-142734AGGUCUGGUCAAGCUGUUG8531991-2009AD-71178A-142735CCAGACCUAGACCUGGGCA646A-142736UGCCCAGGUCUAGGUCUGG8542002-2020AD-71179A-142737CUGGGCCAAAAGGGCAGCA647A-142738UGCUGCCCUUUUGGCCCAG8552014-2032AD-71180A-142739AGGGCAGCCAGGGGCUGCU648A-142740AGCAGCCCCUGGCUGCCCU8562024-2042AD-71181A-142741GGGCUGCUCAUCACCCAGU649A-142742ACUGGGUGAUGAGCAGCCC8572035-2053AD-71182A-142743ACCCAGUCCUGGCCAUUUU650A-142744AAAAUGGCCAGGACUGGGU8582047-2065AD-71183A-142745GCCAUUUUCUUGCCUGAGA651A-142746UCUCAGGCAAGAAAAUGGC8592058-2076AD-71184A-142747CCUGAGGCUCAAGAGGCCA652A-142748UGGCCUCUUGAGCCUCAGG8602070-2088AD-71185A-142749AAGAGGCCCAGGGAGCAAU653A-142750AUUGCUCCCUGGGCCUCUU8612080-2098AD-71186A-142751GGAGCAAUGGGAGGGGGCU654A-142752AGCCCCCUCCCAUUGCUCC8622091-2109AD-71187A-142753AGGGGGCUCCAUGGAGGAA655A-142754UUCCUCCAUGGAGCCCCCU8632102-2120AD-71188A-142755GGAGGAGGUGUCCCAAGCU656A-142756AGCUUGGGACACCUCCUCC8642114-2132AD-71189A-142757UCCCAAGCUUUGAAUACCA657A-142758UGGUAUUCAAAGCUUGGGA8652124-2142AD-71190A-142759AAUACCCCCAGAGACCUUU658A-142760AAAGGUCUCUGGGGGUAUU8662136-2154AD-71191A-142761AGAGACCUUUUCUCUCCCA659A-142762UGGGAGAGAAAAGGUCUCU8672145-2163AD-71192A-142763UCUCCCAUACCAUCACUGA660A-142764UCAGUGAUGGUAUGGGAGA8682157-2175AD-71193A-142765UCACUGAGUGGCUUGUGAU661A-142766AUCACAAGCCACUCAGUGA8692169-2187AD-71194A-142767GGCUUGUGAUUCUGGGAUA662A-142768UAUCCCAGAAUCACAAGCC8702178-2196AD-71195A-142769UGGGAUGGACCCUCGCAGA663A-142770UCUGCGAGGGUCCAUCCCA8712190-2208AD-71196A-142771UCGCAGCAGGUGCAAGAGA664A-142772UCUCUUGCACCUGCUGCGA8722202-2220AD-71197A-142773UGCAAGAGACAGAGCCCCA665A-142774UGGGGCUCUGUCUCUUGCA8732212-2230AD-71198A-142775AGAGCCCCCAAGCCUCUGA666A-142776UCAGAGGCUUGGGGGCUCU8742222-2240AD-71199A-142777CUCUGCCCCAAGGGGCCCA667A-142778UGGGCCCCUUGGGGCAGAG8752235-2253AD-71200A-142779AAGGGGCCCACAAAGGGGA668A-142780UCCCCUUUGUGGGCCCCUU8762244-2262AD-71201A-142781AAAGGGGAGAAGGGCCAGA669A-142782UCUGGCCCUUCUCCCCUUU8772255-2273AD-71202A-142783GGGCCAGCCCUACAUCUUA670A-142784UAAGAUGUAGGGCUGGCCC8782266-2284AD-71203A-142785AUCUUCAGCUCCCAUAGCA671A-142786UGCUAUGGGAGCUGAAGAU8792279-2297AD-71204A-142787UCCCAUAGCGCUGGCUCAA672A-142788UUGAGCCAGCGCUAUGGGA8802288-2306AD-71205A-142789UGGCUCAGGAAGAAACCCA673A-142790UGGGUUUCUUCCUGAGCCA8812299-2317AD-71206A-142791AACCCCAAGCAGCAUUCAA674A-142792UUGAAUGCUGCUUGGGGUU8822312-2330AD-71207A-142793CAGCAUUCAGCACACCCCA675A-142794UGGGGUGUGCUGAAUGCUG8832321-2339AD-71208A-142795CACCCCAAGGGACAACCCA676A-142796UGGGUUGUCCCUUGGGGUG8842333-2351AD-71209A-142797ACAACCCCAUCAUAUGACA677A-142798UGUCAUAUGAUGGGGUUGU8852344-2362AD-71210A-142801ACCCUCUCCAUGCCCAACA678A-142802UGUUGGGCAUGGAGAGGGU8862367-2385AD-71211A-142803UGCCCAACCUAAGAUUGUA679A-142804UACAAUCUUAGGUUGGGCA8872377-2395AD-71212A-142805AAGAUUGUGUGGGUUUUUU680A-142806AAAAAACCCACACAAUCUU8882387-2405AD-71213A-142807UUUUUUAAUUAAAAAUGUU681A-142808AACAUUUUUAAUUAAAAAA8892400-2418AD-71214A-142809UAAAAAUGUUAAAAGUUUU682A-142810AAAACUUUUAACAUUUUUA8902409-2427AD-71215A-142811AAAGUUUUAAACAUGAAAA683A-142812UUUUCAUGUUUAAAACUUU8912420-2438 TABLE 7GCK Modified SequencesAnti-SenseSenseSEQssenseAntisenseSEQmRNASEQDuplexOligoSequenceIDOligoSequenceIDtargetIDNameName(5′-3′)NO:Name(5′-3′)NO:sequenceNO:AD-71009A-142377CUGCCAGCCUCA892A-142378AGCUGCCUGAGG1100CUGCCAGCCU1308GGCAGCUdTdTCUGGCAGdTdTCAGGCAGCUAD-71010A-142379UCAGGCAGCUCU893A-142380UGGAUGGAGAGC1101UCAGGCAGCU1309CCAUCCAdTdTUGCCUGAdTdTCUCCAUCCAAD-71011A-142381CCAUCCAAGCAG894A-142382UCAACGGCUGCU1102CCAUCCAAGC1310CCGUUGAdTdTUGGAUGGdTdTAGCCGUUGCAD-71012A-142383AGCCGUUGCUGC895A-142384UCCUGUGGCAGC1103AGCCGUUGCU1311CACAGGAdTdTAACGGCUdTdTGCCACAGGCAD-71013A-142385ACAGGCGGGCCU896A-142386UAGCGUAAGGCC1104ACAGGCGGGC1312UACGCUAdTdTCGCCUGUdTdTCUUACGCUCAD-71014A-142387UUACGCUCCAAG897A-142388UUGUAGCCUUGG1105UUACGCUCCA1313GCUACAAdTdTAGCGUAAdTdTAGGCUACAGAD-71015A-142389AAGGCUACAGCA898A-142390UAGCACAUGCUG1106AAGGCUACAG1314UGUGCUAdTdTUAGCCUUdTdTCAUGUGCUAAD-71016A-142391UGUGCUAGGCCU899A-142392UCUGCUGAGGCC1107UGUGCUAGGC1315CAGCAGAdTdTUAGCACAdTdTCUCAGCAGGAD-71017A-142393UCAGCAGGCAGG900A-142394AGAUGCUCCUGC1108UCAGCAGGCA1316AGCAUCUdTdTCUGCUGAdTdTGGAGCAUCUAD-71018A-142395AGCAUCUCUGCC901A-142396UUUGGGAGGCAG1109AGCAUCUCUG1317UCCCAAAdTdTAGAUGCUdTdTCCUCCCAAAAD-71019A-142397CCUCCCAAAGCA902A-142398AGGUAGAUGCUU1110CCUCCCAAAG1318UCUACCUdTdTUGGGAGGdTdTCAUCUACCUAD-71020A-142401UAGCCCCUCGGA903A-142402UCAUCUCUCCGA1111UAGCCCCUCG1319GAGAUGAdTdTGGGGCUAdTdTGAGAGAUGGAD-71021A-142403AGAGAUGGCGAU904A-142404UACAUCCAUCGC1112AGAGAUGGCG1320GGAUGUAdTdTCAUCUCUdTdTAUGGAUGUCAD-71022A-142405UGGAUGUCACAA905A-142406UGGCUCCUUGUG1113UGGAUGUCAC1321GGAGCCAdTdTACAUCCAdTdTAAGGAGCCAAD-71023A-142407AGGAGCCAGGCC906A-142408UUGUCUGGGCCU1114AGGAGCCAGG1322CAGACAAdTdTGGCUCCUdTdTCCCAGACAGAD-71024A-142411ACUCUGGUAGAG907A-142412UGAUCUGCUCUA1115ACUCUGGUAG1323CAGAUCAdTdTCCAGAGUdTdTAGCAGAUCCAD-71025A-142413AGCAGAUCCUGG908A-142414AACUCUGCCAGG1116AGCAGAUCCU1324CAGAGUUdTdTAUCUGCUdTdTGGCAGAGUUAD-71026A-142415CAGAGUUCCAGC909A-142416UCCUGCAGCUGG1117CAGAGUUCCA1325UGCAGGAdTdTAACUCUGdTdTGCUGCAGGAAD-71027A-142417AGCUGCAGGAGG910A-142418AGGUCCUCCUCC1118AGCUGCAGGA1326AGGACCUdTdTUGCAGCUdTdTGGAGGACCUAD-71028A-142419AGGACCUGAAGA911A-142420AUCACCUUCUUC1119AGGACCUGAA1327AGGUGAUdTdTAGGUCCUdTdTGAAGGUGAUAD-71029A-142421AAGGUGAUGAGA912A-142422UCAUCCGUCUCA1120AAGGUGAUGA1328CGGAUGAdTdTUCACCUUdTdTGACGGAUGCAD-71030A-142423CGGAUGCAGAAG913A-142424UCAUCUCCUUCU1121CGGAUGCAGA1329GAGAUGAdTdTGCAUCCGdTdTAGGAGAUGGAD-71031A-142425AAGGAGAUGGAC914A-142426UGCCGCGGUCCA1122AAGGAGAUGG1330CGCGGCAdTdTUCUCCUUdTdTACCGCGGCCAD-71032A-142427CGCGGCCUGAGG915A-142428UCUCCAGCCUCA1123CGCGGCCUGA1331CUGGAGAdTdTGGCCGCGdTdTGGCUGGAGAAD-71033A-142429CUGGAGACCCAU916A-142430UCUCUUCAUGGG1124CUGGAGACCC1332GAAGAGAdTdTUCUCCAGdTdTAUGAAGAGGAD-71034A-142431CAUGAAGAGGCC917A-142432UCACACUGGCCU1125CAUGAAGAGG1333AGUGUGAdTdTCUUCAUGdTdTCCAGUGUGAAD-71035A-142433CAGUGUGAAGAU918A-142434UGGCAGCAUCUU1126CAGUGUGAAG1334GCUGCCAdTdTCACACUGdTdTAUGCUGCCCAD-71036A-142435UGCUGCCCACCU919A-142436UGCACGUAGGUG1127UGCUGCCCAC1335ACGUGCAdTdTGGCAGCAdTdTCUACGUGCGAD-71037A-142437UACGUGCGCUCC920A-142438UUGGGGUGGAGC1128UACGUGCGCU1336ACCCCAAdTdTGCACGUAdTdTCCACCCCAGAD-71038A-142439ACCCCAGAAGGC921A-142440UUUCUGAGCCUU1129ACCCCAGAAG1337UCAGAAAdTdTCUGGGGUdTdTGCUCAGAAGAD-71039A-142441UCAGAAGUCGGG922A-142442UGAAGUCCCCGA1130UCAGAAGUCG1338GACUUCAdTdTCUUCUGAdTdTGGGACUUCCAD-71040A-142443GGGGACUUCCUC923A-142444UCAGGGAGAGGA1131GGGGACUUCC1339UCCCUGAdTdTAGUCCCCdTdTUCUCCCUGGAD-71041A-142445UCCCUGGACCUG924A-142446UGCCACCCAGGU1132UCCCUGGACC1340GGUGGCAdTdTCCAGGGAdTdTUGGGUGGCAAD-71042A-142447UGGGUGGCACUA925A-142448UUGAAGUUAGUG1133UGGGUGGCAC1341ACUUCAAdTdTCCACCCAdTdTUAACUUCAGAD-71043A-142449ACUUCAGGGUGA926A-142450ACCAGCAUCACC1134ACUUCAGGGU1342UGCUGGUdTdTCUGAAGUdTdTGAUGCUGGUAD-71044A-142453AGGUGGGAGAAG927A-142454UCCUCACCUUCU1135AGGUGGGAGA1343GUGAGGAdTdTCCCACCUdTdTAGGUGAGGAAD-71045A-142457CAGUGGAGCGUG928A-142458UGGUCUUCACGC1136CAGUGGAGCG1344AAGACCAdTdTUCCACUGdTdTUGAAGACCAAD-71046A-142461CCAGAUGUACUC929A-142462UGGGAUGGAGUA1137CCAGAUGUAC1345CAUCCCAdTdTCAUCUGGdTdTUCCAUCCCCAD-71047A-142467ACCGGCACUGCU930A-142468UCAUCUCAGCAG1138ACCGGCACUG1346GAGAUGAdTdTUGCCGGUdTdTCUGAGAUGCAD-71048A-142469AGAUGCUCUUCG931A-142470AUGUAGUCGAAG1139AGAUGCUCUU1347ACUACAUdTdTAGCAUCUdTdTCGACUACAUAD-71049A-142471UCGACUACAUCU932A-142472UACUCAGAGAUG1140UCGACUACAU1348CUGAGUAdTdTUAGUCGAdTdTCUCUGAGUGAD-71050A-142473UCUGAGUGCAUC933A-142474AGUCGGAGAUGC1141UCUGAGUGCA1349UCCGACUdTdTACUCAGAdTdTUCUCCGACUAD-71051A-142475UCCGACUUCCUG934A-142476UCUUGUCCAGGA1142UCCGACUUCC1350GACAAGAdTdTAGUCGGAdTdTUGGACAAGCAD-71052A-142477GACAAGCAUCAG935A-142478GUUUCAUCUGAU1143GACAAGCAUC1351AUGAAACdTdTGCUUGUCdTdTAGAUGAAACAD-71053A-142479AGAUGAAACACA936A-142480AGCUUCUUGUGU1144AGAUGAAACA1352AGAAGCUdTdTUUCAUCUdTdTCAAGAAGCUAD-71054A-142481AGAAGCUGCCCC937A-142482AAGCCCAGGGGC1145AGAAGCUGCC1353UGGGCUUdTdTAGCUUCUdTdTCCUGGGCUUAD-71055A-142483CCUGGGCUUCAC938A-142484UGAGAAGGUGAA1146CCUGGGCUUC1354CUUCUCAdTdTGCCCAGGdTdTACCUUCUCCAD-71056A-142485ACCUUCUCCUUU939A-142486UCACAGGAAAGG1147ACCUUCUCCU1355CCUGUGAdTdTAGAAGGUdTdTUUCCUGUGAAD-71057A-142487CUGUGAGGCACG940A-142488AUGUCUUCGUGC1148CUGUGAGGCA1356AAGACAUdTdTCUCACAGdTdTCGAAGACAUAD-71058A-142489GAAGACAUCGAU941A-142490UGCCCUUAUCGA1149GAAGACAUCG1357AAGGGCAdTdTUGUCUUCdTdTAUAAGGGCAAD-71059A-142491GAUAAGGGCAUC942A-142492UGAGAAGGAUGC1150GAUAAGGGCA1358CUUCUCAdTdTCCUUAUCdTdTUCCUUCUCAAD-71060A-142493UUCUCAACUGGA943A-142494UCCUUGGUCCAG1151UUCUCAACUG1359CCAAGGAdTdTUUGAGAAdTdTGACCAAGGGAD-71061A-142495ACCAAGGGCUUC944A-142496AGGCCUUGAAGC1152ACCAAGGGCU1360AAGGCCUdTdTCCUUGGUdTdTUCAAGGCCUAD-71062A-142497CAAGGCCUCAGG945A-142498UUCUGCUCCUGA1153CAAGGCCUCA1361AGCAGAAdTdTGGCCUUGdTdTGGAGCAGAAAD-71063A-142499AGGAGCAGAAGG946A-142500AUUGUUCCCUUC1154AGGAGCAGAA1362GAACAAUdTdTUGCUCCUdTdTGGGAACAAUAD-71064A-142501AACAAUGUCGUG947A-142502UAAGCCCCACGA1155AACAAUGUCG1363GGGCUUAdTdTCAUUGUUdTdTUGGGGCUUCAD-71065A-142503UGGGGCUUCUGC948A-142504UCGUCUCGCAGA1156UGGGGCUUCU1364GAGACGAdTdTAGCCCCAdTdTGCGAGACGCAD-71066A-142505CGAGACGCUAUC949A-142506UCCGUUUGAUAG1157CGAGACGCUA1365AAACGGAdTdTCGUCUCGdTdTUCAAACGGAAD-71067A-142507AAACGGAGAGGG950A-142508UAAAGUCCCCUC1158AAACGGAGAG1366GACUUUAdTdTUCCGUUUdTdTGGGACUUUGAD-71068A-142509GGGACUUUGAAA951A-142510ACAUCCAUUUCA1159GGGACUUUGA1367UGGAUGUdTdTAAGUCCCdTdTAAUGGAUGUAD-71069A-142513GCAAUGGUGAAU952A-142514UCGUGUCAUUCA1160GCAAUGGUGA1368GACACGAdTdTCCAUUGCdTdTAUGACACGGAD-71070A-142515AAUGACACGGUG953A-142516UCGUGGCCACCG1161AAUGACACGG1369GCCACGAdTdTUGUCAUUdTdTUGGCCACGAAD-71071A-142517GCCACGAUGAUC954A-142518AGCAGGAGAUCA1162GCCACGAUGA1370UCCUGCUdTdTUCGUGGCdTdTUCUCCUGCUAD-71072A-142519UCCUGCUACUAC955A-142520UGUCUUCGUAGU1163UCCUGCUACU1371GAAGACAdTdTAGCAGGAdTdTACGAAGACCAD-71073A-142523AGUGCGAGGUCG956A-142524AUCAUGCCGACC1164AGUGCGAGGU1372GCAUGAUdTdTUCGCACUdTdTCGGCAUGAUAD-71074A-142525GGCAUGAUCGUG957A-142526UCGUGCCCACGA1165GGCAUGAUCG1373GGCACGAdTdTUCAUGCCdTdTUGGGCACGGAD-71075A-142527GUGGGCACGGGC958A-142528UAUUGCAGCCCG1166GUGGGCACGG1374UGCAAUAdTdTUGCCCACdTdTGCUGCAAUGAD-71076A-142529UGCAAUGCCUGC959A-142530UCAUGUAGCAGG1167UGCAAUGCCU1375UACAUGAdTdTCAUUGCAdTdTGCUACAUGGAD-71077A-142531UACAUGGAGGAG960A-142532UCUGCAUCUCCU1168UACAUGGAGG1376AUGCAGAdTdTCCAUGUAdTdTAGAUGCAGAAD-71078A-142533AGAUGCAGAAUG961A-142534AGCUCCACAUUC1169AGAUGCAGAA1377UGGAGCUdTdTUGCAUCUdTdTUGUGGAGCUAD-71079A-142535UGUGGAGCUGGU962A-142536UCCCUCCACCAG1170UGUGGAGCUG1378GGAGGGAdTdTCUCCACAdTdTGUGGAGGGGAD-71080A-142537UGGAGGGGGACG963A-142538UGGCCCUCGUCC1171UGGAGGGGGA1379AGGGCCAdTdTCCCUCCAdTdTCGAGGGCCGAD-71081A-142539GAGGGCCGCAUG964A-142540UGACGCACAUGC1172GAGGGCCGCA1380UGCGUCAdTdTGGCCCUCdTdTUGUGCGUCAAD-71082A-142541UGCGUCAAUACC965A-142542UCCACUCGGUAU1173UGCGUCAAUA1381GAGUGGAdTdTUGACGCAdTdTCCGAGUGGGAD-71083A-142543CGAGUGGGGCGC966A-142544UCCGAAGGCGCC1174CGAGUGGGGC1382CUUCGGAdTdTCCACUCGdTdTGCCUUCGGGAD-71084A-142545GCCUUCGGGGAC967A-142546UGCCGGAGUCCC1175GCCUUCGGGG1383UCCGGCAdTdTCGAAGGCdTdTACUCCGGCGAD-71085A-142547UCCGGCGAGCUG968A-142548ACUCGUCCAGCU1176UCCGGCGAGC1384GACGAGUdTdTCGCCGGAdTdTUGGACGAGUAD-71086A-142549GACGAGUUCCUG969A-142550ACUCCAGCAGGA1177GACGAGUUCC1385CUGGAGUdTdTACUCGUCdTdTUGCUGGAGUAD-71087A-142551UGCUGGAGUAUG970A-142552AGGCGGUCAUAC1178UGCUGGAGUA1386ACCGCCUdTdTUCCAGCAdTdTUGACCGCCUAD-71088A-142553GACCGCCUGGUG971A-142554UCUCGUCCACCA1179GACCGCCUGG1387GACGAGAdTdTGGCGGUCdTdTUGGACGAGAAD-71089A-142555GGACGAGAGCUC972A-142556GUUUGCAGAGCU1180GGACGAGAGC1388UGCAAACdTdTCUCGUCCdTdTUCUGCAAACAD-71090A-142557UCUGCAAACCCC973A-142558UCUGACCGGGGU1181UCUGCAAACC1389GGUCAGAdTdTUUGCAGAdTdTCCGGUCAGCAD-71091A-142559GGUCAGCAGCUG974A-142560UCUCAUACAGCU1182GGUCAGCAGC1390UAUGAGAdTdTGCUGACCdTdTUGUAUGAGAAD-71092A-142561UAUGAGAAGCUC975A-142562UACCUAUGAGCU1183UAUGAGAAGC1391AUAGGUAdTdTUCUCAUAdTdTUCAUAGGUGAD-71093A-142563UCAUAGGUGGCA976A-142564AUGUACUUGCCA1184UCAUAGGUGG1392AGUACAUdTdTCCUAUGAdTdTCAAGUACAUAD-71094A-142565AAGUACAUGGGC977A-142566UCAGCUCGCCCA1185AAGUACAUGG1393GAGCUGAdTdTUGUACUUdTdTGCGAGCUGGAD-71095A-142567GCGAGCUGGUGC978A-142568ACAAGCCGCACC1186GCGAGCUGGU1394GGCUUGUdTdTAGCUCGCdTdTGCGGCUUGUAD-71096A-142569GGCUUGUGCUGC979A-142570AGCCUGAGCAGC1187GGCUUGUGCU1395UCAGGCUdTdTACAAGCCdTdTGCUCAGGCUAD-71097A-142571UCAGGCUCGUGG980A-142572UUUUCGUCCACG1188UCAGGCUCGU1396ACGAAAAdTdTAGCCUGAdTdTGGACGAAAAAD-69448A-139832UGGACGAAAACC981A-139833AAGAGCAGGUUU1189UGGACGAAAA1397UGCUCUUdTdTUCGUCCAdTdTCCUGCUCUUAD-71098A-142573UGCUCUUCCACG982A-142574UCCUCCCCGUGG1190UGCUCUUCCA1398GGGAGGAdTdTAAGAGCAdTdTCGGGGAGGCAD-71099A-142575GGGAGGCCUCCG983A-142576AGCUGCUCGGAG1191GGGAGGCCUC1399AGCAGCUdTdTGCCUCCCdTdTCGAGCAGCUAD-71100A-142577CGAGCAGCUGCG984A-142578UCGUGUGCGCAG1192CGAGCAGCUG1400CACACGAdTdTCUGCUCGdTdTCGCACACGCAD-71101A-142581AGCCUUCGAGAC985A-142582UAAGCGCGUCUC1193AGCCUUCGAG1401GCGCUUAdTdTGAAGGCUdTdTACGCGCUUCAD-71102A-142583CGCUUCGUGUCG986A-142584UCACCUGCGACA1194CGCUUCGUGU1402CAGGUGAdTdTCGAAGCGdTdTCGCAGGUGGAD-71103A-142585UCGCAGGUGGAG987A-142586UGUCGCUCUCCA1195UCGCAGGUGG1403AGCGACAdTdTCCUGCGAdTdTAGAGCGACAAD-71104A-142587AGCGACACGGGC988A-142588UGCGGUCGCCCG1196AGCGACACGG1404GACCGCAdTdTUGUCGCUdTdTGCGACCGCAAD-71105A-142589CGACCGCAAGCA989A-142590UUAGAUCUGCUU1197CGACCGCAAG1405GAUCUAAdTdTGCGGUCGdTdTCAGAUCUACAD-71106A-142591CAGAUCUACAAC990A-142592UCAGGAUGUUGU1198CAGAUCUACA1406AUCCUGAdTdTAGAUCUGdTdTACAUCCUGAAD-71107A-142593UCCUGAGCACGC991A-142594AGCCCCAGCGUG1199UCCUGAGCAC1407UGGGGCUdTdTCUCAGGAdTdTGCUGGGGCUAD-71108A-142595CUGGGGCUGCGA992A-142596UCGAGGGUCGCA1200CUGGGGCUGC1408CCCUCGAdTdTGCCCCAGdTdTGACCCUCGAAD-71109A-142597CGACCCUCGACC993A-142598AGUCGGUGGUCG1201CGACCCUCGA1409ACCGACUdTdTAGGGUCGdTdTCCACCGACUAD-71110A-142599CACCGACUGCGA994A-142600UACGAUGUCGCA1202CACCGACUGC1410CAUCGUAdTdTGUCGGUGdTdTGACAUCGUGAD-71111A-142601CAUCGUGCGCCG995A-142602UCAGGCGCGGCG1203CAUCGUGCGC1411CGCCUGAdTdTCACGAUGdTdTCGCGCCUGCAD-71112A-142603CGCGCCUGCGAG996A-142604ACACGCUCUCGC1204CGCGCCUGCG1412AGCGUGUdTdTAGGCGCGdTdTAGAGCGUGUAD-71113A-142605AGCGUGUCUACG997A-142606UAGCGCGCGUAG1205AGCGUGUCUA1413CGCGCUAdTdTACACGCUdTdTCGCGCGCUGAD-71114A-142607CGCGCUGCGCAC998A-142608AGCACAUGUGCG1206CGCGCUGCGC1414AUGUGCUdTdTCAGCGCGdTdTACAUGUGCUAD-71115A-142609ACAUGUGCUCGG999A-142610AGCCCCGCCGAG1207ACAUGUGCUC1415CGGGGCUdTdTCACAUGUdTdTGGCGGGGCUAD-71116A-142611CGGGGCUGGCGG1000A-142612AUGACGCCCGCC1208CGGGGCUGGC1416GCGUCAUdTdTAGCCCCGdTdTGGGCGUCAUAD-71117A-142613CGGGCGUCAUCA1001A-142614AUGCGGUUGAUG1209CGGGCGUCAU1417ACCGCAUdTdTACGCCCGdTdTCAACCGCAUAD-71118A-142615AACCGCAUGCGC1002A-142616UGCUCUCGCGCA1210AACCGCAUGC1418GAGAGCAdTdTUGCGGUUdTdTGCGAGAGCCAD-71119A-142617AGAGCCGCAGCG1003A-142618ACGUCCUCGCUG1211AGAGCCGCAG1419AGGACGUdTdTCGGCUCUdTdTCGAGGACGUAD-71120A-142619CGAGGACGUAAU1004A-142620UAUGCGCAUUAC1212CGAGGACGUA1420GCGCAUAdTdTGUCCUCGdTdTAUGCGCAUCAD-71121A-142621UGCGCAUCACUG1005A-142622ACGCCCACAGUG1213UGCGCAUCAC1421UGGGCGUdTdTAUGCGCAdTdTUGUGGGCGUAD-71122A-142623UGGGCGUGGAUG1006A-142624ACGGAGCCAUCC1214UGGGCGUGGA1422GCUCCGUdTdTACGCCCAdTdTUGGCUCCGUAD-71123A-142625UGGCUCCGUGUA1007A-142626UAGCUUGUACAC1215UGGCUCCGUG1423CAAGCUAdTdTGGAGCCAdTdTUACAAGCUGAD-71124A-142627UACAAGCUGCAC1008A-142628AGCUGGGGUGCA1216UACAAGCUGC1424CCCAGCUdTdTGCUUGUAdTdTACCCCAGCUAD-71125A-142629CCAGCUUCAAGG1009A-142630AACCGCUCCUUG1217CCAGCUUCAA1425AGCGGUUdTdTAAGCUGGdTdTGGAGCGGUUAD-71126A-142631AGGAGCGGUUCC1010A-142632UUGGCAUGGAAC1218AGGAGCGGUU1426AUGCCAAdTdTCGCUCCUdTdTCCAUGCCAGAD-71127A-142633AUGCCAGCGUGC1011A-142634AGCCUGCGCACG1219AUGCCAGCGU1427GCAGGCUdTdTCUGGCAUdTdTGCGCAGGCUAD-71128A-142635CGCAGGCUGACG1012A-142636AGCUGGGCGUCA1220CGCAGGCUGA1428CCCAGCUdTdTGCCUGCGdTdTCGCCCAGCUAD-71129A-142637CCCAGCUGCGAG1013A-142638AGGUGAUCUCGC1221CCCAGCUGCG1429AUCACCUdTdTAGCUGGGdTdTAGAUCACCUAD-71130A-142639GAGAUCACCUUC1014A-142640ACUCGAUGAAGG1222GAGAUCACCU1430AUCGAGUdTdTUGAUCUCdTdTUCAUCGAGUAD-71131A-142641AUCGAGUCGGAG1015A-142642UGCCCUCCUCCG1223AUCGAGUCGG1431GAGGGCAdTdTACUCGAUdTdTAGGAGGGCAAD-71132A-142643AGGAGGGCAGUG1016A-142644UCCCGGCCACUG1224AGGAGGGCAG1432GCCGGGAdTdTCCCUCCUdTdTUGGCCGGGGAD-71133A-142645CCGGGGCGCGGC1017A-142646UACCAGGGCCGC1225CCGGGGCGCG1433CCUGGUAdTdTGCCCCGGdTdTGCCCUGGUCAD-71134A-142647CCCUGGUCUCGG1018A-142648UCCACCGCCGAG1226CCCUGGUCUC1434CGGUGGAdTdTACCAGGGdTdTGGCGGUGGCAD-71135A-142649GCGGUGGCCUGU1019A-142650UCUUCUUACAGG1227GCGGUGGCCU1435AAGAAGAdTdTCCACCGCdTdTGUAAGAAGGAD-71136A-142651UAAGAAGGCCUG1020A-142652UAGCAUACAGGC1228UAAGAAGGCC1436UAUGCUAdTdTCUUCUUAdTdTUGUAUGCUGAD-71137A-142653CUGUAUGCUGGG1021A-142654UCACUGGCCCAG1229CUGUAUGCUG1437CCAGUGAdTdTCAUACAGdTdTGGCCAGUGAAD-71138A-142655CAGUGAGAGCAG1022A-142656UCGGCCACUGCU1230CAGUGAGAGC1438UGGCCGAdTdTCUCACUGdTdTAGUGGCCGCAD-71139A-142657CAGUGGCCGCAA1023A-142658UCUGCGCUUGCG1231CAGUGGCCGC1439GCGCAGAdTdTGCCACUGdTdTAAGCGCAGGAD-71140A-142659AGCGCAGGGAGG1024A-142660UUGGCAUCCUCC1232AGCGCAGGGA1440AUGCCAAdTdTCUGCGCUdTdTGGAUGCCACAD-71141A-142661UGCCACAGCCCC1025A-142662UUGCUGUGGGGC1233UGCCACAGCC1441ACAGCAAdTdTUGUGGCAdTdTCCACAGCACAD-71142A-142663CACAGCACCCAG1026A-142664AUGGAGCCUGGG1234CACAGCACCC1442GCUCCAUdTdTUGCUGUGdTdTAGGCUCCAUAD-71143A-142665AGGCUCCAUGGG1027A-142666UCACUUCCCCAU1235AGGCUCCAUG1443GAAGUGAdTdTGGAGCCUdTdTGGGAAGUGCAD-71144A-142667GGAAGUGCUCCC1028A-142668ACGUGUGGGGAG1236GGAAGUGCUC1444CACACGUdTdTCACUUCCdTdTCCCACACGUAD-71145A-142669CCACACGUGCUC1029A-142670AGGCUGCGAGCA1237CCACACGUGC1445GCAGCCUdTdTCGUGUGGdTdTUCGCAGCCUAD-71146A-142671UCGCAGCCUGGC1030A-142672UUGCCCCGCCAG1238UCGCAGCCUG1446GGGGCAAdTdTGCUGCGAdTdTGCGGGGCAGAD-71147A-142673CGGGGCAGGAGG1031A-142674UGCCAGGCCUCC1239CGGGGCAGGA1447CCUGGCAdTdTUGCCCCGdTdTGGCCUGGCCAD-71148A-142675CCUGGCCUUGUC1032A-142676UGGUCCUGACAA1240CCUGGCCUUG1448AGGACCAdTdTGGCCAGGdTdTUCAGGACCCAD-71149A-142677CAGGACCCAGGC1033A-142678UCAGGCGGCCUG1241CAGGACCCAG1449CGCCUGAdTdTGGUCCUGdTdTGCCGCCUGCAD-71150A-142679CCGCCUGCCAUA1034A-142680UCAGCGGUAUGG1242CCGCCUGCCA1450CCGCUGAdTdTCAGGCGGdTdTUACCGCUGGAD-71151A-142681UACCGCUGGGGA1035A-142682UCUCUGUUCCCC1243UACCGCUGGG1451ACAGAGAdTdTAGCGGUAdTdTGAACAGAGCAD-71152A-142683AACAGAGCGGGC1036A-142684UGAAGAGGCCCG1244AACAGAGCGG1452CUCUUCAdTdTCUCUGUUdTdTGCCUCUUCCAD-71153A-142685CUCUUCCCUCAG1037A-142686UGAAAAACUGAG1245CUCUUCCCUC1453UUUUUCAdTdTGGAAGAGdTdTAGUUUUUCGAD-71154A-142687UUUUUCGGUGGG1038A-142688UGGCUGUCCCAC1246UUUUUCGGUG1454ACAGCCAdTdTCGAAAAAdTdTGGACAGCCCAD-71155A-142689GGGACAGCCCCA1039A-142690AGGGCCCUGGGG1247GGGACAGCCC1455GGGCCCUdTdTCUGUCCCdTdTCAGGGCCCUAD-71156A-142691AGGGCCCUAACG1040A-142692UCACCCCCGUUA1248AGGGCCCUAA1456GGGGUGAdTdTGGGCCCUdTdTCGGGGGUGCAD-71157A-142693GGGUGCGGCAGG1041A-142694UCCUGCUCCUGC1249GGGUGCGGCA1457AGCAGGAdTdTCGCACCCdTdTGGAGCAGGAAD-71158A-142695AGGAGCAGGAAC1042A-142696AGUCUCUGUUCC1250AGGAGCAGGA1458AGAGACUdTdTUGCUCCUdTdTACAGAGACUAD-71159A-142697AGAGACUCUGGA1043A-142698UGGGGCUUCCAG1251AGAGACUCUG1459AGCCCCAdTdTAGUCUCUdTdTGAAGCCCCCAD-71160A-142699AAGCCCCCCACC1044A-142700UGAGAAAGGUGG1252AAGCCCCCCA1460UUUCUCAdTdTGGGGCUUdTdTCCUUUCUCGAD-71161A-142701UUUCUCGCUGGA1045A-142702AAUUGAUUCCAG1253UUUCUCGCUG1461AUCAAUUdTdTCGAGAAAdTdTGAAUCAAUUAD-71162A-142703AAUCAAUUUCCC1046A-142704UCCUUCUGGGAA1254AAUCAAUUUC1462AGAAGGAdTdTAUUGAUUdTdTCCAGAAGGGAD-71163A-142705CCCAGAAGGGAG1047A-142706UGAGCAACUCCC1255CCCAGAAGGG1463UUGCUCAdTdTUUCUGGGdTdTAGUUGCUCAAD-71164A-142707UUGCUCACUCAG1048A-142708UAAAGUCCUGAG1256UUGCUCACUC1464GACUUUAdTdTUGAGCAAdTdTAGGACUUUGAD-71165A-142709AGGACUUUGAUG1049A-142710UGAAAUGCAUCA1257AGGACUUUGA1465CAUUUCAdTdTAAGUCCUdTdTUGCAUUUCCAD-71166A-142711AUUUCCACACUG1050A-142712UCUCUGACAGUG1258AUUUCCACAC1466UCAGAGAdTdTUGGAAAUdTdTUGUCAGAGCAD-71167A-142713UGUCAGAGCUGU1051A-142714UAGGCCAACAGC1259UGUCAGAGCU1467UGGCCUAdTdTUCUGACAdTdTGUUGGCCUCAD-71168A-142715UUGGCCUCGCCU1052A-142716UGGGCCCAGGCG1260UUGGCCUCGC1468GGGCCCAdTdTAGGCCAAdTdTCUGGGCCCAAD-71169A-142717CUGGGCCCAGGC1053A-142718UCCCAGAGCCUG1261CUGGGCCCAG1469UCUGGGAdTdTGGCCCAGdTdTGCUCUGGGAAD-71170A-142719CUCUGGGAAGGG1054A-142720AGGGCACCCCUU1262CUCUGGGAAG1470GUGCCCUdTdTCCCAGAGdTdTGGGUGCCCUAD-71171A-142721UGCCCUCUGGAU1055A-142722UAGCAGGAUCCA1263UGCCCUCUGG1471CCUGCUAdTdTGAGGGCAdTdTAUCCUGCUGAD-71172A-142723UCCUGCUGUGGC1056A-142724AAGUGAGGCCAC1264UCCUGCUGUG1472CUCACUUdTdTAGCAGGAdTdTGCCUCACUUAD-71173A-142725CCUCACUUCCCU1057A-142726AGUUCCCAGGGA1265CCUCACUUCC1473GGGAACUdTdTAGUGAGGdTdTCUGGGAACUAD-71174A-142727CUGGGAACUCAU1058A-142728ACACAGGAUGAG1266CUGGGAACUC1474CCUGUGUdTdTUUCCCAGdTdTAUCCUGUGUAD-71175A-142729CCUGUGUGGGGA1059A-142730AGCUGCCUCCCC1267CCUGUGUGGG1475GGCAGCUdTdTACACAGGdTdTGAGGCAGCUAD-71176A-142731GGAGGCAGCUCC1060A-142732AGCUGUUGGAGC1268GGAGGCAGCU1476AACAGCUdTdTUGCCUCCdTdTCCAACAGCUAD-71177A-142733CAACAGCUUGAC1061A-142734AGGUCUGGUCAA1269CAACAGCUUG1477CAGACCUdTdTGCUGUUGdTdTACCAGACCUAD-71178A-142735CCAGACCUAGAC1062A-142736UGCCCAGGUCUA1270CCAGACCUAG1478CUGGGCAdTdTGGUCUGGdTdTACCUGGGCCAD-71179A-142737CUGGGCCAAAAG1063A-142738UGCUGCCCUUUU1271CUGGGCCAAA1479GGCAGCAdTdTGGCCCAGdTdTAGGGCAGCCAD-71180A-142739AGGGCAGCCAGG1064A-142740AGCAGCCCCUGG1272AGGGCAGCCA1480GGCUGCUdTdTCUGCCCUdTdTGGGGCUGCUAD-71181A-142741GGGCUGCUCAUC1065A-142742ACUGGGUGAUGA1273GGGCUGCUCA1481ACCCAGUdTdTGCAGCCCdTdTUCACCCAGUAD-71182A-142743ACCCAGUCCUGG1066A-142744AAAAUGGCCAGG1274ACCCAGUCCU1482CCAUUUUdTdTACUGGGUdTdTGGCCAUUUUAD-71183A-142745GCCAUUUUCUUG1067A-142746UCUCAGGCAAGA1275GCCAUUUUCU1483CCUGAGAdTdTAAAUGGCdTdTUGCCUGAGGAD-71184A-142747CCUGAGGCUCAA1068A-142748UGGCCUCUUGAG1276CCUGAGGCUC1484GAGGCCAdTdTCCUCAGGdTdTAAGAGGCCCAD-71185A-142749AAGAGGCCCAGG1069A-142750AUUGCUCCCUGG1277AAGAGGCCCA1485GAGCAAUdTdTGCCUCUUdTdTGGGAGCAAUAD-71186A-142751GGAGCAAUGGGA1070A-142752AGCCCCCUCCCA1278GGAGCAAUGG1486GGGGGCUdTdTUUGCUCCdTdTGAGGGGGCUAD-71187A-142753AGGGGGCUCCAU1071A-142754UUCCUCCAUGGA1279AGGGGGCUCC1487GGAGGAAdTdTGCCCCCUdTdTAUGGAGGAGAD-71188A-142755GGAGGAGGUGUC1072A-142756AGCUUGGGACAC1280GGAGGAGGUG1488CCAAGCUdTdTCUCCUCCdTdTUCCCAAGCUAD-71189A-142757UCCCAAGCUUUG1073A-142758UGGUAUUCAAAG1281UCCCAAGCUU1489AAUACCAdTdTCUUGGGAdTdTUGAAUACCCAD-71190A-142759AAUACCCCCAGA1074A-142760AAAGGUCUCUGG1282AAUACCCCCA1490GACCUUUdTdTGGGUAUUdTdTGAGACCUUUAD-71191A-142761AGAGACCUUUUC1075A-142762UGGGAGAGAAAA1283AGAGACCUUU1491UCUCCCAdTdTGGUCUCUdTdTUCUCUCCCAAD-71192A-142763UCUCCCAUACCA1076A-142764UCAGUGAUGGUA1284UCUCCCAUAC1492UCACUGAdTdTUGGGAGAdTdTCAUCACUGAAD-71193A-142765UCACUGAGUGGC1077A-142766AUCACAAGCCAC1285UCACUGAGUG1493UUGUGAUdTdTUCAGUGAdTdTGCUUGUGAUAD-71194A-142767GGCUUGUGAUUC1078A-142768UAUCCCAGAAUC1286GGCUUGUGAU1494UGGGAUAdTdTACAAGCCdTdTUCUGGGAUGAD-71195A-142769UGGGAUGGACCC1079A-142770UCUGCGAGGGUC1287UGGGAUGGAC1495UCGCAGAdTdTCAUCCCAdTdTCCUCGCAGCAD-71196A-142771UCGCAGCAGGUG1080A-142772UCUCUUGCACCU1288UCGCAGCAGG1496CAAGAGAdTdTGCUGCGAdTdTUGCAAGAGAAD-71197A-142773UGCAAGAGACAG1081A-142774UGGGGCUCUGUC1289UGCAAGAGAC1497AGCCCCAdTdTUCUUGCAdTdTAGAGCCCCCAD-71198A-142775AGAGCCCCCAAG1082A-142776UCAGAGGCUUGG1290AGAGCCCCCA1498CCUCUGAdTdTGGGCUCUdTdTAGCCUCUGCAD-71199A-142777CUCUGCCCCAAG1083A-142778UGGGCCCCUUGG1291CUCUGCCCCA1499GGGCCCAdTdTGGCAGAGdTdTAGGGGCCCAAD-71200A-142779AAGGGGCCCACA1084A-142780UCCCCUUUGUGG1292AAGGGGCCCA1500AAGGGGAdTdTGCCCCUUdTdTCAAAGGGGAAD-71201A-142781AAAGGGGAGAAG1085A-142782UCUGGCCCUUCU1293AAAGGGGAGA1501GGCCAGAdTdTCCCCUUUdTdTAGGGCCAGCAD-71202A-142783GGGCCAGCCCUA1086A-142784UAAGAUGUAGGG1294GGGCCAGCCC1502CAUCUUAdTdTCUGGCCCdTdTUACAUCUUCAD-71203A-142785AUCUUCAGCUCC1087A-142786UGCUAUGGGAGC1295AUCUUCAGCU1503CAUAGCAdTdTUGAAGAUdTdTCCCAUAGCGAD-71204A-142787UCCCAUAGCGCU1088A-142788UUGAGCCAGCGC1296UCCCAUAGCG1504GGCUCAAdTdTUAUGGGAdTdTCUGGCUCAGAD-71205A-142789UGGCUCAGGAAG1089A-142790UGGGUUUCUUCC1297UGGCUCAGGA1505AAACCCAdTdTUGAGCCAdTdTAGAAACCCCAD-71206A-142791AACCCCAAGCAG1090A-142792UUGAAUGCUGCU1298AACCCCAAGC1506CAUUCAAdTdTUGGGGUUdTdTAGCAUUCAGAD-71207A-142793CAGCAUUCAGCA1091A-142794UGGGGUGUGCUG1299CAGCAUUCAG1507CACCCCAdTdTAAUGCUGdTdTCACACCCCAAD-71208A-142795CACCCCAAGGGA1092A-142796UGGGUUGUCCCU1300CACCCCAAGG1508CAACCCAdTdTUGGGGUGdTdTGACAACCCCAD-71209A-142797ACAACCCCAUCA1093A-142798UGUCAUAUGAUG1301ACAACCCCAU1509UAUGACAdTdTGGGUUGUdTdTCAUAUGACAAD-71210A-142801ACCCUCUCCAUG1094A-142802UGUUGGGCAUGG1302ACCCUCUCCA1510CCCAACAdTdTAGAGGGUdTdTUGCCCAACCAD-71211A-142803UGCCCAACCUAA1095A-142804UACAAUCUUAGG1303UGCCCAACCU1511GAUUGUAdTdTUUGGGCAdTdTAAGAUUGUGAD-71212A-142805AAGAUUGUGUGG1096A-142806AAAAAACCCACA1304AAGAUUGUGU1512GUUUUUUdTdTCAAUCUUdTdTGGGUUUUUUAD-71213A-142807UUUUUUAAUUAA1097A-142808AACAUUUUUAAU1305UUUUUUAAUU1513AAAUGUUdTdTUAAAAAAdTdTAAAAAUGUUAD-71214A-142809UAAAAAUGUUAA1098A-142810AAAACUUUUAAC1306UAAAAAUGUU1514AAGUUUUdTdTAUUUUUAdTdTAAAAGUUUUAD-71215A-142811AAAGUUUUAAAC1099A-142812UUUUCAUGUUUA1307AAAGUUUUAA1515AUGAAAAdTdTAAACUUUdTdTACAUGAAAA TABLE 8GCK Single Dose Screen in Primary Cynomolgus HepatocytesDuplexID20 nM_AVG20 nM_STDEVAD-7100991.78.2AD-7101085.511.8AD-71011101.419.6AD-7101296.320.8AD-71013102.821.7AD-71014105.347.3AD-7101532.66.6AD-7101698.719.1AD-7101764.116.4AD-7101845.912.8AD-7101939.68.5AD-7102089.223.9AD-7102160.47.7AD-71022100.812.7AD-7102397.433AD-7102460.618.8AD-7102531.19AD-7102633.610.5AD-7102746.314.6AD-7102835.69.6AD-7102935.615.9AD-7103030.47.9AD-71031107.229AD-7103278.415.7AD-7103371.813.9AD-7103439.714.3AD-7103546.811.4AD-7103677.621.3AD-7103737.415.2AD-7103848.819.6AD-7103970.19.5AD-7104065.916.2AD-7104194.818.6AD-71042108.424.2AD-7104335.812.2AD-7104446.610.9AD-7104539.49.5AD-7104635.33.9AD-710478224.4AD-7104839.810.1AD-7104995.36.8AD-71050151.222.4AD-710515413.5AD-7105248.78.1AD-710534410.7AD-7105453.48.8AD-7105539.89.4AD-7105651.834AD-71057717.1AD-7105838.93.6AD-7105978.117.8AD-710605414.4AD-71061108.427.2AD-7106269.46.7AD-7106335.111AD-7106453.113.8AD-710659411.6AD-71066149.213.3AD-7106750.815.2AD-71068113.423.7AD-7106944.96.4AD-71070112.324.3AD-7107132.75.3AD-7107240.110.2AD-710735312.5AD-71074135.425.3AD-71075100.831.5AD-7107635.67.3AD-7107726.94.7AD-7107854.411.8AD-71079486.2AD-7108096.16AD-71081105.47.9AD-7108212720AD-71083117.233.2AD-71084124.420.7AD-7108578.56.6AD-7108630.212.7AD-7108744.22AD-7108892.921.9AD-7108936.113.7AD-71090403.6AD-71091583.3AD-7109262.314.9AD-7109358.618.2AD-7109482.722.9AD-71095116.827.6AD-7109640.311.8AD-7109726.66.9AD-6944834.99.3AD-7109850.47.4AD-7109950.718.4AD-7110023.81.7AD-7110170.718.1AD-7110237.25.5AD-7110398.317.6AD-7110478.822.8AD-7110529.66.1AD-7110626.18.1AD-7110754.49.2AD-71108143.228.5AD-71109116.615.9AD-71110107.321.5AD-7111136.89AD-7111276.419.7AD-7111371.712.9AD-7111494.822.5AD-7111543.312.3AD-7111658.77.9AD-7111726.97.1AD-711187415.8AD-7111933.812.9AD-7112030.54.3AD-7112151.87.6AD-7112271.820.4AD-7112345.612.4AD-71124156.46.6AD-7112555.46.2AD-71126102.38.5AD-71127107.818.2AD-7112895.619AD-7112951.616.7AD-7113028.410.3AD-71131496.5AD-71132127.725.6AD-71133157.517.4AD-711343811.1AD-7113562.67.6AD-71136141.620.3AD-7113755.96.4AD-7113837.95AD-71139125.827.6AD-7114041.82.5AD-7114132.86.7AD-7114240.411.5AD-71143177.427.3AD-7114453.59.1AD-7114541.327AD-71146105.913.9AD-7114798.227.5AD-7114873.68.9AD-7114915924.3AD-71150157.931.1AD-71151131.44.3AD-7115298.225.2AD-7115366.623AD-71154134.811.9AD-7115552.84.2AD-71156111.443.9AD-7115737.67.7AD-7115883.316.9AD-7115933.77.3AD-7116044.27.2AD-71161159.128.3AD-71162137.220.9AD-7116323.45.4AD-7116427.72.6AD-7116538.18AD-7116646.411.4AD-7116753.117.6AD-71168130.314AD-7116995.824.4AD-71170108.815.4AD-7117157.65.3AD-711727526.8AD-7117387.324AD-7117462.724.2AD-7117583.625.8AD-7117626.73.5AD-71177314.7AD-71178114.620AD-71179101.925.9AD-7118011412.4AD-7118138.210.1AD-7118232.33.2AD-7118349.97.4AD-7118460.66.7AD-71185303AD-7118655.716.3AD-7118772.815.2AD-7118832.12.2AD-7118976.413.9AD-7119034.29.1AD-7119151.79.5AD-7119287.58.3AD-71193110.220.9AD-7119456.912.6AD-7119563.125.2AD-7119638.110.3AD-71197349.8AD-71198108.112AD-71199117.911.5AD-7120050.112.3AD-7120138.25.8AD-7120273.93.6AD-71203110.39.2AD-7120414011AD-7120535.84.5AD-712062812.1AD-7120722.411AD-7120854.610.9AD-7120940.614.9AD-7121041.56.3AD-7121154.416AD-71212122.420.7AD-71213111.413.7AD-71214120.119.5AD-712159811.7 | 298,070 |
11859186 | DETAILED DESCRIPTION The principle behind antisense technology is that an antisense compound, which hybridizes to a target nucleic acid, modulates gene expression activities such as transcription, splicing or translation. This sequence specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes or gene products involved in disease. Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence, resulting in exon-exon junctions at the site where exons are joined. Targeting exon-exon junctions can be useful in situations where aberrant levels of a normal splice product are implicated in disease, or where aberrant levels of an aberrant splice product are implicated in disease. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions can also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also suitable targets. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts” and are also suitable targets. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA. Single-stranded antisense compounds such as oligonucleotide compounds that work via an RNase H mechanism are effective for targeting pre-mRNA. Antisense compounds that function via an occupancy-based mechanism are effective for redirecting splicing as they do not, for example, elicit RNase H cleavage of the mRNA, but rather leave the mRNA intact and promote the yield of desired splice product(s). It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants.” More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence. Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants.” Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants.” If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant. It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. As used herein, “antisense mechanisms” are all those involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing. As used herein, “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The terms “individual”, “patient”, and “subject” are used interchangeably herein and refer to mammals, in particular primates and preferably humans. The term “exon” refers to a portion of a gene that is present in the mature form of mRNA. Exons include the ORF (open reading frame), i.e., the sequence which encodes protein, as well as the 5′ and 3′ UTRs (untranslated regions). The UTRs are important for translation of the protein. Algorithms and computer programs are available for predicting exons in DNA sequences (Grail, Grail 2 and Genscan and US 20040219522 for determining an exon-intron junctions). As used herein, the term “protein coding exon” refers to an exon which codes (or at least partially codes) for a protein (or part of a protein). The first protein coding exon in an mRNA is the exon which contains the start codon. The last protein encoding exon in an mRNA is the exon which contains the stop codon. The start and stop codons can be predicted using any number of well-known programs in the art. As used herein, the term “internal exon” refers to an exon that is flanked on both its 5′ and 3′ end by another exon. For an mRNA comprising n exons, exon 2 to exon (n−1) are the internal exons. The first and last exons of an mRNA are referred to herein as “external exons”. The term “intron” refers to a portion of a gene that is not translated into protein and while present in genomic DNA and pre-mRNA, it is removed in the formation of mature mRNA. The term “messenger RNA” or “mRNA” refers to RNA that is transcribed from genomic DNA and that carries the coding sequence for protein synthesis. Pre-mRNA (precursor mRNA) is transcribed from genomic DNA. In eukaryotes, pre-mRNA is processed into mRNA, which includes removal of the introns, i.e., “splicing”, and modifications to the 5′ and 3′ end (e.g., polyadenylation). mRNA typically comprises from 5′ to 3′; a 5′ cap (modified guanine nucleotide), 5′ UTR (untranslated region), the coding sequence (beginning with a start codon and ending with a stop codon), the 3′ UTR, and the poly(A) tail. The term “nucleic acid sequence” or “nucleic acid molecule” or polynucleotide are used interchangeably and refer to a DNA or RNA molecule in single or double stranded form. An “isolated nucleic acid sequence” refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a cell. A “mutation” in a nucleic acid molecule is a change of one or more nucleotides compared to the wild type sequence, e.g. by replacement, deletion or insertion of one or more nucleotides. A “point mutation” is the replacement of a single nucleotide, or the insertion or deletion of a single nucleotide. Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned by for example the programs GAP or BESTFIT or the Emboss program “Needle” (using default parameters, see below) share at least a certain minimal percentage of sequence identity (as defined further below). These programs use the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximising the number of matches and minimises the number of gaps. Generally, the default parameters are used, with a gap creation penalty=10 and gap extension penalty=0.5 (both for nucleotide and protein alignments). For nucleotides the default scoring matrix used is DNAFULL and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Sequence alignments and scores for percentage sequence identity may for example be determined using computer programs, such as EMBOSS (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). Alternatively sequence similarity or identity may be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more (as determined by Emboss “needle” using default parameters, i.e. gap creation penalty=10, gap extension penalty=0.5, using scoring matrix DNAFULL for nucleic acids an Blosum62 for proteins). Such sequences are also referred to as ‘variants’ herein, e.g. other variants of antisense oligomeric compounds. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3′- and/or 5′-side are 100% identical. The term “hybridisation” as used herein is generally used to mean hybridisation of nucleic acids at appropriate conditions of stringency as would be readily evident to those skilled in the art depending upon the nature of the probe sequence and target sequences. Conditions of hybridisation and washing are well known in the art, and the adjustment of conditions depending upon the desired stringency by varying incubation time, temperature and/or ionic strength of the solution are readily accomplished. See, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989. The choice of conditions is dictated by the length of the sequences being hybridised, in particular, the length of the probe sequence, the relative G-C content of the nucleic acids and the amount of mismatches to be permitted. Low stringency conditions are preferred when partial hybridisation between strands that have lesser degrees of complementarity is desired. When perfect or near perfect complementarity is desired, high stringency conditions are preferred. For typical high stringency conditions, the hybridisation solution contains 6×S.S.C., 0.01 M EDTA, 1×Denhardt's solution and 0.5% SOS. hybridisation is carried out at about 68° C. for about 3 to 4 hours for fragments of cloned DNA and for about 12 to about 16 hours for total eukaryotic DNA. For lower stringencies the temperature of hybridisation is reduced to about 42° C. below the melting temperature (TM) of the duplex. The TM is known to be a function of the G-C content and duplex length as well as the ionic strength of the solution. The term “allele(s)” means any of one or more alternative forms of a gene at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus. One allele is present on each chromosome of the pair of homologous chromosomes. These may be identical alleles of the gene (homozygous) or two different alleles (heterozygous). Mutant allele” refers herein to an allele comprising one or more mutations in the coding sequence (mRNA, cDNA or genomic sequence) compared to the wild type allele. Such mutation(s) (e.g. insertion, inversion, deletion and/or replacement of one or more nucleotide(s)) may lead to the encoded protein having reduced in vitro and/or in vivo functionality (reduced function) or no in vitro and/or in vivo functionality (loss-of-function), e.g. due to the protein e.g. being truncated or having an amino acid sequence wherein one or more amino acids are deleted, inserted or replaced. Such changes may lead to the protein having a different conformation, being targeted to a different sub-cellular compartment, having a modified catalytic domain, having a modified binding activity to nucleic acids or proteins, etc, it may also lead to a different splicing event. A “fragment” of the gene or nucleotide sequence or antisense oligomeric compound refers to any subset of the molecule, e.g., a shorter polynucleotide or oligonucleotide. A “variant” refers to a molecule substantially similar to the antisense oligomeric compound or a fragment thereof, such as a nucleotide substitution variant having one or more substituted nucleotides, but which maintains the ability to hybridize with the particular gene. Preferably the variant comprises the mutations as identified by the invention. Variants also include longer sequences. An “analogue” refers to a non-natural molecule substantially similar to or functioning in relation to either the entire molecule, a variant or a fragment thereof. As used herein, the terms “precursor mRNA” or “pre-mRNA” refer to an immature single strand of messenger ribonucleic acid (mRNA) that contains one or more intervening sequence(s) (introns). Pre-mRNA is transcribed by an RNA polymerase from a DNA template in the cell nucleus and is comprised of alternating sequences of introns and coding regions (exons). Once a pre-mRNA has been completely processed by the splicing out of introns and joining of exons, it is referred to as “messenger RNA” or “mRNA,” which is an RNA that is comprised exclusively of exons. Eukaryotic pre-mRNAs exist only transiently before being fully processed into mRNA. When a pre-mRNA has been properly processed to an mRNA sequence, it is exported out of the nucleus and eventually translated into a protein by ribosomes in the cytoplasm. As used herein, the terms “splicing” and “processing” refers to the modification of a pre-mRNA following transcription, in which introns are removed and exons are joined. Pre-mRNA splicing involves two sequential biochemical reactions. Both reactions involve the spliceosomal transesterification between RNA nucleotides. In a first reaction, the 2′-OH of a specific branch-point nucleotide within an intron, which is defined during spliceosome assembly, performs a nucleophilic attack on the first nucleotide of the intron at the 5′ splice site forming a lariat intermediate. In a second reaction, the 3′-OH of the released 5′ exon performs a nucleophilic attack at the last nucleotide of the intron at the 3′ splice site thus joining the exons and releasing the intron lariat. Pre-mRNA splicing is regulated by intronic silencer sequence (ISS), exonic silencer sequences (ESS) and terminal stem loop (TSL) sequences. As used herein, the terms “intronic silencer sequences (ISS)” and “exonic silencer sequences (TSL)” refer to sequence elements within introns and exons, respectively, that control alternative splicing by the binding of trans-acting protein factors within a pre-mRNA thereby resulting in differential use of splice sites. Typically, intronic silencer sequences are less conserved than the splice sites at exon-intron junctions. As used herein, “modulation of splicing” refers to altering the processing of a pre-mRNA transcript such that there is an increase or decrease of one or more splice products, or a change in the ratio of two or more splice products. Modulation of splicing can also refer to altering the processing of a pre-mRNA transcript such that a spliced mRNA molecule contains either a different combination of exons as a result of exon skipping or exon inclusion, a deletion in one or more exons, or additional sequence not normally found in the spliced mRNA (e.g., intron sequence). As used herein, “splice site” refers to the junction between an exon and an intron in a pre-mRNA (unspliced RNA) molecule (also known as a “splice junction”). A “cryptic splice site” is a splice site that is not typically used but may be used when the usual splice site is blocked or unavailable or when a mutation causes a normally dormant site to become an active splice site. An “aberrant splice site” is a splice site that results from a mutation in the native DNA and pre-mRNA. As used herein, “splice products” or “splicing products” are the mature mRNA molecules generated from the process of splicing a pre-mRNA. Alternatively spliced pre-mRNAs have at least two different splice products. For example, a first splicing product may contain an additional exon, or portion of an exon, relative to a second splicing product. Splice products of a selected pre-mRNA can be identified by a variety of different techniques well known to those of skill in the art. As used herein “splice donor site” refers to a splice site found at the 5′ end of an intron, or alternatively, the 3′ end of an exon. Splice donor site is used interchangeably with “5′ splice site.” As used herein “splice acceptor site” refers to a splice site found at the 3′ end of an intron, or alternatively, the 5′ end of an exon. Splice acceptor site is used interchangeably with “3′ splice site.” As used herein, “targeting” or “targeted to” refer to the process of designing an oligomeric compound such that the compound hybridizes with a selected nucleic acid molecule or region of a nucleic acid molecule. Targeting an oligomeric compound to a particular target nucleic acid molecule can be a multistep process. The process usually begins with the identification of a target nucleic acid whose expression is to be modulated. As used herein, the terms “target nucleic acid” and “nucleic acid encoding GAA” encompass DNA encoding GAA, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. As disclosed herein, the target nucleic acid encodes GAA. The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. As used herein, “target mRNA” refers to the nucleic acid molecule to which the oligomeric compounds provided herein are designed to hybridize. In the context of the present disclosure, target mRNA is usually unspliced mRNA, or pre-mRNA. In the context of the present invention, the target mRNA is GAA mRNA or GAA pre-mRNA. “Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Target regions may include, for example, a particular exon or intron, or may include only selected nucleotides within an exon or intron which are identified as appropriate target regions. Target regions may also be splicing repressor sites. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as unique nucleobase positions within a target nucleic acid. As used herein, the “target site” of an oligomeric compound is the 5′-most nucleotide of the target nucleic acid to which the compound binds. Target degradation can include an RNase H, which is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit cleavage by RNAse H. Occupancy-based antisense mechanisms, whereby antisense compounds hybridize yet do not elicit cleavage of the target, include inhibition of translation, modulation of splicing, modulation of poly(A) site selection and disruption of regulatory RNA structure. For the present invention “RNA-like” antisense compounds for use in occupancy-based antisense mechanisms are preferred. In the context of the present disclosure, an oligomeric compound “targeted to a splice site” refers to a compound that hybridizes with at least a portion of a region of nucleic acid encoding a splice site or a compound that hybridizes with an intron or exon in proximity to a splice site, such that splicing of the mRNA is modulated. The term “oligomeric compound” refers to a polymeric structure capable of hybridizing to a region of a nucleic acid molecule. This term includes oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and chimeric combinations of these. Oligomeric compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular. Moreover, branched structures are known in the art. Oligomeric compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops. Oligomeric double-stranded compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound. The term “antisense oligonucleotide, AON, or antisense oligomeric compound” refers to an oligonucleotide that is capable of interacting with and/or hybridizing to a pre-mRNA or an mRNA having a complementary nucleotide sequence thereby modifying gene expression and/or splicing. Enzyme-dependent antisense oligonucleotides include forms that are dependent on RNase H activity to degrade target mRNA, and include single-stranded DNA, RNA, and phosphorothioate antisense. Steric blocking antisense oligonucleotides (RNase-H independent antisense) interfere with gene expression or other mRNA-dependent cellular processes by binding to a target sequence of mRNA. Steric blocking antisense includes 2′-0 alkyl antisense oligonucleotides, Morpholino antisense oligonucleotides, and tricyclo-DNA antisense oligonucleotides. Steric blocking antisense oligonucleotides are preferred in the present invention. As used herein, antisense oligonucleotides that are “RNase H-independent” are those compounds which do not elicit cleavage by RNase H when hybridized to a target nucleic acid. RNase H-independent oligomeric compounds modulate gene expression, such as splicing, by a target occupancy-based mechanism. Rnase H-independent antisense oligonucleotides are preferred in the present invention. As used herein, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the context of the present disclosure, an oligomeric compound is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target nucleic acid sequences. One of skill in the art will be able to determine when an oligomeric compound is specifically hybridizable. As used herein, “complementary” refers to a nucleic acid molecule that can form hydrogen bond(s) with another nucleic acid molecule by either traditional Watson-Crick base pairing or other non-traditional types of pairing (e.g., Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleosides or nucleotides. In reference to the antisense oligomeric compound of the present disclosure, the binding free energy for a antisense oligomeric compound with its complementary sequence is sufficient to allow the relevant function of the antisense oligomeric compound to proceed and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of ex vivo or in vivo therapeutic treatment. Determination of binding free energies for nucleic acid molecules is well known in the art (see e.g., Turner et ah, CSH Symp. Quant. Biol. 1/7:123-133 (1987); Frier et al, Proc. Nat. Acad. Sci. USA 83:9373-77 (1986); and Turner et al, J. Am. Chem. Soc. 109:3783-3785 (1987)). Thus, “complementary” (or “specifically hybridizable”) are terms that indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between a antisense oligomeric compound and a pre-mRNA or mRNA target. It is understood in the art that a nucleic acid molecule need not be 100% complementary to a target nucleic acid sequence to be specifically hybridizable. That is, two or more nucleic acid molecules may be less than fully complementary. Complementarity is indicated by a percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule. For example, if a first nucleic acid molecule has 10 nucleotides and a second nucleic acid molecule has 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules represents 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively. Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). “Perfectly” or “fully” complementary nucleic acid molecules means those in which all the contiguous residues of a first nucleic acid molecule will hydrogen bond with the same number of contiguous residues in a second nucleic acid molecule, wherein the nucleic acid molecules either both have the same number of nucleotides (i.e., have the same length) or the two molecules have different lengths. As used herein, “uniformly modified” or “fully modified” refers to an oligomeric compound, an antisense oligonucleotide, or a region of nucleotides wherein essentially each nucleoside is a sugar modified nucleoside having uniform modification. As used herein, a “chimeric oligomeric compound”, “chimeric antisense compound” or “chimeric antisense oligonucleotide compound” is a compound containing two or more chemically distinct regions, each comprising at least one monomer unit (i.e., a nucleotide in the case of an oligonucleotide compound). The term “chimeric antisense compound” specifically refers to an antisense compound, having at least one sugar, nucleobase and/or internucleoside linkage that is differentially modified as compared to the other sugars, nucleotides and internucleoside linkages within the same oligomeric compound. The remainder of the sugars, nucleotides and internucleoside linkages can be independently modified or unmodified. In general a chimeric oligomeric compound will have modified nucleosides that can be in isolated positions or grouped together in regions that will define a particular motif. Chimeric oligomeric compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. In the context of the present disclosure, a “chimeric RNase H-independent antisense compound” is an antisense compound with at least two chemically distinct regions, but which is not susceptible to cleavage by RNase H when hybridized to a target nucleic acid. As used herein, a “nucleoside” is a base-sugar combination and “nucleotides” are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. As used herein, a nucleoside with a modified sugar residue is any nucleoside wherein the ribose sugar of the nucleoside has been substituted with a chemically modified sugar moiety. In the context of the present disclosure, the chemically modified sugar moieties include, but are not limited to, 2′-O-methoxyethyl, 2′-fluoro, 2′-dimethylaminooxyethoxy, 2′-dimethylaminoethoxyethoxy, 2′-guanidinium, 2′-O-guanidinium ethyl, 2′-carbamate, 2′-aminooxy, 2′-acetamido and locked nucleic acid. As used herein, compounds “resistant to RNase H degradation” are antisense compounds having a least one chemical modification that increases resistance of the compound to RNase H cleavage. Such modifications include, but are not limited to, nucleotides with sugar modifications. As used herein, a nucleotide with a modified sugar includes, but is not limited to, any nucleotide wherein the 2′-deoxyribose sugar has been substituted with a chemically modified sugar moiety. In the context of the present invention, chemically modified sugar moieties include, but are not limited to, 2′-O-(2-methoxyethyl), 2′-fluoro, 2′-dimethylaminooxyethoxy, 2′-dimethylaminoethoxyethoxy, 2′-guanidinium, 2′-O-guanidinium ethyl, 2′-carbamate, 2′-aminooxy, 2′-acetamido, locked nucleic acid (LNA) and ethylene bridged nucleic acid (ENA). Modified compounds resistant to RNase H cleavage are thoroughly described herein and are well know to those of skill in the art. In the context of the present disclosure, “cellular uptake” refers to delivery and internalization of oligomeric compounds into cells. The oligomeric compounds can be internalized, for example, by cells grown in culture (in vitro), cells harvested from an animal (ex vivo) or by tissues following administration to an animal (in vivo). By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of this disclosure can be administered. In one embodiment of the invention and/or embodiments thereof, a subject is a mammal or mammalian cell. In another embodiment, a subject is a human or human cell. As used herein, the term “therapeutically effective amount” means an amount of antisense oligomeric compound that is sufficient, in the subject (e.g., human) to which it is administered, to treat or prevent the stated disease, disorder, or condition. The antisense oligomeric compound of the instant disclosure, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein. For example, to treat a particular disease, disorder, or condition, the antisense oligomeric compound can be administered to a patient or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs, under conditions suitable for treatment. In the present invention the disease is preferably Pompe disease. As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. As used herein, the term “isolated” means that the referenced material is removed from its native environment, e.g., a cell. Thus, an isolated biological material can be free of some or all cellular components, i.e. components of the cells in which the native material occurs naturally (e.g., cytoplasmic or membrane component). The term “purified” as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e. contaminants, including native materials from which the material is obtained. For example, a purified tc-DNA antisense oligomeric compound is preferably substantially free of cell or culture components, including tissue culture components, contaminants, and the like. As used herein, the term “substantially free” is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art. In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, “about” or “consisting essentially of mean+−20% of the indicated range, value, or structure, unless otherwise indicated. As used herein, the terms “include” and “comprise” are used synonymously. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “about” or “approximately” means within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. In one aspect, the invention is directed to an antisense oligomeric compound targeting SEQ ID NO: 1 and single nucleotide polymorphism of SEQ ID NO:1. Previous work by others has resulted in the design of antisense oligomeric compounds that promote exon exclusion in several human disorders including Duchenne Muscular Dystrophy (DMD). The strategy is simple and straightforward and relies on blocking a well-defined splice site. This results in exon skipping, thereby removing the exon containing the pathogenic gene variant. The resulting mRNA is a little bit shorter resulting in expression of a truncated protein with considerable residual activity, sufficient to at least partially alleviate the disease. The strategy is simple because canonical splice sites are known for virtually all genes. The only requirement is to design an antisense oligomeric compound that binds to the canonical splice site in the pre-mRNA, which will result in blocking of that site and skipping of the exon involved. A much more difficult task is the reverse process: to promote inclusion rather than exclusion of an exon. To promote exon inclusion, a splice repressor may be blocked using an antisense oligomeric compound. It is however unknown where splice repressors are located. These can be present in introns or in exons and are named intronic or exonic splice silencers (ISSs or ESSs, respectively). There is software available to predict the presence of such silences but these are very unreliable. This is further illustrated by our own experience using the minigene system containing GAA exon 1-3, which failed to confirm activity of predicted splice silencer motifs. The idea to promote exon 2 inclusion of GAA with an antisense oligomeric compound to treat Pompe disease is entirely novel. We show in this in the accompanying patent application (PCT/NL2014/050375) that splice repressor sequences can be identified by two screens: the U7-snRNA antisense oligomeric compound screen, and the random mutagenesis/minigene screen. One target sequence from this screen was successfully targeted with an antisense oligomeric compound, resulting in enhanced inclusion of GAA exon 2 in the context of the IVS1 variant. This corrected the aberrant splicing of exon 2 caused by the IVS1 variant, as visualized by the enhanced abundance of wild type GAA mRNA. It was found that sequences targeting SEQ ID NO: 1 are able to enhance inclusion of GAA exon 2. Also sequences targeting SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, were found to be able to enhance inclusion of GAA exon 2. It is to be noted that targeting means that at least part of the sequence SEQ ID NO: 1 is targeted, e.g. by a sequence that hybridizes with at least a part or by the sequence SEQ ID NO: 1, or that binds to at least a part of SEQ ID NO: 1. Sequences that target may be shorter or longer than the target sequence. Sequence incDNA to whichSEQ IDAON anneals*sequence of AON (5′→3′):NO:c-32-156_-210GCTCTGCACTCCCCTGCTGGAGCTTTT1CTCGCCCTTCCTTCTGGCCCTCTCCCCAc-32-156_-200GCTCTGCACTCCCCTGCTGGAGCTTTT37CTCGCCCTTCCTTCTGGCc-32-160_-190TGCACTCCCCTGCTGGAGCTTTTCTCG38CCCTc-32-160_195TGCACTCCCCTGCTGGAGCTTTTCTCG39CCCTTCCTTc-32-165_-195TCCCCTGCTGGAGCTTTTCTCGCCCTT40CCTT Suitably the sequences targeting SEQ ID NO: 1 hybridize with at least a part of SEQ ID NO: 1. Sequences that hybridize may be shorter or longer than the target sequence. Nucleotide sequences SEQ ID NO: 2-33 are oligomers that are able to enhance GAA exon 2 inclusion. Two variant antisense oligomeric compounds, one of 21 nucleotides (SEQ ID NO: 33) and one of 25 nucleotides (SEQ ID NO: 12), were tested and both were found to enhance exon 2 inclusion. This was accompanied by enhanced GAA enzyme activity of at least 2 fold. It is known that patients with the IVS1 variant have ˜15% leaky wild type splicing. The enhancement of 2 fold results in enzyme activities of ˜30%, which are known to be above the disease threshold of 20% and thus are anticipated to restore at least a part, or even fully the lysosomal glycogen degradation. In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound selected from the group comprising SEQ ID NO: 2-33 and variants and fragments having at least 80% identity thereof. The antisense oligomeric compound may also target single nucleotide polymorphism of SEQ ID NO: 1, 37, 38, 39, 40. It should be noted that it may not necessary to have the full length of SEQ ID NO: 2-33, fragments having a shorter or longer sequence are also envisioned. The inventors have found the target genomic sequence which enables the inclusion of exon 2 of GAA and a skilled person is capable of finding suitable sequences that target this target genomic sequence, such as SEQ ID NO: 1, 37, 38, 39, 40 and single nucleotide polymorphisms thereof. Exemplary sequences that target this target genomic sequence, such as SEQ ID NO: 1, 37, 38, 39, or 41 may be SEQ ID NO: 2-33, but also variants and fragments having at least 80% identity thereof. In particular shorter fragments such as fragments with 18, 19, 20, 21, 22, 23, or 24 nucleotides of SEQ ID NO: 2-33 are envisioned. In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound complementary to a polynucleotide having a sequence selected from the group comprising SEQ ID NO: 1, 37-40 and single nucleotide polymorphisms thereof. Also sequences having at least 80% identity to antisense oligomeric compound complementary to a polynucleotide having a sequence selected from the group comprising SEQ ID NO: 1, 37-40 are envisioned. Antisense oligomeric compound that target one or more than one single nucleotide polymorphisms may be designed. In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound targeting a sequence selected from the group comprising the genomic sequence c−32−156_−210. In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound comprising sequences selected from the group comprising SEQ ID NO: 2-33, 41-1583 and sequences having at least 80% identity thereof. In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to antisense oligomeric compound comprising a sequences selected from the group comprising SEQ ID NO: 2-33, and 41-540. In one aspect or embodiment of aspects and/or embodiments thereof the invention is directed to an antisense oligomeric compound complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of the following mutation c.−32−13T>G, c.−32−3C>G c.−32−102T>C, c.−32−56C>T, c.−32−46G>A, c.−32−28C>A, c.−32−28C>T, c.−32−21G>A, c.7G>A, c.11G>A, c.15_17AAA, c.17C>T, c.19_21AAA, c.26_28AAA, c.33_35AAA, c.39G>A, c.42C>T, c.90C>T, c.112G>A, c.137C>T, c.164C>T, c.348G>A, c.373C>T, c.413T>A, c.469C>T, c.476T>C, c.476T>G, c.478T>G, c.482C>T, c.510C>T, c.515T>A, c.520G>A, c.546+11C>T, c.546+14G>A, c.546+19G>A, c.546+23C>A, c.547−6, c.1071, c.1254, c.1552−30, c.1256A>T, c.1551+1G>T, c.546G>T, 0.17C>T, c.469C>T, c.546+23C>A, c.−32−102T>C, c.−32−56C>T, c.11G>A, c.112G>A, c.137C>T. The above identified mutations have been found to modulate splicing. Targeting the location of the mutation may also modulate the splicing. It is therefore understood that the antisense oligomeric compound targets the location the mutation. The nomenclature of the mutation identifies the location and the mutation. It is understood that the antisense oligomeric compound targets the location of the mutation, and the mutation does not need to be present in the genomic sequence or in the pre-mRNA. The location of the mutation is thus the location of the mutated nucleotide, or the location of the wild type nucleotide of the mutation. The antisense oligomeric compound may be targeted to a sequence comprising nucleotides upstream and nucleotides downstream of the location of the mutation. Suitably the antisense oligomeric compound target a sequence comprising 2-50 nucleotides upstream, and/or 2-50 nucleotides downstream of the location of the mutation, more suitably the antisense oligomeric compound target a sequence comprising 3-45 nucleotides upstream, and/or 3-45 nucleotides downstream of the location of the mutation, more suitably the antisense oligomeric compound target a sequence comprising 5-40 nucleotides upstream, and/or 5-40 nucleotides downstream of the location of the mutation, more suitably the antisense oligomeric compound target a sequence comprising 6-35 nucleotides upstream, and/or 6-35 nucleotides downstream of the location of the mutation, more suitably the antisense oligomeric compound target a sequence comprising 7-33 nucleotides upstream, and/or 7-33 nucleotides downstream of the location of the mutation, more suitably the antisense oligomeric compound target a sequence comprising 8-30 nucleotides upstream, and/or 8-30 nucleotides downstream of the location of the mutation, more suitably the antisense oligomeric compound target a sequence comprising 9-28 nucleotides upstream, and/or 9-28 nucleotides downstream of the location of the mutation, more suitably the antisense oligomeric compound target a sequence comprising 10-25 nucleotides upstream, and/or 10-25 nucleotides downstream of the location of the mutation, more suitably the antisense oligomeric compound target a sequence comprising 11-22 nucleotides upstream, and/or 11-22 nucleotides downstream of the location of the mutation, more suitably the antisense oligomeric compound target a sequence comprising 12-20 nucleotides upstream, and/or 12-20 nucleotides downstream of the location of the mutation, more suitably the antisense oligomeric compound target a sequence comprising 13-18 nucleotides upstream, and/or 13-18 nucleotides downstream of the location of the mutation, more suitably the antisense oligomeric compound target a sequence comprising 14-16 nucleotides upstream, and/or 14-16 nucleotides downstream of the location of the mutation. The nomenclature is well known to a skilled person and can be found in Dunnen and Antonarakis Human mutation 15:7-12(2000) and Antonarakis SE, the Nomenclature Working Group. 1998. Recommendations for a nomenclature system for human gene mutations. Hum Mutat 11:1-3 and on the website (http://www.dmd.nl/mutnomen.html. Genomic positions may also be found on www.pompecenter.nl. All of these are incorporated by reference. Preferably the genomic nucleic acid sequence is pre-mRNA. These antisense oligomeric compound are useful in the treatment of glycogen storage disease type II/Pompe disease. In one aspect or the target sequence is an intronic splicing silencer or ISS. In a preferred embodiment of the invention and/or embodiments thereof of an aspect and/or embodiments of the invention the target sequence is the GCTCTGCACTCCCCTGCTGGAGCTTTTCTCGCCCTTCCTTCTGGCCCTCTCCCC A (SEQ ID NO: 1). It should be noted that also naturally occurring single nucleotide polymorphism are included. Antisense oligomeric compounds targeting SEQ ID NO: 1 are a very suitable to treat Pompe patients. Exemplary antisense oligomeric compounds targeting SEQ ID NO: 1 are SEQ ID NO: 2-33 and in particular SEQ ID NO: 12 and SEQ ID NO 33. However the invention is not limited to these two sequences. A skilled person is capable of designing antisense oligomeric compounds against target sequence SEQ ID NO: 1, 37, 38, 39, or 40. The antisense oligomeric compounds against target sequenced SEQ ID NO: 1 may have length of 10 to 100 nucleotides, preferably 11 to 75 nucleotides, preferably 12 to 73 nucleotides, preferably 13 to 70 nucleotides, preferably 14 to 65 nucleotides, preferably 15 to 60 nucleotides, preferably 16 to 55 nucleotides, preferably 17 to 50 nucleotides, preferably 18 to 45 nucleotides, preferably 19 to 40 nucleotides, preferably 20 to 38 nucleotides, preferably 21 to 35 nucleotides, preferably 22 to 33 nucleotides, preferably 23 to 30 nucleotides, preferably 24 to 29 nucleotides, preferably 25 to 28 nucleotides, preferably 26 to 27 nucleotides. Hereunder exemplary antisense oligomeric compounds targeting SEQ ID NO: 1 are given Sequence incDNA to whichAON anneals*sequence of AON (5′→3′):Seq IDc.-32-180_-156TGGGGAGAGGGCCAGAAGGAAGGGC2c.-32-181_-157GGGGAGAGGGCCAGAAGGAAGGGCG3c.-32-182_-158GGGAGAGGGCCAGAAGGAAGGGCGA4c.-32-183_-159GGAGAGGGCCAGAAGGAAGGGCGAG5c.-32-184_-160GAGAGGGCCAGAAGGAAGGGCGAGA6c.-32-185_-161AGAGGGCCAGAAGGAAGGGCGAGAA7c.-32-186_-162GAGGGCCAGAAGGAAGGGCGAGAAA8c.-32-187_-163AGGGCCAGAAGGAAGGGCGAGAAAA9c.-32-188_-164GGGCCAGAAGGAAGGGCGAGAAAAG10c.-32-189_-165GGCCAGAAGGAAGGGCGAGAAAAGC11c.-32-190_-166GCCAGAAGGAAGGGCGAGAAAAGCT12c.-32-191_-167CCAGAAGGAAGGGCGAGAAAAGCTC13c.-32-192_-168CAGAAGGAAGGGCGAGAAAAGCTCC14c.-32-193_-169AGAAGGAAGGGCGAGAAAAGCTCCA15c.-32-194_-170GAAGGAAGGGCGAGAAAAGCTCCAG16c.-32-195_-171AAGGAAGGGCGAGAAAAGCTCCAGC17c.-32-196_-172AGGAAGGGCGAGAAAAGCTCCAGCA18c.-32-197_-173GGAAGGGCGAGAAAAGCTCCAGCAG19c.-32-198_-174GAAGGGCGAGAAAAGCTCCAGCAGG20c.-32-199_-175AAGGGCGAGAAAAGCTCCAGCAGGG21c.-32-200_-176AGGGCGAGAAAAGCTCCAGCAGGGG22c.-32-201_-177GGGCGAGAAAAGCTCCAGCAGGGGA23c.-32-202_-178GGCGAGAAAAGCTCCAGCAGGGGAG24c.-32-203_-179GCGAGAAAAGCTCCAGCAGGGGAGT25c.-32-204_-180CGAGAAAAGCTCCAGCAGGGGAGTG26c.-32-205_-181GAGAAAAGCTCCAGCAGGGGAGTGC27c.-32-206_-182AGAAAAGCTCCAGCAGGGGAGTGCA28c.-32-207_-183GAAAAGCTCCAGCAGGGGAGTGCAG29c.-32-208_-184AAAAGCTCCAGCAGGGGAGTGCAGA30c.-32-209_-185AAAGCTCCAGCAGGGGAGTGCAGAG31c.-32-210_-186AAGCTCCAGCAGGGGAGTGCAGAGC32c.-32-187_-167CCAGAAGGAAGGGCGAGAAAA33 In the above examples the sequences are 25 nucleotides long however longer variants or shorter fragment are also envisioned. Exemplary is SEQ ID NO: 33 which is only 21 nucleotides long and comprises the same nucleotides as SEQ ID NO: 12 but is shorter. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO: 2-33 and fragments and variants thereof having at least 80% sequence identity. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO: 2-33 and fragments and variants thereof having at least 80%, 83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% sequence identity to SEQ ID NO: 2-33. The present invention is also directed to sequences that are at least 80% identical to SEQ ID NO: 2-33. Preferably at least 85% identical to SEQ ID NO: 2-33, more preferably at least 88% identical to SEQ ID NO: 2-33, more preferably at least 90% identical to SEQ ID NO: 2-33. more preferably at least 91% identical to SEQ ID NO: 2-33, more preferably at least 92% identical to SEQ ID NO: 2-33, more preferably at least 93% identical to SEQ ID NO: 2-33, more preferably at least 94% identical to SEQ ID NO: 2-33, more preferably at least 95% identical to SEQ ID NO: 2-33, more preferably at least 96% identical to SEQ ID NO: 2-33, more preferably at least 97% identical to SEQ ID NO: 2-33, more preferably at least 98% identical to SEQ ID NO: 2-33, more preferably at least 99% identical to SEQ ID NO: 2-33. Preferred antisense sequences are SEQ ID NO: 12, and SEQ ID NO:33 or sequences that are at least 80% identical thereto, preferably at least 85% identical, more preferably at least 88% identical, more preferably at least 90% identical, more preferably at least 91% identical, more preferably at least 92% identical, more preferably at least 93% identical, more preferably at least 94% identical, more preferably at least 95% identical, more preferably at least 96% identical, more preferably at least 97% identical, more preferably at least 98% identical, more preferably at least 99% identical to SEQ ID NO: 12, and/or 33. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO:2-33, wherein the fragment is 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO:2-33, wherein the fragment is 17, 18, 19, 20, 21, or 22 nucleotides long. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO:2-33, wherein the fragment is 19, 20, or 21 nucleotides long. The antisense oligomeric compounds may be selected from the group of SEQ ID NO: 41-540: Sequences Identified with U7 Screen: SEQ ID NO 41-97 Sequence in GAAcDNA to whichSeqAON annealsAON sequence (5′→3′)IDc.-32-319_-300CCAAACAGCTGTCGCCTGGG41c.-32-299_-280AGGTAGACACTTGAAACAGG42c.-32-279_-260CCCAGGAAGACCAGCAAGGC43c.-32-259_-240TCAAACACGCTTAGAATGTC44c.-32-239_-220GTCTGCTAAAATGTTACAAA45c.-32-219_-200GAGTGCAGAGCACTTGCACA46c.-32-199_-180CGAGAAAAGCTCCAGCAGGG47c.-32-179_-160GAGAGGGCCAGAAGGAAGGG48c.-32-159_-140GCCCTGCTGTCTAGACTGGG49c.-32-139_-120AGGTGGCCAGGGTGGGTGTT50c.-32-119_-100GCACCCAGGCAGGTGGGGTA51c.-32-99_-80CAACCGCGGCTGGCACTGCA52c.-32-79_-60TCAAAGCAGCTCTGAGACAT53c.-32-59_-40GGGCGGCACTCACGGGGCTC54c.-32-39_-20GCTCAGCAGGGAGGCGGGAG55c.-32-19_-0CCTGCGGGAGAAGAAAGCGG56c.-30_-12GCCTGGACAGCTCCTACAGG57c.-10_+9CACTCCCATGGTTGGAGATG58c.10_+29TGGGAGCAGGGCGGGTGCCT59c.30_+49CGCAGACGGCCAGGAGCCGG60c.50_+69GGTTGCCAAGGACACGAGGG61c.70_+89ATGTGCCCCAGGAGTGCAGC62c.90_+109GCAGGAAATCATGGAGTAGG63c.110_+129ACTCAGCTCTCGGGGAACCA64c.130_+149TCCAGGACTGGGGAGGAGCC65c.150_+169GGTGAGCTGGGTGAGTCTCC66c.170_+189TGGTCTGCTGGCTCCCTGCT67c.190_+209GCCTGGGCATCCCGGGGCCC68c.210_+229CTCTGGGACGGCCGGGGTGT69c.230_+249GTCGCACTGTGTGGGCACTG70c.250_+269AAGCGGCTGTTGGGGGGGAC71c.270_+289CCTTGTCAGGGGCGCAATCG72c.290_+309GCACTGTTCCTGGGTGATGG73c.310_+329TAGCAACAGCCGCGGGCCTC74c.330_+349GCCCCTGCTTTGCAGGGATG75c.350_+369CCCCATCTGGGCTCCCTGCA76c.370_+389GGGAAGAAGCACCAGGGCTG77c.390_+409TGTAGCTGGGGTAGCTGGGT78c.410_+429GGAGCTCAGGTTCTCCAGCT79c.430_+449GCCGTGTAGCCCATTTCAGA80c.450_+469GGGTGGTACGGGTCAGGGTG81c.470_+489GTCCTTGGGGAAGAAGGTGG82c.490_+509TCCAGCCGCAGGGTCAGGAT83c.510_+529TCTCAGTCTCCATCATCACG84c.530_+546GTGAAGTGGAGGCGGT85c.-32-225_-206AGAGCACTTGCACAGTCTGC86c.-32-223_-204GCAGAGCACTTGCACAGTCT87c.-32-221_-202GTGCAGAGCACTTGCACAGT88c.-32-217_-198GGGAGTGCAGAGCACTTGCA89c.-32-215_-196AGGGGAGTGCAGAGCACTTG90c.-32-213_-194GCAGGGGAGTGCAGAGCACT91c.-32-185_-166GCCAGAAGGAAGGGCGAGAA92c.-32-183_-164GGGCCAGAAGGAAGGGCGAG93c.-32-181_-162GAGGGCCAGAAGGAAGGGCG94c.-32-177_-158GGGAGAGGGCCAGAAGGAAG95c.-32-175_-156TGGGGAGAGGGCCAGAAGGA96c.-32-173_-154ACTGGGGAGAGGGCCAGAAG97variants that affectaberrant splicing ofAON sequence designed toexon 2 caused byblock the region surroundingIVS1 in GAA exon 1-3the identifiedSeqminigene systemsplice element (5′→3′)IDc.-32-102C > TCACCCAGGCAGGTGGGGTAAGGTGG98AGCACCCAGGCAGGTGGGGTAAGGT99GCAGCACCCAGGCAGGTGGGGTAAG100CTGCAGCACCCAGGCAGGTGGGGTA101CACTGCAGCACCCAGGCAGGTGGGG102GGCACTGCAGCACCCAGGCAGGTGG103CTGGCACTGCAGCACCCAGGCAGGT104GGCTGGCACTGCAGCACCCAGGCAG105GCGGCTGGCACTGCAGCACCCAGGC106CCGCGGCTGGCACTGCAGCACCCAG107TCAACCGCGGCTGGCACTGCAGCAC108ACCCAGGCAGGTGGGGTAAGGTGGC109GCACCCAGGCAGGTGGGGTAAGGTG110CAGCACCCAGGCAGGTGGGGTAAGG111TGCAGCACCCAGGCAGGTGGGGTAA112ACTGCAGCACCCAGGCAGGTGGGGT113GCACTGCAGCACCCAGGCAGGTGGG114TGGCACTGCAGCACCCAGGCAGGTG115GCTGGCACTGCAGCACCCAGGCAGG116CGGCTGGCACTGCAGCACCCAGGCA117CGCGGCTGGCACTGCAGCACCCAGG118ACCGCGGCTGGCACTGCAGCACCCA119CAACCGCGGCTGGCACTGCAGCACC120ATCAACCGCGGCTGGCACTGCAGCA121c.-32-56C > T, c-32-GGCTCTCAAAGCAGCTCTGAGACAT12246G > A, c.-32-28C > A, c.-GGGGCTCTCAAAGCAGCTCTGAGAC12332-28C > T, c.-32-21G > AACGGGGCTCTCAAAGCAGCTCTGAG124TCACGGGGCTCTCAAAGCAGCTCTG125ACTCACGGGGCTCTCAAAGCAGCTC126GCACTCACGGGGCTCTCAAAGCAGC127CGGCACTCACGGGGCTCTCAAAGCA128GGCGGCACTCACGGGGCTCTCAAAG129GGGGCGGCACTCACGGGGCTCTCAA130GAGGGGCGGCACTCACGGGGCTCTC131GGGAGGGGCGGCACTCACGGGGCTC132GCGGGAGGGGCGGCACTCACGGGGC133AGGCGGGAGGGGCGGCACTCACGGG134GGAGGCGGGAGGGGCGGCACTCACG135AGGGAGGCGGGAGGGGCGGCACTCA136GCAGGGAGGCGGGAGGGGCGGCACT137CAGCAGGGAGGCGGGAGGGGCGGCA138CTCAGCAGGGAGGCGGGAGGGGCGG139GGCTCAGCAGGGAGGCGGGAGGGGC140CGGGCTCAGCAGGGAGGCGGGAGGG141AGCGGGCTCAGCAGGGAGGCGGGAG142AAAGCGGGCTCAGCAGGGAGGCGGG143AGAAAGCGGGCTCAGCAGGGAGGCG144GAAGAAAGCGGGCTCAGCAGGGAGG145GAGAAGAAAGCGGGCTCAGCAGGGA146GGGAGAAGAAAGCGGGCTCAGCAGG147GCGGGAGAAGAAAGCGGGCTCAGCA148CTGCGGGAGAAGAAAGCGGGCTCAG149GCCTGCGGGAGAAGAAAGCGGGCTC150AGGCCTGCGGGAGAAGAAAGCGGGC151ACTCCCATGGTTGGAGATGGCCTGG152TCACTCCCATGGTTGGAGATGGCCT153CCTCACTCCCATGGTTGGAGATGGC154TGCCTCACTCCCATGGTTGGAGATG155GGTGCCTCACTCCCATGGTTGGAGA156CGGGTGCCTCACTCCCATGGTTGGA157GGCGGGTGCCTCACTCCCATGGTTG158AGGGCGGGTGCCTCACTCCCATGGT159GCAGGGCGGGTGCCTCACTCCCATG160GAGCAGGGCGGGTGCCTCACTCCCA161GGGAGCAGGGCGGGTGCCTCACTCC162GTGGGAGCAGGGCGGGTGCCTCACT163CGGTGGGAGCAGGGCGGGTGCCTCA164GCCGGTGGGAGCAGGGCGGGTGCCT165GAGCCGGTGGGAGCAGGGCGGGTGC166AGGAGCCGGTGGGAGCAGGGCGGGT167CCAGGAGCCGGTGGGAGCAGGGCGG168GGCCAGGAGCCGGTGGGAGCAGGGC169ACGGCCAGGAGCCGGTGGGAGCAGG170AGACGGCCAGGAGCCGGTGGGAGCA171GCAGACGGCCAGGAGCCGGTGGGAG172GCGCAGACGGCCAGGAGCCGGTGGG173GGGCGCAGACGGCCAGGAGCCGGTG174GAGGGCGCAGACGGCCAGGAGCCGG175ACGAGGGCGCAGACGGCCAGGAGCC176ACACGAGGGCGCAGACGGCCAGGAG177GGACACGAGGGCGCAGACGGCCAGG178AAGGACACGAGGGCGCAGACGGCCA179CCAAGGACACGAGGGCGCAGACGGC180TGCCAAGGACACGAGGGCGCAGACG181GCTCTCAAAGCAGCTCTGAGACATC182GGGCTCTCAAAGCAGCTCTGAGACA183CTCACGGGGCTCTCAAAGCAGCTCT184CACTCACGGGGCTCTCAAAGCAGCT185GGCACTCACGGGGCTCTCAAAGCAG186GCGGCACTCACGGGGCTCTCAAAGC187GGGCGGCACTCACGGGGCTCTCAAA188AGGGGCGGCACTCACGGGGCTCTCA189GGAGGGGCGGCACTCACGGGGCTCT190CGGGAGGGGCGGCACTCACGGGGCT191GGCGGGAGGGGCGGCACTCACGGGG192GAGGCGGGAGGGGCGGCACTCACGG193GGGAGGCGGGAGGGGCGGCACTCAC194CAGGGAGGCGGGAGGGGCGGCACTC195AGCAGGGAGGCGGGAGGGGCGGCAC196TCAGCAGGGAGGCGGGAGGGGCGGC197GCTCAGCAGGGAGGCGGGAGGGGCG198GGGCTCAGCAGGGAGGCGGGAGGGG199GCGGGCTCAGCAGGGAGGCGGGAGG200AAGCGGGCTCAGCAGGGAGGCGGGA201GAAAGCGGGCTCAGCAGGGAGGCGG202AAGAAAGCGGGCTCAGCAGGGAGGC203AGAAGAAAGCGGGCTCAGCAGGGAG204GGAGAAGAAAGCGGGCTCAGCAGGG205CGGGAGAAGAAAGCGGGCTCAGCAG206TGCGGGAGAAGAAAGCGGGCTCAGC207CCTGCGGGAGAAGAAAGCGGGCTCA208GGCCTGCGGGAGAAGAAAGCGGGCT209CAGGCCTGCGGGAGAAGAAAGCGGG210CGGGGCTCTCAAAGCAGCTCTGAGA211CACGGGGCTCTCAAAGCAGCTCTGA212c.7G > A, c.11G > A,CTCCCATGGTTGGAGATGGCCTGGA213c.15_17AAA, c.17C > T,CACTCCCATGGTTGGAGATGGCCTG214c.19_21AAA,CTCACTCCCATGGTTGGAGATGGCC215c.26_28AAA,GCCTCACTCCCATGGTTGGAGATGG216c.33_35AAA, c.39G > A,GTGCCTCACTCCCATGGTTGGAGAT217c.42C > TGGGTGCCTCACTCCCATGGTTGGAG218GCGGGTGCCTCACTCCCATGGTTGG219GGGCGGGTGCCTCACTCCCATGGTT220CAGGGCGGGTGCCTCACTCCCATGG221AGCAGGGCGGGTGCCTCACTCCCAT222GGAGCAGGGCGGGTGCCTCACTCCC223TGGGAGCAGGGCGGGTGCCTCACTC224GGTGGGAGCAGGGCGGGTGCCTCAC225CCGGTGGGAGCAGGGCGGGTGCCTC226AGCCGGTGGGAGCAGGGCGGGTGCC227GGAGCCGGTGGGAGCAGGGCGGGTG228CAGGAGCCGGTGGGAGCAGGGCGGG229GCCAGGAGCCGGTGGGAGCAGGGCG230CGGCCAGGAGCCGGTGGGAGCAGGG231GACGGCCAGGAGCCGGTGGGAGCAG232CAGACGGCCAGGAGCCGGTGGGAGC233CGCAGACGGCCAGGAGCCGGTGGGA234GGCGCAGACGGCCAGGAGCCGGTGG235AGGGCGCAGACGGCCAGGAGCCGGT236CGAGGGCGCAGACGGCCAGGAGCCG237CACGAGGGCGCAGACGGCCAGGAGC238GACACGAGGGCGCAGACGGCCAGGA239AGGACACGAGGGCGCAGACGGCCAG240CAAGGACACGAGGGCGCAGACGGCC241GCCAAGGACACGAGGGCGCAGACGG242TTGCCAAGGACACGAGGGCGCAGAC243c.90C > T, c.112G > A,GGATGTGCCCCAGGAGTGCAGCGGT244c.137C > T, c.164C > TTAGGATGTGCCCCAGGAGTGCAGCG245AGTAGGATGTGCCCCAGGAGTGCAG246GGAGTAGGATGTGCCCCAGGAGTGC247ATGGAGTAGGATGTGCCCCAGGAGT248TCATGGAGTAGGATGTGCCCCAGGA249AATCATGGAGTAGGATGTGCCCCAG250GAAATCATGGAGTAGGATGTGCCCC251AGGAAATCATGGAGTAGGATGTGCC252GCAGGAAATCATGGAGTAGGATGTG253CAGCAGGAAATCATGGAGTAGGATG254ACCAGCAGGAAATCATGGAGTAGGA255GAACCAGCAGGAAATCATGGAGTAG256GGGAACCAGCAGGAAATCATGGAGT257CGGGGAACCAGCAGGAAATCATGGA258CTCGGGGAACCAGCAGGAAATCATG259CTCTCGGGGAACCAGCAGGAAATCA260AGCTCTCGGGGAACCAGCAGGAAAT261TCAGCTCTCGGGGAACCAGCAGGAA262ACTCAGCTCTCGGGGAACCAGCAGG263CCACTCAGCTCTCGGGGAACCAGCA264AGCCACTCAGCTCTCGGGGAACCAG265GGAGCCACTCAGCTCTCGGGGAACC266GAGGAGCCACTCAGCTCTCGGGGAA267GGGAGGAGCCACTCAGCTCTCGGGG268TGGGGAGGAGCCACTCAGCTCTCGG269ACTGGGGAGGAGCCACTCAGCTCTC270GGACTGGGGAGGAGCCACTCAGCTC271CAGGACTGGGGAGGAGCCACTCAGC272TCCAGGACTGGGGAGGAGCCACTCA273CCTCCAGGACTGGGGAGGAGCCACT274CTCCTCCAGGACTGGGGAGGAGCCA275GTCTCCTCCAGGACTGGGGAGGAGC276GAGTCTCCTCCAGGACTGGGGAGGA277GTGAGTCTCCTCCAGGACTGGGGAG278GGGTGAGTCTCCTCCAGGACTGGGG279CTGGGTGAGTCTCCTCCAGGACTGG280AGCTGGGTGAGTCTCCTCCAGGACT281TGAGCTGGGTGAGTCTCCTCCAGGA282GGTGAGCTGGGTGAGTCTCCTCCAG283CTGGTGAGCTGGGTGAGTCTCCTCC284TGCTGGTGAGCTGGGTGAGTCTCCT285CCTGCTGGTGAGCTGGGTGAGTCTC286TCCCTGCTGGTGAGCTGGGTGAGTC287GCTCCCTGCTGGTGAGCTGGGTGAG288TGGCTCCCTGCTGGTGAGCTGGGTG289GCTGGCTCCCTGCTGGTGAGCTGGG290CTGCTGGCTCCCTGCTGGTGAGCTG291GTCTGCTGGCTCCCTGCTGGTGAGC292GATGTGCCCCAGGAGTGCAGCGGTT293AGGATGTGCCCCAGGAGTGCAGCGG294GTAGGATGTGCCCCAGGAGTGCAGC295GAGTAGGATGTGCCCCAGGAGTGCA296TGGAGTAGGATGTGCCCCAGGAGTG297CATGGAGTAGGATGTGCCCCAGGAG298ATCATGGAGTAGGATGTGCCCCAGG299AAATCATGGAGTAGGATGTGCCCCA300GGAAATCATGGAGTAGGATGTGCCC301CAGGAAATCATGGAGTAGGATGTGC302AGCAGGAAATCATGGAGTAGGATGT303CCAGCAGGAAATCATGGAGTAGGAT304AACCAGCAGGAAATCATGGAGTAGG305GGAACCAGCAGGAAATCATGGAGTA306GGGGAACCAGCAGGAAATCATGGAG307TCGGGGAACCAGCAGGAAATCATGG308TCTCGGGGAACCAGCAGGAAATCAT309GCTCTCGGGGAACCAGCAGGAAATC310CAGCTCTCGGGGAACCAGCAGGAAA311CTCAGCTCTCGGGGAACCAGCAGGA312CACTCAGCTCTCGGGGAACCAGCAG313GCCACTCAGCTCTCGGGGAACCAGC314GAGCCACTCAGCTCTCGGGGAACCA315AGGAGCCACTCAGCTCTCGGGGAAC316GGAGGAGCCACTCAGCTCTCGGGGA317GGGGAGGAGCCACTCAGCTCTCGGG318CTGGGGAGGAGCCACTCAGCTCTCG319GACTGGGGAGGAGCCACTCAGCTCT320AGGACTGGGGAGGAGCCACTCAGCT321CCAGGACTGGGGAGGAGCCACTCAG322CTCCAGGACTGGGGAGGAGCCACTC323TCCTCCAGGACTGGGGAGGAGCCAC324TCTCCTCCAGGACTGGGGAGGAGCC325AGTCTCCTCCAGGACTGGGGAGGAG326TGAGTCTCCTCCAGGACTGGGGAGG327GGTGAGTCTCCTCCAGGACTGGGGA328TGGGTGAGTCTCCTCCAGGACTGGG329GCTGGGTGAGTCTCCTCCAGGACTG330GAGCTGGGTGAGTCTCCTCCAGGAC331GTGAGCTGGGTGAGTCTCCTCCAGG332TGGTGAGCTGGGTGAGTCTCCTCCA333GCTGGTGAGCTGGGTGAGTCTCCTC334CTGCTGGTGAGCTGGGTGAGTCTCC335CCCTGCTGGTGAGCTGGGTGAGTCT336CTCCCTGCTGGTGAGCTGGGTGAGT337GGCTCCCTGCTGGTGAGCTGGGTGA338CTGGCTCCCTGCTGGTGAGCTGGGT339TGCTGGCTCCCTGCTGGTGAGCTGG340TCTGCTGGCTCCCTGCTGGTGAGCT341GGTCTGCTGGCTCCCTGCTGGTGAG342c.348G > A, c.373C > TAGCCCCTGCTTTGCAGGGATGTAGC343GCAGCCCCTGCTTTGCAGGGATGTA344CTGCAGCCCCTGCTTTGCAGGGATG345CCCTGCAGCCCCTGCTTTGCAGGGA346CTCCCTGCAGCCCCTGCTTTGCAGG347GGCTCCCTGCAGCCCCTGCTTTGCA348TGGGCTCCCTGCAGCCCCTGCTTTG349TCTGGGCTCCCTGCAGCCCCTGCTT350CATCTGGGCTCCCTGCAGCCCCTGC351CCCATCTGGGCTCCCTGCAGCCCCT352GCCCCATCTGGGCTCCCTGCAGCCC353CTGCCCCATCTGGGCTCCCTGCAGC354GGCTGCCCCATCTGGGCTCCCTGCA355AGGGCTGCCCCATCTGGGCTCCCTG356CCAGGGCTGCCCCATCTGGGCTCCC357CACCAGGGCTGCCCCATCTGGGCTC358AGCACCAGGGCTGCCCCATCTGGGC359GAAGCACCAGGGCTGCCCCATCTGG360AAGAAGCACCAGGGCTGCCCCATCT361GGAAGAAGCACCAGGGCTGCCCCAT362TGGGAAGAAGCACCAGGGCTGCCCC363GGTGGGAAGAAGCACCAGGGCTGCC364TGGGTGGGAAGAAGCACCAGGGCTG365GCTGGGTGGGAAGAAGCACCAGGGC366GCCCCTGCTTTGCAGGGATGTAGCA367CAGCCCCTGCTTTGCAGGGATGTAG368TGCAGCCCCTGCTTTGCAGGGATGT369CCTGCAGCCCCTGCTTTGCAGGGAT370TCCCTGCAGCCCCTGCTTTGCAGGG371GCTCCCTGCAGCCCCTGCTTTGCAG372GGGCTCCCTGCAGCCCCTGCTTTGC373CTGGGCTCCCTGCAGCCCCTGCTTT374ATCTGGGCTCCCTGCAGCCCCTGCT375CCATCTGGGCTCCCTGCAGCCCCTG376CCCCATCTGGGCTCCCTGCAGCCCC377TGCCCCATCTGGGCTCCCTGCAGCC378GCTGCCCCATCTGGGCTCCCTGCAG379GGGCTGCCCCATCTGGGCTCCCTGC380CAGGGCTGCCCCATCTGGGCTCCCT381ACCAGGGCTGCCCCATCTGGGCTCC382GCACCAGGGCTGCCCCATCTGGGCT383AAGCACCAGGGCTGCCCCATCTGGG384AGAAGCACCAGGGCTGCCCCATCTG385GAAGAAGCACCAGGGCTGCCCCATC386GGGAAGAAGCACCAGGGCTGCCCCA387GTGGGAAGAAGCACCAGGGCTGCCC388GGGTGGGAAGAAGCACCAGGGCTGC389CTGGGTGGGAAGAAGCACCAGGGCT390AGCTGGGTGGGAAGAAGCACCAGGG391c.413T > ACAGCTTGTAGCTGGGGTAGCTGGGT392TCCAGCTTGTAGCTGGGGTAGCTGG393TCTCCAGCTTGTAGCTGGGGTAGCT394GTTCTCCAGCTTGTAGCTGGGGTAG395AGGTTCTCCAGCTTGTAGCTGGGGT396TCAGGTTCTCCAGCTTGTAGCTGGG397GCTCAGGTTCTCCAGCTTGTAGCTG398GAGCTCAGGTTCTCCAGCTTGTAGC399AGGAGCTCAGGTTCTCCAGCTTGTA400AGAGGAGCTCAGGTTCTCCAGCTTG401TCAGAGGAGCTCAGGTTCTCCAGCT402TTTCAGAGGAGCTCAGGTTCTCCAG403AGCTTGTAGCTGGGGTAGCTGGGTG404CCAGCTTGTAGCTGGGGTAGCTGGG405CTCCAGCTTGTAGCTGGGGTAGCTG406TTCTCCAGCTTGTAGCTGGGGTAGC407GGTTCTCCAGCTTGTAGCTGGGGTA408CAGGTTCTCCAGCTTGTAGCTGGGG409CTCAGGTTCTCCAGCTTGTAGCTGG410AGCTCAGGTTCTCCAGCTTGTAGCT411GGAGCTCAGGTTCTCCAGCTTGTAG412GAGGAGCTCAGGTTCTCCAGCTTGT413CAGAGGAGCTCAGGTTCTCCAGCTT414TTCAGAGGAGCTCAGGTTCTCCAGC415ATTTCAGAGGAGCTCAGGTTCTCCA416c.469C > T, c.476T > C,GGGGTGGTACGGGTCAGGGTGGCCG417c.476T > G, c.478T > G,TGGGGGTGGTACGGGTCAGGGTGGC418c.482C > TGGTGGGGGTGGTACGGGTCAGGGTG419AAGGTGGGGGTGGTACGGGTCAGGG420AGAAGGTGGGGGTGGTACGGGTCAG421GAAGAAGGTGGGGGTGGTACGGGTC422GGGAAGAAGGTGGGGGTGGTACGGG423TGGGGAAGAAGGTGGGGGTGGTACG424CTTGGGGAAGAAGGTGGGGGTGGTA425TCCTTGGGGAAGAAGGTGGGGGTGG426TGTCCTTGGGGAAGAAGGTGGGGGT427GATGTCCTTGGGGAAGAAGGTGGGG428AGGATGTCCTTGGGGAAGAAGGTGG429TCAGGATGTCCTTGGGGAAGAAGGT430GGTCAGGATGTCCTTGGGGAAGAAG431AGGGTCAGGATGTCCTTGGGGAAGA432GCAGGGTCAGGATGTCCTTGGGGAA433CCGCAGGGTCAGGATGTCCTTGGGG434AGCCGCAGGGTCAGGATGTCCTTGG435GGGTGGTACGGGTCAGGGTGGCCGT436GGGGGTGGTACGGGTCAGGGTGGCC437GTGGGGGTGGTACGGGTCAGGGTGG438AGGTGGGGGTGGTACGGGTCAGGGT439GAAGGTGGGGGTGGTACGGGTCAGG440AAGAAGGTGGGGGTGGTACGGGTCA441GGAAGAAGGTGGGGGTGGTACGGGT442GGGGAAGAAGGTGGGGGTGGTACGG443TTGGGGAAGAAGGTGGGGGTGGTAC444CCTTGGGGAAGAAGGTGGGGGTGGT445GTCCTTGGGGAAGAAGGTGGGGGTG446ATGTCCTTGGGGAAGAAGGTGGGGG447GGATGTCCTTGGGGAAGAAGGTGGG448CAGGATGTCCTTGGGGAAGAAGGTG449GTCAGGATGTCCTTGGGGAAGAAGG450GGGTCAGGATGTCCTTGGGGAAGAA451CAGGGTCAGGATGTCCTTGGGGAAG452CGCAGGGTCAGGATGTCCTTGGGGA453GCCGCAGGGTCAGGATGTCCTTGGG454CAGCCGCAGGGTCAGGATGTCCTTG455c.510C > T, c.515T > A,CGTCCAGCCGCAGGGTCAGGATGTC456c.520G > ACACGTCCAGCCGCAGGGTCAGGATG457ATCACGTCCAGCCGCAGGGTCAGGA458TCATCACGTCCAGCCGCAGGGTCAG459CATCATCACGTCCAGCCGCAGGGTC460TCCATCATCACGTCCAGCCGCAGGG461TCTCCATCATCACGTCCAGCCGCAG462AGTCTCCATCATCACGTCCAGCCGC463TCAGTCTCCATCATCACGTCCAGCC464TCTCAGTCTCCATCATCACGTCCAG465GTTCTCAGTCTCCATCATCACGTCC466CGGTTCTCAGTCTCCATCATCACGT467GGCGGTTCTCAGTCTCCATCATCAC468GAGGCGGTTCTCAGTCTCCATCATC469TGGAGGCGGTTCTCAGTCTCCATCA470AGTGGAGGCGGTTCTCAGTCTCCAT471GAAGTGGAGGCGGTTCTCAGTCTCC472GTCCAGCCGCAGGGTCAGGATGTCC473ACGTCCAGCCGCAGGGTCAGGATGT474TCACGTCCAGCCGCAGGGTCAGGAT475CATCACGTCCAGCCGCAGGGTCAGG476ATCATCACGTCCAGCCGCAGGGTCA477CCATCATCACGTCCAGCCGCAGGGT478CTCCATCATCACGTCCAGCCGCAGG479GTCTCCATCATCACGTCCAGCCGCA480CAGTCTCCATCATCACGTCCAGCCG481CTCAGTCTCCATCATCACGTCCAGC482TTCTCAGTCTCCATCATCACGTCCA483GGTTCTCAGTCTCCATCATCACGTC484GCGGTTCTCAGTCTCCATCATCACG485AGGCGGTTCTCAGTCTCCATCATCA486GGAGGCGGTTCTCAGTCTCCATCAT487GTGGAGGCGGTTCTCAGTCTCCATC488AAGTGGAGGCGGTTCTCAGTCTCCA489TGAAGTGGAGGCGGTTCTCAGTCTC490c.546+11C > T,TGCCCTGCCCACCGTGAAGTGGAGG491c.546+14G > A,CCTGCCCTGCCCACCGTGAAGTGGA492c.546+19G > A,CCCCTGCCCTGCCCACCGTGAAGTG493c.546+23C > ACGCCCCTGCCCTGCCCACCGTGAAG494CCCGCCCCTGCCCTGCCCACCGTGA495GCCCTGCCCACCGTGAAGTGGAGGC496CTGCCCTGCCCACCGTGAAGTGGAG497CCCTGCCCTGCCCACCGTGAAGTGG498GCCCCTGCCCTGCCCACCGTGAAGT499CCGCCCCTGCCCTGCCCACCGTGAA500CCCCGCCCCTGCCCTGCCCACCGTG501GCCCCCGCCCCTGCCCTGCCCACCG502CCGCCCCCGCCCCTGCCCTGCCCAC503CGCCGCCCCCGCCCCTGCCCTGCCC504GCCGCCGCCCCCGCCCCTGCCCTGC505TGGCCGCCGCCCCCGCCCCTGCCCT506CCTGGCCGCCGCCCCCGCCCCTGCC507GCCCTGGCCGCCGCCCCCGCCCCTG508CTGCCCTGGCCGCCGCCCCCGCCCC509CTCTGCCCTGGCCGCCGCCCCCGCC510CCCTCTGCCCTGGCCGCCGCCCCCG511CACCCTCTGCCCTGGCCGCCGCCCC512CGCACCCTCTGCCCTGGCCGCCGCC513CGCGCACCCTCTGCCCTGGCCGCCG514CCCCCGCCCCTGCCCTGCCCACCGT515CGCCCCCGCCCCTGCCCTGCCCACC516GCCGCCCCCGCCCCTGCCCTGCCCA517CCGCCGCCCCCGCCCCTGCCCTGCC518GGCCGCCGCCCCCGCCCCTGCCCTG519CTGGCCGCCGCCCCCGCCCCTGCCC520CCCTGGCCGCCGCCCCCGCCCCTGC521TGCCCTGGCCGCCGCCCCCGCCCCT522TCTGCCCTGGCCGCCGCCCCCGCCC523CCTCTGCCCTGGCCGCCGCCCCCGC524ACCCTCTGCCCTGGCCGCCGCCCCC525GCACCCTCTGCCCTGGCCGCCGCCC526GCGCACCCTCTGCCCTGGCCGCCGC527c.547-6AGAGATGGGGGTTTATTGATGTTCC528GAAGAGATGGGGGTTTATTGATGTT529TAGAAGAGATGGGGGTTTATTGATG530TCTAGAAGAGATGGGGGTTTATTGA531GATCTAGAAGAGATGGGGGTTTATT532TTGATCTAGAAGAGATGGGGGTTTA533CTTTGATCTAGAAGAGATGGGGGTT534ATCTTTGATCTAGAAGAGATGGGGG535GGATCTTTGATCTAGAAGAGATGGG536CTGGATCTTTGATCTAGAAGAGATG537AGCTGGATCTTTGATCTAGAAGAGA538TTAGCTGGATCTTTGATCTAGAAGA539TGTTAGCTGGATCTTTGATCTAGAA540 In the above examples the sequences are 25 nucleotides long however longer variants or shorter fragment are also envisioned. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO:41-540 and fragments and variants thereof having at least 80% sequence identity. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO: 41-540 and fragments and variants thereof having at least 80%, 83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% sequence identity to SEQ ID NO: 41-540. The present invention is also directed to sequences that are at least 80% identical to SEQ ID NO: 41-540. Preferably at least 85% identical to SEQ ID NO: 41-540, more preferably at least 88% identical to SEQ ID NO: 41-540, more preferably at least 90% identical to SEQ ID NO: 41-540. more preferably at least 91% identical to SEQ ID NO: 41-540, more preferably at least 92% identical to SEQ ID NO: 41-540, more preferably at least 93% identical to SEQ ID NO: 41-540, more preferably at least 94% identical to SEQ ID NO: 41-540, more preferably at least 95% identical to SEQ ID NO: 41-540, more preferably at least 96% identical to SEQ ID NO: 41-540, more preferably at least 97% identical to SEQ ID NO: 41-540, more preferably at least 98% identical to SEQ ID NO: 41-540, more preferably at least 99% identical to SEQ ID NO: 41-540. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 41-540, wherein the fragment is 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 41-540, wherein the fragment is 17, 18, 19, 20, 21, or 22 nucleotides long. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 41-540, wherein the fragment is 19, 20, or 21 nucleotides long. In a preferred embodiment of the invention and/or embodiments thereof the target sequence provides exclusion of intron 6. It was found that SEQ ID NO: 1584 provides the target sequence for exclusion of intron 6. In a preferred embodiment of the invention and/or embodiments thereof of an aspect and/or embodiments of the invention the target sequence is the AACCCCAGAGCTGCTTCCCTTCCAGATGTGGTCCTGCAGCCGAGCCCTGCCCT TAGCTGGAGGTCGACAGGTGGGATCCTGGATGTCTACATCTTCCTGGGCCCAG AGCCCAAGAGCGTGGTGCAGCAGTACCTGGACGTTGTGGGTAGGGCCTGCTC CCTGGCCGCGGCCCCCGCCCCAAGGCTCCCTCCTCCCTCCCTCATGAAGTCGG CGTTGGCCTGCAGGATACCCGTTCATGCCGCCATACTGGGGCCTGGGCTTCCA CCTGTGCCGCTGGGGCTACTCCTCCACCGCTATCACCCGCCAGGTGGTGGAGA ACATGACCAGGGCCCACTTCCCCCTGGTGAGTTGGGGTGGTGGCAGGGGAG (SEQ ID NO: 1584). It should be noted that also naturally occurring single nucleotide polymorphism are included. Also the following genomic sequences are target sequences for exclusion of intron 6 of GAA: Sequence incDNA to whichAON anneals*sequence of region (5′→3′):Seq IDc.956-AACCCCAGAGCTGCTTCCCTTCCAGATGTGGTCCTGC158425_1194+25AGCCGAGCCCTGCCCTTAGCTGGAGGTCGACAGGTGGGATCCTGGATGTCTACATCTTCCTGGGCCCAGAGCCCAAGAGCGTGGTGCAGCAGTACCTGGACGTTGTGGGTAGGGCCTGCTCCCTGGCCGCGGCCCCCGCCCCAAGGCTCCCTCCTCCCTCCCTCATGAAGTCGGCGTTGGCCTGCAGGATACCCGTTCATGCCGCCATACTGGGGCCTGGGCTTCCACCTGTGCCGCTGGGGCTACTCCTCCACCGCTATCACCCGCCAGGTGGTGGAGAACATGACCAGGGCCCACTTCCCCCTGGTGAGTTGGGGTGGTGGCAGGGGAGc.956-25_1004AACCCCAGAGCTGCTTCCCTTCCAGATGTGGTCCTGC1585AGCCGAGCCCTGCCCTTAGCTGGAGGTCGACAGGTGGc.1005_1075+3GATCCTGGATGTCTACATCTTCCTGGGCCCAGAGCC1586CAAGAGCGTGGTGCAGCAGTACCTGGACGTTGTGGGTAc.1075+4_1076-2GGGCCTGCTCCCTGGCCGCGGCCCCCGCCCCAAGGC1587TCCCTCCTCCCTCCCTCATGAAGTCGGCGTTGGCCTGCc.1076-2_1147AGGATACCCGTTCATGCCGCCATACTGGGGCCTGGG1588CTTCCACCTGTGCCGCTGGGGCTACTCCTCCACCGCTAc.1148_1194+25TCACCCGCCAGGTGGTGGAGAACATGACCAGGGCCC1589ACTTCCCCCTGGTGAGTTGGGGTGGTGGCAGGGGAG It is to be noted that targeting means that at least part of the sequence SEQ ID NO: 1584-1589 is targeted, e.g. by a sequence that hybridizes with at least a part or by the sequence SEQ ID NO: 1584-1589, or that binds to at least a part of SEQ ID NO: 1584-1589. Sequences that target may be shorter or longer than the target sequence. Suitably the sequences targeting SEQ ID NO: 1584-1589 hybridize with at least a part of SEQ ID NO: 1584-1589. Sequences that hybridize may be shorter or longer than the target sequence. Nucleotide sequences SEQ ID NO: 541-1583 are oligomers that are able to enhance GAA intron 6 exclusion. In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound selected from the group comprising SEQ ID NO: 541-1583 and variants and fragments having at least 80% identity thereof. The antisense oligomeric compound may also target single nucleotide polymorphism of SEQ ID NO: 1584-1589. It should be noted that it may not necessary to have the full length of SEQ ID NO: 541-1583, fragments having a shorter or longer sequence are also envisioned. The inventors have found the target genomic sequence which enables the exclusion of intron 6 and a skilled person is capable of finding suitable sequences that target this target genomic sequence, such as SEQ ID NO: 1584-1589 and single nucleotide polymorphisms thereof. Exemplary sequences that target this target genomic sequence, such as SEQ ID NO: 1584-1589 may be SEQ ID NO: 541-1583, but also variants and fragments having at least 80% identity thereof. In particular shorter fragments such as fragments with 18, 19, 20, 21, 22, 23, or 24 nucleotides of SEQ ID NO: 541-1583 are envisioned. In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound complementary to a polynucleotide having a sequence selected from the group comprising SEQ ID NO: 1584-1589 and single nucleotide polymorphisms thereof. Also sequences having at least 80% identity to antisense oligomeric compound complementary to a polynucleotide having a sequence selected from the group comprising SEQ ID NO: 1584-1589 are envisioned. Antisense oligomeric compound that target one or more than one single nucleotide polymorphisms may be designed. In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound targeting a sequence selected from the group comprising the genomic sequence c.956−25_1194+25. In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound comprising sequences selected from the group comprising SEQ ID NO: 41-1583 and sequences having at least 80% identity thereof. In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to antisense oligomeric compound comprising a sequences selected from the group comprising SEQ ID NO: 541-1583. Antisense oligomeric compounds targeting SEQ ID NO: 1584 are a very suitable to treat Pompe patients. Exemplary antisense oligomeric compounds targeting SEQ ID NO: 1584 are SEQ ID NO: 541-1853. However the invention is not limited to these sequences. A skilled person is capable of designing antisense oligomeric compounds against target sequence SEQ ID NO: 1584, 1885, 1586, 1587, 1588, 1589. The antisense oligomeric compounds against target sequenced SEQ ID NO: 1584, 1885, 1586, 1587, 1588, or 1589 may have length of 10 to 100 nucleotides, preferably 11 to 75 nucleotides, preferably 12 to 73 nucleotides, preferably 13 to 70 nucleotides, preferably 14 to 65 nucleotides, preferably 15 to 60 nucleotides, preferably 16 to 55 nucleotides, preferably 17 to 50 nucleotides, preferably 18 to 45 nucleotides, preferably 19 to 40 nucleotides, preferably 20 to 38 nucleotides, preferably 21 to 35 nucleotides, preferably 22 to 33 nucleotides, preferably 23 to 30 nucleotides, preferably 24 to 29 nucleotides, preferably 25 to 28 nucleotides, preferably 26 to 27 nucleotides. The antisense oligomeric compounds may be selected from the group of SEQ ID NO541-1583: Sequence in cDNAto which AONanneals forSeqintron 6 exclusionAON sequence (5′→3′)IDc.956-25_-1CTGGAAGGGAAGCAGCTCTGGGGTT541c.956-24_956TCTGGAAGGGAAGCAGCTCTGGGGT542c.956-23_957ATCTGGAAGGGAAGCAGCTCTGGGG543c.956-22_958CATCTGGAAGGGAAGCAGCTCTGGG544c.956-21_959ACATCTGGAAGGGAAGCAGCTCTGG545c.956-20_960CACATCTGGAAGGGAAGCAGCTCTG546c.956-19_961CCACATCTGGAAGGGAAGCAGCTCT547c.956-18_962ACCACATCTGGAAGGGAAGCAGCTC548c.956-17_963GACCACATCTGGAAGGGAAGCAGCT549c.956-16_964GGACCACATCTGGAAGGGAAGCAGC550c.956-15_965AGGACCACATCTGGAAGGGAAGCAG551c.956-14_966CAGGACCACATCTGGAAGGGAAGCA552c.956-13_967GCAGGACCACATCTGGAAGGGAAGC553c.956-12_968TGCAGGACCACATCTGGAAGGGAAG554c.956-11_969CTGCAGGACCACATCTGGAAGGGAA555c.956-10_970GCTGCAGGACCACATCTGGAAGGGA556c.956-9_971GGCTGCAGGACCACATCTGGAAGGG557c.956-8_972CGGCTGCAGGACCACATCTGGAAGG558c.956-7_973TCGGCTGCAGGACCACATCTGGAAG559c.956-6_974CTCGGCTGCAGGACCACATCTGGAA560c.956-5_975GCTCGGCTGCAGGACCACATCTGGA561c.956-4_976GGCTCGGCTGCAGGACCACATCTGG562c.956-3_977GGGCTCGGCTGCAGGACCACATCTG563c.956-2_978AGGGCTCGGCTGCAGGACCACATCT564c.956-1_979CAGGGCTCGGCTGCAGGACCACATC565c.956_980GCAGGGCTCGGCTGCAGGACCACAT566c.957_981GGCAGGGCTCGGCTGCAGGACCACA567c.958_982GGGCAGGGCTCGGCTGCAGGACCAC568c.959_983AGGGCAGGGCTCGGCTGCAGGACCA569c.960_984AAGGGCAGGGCTCGGCTGCAGGACC570c.961_985TAAGGGCAGGGCTCGGCTGCAGGAC571c.962_986CTAAGGGCAGGGCTCGGCTGCAGGA572c.963_987GCTAAGGGCAGGGCTCGGCTGCAGG573c.964_988AGCTAAGGGCAGGGCTCGGCTGCAG574c.965_989CAGCTAAGGGCAGGGCTCGGCTGCA575c.966_990CCAGCTAAGGGCAGGGCTCGGCTGC576c.967_991TCCAGCTAAGGGCAGGGCTCGGCTG577c.968_992CTCCAGCTAAGGGCAGGGCTCGGCT578c.969_993CCTCCAGCTAAGGGCAGGGCTCGGC579c.970_994ACCTCCAGCTAAGGGCAGGGCTCGG580c.971_995GACCTCCAGCTAAGGGCAGGGCTCG581c.972_996CGACCTCCAGCTAAGGGCAGGGCTC582c.973_997TCGACCTCCAGCTAAGGGCAGGGCT583c.974_998GTCGACCTCCAGCTAAGGGCAGGGC584c.975_999TGTCGACCTCCAGCTAAGGGCAGGG585c.976_1000CTGTCGACCTCCAGCTAAGGGCAGG586c.977_1001CCTGTCGACCTCCAGCTAAGGGCAG587c.978_1002ACCTGTCGACCTCCAGCTAAGGGCA588c.979_1003CACCTGTCGACCTCCAGCTAAGGGC589c.980_1004CCACCTGTCGACCTCCAGCTAAGGG590c.981_1005CCCACCTGTCGACCTCCAGCTAAGG591c.982_1006TCCCACCTGTCGACCTCCAGCTAAG592c.983_1007ATCCCACCTGTCGACCTCCAGCTAA593c.984_1008GATCCCACCTGTCGACCTCCAGCTA594c.985_1009GGATCCCACCTGTCGACCTCCAGCT595c.986_1010AGGATCCCACCTGTCGACCTCCAGC596c.987_1011CAGGATCCCACCTGTCGACCTCCAG597c.988_1012CCAGGATCCCACCTGTCGACCTCCA598c.989_1013TCCAGGATCCCACCTGTCGACCTCC599c.990_1014ATCCAGGATCCCACCTGTCGACCTC600c.991_1015CATCCAGGATCCCACCTGTCGACCT601c.992_1016ACATCCAGGATCCCACCTGTCGACC602c.993_1017GACATCCAGGATCCCACCTGTCGAC603c.994_1018AGACATCCAGGATCCCACCTGTCGA604c.995_1019TAGACATCCAGGATCCCACCTGTCG605c.996_1020GTAGACATCCAGGATCCCACCTGTC606c.997_1021TGTAGACATCCAGGATCCCACCTGT607c.998_1022ATGTAGACATCCAGGATCCCACCTG608c.999_1023GATGTAGACATCCAGGATCCCACCT609c.1000_1024AGATGTAGACATCCAGGATCCCACC610c.1001_1025AAGATGTAGACATCCAGGATCCCAC611c.1002_1026GAAGATGTAGACATCCAGGATCCCA612c.1003_1027GGAAGATGTAGACATCCAGGATCCC613c.1004_1028AGGAAGATGTAGACATCCAGGATCC614c.1005_1029CAGGAAGATGTAGACATCCAGGATC615c.1006_1030CCAGGAAGATGTAGACATCCAGGAT616c.1007_1031CCCAGGAAGATGTAGACATCCAGGA617c.1008_1032GCCCAGGAAGATGTAGACATCCAGG618c.1009_1033GGCCCAGGAAGATGTAGACATCCAG619c.1010_1034GGGCCCAGGAAGATGTAGACATCCA620c.1011_1035TGGGCCCAGGAAGATGTAGACATCC621c.1012_1036CTGGGCCCAGGAAGATGTAGACATC622c.1013_1037TCTGGGCCCAGGAAGATGTAGACAT623c.1014_1038CTCTGGGCCCAGGAAGATGTAGACA624c.1015_1039GCTCTGGGCCCAGGAAGATGTAGAC625c.1016_1040GGCTCTGGGCCCAGGAAGATGTAGA626c.1017_1041GGGCTCTGGGCCCAGGAAGATGTAG627c.1018_1042TGGGCTCTGGGCCCAGGAAGATGTA628c.1019_1043TTGGGCTCTGGGCCCAGGAAGATGT629c.1020_1044CTTGGGCTCTGGGCCCAGGAAGATG630c.1021_1045TCTTGGGCTCTGGGCCCAGGAAGAT631c.1022_1046CTCTTGGGCTCTGGGCCCAGGAAGA632c.1023_1047GCTCTTGGGCTCTGGGCCCAGGAAG633c.1024_1048CGCTCTTGGGCTCTGGGCCCAGGAA634c.1025_1049ACGCTCTTGGGCTCTGGGCCCAGGA635c.1026_1050CACGCTCTTGGGCTCTGGGCCCAGG636c.1027_1051CCACGCTCTTGGGCTCTGGGCCCAG637c.1028_1052ACCACGCTCTTGGGCTCTGGGCCCA638c.1029_1053CACCACGCTCTTGGGCTCTGGGCCC639c.1030_1054GCACCACGCTCTTGGGCTCTGGGCC640c.1031_1055TGCACCACGCTCTTGGGCTCTGGGC641c.1032_1056CTGCACCACGCTCTTGGGCTCTGGG642c.1033_1057GCTGCACCACGCTCTTGGGCTCTGG643c.1034_1058TGCTGCACCACGCTCTTGGGCTCTG644c.1035_1059CTGCTGCACCACGCTCTTGGGCTCT645c.1036_1060ACTGCTGCACCACGCTCTTGGGCTC646c.1037_1061TACTGCTGCACCACGCTCTTGGGCT647c.1038_1062GTACTGCTGCACCACGCTCTTGGGC648c.1039_1063GGTACTGCTGCACCACGCTCTTGGG649c.1040_1064AGGTACTGCTGCACCACGCTCTTGG650c.1041_1065CAGGTACTGCTGCACCACGCTCTTG651c.1042_1066CCAGGTACTGCTGCACCACGCTCTT652c.1043_1067TCCAGGTACTGCTGCACCACGCTCT653c.1044_1068GTCCAGGTACTGCTGCACCACGCTC654c.1045_1069CGTCCAGGTACTGCTGCACCACGCT655c.1046_1070ACGTCCAGGTACTGCTGCACCACGC656c.1047_1071AACGTCCAGGTACTGCTGCACCACG657c.1048_1072CAACGTCCAGGTACTGCTGCACCAC658c.1049_1073ACAACGTCCAGGTACTGCTGCACCA659c.1050_1074CACAACGTCCAGGTACTGCTGCACC660c.1051_1075CCACAACGTCCAGGTACTGCTGCAC661c.1052_1075+1CCCACAACGTCCAGGTACTGCTGCA662c.1053_1075+2ACCCACAACGTCCAGGTACTGCTGC663c.1054_1075+3TACCCACAACGTCCAGGTACTGCTG664c.1055_1075+4CTACCCACAACGTCCAGGTACTGCT665c.1056_1075+5CCTACCCACAACGTCCAGGTACTGC666c.1057_1075+6CCCTACCCACAACGTCCAGGTACTG667c.1058_1075+7GCCCTACCCACAACGTCCAGGTACT668c.1059_1075+8GGCCCTACCCACAACGTCCAGGTAC669c.1060_1075+9AGGCCCTACCCACAACGTCCAGGTA670c.1061_1075+10CAGGCCCTACCCACAACGTCCAGGT671c.1062_1075+11GCAGGCCCTACCCACAACGTCCAGG672c.1063_1075+12AGCAGGCCCTACCCACAACGTCCAG673c.1064_1075+13GAGCAGGCCCTACCCACAACGTCCA674c.1065_1075+14GGAGCAGGCCCTACCCACAACGTCC675c.1066_1075+15GGGAGCAGGCCCTACCCACAACGTC676c.1067_1075+16AGGGAGCAGGCCCTACCCACAACGT677c.1068_1075+17CAGGGAGCAGGCCCTACCCACAACG678c.1069_1075+18CCAGGGAGCAGGCCCTACCCACAAC679c.1070_1075+19GCCAGGGAGCAGGCCCTACCCACAA680c.1071_1075+20GGCCAGGGAGCAGGCCCTACCCACA681c.1072_1075+21CGGCCAGGGAGCAGGCCCTACCCAC682c.1073_1075+22GCGGCCAGGGAGCAGGCCCTACCCA683c.1074_1075+23CGCGGCCAGGGAGCAGGCCCTACCC684c.1075_1075+24CCGCGGCCAGGGAGCAGGCCCTACC685C.1075+1_+25GCCGCGGCCAGGGAGCAGGCCCTAC686C.1075+2_+26GGCCGCGGCCAGGGAGCAGGCCCTA687C.1075+3_+27GGGCCGCGGCCAGGGAGCAGGCCCT688C.1075+4_+28GGGGCCGCGGCCAGGGAGCAGGCCC689C.1075+5_+29GGGGGCCGCGGCCAGGGAGCAGGCC690C.1075+6_+30CGGGGGCCGCGGCCAGGGAGCAGGC691C.1075+7_+31GCGGGGGCCGCGGCCAGGGAGCAGG692C.1075+8_+32GGCGGGGGCCGCGGCCAGGGAGCAG693C.1075+9_+33GGGCGGGGGCCGCGGCCAGGGAGCA694C.1075+10_+34GGGGCGGGGGCCGCGGCCAGGGAGC695C.1075+11_+35TGGGGCGGGGGCCGCGGCCAGGGAG696C.1075+12_+36TTGGGGCGGGGGCCGCGGCCAGGGA697C.1075+13_+37CTTGGGGCGGGGGCCGCGGCCAGGG698C.1075+14_+38CCTTGGGGCGGGGGCCGCGGCCAGG699C.1075+15_+39GCCTTGGGGCGGGGGCCGCGGCCAG700C.1075+16_+40AGCCTTGGGGCGGGGGCCGCGGCCA701C.1075+17_1076-39GAGCCTTGGGGCGGGGGCCGCGGCC702C.1075+18_1076-38GGAGCCTTGGGGCGGGGGCCGCGGC703C.1075+19_1076-37GGGAGCCTTGGGGCGGGGGCCGCGG704C.1075+20_1076-36AGGGAGCCTTGGGGCGGGGGCCGCG705C.1075+21_1076-35GAGGGAGCCTTGGGGCGGGGGCCGC706C.1075+22_1076-34GGAGGGAGCCTTGGGGCGGGGGCCG707C.1075+23_1076-33AGGAGGGAGCCTTGGGGCGGGGGCC708C.1075+24_1076-32GAGGAGGGAGCCTTGGGGCGGGGGC709C.1075+25_1076-31GGAGGAGGGAGCCTTGGGGCGGGGG710C.1075+26_1076-30GGGAGGAGGGAGCCTTGGGGCGGGG711C.1075+27_1076-29AGGGAGGAGGGAGCCTTGGGGCGGG712C.1075+28_1076-28GAGGGAGGAGGGAGCCTTGGGGCGG713C.1075+29_1076-27GGAGGGAGGAGGGAGCCTTGGGGCG714C.1075+30_1076-26GGGAGGGAGGAGGGAGCCTTGGGGC715C.1075+31_1076-25AGGGAGGGAGGAGGGAGCCTTGGGG716C.1075+32_1076-24GAGGGAGGGAGGAGGGAGCCTTGGG717C.1075+33_1076-23TGAGGGAGGGAGGAGGGAGCCTTGG718C.1075+34_1076-22ATGAGGGAGGGAGGAGGGAGCCTTG719C.1075+35_1076-21CATGAGGGAGGGAGGAGGGAGCCTT720C.1075+36_1076-20TCATGAGGGAGGGAGGAGGGAGCCT721C.1075+37_1076-19TTCATGAGGGAGGGAGGAGGGAGCC722C.1075+38_1076-18CTTCATGAGGGAGGGAGGAGGGAGC723C.1075+39_1076-17ACTTCATGAGGGAGGGAGGAGGGAG724C.1075+40_1076-16GACTTCATGAGGGAGGGAGGAGGGA725c.1076-39_-15CGACTTCATGAGGGAGGGAGGAGGG726c.1076-38_-14CCGACTTCATGAGGGAGGGAGGAGG727c.1076-37_-13GCCGACTTCATGAGGGAGGGAGGAG728c.1076-36_-12CGCCGACTTCATGAGGGAGGGAGGA729c.1076-35_-11ACGCCGACTTCATGAGGGAGGGAGG730c.1076-34_-10AACGCCGACTTCATGAGGGAGGGAG731c.1076-33_-9CAACGCCGACTTCATGAGGGAGGGA732c.1076-32_-8CCAACGCCGACTTCATGAGGGAGGG733c.1076-31_-7GCCAACGCCGACTTCATGAGGGAGG734c.1076-30_-6GGCCAACGCCGACTTCATGAGGGAG735c.1076-29_-5AGGCCAACGCCGACTTCATGAGGGA736c.1076-28_-4CAGGCCAACGCCGACTTCATGAGGG737c.1076-27_-3GCAGGCCAACGCCGACTTCATGAGG738c.1076-26_-2TGCAGGCCAACGCCGACTTCATGAG739c.1076-25_-1CTGCAGGCCAACGCCGACTTCATGA740c.1076-24_1076CCTGCAGGCCAACGCCGACTTCATG741c.1076-23_1077TCCTGCAGGCCAACGCCGACTTCAT742c.1076-22_1078ATCCTGCAGGCCAACGCCGACTTCA743c.1076-21_1079TATCCTGCAGGCCAACGCCGACTTC744c.1076-20_1080GTATCCTGCAGGCCAACGCCGACTT745c.1076-19_1081GGTATCCTGCAGGCCAACGCCGACT746c.1076-18_1082GGGTATCCTGCAGGCCAACGCCGAC747c.1076-17_1083CGGGTATCCTGCAGGCCAACGCCGA748c.1076-16_1084ACGGGTATCCTGCAGGCCAACGCCG749c.1076-15_1085AACGGGTATCCTGCAGGCCAACGCC750c.1076-14_1086GAACGGGTATCCTGCAGGCCAACGC751c.1076-13_1087TGAACGGGTATCCTGCAGGCCAACG752c.1076-12_1088ATGAACGGGTATCCTGCAGGCCAAC753c.1076-11_1089CATGAACGGGTATCCTGCAGGCCAA754c.1076-10_1090GCATGAACGGGTATCCTGCAGGCCA755c.1076-9_1091GGCATGAACGGGTATCCTGCAGGCC756c.1076-8_1092CGGCATGAACGGGTATCCTGCAGGC757c.1076-7_1093GCGGCATGAACGGGTATCCTGCAGG758c.1076-6_1094GGCGGCATGAACGGGTATCCTGCAG759c.1076-5_1095TGGCGGCATGAACGGGTATCCTGCA760c.1076-4_1096ATGGCGGCATGAACGGGTATCCTGC761c.1076-3_1097TATGGCGGCATGAACGGGTATCCTG762c.1076-2_1098GTATGGCGGCATGAACGGGTATCCT763c.1076-1_1099AGTATGGCGGCATGAACGGGTATCC764c.1076_1100CAGTATGGCGGCATGAACGGGTATC765c.1077_1101CCAGTATGGCGGCATGAACGGGTAT766c.1078_1102CCCAGTATGGCGGCATGAACGGGTA767c.1079_1103CCCCAGTATGGCGGCATGAACGGGT768c.1080_1104GCCCCAGTATGGCGGCATGAACGGG769c.1081_1105GGCCCCAGTATGGCGGCATGAACGG770c.1082_1106AGGCCCCAGTATGGCGGCATGAACG771c.1083_1107CAGGCCCCAGTATGGCGGCATGAAC772c.1084_1108CCAGGCCCCAGTATGGCGGCATGAA773c.1085_1109CCCAGGCCCCAGTATGGCGGCATGA774c.1086_1110GCCCAGGCCCCAGTATGGCGGCATG775c.1087_1111AGCCCAGGCCCCAGTATGGCGGCAT776c.1088_1112AAGCCCAGGCCCCAGTATGGCGGCA777c.1089_1113GAAGCCCAGGCCCCAGTATGGCGGC778c.1090_1114GGAAGCCCAGGCCCCAGTATGGCGG779c.1091_1115TGGAAGCCCAGGCCCCAGTATGGCG780c.1092_1116GTGGAAGCCCAGGCCCCAGTATGGC781c.1093_1117GGTGGAAGCCCAGGCCCCAGTATGG782c.1094_1118AGGTGGAAGCCCAGGCCCCAGTATG783c.1095_1119CAGGTGGAAGCCCAGGCCCCAGTAT784c.1096_1120ACAGGTGGAAGCCCAGGCCCCAGTA785c.1097_1121CACAGGTGGAAGCCCAGGCCCCAGT786c.1098_1122GCACAGGTGGAAGCCCAGGCCCCAG787c.1099_1123GGCACAGGTGGAAGCCCAGGCCCCA788c.1100_1124CGGCACAGGTGGAAGCCCAGGCCCC789c.1101_1125GCGGCACAGGTGGAAGCCCAGGCCC790c.1102_1126AGCGGCACAGGTGGAAGCCCAGGCC791c.1103_1127CAGCGGCACAGGTGGAAGCCCAGGC792c.1104_1128CCAGCGGCACAGGTGGAAGCCCAGG793c.1105_1129CCCAGCGGCACAGGTGGAAGCCCAG794c.1106_1130CCCCAGCGGCACAGGTGGAAGCCCA795c.1107_1131GCCCCAGCGGCACAGGTGGAAGCCC796c.1108_1132AGCCCCAGCGGCACAGGTGGAAGCC797c.1109_1133TAGCCCCAGCGGCACAGGTGGAAGC798c.1110_1134GTAGCCCCAGCGGCACAGGTGGAAG799c.1111_1135AGTAGCCCCAGCGGCACAGGTGGAA800c.1112_1136GAGTAGCCCCAGCGGCACAGGTGGA801c.1113_1137GGAGTAGCCCCAGCGGCACAGGTGG802c.1114_1138AGGAGTAGCCCCAGCGGCACAGGTG803c.1115_1139GAGGAGTAGCCCCAGCGGCACAGGT804c.1116_1140GGAGGAGTAGCCCCAGCGGCACAGG805c.1117_1141TGGAGGAGTAGCCCCAGCGGCACAG806c.1118_1142GTGGAGGAGTAGCCCCAGCGGCACA807c.1119_1143GGTGGAGGAGTAGCCCCAGCGGCAC808c.1120_1144CGGTGGAGGAGTAGCCCCAGCGGCA809c.1121_1145GCGGTGGAGGAGTAGCCCCAGCGGC810c.1122_1146AGCGGTGGAGGAGTAGCCCCAGCGG811c.1123_1147TAGCGGTGGAGGAGTAGCCCCAGCG812c.1124_1148ATAGCGGTGGAGGAGTAGCCCCAGC813c.1125_1149GATAGCGGTGGAGGAGTAGCCCCAG814c.1126_1150TGATAGCGGTGGAGGAGTAGCCCCA815c.1127_1151GTGATAGCGGTGGAGGAGTAGCCCC816c.1128_1152GGTGATAGCGGTGGAGGAGTAGCCC817c.1129_1153GGGTGATAGCGGTGGAGGAGTAGCC818c.1130_1154CGGGTGATAGCGGTGGAGGAGTAGC819c.1131_1155GCGGGTGATAGCGGTGGAGGAGTAG820c.1132_1156GGCGGGTGATAGCGGTGGAGGAGTA821c.1133_1157TGGCGGGTGATAGCGGTGGAGGAGT822c.1134_1158CTGGCGGGTGATAGCGGTGGAGGAG823c.1135_1159CCTGGCGGGTGATAGCGGTGGAGGA824c.1136_1160ACCTGGCGGGTGATAGCGGTGGAGG825c.1137_1161CACCTGGCGGGTGATAGCGGTGGAG826c.1138_1162CCACCTGGCGGGTGATAGCGGTGGA827c.1139_1163ACCACCTGGCGGGTGATAGCGGTGG828c.1140_1164CACCACCTGGCGGGTGATAGCGGTG829c.1141_1165CCACCACCTGGCGGGTGATAGCGGT830c.1142_1166TCCACCACCTGGCGGGTGATAGCGG831c.1143_1167CTCCACCACCTGGCGGGTGATAGCG832c.1144_1168TCTCCACCACCTGGCGGGTGATAGC833c.1145_1169TTCTCCACCACCTGGCGGGTGATAG834c.1146_1170GTTCTCCACCACCTGGCGGGTGATA835c.1147_1171TGTTCTCCACCACCTGGCGGGTGAT836c.1148_1172ATGTTCTCCACCACCTGGCGGGTGA837c.1149_1173CATGTTCTCCACCACCTGGCGGGTG838c.1150_1174TCATGTTCTCCACCACCTGGCGGGT839c.1151_1175GTCATGTTCTCCACCACCTGGCGGG840c.1152_1176GGTCATGTTCTCCACCACCTGGCGG841c.1153_1177TGGTCATGTTCTCCACCACCTGGCG842c.1154_1178CTGGTCATGTTCTCCACCACCTGGC843c.1155_1179CCTGGTCATGTTCTCCACCACCTGG844c.1156_1180CCCTGGTCATGTTCTCCACCACCTG845c.1157_1181GCCCTGGTCATGTTCTCCACCACCT846c.1158_1182GGCCCTGGTCATGTTCTCCACCACC847c.1159_1183GGGCCCTGGTCATGTTCTCCACCAC848c.1160_1184TGGGCCCTGGTCATGTTCTCCACCA849c.1161_1185GTGGGCCCTGGTCATGTTCTCCACC850c.1162_1186AGTGGGCCCTGGTCATGTTCTCCAC851c.1163_1187AAGTGGGCCCTGGTCATGTTCTCCA852c.1164_1188GAAGTGGGCCCTGGTCATGTTCTCC853c.1165_1189GGAAGTGGGCCCTGGTCATGTTCTC854c.1166_1190GGGAAGTGGGCCCTGGTCATGTTCT855c.1167_1191GGGGAAGTGGGCCCTGGTCATGTTC856c.1168_1192GGGGGAAGTGGGCCCTGGTCATGTT857c.1169_1193AGGGGGAAGTGGGCCCTGGTCATGT858c.1170_1194CAGGGGGAAGTGGGCCCTGGTCATG859c.1171_1194+1CCAGGGGGAAGTGGGCCCTGGTCAT860c.1172_1194+2ACCAGGGGGAAGTGGGCCCTGGTCA861c.1173_1194+3CACCAGGGGGAAGTGGGCCCTGGTC862c.1174_1194+4TCACCAGGGGGAAGTGGGCCCTGGT863c.1175_1194+5CTCACCAGGGGGAAGTGGGCCCTGG864c.1176_1194+6ACTCACCAGGGGGAAGTGGGCCCTG865c.1177_1194+7AACTCACCAGGGGGAAGTGGGCCCT866c.1178_1194+8CAACTCACCAGGGGGAAGTGGGCCC867c.1179_1194+9CCAACTCACCAGGGGGAAGTGGGCC868c.1180_1194+10CCCAACTCACCAGGGGGAAGTGGGC869c.1181_1194+11CCCCAACTCACCAGGGGGAAGTGGG870c.1182_1194+12ACCCCAACTCACCAGGGGGAAGTGG871c.1183_1194+13CACCCCAACTCACCAGGGGGAAGTG872c.1184_1194+14CCACCCCAACTCACCAGGGGGAAGT873c.1185_1194+15ACCACCCCAACTCACCAGGGGGAAG874c.1186_1194+16CACCACCCCAACTCACCAGGGGGAA875c.1187_1194+17CCACCACCCCAACTCACCAGGGGGA876c.1188_1194+18GCCACCACCCCAACTCACCAGGGGG877c.1189_1194+19TGCCACCACCCCAACTCACCAGGGG878c.1190_1194+20CTGCCACCACCCCAACTCACCAGGG879c.1191_1194+21CCTGCCACCACCCCAACTCACCAGG880c.1192_1194+22CCCTGCCACCACCCCAACTCACCAG881c.1193_1194+23CCCCTGCCACCACCCCAACTCACCA882c.1194_1194+24TCCCCTGCCACCACCCCAACTCACC883c.1194+1_+25CTCCCCTGCCACCACCCCAACTCAC884c.956-25_-5AAGGGAAGCAGCTCTGGGGTT885c.956-24_-4GAAGGGAAGCAGCTCTGGGGT886c.956-23_-3GGAAGGGAAGCAGCTCTGGGG887c.956-22_-2TGGAAGGGAAGCAGCTCTGGG888c.956-21_-1CTGGAAGGGAAGCAGCTCTGG889c.956-20_956TCTGGAAGGGAAGCAGCTCTG890c.956-19_957ATCTGGAAGGGAAGCAGCTCT891c.956-18_958CATCTGGAAGGGAAGCAGCTC892c.956-17_959ACATCTGGAAGGGAAGCAGCT893c.956-16_960CACATCTGGAAGGGAAGCAGC894c.956-15_961CCACATCTGGAAGGGAAGCAG895c.956-14_962ACCACATCTGGAAGGGAAGCA896c.956-13_963GACCACATCTGGAAGGGAAGC897c.956-12_964GGACCACATCTGGAAGGGAAG898c.956-11_965AGGACCACATCTGGAAGGGAA899c.956-10_966CAGGACCACATCTGGAAGGGA900c.956-9_967GCAGGACCACATCTGGAAGGG901c.956-8_968TGCAGGACCACATCTGGAAGG902c.956-7_969CTGCAGGACCACATCTGGAAG903c.956-6_970GCTGCAGGACCACATCTGGAA904c.956-5_971GGCTGCAGGACCACATCTGGA905c.956-4_972CGGCTGCAGGACCACATCTGG906c.956-3_973TCGGCTGCAGGACCACATCTG907c.956-2_974CTCGGCTGCAGGACCACATCT908c.956-1_975GCTCGGCTGCAGGACCACATC909c.956_976GGCTCGGCTGCAGGACCACAT910c.957_977GGGCTCGGCTGCAGGACCACA911c.958_978AGGGCTCGGCTGCAGGACCAC912c.959_979CAGGGCTCGGCTGCAGGACCA913c.960_980GCAGGGCTCGGCTGCAGGACC914c.961_981GGCAGGGCTCGGCTGCAGGAC915c.962_982GGGCAGGGCTCGGCTGCAGGA916c.963_983AGGGCAGGGCTCGGCTGCAGG917c.964_984AAGGGCAGGGCTCGGCTGCAG918c.965_985TAAGGGCAGGGCTCGGCTGCA919c.966_986CTAAGGGCAGGGCTCGGCTGC920c.967_987GCTAAGGGCAGGGCTCGGCTG921c.968_988AGCTAAGGGCAGGGCTCGGCT922c.969_989CAGCTAAGGGCAGGGCTCGGC923c.970_990CCAGCTAAGGGCAGGGCTCGG924c.971_991TCCAGCTAAGGGCAGGGCTCG925c.972_992CTCCAGCTAAGGGCAGGGCTC926c.973_993CCTCCAGCTAAGGGCAGGGCT927c.974_994ACCTCCAGCTAAGGGCAGGGC928c.975_995GACCTCCAGCTAAGGGCAGGG929c.976_996CGACCTCCAGCTAAGGGCAGG930c.977_997TCGACCTCCAGCTAAGGGCAG931c.978_998GTCGACCTCCAGCTAAGGGCA932c.979_999TGTCGACCTCCAGCTAAGGGC933c.980_1000CTGTCGACCTCCAGCTAAGGG934c.981_1001CCTGTCGACCTCCAGCTAAGG935c.982_1002ACCTGTCGACCTCCAGCTAAG936c.983_1003CACCTGTCGACCTCCAGCTAA937c.984_1004CCACCTGTCGACCTCCAGCTA938c.985_1005CCCACCTGTCGACCTCCAGCT939c.986_1006TCCCACCTGTCGACCTCCAGC940c.987_1007ATCCCACCTGTCGACCTCCAG941c.988_1008GATCCCACCTGTCGACCTCCA942c.989_1009GGATCCCACCTGTCGACCTCC943c.990_1010AGGATCCCACCTGTCGACCTC944c.991_1011CAGGATCCCACCTGTCGACCT945c.992_1012CCAGGATCCCACCTGTCGACC946c.993_1013TCCAGGATCCCACCTGTCGAC947c.994_1014ATCCAGGATCCCACCTGTCGA948c.995_1015CATCCAGGATCCCACCTGTCG949c.996_1016ACATCCAGGATCCCACCTGTC950c.997_1017GACATCCAGGATCCCACCTGT951c.998_1018AGACATCCAGGATCCCACCTG952c.999_1019TAGACATCCAGGATCCCACCT953c.1000_1020GTAGACATCCAGGATCCCACC954c.1001_1021TGTAGACATCCAGGATCCCAC955c.1002_1022ATGTAGACATCCAGGATCCCA956c.1003_1023GATGTAGACATCCAGGATCCC957c.1004_1024AGATGTAGACATCCAGGATCC958c.1005_1025AAGATGTAGACATCCAGGATC959c.1006_1026GAAGATGTAGACATCCAGGAT960c.1007_1027GGAAGATGTAGACATCCAGGA961c.1008_1028AGGAAGATGTAGACATCCAGG962c.1009_1029CAGGAAGATGTAGACATCCAG963c.1010_1030CCAGGAAGATGTAGACATCCA964c.1011_1031CCCAGGAAGATGTAGACATCC965c.1012_1032GCCCAGGAAGATGTAGACATC966c.1013_1033GGCCCAGGAAGATGTAGACAT967c.1014_1034GGGCCCAGGAAGATGTAGACA968c.1015_1035TGGGCCCAGGAAGATGTAGAC969c.1016_1036CTGGGCCCAGGAAGATGTAGA970c.1017_1037TCTGGGCCCAGGAAGATGTAG971c.1018_1038CTCTGGGCCCAGGAAGATGTA972c.1019_1039GCTCTGGGCCCAGGAAGATGT973c.1020_1040GGCTCTGGGCCCAGGAAGATG974c.1021_1041GGGCTCTGGGCCCAGGAAGAT975c.1022_1042TGGGCTCTGGGCCCAGGAAGA976c.1023_1043TTGGGCTCTGGGCCCAGGAAG977C.1024_1044CTTGGGCTCTGGGCCCAGGAA978c.1025_1045TCTTGGGCTCTGGGCCCAGGA979c.1026_1046CTCTTGGGCTCTGGGCCCAGG980c.1027_1047GCTCTTGGGCTCTGGGCCCAG981c.1028_1048CGCTCTTGGGCTCTGGGCCCA982c.1029_1049ACGCTCTTGGGCTCTGGGCCC983c.1030_1050CACGCTCTTGGGCTCTGGGCC984c.1031_1051CCACGCTCTTGGGCTCTGGGC985c.1032_1052ACCACGCTCTTGGGCTCTGGG986c.1033_1053CACCACGCTCTTGGGCTCTGG987c.1034_1054GCACCACGCTCTTGGGCTCTG988c.1035_1055TGCACCACGCTCTTGGGCTCT989c.1036_1056CTGCACCACGCTCTTGGGCTC990c.1037_1057GCTGCACCACGCTCTTGGGCT991c.1038_1058TGCTGCACCACGCTCTTGGGC992c.1039_1059CTGCTGCACCACGCTCTTGGG993c.1040_1060ACTGCTGCACCACGCTCTTGG994c.1041_1061TACTGCTGCACCACGCTCTTG995c.1042_1062GTACTGCTGCACCACGCTCTT996c.1043_1063GGTACTGCTGCACCACGCTCT997c.1044_1064AGGTACTGCTGCACCACGCTC998c.1045_1065CAGGTACTGCTGCACCACGCT999c.1046_1066CCAGGTACTGCTGCACCACGC1000c.1047_1067TCCAGGTACTGCTGCACCACG1001c.1048_1068GTCCAGGTACTGCTGCACCAC1002c.1049_1069CGTCCAGGTACTGCTGCACCA1003c.1050_1070ACGTCCAGGTACTGCTGCACC1004c.1051_1071AACGTCCAGGTACTGCTGCAC1005c.1052_1072CAACGTCCAGGTACTGCTGCA1006c.1053_1073ACAACGTCCAGGTACTGCTGC1007c.1054_1074CACAACGTCCAGGTACTGCTG1008c.1055_1075CCACAACGTCCAGGTACTGCT1009c.1056_1075+1CCCACAACGTCCAGGTACTGC1010c.1057_1075+2ACCCACAACGTCCAGGTACTG1011c.1058_1075+3TACCCACAACGTCCAGGTACT1012c.1059_1075+4CTACCCACAACGTCCAGGTAC1013c.1060_1075+5CCTACCCACAACGTCCAGGTA1014c.1061_1075+6CCCTACCCACAACGTCCAGGT1015c.1062_1075+7GCCCTACCCACAACGTCCAGG1016c.1063_1075+8GGCCCTACCCACAACGTCCAG1017c.1064_1075+9AGGCCCTACCCACAACGTCCA1018c.1065_1075+10CAGGCCCTACCCACAACGTCC1019c.1066_1075+11GCAGGCCCTACCCACAACGTC1020c.1067_1075+12AGCAGGCCCTACCCACAACGT1021c.1068_1075+13GAGCAGGCCCTACCCACAACG1022c.1069_1075+14GGAGCAGGCCCTACCCACAAC1023c.1070_1075+15GGGAGCAGGCCCTACCCACAA1024c.1071_1075+16AGGGAGCAGGCCCTACCCACA1025c.1072_1075+17CAGGGAGCAGGCCCTACCCAC1026c.1073_1075+18CCAGGGAGCAGGCCCTACCCA1027c.1074_1075+19GCCAGGGAGCAGGCCCTACCC1028c.1075_1075+20GGCCAGGGAGCAGGCCCTACC1029c.1075+1_+21CGGCCAGGGAGCAGGCCCTAC1030c.1075+2_+22GCGGCCAGGGAGCAGGCCCTA1031c.1075+3_+23CGCGGCCAGGGAGCAGGCCCT1032c.1075+4_+24CCGCGGCCAGGGAGCAGGCCC1033c.1075+5_+25GCCGCGGCCAGGGAGCAGGCC1034c.1075+6_+26GGCCGCGGCCAGGGAGCAGGC1035c.1075+7_+27GGGCCGCGGCCAGGGAGCAGG1036c.1075+8_+28GGGGCCGCGGCCAGGGAGCAG1037c.1075+9_+29GGGGGCCGCGGCCAGGGAGCA1038c.1075+10_+30CGGGGGCCGCGGCCAGGGAGC1039c.1075+11_+31GCGGGGGCCGCGGCCAGGGAG1040c.1075+12_+32GGCGGGGGCCGCGGCCAGGGA1041c.1075+13_+33GGGCGGGGGCCGCGGCCAGGG1042c.1075+14_+34GGGGCGGGGGCCGCGGCCAGG1043c.1075+15_+35TGGGGCGGGGGCCGCGGCCAG1044c.1075+16_+36TTGGGGCGGGGGCCGCGGCCA1045c.1075+17_+37CTTGGGGCGGGGGCCGCGGCC1046c.1075+18_+38CCTTGGGGCGGGGGCCGCGGC1047c.1075+19_+39GCCTTGGGGCGGGGGCCGCGG1048c.1075+20_+40AGCCTTGGGGCGGGGGCCGCG1049c.1075+21_1076-39GAGCCTTGGGGCGGGGGCCGC1050c.1075+22_1076-38GGAGCCTTGGGGCGGGGGCCG1051c.1075+23_1076-37GGGAGCCTTGGGGCGGGGGCC1052c.1075+24_1076-36AGGGAGCCTTGGGGCGGGGGC1053c.1075+25_1076-35GAGGGAGCCTTGGGGCGGGGG1054c.1075+26_1076-34GGAGGGAGCCTTGGGGCGGGG1055c.1075+27_1076-33AGGAGGGAGCCTTGGGGCGGG1056c.1075+28_1076-32GAGGAGGGAGCCTTGGGGCGG1057c.1075+29_1076-31GGAGGAGGGAGCCTTGGGGCG1058c.1075+30_1076-30GGGAGGAGGGAGCCTTGGGGC1059c.1075+31_1076-29AGGGAGGAGGGAGCCTTGGGG1060c.1075+32_1076-28GAGGGAGGAGGGAGCCTTGGG1061c.1075+33_1076-27GGAGGGAGGAGGGAGCCTTGG1062c.1075+34_1076-26GGGAGGGAGGAGGGAGCCTTG1063c.1075+35_1076-25AGGGAGGGAGGAGGGAGCCTT1064c.1075+36_1076-24GAGGGAGGGAGGAGGGAGCCT1065c.1075+37_1076-23TGAGGGAGGGAGGAGGGAGCC1066c.1075+38_1076-22ATGAGGGAGGGAGGAGGGAGC1067c.1075+39_1076-21CATGAGGGAGGGAGGAGGGAG1068c.1075+40_1076-20TCATGAGGGAGGGAGGAGGGA1069c.1076-39_-19TTCATGAGGGAGGGAGGAGGG1070c.1076-38_-18CTTCATGAGGGAGGGAGGAGG1071c.1076-37_-17ACTTCATGAGGGAGGGAGGAG1072c.1076-36_-16GACTTCATGAGGGAGGGAGGA1073c.1076-35_-15CGACTTCATGAGGGAGGGAGG1074c.1076-34_-14CCGACTTCATGAGGGAGGGAG1075c.1076-33_-13GCCGACTTCATGAGGGAGGGA1076c.1076-32_-12CGCCGACTTCATGAGGGAGGG1077c.1076-31_-11ACGCCGACTTCATGAGGGAGG1078c.1076-30_-10AACGCCGACTTCATGAGGGAG1079c.1076-29_-9CAACGCCGACTTCATGAGGGA1080c.1076-28_-8CCAACGCCGACTTCATGAGGG1081c.1076-27_-7GCCAACGCCGACTTCATGAGG1082c.1076-26_-6GGCCAACGCCGACTTCATGAG1083c.1076-25_-5AGGCCAACGCCGACTTCATGA1084c.1076-24_-4CAGGCCAACGCCGACTTCATG1085c.1076-23_-3GCAGGCCAACGCCGACTTCAT1086c.1076-22_-2TGCAGGCCAACGCCGACTTCA1087c.1076-21_-1CTGCAGGCCAACGCCGACTTC1088c.1076-20_1076CCTGCAGGCCAACGCCGACTT1089c.1076-19_1077TCCTGCAGGCCAACGCCGACT1090c.1076-18_1078ATCCTGCAGGCCAACGCCGAC1091c.1076-17_1079TATCCTGCAGGCCAACGCCGA1092c.1076-16_1080GTATCCTGCAGGCCAACGCCG1093c.1076-15_1081GGTATCCTGCAGGCCAACGCC1094c.1076-14_1082GGGTATCCTGCAGGCCAACGC1095c.1076-13_1083CGGGTATCCTGCAGGCCAACG1096c.1076-12_1084ACGGGTATCCTGCAGGCCAAC1097c.1076-11_1085AACGGGTATCCTGCAGGCCAA1098c.1076-10_1086GAACGGGTATCCTGCAGGCCA1099c.1076-9_1087TGAACGGGTATCCTGCAGGCC1100c.1076-8_1088ATGAACGGGTATCCTGCAGGC1101c.1076-7_1089CATGAACGGGTATCCTGCAGG1102c.1076-6_1090GCATGAACGGGTATCCTGCAG1103c.1076-5_1091GGCATGAACGGGTATCCTGCA1104c.1076-4_1092CGGCATGAACGGGTATCCTGC1105c.1076-3_1093GCGGCATGAACGGGTATCCTG1106c.1076-2_1094GGCGGCATGAACGGGTATCCT1107c.1076-1_1095TGGCGGCATGAACGGGTATCC1108c.1076_1096ATGGCGGCATGAACGGGTATC1109c.1077_1097TATGGCGGCATGAACGGGTAT1110c.1078_1098GTATGGCGGCATGAACGGGTA1111c.1079_1099AGTATGGCGGCATGAACGGGT1112c.1080_1100CAGTATGGCGGCATGAACGGG1113c.1081_1101CCAGTATGGCGGCATGAACGG1114c.1082_1102CCCAGTATGGCGGCATGAACG1115c.1083_1103CCCCAGTATGGCGGCATGAAC1116c.1084_1104GCCCCAGTATGGCGGCATGAA1117c.1085_1105GGCCCCAGTATGGCGGCATGA1118c.1086_1106AGGCCCCAGTATGGCGGCATG1119c.1087_1107CAGGCCCCAGTATGGCGGCAT1120c.1088_1108CCAGGCCCCAGTATGGCGGCA1121c.1089_1109CCCAGGCCCCAGTATGGCGGC1122c.1090_1110GCCCAGGCCCCAGTATGGCGG1123c.1091_1111AGCCCAGGCCCCAGTATGGCG1124c.1092_1112AAGCCCAGGCCCCAGTATGGC1125c.1093_1113GAAGCCCAGGCCCCAGTATGG1126c.1094_1114GGAAGCCCAGGCCCCAGTATG1127c.1095_1115TGGAAGCCCAGGCCCCAGTAT1128c.1096_1116GTGGAAGCCCAGGCCCCAGTA1129c.1097_1117GGTGGAAGCCCAGGCCCCAGT1130c.1098_1118AGGTGGAAGCCCAGGCCCCAG1131c.1099_1119CAGGTGGAAGCCCAGGCCCCA1132c.1100_1120ACAGGTGGAAGCCCAGGCCCC1133c.1101_1121CACAGGTGGAAGCCCAGGCCC1134c.1102_1122GCACAGGTGGAAGCCCAGGCC1135c.1103_1123GGCACAGGTGGAAGCCCAGGC1136c.1104_1124CGGCACAGGTGGAAGCCCAGG1137c.1105_1125GCGGCACAGGTGGAAGCCCAG1138c.1106_1126AGCGGCACAGGTGGAAGCCCA1139c.1107_1127CAGCGGCACAGGTGGAAGCCC1140c.1108_1128CCAGCGGCACAGGTGGAAGCC1141c.1109_1129CCCAGCGGCACAGGTGGAAGC1142c.1110_1130CCCCAGCGGCACAGGTGGAAG1143c.1111_1131GCCCCAGCGGCACAGGTGGAA1144c.1112_1132AGCCCCAGCGGCACAGGTGGA1145c.1113_1133TAGCCCCAGCGGCACAGGTGG1146c.1114_1134GTAGCCCCAGCGGCACAGGTG1147c.1115_1135AGTAGCCCCAGCGGCACAGGT1148c.1116_1136GAGTAGCCCCAGCGGCACAGG1149c.1117_1137GGAGTAGCCCCAGCGGCACAG1150c.1118_1138AGGAGTAGCCCCAGCGGCACA1151c.1119_1139GAGGAGTAGCCCCAGCGGCAC1152c.1120_1140GGAGGAGTAGCCCCAGCGGCA1153c.1121_1141TGGAGGAGTAGCCCCAGCGGC1154c.1122_1142GTGGAGGAGTAGCCCCAGCGG1155c.1123_1143GGTGGAGGAGTAGCCCCAGCG1156c.1124_1144CGGTGGAGGAGTAGCCCCAGC1157c.1125_1145GCGGTGGAGGAGTAGCCCCAG1158c.1126_1146AGCGGTGGAGGAGTAGCCCCA1159c.1127_1147TAGCGGTGGAGGAGTAGCCCC1160c.1128_1148ATAGCGGTGGAGGAGTAGCCC1161c.1129_1149GATAGCGGTGGAGGAGTAGCC1162c.1130_1150TGATAGCGGTGGAGGAGTAGC1163c.1131_1151GTGATAGCGGTGGAGGAGTAG1164c.1132_1152GGTGATAGCGGTGGAGGAGTA1165c.1133_1153GGGTGATAGCGGTGGAGGAGT1166c.1134_1154CGGGTGATAGCGGTGGAGGAG1167c.1135_1155GCGGGTGATAGCGGTGGAGGA1168c.1136_1156GGCGGGTGATAGCGGTGGAGG1169c.1137_1157TGGCGGGTGATAGCGGTGGAG1170c.1138_1158CTGGCGGGTGATAGCGGTGGA1171c.1139_1159CCTGGCGGGTGATAGCGGTGG1172c.1140_1160ACCTGGCGGGTGATAGCGGTG1173c.1141_1161CACCTGGCGGGTGATAGCGGT1174c.1142_1162CCACCTGGCGGGTGATAGCGG1175c.1143_1163ACCACCTGGCGGGTGATAGCG1176c.1144_1164CACCACCTGGCGGGTGATAGC1177c.1145_1165CCACCACCTGGCGGGTGATAG1178c.1146_1166TCCACCACCTGGCGGGTGATA1179c.1147_1167CTCCACCACCTGGCGGGTGAT1180c.1148_1168TCTCCACCACCTGGCGGGTGA1181c.1149_1169TTCTCCACCACCTGGCGGGTG1182c.1150_1170GTTCTCCACCACCTGGCGGGT1183c.1151_1171TGTTCTCCACCACCTGGCGGG1184c.1152_1172ATGTTCTCCACCACCTGGCGG1185c.1153_1173CATGTTCTCCACCACCTGGCG1186c.1154_1174TCATGTTCTCCACCACCTGGC1187c.1155_1175GTCATGTTCTCCACCACCTGG1188c.1156_1176GGTCATGTTCTCCACCACCTG1189c.1157_1177TGGTCATGTTCTCCACCACCT1190c.1158_1178CTGGTCATGTTCTCCACCACC1191c.1159_1179CCTGGTCATGTTCTCCACCAC1192c.1160_1180CCCTGGTCATGTTCTCCACCA1193c.1161_1181GCCCTGGTCATGTTCTCCACC1194c.1162_1182GGCCCTGGTCATGTTCTCCAC1195c.1163_1183GGGCCCTGGTCATGTTCTCCA1196c.1164_1184TGGGCCCTGGTCATGTTCTCC1197c.1165_1185GTGGGCCCTGGTCATGTTCTC1198c.1166_1186AGTGGGCCCTGGTCATGTTCT1199c.1167_1187AAGTGGGCCCTGGTCATGTTC1200c.1168_1188GAAGTGGGCCCTGGTCATGTT1201c.1169_1189GGAAGTGGGCCCTGGTCATGT1202c.1170_1190GGGAAGTGGGCCCTGGTCATG1203c.1171_1191GGGGAAGTGGGCCCTGGTCAT1204c.1172_1192GGGGGAAGTGGGCCCTGGTCA1205c.1173_1193AGGGGGAAGTGGGCCCTGGTC1206c.1174_1194CAGGGGGAAGTGGGCCCTGGT1207c.1175_1194+1CCAGGGGGAAGTGGGCCCTGG1208c.1176_1194+2ACCAGGGGGAAGTGGGCCCTG1209c.1177_1194+3CACCAGGGGGAAGTGGGCCCT1210c.1178_1194+4TCACCAGGGGGAAGTGGGCCC1211c.1179_1194+5CTCACCAGGGGGAAGTGGGCC1212c.1180_1194+6ACTCACCAGGGGGAAGTGGGC1213c.1181_1194+7AACTCACCAGGGGGAAGTGGG1214c.1182_1194+8CAACTCACCAGGGGGAAGTGG1215c.1183_1194+9CCAACTCACCAGGGGGAAGTG1216c.1184_1194+10CCCAACTCACCAGGGGGAAGT1217c.1185_1194+11CCCCAACTCACCAGGGGGAAG1218c.1186_1194+12ACCCCAACTCACCAGGGGGAA1219c.1187_1194+13CACCCCAACTCACCAGGGGGA1220c.1188_1194+14CCACCCCAACTCACCAGGGGG1221c.1189_1194+15ACCACCCCAACTCACCAGGGG1222c.1190_1194+16CACCACCCCAACTCACCAGGG1223c.1191_1194+17CCACCACCCCAACTCACCAGG1224c.1192_1194+18GCCACCACCCCAACTCACCAG1225c.1193_1194+19TGCCACCACCCCAACTCACCA1226c.1194_1194+20CTGCCACCACCCCAACTCACC1227c.1194+1_+21CCTGCCACCACCCCAACTCAC1228c.1194+2_+22CCCTGCCACCACCCCAACTCA1229c.1194+3_+23CCCCTGCCACCACCCCAACTC1230c.1194+4_+24TCCCCTGCCACCACCCCAACT1231c.1194+5_+25CTCCCCTGCCACCACCCCAAC1232c.956-25_-8GGAAGCAGCTCTGGGGTT1233c.956-24_-7GGGAAGCAGCTCTGGGGT1234c.956-23_-6AGGGAAGCAGCTCTGGGG1235c.956-22_-5AAGGGAAGCAGCTCTGGG1236c.956-21_-4GAAGGGAAGCAGCTCTGG1237c.956-20_-3GGAAGGGAAGCAGCTCTG1238c.956-19_-2TGGAAGGGAAGCAGCTCT1239c.956-18_-1CTGGAAGGGAAGCAGCTC1240c.956-17_956TCTGGAAGGGAAGCAGCT1241c.956-16_957ATCTGGAAGGGAAGCAGC1242c.956-15_958CATCTGGAAGGGAAGCAG1243c.956-14_959ACATCTGGAAGGGAAGCA1244c.956-13_960CACATCTGGAAGGGAAGC1245c.956-12_961CCACATCTGGAAGGGAAG1246c.956-11_962ACCACATCTGGAAGGGAA1247c.956-10_963GACCACATCTGGAAGGGA1248c.956-9_964GGACCACATCTGGAAGGG1249c.956-8_965AGGACCACATCTGGAAGG1250c.956-7_966CAGGACCACATCTGGAAG1251c.956-6_967GCAGGACCACATCTGGAA1252c.956-5_968TGCAGGACCACATCTGGA1253c.956-4_969CTGCAGGACCACATCTGG1254c.956-3_970GCTGCAGGACCACATCTG1255c.956-2_971GGCTGCAGGACCACATCT1256c.956-1_972CGGCTGCAGGACCACATC1257c.956_973TCGGCTGCAGGACCACAT1258c.957_974CTCGGCTGCAGGACCACA1259c.958_975GCTCGGCTGCAGGACCAC1260c.959_976GGCTCGGCTGCAGGACCA1261c.960_977GGGCTCGGCTGCAGGACC1262c.961_978AGGGCTCGGCTGCAGGAC1263c.962_979CAGGGCTCGGCTGCAGGA1264c.963_980GCAGGGCTCGGCTGCAGG1265c.964_981GGCAGGGCTCGGCTGCAG1266c.965_982GGGCAGGGCTCGGCTGCA1267c.966_983AGGGCAGGGCTCGGCTGC1268c.967_984AAGGGCAGGGCTCGGCTG1269c.968_985TAAGGGCAGGGCTCGGCT1270c.969_986CTAAGGGCAGGGCTCGGC1271c.970_987GCTAAGGGCAGGGCTCGG1272c.971_988AGCTAAGGGCAGGGCTCG1273c.972_989CAGCTAAGGGCAGGGCTC1274c.973_990CCAGCTAAGGGCAGGGCT1275c.974_991TCCAGCTAAGGGCAGGGC1276c.975_992CTCCAGCTAAGGGCAGGG1277c.976_993CCTCCAGCTAAGGGCAGG1278c.977_994ACCTCCAGCTAAGGGCAG1279c.978_995GACCTCCAGCTAAGGGCA1280c.979_996CGACCTCCAGCTAAGGGC1281c.980_997TCGACCTCCAGCTAAGGG1282c.981_998GTCGACCTCCAGCTAAGG1283c.982_999TGTCGACCTCCAGCTAAG1284c.983_1000CTGTCGACCTCCAGCTAA1285c.984_1001CCTGTCGACCTCCAGCTA1286c.985_1002ACCTGTCGACCTCCAGCT1287c.986_1003CACCTGTCGACCTCCAGC1288c.987_1004CCACCTGTCGACCTCCAG1289c.988_1005CCCACCTGTCGACCTCCA1290c.989_1006TCCCACCTGTCGACCTCC1291c.990_1007ATCCCACCTGTCGACCTC1292c.991_1008GATCCCACCTGTCGACCT1293c.992_1009GGATCCCACCTGTCGACC1294c.993_1010AGGATCCCACCTGTCGAC1295c.994_1011CAGGATCCCACCTGTCGA1296c.995_1012CCAGGATCCCACCTGTCG1297c.996_1013TCCAGGATCCCACCTGTC1298c.997_1014ATCCAGGATCCCACCTGT1299c.998_1015CATCCAGGATCCCACCTG1300c.999_1016ACATCCAGGATCCCACCT1301c.1000_1017GACATCCAGGATCCCACC1302c.1001_1018AGACATCCAGGATCCCAC1303c.1002_1019TAGACATCCAGGATCCCA1304c.1003_1020GTAGACATCCAGGATCCC1305C.1004_1021TGTAGACATCCAGGATCC1306c.1005_1022ATGTAGACATCCAGGATC1307c.1006_1023GATGTAGACATCCAGGAT1308c.1007_1024AGATGTAGACATCCAGGA1309c.1008_1025AAGATGTAGACATCCAGG1310c.1009_1026GAAGATGTAGACATCCAG1311c.1010_1027GGAAGATGTAGACATCCA1312c.1011_1028AGGAAGATGTAGACATCC1313c.1012_1029CAGGAAGATGTAGACATC1314c.1013_1030CCAGGAAGATGTAGACAT1315c.1014_1031CCCAGGAAGATGTAGACA1316c.1015_1032GCCCAGGAAGATGTAGAC1317c.1016_1033GGCCCAGGAAGATGTAGA1318c.1017_1034GGGCCCAGGAAGATGTAG1319c.1018_1035TGGGCCCAGGAAGATGTA1320c.1019_1036CTGGGCCCAGGAAGATGT1321c.1020_1037TCTGGGCCCAGGAAGATG1322c.1021_1038CTCTGGGCCCAGGAAGAT1323c.1022_1039GCTCTGGGCCCAGGAAGA1324c.1023_1040GGCTCTGGGCCCAGGAAG1325c.1024_1041GGGCTCTGGGCCCAGGAA1326c.1025_1042TGGGCTCTGGGCCCAGGA1327c.1026_1043TTGGGCTCTGGGCCCAGG1328c.1027_1044CTTGGGCTCTGGGCCCAG1329c.1028_1045TCTTGGGCTCTGGGCCCA1330c.1029_1046CTCTTGGGCTCTGGGCCC1331c.1030_1047GCTCTTGGGCTCTGGGCC1332c.1031_1048CGCTCTTGGGCTCTGGGC1333c.1032_1049ACGCTCTTGGGCTCTGGG1334c.1033_1050CACGCTCTTGGGCTCTGG1335c.1034_1051CCACGCTCTTGGGCTCTG1336c.1035_1052ACCACGCTCTTGGGCTCT1337c.1036_1053CACCACGCTCTTGGGCTC1338c.1037_1054GCACCACGCTCTTGGGCT1339c.1038_1055TGCACCACGCTCTTGGGC1340c.1039_1056CTGCACCACGCTCTTGGG1341c.1040_1057GCTGCACCACGCTCTTGG1342c.1041_1058TGCTGCACCACGCTCTTG1343c.1042_1059CTGCTGCACCACGCTCTT1344c.1043_1060ACTGCTGCACCACGCTCT1345c.1044_1061TACTGCTGCACCACGCTC1346c.1045_1062GTACTGCTGCACCACGCT1347c.1046_1063GGTACTGCTGCACCACGC1348c.1047_1064AGGTACTGCTGCACCACG1349c.1048_1065CAGGTACTGCTGCACCAC1350c.1049_1066CCAGGTACTGCTGCACCA1351c.1050_1067TCCAGGTACTGCTGCACC1352c.1051_1068GTCCAGGTACTGCTGCAC1353c.1052_1069CGTCCAGGTACTGCTGCA1354c.1053_1070ACGTCCAGGTACTGCTGC1355c.1054_1071AACGTCCAGGTACTGCTG1356c.1055_1072CAACGTCCAGGTACTGCT1357c.1056_1073ACAACGTCCAGGTACTGC1358c.1057_1074CACAACGTCCAGGTACTG1359c.1058_1075CCACAACGTCCAGGTACT1360c.1059_1075+1CCCACAACGTCCAGGTAC1361c.1060_1075+2ACCCACAACGTCCAGGTA1362c.1061_1075+3TACCCACAACGTCCAGGT1363c.1062_1075+4CTACCCACAACGTCCAGG1364c.1063_1075+5CCTACCCACAACGTCCAG1365c.1064_1075+6CCCTACCCACAACGTCCA1366c.1065_1075+7GCCCTACCCACAACGTCC1367c.1066_1075+8GGCCCTACCCACAACGTC1368c.1067_1075+9AGGCCCTACCCACAACGT1369c.1068_1075+10CAGGCCCTACCCACAACG1370c.1069_1075+11GCAGGCCCTACCCACAAC1371c.1070_1075+12AGCAGGCCCTACCCACAA1372c.1071_1075+13GAGCAGGCCCTACCCACA1373c.1072_1075+14GGAGCAGGCCCTACCCAC1374c.1073_1075+15GGGAGCAGGCCCTACCCA1375c.1074_1075+16AGGGAGCAGGCCCTACCC1376c.1075_1075+17CAGGGAGCAGGCCCTACC1377c.1075+1_+18CCAGGGAGCAGGCCCTAC1378c.1075+2_+19GCCAGGGAGCAGGCCCTA1379c.1075+3_+20GGCCAGGGAGCAGGCCCT1380c.1075+4_+21CGGCCAGGGAGCAGGCCC1381c.1075+5_+22GCGGCCAGGGAGCAGGCC1382c.1075+6_+23CGCGGCCAGGGAGCAGGC1383c.1075+7_+24CCGCGGCCAGGGAGCAGG1384c.1075+8_+25GCCGCGGCCAGGGAGCAG1385c.1075+9_+26GGCCGCGGCCAGGGAGCA1386c.1075+10_+27GGGCCGCGGCCAGGGAGC1387c.1075+11_+28GGGGCCGCGGCCAGGGAG1388c.1075+12_+29GGGGGCCGCGGCCAGGGA1389c.1075+13_+30CGGGGGCCGCGGCCAGGG1390c.1075+14_+31GCGGGGGCCGCGGCCAGG1391c.1075+15_+32GGCGGGGGCCGCGGCCAG1392c.1075+16_+33GGGCGGGGGCCGCGGCCA1393c.1075+17_+34GGGGCGGGGGCCGCGGCC1394c.1075+18_+35TGGGGCGGGGGCCGCGGC1395c.1075+19_+36TTGGGGCGGGGGCCGCGG1396c.1075+20_+37CTTGGGGCGGGGGCCGCG1397c.1075+21_+38CCTTGGGGCGGGGGCCGC1398c.1075+22_+39GCCTTGGGGCGGGGGCCG1399c.1075+23_+40AGCCTTGGGGCGGGGGCC1400c.1075+24_1076-39GAGCCTTGGGGCGGGGGC1401c.1075+25_1076-38GGAGCCTTGGGGCGGGGG1402c.1075+26_1076-37GGGAGCCTTGGGGCGGGG1403c.1075+27_1076-36AGGGAGCCTTGGGGCGGG1404c.1075+28_1076-35GAGGGAGCCTTGGGGCGG1405c.1075+29_1076-34GGAGGGAGCCTTGGGGCG1406c.1075+30_1076-33AGGAGGGAGCCTTGGGGC1407c.1075+31_1076-32GAGGAGGGAGCCTTGGGG1408c.1075+32_1076-31GGAGGAGGGAGCCTTGGG1409c.1075+33_1076-30GGGAGGAGGGAGCCTTGG1410c.1075+34_1076-29AGGGAGGAGGGAGCCTTG1411c.1075+35_1076-28GAGGGAGGAGGGAGCCTT1412c.1075+36_1076-27GGAGGGAGGAGGGAGCCT1413c.1075+37_1076-26GGGAGGGAGGAGGGAGCC1414c.1075+38_1076-25AGGGAGGGAGGAGGGAGC1415c.1075+39_1076-24GAGGGAGGGAGGAGGGAG1416c.1075+40_1076-23TGAGGGAGGGAGGAGGGA1417c.1076-39_-22ATGAGGGAGGGAGGAGGG1418c.1076-38_-21CATGAGGGAGGGAGGAGG1419c.1076-37_-20TCATGAGGGAGGGAGGAG1420c.1076-36_-19TTCATGAGGGAGGGAGGA1421c.1076-35_-18CTTCATGAGGGAGGGAGG1422c.1076-34_-17ACTTCATGAGGGAGGGAG1423c.1076-33_-16GACTTCATGAGGGAGGGA1424c.1076-32_-15CGACTTCATGAGGGAGGG1425c.1076-31_-14CCGACTTCATGAGGGAGG1426c.1076-30_-13GCCGACTTCATGAGGGAG1427c.1076-29_-12CGCCGACTTCATGAGGGA1428c.1076-28_-11ACGCCGACTTCATGAGGG1429c.1076-27_-10AACGCCGACTTCATGAGG1430c.1076-26_-9CAACGCCGACTTCATGAG1431c.1076-25_-8CCAACGCCGACTTCATGA1432c.1076-24_-7GCCAACGCCGACTTCATG1433c.1076-23_-6GGCCAACGCCGACTTCAT1434c.1076-22_-5AGGCCAACGCCGACTTCA1435c.1076-21_-4CAGGCCAACGCCGACTTC1436c.1076-20_-3GCAGGCCAACGCCGACTT1437c.1076-19_-2TGCAGGCCAACGCCGACT1438c.1076-18_-1CTGCAGGCCAACGCCGAC1439c.1076-17_1076CCTGCAGGCCAACGCCGA1440c.1076-16_1077TCCTGCAGGCCAACGCCG1441c.1076-15_1078ATCCTGCAGGCCAACGCC1442c.1076-14_1079TATCCTGCAGGCCAACGC1443c.1076-13_1080GTATCCTGCAGGCCAACG1444c.1076-12_1081GGTATCCTGCAGGCCAAC1445c.1076-11_1082GGGTATCCTGCAGGCCAA1446c.1076-10_1083CGGGTATCCTGCAGGCCA1447c.1076-9_1084ACGGGTATCCTGCAGGCC1448c.1076-8_1085AACGGGTATCCTGCAGGC1449c.1076-7_1086GAACGGGTATCCTGCAGG1450c.1076-6_1087TGAACGGGTATCCTGCAG1451c.1076-5_1088ATGAACGGGTATCCTGCA1452c.1076-4_1089CATGAACGGGTATCCTGC1453c.1076-3_1090GCATGAACGGGTATCCTG1454c.1076-2_1091GGCATGAACGGGTATCCT1455c.1076-1_1092CGGCATGAACGGGTATCC1456c.1076_1093GCGGCATGAACGGGTATC1457c.1077_1094GGCGGCATGAACGGGTAT1458c.1078_1095TGGCGGCATGAACGGGTA1459c.1079_1096ATGGCGGCATGAACGGGT1460c.1080_1097TATGGCGGCATGAACGGG1461c.1081_1098GTATGGCGGCATGAACGG1462c.1082_1099AGTATGGCGGCATGAACG1463c.1083_1100CAGTATGGCGGCATGAAC1464c.1084_1101CCAGTATGGCGGCATGAA1465c.1085_1102CCCAGTATGGCGGCATGA1466c.1086_1103CCCCAGTATGGCGGCATG1467c.1087_1104GCCCCAGTATGGCGGCAT1468c.1088_1105GGCCCCAGTATGGCGGCA1469c.1089_1106AGGCCCCAGTATGGCGGC1470c.1090_1107CAGGCCCCAGTATGGCGG1471c.1091_1108CCAGGCCCCAGTATGGCG1472c.1092_1109CCCAGGCCCCAGTATGGC1473c.1093_1110GCCCAGGCCCCAGTATGG1474c.1094_1111AGCCCAGGCCCCAGTATG1475c.1095_1112AAGCCCAGGCCCCAGTAT1476c.1096_1113GAAGCCCAGGCCCCAGTA1477c.1097_1114GGAAGCCCAGGCCCCAGT1478c.1098_1115TGGAAGCCCAGGCCCCAG1479c.1099_1116GTGGAAGCCCAGGCCCCA1480c.1100_1117GGTGGAAGCCCAGGCCCC1481c.1101_1118AGGTGGAAGCCCAGGCCC1482c.1102_1119CAGGTGGAAGCCCAGGCC1483c.1103_1120ACAGGTGGAAGCCCAGGC1484c.1104_1121CACAGGTGGAAGCCCAGG1485c.1105_1122GCACAGGTGGAAGCCCAG1486c.1106_1123GGCACAGGTGGAAGCCCA1487c.1107_1124CGGCACAGGTGGAAGCCC1488c.1108_1125GCGGCACAGGTGGAAGCC1489c.1109_1126AGCGGCACAGGTGGAAGC1490c.1110_1127CAGCGGCACAGGTGGAAG1491c.1111_1128CCAGCGGCACAGGTGGAA1492c.1112_1129CCCAGCGGCACAGGTGGA1493c.1113_1130CCCCAGCGGCACAGGTGG1494c.1114_1131GCCCCAGCGGCACAGGTG1495c.1115_1132AGCCCCAGCGGCACAGGT1496c.1116_1133TAGCCCCAGCGGCACAGG1497c.1117_1134GTAGCCCCAGCGGCACAG1498c.1118_1135AGTAGCCCCAGCGGCACA1499c.1119_1136GAGTAGCCCCAGCGGCAC1500c.1120_1137GGAGTAGCCCCAGCGGCA1501c.1121_1138AGGAGTAGCCCCAGCGGC1502c.1122_1139GAGGAGTAGCCCCAGCGG1503c.1123_1140GGAGGAGTAGCCCCAGCG1504c.1124_1141TGGAGGAGTAGCCCCAGC1505c.1125_1142GTGGAGGAGTAGCCCCAG1506c.1126_1143GGTGGAGGAGTAGCCCCA1507c.1127_1144CGGTGGAGGAGTAGCCCC1508c.1128_1145GCGGTGGAGGAGTAGCCC1509c.1129_1146AGCGGTGGAGGAGTAGCC1510c.1130_1147TAGCGGTGGAGGAGTAGC1511c.1131_1148ATAGCGGTGGAGGAGTAG1512c.1132_1149GATAGCGGTGGAGGAGTA1513c.1133_1150TGATAGCGGTGGAGGAGT1514c.1134_1151GTGATAGCGGTGGAGGAG1515c.1135_1152GGTGATAGCGGTGGAGGA1516c.1136_1153GGGTGATAGCGGTGGAGG1517c.1137_1154CGGGTGATAGCGGTGGAG1518c.1138_1155GCGGGTGATAGCGGTGGA1519c.1139_1156GGCGGGTGATAGCGGTGG1520c.1140_1157TGGCGGGTGATAGCGGTG1521c.1141_1158CTGGCGGGTGATAGCGGT1522c.1142_1159CCTGGCGGGTGATAGCGG1523c.1143_1160ACCTGGCGGGTGATAGCG1524c.1144_1161CACCTGGCGGGTGATAGC1525c.1145_1162CCACCTGGCGGGTGATAG1526c.1146_1163ACCACCTGGCGGGTGATA1527c.1147_1164CACCACCTGGCGGGTGAT1528c.1148_1165CCACCACCTGGCGGGTGA1529c.1149_1166TCCACCACCTGGCGGGTG1530c.1150_1167CTCCACCACCTGGCGGGT1531c.1151_1168TCTCCACCACCTGGCGGG1532c.1152_1169TTCTCCACCACCTGGCGG1533c.1153_1170GTTCTCCACCACCTGGCG1534c.1154_1171TGTTCTCCACCACCTGGC1535c.1155_1172ATGTTCTCCACCACCTGG1536c.1156_1173CATGTTCTCCACCACCTG1537c.1157_1174TCATGTTCTCCACCACCT1538c.1158_1175GTCATGTTCTCCACCACC1539c.1159_1176GGTCATGTTCTCCACCAC1540c.1160_1177TGGTCATGTTCTCCACCA1541c.1161_1178CTGGTCATGTTCTCCACC1542c.1162_1179CCTGGTCATGTTCTCCAC1543c.1163_1180CCCTGGTCATGTTCTCCA1544c.1164_1181GCCCTGGTCATGTTCTCC1545c.1165_1182GGCCCTGGTCATGTTCTC1546c.1166_1183GGGCCCTGGTCATGTTCT1547c.1167_1184TGGGCCCTGGTCATGTTC1548c.1168_1185GTGGGCCCTGGTCATGTT1549c.1169_1186AGTGGGCCCTGGTCATGT1550c.1170_1187AAGTGGGCCCTGGTCATG1551c.1171_1188GAAGTGGGCCCTGGTCAT1552c.1172_1189GGAAGTGGGCCCTGGTCA1553c.1173_1190GGGAAGTGGGCCCTGGTC1554c.1174_1191GGGGAAGTGGGCCCTGGT1555c.1175_1192GGGGGAAGTGGGCCCTGG1556c.1176_1193AGGGGGAAGTGGGCCCTG1557c.1177_1194CAGGGGGAAGTGGGCCCT1558c.1178_1194+1CCAGGGGGAAGTGGGCCC1559c.1179_1194+2ACCAGGGGGAAGTGGGCC1560c.1180_1194+3CACCAGGGGGAAGTGGGC1561c.1181_1194+4TCACCAGGGGGAAGTGGG1562c.1182_1194+5CTCACCAGGGGGAAGTGG1563c.1183_1194+6ACTCACCAGGGGGAAGTG1564c.1184_1194+7AACTCACCAGGGGGAAGT1565c.1185_1194+8CAACTCACCAGGGGGAAG1566c.1186_1194+9CCAACTCACCAGGGGGAA1567c.1187_1194+10CCCAACTCACCAGGGGGA1568c.1188_1194+11CCCCAACTCACCAGGGGG1569c.1189_1194+12ACCCCAACTCACCAGGGG1570c.1190_1194+13CACCCCAACTCACCAGGG1571c.1191_1194+14CCACCCCAACTCACCAGG1572c.1192_1194+15ACCACCCCAACTCACCAG1573c.1193_1194+16CACCACCCCAACTCACCA1574c.1194_1194+17CCACCACCCCAACTCACC1575c.1194+1_+18GCCACCACCCCAACTCAC1576c.1194+2_+19TGCCACCACCCCAACTCA1577c.1194+3_+20CTGCCACCACCCCAACTC1578c.1194+4_+21CCTGCCACCACCCCAACT1579c.1194+5_+22CCCTGCCACCACCCCAAC1580c.1194+6_+23CCCCTGCCACCACCCCAA1581c.1194+7_+24TCCCCTGCCACCACCCCA1582c.1194+8_+25CTCCCCTGCCACCACCCC1583 In the above examples the sequences are 18, 21 and 25 nucleotides long however longer variants or shorter fragment are also envisioned. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO: 541-1583 and fragments and variants thereof having at least 80% sequence identity. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO: 541-1583 and fragments and variants thereof having at least 80%, 83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% sequence identity to SEQ ID NO: 541-1583. Or sequences that are at least 80% identical to SEQ ID NO: 541-1583. Preferably at least 85% identical to SEQ ID NO: 541-1583, more preferably at least 88% identical to SEQ ID NO: 541-1583, more preferably at least 90% identical to SEQ ID NO: 541-1583. more preferably at least 91% identical to SEQ ID NO: 541-1583, more preferably at least 92% identical to SEQ ID NO: 541-1583, more preferably at least 93% identical to SEQ ID NO: 541-1583, more preferably at least 94% identical to SEQ ID NO: 541-1583, more preferably at least 95% identical to SEQ ID NO: 541-1583, more preferably at least 96% identical to SEQ ID NO: 541-1583, more preferably at least 97% identical to SEQ ID NO: 541-1583, more preferably at least 98% identical to SEQ ID NO: 541-1583, more preferably at least 99% identical to SEQ ID NO: 541-1583. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 541-1583, wherein the fragment is 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 541-1583, wherein the fragment is 17, 18, 19, 20, 21, or 22 nucleotides long. In a preferred embodiment of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 541-1583, wherein the fragment is 19, 20, or 21 nucleotides long. The antisense oligomeric compound may be also be complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation selected from the groupc.−32−13T>G (IVS1), c.1636+5G>T, c.525delT, c.−32−3C>G, c. 1551+1G>A, c.1075G>A, c.1552−3C>G, c.1437G>A, c.1256A>T, c.1551+1G>T. Preferably the genomic nucleic acid sequence is pre-mRNA. In a preferred embodiment of the invention and/or embodiments thereof, the antisense oligomeric compound may be also be complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation selected from the group comprisingc.−32−3C>G, c.−32−13T>G, c.−32−102T>C, c.−32−56C>T, c.−32−46G>A, c.−32−28C>A, c.−32−28C>T, c.−32−21G>A, c.7G>A, c.11G>A, c.15_17AAA, c.17C>T, c.19_21AAA, c.26_28AAA, c.33_35AAA, c.39G>A, c.42C>T, c.90C>T, c.112G>A, c.137C>T, c.164C>T, c.348G>A, c.373C>T, c.413T>A, c.469C>T, c.476T>C, c.476T>G, c.478T>G, c.482C>T, c.510C>T, c.515T>A, c.520G>A, c.546+11C>T, c.546+14G>A, c.546+19G>A, c.546+23C>A, c.547−6, c.1071, c.1254, and c.1552−30. Preferably the genomic nucleic acid sequence is pre-mRNA In a preferred embodiment of the invention and/or embodiments thereof, the antisense oligomeric compound may be also be complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation selected from the group comprising c.17C>T c.469C>T c.546+23C>A, c.−32−102T>C c.−32−56C>T c.11G>A c.112G>A c.137C>T. In a preferred embodiment of the invention and/or embodiments thereof, the antisense oligomeric compound may be also be complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation selected from the group comprising c.17C>T c.469C>T c.546+23C>A. In a preferred embodiment of the invention and/or embodiments thereof, the antisense oligomeric compound may be also be complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation selected from the group comprising c.−32−102T>C c.−32−56C>T c.11G>A c.112G>A c.137C>T. Most preferred are antisense oligomeric compounds that are complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation c.−32−13T>G (IVS1). Most preferred are antisense oligomeric compounds that are complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation c.−32−3C>G, c.1256A>T, c.1551+1G>T, c.546G>T. Most preferred are antisense oligomeric compounds that are complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation c.−32−3C>G. Most preferred are antisense oligomeric compounds that are complementary to a genomic nucleic acid sequence of GAA gene targeting SEQ ID NO: 1. (SEQ ID NO: 1)GCTCTGCACTCCCCTGCTGGAGCTTTTCTCGCCCTTCCTTCTGGCCCTCTCCCCA. In a preferred embodiment of the invention and/or embodiments thereof, the antisense oligomeric compound are 8 to 80 nucleotides in length, 9 to 50 nucleotides in length, 10 to 30 nucleotides in length, 12 to 30 nucleotides in length, 15 to 25 nucleotides in length or about 20 nucleotides in length. One of ordinary skill in the art will appreciate that this comprehends antisense compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 13 to 80 nucleotides. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 13 to 50 nucleotides. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 13 to 30 nucleotides. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 20 to 30 nucleotides. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 15 to 25 nucleotides. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 20 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 19 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 18 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 17 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 16 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 15 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 14 nucleotides. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 13 nucleotides. In one embodiment of the invention and/or embodiments thereof, compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleotides from one of the antisense compounds as claimed. Preferably at least 9 consecutive nucleotides from one of the antisense compounds as claimed, more preferably at least 10 consecutive nucleotides from one of the antisense compounds as claimed, more preferably at least 11 consecutive nucleotides from one of the antisense compounds as claimed, more preferably at least 12 consecutive nucleotides from one of the antisense compounds as claimed, more preferably at least 13 consecutive nucleotides from one of the antisense compounds as claimed, more preferably at least 14 consecutive nucleotides from one of the antisense compounds as claimed, more preferably at least 15 consecutive nucleotides from one of the antisense compounds as claimed, more preferably at least 16 consecutive nucleotides from one of the antisense compounds as claimed, more preferably at least 17 consecutive nucleotides from one of the antisense compounds as claimed, more preferably at least 18 consecutive nucleotides from one of the antisense compounds as claimed, more preferably at least 19 consecutive nucleotides from one of the antisense compounds as claimed, more preferably at least 20 consecutive nucleotides from one of the antisense compounds as claimed. Any remaining nucleotides from the oligonuclotides may be oligonucleotides that improve resistance to Rnase H, cell-targeting sequences, cell penetrating sequences, marker sequences or any other sequences. One having skill in the art armed with the antisense compounds disclosed herein will be able, without undue experimentation, to identify further antisense compounds. In order for an antisense oligonucleotide to achieve therapeutic success, oligonucleotide chemistry must allow for adequate cellular uptake (Kurreck, J. (2003) Eur. J. Biochem. 270:1628-1644). Splicing oligonucleotides have traditionally been comprised of uniform modifications that render the oligonucleotide RNA-like, and thus resistant to cleavage by RNase H, which is critical to achieve modulation of splicing. Provided herein are antisense compounds for modulation of splicing. In a preferred embodiment of the invention and/or embodiments thereof, the antisense compounds are chimeric, with regions of RNA-like and DNA-like chemistry. Despite regions of DNA-like chemistry, the chimeric compounds are preferably RNase H-resistant and effectively modulate splicing of target mRNA in vitro and in vivo. In another preferred embodiment the disclosed antisense oligomeric compounds show enhanced cellular uptake and greater pharmacologic activity compared with uniformly modified oligonucleotides. Contemplated herein are antisense oligomeric compound which are targeted to a splice site of a target mRNA or to splicing repressor sequences, or to splicing enhancer sequences, preferably to splicing repressor sequences. Splice sites include aberrant and cryptic splice sites. One skilled in the art recognizes that the inclusion of mismatches is possible without eliminating the activity of the antisense compound. Compounds provided herein are therefore directed to those antisense compounds that may contain up to about 20% nucleotides that disrupt base pairing of the antisense compound to the target. Preferably the compounds contain no more than about 15%, more preferably not more than about 10%, most preferably not more than 5% or no mismatches. The remaining nucleotides do not disrupt hybridization (e.g., universal bases). It is understood in the art that incorporation of nucleotide affinity modifications may allow for a greater number of mismatches compared to an unmodified compound. Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences. One of the skill in the art is capable of determining an appropriate number of mismatches between oligonucleotides, or between an oligonucleotide and a target nucleic acid, such as by determining melting temperature. It is known by a skilled person that hybridization to a target mRNA depends on the conditions. “Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated. Antisense compounds, or a portion thereof, may have a defined percent identity to a SEQ ID NO, or a compound having a specific Isis number. As used herein, a sequence is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in the disclosed sequences would be considered identical as they both pair with adenine. This identity may be over the entire length of the oligomeric compound, or in a portion of the antisense compound (e.g., nucleotides 1-20 of a 27-mer may be compared to a 20-mer to determine percent identity of the oligomeric compound to the SEQ ID NO.) It is understood by those skilled in the art that an antisense compound need not have an identical sequence to those described herein to function similarly to the antisense compound described herein. Shortened versions of antisense compound taught herein, or non-identical versions of the antisense compound taught herein are also contemplated. Non-identical versions are those wherein each base does not have the same pairing activity as the antisense compounds disclosed herein. Bases do not have the same pairing activity by being shorter or having at least one abasic site. Alternatively, a non-identical version can include at least one base replaced with a different base with different pairing activity (e.g., G can be replaced by C, A, or T). Percent identity is calculated according to the number of bases that have identical base pairing corresponding to the SEQ ID NO or antisense compound to which it is being compared. The non-identical bases may be adjacent to each other, dispersed through out the oligonucleotide, or both. For example, a 16-mer having the same sequence as nucleotides 2-17 of a 20-mer is 80% identical to the 20-mer. Alternatively, a 20-mer containing four nucleotides not identical to the 20-mer is also 80% identical to the 20-mer. A 14-mer having the same sequence as nucleotides 1-14 of an 18-mer is 78% identical to the 18-mer. Such calculations are well within the ability of those skilled in the art. The percent identity is based on the percent of nucleotides in the original sequence present in a portion of the modified sequence. Therefore, a 30 nucleobase antisense compound comprising the full sequence of the complement of a 20 nucleobase active target segment would have a portion of 100% identity with the complement of the 20 nucleobase active target segment, while further comprising an additional 10 nucleobase portion. The complement of an active target segment may constitute a single portion. In a preferred embodiment of the invention and/or embodiments thereof, the oligonucleotides are at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, most preferably at least 95% identical to at least a portion of the complement of the active target segments presented herein. It is well known by those skilled in the art that it is possible to increase or decrease the length of an antisense compound and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7310, 1992, incorporated herein by reference), a series of antisense oligomeric compounds of 13-25 nucleotides in length were tested for their ability to induce cleavage of a target RNA. Antisense oligomeric compounds of 25 nucleotides in length with 8 or 11 mismatch bases near the ends of the antisense oligomeric compounds were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligomeric compounds that contained no mismatches. Similarly, target specific cleavage was achieved using a 13 nucleobase antisense oligomeric compounds, including those with 1 or 3 mismatches. Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988, incorporated herein by reference) tested a series of tandem 14 nucleobase antisense oligomeric compounds, and a 28 and 42 nucleobase antisense oligomeric compounds comprised of the sequence of two or three of the tandem antisense oligomeric compounds, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligomeric compounds alone were able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligomeric compounds. It is understood that antisense compounds can vary in length and percent complementarity to the target provided that they maintain the desired activity. Methods to determine desired activity are disclosed herein and well known to those skilled in the art. In a preferred embodiment of the invention and/or embodiments thereof, the antisense oligomeric compounds have at least 80% complementarity to the target mRNA, more preferably at least 85% complementarity to the target mRNA, more preferably at least 90% complementarity to the target mRNA, more preferably at least 95% complementarity to the target mRNA, more preferably at least 96% complementarity to the target mRNA, more preferably at least 97% complementarity to the target mRNA, more preferably at least 98% complementarity to the target mRNA, more preferably at least 99% complementarity to the target mRNA, more preferably at least 100% complementarity to the target mRNA. As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base (sometimes referred to as a “nucleobase” or simply a “base”). The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage. It is often preferable to include chemical modifications in oligonucleotides to alter their activity. Chemical modifications can alter oligonucleotide activity by, for example: increasing affinity of an antisense oligonucleotide for its target RNA, increasing nuclease resistance, and/or altering the pharmacokinetics of the oligonucleotide. The use of chemistries that increase the affinity of an oligonucleotide for its target can allow for the use of shorter oligonucleotide compounds. Antisense compounds provided herein may also contain one or more nucleosides having modified sugar moieties. The furanosyl sugar ring of a nucleoside can be modified in a number of ways including, but not limited to, addition of a substituent group, bridging of two non-geminal ring atoms to form a bicyclic nucleic acid (BNA) and substitution of an atom or group such as —S—, —N(R)— or —C(R1)(R2) for the ring oxygen at the 4′-position. Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of the antisense compound for its target and/or increase nuclease resistance. A representative list of preferred modified sugars includes but is not limited to bicyclic modified sugars (BNA's), including LNA and ENA (4′-(CH2)2-O-2′ bridge); and substituted sugars, especially 2′-substituted sugars having a 2′-F, 2′-OCH2 or a 2′-O(CH2)2-OCH3 substituent group. Sugars can also be replaced with sugar mimetic groups among others. Methods for the preparations of modified sugars are well known to those skilled in the art. Suitable compounds can comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Also suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Other oligonucleotides comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, poly-alkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. One modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group. A further modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)20N(CH3)2 group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—(CH2)2-O—(CH2)2-N(CH3)2. Other modifications include 2′-methoxy (2′-O—CH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2), 2′-allyl (2′-CH2-CH—CH2), 2′-O-allyl (2′-O-CH2-CH—CH2) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. One 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3 position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Antisense compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; and, 6,147,200. In one aspect of the present invention oligomeric compounds include nucleosides modified to induce a 3-endo sugar conformation. A nucleoside can incorporate modifications of the heterocyclic base, the sugar moiety or both to induce a desired 3-endo sugar conformation. These modified nucleosides are used to mimic RNA-like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3-endo conformational geometry. In the present invention there is a preference for an RNA type duplex (A form helix, predominantly 3-endo) as they are RnasH resistant. Properties that are enhanced by using more stable 3-endo nucleosides include but are not limited to: modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage. Nucleoside conformation is influenced by various factors including substitution at the 2′, 3′ or 4-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2′ position to favor the 3-endo conformation can be achieved while maintaining the 2′-OH as a recognition element (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference for the 3-endo conformation can be achieved by deletion of the 2′-OH as exemplified by 2′ deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3-endo conformation positioning the electronegative fluorine atom in the axial position. Representative 2′-substituent groups amenable to the present invention that give A-form conformational properties (3-endo) to the resultant duplexes include 2′-O-alkyl, 2′-O-substituted alkyl and 2′-fluoro substituent groups. Other suitable substituent groups are various alkyl and aryl ethers and thioethers, amines and monoalkyl and dialkyl substituted amines. Other modifications of the ribose ring, for example substitution at the 4′-position to give 4′-F modified nucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example modification to yield methanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) also induce preference for the 3-endo conformation. Along similar lines, one or more nucleosides may be modified in such a way that conformation is locked into a C3′-endo type conformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENA(TM), Morita et al, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.) Preferred modification of the sugar are selected from the group consisting of 2′-O-methyl 2′-O-methoxyethyl, 2′-fluoro, 2′-dimethylaminooxyethoxy, 2′-dimethylaminoethoxyethoxy, 2′-guanidinium, 2′-O-guanidinium ethyl, 2′-carbamate, 2′-aminooxy, 2′-acetamido and locked nucleic acid. In one preferred embodiment, the sugar modification is 2′-O-methyl or 2′-O-methoxyethyl. Oligomeric compounds can also include nucleobase (often referred to in the art as heterocyclic base or simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). A “substitution” is the replacement of an unmodified or natural base with another unmodified or natural base. “Modified” nucleotides mean other synthetic and natural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C[identical to]C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleotides include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3′,2′:4,5)pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleotides may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleotides include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleotides are known to those skilled in the art as suitable for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently suitable base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications. It is understood in the art that modification of the base does not entail such chemical modifications as to produce substitutions in a nucleic acid sequence. Representative United States patents that teach the preparation of certain of the above noted modified nucleotides as well as other modified nucleotides include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,681,941; and 5,750,692. Oligomeric compounds of the present invention may also include polycyclic heterocyclic compounds in place of one or more of the naturally-occurring heterocyclic base moieties. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G-clamps or cytidine analogs. Representative cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand include 1,3-diazaphenoxazine-2-one (Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16, 1837-1846), 1,3-diazaphenothiazine-2-one, (Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388). Incorporated into oligonucleotides these base modifications were shown to hybridize with complementary guanine and the latter was also shown to hybridize with adenine and to enhance helical thermal stability by extended stacking interactions (also see U.S. Pre-Grant Publications 20030207804 and 20030175906). Further helix-stabilizing properties have been observed when a cytosine analog/substitute has an aminoethoxy moiety attached to the rigid 1,3-diazaphenoxazine-2-one scaffold (Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). Binding studies demonstrated that a single incorporation could enhance the binding affinity of a model oligonucleotide to its complementary target DNA or RNA with a ATm of up to 18° C. relative to 5-methyl cytosine, which is a high affinity enhancement for a single modification. On the other hand, the gain in helical stability does not compromise the specificity of the oligonucleotides. Further tricyclic heterocyclic compounds and methods of using them that are amenable to use in the present invention are disclosed in U.S. Pat. Nos. 6,028,183, and 6,007,992. The enhanced binding affinity of the phenoxazine derivatives together with their uncompromised sequence specificity makes them valuable nucleobase analogs for the development of more potent antisense-based drugs. In fact, promising data have been derived from in vitro experiments demonstrating that heptanucleotides containing phenoxazine substitutions are capable to activate RNase H, enhance cellular uptake and exhibit an increased antisense activity (Lin, K-Y; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). The activity enhancement was even more pronounced in case of G-clamp, as a single substitution was shown to significantly improve the in vitro potency of a 20 mer 2′-deoxyphosphorothioate oligonucleotides (Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518). Further modified polycyclic heterocyclic compounds useful as heterocyclic bases are disclosed in but not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. Pre-Grant Publication 20030158403. The compounds described herein may include internucleoside linking groups that link the nucleosides or otherwise modified monomer units together thereby forming an antisense compound. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (−CH2-N(CH3)-O-CH2-), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2-O—); and N,N′-dimethylhydrazine (˜CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the antisense compound. Internucleoside linkages having a chiral atom may be prepared racemic, chiral, or as a mixture. Representative chiral internucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known to those skilled in the art. Suitable modified internucleoside linking groups are for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl-phosphonates, thionoalkylphosphotriesters, phosphonoacetate and thiophosphonoacetate (see Sheehan et al., Nucleic Acids Research, 2003, 31(14), 4109-4118 and Dellinger et al., J. Am. Chem. Soc., 2003, 125, 940-950), selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage, i.e., a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included. N3′-P5′-phosphoramidates have been reported to exhibit both a high affinity towards a complementary RNA strand and nuclease resistance (Gryaznov et al., J. Am. Chem. Soc., 1994, 116, 3143-3144). N3′-P5′-phosphoramidates have been studied with some success in vivo to specifically down regulate the expression of the c-myc gene (Skorski et al., Proc. Natl. Acad. Sci., 1997, 94, 3966-3971; and Faira et al., Nat. Biotechnol., 2001, 19, 40-44). Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050. In some embodiments of the invention, oligomeric compounds may have one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH2-NH—O-CH2-, —CH2-N(CH3)-O—CH2- (known as a methylene (methylimino) or MMI backbone), —CH2-O—N(CH3)-CH2-, —CH2-N(CH3)-N(CH3)-CH2- and —O—N(CH3)-CH2-CH2- (wherein the native phosphodiester internucleotide linkage is represented as —O—P(—O)(OH)—O-CH2-). The MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Amide internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,602,240. Some oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439. In a preferred embodiment of the invention and/or embodiments thereof the internucleoside linkage is phosphorothioate, or phosphorodiamidate It is further intended that multiple modifications can be made to one or more of the oligomeric compounds of the invention at multiple sites of one or more monomeric subunits (nucleosides are suitable) and/or internucleoside linkages to enhance properties such as but not limited to activity in a selected application. The synthesis of numerous of the modified nucleosides amenable to the present invention are known in the art (see for example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988, Plenum press). The conformation of modified nucleosides and their oligomers can be estimated by various methods routine to those skilled in the art such as molecular dynamics calculations, nuclear magnetic resonance spectroscopy and CD measurements. In a preferred embodiment of the invention and/or embodiments thereof, the oligomeric compounds of the present invention are morpholino phosphorothioates, or phosphorodiamidate morpholino. Another group of oligomeric compounds includes oligonucleotide mimetics. As used herein the term “mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target. Representative examples of a sugar mimetic include, but are not limited to, cyclohexenyl or morpholino. Representative examples of a mimetic for a sugar-internucleoside linkage combination include, but are not limited to, peptide nucleic acids (PNA) and morpholino groups linked by uncharged achiral linkages. In some instances a mimetic is used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods of synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art. The heterocyclic base moiety or a modified heterocyclic base moiety is preferably maintained for hybridization with an appropriate target nucleic acid. The compounds described herein may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), [alpha] or [beta], or as (D) or (L) such as for amino acids et al. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA) (Nielsen et al., Science, 1991, 254, 1497-1500). PNAs have favorable hybridization properties, high biological stability and are electrostatically neutral molecules. PNA compounds have been used to correct aberrant splicing in a transgenic mouse model (Sazani et al., Nat. Biotechnol., 2002, 20, 1228-1233). In PNA oligomeric compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA oligomeric compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. PNA compounds can be obtained commercially from Applied Biosystems (Foster City, Calif., USA). Numerous modifications to the basic PNA backbone are known in the art; particularly useful are PNA compounds with one or more amino acids conjugated to one or both termini. For example, 1-8 lysine or arginine residues are useful when conjugated to the end of a PNA molecule. A polyarginine tail may be a suitable for enhancing cell penetration. Another class of oligonucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups have been selected to give a non-ionic oligomeric compound. Morpholino-based oligomeric compounds are non-ionic mimetics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based oligomeric compounds have been studied in zebrafish embryos (see: Genesis, volume 30, issue 3, 2001 and Heasman, J., Dev. Biol., 2002, 243, 209-214). Further studies of morpholino-based oligomeric compounds have also been reported (Nasevicius et al., Nat. Genet., 2000, 26, 216-220; and Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596). Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No. 5,034,506. The morpholino class of oligomeric compounds have been prepared having a variety of different linking groups joining the monomeric subunits. Linking groups can be varied from chiral to achiral, and from charged to neutral. U.S. Pat. No. 5,166,315 discloses linkages including —O—P(—O)(N(CH3)2)-O—; U.S. Pat. No. 5,034,506 discloses achiral intermorpholino linkages; and U.S. Pat. No. 5,185,444 discloses phosphorus containing chiral intermorpholino linkages. A further class of oligonucleotide mimetic is referred to as cyclohexene nucleic acids (CeNA). In CeNA oligonucleotides, the furanose ring normally present in a DNA or RNA molecule is replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation. Furthermore the incorporation of CeNA into a sequence targeting RNA was stable to serum and able to activateE. coliRNase H resulting in cleavage of the target RNA strand. A further modification includes bicyclic sugar moieties such as “Locked Nucleic Acids” (LNAs) in which the 2′-hydroxyl group of the ribosyl sugar ring is linked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage to form the bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (˜CH2-) group bridging the 2′ oxygen atom and the 4′ carbon atom, for which the term LNA is used for the bicyclic moiety; in the case of an ethylene group in this position, the term ENA(TM) is used (Singh et al., Chem. Commun., 1998, 4, 455-456; ENA(TM): Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). LNA and other bicyclic sugar analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10[deg.] C.), stability towards 3′-exonucleolytic degradation and good solubility properties. LNAs are commercially available from ProLigo (Paris, France and Boulder, Colo., USA). An isomer of LNA that has also been studied is alpha-L-LNA which has been shown to have superior stability against a 3-exonuclease. The alpha-L-LNAs were incorporated into antisense gapmers and chimeras that showed potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Another similar bicyclic sugar moiety that has been prepared and studied has the bridge going from the 3-hydroxyl group via a single methylene group to the 4 carbon atom of the sugar ring thereby forming a 3′-C,4′-C-oxymethylene linkage (see U.S. Pat. No. 6,043,060). LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNA hybridization was shown to be the most thermally stable nucleic acid type duplex system, and the RNA-mimicking character of LNA was established at the duplex level. Introduction of 3 LNA monomers (T or A) significantly increased melting points (Tm=+15/+11[deg.] C.) toward DNA complements. The universality of LNA-mediated hybridization has been stressed by the formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking of LNA was reflected with regard to the N-type conformational restriction of the monomers and to the secondary structure of the LNA:RNA duplex. LNAs also form duplexes with complementary DNA, RNA or LNA with high thermal affinities. Circular dichroism (CD) spectra show that duplexes involving fully modified LNA (esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination of an LNA:DNA duplex confirmed the 3-endo conformation of an LNA monomer. Recognition of double-stranded DNA has also been demonstrated suggesting strand invasion by LNA. Studies of mismatched sequences show that LNAs obey the Watson-Crick base pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands. DNA-LNA chimeras have been shown to efficiently inhibit gene expression when targeted to a variety of regions (5′-untranslated region, region of the start codon or coding region) within the luciferase mRNA (Braasch et al., Nucleic Acids Research, 2002, 30, 5160-5167). Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sc U.S.A., 2000, 97, 5633-5638). The authors have demonstrated that LNAs confer several desired properties. LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. LNA/DNA copolymers exhibited potent antisense activity in assay systems as disparate as G-protein-coupled receptor signaling in living rat brain and detection of reporter genes inEscherichia coli. Lipofectin-mediated efficient delivery of LNA into living human breast cancer cells has also been accomplished. Further successful in vivo studies involving LNA's have shown knock-down of the rat delta opioid receptor without toxicity (Wahlestedt et al., Proc. Natl. Acad. Sci., 2000, 97, 5633-5638) and in another study showed a blockage of the translation of the large subunit of RNA polymerase II (Fluiter et al., Nucleic Acids Res., 2003, 31, 953-962). The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226. Analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-LNA, a novel conformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-Amino- and 2′-methylamino-LNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported. Another oligonucleotide mimetic that has been prepared and studied is threose nucleic acid. This oligonucleotide mimetic is based on threose nucleosides instead of ribose nucleosides. Initial interest in (3′,2′)-alpha-L-threose nucleic acid (TNA) was directed to the question of whether a DNA polymerase existed that would copy the TNA. It was found that certain DNA polymerases are able to copy limited stretches of a TNA template (reported in Chemical and Engineering News, 2003, 81, 9). In another study it was determined that TNA is capable of antiparallel Watson-Crick base pairing with complementary DNA, RNA and TNA oligonucleotides (Chaput et al., J. Am. Chem. Soc., 2003, 125, 856-857). In one study (3′,2′)-alpha-L-threose nucleic acid was prepared and compared to the 2′ and 3′ amidate analogs (Wu et al., Organic Letters, 2002, 4(8), 1279-1282). The amidate analogs were shown to bind to RNA and DNA with comparable strength to that of RNA/DNA. Further oligonucleotide mimetics have been prepared to include bicyclic and tricyclic nucleoside analogs (see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; Renneberg et al., J. Am. Chem. Soc., 2002, 124, 5993-6002; and Renneberg et al., Nucleic acids res., 2002, 30, 2751-2757). These modified nucleoside analogs have been oligomerized using the phosphoramidite approach and the resulting oligomeric compounds containing tricyclic nucleoside analogs have shown increased thermal stabilities (Tm's) when hybridized to DNA, RNA and itself. Oligomeric compounds containing bicyclic nucleoside analogs have shown thermal stabilities approaching that of DNA duplexes. Another class of oligonucleotide mimetic is referred to as phosphonomonoester nucleic acids which incorporate a phosphorus group in the backbone. This class of oligonucleotide mimetic is reported to have useful physical and biological and pharmacological properties in the areas of inhibiting gene expression (antisense oligonucleotides, sense oligonucleotides and triplex-forming oligonucleotides), as probes for the detection of nucleic acids and as auxiliaries for use in molecular biology. Further oligonucleotide mimetics amenable to the present invention have been prepared wherein a cyclobutyl ring replaces the naturally occurring furanosyl ring. Another modification of the oligomeric compounds of the invention involves chemically linking to the oligomeric compound one or more moieties or conjugates which enhance the properties of the oligomeric compound, such as to enhance the activity, cellular distribution or cellular uptake of the oligomeric compound. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. Nos. 6,287,860 and 6,762,169. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligomeric compounds of the invention may also be conjugated to drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)—(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. Pat. No. 6,656,730. Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941. Oligomeric compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of an oligomeric compound to enhance properties such as for example nuclease stability. Included in stabilizing groups are cap structures. By “cap structure or terminal cap moiety” is meant chemical modifications, which have been incorporated at either terminus of oligonucleotides (see for example Wincott et al., WO 97/26270). These terminal modifications protect the oligomeric compounds having terminal nucleic acid molecules from exonuclease degradation, and can improve delivery and/or localization within a cell. The cap can be present at either the 5-terminus (5′-cap) or at the 3-terminus (3-cap) or can be present on both termini of a single strand, or one or more termini of both strands of a double-stranded compound. This cap structure is not to be confused with the inverted methylguanosine “5′ cap” present at the 5′ end of native mRNA molecules. In non-limiting examples, the 5-cap includes inverted abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl riucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al., International PCT publication No. WO 97/26270). Particularly suitable 3′-cap structures include, for example 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925). Further 3′ and 5-stabilizing groups that can be used to cap one or both ends of an oligomeric compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003. In certain embodiments, oligomeric compounds, may be conjugated with a wide variety of different positively charged polymers. Examples of positively charged polymers include peptides, such as argine rich peptides (Examples of positively charged peptides that may be used in the practice of the invention include R9F2C; (RXR)4 XB (where X can be any amino acid); R5F2R4c; (RFF)3; Tat proteins, such as TAT sequence CYGRKKRRQRRR; and (RFF)3R), cationic polymers, such as dendrimeric octaguanindine polymer, and other positively charged molecules as known in the art for conjugation to antisense oligonucleotide compounds. In one embodiment of the invention and/or embodiments thereof, the antisense oligonucleotides are conjugated with positively charged polymer comprising a polymer having a molecular weight that is from about 1,000 to 20,000 Daltons, and preferably from about 5,000 to 10,000 Daltons. Another example of positively charged polymers is polyethylenimine (PEI) with multiple positively charged amine groups in its branched or unbranched chains. PEI has else been widely used as gene and oligomer delivery vesicle. In a preferred embodiment of the invention and/or embodiments thereof the oligomeric compounds are modified with cell penetrating sequences. Suitable cell penetrating sequences include cell penetrating peptides, such as TAT peptide, MPG, Pep-1, MAP, fusogenic, antimicrobial peptides (AMPs), bacteriocidal peptides, fungicidal peptides, virucidal peptides, Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular uptake of the particles of the invention. The particle of the invention is associated with the CPP peptides either through chemical linkage via covalent bonds or through non-covalent interactions. The function of the CPPs are to deliver the particles into cells, a process that commonly occurs through endocytosis with the cargo delivered to the endosomes of living mammalian cells. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of CPPs are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for cellular uptake. An exemplary cell penetrating peptide is the trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) could be efficiently taken up from the surrounding media by numerous cell types in culture. Other cell penetrating peptides are MPG, Pep-1, transportan, penetratin, CADY, TP, TP10, arginine octamer. polyarginine sequences, Arg8, VP22 HSV-1 structural protein, SAP Proline-rich motifs, Vectocell@ peptides, hCT (9-32), SynB, Pvec, and PPTG1. Cell penetrating peptides may be cationic, essentially containing clusters of polyarginine in their primary sequence or amphipathic. CPPs are generally peptides of less than 30 amino acids, derived from natural or unnatural protein or chimeric sequences. In suitable embodiments, the oligomeric compounds are incorporated or otherwise associated with nanoparticles. Nanoparticles may suitably modified for targeting specific cells and optimised for penetrating cells. A skilled person is aware of methods to employ nanoparticles for oligomeric compounds delivery to cells. In suitable embodiments of the present invention, the oligomeric compounds are modified with an endosomal escape agent moiety. The endocytic pathway is a major uptake mechanism of cells. Compounds taken up by the endocytic pathway become entrapped in endosomes and may be degraded by specific enzymes in the lysosome. This may be desired or not desired depending on the purpose. If taken up by the endosomes is not desired, endosomal escape agent may be used. Suitable endosomal escape agents may be chloroquine, TAT peptide. It is not necessary for all positions in a given oligomeric compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even within a single nucleoside within an oligomeric compound. The present invention also includes oligomeric compounds which are chimeric compounds. “Chimeric” oligomeric compounds or “chimeras,” in the context of this invention, are single- or double-stranded oligomeric compounds, such as oligonucleotides, which contain two or more chemically distinct regions, each comprising at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. Chimeric antisense oligonucleotides are one form of oligomeric compound. These oligonucleotides typically contain at least one region which is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, alteration of charge, increased stability and/or increased binding affinity for the target nucleic acid. Chimeric oligomeric compounds of the invention can be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, oligonucleotide mimetics, or regions or portions thereof. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922. Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713). Oligomeric compounds of the present invention can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The following precursor compounds, including amidites and their intermediates can be prepared by methods routine to those skilled in the art; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N4-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N<4>-benzoyl-5-methylcytidine penultimate intermediate, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N<4>-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N<6>-benzoyladenosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N<4>-isobutyrylguanosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O<2>-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-((2-phthalimidoxy)ethyl)-5′-t-butyldiphenylsilyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-((2-formadoximinooxy)ethyl)-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O—(N,N dimethylaminooxyethyl)-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite), 2′-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite), 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite. The preparation of such precursor compounds for oligonucleotide synthesis are routine in the art and disclosed in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743. 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites can be purchased from commercial sources (e.g. Chemgenes, Needham, Mass. or Glen Research, Inc. Sterling, Va.). Other 2′-O-alkoxy substituted nucleoside amidites can be prepared as described in U.S. Pat. No. 5,506,351. Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C) nucleotides can be synthesized routinely according to published methods (Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham, Mass.). 2′-fluoro oligonucleotides can be synthesized routinely as described (Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841) and U.S. Pat. No. 5,670,633. 2′-O-Methoxyethyl-substituted nucleoside amidites can be prepared routinely as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504. Aminooxyethyl and dimethylaminooxyethyl amidites can be prepared routinely as per the methods of U.S. Pat. No. 6,127,533. Phosphorothioate-containing oligonucleotides (P—S) can be synthesized by methods routine to those skilled in the art (see, for example, Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press). Phosphinate oligonucleotides can be prepared as described in U.S. Pat. No. 5,508,270. Alkyl phosphonate oligonucleotides can be prepared as described in U.S. Pat. No. 4,469,863. 3′-Deoxy-3-methylene phosphonate oligonucleotides can be prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050. Phosphoramidite oligonucleotides can be prepared as described in U.S. Pat. No. 5,256,775 or 5,366,878. Alkylphosphonothioate oligonucleotides can be prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively). 3′-Deoxy-3′-amino phosphoramidate oligonucleotides can be prepared as described in U.S. Pat. No. 5,476,925. Phosphotriester oligonucleotides can be prepared as described in U.S. Pat. No. 5,023,243. Borano phosphate oligonucleotides can be prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198. 4′-thio-containing oligonucleotides can be synthesized as described in U.S. Pat. No. 5,639,873. Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P—O or P—S linkages can be prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289. Formacetal and thioformacetal linked oligonucleosides can be prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxide linked oligonucleosides can be prepared as described in U.S. Pat. No. 5,223,618. Peptide nucleic acids (PNAs) can be prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, 5,719,262, 6,559,279 and 6,762,281. Oligomeric compounds incorporating at least one 2′-O-protected nucleoside by methods routine in the art. After incorporation and appropriate deprotection the 2′-O-protected nucleoside will be converted to a ribonucleoside at the position of incorporation. The number and position of the 2-ribonucleoside units in the final oligomeric compound may vary from one at any site or the strategy can be used to prepare up to a full 2′-OH modified oligomeric compound. The main RNA synthesis strategies that are presently being used commercially include 5′-[beta]-DMT-2′-O-t-butyldimethylsilyl (TBDMS), 5′-O-DMT-2′-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP), 2′-O-[(triisopropylsilyl)oxy]methyl (2′-O-CH2-O—Si(iPr)3 (TOM), and the 5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). Some companies currently offering RNA products include Pierce Nucleic Acid Technologies (Milwaukee, Wis.), Dharmacon Research Inc. (a subsidiary of Fisher Scientific, Lafayette, Colo.), and Integrated DNA Technologies, Inc. (Coralville, Iowa). One company, Princeton Separations, markets an RNA synthesis activator advertised to reduce coupling times especially with TOM and TBDMS chemistries. Such an activator would also be amenable to the oligomeric compounds of the present invention. All of the aforementioned RNA synthesis strategies are amenable to the oligomeric compounds of the present invention. Strategies that would be a hybrid of the above e.g. using a 5′-protecting group from one strategy with a 2′-O-protecting from another strategy is also contemplated herein. Chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides can be synthesized according to U.S. Pat. No. 5,623,065. Chimeric oligomeric compounds exhibitting enhanced cellular uptake and greater pharmacologic activity may be made in accordance to U.S. Pat. No. 8,501,703. Another form of oligomeric compounds comprise tricyclo-DNA (tc-DNA) antisense oligonucleotides. Tricyclo-DNA nucleotides are nucleotides modified by the introduction of a cyclopropane ring to restrict conformational flexibility of the backbone and to optimize the backbone geometry of the torsion angle γ. Homobasic adenine- and thymine-containing tc-DNAs form extraordinarily stable A-T base pairs with complementary RNAs. Antisense oligomeric compound that contains between 6-22 tricyclo nucleotides in length, in particular between 8-20 tricyclo nucleotides, more particularly between 10 and 18 or between 11 and 18 tricyclo nucleotides are suitable. See e.g. WO2010115993 for examples of tricyclo-DNA (tc-DNA) antisense oligonucleotides. Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713). Antisense compounds can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The disclosure is not limited by the method of antisense compound synthesis. Methods of oligonucleotide purification and analysis are known to those skilled in the art. Analysis methods include capillary electrophoresis (CE) and electrospray-mass spectroscopy. Such synthesis and analysis methods can be performed in multi-well plates. The methods described herein are not limited by the method of oligomer purification. In a preferred embodiment of the invention and/or embodiments thereof, the antisense compounds provided herein are resistant to RNase H degradation. In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise at least one modified nucleotide. In another embodiment, the antisense compounds comprise a modified nucleotide at each position. In yet another embodiment, the antisense compounds are uniformly modified at each position. Modulation of splicing can be assayed in a variety of ways known in the art. Target mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA by methods known in the art. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions. Levels of a protein encoded by a target mRNA can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to a protein encoded by a target mRNA can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997. Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991. The effect of the oligomeric compounds of the present invention may be analysed by RT PCT, qPCR, flanking exon PCR and/or a method comprisingflanking exon PCR on each internal exon corresponding to the mRNA to obtain one or more flanking exon amplification products, and detecting the presence and length of the said flanking exon amplification products,quantifying of each protein encoding exon of said mRNA. The oligomeric compounds provided herein may be utilized for therapeutics or research. Furthermore, antisense compounds, which are able to inhibit gene expression or modulate splicing with specificity, may be used to elucidate the function of particular genes or gene products or to distinguish between functions of various members of a biological pathway. In a preferred embodiment of the invention and/or embodiments thereof the oligomeric compounds are used for the treatment of Pompe disease. In a preferred embodiment of the invention and/or embodiments thereof the oligomeric compounds are used in research of the function of the GAA gene. Compounds described herein can be used to modulate splicing of a target mRNA in an metazoans, preferably mammals preferably human. In one non-limiting embodiment of the invention and/or embodiments thereof, the methods comprise the step of administering to said animal an effective amount of an antisense compound that modulates splicing of a target mRNA. For example, modulation of splicing of a target mRNA can be measured by determining levels of mRNA splicing products in a bodily fluid, tissue, organ of cells of the animal. Bodily fluids include, but are not limited to, blood (serum or plasma), lymphatic fluid, cerebrospinal fluid, semen, urine, synovial fluid and saliva and can be obtained by methods routine to those skilled in the art. Tissues, organs or cells include, but are not limited to, blood (e.g., hematopoietic cells, such as human hematopoietic progenitor cells, human hematopoietic stem cells, CD34+ cells CD4+ cells), lymphocytes and other blood lineage cells, skin, bone marrow, spleen, thymus, lymph node, brain, spinal cord, heart, skeletal muscle, liver, connective tissue, pancreas, prostate, kidney, lung, oral mucosa, esophagus, stomach, ilium, small intestine, colon, bladder, cervix, ovary, testis, mammary gland, adrenal gland, and adipose (white and brown). Samples of tissues, organs and cells can be routinely obtained by biopsy. In some alternative situations, samples of tissues or organs can be recovered from an animal after death. In a preferred embodiment of the invention and/or embodiments thereof modulation of splicing is measured in fibroblast, preferably primary fibroblasts, preferably primary fibroblasts from patients suffering from Pompe disease. The effects of treatment with the oligomeric compounds can be assessed by measuring biomarkers associated with modulation of splicing of a target mRNA in the aforementioned fluids, tissues or organs, collected from an animal contacted with one or more compounds, by routine clinical methods known in the art. These biomarkers include but are not limited to: glucose, cholesterol, lipoproteins, triglycerides, free fatty acids and other markers of glucose and lipid metabolism; liver transaminases, bilirubin, albumin, blood urea nitrogen, creatine and other markers of kidney and liver function; interleukins, tumor necrosis factors, intracellular adhesion molecules, C-reactive protein and other markers of inflammation; testosterone, estrogen and other hormones; tumor markers; vitamins, minerals and electrolytes. In a preferred embodiment of the invention and/or embodiments thereof the biomarker is glycogen. The compounds disclosed herein can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. The compounds can also be used in the manufacture of a medicament for the treatment of diseases and disorders related to alterations in splicing. In a preferred embodiment of the invention and/or embodiments thereof, the disease is Pompe disease. Methods whereby bodily fluids, organs or tissues are contacted with an effective amount of one or more of the antisense compounds or compositions of the disclosure are also contemplated. Bodily fluids, organs or tissues can be contacted with one or more of the compounds of the disclosure resulting in modulation of splicing of target mRNA in the cells of bodily fluids, organs or tissues. An effective amount can be determined by monitoring the modulatory effect of the antisense compound or compounds or compositions on target nucleic acids or their products by methods routine to the skilled artisan. Further contemplated are ex vivo methods of treatment whereby cells or tissues are isolated from a subject, contacted with an effective amount of the antisense compound or compounds or compositions and reintroduced into the subject by routine methods known to those skilled in the art. A sufficient amount of an antisense oligomeric compound to be administered will be an amount that is sufficient to induce amelioration of unwanted disease symptoms. Such an amount may vary inter alia depending on such factors as the gender, age, weight, overall physical condition, of the patient, etc. and may be determined on a case by case basis. The amount may also vary according to the type of condition being treated, and the other components of a treatment protocol (e.g. administration of other medicaments such as steroids, etc.). The amount may also vary according to the method of administration such as systemically or locally. Typical dosage amounts of the antisense oligonucleotide molecules in pharmaceutical formulations may range from about 0.05 to 1000 mg/kg body weight, and in particular from about 5 to 500 mg/kg body weight. In one embodiment of the invention and/or embodiments thereof, the dosage amount is from about 50 to 300 mg/kg body weight once in 2 weeks, or once or twice a week, or any frequency required to achieve therapeutic effect. Suitably amounts are from 3-50 mg/kg, more suitably 10-40 mg/kg, more suitably 15-25 mg/kg. The dosage administered will, of course, vary depending on the use and known factors such as the pharmacodynamic characteristics of the active ingredient; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. The recipient may be any type of mammal, but is preferably a human. In one embodiment of the invention and/or embodiments thereof, dosage forms (compositions) of the inventive pharmaceutical composition may contain about 1 microgram to 50,000 micrograms of active ingredient per unit, and in particular, from about 10 to 10,000 micrograms of active ingredient per unit. (if here a unit means a vial or one package for one injection, then it will be much higher, up to 15 g if the weight of a patient is 50 kg) For intravenous delivery, a unit dose of the pharmaceutical formulation will generally contain from 0.5 to 500 micrograms per kg body weight and preferably will contain from 5 to 300 micrograms, in particular 10, 15, 20, 30, 40, 50, 100, 200, or 300 micrograms per kg body weight ([mu]g/kg body weight) of the antisense oligonucleotide molecule. Preferred intravenous dosage ranges from 10 ng to 2000 microg, preferably 3 to 300 [mg, more preferably 10 to 100 [mu]g of compound per kg of body weight. Alternatively the unit dose may contain from 2 to 20 milligrams of the antisense oligonucleotide molecule and be administered in multiples, if desired, to give the preceding daily dose. In these pharmaceutical compositions, the antisense oligonucleotide molecule will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition. In one particular embodiment, it should be recognized that the dosage can be raised or lowered based on individual patient response. It will be appreciated that the actual amounts of antisense oligonucleotide molecule used will vary according to the specific antisense oligonucleotide molecule being utilized, the particular compositions formulated, the mode of application, and the particular site of administration. Preferably the compounds are administered daily, once every 2 days, once every 3 days, once a week, once every two weeks, or once every month. In another preferred embodiment the administration is only one time, e.g. when using a viral vector. If a viral-based delivery of antisense oligomeric compounds is chosen, suitable doses will depend on different factors such as the viral strain that is employed, the route of delivery (intramuscular, intravenous, intra-arterial or other), Those of skill in the art will recognize that such parameters are normally worked out during clinical trials. Further, those of skill in the art will recognize that, while disease symptoms may be completely alleviated by the treatments described herein, this need not be the case. Even a partial or intermittent relief of symptoms may be of great benefit to the recipient. In addition, treatment of the patient is usually not a single event. Rather, the antisense oligomeric compounds of the invention will likely be administered on multiple occasions, that may be, depending on the results obtained, several days apart, several weeks apart, or several months apart, or even several years apart. Those of skill in the art will recognize that there are many ways to determine or measure a level of functionality of a protein, and to determine a level of increase or decrease of functionality e.g. in response to a treatment protocol. Such methods include but are not limited to measuring or detecting an activity of the protein, etc. Such measurements are generally made in comparison to a standard or control or “normal” sample. In addition, when the protein's lack of functionality is involved in a disease process, disease symptoms may be monitored and/or measured in order to indirectly detect the presence or absence of a correctly functioning protein, or to gauge the success of a treatment protocol intended to remedy the lack of functioning of the protein. In preferred embodiment the functionality of the GAA protein is measured. This is suitably performed with an enzymatic activity assays as is well known to a skilled person. In a particular embodiment of the invention and/or embodiments thereof; antisense oligonucleotides of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a “vector” is any vehicle capable of facilitating the transfer of the antisense oligonucleotide of the invention to the cells. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, naked plasmids, non viral delivery systems (electroporation, sonoporation, cationic transfection agents, liposomes, etc. . . . ), phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: R A viruses such as a retrovirus (as for example moloney murine leukemia virus and lentiviral derived vectors), harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus. One can readily employ other vectors not named but known to the art. Preferred viral vectors according to the invention include adenoviruses and adeno-associated (AAV) viruses, which are DNA viruses that have already been approved for human use in gene therapy. Actually 12 different AAV serotypes (AAV1 to 12) are known, each with different tissue tropisms (Wu, Z Mol Ther 2006; 14:316-27). Recombinant AAV are derived from the dependent parvovirus AAV (Choi, V W J Virol 2005; 79:6801-07). The adeno-associated virus type 1 to 12 can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species (Wu, Z Mol Ther 2006; 14:316-27). It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion. Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by, intranasal sprays or drops, rectal suppository and orally. Preferably, said DNA plasmid is injected intramuscular, or intravenous. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation. In a preferred embodiment of the invention and/or embodiments thereof, the antisense oligonucleotide nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters. In a preferred embodiment of the invention and/or embodiments thereof, the vector may code for more than one antisense oligomeric compound. Each antisense oligomeric compound is directed to different targets. Pharmaceutical composition comprising the antisense compounds described herein may comprise any pharmaceutically acceptable salts, esters, or salts of such esters, or any other functional chemical equivalent which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the antisense compounds, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. The term “prodrug” indicates a therapeutic agent that is prepared in an inactive or less active form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes, chemicals, and/or conditions. In particular, prodrug versions of the oligonucleotides are prepared as SATE ((S-acetyl-2-thioethyl) phosphate) derivatives according to the methods disclosed in WO 93/24510 or WO 94/26764. Prodrugs can also include antisense compounds wherein one or both ends comprise nucleotides that are cleaved (e.g., by incorporating phosphodiester backbone linkages at the ends) to produce the active compound. The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to humans. In another embodiment of the invention and/or embodiments thereof, sodium salts of dsRNA compounds are also provided. The antisense compounds described herein may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds. The present disclosure also includes pharmaceutical compositions and formulations which include the antisense compounds described herein. The pharmaceutical compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. In a preferred embodiment of the invention and/or embodiments thereof, administration is intramuscular or intravenous. The pharmaceutical formulations, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product (e.g., into a specific particle size for delivery). In a preferred embodiment of the invention and/or embodiments thereof, the pharmaceutical formulations are prepared for intramuscular administration in an appropriate solvent, e.g., water or normal saline, possibly in a sterile formulation, with carriers or other agents. A “pharmaceutical carrier” or “excipient” can be a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal and are known in the art. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Compositions provided herein may contain two or more antisense compounds. In another related embodiment, compositions may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions provided herein can contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Two or more combined compounds may be used together or sequentially. Compositions can also be combined with other non-antisense compound therapeutic agents. The antisense oligomeric compound described herein may be in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. antisense oligomeric compound compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. Suspensions may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The present disclosure also includes antisense oligomeric compound compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co., A. R. Gennaro edit., 1985). For example, preservatives and stabilizers can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used. Pharmaceutical compositions of this disclosure can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxy ethylene sorbitan monooleate. The antisense oligomeric compound of this disclosure may be administered to a patient by any standard means, with or without stabilizers, buffers, or the like, to form a composition suitable for treatment. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. Thus the antisense oligomeric compound of the present disclosure may be administered in any form, for example intramuscular or by local, systemic, or intrathecal injection. This disclosure also features the use of antisense oligomeric compound compositions comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modif[iota]ed, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of antisense oligomeric compound in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated antisense oligomeric compound (Lasic et al, Chem. Rev. 95:2601-2627 (1995) and Ishiwata et al, Chem. Pharm. Bull. 43:1005-1011 (1995). Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of antisense oligomeric compound, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol. Chem. 42:24864-24870 (1995); Choi et al, PCT Publication No. WO 96/10391; Ansell et al, PCT Publication No. WO 96/10390; Holland et al, PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect antisense oligomeric compound from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen. Following administration of the antisense oligomeric compound compositions according to the formulations and methods of this disclosure, test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated, as compared to placebo-treated or other suitable control subjects. EXAMPLES Example 1 Mutations affecting pre-mRNA splicing are difficult to predict due to the complex mechanism of splicing regulation. A generic approach to systemically detect and characterize effects of sequence variants on splicing would improve current diagnostic practice. Here, we show that such approach is feasible by combining flanking exon RT-PCR, sequence analysis of PCR products, and exon-internal quantitative RT-PCR for all coding exons. It has been applied to uncharacterized mutations in the acid-alpha glucosidase gene causing Pompe disease, a monogenic autosomal recessive disease. Effects on splicing included cryptic splice site usage, intron retention and exon skipping. These differed from in silico predictions, highlighting the need for experimental testing. Quantification of the extent of leaky wild type splicing correlated with disease severity. Materials and Methods Patients and Healthy Control Patients were diagnosed with Pompe disease based on clinical symptoms and GAA enzyme activity. All patients and the healthy control provided informed consent for molecular analysis. Nomenclature The positions of the mutations described are aligned against Ensembl GAA cDNA association number ENST00000302262.3. c.1 indicates the first nucleotide of the coding region of GAA mRNA. Further numbering is according to HGVS standards [14]. Cell Culture and cDNA Preparation Fibroblasts were isolated from skin biopsies of patients and a healthy individual. Cells were cultured in DMEM High Glucose (Lonza)+10% Fetal bovine serum (HyClone, Thermo Scientific)+1% penicillin/streptomycin (Lonza). RNA was isolated using the RNAeasy miniprep kit (Qiagen). 800 ng of RNA was used for generation of cDNA using the iScript cDNA synthesis kit (Biorad). cDNA was diluted 10 times before use. Flanking Exon PCR Analysis cDNA was amplified using FastStart Taq Polymerase (Roche). Primers were used at a final concentration of 0.333 μM each, dNTPs at 0,333 mM each. The PCR program was performed on a Biorad s1000 thermal cycler (96° C. for 4 min., 35× [96° C. 20 sec., 60° C. 30 sec., 72° C. 1 min.], 72° C. 5 min.) 5 μl of each PCR reaction was run on a 1,5% agarose gel containing ethidium bromide. Gel were photographed on a Typhoon FLA 9000 gel imager (G&E Healthcare). The primers used are listed inFIG.15. Exon-Internal qPCR Analysis To determine the relative concentration of each sample, 4 μl of each cDNA sample (10 times diluted in H2O) was processed in a 15 μl PCR reaction containing IQ Mastermix (Biorad) and 0,333 μM of each primer. To account for the efficiency of each specific primer set, all samples were related to a standard curve from the healthy control sample. All samples were measured in triplicate. The primers used are listed inFIG.16. Sanger Sequencing Genomic DNA mutations were identified at the diagnostic department of Clinical Genetics at the Erasmus MC, Rotterdam, The Netherlands. Direct sequencing of flanking exon PCR products was performed using the Big Dye Terminator kit v3.1 (Applied Biosystems). To obtain pure DNA samples, PCR products visible on gel in the splicing assay were stabbed with a 20 μl pipet tip and DNA on the tip was resuspended in 10 μl H2O. 1 μl was subsequently used in a new PCR (as described in the splicing assay) to obtain DNA from a single template. Excess primers and dNTPs were removed using FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), according to the manufacturer's protocol. Samples were purified with sephadex G-50 (GE Healthcare) and the sequence was determined on an AB3130 Genetic Analyzer (Applied Biosystems, Hitachi). GAA Enzyme Activity The activity of GAA in fibroblasts was measured with 4-methylumbelliferyl-α-gluocpyranoside (4-MU) or with glycogen as substrate as described [15]. Results Generic Assay to Detect Splicing Mutations The approach consists of two parts. First (FIG.1, left), a generic RT-PCR is performed of the mRNA of interest using standard primers that flank each individual canonical exon (flanking exon PCR). The products are separated by agarose gel electrophoresis. Changes in product size are indicative of alternative/aberrant splicing. Splicing junctions can be precisely determined using sequencing of products isolated from gel or by direct sequencing of the PCR reaction. Second (FIG.1, right), a standard qPCR is performed to quantify each individual exon (exon-internal qPCR). Primers that anneal within each exon are used. Results are normalized for beta-actin mRNA and for expression in a healthy control. The results quantify exon skipping/inclusion, and may also indicate whether a splicing mutation allows leaky wild type splicing. Development and Validation of the Assay Healthy Control The assay was developed using a healthy control. To detect splicing junctions and exon sizes, flanking exon PCR analysis was performed on cDNA prepared from primary fibroblasts using primers that annealed to flanking exons (FIG.2A). Gel electrophoresis and ethidium bromide staining showed the correct molecular weight products in all cases. This indicated canonical splicing for all exons in these cells. Some additional products were observed in at minor amounts, notably, just above exon 6 and 7. Sequence analysis indicated that these represent products in which intron 6 was retained. The products were observed in this healthy control and in many Pompe patients and may indicate noisy aberrant splicing, which is a known phenomenon [16]. Individual exons were quantified using exon-internal qPCR (FIG.1B). Values were normalized for 6-actin expression (as measured by qPCR analysis), and were then ready to use for normalization of test samples. Patient 1 This patient was used to validate whether a well described splicing mutation could be accurately detected in primary fibroblasts using the assay described above. The c.−32−13T>G (IVS1) mutation was chosen because it is a frequent mutation causing juvenile/adult onset of Pompe disease. It is located in intron 1 close to the splice acceptor site of exon 2, and it causes aberrant splicing of exon 2 but also allows leaky wild type splicing [17, 18]. The second allele is known to be expressed at very low levels due to NMD [19]. This is caused by the c.1636+5G>T mutation, which leads to intron 11 inclusion and a premature termination codon. For this reason, the allele containing the IVS1 mutation dominates in the splicing assay described below. Flanking exon PCR analysis yielded three major products from exon 2 amplification (FIG.2A). These products were analyzed by DNA sequencing, which indicated that product 1 represented full exon 2 with canonical splicing junctions (FIG.9). Product 2 contained partially skipped exon 2 due to the utilization of a cryptic splice acceptor site at c.486 while product 3 represented fully skipped exon 2 (FIGS.2Aand S2). These products correspond to the major splicing variants reported for the IVS1 mutation, namely normal (N) (product 1), splicing variant (SV) 1 (product 2) and SV2 (product 3) [18]. Exon-internal qPCR analysis showed 10-15% expression of exon 2 and all other exons (FIG.2). This can be explained as follows. The IVS1 mutation allows leaky wild type splicing of exon 2 (product 1 inFIG.2A) yielding a normal mRNA containing all exons, as noted previously ([18, 20]. The 2 other major products 2 and 3 both result in the deletion of the canonical start of translation, which is located in exon 2. This leads to in mRNA degradation, resulting in minor contribution in the quantitative exon-internal qPCR assay, and predominant detection of the leaky wild type GAA mRNA from the IVS1 allele. In conclusion, the known effects of the IVS1 mutation on splicing were faithfully detected using the generic splicing assay for GAA. Leaky wild type splicing were 10-15% of healthy control levels and explained the juvenile/adult onset of Pompe disease. It is of note that all five splicing prediction programs used here (SpliceSiteFinder-like (SSF), MaxEntScan (MES), NNSplice (NNS), GeneSplicer (GS) and Human Splicing Finder (HSF)) failed to detect an effect of the IVS1 mutation on splicing (FIG.14A). Patient 2 This patient was chosen to test the sensitivity of the assay. Due to a homozygous c.525delT mutation, GAA mRNA expression is very low due to NMD [21]. Surprisingly, flanking exon PCR analysis showed that all exons could still be detected at the correct sizes, although at reduced levels (FIG.8). Higher molecular weight products were also observed at even lower levels. These may represent unspliced pre-mRNA species, amplified due to the reduced abundance of competing spliced mRNA in the PCR reaction. To quantify the amount of residual mRNA, exon-internal qPCR was performed and showed 5-10% expression of all exons relative to the healthy control (FIG.8B). In conclusion, the generic splicing assays for GAA allow analysis and quantification of very low mRNA expression. This is particularly relevant for mRNAs that are subject to degradation as the result of reading frame alterations. Patient 3 A third validation was performed on a patient carrying a well-known deletion removing the entire exon 18 plus its flanking sequences (del ex18, or c.2481+102_2646+31del) (FIG.2A). This case is interesting because the splice sites of exon 18 are removed. Previous work has shown that a new mRNA is formed in which exon 17 is neatly spliced to exon 19 via canonical splice sites [17]. The translation reading frame of the resulting mRNA remains intact, suggesting that this mRNA is not susceptible to degradation via the NMD pathway (FIG.7—Table 2). The second mutation in this patient, c.1548G>A, generates a termination codon in exon 10 [22]. Its effects on mRNA expression have not been reported so far. The premature termination codon is likely to result in low mRNA abundance from this allele. Flanking exon PCR indicated changes for amplification of exons 17, 18, and 19 (FIG.3A). Exon 18 amplification yielded two products instead of one. Sequence analysis indicated that the highest MW product (number 4) represented wild type spliced exon 18, while the lower MW product (number 5) lacked the entire exon 18, and exon 17 and exon 19 were joined via their canonical splice sites (FIG. S3A). Amplification of exons 17 and 19 yielded lower amounts of the correct products compared to the healthy control. The primers used for their amplification anneal to exon 18, indicating that their detection could not be derived from the delex18 allele but must have come from the c.1548G>A allele. This indicates that the c.1548G>A allele is expressed to some extent, and it explains the detection of moderate levels of wild type spliced exon 18 by flanking exon PCR. To quantify expression from the c.1548G>A allele, exon-internal qPCR was performed and indicated 3% expression of exon 18, while all other exons were expressed at ˜40-50% of healthy control levels (FIG.3F). This shows that the c.1548G>A mutation results in very low mRNA expression, as measured by the low level of exon 18 detection. Expression of all other exons is derived from the delex18 allele, which produces a stable mRNA in which exon 18 is precisely deleted. In summary, the generic splicing assay also allows detection and characterization of exonic deletions. A dissection can be made between two alleles by comparing the results of the flanking exon PCR and the exon-internal qPCR assays. Characterization of Novel Splicing Mutations Next, a number of patients were analyzed that contained partially characterized or uncharacterized mutations. Patient 4 Patient 4 contained a novel mutation at c.−32−3C>G located in intron 1 close to the splice acceptor site of exon 2 (FIG.3D). This mutation is suspected to affect splicing of exon 2 based on its similarity to the published c.−32−3C>A mutation [19]. In this study, a perfect skip of exon 2 was reported. Splicing prediction programs indicated that the c.−32−3C>G mutation weakens the splice acceptor site of exon 2 for some but not all programs (FIG.14C). The second allele contained a previously reported [23] but uncharacterized mutation at c.1551+1G>A which is located in intron 10 close to the splice donor site of exon 10 (FIG.3E). Based on the similarity to the published c.1551+1G>C mutation [17, 24], the c.1551+1G>A mutation is suspected to affect exon 10 splicing. Splicing prediction programs indicated loss of the splice donor site of exon 10 (FIG.14C). The results of the flanking exon PCR analysis indicated aberrant splicing of two exons: exon 2 and exon 10 (FIG.3C). Amplification of exon 2 resulted in 3 major products, number 6-8, and sequence analysis indicated that these products included wild type splicing, partial skipping of exon 2 via the cryptic splice acceptor site at c.486 in exon 2, and perfect skipping of exon 2, respectively (FIG.3DandFIG.10B). This indicates that two independent mutations in intron 1, namely c.−32−13T>G, which is located in the polypyrimidine tract, and c.−32−3C>G, located near the splice acceptor site, have the same qualitative outcome with respect to exon 2 splicing. Splicing prediction programs were insufficient to accurately predict this outcome. Flanking exon PCR amplification of exon 10 resulted in two major products, 9 and 10 (FIG.3C). Sequence analysis showed that product 9 contained wild type junctions between exons 9, 10, and 11, and that product 10 represented precise skipping of exon 10 mRNA (FIG.3EandFIG.10) in which the reading frame remains intact. This was surprising because the most straightforward result of a weakening of the splice donor site of exon 10 would be a failure to remove intron 10 rather than a skipping of exon 10. To determine the extent of splicing defects, exon-internal qPCR was performed. Exon 10 was expressed at ˜6%, while all other exons were expressed at ˜50% of healthy control levels (FIG.3F). This is consistent with the idea that the majority of mRNA is derived from the c.1551+1G>A allele in which exon 10 is skipped. The shorter product has an unchanged reading frame and is expected to be stable. In contrast, the c.−32−3C>G allele results in (partial) exon 2 skipping, which is known to result in mRNA degradation analogous to the IVS1 mutation. The c.−32−3C>G allele has only a minor contribution to the exon-internal qPCR results. Its contribution can be judged from exon 10 expression, which can result from leaky wild type splicing of the c.−32−3C>G mutation. However, an alternative source for exon 10 expression is leaky wild type expression of the c.1551+1G>A allele. The very low level of exon 10 expression indicates that both the c.−32−3C>G and the c.1551+1G>A have low or absent levels of leaky wild type expression. This indicates that the c.−32−3C>G mutation may be more severe compared to the IVS1 mutation, as the IVS1 mutation allows a higher level of wild type splicing of 10-15% (FIG.2D). The clinical course of Pompe disease indicates a juvenile onset for this patient, consistent with a low level of wild type GAA expression and GAA enzyme activity levels that were lower compared to adult onset patients (FIG.6—Table 1). Patient 5 Patient 5 was homozygous for c.1075G>A, which is a p.Gly359Arg missense mutation located at the last basepair of exon 6 (FIG.4B) [25]. This mutation has been classified as presumably nonpathogenic with possible effects on splicing [26]. It is located near the splice donor site of exon 6, and splicing prediction analysis indicated weakening of this site and strengthening of a cryptic splice donor site 4 nucleotides upstream (FIG.14D). Flanking exon PCR analysis showed absence of a product for exon 7, low levels of the other exons, and a low level of a low MW product for exon 2 (FIG.4A). Based on the predictions and on the location of this mutation in exon 6, we suspected that splicing junctions around exon 6 and 7 may be altered. In agreement, sequencing of the exon 6 PCR product (product 11) showed that the cryptic splice donor site in exon 6 located 4 nucleotides upstream at c.1071 was used instead (FIG.4BandFIG. S4B). This explains the absence of a product for exon 7, as the forward primer for exon 7 amplification has 4 mismatches due to the changed splice donor site. Remarkably, the flanking exon PCR assay failed to detect leaky wild type splicing for this mutation. This would have resulted in the presence of a wild type band for exon 7 amplification, which was not observed. To further investigate splicing of exon 7, an alternative forward primer located in exon 5 was used. The expected product was now obtained, and showed splicing from c.1071 in exon 6 to the canonical splice acceptor site of exon 7 (FIG.11A), as was observed for sequence analysis of product 11. The reading frame of the resulting mRNA has been changed leading to a premature termination codon (Table 2). The low MW product obtained with exon 2 amplification has not been pursued further. It may be caused by a yet unidentified intronic mutation. Alternatively, wild type GAA mRNA is known to have leaky exon 2 skipping, the product of which may be preferentially amplified because of mRNA degradation due to the c.1071 mutation. Quantification of GAA mRNA expression using the exon-internal qPCR assay showed that all GAA exons were expressed at very low levels, well below levels observed for the IVS1 mutation but just above the levels observed for the c.525delT mutation (FIG.4G). This confirmed the notion that leaky wild type splicing levels in this patient are very low or absent, while the majority of the mRNA is unstable. In agreement, very low GAA activity in fibroblasts was measured and the diagnosis of this patient was the most severe classic infantile form of Pompe disease. Patient 6 Patient 6 carried a homozygous c.1552−3C>G mutation. This mutation is located in intron 10 close to exon 11 (FIG.4D). Flanking exon PCR analysis showed aberrant splicing of exon 10 with three major products (12-14;FIG.4E). Sequence analysis indicated that in product 14, exon 10 was completely skipped while a novel splice acceptor site near exon 11 at c.1552−30 was utilized (FIGS.4D and11C). This mRNA leaves the reading frame intact (Table 2). Product 13 was identified as wild type spliced mRNA. Product 12 consisted of mRNA in which the complete intron 10 was retained. The reading frame is disrupted in this splicing product. While products 13 and 14 have been detected previously [27], product 12 is novel. Interestingly, splicing prediction programs were ambivalent on predicting the extent of utilization of the canonical or the cryptic splice acceptor sites of exon 11 (FIG.14F). Moreover, the outcome was unexpected in any case: weakening of the splice acceptor site of exon 11 would not be expected to result in the skipping of exon 10. Instead, two products could be envisioned: one in which the splice donor site of exon 10 splices to the cryptic acceptor at c.1552−30, resulting in extension of exon 11 with a part of intron 10 and further normal splicing. The other expected product would be a perfect skipping of exon 11. The completely different outcome illustrates that experimental validation is required to analyze the molecular consequences of potential splicing mutations. Quantification of splicing defects was performed with the exon-internal qPCR assay. This showed expression of all exons at ˜20% of healthy control levels (FIG.4G). No extra reduction of exon 10 expression was observed, suggesting that the majority of mRNA included exon 10, favoring products 12 and 13 above 14. The presence of leaky wild type splicing (product 13) is consistent with residual GAA enzyme activity and the milder phenotype with adult onset of Pompe disease in this patient (table 1). In conclusion, c.1552−3C>G results in several splicing defects around exon 10 and intron 10, and it allows leaky wild type splicing compatible with adult disease onset. Patient 7 Patient 7 was homozygous for c.1437G>A, a silent mutation located at the splice donor site of exon 9 (FIG.4F). Flanking exon PCR analysis showed two products instead of one for exon 9 amplification, and low yields for exon 8 and exon 10 amplification (FIG.4E). Sequence analysis indicated that product 15 represented wild type spliced exon 9, while in product 16, exon 9 was perfectly skipped, resulting in a shorter transcript in which the reading frame was unchanged (FIG.4F andFIG.11D). As expected from its location, the c.1437G>A mutation was predicted in silico to weaken to splice donor site of exon 9 (FIG.14E). However, the experimental result was surprising as failure of the splice donor site of exon 9 would be expected to result in inclusion of intron 9 rather than skipping of exon 9. Products of exon 8 and exon 10 amplification had correct sizes but lower yield because exon 9 had reduced availability to serve as template for annealing of the reverse PCR primer (for exon 8) or the forward PCR primer (for exon 10). Quantification using exon-internal qPCR showed near-normal (70-80% of control) expression levels for all exons except for exon 9, which showed expression of only 5% of healthy control. The juvenile/adult disease onset of this patient is consistent with the leaky nature of the splice site mutation (Table 1). In summary, the c.1437G>A mutation results in precise skipping of exon 9 leaving the reading frame intact, and allows a low level of leaky wild type GAA splicing. Characterization of a Complex Case: Patient 8 Genotype Patient 8 contained the missense mutation c.1256A>T on allele 1. It is located in the middle of exon 8, results in p.Asp419Val, and has been classified as mildly pathogenic (FIG.5B) [26]. The 2nd allele contained a c.1551+1G>T mutation, which is located in intron 10 close to the splice donor site of exon 10[26]. It resembles the c.1551+1G>A mutation described above for patient 4. Analysis of Splicing Products Flanking exon PCR analysis indicated multiple PCR products from amplification of exons 8, 9, and 10 (FIG.5A). All these products were analyzed by sequencing (FIG.12). This indicated the presence of wild type exon 8 splicing (product 17) and utilization of a novel splice donor site in exon 8 at c.1254, which is located 2 nt upstream of the c.1256A>T mutation (product 18;FIG.5B-C). This donor spliced to the canonical splicing acceptor site of exon 9 and the resulting reading frame was unchanged (Table 2). Splicing prediction programs indeed showed that c.1254 turned into a splice donor site due to the c.1256A>T mutation (FIG.14G). The canonical splice donor site of exon 8 remained unchanged, and it was unclear which of the two sites would be preferred from in silico predictions. Product 21 represented wild type splicing of exon 10, while product 22 was the result of perfect exon 10 skipping in which the reading frame remained intact (FIG.5D andFIG.12). Loss of the exon 10 splice donor site by the c.1551+1G>T mutation was consistent with splicing predictions (FIG.14G), but the outcome was not anticipated, as intron 10 inclusion rather than exon 10 skipping seemed the most logical consequence. Evidence for Low Levels of Leaky Wild Type Splicing Along with the exon-internal qPCR analysis described below, the flanking exon PCR assay provides information on the severity of the mutations via the relative intensities of the products. These can be explained based on the identification of the splicing products (FIG.5B-D) and on the locations of the primers used for amplification (FIG.13). Exon 7 Detection of exon 7 is performed with a forward primer that anneals to the 3′ end of exon 6 and a reverse primer to the 5′ end of exon 8 (FIG.13). The 5′ end of exon 8 is retained in all cases while the 3′ part is spliced out in the c.1256A>T allele. Flanking exon PCR detection of exon 7 should therefore not be affected in this patient and this was indeed the case (FIG.5A). Exon 8 Flanking exon PCR primers used for detection of exon 8 are anneal to exon 7 and 9 (FIG.13). Both exons are not affected in this patient predicting that all splicing alterations of exon 8 itself should be detected in a semi-quantitative manner. Indeed, a strong wild type product (number 17) was detected, dominated by allele 2, and a slightly weaker smaller product 18 was detected due to the novel cryptic splice donor site at c.1254 in allele 1. Maximal 50% of product 17 is expected to be derived from allele 2 and its stronger abundance compared to product 18 therefore suggests that allele 1 has leaky wild type splicing. Exon 9 PCR primers for detection of exon 9 by flanking exon PCR anneal to the 5′ part of exon 8, which is the part that is not skipped in allele 1, and to exon 10, which is completely skipped in allele 2 (FIG.12). This complicates detection of exon 9 from these two alleles: a product from allele 1 would be shorter than normal due to the partial skipping of exon 8. A product from allele 2 is not possible due to the precise skipping of exon 10, while this exon is required for primer annealing. The predominant product obtained was the shorter product number 20 which was derived from allele 1. However, a small amount of wild type product number 19 was also observed. This indicates that at least one of the two alleles allows leaky wild type splicing. Exon 10 Flanking exon PCR analysis of exon 10 is performed with primers annealing in exon 9 and exon 11, both of which are unaffected. The result therefore reflects the splicing alterations of exon 10 in a semi-quantitative way. Product 21 representing wild type splicing was the most abundant, while product 22 in which exon 10 was perfectly skipped was slightly less abundant. Because exon 10 splicing of allele 1 is unaffected and can account for 50% of wild type product, this result suggests that allele 2 also has leaky wild type splicing similar to allele 1. Quantification Using Exon-Internal qPCR Analysis Quantification of mRNA expression of each exon revealed that all exons except exons 8 and 10 showed ˜2 fold higher abundance compared to the healthy control. Exons 8 and 10 were expressed at 2-fold lower levels with respect to the other exons but still at 80-120% of the levels of the healthy control. This indicates abnormally high mRNA expression in this patient. Allele 1 (1256A>T) suffers from partial skipping of exon 8 resulting in failure in detection of a qPCR product. The residual detection of exon 8 is therefore derived from allele 2 (c.1551+1G>T), expected to contribute 50%, and the remaining expression is likely derived from leaky wild type splicing from allele 1. The same rationale applies to detection of exon 10. In this case, expression was close to 50% relative to other exons, suggesting that the c.1551+1G>T mutation allowed much lower levels of wild type splicing. It should be noted that it is unclear why this patient shows 2-fold higher GAA expression relative to the healthy control, and whether this increase applies to both alleles to similar extents. This patient has a childhood/juvenile disease onset but is clearly less affected compared to classic infantile Pompe patients, consistent with low levels of residual wild type expression of GAA (table 1). In summary, patient 8 contained two splicing mutations. c.1256A>T is a missense mutation in exon 8 that causes p.Asp419Val and in addition generates a novel splice donor site at c.1254, resulting in partial skipping of exon 8 and in leaky wild type splicing. c.1551+1G>T is located in intron 10 and causes perfect skipping of exon 10 and in leaky wild type splicing. The childhood/juvenile onset of Pompe disease suggests that both mutations are moderately to severely pathogenic. This is consistent with the GAA enzyme activity levels, which are lower compared to adult onset patients. Mucopolycaccharidosis type VI (Maroteaux-Lamy syndrome) is a autosomal recessive monogenic disorder caused by defects in the gene coding for N-acetylgalactosamine 4-sulfatase (arylsulfatase B; ARSB). To demonstrate the generic nature of the splicing assay, the assay was adapted for MPSVI. To this end, flanking exon primers were designed for all coding exons of the ARSB gene (exons 2-7; the first and the last exons cannot beflanked). The following primer sequences and the expected product sizes (column “WT product size”) were used: SEQWTIDprod-1142 +ExonprimerNO:uct2T > C2Forward1590378378GGGTGCTCCTGGACAACTACReverse1591CCTGTTGCAACTTCTTCGCC3Forward1592444444ATGGCACCTGGGAATGTACCReverse1593GTGTTGTTCCAGAGCCCACT4Forward1594514514ACGCTCTGAATGTCACACGAReverse1595GTTGGCAGCCAGTCAGAGAT5Forward1596361117AAAAAGCAGTGGGCTCTGGAReverse1597CGGTGAAGAGTCCACGAAGT6Forward1598314314CAGAAGGGCGTGAAGAACCGReverse1599CCCGTGAGGAGTTTCCAATTTC7Forward1600348348ACTTCGTGGACTCTTCACCGReverse1601AGTACACGGGGACTGAGTGT Primary fibroblasts from a healthy control were grown, total RNA was harvested, cDNA was synthesized, and exons 2-7 were amplified by PCR, seeFIG.31. Products were separated on an agarose gel and visualized using ethidium bromide.FIG.31shows that all exons gave a predominant single band at the expected size (size markers are indicated on the left and numbers refer to sizes in bp). Next, fibroblasts were grown from a patient homozygous for the ARSB variant c.1142+2T>C. This patient has been described previously in Brands et al. (Orphanet J Rare Dis. 2013 Apr. 4; 8:51). While a splicing defect was suspected, it has not been demonstrated. In addition, it was not known how severe the potential splicing defect may be. Application of the splicing assay to analyze the nature of this variant revealed a severe splicing defect with two major outcomes, as shown inFIG.32, left part: 1) The product for amplification of exon 5 was lower compared to the healthy control: now a single product of 117 bp instead of 361 bp was obtained, which is consistent with a skipping of exon 5 and a deletion of 244 nucleotides in the mRNA, see above, all products had a lower abundance compared to the healthy control. This is consistent with the idea that the deletion of 244 nucleotides results in a reading frame shift, resulting in activation of the nonsense mediated decay pathway and degradation of the mRNA. Interestingly, no leaky wild type splicing could be detected. This is consistent with the severe and fast disease progression in this patient as described in Brands et al. (Orphanet J Rare Dis. 2013 Apr. 4; 8:51). Taken together, the expression and splicing assay was successfully applied to MPSVI, in which is resulted in the identification of the splicing defect caused by the c.1142+2T>C ARSB variant. The absence of leaky wild type splicing was consistent with the severe phenotype of the patient involved. Example 2 1 Generation of the SF-U7 snRNA Antisense Vector The U7snRNA gene with promoter was obtained from female mouse genomic DNA by using Fw-GCGCctgcagTAACAACATAGGAGCTGTG (SEQ ID NO: 1602) and Rv-GCGCgtcgacCAGATACGCGTTTCCTAGGA (SEQ ID NO: 1603) primers with PstI and SalI overhang (indicated in bold regular letter type) in a PCR amplification. The whole PCR reaction was loaded on a 1% gel and the PCR fragment (425 bp) was cloned into a Topo-II-vector according to the manufacture's manual (Invitrogen). SMopt and StuI sites were generated by using site directed mutagenesis according to an inner and outer primer design with Fw-(GCTCTTTTAGAATTTTTGGAGCAGGTTTTCTGACTTCG (SEQ ID NO: 1604) and Rv-U7snRNA-SmOPT (CGAAGTCAGAAAACCTGCTCCAAAAATTCTAAAAGAGC (SEQ ID NO: 1605) or Fw-(CCTGGCTCGCTACAGAGGCCTTTCCGCAAGTGTTACAGC (SEQ ID NO: 1606) and Rv-U7snRNA-StuI (GCTGTAACACTTGCGGAAAGGCCTCTGTAGCGAGCCAGG (SEQ ID NO: 1607) as inner primers and with Fw-M13 (GTAAAACGACGGCCAG) (SEQ ID NO: 1608) and Rv-M13 (CAGGAAACAGCTATGAC) (SEQ ID NO: 1609) as outer primers [Heckman, K. L. and L. R. Pease, Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc, 2007. 2(4): p. 924-32]. The modified U7 snRNA sequence was cloned back into pRRL.PPT.SF.pre vector [Warlich E et al., Lentiviral vector design and imaging approaches to visualize the early stages of cellular reprogramming. Mol Ther. 2011 April; 19(4):782-9.] by using PstI and SalI sites and replaced the original SFFV promoter. This is the procedure for generating the SF_U7snRNA vector. 2 Optimization of the SF-U7 snRNA Antisense Vector for High Throughput Screening The originally used StuI site is not unique in the lentiviral vector of Warlich et al and was replaced by a NsiI restriction site by site directed mutagenesis by using Fw-cctggctcgctacagatgcaTaggaggacggaggacg (SEQ ID NO: 1610) and Rv-cgtcctccgtcctcctAtgcatctgtagcgagccagg (SEQ ID NO: 1611) primers. Capital letters indicate mutated residues. 3 Insertion of Antisense Sequences New antisense sequences were inserted with an overhang PCR by using overhang forward primers containing the desired antisense sequences (gcgcATGCAT-antisense sequence-ttggagcagg) (SEQ ID NO:1612). Bold capital letters indicate the NsiI restriction site. The reverse primer Rv_ms_U7snRNA_SalI is (GCGCgtcgacCAGATACGCGTTTCCTAGGA) (SEQ ID NO: 1613) and was the same for every construct., the small letters indicate the SalI restriction site. Overhang PCR was performed on the modified vector (SF_U7snRNA_NSI) using PfuUltra HF (Agilent Technologies) The PCR program consisted of a 30 second initial denaturation step at 95° C., 35 cycles at 95° C. for 10 seconds, 60° C. for 30 seconds and 72° C. for 10 seconds. Final extension step was at 72° C. for 10 minutes. The PCR reaction containing the desired antisense sequence and U7 snRNA loaded on a 2% agarose gel with 0.2% ethidiumbromide staining. Bands were then visualized under a transilluminator (UVP, LLC) excised and extracted using the QIAquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany). After gel extraction, 16 μl of purified product was digested using SalI and NsiI (Roche) for 1 hour at 37° C. and purified using the QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany). Meanwhile the original vector was digested with SalI and NsiI for 1 hour at 37° C., resulting in a vector without antisense sequence. The digested vector was loaded on a 1% agarose gel with ethidiumbromide staining. Bands were visualized under a transilluminator and the band corresponding with the digested vector (6358 bp) was excised and purified using the QIAquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany). Purified digested vector and digested PCR products were ligated with T4 DNA ligase with ATP (New England BioLabs) for 1 hour at room temperature. The ligation products were transformed inE. coli(TOP10) and inoculated on LB agar plates containing 100 μg/ml ampicillin (Sigma). After overnight incubation, three colonies were picked per ligation product for miniprep cultures. Picked colonies were grown overnight in 2 ml LB containing 100 μg/ml ampicillin at 37° C. Purification of the plasmids was carried out using the QIAprep Spin Miniprep Kit (Qiagen GmbH, Hilden, Germany). After extraction, DNA concentration was measured with the Nanovue Spectrophotometer. Sequences of newly generated constructs were validated with Sanger Sequencing using BigDye Terminator v3.1 (Applied Biosystems) for the sequence reaction and were then purified with Sephadex G-50 (Sigma) according to manufacturer's protocol. Sequences SEQ ID NO: 41-97 are antisense compounds identified with the U7 screen. The antisense sequence above is depicted as DNA as it is cloned into a vector, however in the cell it is transcribed as a RNA molecule. The skilled person knows then that T is U. FIG.22shows examples of positions of antisense sequences targeting GAA for the unbiased intron 1 and exon 2 screen. Enzyme Activity Assay Enzyme activity was measured using the 4-methylumbelliferone assay. Samples were harvested after twelve days of transduction. The lysis buffer consisted of 50 mM Tris (pH 7.5), 100 mM NaCl, 50 mM NaF, 1% Tx-100 and one tablet protease inhibitor with EDTA (Roche). Lysis buffer was incubated on transduced fibroblasts for 5 minutes on ice before harvesting. Samples were either directly used or snap-freezed using liquid nitrogen and stored at −80° C. Otherwise, samples were kept on ice for further use in 4-methylumbelliferone assay. GAA activity was measured using the substrate 4-methylumbelliferyl-α-D-glucopyranoside, which is fluorogenic in nature. Protein concentrations of the samples was determined by the Lowry protein method using the BCA Protein Assay Kit (Pierce, Thermo Scientific). Bovine serum albumin (BSA) standards consisted of 0, 0.1, 0.2, 0.4, 0.5, 0.6, 1.0, 2.0 mg/ml. Absorbance was measured at 562 nm for the BCA Protein Assay, and for the 4-methylumbelliferone assay excitation was at 365 nm and emission at 448 nm, using the Varioskan (Thermo Scientific) microplate reader. GAA enzyme activity was expressed as nanomoles of substrate hydrolyzed per hour per milligram of total protein. Lentiviral Vector Production For lentiviral vector production, 293T cells 90% confluent growing on 10 cm culture dishes were seeded 1/8 on 10 cm culture dishes. After 16-24 hours, a total of 3 μg U7 snRNA construct, 2 μg Pax2 and 1 μg VSV were cotransfected using Fugene 6 Transfection Agent (Promega). Viral supernatants (9 ml) were harvested 72 hours post-transfection, filtered over 0.45 μm filters (MillexHV, Millipore) and concentrated by ultra-centrifugation in a Beckman Ultracentrifuge (Beckman Coulter) at 20.000 rpm, 4° C. for 2 hours. Viral pellets were resuspended in 100 μl Dulbecco's modified Eagle's medium Low Glucose (Gibco, Paisley, UK), aliquoted in CryoTubes (Thermo Scientific) and stored at −80° C. Lentiviral titers were determined after concentration by ultracentrifugation with the HIV p24 Antigen ELISA Kit (Retrotek, ZeptroMetrix Corporation). The assay was measured with a Varioskan microplate reader (Thermo Scientific) Transduction of Cells Culture media was replaced with new culture media containing 6 ng/ml protamine sulphate (sigma) 24 hours after seeding. The cells were transduced with equal titers of lentiviruses (see above). Primary fibroblasts from patient were transduced, see above with lentivirus containing the U7snRNA AON construct and splicing was allowed to occur. The screen on fibroblasts was performed by infection of individual wells containing primary fibroblasts with lentiviruses expressing a single type of U7 snRNA AONs. RNA was analysed 5 days after infection. Splicing products were analysed with RT-qPCR. GAA enzyme activity was analysed 12 days after infection (see above: enzyme activity assay).FIG.19shows changes in exon 2 inclusion by different AONs. RNA expression analysis using RT-qPCR of a screen on intron 1 and exon 2 of GAA with antisense sequences with the use of the U7 small nuclear RNA system. Numbers indicate antisense sequence positions according to table 1. The control is the patient fibroblast without added AON vector. FIG.20shows RNA analysis with RT-PCR of a screen on intron 1 and exon 2 of GAA with antisense sequences used in the U7 small nuclear RNA system. Numbers indicate antisense sequence positions according to table 1. In the GAA RT-PCR, three major products are observed. The upper product represents exon 2 inclusion, the lower doublet represents partial skipping of exon 2 (upper band of the doublet) and complete skipping of exon 2 (lower band of the doublet. Beta-actin RT-PCR was used as loading control. FIG.21shows GAA enzyme activity of the screen on intron 1 and exon 2 of GAA with antisense sequences in the U7 small nuclear RNA system. Numbers indicate antisense sequence positions according to table 1. The control is the patient fibroblast without added AON vector. It is clear that some clones significantly increase the inclusion of exon 2 and thereby provide potential candidates for a therapy for pompe patients having the IVS1 mutation.FIG.23shows an example illustrating that the identified sequence could not be predicted as the identified sequence was identified both as enhancer and as silencer motif. Example 3 By far the most common mutation causing Pompe disease is the c.−32−13T>G (IVS1) mutation. This mutation in the GAA gene is located in an intron 13 basebairs upstream of exon 2, the exon that contains the start codon for translation of the GAA mRNA. The IVS1 mutation causes miss-splicing of exon 2 in approximately 90% of GAA transcripts because it disrupts the polypyrimidine tract which reduces the strength of the exon 2 splice acceptor site. To counteract this reduced strength of the splice site, we want to identify sequences that bind splicing factors that have a negative effect on splicing of GAA exon 2. By integration of random mutations in and around exon 2 we could be able to find these sequences. For quick screening of a large number of mutations we generated a minigene containing GAA exon 1, intron 1, exon 2, intron 2, exon 3 and a part of intron 3 (FIG.24, part 1). By integration of 2 unique restriction sites, we are able to quickly exchange part of the minigene surrounding exon 2 with mutant sequences (FIG.24, part 2). A PCR is carried out at suboptimal conditions to integrate random mutations in the PCR products (FIG.24, part 3). These PCR products, which also contain the restriction sites located around exon 2, can then be ligated directly into the destination vector. After transformation of the ligated products, clones can be picked and the plasmid can be isolated from the clone, containing a random mutation (FIG.24, part 4). Separate transfection of these clones into HEK293 cells generate RNA-transcripts from the GAA minigene that result in differential splicing compared to the control. An example is shown in figure part 5, were a flanking exon RT-PCR and an exon internal qPCR is carried out against cDNA generated from 3 clones (indicated inFIG.24, part 5). Sequencing of the plasmids that yield a higher inclusion of exon 2 results in identification on an important sequence that influences splicing in a negative manner. These sequences can sequentially be used to test as a potential target for antisense therapy or to screen for compounds that bind to this area. FIG.25provides the results of two of the clones. Clone 115 and clone 97 demonstrate a 118% and a 297% increase of exon 2 inclusion, respectively, in comparison to the IVS1 mutation. Clone 115 contains the mutations: c.17C>T, c.469C>T, and c.546+23C>A. It results in increased wild type splicing (band 1) and decreased perfect skipping (band 3). Clone 97 contains the mutations: c.−32−102T>C, c.−32−56C>T, c.11G>A, c.112G>A, and c.137C>T. This clone also misses c.−32−553 to c.−32−122, however, this does not affect exon 2 exclusion (as determined by us by comparing splicing from minigene constructs that do or do not contain this region). Wild type splicing (band 3) is strongly increased, while both partial (band 2) and perfect (band 3) skipping are decreased. Apart from the minigene for Exon 1-Exon 3, we also generated a minigene containing the genomic region from GAA exon 5 to GAA exon 8. With this minigene we can test other mutations that influence splicing much like the IVS1 mutation. FIG.37shows the result of inhibition of the nonsense mediated decay (NMD) pathway on inclusion of intron 6 of the GAA mRNA. Cyclohexamide treatment of primary fibroblasts from a healthy control (upper gel), a Pompe patient with the genotype c.−32−13T>G, c.525delT (middle gel), and a Pompe patient with the genotype c.525deT, c.525delT (lower gel) was performed. Without inhibition of the NMD pathway (lanes labelled with 0 hr), a strong band was detected using RT-PCR representing canonical splicing of exon 6 and exon 7. A faint band just above the canonical band was observed. This band was determined by DNA sequence analysis to represent inclusion of intron 6. Because such product changes the reading frame resulting in activation of the NMD pathway, we speculated that intron 6 inclusion may in fact be a frequent event that escapes proper detection. This idea was confirmed by inhibition of the NMD pathway: this resulted in the detection of a strong band representing intron 6 inclusion. This indicated that many GAA pre-mRNA species escape canonical splicing in both healthy controls and in Pompe patients. The minigene containing GAA exon 5-8 mentioned above and the U7 snRNA screen will be used to identify sequences that can prevent inclusion of intron 6 in the final mRNA by blocking a repressor of exon 6/7 splicing. This would represent a generic therapy for all splicing mutations with leaky wild type splicing causing Pompe disease, because correct splicing of exons 6/7 will be enhanced thereby also enhancing the levels of leaky wild type splicing. The following mutations give an increased RNA expression: c.17C>T, c.469C>T, and c.546+23C>A., c.−32−102T>C, c.−32−56C>T, c.11G>A, c.112G>A, and c.137C>T.AONs that target mRNA sequences where these mutations are located may be useful for treating patients. SEQ ID NO: 98-540 are exemplary sequences found with the minigene approach. The table above shows SEQ ID NO: 98-540 and the mutation or genomic sequence it targets. TheFIG.26shows a dose-response curve for SEQ ID NO: 12 (AON 1) (upper panels) and SEQ ID NO: 33 (AON 2) (lower panels). Patient-derived fibroblasts with the genotype c.−32−13T>G (IVS1) on one allele and c.525delT on the other allele were either untreated (‘no transfection’) or incubated with antisense oligomeric compound at 0-20 μM. Please note that the c.525delT undergoes nonsense-mediated decay, which explains why the effects at the RNA level are derived primarily from the IVS1 allele. Cells were harvested for RNA analysis after 3 days (A, C), and for protein analysis after 5 days (B, D). Both SEQ ID NO: 12 AON 1 and SEQ ID NO: 33 (AON 2) bind to a sequence present in intron 1 of the GAA pre-mRNA, which was identified using the U7 snRNA assay. This results in promotion of exon 2 inclusion, yielding higher expression of wild type GAA mRNA. This is measured at the mRNA level (using primers that specifically detect wild type GAA) and at the protein level (using an assay for GAA enzymatic activity). RNA analysis: total RNA was isolated, cDNA was synthesized, and RT-qPCR analysis was performed to detect GAA exon 2 inclusion (using a forward primer specific for exon 1 and a reverse primer specific for exon 2). Protein analysis: GAA enzyme activity was measured using the 4-MU assay. Activities were normalized for total protein as measured using the BCA assay. Antisense oligomeric compound treatment: Antisense oligomeric compound used herein are morpholino's obtained from gene tools. Antisense oligomeric compound were transfected into the cells using endoporter (gene tools) according to the manufactor's instructions. This following experiment is similar to that of patient fibroblast line 1 (FIG.26) and served to demonstrate that the antisense oligomeric compounds also work in an independent cell line 2 from another patient. In this case, the genotype was IVS1 on one allele and a missense variant (c.923A>C) on the other allele. Please note that the c.923A>C allele does not undergo nonsense-mediated decay, and mRNA levels represent a mix of both alleles, making the effects on the IVS1 allele less pronounced compared to patient 1. TheFIG.27shows a dose-response curve for SEQ ID NO: 12 (AON 1) (upper panels) and SEQ ID NO: 33 (AON 2) (lower panels). FIG.28shows the specificity of antisense oligomeric compounds SEQ ID NO: 12 (AON 1) and SEQ ID NO: 33 (AON 2) for promoting exon 2 inclusion. SEQ ID NO: 35 (control AON 2) and SEQ ID NO: 36 (control AON 3) target another region in intron 1 of GAA but is ineffective in promoting exon 2 inclusion. An unrelated AON targeting the CypA mRNA (control AON 1; SEQ ID NO: 34) does not affect GAA exon 2 inclusion. SEQ ID NO: 12 (AON 1) and SEQ ID NO: 33 (AON 2) efficiently promote inclusion of GAA exon 2 as shown by RT-qPCR analysis (A) and concomitant GAA enzyme activity assay (B). This shows that only when the in the U7 snRNA assay identified intronic splice silencing (ISS) sequence is targeted, as with SEQ ID NO: 12 (AON 1) and SEQ ID NO: 33 (AON 2), GAA exon 2 inclusion is promoted. Se-Tar-Sequence insequencequencegetcDNA to whichof AONSeqnumberGeneAON anneals(5′->3′):IDControlCypAc.354_362 + 11*TGTACCCTTAC34AON 1CACTCAGTCControlGAAc.−32-224_−200**GAGTGCAGAGCAC35AON 2TTGCACAGTCTGControlGAAc.−32-219_−200**GAGTGCAGAGCAC36AON 3TTGCACAGTCTG*CypA cDNA sequence is Refseq entry NM_021130.4**GAA cDNA sequence is Refseq entry NM_000152.3 FIG.29shows the time course of the effect of the SEQ ID NO: 33 (AON 2) on patient fibroblast line 1. Cells were assayed for GAA activity at 3-7 days after the addition of antisense oligomeric compound. Antisense oligomeric compound was continuously present in the medium throughout the experiment. The figure shows that the effect on GAA activity starts after 3 days and reaches a maximum at 5 days after AON addition. REFERENCES 1. 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11859187 | DETAILED DESCRIPTION OF THE INVENTION I. Definitions As used herein, the term “aptamer” refers to a single stranded oligonucleotide or a peptide that has a binding affinity for a specific target. As used herein, the term “nucleic acid” refers to a polymer or oligomer of nucleotides. Nucleic acids are also referred as “ribonucleic acids” when the sugar moiety of the nucleotides is D-ribose and as “deoxyribonucleic acids” when the sugar moiety is 2-deoxy-D-ribose. As used herein, the term “nucleotide” refers to a compound consisting of a nucleoside esterified to a monophosphate, polyphosphate, or phosphate-derivative group via the hydroxyl group of the 5-carbon of the sugar moiety. Nucleotides are also referred as “ribonucleotides” when the sugar moiety is D-ribose and as “deoxyribonucleotides” when the sugar moiety is 2-deoxy-D-ribose. As used herein, the term “nucleoside” refers to a glycosylamine consisting of a nucleobase, such as a purine or pyrimidine, usually linked to a 5-carbon sugar (e.g. D-ribose or 2-deoxy-D-ribose) via a β-glycosidic linkage. Nucleosides are also referred as “ribonucleosides” when the sugar moiety is D-ribose and as “deoxyribonucleosides” when the sugar moiety is 2-deoxy-D-ribose. As used herein, the term “nucleobase” refers to a compound containing a nitrogen atom that has the chemical properties of a base. Non-limiting examples of nucleobases are compounds comprising pyridine, purine, or pyrimidine moieties, including but not limited to, adenine, guanine, hypoxanthine, thymine, cytosine, and uracil. As used herein, the term “oligonucleotide” refers to an oligomer composed of nucleotides. As used herein, the term “identical” or “sequence identity”, in the context of two or more oligonucleotides, nucleic acids, or aptamers, refers to two or more sequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection. As used herein, the term “substantially homologous” or “substantially identical”, in the context of two or more oligonucleotides, nucleic acids, or aptamers, generally refers to two or more sequences or subsequences that have at least 40%, 60%, 80%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide identity, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection. As used herein, the term “epitope” refers to the region of a target that interacts with the aptamer. An epitope can be a contiguous stretch within the target or can be represented by multiple points that are physically proximal in a folded form of the target. As used herein, the term “motif” refers to the sequence of contiguous, or series of contiguous, nucleotides occurring in a library of aptamers with binding affinity towards a specific target and that exhibits a statistically significant higher probability of occurrence than would be expected compared to a library of random oligonucleotides. The motif sequence is frequently the result or driver of the aptamer selection process. As used herein, the term “personal health care compositions” refers to compositions in a form that is directly deliverable to the upper respiratory tract. As used herein, “a pharmaceutically effective amount” refers to an amount sufficient to confer a therapeutic effect on the subject. In some aspects the therapeutic effect is reduced rhinovirus binding to cellular membrane glycoproteins such as ICAM-1, reduced severity and/or duration of a cold, or reduced incidence of respiratory illness due to rhinovirus. II. Aptamer Composition The human rhinoviruses (RV) are the predominant cause of the common cold. They are classified in three groups (RV-A, RV-B, and RV-C), including around 160 types that express different surface proteins. Despite this diversity, rhinoviruses utilize mostly three glycoproteins of epithelial cells to cross the cellular membrane and access the host cell replication machinery: intercellular adhesion molecule 1 or ICAM-1 protein, utilized by the majority of RV-A and all RV-B types; low-density lipoprotein receptor or LDLR family members, utilized by at least twelve RV-A types; and cadherin-related family member 3 or CADHR3 proteins, utilized mostly by RV-C types. An aptamer composition may comprise at least one oligonucleotide selected from the group consisting of deoxyribonucleotides, ribonucleotides, derivatives of deoxyribonucleotides, derivatives of ribonucleotides, and mixtures thereof, wherein the aptamer composition has a binding affinity for intercellular adhesion molecule 1 (ICAM-1). In one aspect, the aptamer composition may have a binding affinity for one or more cellular membrane glycoproteins selected from the group consisting of intercellular adhesion molecule 1 (ICAM-1), low-density lipoprotein receptor (LDLR) family members, and cadherin-related family member 3 (CDHR3) and combinations thereof. Preferably the one or more cellular membrane glycoprotein is intercellular adhesion molecule 1 (ICAM-1). The aptamer composition can reduce the binding of one or more human rhinoviruses to the intercellular adhesion molecule 1 (ICAM-1). The aptamer composition may comprise at least one oligonucleotide selected from the group consisting of oligonucleotides with at least 80% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 200. The aptamer composition may comprise at least one oligonucleotide selected from the group consisting of oligonucleotides with at least 90% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 200. The aptamer composition may comprise at least one oligonucleotide selected from the group consisting of oligonucleotides with at least 95% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 200. The aptamer composition may comprise at least one oligonucleotide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 200. A non-limiting example of oligonucleotide with at least 90% nucleotide sequence identity to SEQ ID NO: 3 is SEQ ID NO: 88. The aptamer composition may comprise at least one oligonucleotide selected from the group consisting of oligonucleotides containing at least 10 contiguous nucleotides from sequences selected from the group consisting of SEQ ID NO: 201 to SEQ ID NO: 212. The aptamer composition may comprise at least one oligonucleotide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8. The aptamer composition may comprise at least one oligonucleotide selected from the group consisting of oligonucleotides with at least 50% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8. The aptamer composition may comprise at least one oligonucleotide selected from the group consisting of oligonucleotides with at least 70% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8. The aptamer composition may comprise at least one oligonucleotide selected from the group consisting of oligonucleotides with at least 90% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8. A non-limiting example of oligonucleotide with at least 50% nucleotide sequence identity to SEQ ID NO: 4 is SEQ ID NO: 35. Non-limiting examples of oligonucleotides with at least 50% nucleotide sequence identity to SEQ ID NO: 7 are SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 77, and SEQ ID NO: 97. Non-limiting examples of oligonucleotides with at least 50% nucleotide sequence identity to SEQ ID NO: 8 are SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 74, and SEQ ID NO: 89. The at least one oligonucleotide can comprise one or more motifs selected from the group consisting of SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, and SEQ ID NO: 212. The aptamer composition may comprise at least one oligonucleotide comprising a sequence of nucleotides with at least 80% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, and SEQ ID NO: 212. The aptamer composition may comprise at least one oligonucleotide comprising a sequence of nucleotides with at least 90% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, and SEQ ID NO: 212. The aptamer composition may comprise at least one oligonucleotide comprising a sequence of nucleotides with at least 95% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, and SEQ ID NO: 212. In one aspect, the aptamer composition has a binding affinity for the human intercellular adhesion molecule 1 (ICAM-1) (SEQ ID NO: 213), its natural variants, polymorphic variants, or any post-translationally modified versions of said protein. Non-limiting examples of posttranslational modifications of ICAM-1 are disulfide bonds (e.g. between Cys48 and Cys92, Cys52 and Cys96, Cys135 and Cys186, Cys237 and Cys290, Cys332 and Cys371, Cys403 and Cys419, Cys431 and Cys457), glycosylations (e.g. at Asn130, Asn145, Asn183, Asn202, Asn267, Asn296, Asn385, and Asn406), phosphorylations (e.g. at Thr521 or Thr530), and ubiquitination. In one aspect, the aptamer composition has a binding affinity for the extracellular domain of human intercellular adhesion molecule 1 (ICAM-1) (SEQ ID NO: 214) or any post-translationally modified versions of said domain. In one aspect, the aptamer composition has a binding affinity for one or more domains of the intercellular adhesion molecule 1 (ICAM-1) selected from the group consisting of: Ig-like C2-type 1 domain (SEQ ID NO: 215), Ig-like C2-type 2 domain (SEQ ID NO: 216), Ig-like C2-type 3 domain (SEQ ID NO: 217), Ig-like C2-type 4 domain (SEQ ID NO: 218), Ig-like C2-type 5 domain (SEQ ID NO: 219), any post-translationally modified versions of said domains, and mixtures thereof. In one aspect, the aptamer composition has a binding affinity for the Ig-like C2-type 1 domain (SEQ ID NO: 215) of the intercellular adhesion molecule 1 (ICAM-1), any post-translationally modified versions of said domain, and mixtures thereof. In one aspect, the aptamer composition has a binding affinity for one or more regions of the human intercellular adhesion molecule 1, wherein said regions comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, and fragments of said sequences. Chemical modifications can introduce new features into the aptamers such as different molecular interactions with the target, improved binding capabilities, enhanced stability of oligonucleotide conformations, or increased resistance to nucleases. In one aspect, the at least one oligonucleotide of the aptamer composition may comprise natural or non-natural nucleobases. Natural nucleobases are adenine, cytosine, guanine, thymine, and uracil. Non-limiting examples of non-natural nucleobases can include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-5-methylcytosine, 5-hydroxymethylcytosine, thiouracil, 1-methylhypoxanthine, 6-methylisoquinoline-1-thione-2-yl, 3-methoxy-2-naphthyl, 5-propynyluracil-1-yl, 5-methylcytosin-1-yl, 2-aminoadenin-9-yl, 7-deaza-7-iodoadenin-9-yl, 7-deaza-7-propynyl-2-aminoadenin-9-yl, phenoxazinyl, phenoxazinyl-G-clam, bromouracil, 5-iodouracil, and mixtures thereof. Modifications of the phosphate backbone of the oligonucleotides can also increase the resistance against nuclease digestion. In one aspect, the nucleosides of the oligonucleotides may be linked by a chemical motif selected from the group consisting of natural phosphate diester, chiral phosphorothionate, chiral methyl phosphonate, chiral phosphoramidate, chiral phosphate chiral triester, chiral boranophosphate, chiral phosphoroselenoate, phosphorodithioate, phosphorothionate amidate, methylenemethylimino, 3′-amide, 3′ achiral phosphoramidate, 3′ achiral methylene phosphonates, thioformacetal, thioethyl ether, fluorophosphate, and mixtures thereof. In one aspect, the nucleosides of the oligonucleotides may be linked by natural phosphate diesters. In one aspect, the sugar moiety of the nucleosides of the oligonucleotides may be selected from the group consisting of ribose, deoxyribose, 2′-fluoro deoxyribose, 2′-O-methyl ribose, 2′-O-(3-amino)propyl ribose, 2′-O-(2-methoxy)ethyl ribose, 2′-O-2-(N,N-dimethylaminooxy)ethyl ribose, 2′-O-2-[2-(N,N-dimethylamino)ethyloxy]ethyl ribose, 2′-O—N,N-dimethylacetamidyl ribose, N-morpholinophosphordiamidate, α-deoxyribofuranosyl, other pentoses, hexoses, and mixtures thereof. In one aspect, the derivatives of ribonucleotides or said derivatives of deoxyribonucleotides may be selected from the group consisting of locked oligonucleotides, peptide oligonucleotides, glycol oligonucleotides, threose oligonucleotides, hexitol oligonucleotides, altritol oligonucleotides, butyl oligonucleotides, L-ribonucleotides, arabino oligonucleotides, 2′-fluoroarabino oligonucleotides, cyclohexene oligonucleotides, phosphorodiamidate morpholino oligonucleotides, and mixtures thereof. In one aspect, the nucleotides at the 5′- and 3′-ends of the at least one oligonucleotide may be inverted. In one aspect, at least one nucleotide of the at least one oligonucleotide may be fluorinated at the 2′ position of the pentose group. In one aspect, the pyrimidine nucleotides of said at least one oligonucleotide may be fluorinated at the 2′ position of the pentose group. In one aspect, said aptamer composition may comprise at least one polymeric material, wherein said at least one polymeric material is covalently linked to said at least one oligonucleotide. In one aspect, said at least one polymeric material may be polyethylene glycol. In one aspect, said at least one oligonucleotide may be between about 10 and about 200 nucleotides in length. In one aspect, said at least one oligonucleotide may be less than about 100 nucleotides in length, alternatively said at least one oligonucleotide may be less than about 50 nucleotides in length. In one aspect, said at least one oligonucleotide may be covalently or non-covalently attached to one or more active ingredients. In one aspect, said one or more active ingredients may be selected from the group comprising respiratory illness treatment agents, cold-treatment agents, flu-treatment agents, antiviral agents, antimicrobial agents, cooling sensates, warming sensates, malodor absorbing agents, natural extracts, peptides, enzymes, pharmaceutical active ingredients, metal compounds, and mixtures thereof. In one aspect, said one or more active ingredients can include, but are not limited to, pharmaceutical active ingredients, menthol, levomenthol, zinc and salts thereof,eucalyptus, camphor, and combinations thereof. Suitable active ingredients include any material that is generally considered as safe and that provides health care benefits. In one aspect, said at least one oligonucleotide may be non-covalently attached to said one or more active ingredients via molecular interactions. Examples of molecular interactions are electrostatic forces, van der Waals interactions, hydrogen bonding, and π-π stacking interactions of aromatic rings. In one aspect, said at least one oligonucleotide may be covalently attached to said one or more active ingredients using one or more linkers or spacers. Non-limiting examples of linkers are chemically labile linkers, enzyme-labile linkers, and non-cleavable linkers. Examples of chemically labile linkers are acid-cleavable linkers and disulfide linkers. Acid-cleavable linkers take advantage of low pH to trigger hydrolysis of an acid-cleavable bond, such as a hydrazone bond, to release the active ingredient or payload. Disulfide linkers can release the active ingredients under reducing environments. Examples of enzyme-labile linkers are peptide linkers that can be cleaved in the presence of proteases and β-glucuronide linkers that are cleaved by glucuronidases releasing the payload. Non-cleavable linkers can also release the active ingredient if the aptamer is degraded by nucleases. In one aspect, said at least one oligonucleotide may be covalently or non-covalently attached to one or more nanomaterials. In the present invention, said at least one oligonucleotide and said one or more active ingredients may be covalently or non-covalently attached to one or more nanomaterials. In one aspect, said one or more active ingredients may be carried by said one or more nanomaterials. Non-limiting examples of nanomaterials can include gold nanoparticles, nano-scale iron oxides, carbon nanomaterials (such as single-walled carbon nanotubes and graphene oxide), mesoporous silica nanoparticles, quantum dots, liposomes, poly (lactide-co-glycolic acids) nanoparticles, polymeric micelles, dendrimers, serum albumin nanoparticles, DNA-based nanomaterials, and combinations thereof. These nanomaterials can serve as carriers for large volumes of active ingredients, while the aptamers can facilitate the delivery of the nanomaterials with the actives to the expected target. Nanomaterials can have a variety of shapes or morphologies. Non-limiting examples of shapes or morphologies can include spheres, rectangles, polygons, disks, toroids, cones, pyramids, rods/cylinders, and fibers. In the context of the present invention, nanomaterials usually have at least one spatial dimension that is less than about 100 μm and more preferably less than about 10 μm. Nanomaterials comprise materials in solid phase, semi-solid phase, or liquid phase.1. Aptamers can also be peptides that bind to targets with high affinity and specificity. These peptide aptamers can be part of a scaffold protein. Peptide aptamers can be isolated from combinatorial libraries and improved by directed mutation or rounds of variable region mutagenesis and selection. In one aspect, said aptamer composition may comprise at least one peptide or protein; wherein said aptamer composition has a binding affinity for one or more cellular membrane glycoproteins, wherein said one or more cellular membrane glycoproteins can be selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), low-density lipoprotein receptor (LDLR) family members, and cadherin-related family member 3 (CDHR3); preferably intercellular adhesion molecule 1 (ICAM-1) and wherein said aptamer is configured to reduce the binding of one or more human rhinoviruses to said cellular membrane glycoproteins, preferably the intercellular adhesion molecule 1 (ICAM-1). In particular said aptamer composition may comprise at least one peptide or protein translated from(a) at least one oligonucleotide selected from the group consisting of oligonucleotides with at least 80% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 200; and/or;(b) at least one oligonucleotide comprising one or more motifs selected from the group consisting of SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, and SEQ ID NO: 212. III. Methods of Designing Aptamer Compositions The method of designing nucleic acid aptamers known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) has been broadly studied and improved for the selection of aptamers against small molecules and proteins (WO 91/19813). In brief, in the conventional version of SELEX, the process starts with the synthesis of a large library of oligonucleotides consisting of randomly generated sequences of fixed length flanked by constant 5′- and 3′-ends that serve as primers. The oligonucleotides in the library are then exposed to the target ligand and those that do not bind the target are removed. The bound sequences are eluted and amplified by PCR (polymerase chain reaction) to prepare for subsequent rounds of selection in which the stringency of the elution conditions is usually increased to identify the tightest-binding oligonucleotides. In addition to conventional SELEX, there are improved versions such as capillary electrophoresis-SELEX, magnetic bead-based SELEX, cell-SELEX, automated SELEX, complex-target SELEX, among others. A review of aptamer screening methods is found in (1) Kim, Y. S. and M. B. Gu, “Advances in Aptamer Screening and Small Molecule Aptasensors”, Adv. Biochem. Eng. Biotechnol., 2014 140:29-67 (Biosensors based on Aptamers and Enzymes) and (2) Stoltenburg, R., et al. (2007) “SELEX-A (r)evolutionary method to generate high-affinity nucleic acid ligands” Biomol. Eng. 2007 24(4): 381-403, the contents of which are incorporated herein by reference. Although the SELEX method has been broadly applied, it is neither predictive nor standardized for every target. Instead, a method must be developed for each particular target in order for the method to lead to viable aptamers. Despite the large number of selected aptamers, SELEX has not been routinely applied for the selection of aptamers with binding affinities towards cellular membrane glycoproteins such as intercellular adhesion molecule 1 (ICAM-1), low-density lipoprotein receptor (LDLR) family members, and cadherin-related family member 3 (CDHR3) and that prevent the binding of human rhinoviruses to such proteins. Unexpectedly, the inventors have found that SELEX can be used for the design of aptamers that prevent the binding of human rhinoviruses to the ICAM-1 receptor. Selection Library In SELEX, the initial candidate library is generally a mixture of chemically synthesized DNA oligonucleotides, each comprising a long variable region of n nucleotides flanked at the 3′ and 5′ ends by conserved regions or primer recognition regions for all the candidates of the library. These primer recognition regions allow the central variable region to be manipulated during SELEX in particular by means of PCR. The length of the variable region determines the diversity of the library, which is equal to 4nsince each position can be occupied by one of four nucleotides A, T, G or C. For long variable regions, huge library complexities arise. For instance, when n=50, the theoretical diversity is 450or 1030, which is an inaccessible value in practice as it corresponds to more than 105 tons of material for a library wherein each sequence is represented once. The experimental limit is around 1015different sequences, which is that of a library wherein all candidates having a variable region of 25 nucleotides are represented. If one chooses to manipulate a library comprising a 30-nucleotide variable region whose theoretical diversity is about 1018, only 1/1000 of the possibilities will thus be explored. In practice, that is generally sufficient to obtain aptamers having the desired properties. Additionally, since the polymerases used are unreliable and introduce errors at a rate on the order of 10−4, they contribute to significantly enrich the diversity of the sequence pool throughout the SELEX process. One candidate in 100 will be modified in each amplification cycle for a library with a random region of 100 nucleotides in length, thus leading to the appearance of 1013new candidates for the overall library. In one aspect, the starting mixture of oligonucleotides may comprise more than about 106different oligonucleotides and more preferably between about 1013to about 1015different oligonucleotides. In one aspect, the length of the variable region may be between about 10 and about 100 nucleotides. In one aspect, the length of the variable region may be between about 20 and about 60 nucleotides. In one aspect, the length of the variable region may be about 40 nucleotides. Random regions shorter than 10 nucleotides may be used but may be constrained in their ability to form secondary or tertiary structures and in their ability to bind to target molecules. Random regions longer than 100 nucleotides may also be used but may present difficulties in terms of cost of synthesis. The randomness of the variable region is not a constraint of the present invention. For instance, if previous knowledge exists regarding oligonucleotides that bind to a given target, libraries spiked with such sequences may work as well or better than completely random ones. In the design of primer recognition sequences, care should be taken to minimize potential annealing among sequences, fold back regions within sequences, or annealing of the same sequence itself. In one aspect, the length of primer recognition sequences may be between about 10 and about 40 nucleotides. In one aspect, the length of primer recognition sequences may be between about 12 and about 30 nucleotides. In one aspect, the length of primer recognition sequences may be between about 18 and about 26 nucleotides, i.e., about 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides. The length and sequence of the primer recognition sequences determine their annealing temperature. In one aspect, the primer recognition sequences of said oligonucleotides may have an annealing temperature between about 60° C. and about 72° C. Aptamers can be ribonucleotides (RNA), deoxynucleotides (DNA), or their derivatives. When aptamers are ribonucleotides, the first SELEX step may consist of transcribing the initial mixture of chemically synthesized DNA oligonucleotides via the primer recognition sequence at the 5′ end. After selection, the candidates are converted back into DNA by reverse transcription before being amplified. RNA and DNA aptamers having comparable characteristics have been selected against the same target and reported in the art. Additionally, both types of aptamers can be competitive inhibitors of one another, suggesting potential overlapping of interaction sites. New functionalities, such as hydrophobicity or photoreactivity, can be incorporated into the oligonucleotides by modifications of the nucleobases before or after selection. Modifications at the C-5 position of pyrimidines or at the C-8 or N-7 positions of purines are especially common and compatible with certain enzymes used during the amplification step in SELEX. In one aspect, said oligonucleotides may comprise natural or non-natural nucleobases. Natural nucleobases are adenine, cytosine, guanine, thymine, and uracil. Non-limiting examples of non-natural nucleobases are hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-5-methylcytosine, 5-hydroxymethylcytosine, thiouracil, 1-methylhypoxanthine, 6-methylisoquinoline-1-thione-2-yl, 3-methoxy-2-naphthyl, 5-propynyluracil-1-yl, 5-methylcytosin-1-yl, 2-aminoadenin-9-yl, 7-deaza-7-iodoadenin-9-yl, 7-deaza-7-propynyl-2-aminoadenin-9-yl, phenoxazinyl, phenoxazinyl-G-clam, 5-bromouracil, 5-iodouracil, and mixtures thereof. Some non-natural nucleobases, such as 5-bromouracil or 5-iodouracil, can be used to generate photo-crosslinkable aptamers, which can be activated by UV light to form a covalent link with the target. In one aspect, the nucleosides of said oligonucleotides may be linked by a chemical motif selected from the group comprising natural phosphate diester, chiral phosphorothionate, chiral methyl phosphonate, chiral phosphoramidate, chiral phosphate chiral triester, chiral boranophosphate, chiral phosphoroselenoate, phosphorodithioate, phosphorothionate amidate, methylenemethylimino, 3′-amide, 3′ achiral phosphoramidate, 3′ achiral methylene phosphonates, thioformacetal, thioethyl ether, fluorophosphate, and mixtures thereof. In one aspect, the nucleosides of said oligonucleotides may be linked by natural phosphate diesters. In one aspect, the sugar moiety of the nucleosides of said oligonucleotides may be selected from the group comprising ribose, deoxyribose, 2′-fluoro deoxyribose, 2′-O-methyl ribose, 2′-O-(3-amino)propyl ribose, 2′-O-(2-methoxy)ethyl ribose, 2′-O-2-(N,N-dimethylaminooxy)ethyl ribose, 2′-O-2-[2-(N,N-dimethylamino)ethyloxy]ethyl ribose, 2′-O—N,N-dimethylacetamidyl ribose, N-morpholinophosphordiamidate, α-deoxyribofuranosyl, other pentoses, hexoses, and mixtures thereof. In one aspect, said derivatives of ribonucleotides or said derivatives of deoxyribonucleotides may be selected from the group comprising locked oligonucleotides, peptide oligonucleotides, glycol oligonucleotides, threose oligonucleotides, hexitol oligonucleotides, altritol oligonucleotides, butyl oligonucleotides, L-ribonucleotides, arabino oligonucleotides, 2′-fluoroarabino oligonucleotides, cyclohexene oligonucleotides, phosphorodiamidate morpholino oligonucleotides, and mixtures thereof. When using modified nucleotides during the SELEX process, they should be compatible with the enzymes used during the amplification step. Non-limiting examples of modifications that are compatible with commercial enzymes include modifications at the 2′ position of the sugar in RNA libraries. The ribose 2′-OH group of pyrimidine nucleotides can be replaced with 2′-amino, 2′-fluoro, 2′-methyl, or 2′-O-methyl, which protect the RNA from degradation by nucleases. Additional modifications in the phosphate linker, such as phosphorothionate and boranophosphate, are also compatible with the polymerases and confer resistance to nucleases. In one aspect, at least one nucleotide of said oligonucleotides may be fluorinated at the 2′ position of the pentose group. In one aspect, the pyrimidine nucleotides of said oligonucleotides may be at least partially fluorinated at the 2′ position of the pentose group. In one aspect, all the pyrimidine nucleotides of said oligonucleotides may be fluorinated at the 2′ position of the pentose group. In one aspect, at least one nucleotide of said oligonucleotides may be aminated at the 2′ position of the pentose group. Another approach, recently described as two-dimensional SELEX, simultaneously applies in vitro oligonucleotide selection and dynamic combinatorial chemistry (DCC), e.g., a reversible reaction between certain groups of the oligonucleotide (amine groups) and a library of aldehyde compounds. The reaction produces imine oligonucleotides, which are selected on the same principles as for conventional SELEX. It is thus possible to identify for a target hairpin RNA modified aptamers that differ from natural aptamers. A very different approach relates to the use of optical isomers. Natural oligonucleotides are D-isomers. L-analogs are resistant to nucleases but cannot be synthesized by polymerases. According to the laws of optical isomerism, an L-series aptamer can form with its target (T) a complex having the same characteristics as the complex formed by the D-series isomer and the enantiomer (T′) of the target (T). Consequently, if compound T′ can be chemically synthesized, it can be used to perform the selection of a natural aptamer (D). Once identified, this aptamer can be chemically synthesized in an L-series. This L-aptamer is a ligand of the natural target (T). Selection Step Single stranded oligonucleotides can fold to generate secondary and tertiary structures, resembling the formation of base pairs. The initial sequence library is thus a library of three-dimensional shapes, each corresponding to a distribution of units that can trigger electrostatic interactions, create hydrogen bonds, etc. Selection becomes a question of identifying in the library the shape suited to the target, i.e., the shape allowing the greatest number of interactions and the formation of the most stable aptamer-target complex. For small targets (dyes, antibiotics, etc.) the aptamers identified are characterized by equilibrium dissociation constants in the micromolar range, whereas for protein targets Kd values below 10−9M are not rare. Selection in each round occurs by means of physical separation of oligonucleotides associated with the target from free oligonucleotides. Multiple techniques may be applied (chromatography, filter retention, electrophoresis, etc.). The selection conditions are adjusted (relative concentration of target/candidates, ion concentration, temperature, washing, etc.) so that a target-binding competition occurs between the oligonucleotides. Generally, stringency is increased as the rounds proceed in order to promote the capture of oligonucleotides with the highest affinity. In addition, counter-selections or negative selections are carried out to eliminate oligonucleotides that recognize the support or unwanted targets (e.g., filter, beads, etc.). The SELEX process for the selection of target-specific aptamers is characterized by repetition of five main steps: (1) binding of oligonucleotides to the target, (2) partition or removal of oligonucleotides with low binding affinity, (3) elution of oligonucleotides with high binding affinity, (4) amplification or replication of oligonucleotides with high binding affinity, and (5) conditioning or preparation of the oligonucleotides for the next cycle. This selection process is designed to identify the oligonucleotides with the greatest affinity and specificity for the target material. In one aspect, a method of designing an aptamer composition may comprise the step of contacting: a) a mixture of oligonucleotides, b) a selection buffer, and c) a target material comprising one or more cellular membrane glycoproteins selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), low-density lipoprotein receptor (LDLR) family members, cadherin-related family member 3 (CDHR3), truncations thereof, and mixtures thereof; preferably intercellular adhesion molecule 1 (ICAM-1) and truncations thereof. In another aspect, the method of designing an aptamer composition may comprise the step of contacting: a) a mixture of oligonucleotides, b) a selection buffer, and c) cells expressing one or more cellular membrane glycoproteins selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), low-density lipoprotein receptor (LDLR) family members, cadherin-related family member 3 (CDHR3), truncations thereof, and mixtures thereof; preferably intercellular adhesion molecule 1 (ICAM-1) and truncations thereof. In yet another aspect, the method of designing an aptamer composition may comprise the step of contacting: a) a mixture of oligonucleotides, b) a selection buffer, and c) human nasal epithelial cells expressing one or more cellular membrane glycoproteins selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), low-density lipoprotein receptor (LDLR) family members, cadherin-related family member 3 (CDHR3), truncations thereof, and mixtures thereof; preferably intercellular adhesion molecule 1 (ICAM-1) and truncations thereof. In one aspect, said mixture of oligonucleotides may comprise oligonucleotides selected from the group consisting of deoxyribonucleotides, ribonucleotides, derivatives of deoxyribonucleotides, derivatives of ribonucleotides, and mixtures thereof. Furthermore, said one or more cellular membrane glycoproteins or truncations thereof can be isolated, in mixture with other materials such as proteins or peptides, or part of a cell expressing said glycoproteins. SELEX cycles are usually repeated several times until oligonucleotides with high binding affinity are identified. The number of cycles depends on multiple variables, including target features and concentration, design of the starting random oligonucleotide library, selection conditions, ratio of target binding sites to oligonucleotides, and the efficiency of the partitioning step. In one aspect, said contacting step may be performed at least 5 times. In one aspect, said contacting step may be performed between 6 and 30 times. In one aspect, said method further may comprise the step of removing the oligonucleotides that do not bind said target material during said contacting step. Oligonucleotides are oligo-anions, each unit having a charge and hydrogen-bond donor/acceptor sites at a particular pH. Thus, the pH and ionic strength of the selection buffer are important and should represent the conditions of the intended aptamer application. In one aspect, the pH of said selection buffer may be between about 2 and about 9, alternatively between about 5 and about 8. Cations do not only facilitate the proper folding of the oligonucleotides, but also can provide benefits. In one aspect, said selection buffer may comprise cations. Non-limiting examples of cations are Na+, K+, Mg2+, Ca2+. In order for the aptamers to maintain their structures and function during their application, the in vitro selection process can be carried out under conditions similar to those for which they are being developed. In one aspect, said selection buffer may comprise a solution or suspension of a personal health care composition selected from the group comprising tablets, lyophilized tablets, lollipops, lozenges, liquid center-filled confectioneries, candies, powders, granular substances, films, liquids, solutions, suspensions, mouth rinses or gargles, saline washes, dispersible fluids, sprays, quick dissolving fibers, vapors, creams, ointments, powders, granular substances, films, and combinations thereof. In one aspect, said selection buffer may comprise at least one surfactant. In one aspect, the at least one surfactant may be selected from the group consisting of anionic surfactants, amphoteric or zwitterionic surfactants, and mixtures thereof. Non-limiting examples of anionic surfactants are alkyl and alkyl ether sulfates or sulfonates, including ammonium lauryl sulfate, ammonium laureth sulfate, triethylamine lauryl sulfate, triethylamine laureth sulfate, triethanolamine lauryl sulfate, triethanolamine laureth sulfate, monoethanolamine lauryl sulfate, monoethanolamine laureth sulfate, diethanolamine lauryl sulfate, diethanolamine laureth sulfate, lauric monoglyceride sodium sulfate, sodium lauryl sulfate, sodium laureth sulfate, potassium lauryl sulfate, potassium laureth sulfate, sodium lauryl sarcosinate, sodium lauroyl sarcosinate, lauryl sarcosine, cocoyl sarcosine, ammonium cocoyl sulfate, ammonium lauroyl sulfate, sodium cocoyl sulfate, sodium lauroyl sulfate, potassium cocoyl sulfate, potassium lauryl sulfate, triethanolamine lauryl sulfate, triethanolamine lauryl sulfate, monoethanolamine cocoyl sulfate, monoethanolamine lauryl sulfate, sodium tridecyl benzene sulfonate, sodium dodecyl benzene sulfonate, sodium cocoyl isethionate, and combinations thereof. Non-limiting amphoteric surfactants include those surfactants broadly described as derivatives of aliphatic secondary and tertiary amines in which the aliphatic radical can be straight or branched chain and wherein one of the aliphatic substituents contains from about 8 to about 18 carbon atoms and one contains an anionic group such as carboxy, sulfonate, sulfate, phosphate, or phosphonate, including cocoamphoacetate, cocoamphodiacetate, lauroamphoacetate, lauroamphodiacetate, and mixtures thereof. Non-limiting examples of zwitterionic surfactants include those surfactants broadly described as derivatives of aliphatic quaternaryammonium, phosphonium, and sulfonium compounds, in which the aliphatic radicals can be straight or branched chain, and wherein one of the aliphatic substituents contains from about 8 to about 18 carbon atoms and one contains an anionic group such as carboxy, sulfonate, sulfate, phosphate or phosphonate, and betaine. The selection buffer may comprise at least one material selected from the group comprising: aqueous carriers, gel matrixes, silicone conditioning agents, organic conditioning materials, non-ionic polymers, deposition aids, rheology modifier/suspending agents, benefit agents, and mixtures thereof. Non-limiting examples of aqueous carriers are water and water solutions of lower alkyl alcohols and polyhydric alcohols, including ethanol, isopropanol, propylene glycol, hexylene glycol, glycerin, and propane diol. Non-limiting examples of gel matrixes include water solutions of fatty alcohols, including cetyl alcohol, stearyl alcohol, behenyl alcohol, and mixtures thereof. Non-limiting examples of silicone conditioning agents include dimethicones, dimethiconols, cyclic silicones, methylphenyl polysiloxane, and modified silicones with various functional groups such as amino groups, quaternary ammonium salt groups, aliphatic groups, alcohol groups, carboxylic acid groups, ether groups, sugar or polysaccharide groups, fluorine-modified alkyl groups, alkoxy groups, or combinations of such groups. Non-limiting examples of organic conditioning materials include hydrocarbon oils, polyolefins, fatty esters, fluorinated conditioning compounds, fatty alcohols, alkyl glucosides and alkyl glucoside derivatives, quaternary ammonium compounds, polyethylene glycols and polypropylene glycols having a molecular weight of up to about 2,000,000 including those with CTFA names PEG-200, PEG-400, PEG-600, PEG-1000, PEG-2M, PEG-7M, PEG-14M, PEG-45M, and mixtures thereof. Non-limiting examples of non-ionic polymers include polyalkylene glycols, such as polyethylene glycols. Non-limiting examples of deposition aids include copolymers of vinyl monomers having cationic amine or quaternary ammonium functionalities with water soluble spacer monomers such as acrylamide, methacrylamide, alkyl and dialkyl acrylamides, alkyl and dialkyl methacrylamides, alkyl acrylate, alkyl methacrylate, vinyl caprolactone, and vinyl pyrrolidone; vinyl esters, vinyl alcohol (made by hydrolysis of polyvinyl acetate), maleic anhydride, propylene glycol, and ethylene glycol, cationic celluloses, cationic starches, and cationic guar gums. Non-limiting examples of rheology modifier/suspending agents include homopolymers based on acrylic acid, methacrylic acid or other related derivatives, alginic acid-based materials, and cellulose derivatives. Non-limiting examples of benefit agents include brightening agents, strengthening agents, anti-fungal agents, anti-bacterial agents, anti-microbial agents, anti-dandruff agents, anti-malodor agents, perfumes, olfactory enhancement agents, anti-itch agents, cooling agents, anti-adherence agents, moisturization agents, smoothness agents, surface modification agents, antioxidants, natural extracts and essential oils, dyes, pigments, bleaches, nutrients, peptides, vitamins, enzymes, chelants, and mixtures thereof. Negative selection or counter-selection steps can minimize the enrichment of oligonucleotides that bind to undesired targets or undesired epitopes within a target. In one aspect, said method of designing an aptamer composition may further comprise the step of contacting: a) a mixture of oligonucleotides, b) a selection buffer, and c) one or more undesired targets. Methods for negative selection or counter-selection of aptamers against unbound targets have been published in WO201735666, the content of which is incorporated herein by reference. The method of designing an aptamer composition may comprise the steps of: a) synthesizing a mixture of oligonucleotides; b) contacting: i. said mixture of oligonucleotides, ii. a selection buffer, and iii. a target material comprising one or more cellular membrane glycoproteins; wherein said glycoproteins are selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), its fragments, and combinations thereof, to produce a target suspension; c) removing the liquid phase from said target suspension to produce a target-oligonucleotide mixture; d) contacting said target-oligonucleotide mixture with a washing buffer and removing the liquid phase to produce a target-aptamer mixture; and e) contacting said target-aptamer mixture with an elution buffer and recovering the liquid phase to produce an aptamer mixture. In one aspect, said steps may be performed repetitively at least 5 times. In one aspect, said steps may be performed between 6 and 30 times, preferably less than 20 times. In another aspect, a method of designing an aptamer composition comprising the steps of: a) synthesizing a random mixture of deoxyribonucleotides comprising oligonucleotides consisting of: i. a T7 promoter sequence at the 5′-end, ii. a variable 40-nucleotide sequence in the middle, and iii. a conserved reverse primer recognition sequence at the 3′end; b) transcribing said random mixture of deoxyribonucleotides using pyrimidine nucleotides fluorinated at the 2′ position of the pentose group and natural purine nucleotides and a mutant T7 polymerase to produce a mixture of fluorinated ribonucleotides; c) contacting: i. said mixture of fluorinated ribonucleotides, ii. a selection buffer, and iii. a target material comprising one or more cellular membrane glycoproteins; wherein said glycoproteins are selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), its fragments, and combinations thereof, to produce a target suspension; d) removing the liquid phase from said target suspension to produce a target-oligonucleotide mixture; e) contacting said target-oligonucleotide mixture with a washing buffer and removing the liquid phase to produce a target-aptamer mixture; f) contacting said target-aptamer mixture with an elution buffer and recovering the liquid phase to produce an RNA aptamer mixture; g) reserve transcribing and amplifying said RNA aptamer mixture to produce a DNA copy of said RNA aptamer mixture; and h) sequencing said DNA copy of said RNA aptamer mixture. Post-Selection Modification To enhance stability of the aptamers, chemical modifications can be introduced in the aptamer after the selection process. For instance, the 2′-OH groups of the ribose moieties can be replaced by 2′-fluoro, 2′-amino, or 2′-O-methyl groups. Furthermore, the 3′- and 5′-ends of the aptamers can be capped with different groups, such as streptavidin-biotin, inverted thymidine, amine, phosphate, polyethylene-glycol, cholesterol, fatty acids, proteins, enzymes, fluorophores, among others, making the oligonucleotides resistant to exonucleases or providing some additional benefits. Other modifications are described in previous sections of the present disclosure. Unlike backbone modifications which can cause aptamer-target interaction properties to be lost, it is possible to conjugate various groups at one of the 3′- or 5′-ends of the oligonucleotide in order to convert it into a delivery vehicle, tool, probe, or sensor without disrupting its characteristics. This versatility constitutes a significant advantage of aptamers, in particular for their application in the current invention. In one aspect, one or more personal care active ingredients may be covalently attached to the 3′-end of said at least one oligonucleotide. In one aspect, one or more personal care active ingredients may be covalently attached to the 5′-end of said at least one oligonucleotide. In one aspect, one or more personal care active ingredients may be covalently attached to random positions of said at least one oligonucleotide. Incorporation of modifications to aptamers can be performed using enzymatic or chemical methods. Non-limiting examples of enzymes used for modification of aptamers are terminal deoxynucleotidyl transferases (TdT), T4 RNA ligases, T4 polynucleotide kinases (PNK), DNA polymerases, RNA polymerases, and other enzymes known by those skilled in the art. TdTs are template-independent polymerases that can add modified deoxynucleotides to the 3′ terminus of deoxyribonucleotides. T4 RNA ligases can be used to label ribonucleotides at the 3′-end by using appropriately modified nucleoside 3′,5′-bisphosphates. PNK can be used to phosphorylate the 5′-end of synthetic oligonucleotides, enabling other chemical transformations (see below). DNA and RNA polymerases are commonly used for the random incorporation of modified nucleotides throughout the sequence, provided such nucleotides are compatible with the enzymes. Non-limiting examples of chemical methods used for modification of aptamers are periodate oxidation of ribonucleotides, EDC activation of 5′-phosphate, random chemical labeling methods, and other chemical methods known by those skilled in the art, incorporated herein. During periodate oxidation, meta- and ortho-periodates cleave the C—C bonds between vicinal diols of 3′-ribonucleotides, creating two aldehyde moieties that enable the conjugation of labels or active ingredients at the 3′-end of RNA aptamers. The resulting aldehydes can be easily reacted with hydrazine- or primary amine-containing molecules. When amines are used, the produced Schiff bases can be reduced to more stable secondary amines with sodium cyanoborohydride (NaCNBH3). When EDC activation of 5′-phosphate is used, the 5′-phosphate of oligonucleotides is frequently activated with EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and imidazole to produce a reactive imidazolide intermediate, followed by reaction with a primary amine to generate aptamers modified at the 5′end. Because the 5′ phosphate group is required for the reaction, synthetic oligonucleotides can be first treated with a kinase (e.g. PNK). Random chemical labeling can be performed with different methods. Because they allow labeling at random sites along the aptamer, a higher degree of modification can be achieved compared to end-labeling methods. However, since the nucleobases are modified, binding of the aptamers to their target can be disrupted. The most common random chemical modification methods involve the use of photoreactive reagents, such as phenylazide-based reagents. When the phenylazide group is exposed to UV light, it forms a labile nitrene that reacts with double bonds and C—H and N—H sites of the aptamers. Additional information about methods for modification of aptamers is summarized in Hermanson G. T., “Bioconjugate Techniques”, pp. 969-1002, 2nd Edition, Academic Press, San Diego, 2008, the content of which is incorporated herein by reference. After selection, in addition to chemical modifications, sequence truncations can be performed to remove regions that are not essential for binding or for folding into the structure. Moreover, aptamers can be linked together to provide different features or better affinity. Thus, any truncations or combinations of the aptamers described herein can also be incorporated in the aptamer composition. IV. Application of Aptamer Compositions in Personal Health Care Products Described herein are personal health care compositions and methods for using such compositions for the prevention and treatment of cold-like symptoms due to respiratory tract viral infections. In some aspects, a personal health care composition comprises at least one aptamer as disclosed herein; wherein the at least one aptamer has a binding affinity for ICAM-1 and is configured to reduce the binding of one or more human rhinoviruses to the intercellular adhesion molecule 1 (ICAM-1). The personal health care composition can be preferably applied to areas of the upper respiratory tract, such as the nasal cavity and throat, to provide a barrier to rhinovirus binding and entrance into cells. The personal health care composition preferably comprises a pharmaceutically effective amount of at least one aptamer. In some aspects, the personal health care composition can comprise between about 0.001% to about 1% of the at least one aptamer, alternatively from about 0.005% to about 0.5%, alternatively from about 0.01% to about 0.1%, all by weight of the composition. The personal health care compositions can be administered orally or intranasally. In one aspect, the personal health care composition can be an oral composition. An oral composition can be in liquid form, semi-solid form, suspension form, or in any solid form that is capable of quickly dissolving in the mouth. Non-limiting examples of oral dosage forms can include tablets, lyophilized tablets, lollipops, lozenges, liquid center-filled confectioneries, candies, powders, granular substances, films, liquids, solutions, suspensions, mouth rinses or gargles, saline washes, dispersible fluids, sprays, quick dissolving fibers, such as polyvinylpyrrolidone and poly(vinyl alcohol), and combinations thereof. Solid oral dosage forms can be of any desired size, shape, weight, consistency or hardness, bearing in mind that it should not be swallowed before it disintegrates and can easily fit inside the mouth. Alternatively, the personal health care composition can be a nasal composition. A nasal composition can be in any dosage form capable of quickly dispersing in the nose. Non-limiting examples of nasal dosage forms can include vapors, creams, ointments, powders, granular substances, films, liquids, dispersible fluids, sprays, and combinations thereof. As used herein, the term “administering” with respect to a human/mammal means that the human/mammal ingests or is directed to ingest, or does ingest, or deliver, or chew, or drink, or spray, or place in mouth or nose, or inhale one or more of the personal health care compositions. Administration may be on an as-needed or as-desired basis, for example, once-weekly, or daily, including multiple times daily, for example, at least once daily, at least twice daily, at least three times daily, or at least four times daily. The personal health care compositions may be administered to prevent and treat cold-like symptoms. As used herein “cold-like symptoms” refer to symptoms typically associated with respiratory tract viral infections. These symptoms include, but are not limited to, nasal congestion, chest congestion, sneezing, rhinorrhea, fatigue or malaise, coughing, fever, sore throat, headache, and other known cold symptoms. As further used herein, “treat” or “treatment” with respect to respiratory illness means that administration of the referenced composition prevents, alleviates, ameliorates, inhibits, or mitigates one or more symptoms of the respiratory illness or the respiratory illness itself, or any like benefit with respect to the respiratory illness in a mammalian subject in need thereof, preferably in humans. As such, this includes, for example: preventing a respiratory illness or its associated symptoms from occurring in a mammal, for example when the mammal is predisposed to acquiring the respiratory illness, but has not yet been diagnosed with the illness; inhibiting the respiratory illness or its associated symptoms; and/or alleviating, reversing, or curing the respiratory illness or its associated symptoms. Insofar as the methods of the present invention are directed to preventing a respiratory illness, it is understood that the term “prevent” does not require that the respiratory illness be completely thwarted. Rather, as used herein, the term “preventing” or the like refers to the ability of the skilled artisan to identify susceptibility to respiratory illness (such as, for example, in humans during winter months), such that administration of the referenced compositions may occur prior to the onset of the symptoms associated with the illness. The personal health care compositions and methods of the present invention can comprise, consist of, or consist essentially of, the essential elements and limitations of the invention described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in personal health care compositions intended for use by a subject. All parts, percentages, and ratios herein are by weight unless otherwise specified. All such weights as they pertain to listed ingredients are based on the active level and, therefore do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified. All measurements referred to herein are made at 25° C. unless otherwise specified. The personal health care compositions of the present invention may include one or more of the following: The personal health care composition can comprise a solvent. Non-limiting examples of solvents include water, propylene glycol, ethanol, glycerin, polyethylene glycol, and combinations thereof. Solvent can be present in an amount of from about 2% to about 99%, by weight of the composition, alternatively from about 5% to about 95%, alternatively from about 10% to about 80, alternatively from about 12% to about 65%, alternatively from about 20% to about 50%. The personal health care composition can comprise a thickening agent. Non-limiting examples of thickening agents can include carboxymethylcellulose (CMC), carboxymethylcellulose sodium; and mixtures thereof. When present, the composition can comprise from about 0.01% to about 60% of a thickening agent, alternatively from about 0.1% to about 40%, alternatively from about 1% to about 30%, alternatively from about 2% to about 20%, alternatively from about 3% to about 15%, all by weight of the composition. In one aspect, the thickening agent can provide a moisturizing and/or hydration benefit that relieves the cough on contact and/or provides aid in healing the mouth and/or throat. The personal health care composition can comprise a diluent. Non-limiting examples of diluents can include microcrystalline cellulose, silicified microcrystalline cellulose, such as ProSolv® SMCC 90 (commercially available from JRS Pharma, Patterson, NY, USA), dextrose, mannitol, sorbitol, maltodextrin, maltitol, and combinations thereof. Suitable diluent levels are from about 20% to about 90% diluent, by weight of the composition, alternatively from about 30% to about 85%, alternatively from about 40% to about 83%, alternatively from about 50% to about 80%, alternatively from about 60% to about 78%. The personal health care composition can comprise a disintegrant. A disintegrant can be included to formulate a rapid disintegration of the solid oral dosage form following administration. Non-limiting examples of disintegrants can include crospovidone, sodium starch glycolate, crosslinked sodium carboxymethyl cellulose, low substituted hydroxypropylcellulose, guar gum, sodium alginate, and mixtures thereof. Suitable disintegrant levels are from about 1% to about 20%, by weight of the composition, alternatively from about 2% to about 15%, alternatively from about 3% to about 10%, alternatively from about 5% to about 8%. In one aspect, a composition can comprise mannitol and crospovidone to provide quick disintegration and dissolution. One advantage to using a soluble sugar, like mannitol, is that it can pick up water and dissolve quickly. One advantage to using a disintegrant, like crospovidone, is that it can absorb water and swell, thus causing the dosage form to break apart. As a dosage form breaks apart it is exposed to liquid, such as saliva in the oral cavity, and can dissolve faster. The ratio of mannitol to crospovidone can be about 15:1, alternatively about 13:1 alternatively about 10:1. The personal health care composition can comprise a lubricant. Non-limiting examples of lubricants can include sodium stearyl fumarate, magnesium stearate, calcium stearate, zinc stearate, stearic acid, glyceryl behenate, hydrogenated vegetable oils, talc, polyethylene glycol, mineral oil, and combinations thereof. Suitable levels of lubricant are from about 0.05% to about 5% lubricant, by weight of the composition, alternatively from about 0.1% to about 3%, alternatively from about 0.25% to about 1.5%, alternatively from about 0.3% to about 1%, alternatively from about 0.4% to about 0.6%. In one aspect, the personal health care composition can be a non-Newtonian, or thixotropic, fluid, exhibiting a reduced apparent viscosity while being subjected to shear forces, but a high apparent viscosity while at rest. One advantage to a non-Newtonian fluid is that it permits application by spraying with a pump spray device or squeeze-type spray bottle immediately following the application of a shearing force (such as those created by vigorously shaking the device) but causes the sprayed material to remain at least temporarily relatively immobile on mucosal membranes or the skin. Preferably, the composition can have a very rapid rate of viscosity recovery following withdrawal of the shearing force. The personal health care composition can comprise a rheology-modifying agent. Non-limiting examples of rheology-modifying agents can include sodium carboxymethyl cellulose, algin, carrageenans (including iota, kappa, lambda carrageenan, and combinations thereof), carbomers, galactomannans, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyethylene glycols, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl chitin, sodium carboxymethyl dextran, sodium carboxymethyl starch, microcrystalline cellulose, mixtures of microcrystalline cellulose and carboxymethylcellulose sodium (commercially available as Avicel® RC-591 from FMC Corporation, Philadelphia, Pa), xanthan gum, and combinations thereof. Suitable levels of rheology-modifying agents can be from about 0.5% to about 15%, alternatively from about 1% to about 12%, alternatively from about 2% to about 6%, all by weight of the composition. Rheology-modifying agents can not only provide viscosity benefits but can also coat the nose and throat longer to sooth and/or deliver an agent of choice. The personal health care composition may further comprise a humectant. Humectants, which can be hygroscopic materials such as glycerin, a polyethylene or other glycol, a polysaccharide, aloe, and the like, act to inhibit water loss from the composition and may add moisturizing qualities. The personal health care composition can comprise an acidic agent. The acidic agent can comprise organic acids, pyroglutamic acid, and combinations thereof. Suitable organic acid can include, but are not limited to, ascorbic acid, monocarboxylic acids, dicarboxylic acids, tricarboxylic acids, and mixtures thereof. Specific non-limiting examples of suitable monocarboxylic, dicarboxylic, or tricarboxylic acids include salicylic, fumaric, benzoic, glutaric, lactic, citric, malonic, acetic, glycolic, malic, adipic, succinic, aspartic, phthalic, tartaric, glutamic, gluconic, and mixtures thereof. Without being limited by theory, it is believed that incorporating acids in a nasal composition can create a hostile environment for viruses without significantly irritating specific areas of the respiratory tract such as the nasal tissues. The composition can comprise from about 0.01% to about 10% organic acid, alternatively from about 0.05% to about 5%, alternatively from about 0.10% to about 2.5%, all by weight of the composition. The personal health care composition can comprise a surfactant spreading aid such as polyoxyethylene (20) sorbitan mono-oleate, commercially sold as Polysorbate 80, Polyoxyethylene (20) sorbitan monolaurate, commercially sold as Polysorbate 20, Polyoxyl 400 stearate, polyethylene glycol, Polyethylene-polypropylene glycol, commercially sold as Poloxamer 407, and combinations thereof. The surfactants can be included in the composition at concentrations ranging from about 0.001% to about 10%, alternatively from about 0.01% to about 5%, alternatively from about 0.1% to about 3%, by weight of the composition. Additional Components The personal health care composition described herein may optionally comprise one or more additional components known for use in personal health care products, provided that the additional components are physically and chemically compatible with the components described herein, or do not otherwise unduly impair product stability, aesthetics, or performance. Optional components suitable for use herein include materials such as preservatives, pH adjusting agents, chelating agents, metal compounds, pharmaceutical active ingredients, vitamins, herbal ingredients, sweeteners, sensates, flavoring agents, natural honey, volatile oils, aromatic components such as camphor, eucalyptol, menthol, fragrances and the like, antioxidants, amino acids, energy boosting ingredients, sleep aids, sodium chloride, and combinations thereof. The optional components can be included in the personal health care composition at concentrations ranging from about 0.001% to about 20%, alternatively from about 0.01% to about 10%, alternatively from about 0.1% to about 5%, all by weight of the composition. In one aspect, the personal health care composition can comprise a preservative. Preservatives can optionally be included to prevent microbial contamination. Non-limiting examples of preservatives can include benzalkonium chloride, chlorhexidine gluconate, phenyl ethyl alcohol, phenoxyethanol, benzyl alcohol, sorbic acid, thimerosal, phenylmercuric acetate, methylparaben, propylparaben, butylparaben, chlorobutanol, and mixtures thereof. In one aspect, the personal health care composition can comprise a pH adjusting agent. Non-limiting examples of pH adjusting agents can include sodium bicarbonate, sodium phosphate, sodium hydroxide, ammonium hydroxide, sodium stannate, triethanolamine, sodium citrate, disodium succinate, and mixtures thereof. Optional pH adjusting agents can be included in the composition to adjust the pH to a value of from about 2 to about 8, alternatively from about 2 to about 5. If present, the pH adjusting agents are generally included at concentrations ranging from about 0.01 to about 5.0%, by weight of the composition. In one aspect, the personal health care composition can comprise a chelating agent. Non-limiting examples of suitable optional chelating agents can include phytic acid, disodium and calcium salts of ethylene diamine tetraacetic acid (EDTA), tetrasodium EDTA, sodium hexametaphosphate (SHMP), di(hydroxyethyl)glycine, 8-hydroxyquinoline, and mixtures thereof. The chelating agents can be included at concentrations ranging from about 0.001% to 10%, preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 2%, by weight of the composition. The personal health care composition can comprise a metal compound. Metal compounds suitable for use herein include those metal compounds containing a metal ion selected from the group consisting of manganese (Mn), silver (Ag), zinc (Zn), tin (Sn), iron (Fe), copper (Cu), aluminum (Al), nickel (Ni), cobalt (Co), and mixtures thereof. Non-limiting examples of a metal compound suitable for use herein include zinc acetate, zinc chloride, zinc ascorbate, zinc gluconate, zinc pidolate, zinc succinate, zinc sulphate, zinc edetate, and mixtures thereof. Zinc acetate is the most preferred metal compound. When the personal health care composition comprises a metal compound containing a zinc ion, it is believed that the zinc ion provides for antiviral properties. Zinc ions have been shown to be both antiviral and antibacterial. They are believed to inhibit cleavage of rhinovirus polypeptides, preventing replication and formation of infective virions. Zinc ions reduce the ability of rhinoviruses to penetrate cell membranes, partly by lowering expression of intercellular adhesion molecule ICAM. Zinc ions have also been shown to stimulate T-cell lyphocytes, including production of the natural antiviral, interferon-gamma. They stabilize cell plasma membranes, protecting cells from cytotoxic agents, and preventing cell leakage. Furthermore, it is known that metal ions such as iron, silver, copper, and zinc can provide antiviral properties for the prevention and treatment of cold and influenza-like symptoms. The concentration of the metal compound in the personal health care compositions can range from about 0.001% to about 20%, alternatively from about 0.01% to about 10%, alternatively from about 0.05% to about 5%, alternatively from about 0.1% to about 2%, alternatively from 0.2% to about 1%, all by weight of the composition. Non-limiting examples of pharmaceutical active ingredients can include menthol; anesthetics such as benzocaine and lidocaine; decongestants such as phenylephrine, pseudoephedrine, xylometazoline, and oxymetazoline; antihistamines such as doxylamine, diphenhydramine, loratadine, and cetirizine; expectorants such as guaifenesin, ambroxol, and bromhexine; pain relievers such as acetaminophen (APAP), ibuprofen, ketoprofen, diclofenac, naproxen, and aspirin; antitussives such as dextromethorphan, codeine, chlophedianol, and levodropropizine; the free and addition salts thereof; and combinations thereof. Pharmaceutical active ingredients can be present at a level from about 0.01% to about 25%, alternatively from about 0.05% to about 15%, alternatively from about 0.1% to about 10%, from about 1% to about 5%, all by weight of the composition. In one aspect, the personal healthcare composition can comprise at least one aptamer and one or more pharmaceutical active ingredients to provide relief of one or more symptoms and inhibit rhinovirus binding. Non-limiting examples of vitamins can include Vitamin A, Vitamin C, Vitamin D2, Vitamin D3, Vitamin E, Vitamin K1, Vitamin K3, Vitamin B1, vitamin B3, folic acid, Vitamin B12, Vitamin B3, Vitamin B7, and combinations thereof. In some aspects, the composition can comprise from about 0.1 to about 10% vitamins, alternatively from about 1 to about 8%, alternatively from about 2 to about 6%, all by weight of the composition. Non-limiting examples of herbal ingredients can include rosemary (leaf), ginger, lemon balm, green tea, holy basil, oregano, thyme, ashwagandha, bacopa, chamomile, valerian, rosemary, turmeric, grapeseed, blueberry, coffee, curcumin, elderberry, marshmallow root, ivy leaf, black tea, white tea, oolong tea, green tea, and combinations thereof. In some aspects, the herbal ingredient can be whole herbs or plant parts, extracts, powders, concentrates, or combinations thereof. In some aspects, the composition can comprise from about 0.1 to about 10% herbal ingredients, alternatively from about 1 to about 8%, alternatively from about 2 to about 6%, all by weight of the composition. In one aspect, the sweetener can be selected from the group comprising sugar alcohols, synthetic sweeteners, high intensity natural sweeteners, and combinations thereof. Non-limiting examples of nutritive sweeteners can include sucrose, dextrose, glucose, fructose, lactose, tagatose, maltose, trehalose, high fructose corn syrup, and combinations thereof. Nutritive sweeteners can be present in an amount from about 1% to about 99%, by weight of the composition, alternatively from about 4% to about 95%, alternatively from about 10% to about 70%, alternatively from about 15% to about 60%, alternatively from about 25% to about 50%, in another example about 35% to about 45%. Non-limiting examples of sugar alcohols can include xylitol, sorbitol, mannitol, maltitol, lactitol, isomalt, erythritol, and combinations thereof. Sugar alcohols can be present in an amount from about 5% to about 70%, by weight of the composition, alternatively from about 10% to about 60%, alternatively from about 15% to about 55%, alternatively from about 25% to about 50%, alternatively from about 30% to about 45%. Non-limiting examples of synthetic sweeteners can include aspartame, acesulfame potassium, alitame, sodium saccharin, sucralose, neotame, cyclamate, and combinations thereof. Synthetic sweeteners can be present in an amount from about 0.01% to about 10%, by weight of the composition, alternatively from about 0.05% to about 5%, alternatively about 0.1% to about 3%, alternatively from about 0.2% to about 1%, alternatively from about 0.1% to about 0.5%. Non-limiting examples of high intensity natural sweeteners can include neohesperidin dihydrochalcone, stevioside, rebaudioside A, rebaudioside C, dulcoside, monoammonium glycrrhizinate, thaumatin, and combinations thereof. High intensity natural sweeteners can be present in an amount from about 0.01% to about 10% by weight of the composition, alternatively about 0.05% to about 5%, alternatively from about 0.1% to about 3%, alternatively from about 0.5% to about 1%. The personal health care composition can comprise a flavoring system comprising sensates, flavoring agents, salivating agents, and combinations thereof. The personal health care composition can comprise a sensate. Non-limiting examples of sensates can include cooling sensates, warming sensates, tingling sensates, and combinations thereof. Sensates can deliver sensory signals to the mouth, throat, nasal, and/or sinus passages so that the personal health care composition may be perceived by the user as immediately acting to alleviate an ailment and/or to provide a soothing sensation. Non-limiting examples of cooling sensates can include WS-23 (2-Isopropyl-N,2,3-trimethylbutyramide), WS-3 (N-ethyl-p-menthane-3-carboxamide), WS-30 (1-glyceryl-p-menthane-3-carboxylate), WS-4 (ethyleneglycol-p-methane-3-carboxylate), WS-14 (N-t-butyl-p-menthane-3-carboxamide), WS-12 (N-(4-,ethoxyphenyl)-p-menthane-3-carboxamide), WS-5 (ethyl 3-(p-menthane-3-carboxamido)acetate), menthol, levomenthol, 1-menthone glycerol ketal (sold as Frescolat® MGA by Symrise, Holzminden, Germany), (−)-Menthyl lactate (sold as Frescolat® ML by Symrise, Holzminden, Germany), (−)-Menthoxypropane-1,2-diol (sold as Coolact® 10 by Vantage Specialty Ingredients, Inc., Warren, NJ), 3-(1-menthoxy)-2-methylpropane-1,2-diol, (−)-Isopulegol (sold as Coolact P® by Takasago International, Tokyo, Japan), cis & trans p-Menthane-3,8-diols (sold Coolact® 38D by Takasago International), menthyl pyrrolidone carboxylate (sold as Questice® by Givaudan Active Beauty, Verbuer, Switzerland), (1R,3R,4S)-3-menthyl-3,6-dioxaheptanoate (available from Firmenich, Geneva, Switzerland), (1R,2S,5R)-3-menthyl methoxyacetate (available from Firmenich), (1R,2S,5R)-3-menthyl 3,6,9-trioxadecanoate (available from Firmenich), (1R,2S,5R)-menthyl 11-hydroxy-3,6,9-trioxaundecanoate (available from Firmenich), (1R,2S,5R)-3-menthyl (2-hydroxyethoxy)acetate (available from Firmenich), Icilin also known as AG-3-5 (chemical name 1-(2-hydroxyphenyl)-4-(3-nitrophenyl)-3,6-dihydropyrimidin-2-one), 4-methyl-3-(1-pyrrolidinyl)-2[5H]-furanone, Peppermint oil, Spearmint oil, L-Monomenthyl succinate, L-monomenthyl glutarate, 2-1-menthoxyethanol (Coolact® 5), 3-1-Menthoxy propane-1,2-diol (sold as TK10 by Takasago International), N-(4-cyanomethylphenyl)-p-menthanecarboxamide (sold as Evercool™ 180 by Givaudan), and combinations thereof. Cooling sensates can be present from about 0.001% to about 1%, by weight of the composition, alternatively from about 0.01% to about 0.5%, alternatively from about 0.02% to about 0.25%, alternatively from about 0.03% to about 0.10%. Non-limiting examples of warming sensates can include vanillyl alcohol n-butyl ether (sold as TK-1000 by Takasago International), Heatenol™ (available from Sensient Pharmaceutical, St. Louis, MO), Optaheat (sold by Symrise, Holzminden, Germany), ginger extract,capsicumtincture, cinnamon, capsaicin, curry, Isobutavan, Nonivamide, vanillyl butyl ether (commercially available as Hotact® VBE), piperine, and combinations thereof. Warming sensates can be present from about 0.005% to about 2%, by weight of the composition, alternatively from about 0.01% to about 1%, and alternatively from about 0.1% to about 0.5%. Non-limiting examples of flavoring agents can include natural flavoring agents, artificial flavoring agents, artificial extracts, natural extracts and combination thereof. Non-limiting examples of flavoring agents can include vanilla, honey, lemon, lemon honey, cherry vanilla, peach, honey ginger, chamomile, cherry, cherry cream, mint, vanilla mint, dark berry, black berry, raspberry, peppermint, spearmint, honey peach, acai berry, cranberry, honey cranberry, tropical fruit, dragon fruit, wolf berry, red stem mint, pomegranate, black current, strawberry, lemon, lime, peach ginger, orange, orange cream, apricot, anethole, ginger, jack fruit, star fruit, blueberry, fruit punch, lemon grass, banana, strawberry banana, grape, blue raspberry, lemon lime, wintergreen mint, bubble gum, tart honey lemon, green apple, apple, tangerine, grapefruit, kiwi, pear, tangerine, tangerine lime, menthol, and combinations thereof. Flavoring agents can be present from about 0.05% to about 10%, by weight of the composition, alternatively from about 0.1% to about 8%, alternatively from about 0.2% to about 6%, alternatively from about 0.4% to about 3%, alternatively from about 0.6% to about 1.5%. Also described herein is a kit comprising the personal health care composition described herein. In one aspect, the kit can comprise a delivery device and the personal health care composition contained in the delivery device. In one aspect, the kit can optionally comprise at least one additional component, such as a supplement or a vitamin composition. Also described herein is a method of providing one or more health benefits comprising administering a personal health care composition as described herein comprising an aptamer to a subject in need thereof, wherein the aptamer has a binding affinity for ICAM-1. Non-limiting examples of the one or more health benefits can include providing a physical barrier to block rhinovirus binding and entering cells, helping to stop a cold caused by rhinovirus from forming, reducing the severity and/or duration of a cold caused by rhinovirus, reducing the chances of getting a cold, and combinations thereof. EXAMPLES The following examples illustrate non-limiting examples of the invention described herein. The exemplified personal health care compositions can be prepared by conventional formulation and mixing techniques. It will be appreciated that other modifications of the personal health care compositions within the skill of those in the formulation art can be undertaken without departing from the spirit and scope of this invention. The following are non-limiting examples of personal health care compositions described herein. Oral Composition Examples Throat Spray Ex. 1Ex. 2(Wt %)(Wt %)Benzocaine5.00Menthol1.01.0Glycerin17.017.0Flavoring system0.150.15Propylene Glycol65.065.0Ethyl Alcohol 95%7.997.99Saccharin Sodium0.130.13Sucralose0.180.18Color0.0050.005Aptamer0.001-1.00.001-1.0WaterQ.S.Q.S. Orally Dissolving Tablet Formula Ex. 3Ex. 4Ex. 5Ex. 6Ex. 7(Wt %)(Wt %)(Wt %)(Wt %)(Wt %)Mannitol59.549.539.539.539.5Sucrose4.04.04.04.04.0Crospovidone4.04.04.04.04.0ProSolv ® SMCCQ.S.Q.S.Q.S.Q.S.Q.S.90Diphenhydramine012.512.512.512.5HCl (Active)Sodium Caprate0001.00Cetylpyridinium00001.0ChlorideMagnesium1.01.01.01.01.0StearateAptamer0.001-1.00.001-1.00.001-1.00.001-1.00.001-1.0 Liquid Composition Ex. 8Ex. 9(Wt %)(Wt %)Phenylephrine HCl0.0310Acetaminophen2.010Dextromethorphan0.060Guaifenesin1.240Propylene glycol23.0223.02Glycerin Solution (96%)8.008.00Sorbitol Solution (70%)13.1513.15Xanthan gum0.150.15Sodium citrate dihydrate0.200.20Citric acid USP0.220.22Sodium benzoate0.100.10Saccharin sodium0.200.20Sucralose0.200.20Flavor0.001-0.60.001-0.6Color0.020.02WaterQ.S.Q.S.Aptamer0.001-1.00.001-1.0 Throat Lozenge Composition Ex. 10(Wt %)Menthol0.2882Color0.1Ascorbic Acid0.26SucroseQ.S.Liquid Glucose33.26Flavor0-0.6Aptamer0.001-1.0 Nasal Compositions Saline Nasal Spray Composition Ex. 11(Wt %)WaterQ.S.Sodium Chloride2.0Aloe0-1.0Sodium Bicarbonate0-2.0Eucalyptus Oil0-0.3Aptamer0.001-1.0 Nasal Spray Compositions Ex. 12Ex. 13Ingredient(Wt %)(Wt %)WaterQ.S.Q.S.Avicel ™ 59133Polyvinylpyrrolidone33Carbowax ™ PEG 145055Sodium phosphate, dibasic0.09750.0975Sodium phosphate, monobasic0.55250.5525Levomenthol0.0270.027Eucalyptol0.0090.009Camphor0.0090.009Benzalkonium Chloride 50%0.14710.1471SolutionBenzyl Alcohol0.350.35Disodium EDTA0.030.03Oxymetazoline HCl0.050Aptamer0.001-1.00.001-1.0 Additional Nasal Spray Compositions Ex. 14Ex. 15Ex. 16(Wt %)(Wt %)(Wt %)Pyroglutamic Acid0.350.701.00Succinic Acid1.000.700.35Zinc Acetate Dihydrate0.120.0120.12Polysorbate 800.050.050.05Carbopol 980——1.20Hydroxypropyl methyl1.20——cellulosePoloxamer 407—15.8—Sodium Saccharin—0.0250.025Sucralose0.025——Phenyl ethyl alcohol0.370.370.35Sodium chloride0.200.200.50Camphor—0.03—Menthol0.020.060.02Eucalyptol—0.02—Aromatic System0.050.380.05Sodium Hydroxide (30%)——0.10Disodium succinate1.000.50—WaterQ.S.Q.S.Q.S.Aptamer0.001-1.00.001-1.00.001-1.0 V. EXAMPLES Example 1. Aptamer Selection and Next Generation Sequence Characterization A. Selection Strategy One objective of this invention was to develop aptamers that would not just specifically bind to ICAM-1 receptors but would do so in a way that would block or inhibit the binding of virus particles to the receptor protein. The selection of aptamers against the extracellular domain of the ICAM-1 receptor alone would not necessarily be sufficient to block virus binding to the same protein as aptamers are relatively small and their blocking footprint will be limited to the epitopes that they bind to. If the epitopes that the aptamer binds to are not involved in virus binding to the ICAM-1 receptor, they will not inhibit binding of the virus particles. This objective was consciously incorporated into the selection strategy, first by including several rounds of positive selection against the exo-cellular domain of the ICAM-1 protein (SEQ ID NO: 214); secondly, by imposing a double positive selection such that aptamers would be enriched for binding to the ICAM-1 extra-cellular domain in the context of nasal cells; thirdly, by imposing counter selection against HEK293 cells that carry similar receptor proteins (ICAM-3 and ICAM-5); and fourthly, by performing selection channels against specific desirable and undesirable aptamer binding outcomes including, specific elution of bound aptamers from nasal cells with the addition of rhinovirus particles, blocking of aptamer binding to ICAM-1 cells by the pre-application of rhinovirus particles, positive selection against HEK293 cells, positive selection against the extra-cellular domain of ICAM-1, and double positive selection against the extra-cellular domain of ICAM-1 and nasal cells. Double positive selection (extra-cellular domain of ICAM-1 and nasal cells) ensures that enriched aptamers are favored that bind to the ICAM-1 receptor as it is presented on nasal cells. If selection was only performed against the extra-cellular domain of ICAM-1, it is possible that epitopes would be present that are not present in vivo. If selection was only performed against nasal cells, it is possible that aptamers would be enriched for binding targets other than ICAM-1 on the surface of such cells. The counter selection against HEK293 cells was implemented to drive enrichment of aptamers that bound to the N-terminus of the ICAM-1 extracellular domain. HEK293 cells express other members of the ICAM receptor family, ICAM-3 and ICAM-5. These receptor proteins differ in their extracellular domain from ICAM-1 predominantly at their N-terminus. The N-terminus of the ICAM-1 receptor is the region of the extra-cellular domain that rhinovirus particles bind to. Thus, this counter selection step was included to drive aptamer selection towards those aptamers that will block or inhibit rhinovirus binding to nasal cells. Finally, once the aptamer library was enriched with double positive selection against the extra-cellular domain of ICAM-1 and nasal cells, and counter selection against HEK293 cells, the enriched library was separated into aliquots and applied to several different targets, including continued double positive selection, positive selection against HEK293 cells, positive selection against the extra-cellular domain alone, selection based on rhinovirus particle elution of aptamers bound to nasal cells, and selection based on blocking aptamer binding to nasal cells through pre-treatment with rhinovirus particles. Each of these selected libraries was characterized by next generation sequencing. Aptamers that exhibit higher levels of enrichment against the double positive selection, the extracellular domain selection, and either of the rhinovirus particle enabled selection processes and lower enrichment against HEK293 alone would be desirable sequences for the blocking or inhibition of rhinovirus binding to nasal cells. B. Growth of Human Cells B.1. Human Nasal Epithelial Cells Growth Conditions Primary human nasal epithelial cells (HNepC; PromoCell, Catalog #C-21060) were grown in airway epithelial cell growth medium (PromoCell, Catalog #C-21160) at 37° C. and 5% CO2. B.2. Growth of HEK293 Cells HEK293 cells purchased from ATCC (CRL-1573) were grown in Eagle's Minimum Essential Medium (EMEM)+10% Fetal Bovine Serum (FBS) at 37° C. and 5% CO2. B.3. Human Rhinovirus A16 Suspension UV inactivated HRV16 virus particles were purchased (Zeptometrix Corporation) and stored at −80° C. until use. The concentration of the virus particles (VPs) was calculated to be 98,700 vp/mL. C. Aptamer Selection C.1. Library Preparation In the first step, a DNA library of about 1015different sequences (TriLink BioTechnologies), containing a random region of 40 nucleotides flanked by two conserved regions, forward primer recognition sequence (5′-GGGTGCATCGTTTACGC-3′; SEQ ID No 224) and a 3′ reverse primer recognition sequence (5′-CTGCTGCTGAGGAAGGATATGAG-3′ SEQ ID No 225) (seeFIG.1), was transcribed to RNA using a mixture of 2′-fluoro pyrimidines nucleotides (2F-UTP and 2F-CTP) and natural purine nucleotides. In brief, about 1.66 nmoles of single stranded DNA were amplified in 390×50 μL PCR reactions for 4 cycles using the primers Lib7_T7 Fwd primer (sequence: 5′-TAATACGACTCACTATAGGGTGCATCGTTTACGC-3′, (SEQ ID No 226) with transcription starting at the first G underlined) and Lib7_Rvs primer (sequence 5′-CTCATATCCTTCCTCAGCAGCAG-3′ SEQ ID No 227). The amplified DNA was purified using the Genejet PCR purification kit (Fisher Scientific, Catalog #K0701). This amplification of the ssDNA library created a dsDNA library with a T7 promoter, which was used as a templated to generate a modified RNA library for selection. Post DNA amplification, 52 μg of purified dsDNA was transcribed in 26×20 μL transcription reactions by using a mutant T7 polymerase (T7 R&DNA polymerase, Lucigen, Catalog #D7P9205K) polymerase and a mixture of rATP, rGTP and the modified nucleotides 2F-UTP and 2F-CTP. The NTPs were mixed together at a ratio of 3:1 modified to non-modified. Each reaction mixture contained 4 μL 5×T7 R&D polymerase, 1 μL NTP 3:1 mix, 2 μL DTT (0.1M), 0.7 μL T7 R&D polymerase, 1.2 μL inorganic pyrophosphatase, 0.5 μL Rnase inhibitor, and 10.6 μL DNA template. The reactions were incubated at 37° C. for 16 hours. The transcribed library was subjected to Dnase treatment by setting up reaction mixtures consisting of 10 μL 10× Dnase buffer, 4 μL Dnase I, 66 μL Rnase free water, and 20 μL transcription reaction. The reaction mixtures were then incubated at 37° C. for 30 min, 1 μL of 0.5 M EDTA was added and mixed, further incubated at 75° C. for 10 minutes and purified using Monarch RNA cleanup kit (New England Biolabs, Catalog #T2040L). C.2. Immobilization of ICAM-1 onto his-Pur Ni-NTA Resin Lyophilized ICAM-1 protein (50 μg Ray-Biotech, Catalog #: 228-21751-2) with a His-tag on the C-terminus region was resuspended in 100 μL of sH2O (final concentration of 0.5 μg/μL or 9.88 μM). The solution was aliquoted and stored at −20° C. until use. The protein sequence was: (SEQ ID No 228)QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQVEGGAPRANLTVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPRVLEVDTQGTVVCSLDGLFPVSEAQVHLALGDQRLNPTVTYGNDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQETLQTVTIYSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATLEVAGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYEVDHHHHHH. An aliquot of His-Pur Ni-NTA (Fisher Scientific, Catalog #PI88221) resin was transferred to a 0.6 mL tube and centrifuged at 700×g for 2 minutes. The supernatant was removed, and the resin was washed 3 times with 500 μL of PBS buffer (pH 7.4). Then, aliquots of ICAM-1 protein in 1×PBS buffer (pH 7.4) were incubated with the His-Pur Ni-NTA resin overnight at 4° C. while mixing. For selection round 1, 300 pmoles of ICAM-1 protein was immobilized onto 50 μL of resin. For subsequent rounds, 50 pmoles of ICAM-1 protein was immobilized onto 25 μL of resin. After protein immobilization, the resin was transferred to a 1 mL cartridge with a frit filter and washed with 2 mL of 1×PBS buffer. Finally, aliquots of 0.5-1 mM imidazole in 1×PBS buffer were added and incubated with the resin for 30 minutes at 4° C. to block unreacted binding sites on the resin. The resin was washed three times with 1 mL aliquots of 1×PBS buffer. For negative selections with imidazole blocked resin, aliquots of the His-Pur Ni-NTA resin were incubated with an appropriate concentration of imidazole in 1×PBS buffer for 30 minutes to block unreacted binding sites on the resin, followed by washing with 1× selection buffer. The selection buffer used for all the examples in this application was Dulbecco's PBS buffer supplemented with calcium chloride (CaCl2), 0.9 mM), magnesium chloride (MgCl2 0.49 mM), potassium chloride (KCl, 2.67 mM), potassium phosphate monobasic (KH2PO4, 1.47 mM), sodium chloride (NaCl, 137.93 mM), and sodium phosphate dibasic (Na2HPO4, 8.06 mM). C.3. Aptamer Selection Overview The aptamer selection was performed in fourteen selection rounds (“SR”), which are illustrated inFIG.2. The selection rounds 1 to 5 enrich the sequences in the aptamer library that bind to ICAM-1 immobilized onto the Ni-NTA Resin. In selection rounds 6 to 9, the aptamer library was subjected to the same ICAM-1 immobilized Ni-NTA Resin procedure and the eluted aptamers were further enriched towards sequences that bind to human nasal epithelial cells (HNepC), this is referred to as double positive selection. In selection rounds 10 to 11, counter selection against HEK293 cells and positive selections against HNepC were performed. Selection rounds 12 to 14, illustrated inFIG.3, break out to different selection conditions and are referred to as splits. Five different splits were performed: split A: nasal epithelial cells, split B: HEK293 cells, split C: ICAM-1 protein, split D: human rhinovirus A16 (HRV16) elution, and split E: HRV16 blocking. C.4. Aptamer Selection Process C.4.1 Selection Round 1 The aptamer selection round 1 was completed by performing a positive selection against ICAM-1 immobilized Ni-NTA resin. The RNA library (produced as described in section C.1) was heated to 45° C. for 10 minutes and allowed to cool to room temperature for 10 minutes. Then, the prepared aptamer library was added to 300 pmol of the ICAM-1 immobilized on Ni-NTA resin (prepared as described in section C.2) and incubated with rotation at room temperature for 30 minutes. Unbound RNA was washed off the resin with 500 μL of selection buffer (pH 7.4). The bound RNA was then eluted twice by adding aliquots of 200 μL of 6 M urea to the resin and incubating the suspension at 85° C. for 5 minutes. The recovered RNA library was collected and purified using Monarch RNA cleanup kit. The collected aptamer library was reverse transcribed following the Protoscript II Reverse Transcriptase manufacturer's protocol. The number of reverse transcription reactions varied depending on the amount of RNA going into that specific round of selection. Then, the reverse transcribed aptamer library was amplified by polymerase chain reaction (PCR) using a standard PCR protocol and the following amplification steps:Step 1: 95° C.—5 minutesStep 2: 95° C. —10 secondsStep 3: 56° C.—15 secondsStep 4: 72° C.—30 secondsRepeat steps 2 to 4 for 4 cyclesStep 5: 95° C.—10 secondsStep 6: 59° C.—15 secondsStep 7: 72° C.—30 secondsRepeat steps 5 to 7 for up to 26 cycles. The PCR amplified dsDNA aptamer library was then transcribed back into RNA and subjected to Dnase treatment using the protocols described in section C.1. C.4.2 Selection Rounds 2 to 5 Selection rounds 2 to 5 incorporate two selection strategies: negative selection against imidazole blocked Ni-NTA resin and positive selection with ICAM-1 immobilized Ni-NTA resin (seeFIG.2). The negative selection was performed to select aptamer sequences that do not bind to the imidazole blocked Ni-NTA resin (prepared as described in Section C.2). First, an aliquot of 50 μL of imidazole blocked resin was transferred to a 1 mL cartridge fitted with a 20 μm frit and washed twice with 1 mL aliquots of selection buffer. Then, the prepared RNA library from the previous selection round was heated to 45° C. for 10 minutes and allowed to cool to room temperature for 10 minutes. The RNA library was added to the cartridge and incubated at room temperature for 30 minutes with the imidazole blocked Ni-NTA resin. Following incubation, the flow through solution was collected. Then, the cartridge was washed using an aliquot of 500 μL of selection buffer and the solution was collected. The flow through solution and column wash collections were pooled together and purified with Monarch RNA cleanup kit following manufacture protocols. The RNA library that was obtained from the negative selection was then subjected to the positive selection, which selects for sequences that bind to ICAM-1 immobilized Ni-NTA resin (prepared as described in Section C.2). In brief, the RNA library was heated to 45° C. for 10 minutes and allowed to cool to room temperature for 10 minutes. Then, the RNA library was added to 50 pmoles of the ICAM-1 immobilized on Ni-NTA resin (prepared as described in Section C.2) and incubated with rotation at room temperature for 30 minutes. Unbound RNA was washed off the resin with aliquots of 500 μL of selection buffer. The number of washes varied depending on the selection round and the number of positive selections completed and was pre-determined by selection modelling. Then, the bound RNA library was eluted twice by adding aliquots of 200 μL of 6 M urea to the resin and incubating the suspension at 85° C. for 5 minutes. The eluted RNA library was collected and purified with the Monarch RNA cleanup kit, followed by reverse transcription, PCR amplification, transcription, and DNAse treatment as described in sections C.1 and C.4.1. C.4.3. Selection Rounds 6 to 9 The RNA aptamer library that was enriched from selection rounds 1 to 5 was further enriched in selection rounds 6 to 9, which utilizes two selection strategies: a positive selection with ICAM-1 immobilized Ni-NTA resin and another positive selection against human nasal epithelial cells (HNepC) that express the ICAM-1 receptor. This group of selection rounds is referred to as “double positive selection”. In selection round 8, two positive selections against HNepC were performed (i.e. “triple positive selection”). In selection rounds 6 and 7, the RNA library was resuspended in 500 μL of 1× selection buffer. The first positive selection (selecting against ICAM-1 immobilized Ni-NTA resin) started by adding the resuspended RNA to the ICAM-1 immobilized on Ni-NTA resin, followed by incubation at 37° C. for 30 minutes. The unbound RNA was discarded and the resin was washed with aliquots of 500 μL of 1× selection buffer. For the elution step, an aliquot of 200 μL of 6 M urea was added to the resin and incubated at 85° C. for 5 minutes and the elution solution was collected. The elution step was repeated and the eluants were pooled together and cleaned up using a Monarch RNA clean up kit. The second positive selection started by preparing the HNepC cells by aspirating the medium from the 6-well plate (˜3 mL) where the cells were grown, followed by washing the cells three times with 3 mL of prewarmed 1× selection buffer. A solution of 1 mL of RNA library in 1× selection buffer was immediately applied to the washed cells and incubated for 30 minutes at 37° C. and 50 revolutions per minute (rpm). After the 30 minute incubation, the supernatant containing ˜50% of the cells was collected, the cells were pelleted at 500×g for 2 minutes and washed twice with 200 μL prewarmed 1× selection buffer. The cell pellet was collected, and the bound RNA was eluted from the cells by the addition of 6 M urea, followed by incubation at 85° C. and RNA purification. The adhered cells (i.e. remaining ˜50% cells) were washed twice with 1 mL of preheated 1× selection buffer. Then, an aliquot of 1 mL of 10 mM EDTA was added and allowed to incubate with the cells at 37° C. for 15 minutes at 50 rpm. The EDTA treated cells were pelleted at 500×g for 2 minutes. Then, an aliquot of 200 μL of 6 M urea was added to the pellet and the suspension was heated to 85° C. for 5 minutes, followed by centrifugation at 13,000 rpm to recover the RNA aptamers in the supernatant. The elution step was repeated one more time, the eluants were combined, and the RNA aptamers were purified. The reverse transcription, PCR amplification, and transcription following the protocol in sections C.1 and C.4.1 was performed on the purified samples. In selection rounds 8 and 9, the EDTA lifting of the cells was removed from the protocol and the RNA bound to the cells was eluted using 6 M urea while they were still attached to the 6-well plate. Additionally, a negative selection step was included in both rounds to remove any RNA sequences that bind to the plastic of the 6-well culture plate. For the negative selection, the RNA library was resuspended in 1 mL of 1× selection buffer, followed by heating to 37° C. for at least 10 minutes. One well in a 6-well culture plate was pre-washed twice with 1 mL of 1× selection buffer. Then, the heated RNA library was added to the well and incubated at 37° C. and 50 rpm for 30 minutes. The solution in the well was collected and brought up to 1 mL volume with selection buffer. The resulting 1 mL solution of RNA library was incubated with HNepC, grown in a 6-well plate, at 37° C. at 50 rpm for 1 hour. The unbound RNA was removed from the cells and the cells were washed twice with 1 mL of 1× selection buffer (prewarmed to 37° C.). The bound RNA was eluted by adding 1 mL of 6 M urea and incubating the cells at 85° C. for 5 minutes. The elution step was repeated. The eluants were pooled together and the RNA was purified using the Monarch RNA clean up kit. The selected RNA was reverse transcribed, PCR amplified, transcribed and DNAse treated as previously described. C.4.4. Selection Rounds 10 and 11 In selection rounds 10 and 11, a negative selection against HEK293 cells was introduced (seeFIG.2). HEK293 cells do not express the ICAM-1 receptor, which allows for the counterselection of sequences that bind elsewhere on the cell surface that is not ICAM-1. The HEK293 cells were grown in a 6-well culture plate and were used at 80% confluency or greater. The cells were prepared by removing and discarding all media from the well and by washing the cells three times with 3 mL of pre-warmed 1× selection buffer. Then, the prepared RNA library was added to the cells and the library and cell solution were incubated for 1 hour at 37° C. with gentle shaking (50 rpm). After incubation, the supernatant with the unbound RNA library was removed and collected. Then, the cells were washed with 1 mL of pre-warmed 1× selection buffer and the solution was also collected. The collected RNA solutions were combined and purified with a Monarch RNA Cleanup Kit. This purified RNA library was then subjected to a positive selection round against HNepC, following the same protocol as described on selection rounds 8 and 9 (see section C.4.3). Two positive selections were performed in selection round 10, while a single positive selection was completed in selection round 11. C.4.5. Selection Rounds 12 to 14: Nasal Epithelial Cell Split In the nasal epithelial cell split of selection rounds 12 to 14 (seeFIG.3), the RNA library collected from selection round 11 was further subjected to the negative selection against the HEK293 cells followed by the positive selection with the HNepC, using the protocol described in section C.4.4. C.4.6. Selection Rounds 12 to 14: HEK293 Cell Split In the HEK293 cell split of selection rounds 12 to 14 (seeFIG.3), the RNA library collected from selection round 11 was enriched towards sequences that bind to HEK293 cells. The protocol for this selection round followed the procedure of selection rounds 10 to 11 described in section C.4.4, excluding the selection with the HNepC. C.4.7. Selection Rounds 12 to 14: ICAM-1 Protein Split In the ICAM-1 split of selection rounds 12 to 14 (seeFIG.3), the RNA library collected from selection round 11 was enriched towards sequences that bind to ICAM-1 immobilized onto the Ni-NTA Resin. The protocol for this selection round followed the procedure of selection round 1 described in sections C.1 and C.4.1. C.4.8. Selection Rounds 12 to 13: Human Rhinovirus A16 (HRV16) Elution Split The HRV16 elution split only occurred during selection rounds 12 and 13 (seeFIG.3). The RNA library collected in selection round 11 was further enriched by a negative selection against HEK293 cells followed by a positive selection on HNepC using Human Rhinovirus A16 (HRV16) particles to elute the aptamer library. The negative selection on HEK293 cells followed the same protocol of selection rounds 10 and 11 described in section C.4.4 but excluding the selection against the HNepC. Following the negative selection with the HEK293 cells, the collected RNA was diluted in 1× selection buffer and heated to 37° C. for 15 minutes. The HNepC cells were washed three times with 1 mL of prewarmed selection buffer and the heated RNA library was added to the cells and incubated for 1 hour at 37° C. and 50 rpm. After incubation, the unbound RNA was removed and discarded. The recovered cells were washed ten times with 1 mL of preheated 1× selection buffer. Then, a suspension of 50% (v/v) virus particles (VPs) (see Section B.3) in 1× selection buffer were mixed with the cells and incubated for 1 hour at 37° C. with 50 rpm mixing. The supernatant was collected, and the RNA was purified and reverse transcribed following the protocol described in sections C.1 and C.4.1. C.4.9. Selection Rounds 12 and 13: HRV16 Blocking Split The HRV16 blocking split was performed during selection rounds 12 and 13 (seeFIG.3). The RNA library of selection round 11 was further enriched by a negative selection against HEK293 cells followed by a positive selection on HNepC with HRV16 bound to the ICAM-1 receptor before exposing the cells to the RNA library. The HEK293 negative selection followed the same protocol of selection rounds 10 and 11 described in section C.4.4, excluding the selection with the HNepC. Following the negative selection on the HEK293 cells, a suspension of 50% (v/v) virus particles (VPs) in 1× selection buffer was prepared. Then, the suspension was heated to 37° C. for 15 minutes and mixed with prewashed HNepC cells, followed by incubation for 1 hour at 37° C. and 50 rpm. After incubation, all unbound VPs were removed and discarded. Then, the RNA library recovered from the negative selection was resuspended in 1× selection buffer, added to the cells, and incubated at 37° C. for 1 hour. The supernatant containing the unbound RNA was collected, purified and reverse transcribed following the protocols described in sections C.1 and C.4.1. D. Aptamers Sequencing After 14 selection rounds, the aptamer libraries were sequenced. In summary, the selection libraries from rounds 10 to 14 were prepared for next generation sequencing (NGS) through a two-step PCR process. In the first step, a different hex code (6 base sequence) and a portion of a universal sequencing primer was added to the 5′ end of each aptamer library. In the second step, complete universal sequencing primers were added to both ends. After the second PCR step, the libraries were purified through acrylamide electrophoresis and balanced for relative quantity. These libraries were then pooled and sent to the Hospital for Sick Children in Toronto for NGS with an Illumina HiSeq 2500 instrument. The sequencing data was tabulated and analyzed. A total of 16,116,086 sequences were analyzed and each library contained more than 200,000 sequences. The sequences from selection round 14 (nasal epithelial cell split) were sorted by copy number and named in descending order with the highest copy number sequence being named Nas.R-1. These top sequences are listed in Table 3. The copy numbers of the top sequences of selection round 14 were determined on the libraries obtained from the other selection rounds. Finally, the frequency was computed for each sequence by dividing observed copy number by the total number of sequences observed in the particular selection library. Enrichment trajectories of the top 20 sequences in terms of frequency across different selection rounds were plotted (seeFIG.4). During the selection, these sequences were enriching at a similar rate. Example 2. Aptamer Binding Specificity It was desired to identify aptamer sequences that bind specifically to the ICAM-1 receptor and block the ability of the rhinovirus from infecting human nasal epithelial cells. The previous section, Example 1, detailed the protocol on the selection process of determining sequences that enriched in the presence ICAM-1. This section will highlight the protocols that were used to determine the sequences discovered in Example 1 that have the highest affinity and specificity towards the ICAM-1 receptor target. Multiple strategies were implemented to determine the top sequences from selection process for RNA aptamers that bind specifically and with high affinity towards human epithelial cells (HNepC), but not towards HEK293 cells that do not express the ICAM-1 target. The first protocol included exposing HNepC and HEK293 cells to some of the selected aptamer sequences, followed by incubation, elution, and quantification of the concentration of aptamers that bound to each cell type. Another strategy implemented included the visualization and identification of fluorescently labeled RNA aptamers that bind to HNepC, but do not visually bind to HEK293 cells. A final strategy included immobilizing the top RNA aptamer sequences, followed by flowing the exo-cellular domain of the ICAM-1 protein and other various proteins across the aptamer and using plasmon resonance to determine binding affinity. The following section describes in detail the strategies that are summarized above. A. Detecting Binding Specificity and Affinity Via qPCR A.1. Synthesis of Aptamer RNA Sequences DNA oligos that corresponded to the RNA aptamer sense and antisense sequences plus the T7 RNA polymerase promoter were purchased (Integrated DNA Technologies). Each of the oligos were mixed at equimolar concentrations in 10 mM Tris buffer (pH 8.3) containing 50 mM KCl and 1.5 mM MgCl2, followed by incubation at 95° C. for 5 minutes. Then, the modified RNA aptamers were synthesized by transcription of the dsDNA template, followed by DNAse treatment, and purification as described in Example 1 Sections C.1 and C.4.1. A.2. RNA Aptamers, HNepC and Hek293 Cell Preparation The modified RNA aptamers were dissolved at a concentration of 28.2 nM in 1× selection buffer. HNepC or HEK293 cells were grown in a well of a 24-well plate at densities ranging from 70-75% (HNepC) or 90-95% (HEK293 cells) following the protocol outlined in Example 1 Sections B.1 and B.2. A.3. qPCR Analysis Procedure For each sample, two 20 μL qPCR reactions were prepared using the Luna qPCR universal mastermix (New England Biolabs, Catalog #M3003L), 0.2 μM of each primer (forward primer: 5′-TAATACGACTCACTATAGGGTGCATCGTTTACGC-3′ (SEQ ID No 226), reverse primer: 5′-CTCATATCCTTCCTCAGCAGCAG-3″ (SEQ ID No 227)), and 5 μL of the cDNA sample. qPCR reactions containing known amounts of the sense DNA template were also prepared. The PCR reactions were performed using the following conditions:Step 1: 95° C. for 3 minutesStep 2: 95° C. for 15 secondsStep 3: 56° C. for 15 secondsStep 4: 60° C. for 30 secondsSteps 2 to 4 were repeated for 40 cycles. The Ct values of the binding assay samples were compared to the Ct values of the known amounts of DNA samples to determine the amount of RNA that bound to the cells. A.4. Human Nasal Epithelial and HEK293 Aptamer Binding Assay Six of the top aptamer sequences (Nas.R-1, Nas.R-2, Nas.R-4, Nas.R-5, Nas.R-7 and Nas.R-8) that were identified in the selection process (Example 1) were tested for their binding specificity and affinity towards HNepC or HEK293 cells. The RNA aptamers, HNepC, and HEK293 cells were prepared as described in Section A.2. The aptamers were incubated with the HNepC for 1 hour at 37° C. and 5% CO2 with gentle shaking every 15 minutes. The unbound RNA was removed and the cells were washed four times with 150 μL of 1× selection buffer prewarmed at 37° C. To elute the bound RNA aptamers, aliquots of 200 μL of 6 M urea were added to the cells, followed by incubation at 85° C. for 5 minutes. The elution step was repeated, the eluants were combined, and the RNA aptamers were purified using a Monarch RNA clean up kit following the manufacture's protocol. Each RNA sample was reverse transcribed in a 20 μL M-MμLV (New England Biolabs, M0253L) reverse transcriptase reaction following the manufacturer's protocol. The reverse transcribed sequences were quantified using qPCR analysis following the protocol described in section A.3. The same procedure was followed for the HEK293 cells. The results are illustrated inFIG.5. For aptamers Nas.R-2, Nas.R-4, Nas.R-5, Nas.R-7, and Nas.R-8, the binding affinity towards HNepC was higher than for HEK293 cells. B.1. Visualizing Aptamer Bound to ICAM-1 on HNepC and HEK293 by Fluorescence B.1.1. Preparation of Fluorescently Tagged RNA Aptamers Modified RNA aptamer Nas.R-4 with a spacer (AAACAAACAAAC; SEQ ID No. 235) and a sense binding sequence (GUAUGGCGGUCUCCAACAGG; SEQ ID No 236) at the 3′ end was synthesized, as previously described in section A.1. (SEQ ID No 229)5′-GGGUGCAUCGUUUACGCGCAACAUAAAAAUUUAAAGUGCUCAGUUGUCAAUCUAUGACUGCUGCUGAGGAAGGAUAUGAGAAACAAACAAACGUAUGGCGGUCUCCAACAGG-3′ The sense binding sequence was added to anneal to a 6-FAM labelled fluorescent antisense oligonucleotide. Before each binding assay, the NAS-FAM antisense oligo (5′ 6-FAM/CCTGTT GGAGACCGCCATAC-3′ (SEQ ID No 230)) was mixed with the modified RNA aptamer at equimolar concentrations in 1× selection buffer, followed by incubation at 37° C. for 15 minutes. B.1.2. HNepC and Hek293 Cell Preparation HNepC and HEK293 cells were prepared following the procedure outlined in Section A.2 but were seeded at densities of about 50% one to two days before the assay, onto 12 mm glass coverslips (Fisher Scientific, Catalog #12-545-82) submersed in medium in wells of 24-well plates. B.1.3. Binding of the Fluorescently Labelled Aptamers to Cells The medium was aspirated from the HNepC culture. Then, an aliquot of 150 μL of the aptamer/NAS-FAM antisense mixture, prepared as described in Section B.1, was applied to the cells, followed by incubation for 15 minutes at 37° C. and 5% CO2and with gentle agitation every 5 minutes. The unbound RNA aptamer was aspirated and the HNepC were washed three times with 150 μL of 1× selection buffer prewarmed at 37° C. The coverslip was removed and submersed into a drop of selection buffer on a glass microscope slide. Fluorescence of the cells was monitored for up to about 1 hour using a Nikon inverted fluorescent microscope and a FITC fluorescence filter. Images (seeFIG.6) were taken using a Nikon D7500 camera at 1/30 sec exposure. The same process was followed using HEK293 cells (seeFIGS.6C and6D). As illustrated inFIGS.6A and6B, significant fluorescence was observed when the labelled aptamers were incubated with HNepC, while no fluorescence was detected with HEK293 cells, confirming the stronger binding affinity of the aptamers towards surface markers on the surface of HNepC (e.g. ICAM-1) compared to markers on HEK293 cells. B.2. Visualizing Virus Inhibition on H1-HeLa Cells by a Viral Inhibition Assay Using Fluorescence DNA aptamers Nas.R-2 and Nas.R-8 that bind to ICAM-1 were tested in a viral inhibition assay compared to a negative control aptamer to demonstrate their efficacy in blocking Rhinovirus infection (FIG.7). B.2.1. Aptamer Incubation and Viral Infection H1-HeLa cells in RPMI+2% Fetal Bovine Serum were seeded onto 24-well plates at 1×105cells/mL and 1.0 mL/well. The seed medium was aspirated, and 0.5 mL of each aptamer at 40 μM was added to the host cell wells. The host cells were incubated for 30±5 minutes at 33±2° C. with 5±3% CO2. 0.5 mL of Rhinovirus Type 14 at 103TCID50/well was added to the host cell wells without aspiration. The host cell wells were incubated 120±10 minutes at 33±2° C. with 5±3% CO2. The host cells wells were aspirated and refed with 1.0 mL of each aptamer in cell culture medium and returned to incubation at 33±2° C. with 5±3% CO2. After 18±1 hours, the cells were refed with 1.0 mL of a 2× concentration of aptamer in cell culture medium and incubated for 12±1 hours at 33±2° C. with 5±3% CO2. B.2.2. Quantification of Viral Inhibition After the total incubation period the host cell plates were frozen at −60 to −90° C. overnight and then thawed at ambient temperature. The contents of each well were individually harvested and centrifuged at 2,000 rpm for 10 minutes. The supernatant of each harvest was collected, serially diluted in cell culture medium and inoculated onto fresh H1-HeLa cells to determine the quantity of infectious virus using a Tissue Culture Infectious Does 50% (TCID50) assay. The average yield of virus from control wells with cells treated with cell culture medium only were used to calculate the viral inhibitory activity (Log10reduction) by each aptamer. TABLE 1B.2.3.: ResultsAptamerLog Viral Titer ReductionReduction (%)Nas.R-22.0899.2Nas.R-81.3395.3 FIG.7shows the result as images. Red labelled cells can be seen in the fluorescent image, if the TRITC-labelled virus was able to infect the cells. The position of the cells in the fluorescent images was marked with an arrow based on the corresponding position in the brightfield image. No infection can be seen using the Nas.R-2 aptamer (FIG.7Afluorescent image;FIG.7Bbrightfield image). Nearly no infection can be seen using the Nas.R-8 aptamer (FIG.7Cfluorescent image;FIG.7Dbrightfield image). The cells were infected and appear red (FIG.7E) using the negative control aptamer (FIG.7Fbrightfield image) andFIGS.7Gand H show the control cells which were not infected with the virus (FIG.7Gfluorescent imageFIG.7Hbrightfield image). C. Determination of Binding Affinity by Surface Plasmon Resonance (SPR) C.1. Immobilization of RNA Aptamers in Gold Chips RNA aptamers Nas.R-1, Nas.R-2, Nas.R-4, Nas.R-8, and a negative control were immobilized on the surface of gold chips. In brief, the RNA aptamer was dissolved in 1×PBS buffer supplemented with 10 mM EDTA. Then, an aliquot of 20 μL of this solution was added to 3.375 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in a 1.5 mL tube. Next, an aliquot of 13.5 μL of cystamine-imidazole solution was added to the RNA aptamer and EDC solution, followed by mixing and centrifugation. The supernatant was removed and an additional aliquot of 54 μL of 100 μM imidazole (pH 6.0) was added. The solution was incubated at room temperature overnight. Finally, an RNA cleanup column was used to remove unincorporated cystamine and imidazole. After conjugation of the cystamine moeities to phosphoramidate bonds at the 5′ phosphate group, the aptamer was immobilized on a gold chip by depositing an aliquot of 10 nL of aptamer solution at a concentration of 10 μM onto the surface of the chip. The gold reduces the cystamine to a pair of thiols and then catalyzes the reduction reaction that results in the covalent bond between the gold surface and the thiol groups of the modified aptamers. C.2. Surface Plasmon Resonance (SPR) Procedure Solutions of 200 μL of ICAM-1 protein or human serum albumin were flown over the gold chip at a concentration of 250 nM and a flow rate of 50 μL/min using an Openplex Surface Plasmon Resonance System (Horiba, Kyoto, Kyoto, Japan). Thus, the association phase lasted for 4 minutes after injection and was immediately followed by the disassociation phase (seeFIGS.8and9). The total resonance of the negative control aptamer was subtracted from the total resonance observed for each of the candidate aptamers. The result corresponds to the resonance contribution due to the binding of the protein to the aptamer. The kd (koff) value was calculated by fitting the curve to equation [1]: x′˜−kd*x [1] wherein x is the resonance due to binding and x′ is the derivative of this value at each time point captured on the disassociation curve. The kd value is then used to determine the ka value by using equation [2]: x′˜ka*Rmax*c−(ka*c+kd)*x [2] where Rmax is the maximum resonance due to binding observed, and c is the concentration of the injectant. Finally, the dissociation equilibrium constant kD was calculated as the ratio of kd over ka (see Table 2). The low nanomolar kD values obtained for the different aptamers confirm the strong binding affinity of such molecules towards ICAM-1 and validate the aptamer selection process described in Example 1. As used herein, “kd” refers to the dissociation rate, “ka” refers to the association rate, and “kD” refers to the dissociation equilibrium constant. TABLE 2Binding Coefficients of Nas.R-1, Nas.R-2, Nas.R-4,and Nas.R-8 on 250 nM Exogenous ICAM-1.AptamerNas.R-1Nas.R-2Nas.R-4Nas.R-8kd, [1/s]1.27E−021.42E−022.25E−022.63E−03ka, [1/M · s]1.97E+052.02E+055.08E+059.27E+04kD, [M]6.44E−087.02E−084.43E−082.84E−08 D. Aptamer Binding Specificity As described in Example 1, in the selection process, a counter selection was performed against with HEK293 cells. HEK293 cells do not express the ICAM-1 receptor, but they do express the related receptor proteins ICAM-3 and ICAM-5. For certain sequences, for instance Nas.R-2 (SEQ ID NO: 2), substantially higher affinity to nasal cells compared to HEK293 cells was observed. Not wishing to be bound by theory, given the presence of ICAM-5 and ICAM-3 on the HEK293 cells, it stands to reason that the selected aptamers are binding to epitopes from regions of the ICAM-1 receptor protein that are different in sequence from those of the ICAM-5 or ICAM-3 receptors.FIG.10illustrates the sequence alignment of ICAM-1, ICAM-3, and ICAM-5 and the regions that are likely to give rise to ICAM-1 specific binding are highlighted. Rhinoviruses bind to the N-terminal Ig-like C2-type 1 domain of ICAM-1 receptor. Given the selection strategy, including elution with human rhinovirus particles, and counter selection against HEK293 cells, it is clear to one trained in the art that the mature selected aptamer library would be enriched in aptamer sequences that not only bind to the extracellular domain of the ICAM-1 receptor but do so specifically to the Ig-like C2-type 1 domain at the N-terminus. FIG.11illustrates a fold comparison in aptamer frequency over the final three selection rounds applied in the aptamer selection process. The data is presented as the frequency of the individual aptamer sequence as selected against nasal cells divided by the frequency of the same sequence observed in selection against HEK293 cells. For aptamers Nas.R-2, Nas.R-1, and Nas.R-17, the sequences were not observed in the selections against HEK293 cells (the legend refers to the selection round). That is, at least in terms of the subsample of sequences observed in the next generation sequencing process, these sequences were observed at high frequency in selection round 14 against the nasal cells but not observed at all in the selections against HEK293 cells. Not wishing to be bound by theory, aptamers that did not exhibit enrichment in frequency when selected on nasal cells compared to HEK293 cells should be considered as aptamers that likely would not block HRV binding.FIG.12depicts sequences that in selection round 14 all exhibited higher enrichment levels with HEK293 positive selection than with positive selection against nasal cells. These aptamers would be expected to bind to regions of the ICAM-1 receptor that are not in the N-terminus and that have considerable sequence identity with regions of ICAM-3 or ICAM-5. Example 3. Analysis of Sequences Similarity Alignment of SEQ ID NO: 1 to SEQ ID NO: 100 was performed using the software Align X, a component of Vector NTI Advanced 11.5.4 by Invitrogen. Several groups of sequences have at least 90%, at least 70%, or at least 50% nucleotide sequence identity as illustrated in the alignments ofFIGS.13,14, and15. In these alignments, only the central variable region of the aptamers was included for simplicity. Thus, oligonucleotides with at least 50%, at least 70%, or at least 90% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 200 are included as part of the current invention. Example 4. Motif Analysis and Predicted Secondary Structure Aptamers bind to target molecules on the basis of the lowest free-energy shape that they form. The lowest free energy shape is a function of homology between regions within the single stranded sequence. These regions of homology fold back onto each other and thus create the secondary and tertiary shape of the aptamer that is crucial to enable binding. We characterized the core characteristics of these aptamers through a combined analysis of conserved motif sequences and their effect on the predicted structure of the whole aptamer. A motif in this context is defined as a contiguous sequence of nucleotides of a defined length. For this example, we considered each possible overlapping six nucleotide motif within the random region of each aptamer characterized. The frequency of motifs of six nucleotides from the random regions of the top aptamers (Nas.R-1, Nas.R-2, Nas.R-4, and Nas.R-8) within all the sequences of selection round 14—Nasal Epithelial Cell Split library was determined. Then, the average motif frequency was subtracted from the frequency of each motif and this value was divided by the standard deviation of all the motifs frequencies in that selection round, resulting in a Z value for every motif. It stands to reason that sequences containing high frequency motifs also bind to the target molecule and are part of the present invention. The prediction of the secondary structures of the aptamers was performed with The Vienna RNA Websuite. (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi. Gruber A R, Lorenz R, Bernhart S H, Neuböck R, Hofacker IL; Nucleic Acids Research, Volume 36, Issue suppl_2, 1 Jul. 2008, Pages W70-W74, DOI: 10.1093/nar/gkn188) and the motifs are highlighted within these structures. A. Analysis of the Role of Conserved Motifs on Structure within the Aptamer Nas.R-1: The results of motif analysis are presented inFIG.16. The overlapping six nucleotide motifs comprising the random region of the aptamer are provided consecutively along the x axis in this figure. The y axis provides a statistical significance (Z value) for each motif in the library. The Z value was computed as the observed frequency of this motif in the library minus the average of the frequency for all motifs in the library and this subtractant was divided by the standard deviation of all motifs in the library to provide the Z value. Thus, a Z value of 2 represents a frequency of this motif in the library that is two standard deviations greater than the average value for all motifs. InFIG.16, it is clear that the sequences AAACAAAAAGA (see, e.g., SEQ ID NO: 201) and UAAAAAUCA (see, e.g., SEQ ID NO: 202) were conserved at a level that represented more than two standard deviations from the average. The lowest free energy predicted structure of the Nas.R-1 aptamer and the consensus sequences are shown inFIG.17. SEQ ID NO: 201:5′-AAACAAAAAGA-3′SEQ ID NO: 202:5′-UAAAAAUCA-3′ Sequences containing any of these motifs are also expected to bind to ICAM-1 and are included as embodiments of the present invention. The conclusions arrived at within this example regarding conserved motifs in the RNA sequence would apply to the DNA sequence as well. Thus, any sequences containing the corresponding deoxyribonucleotide motif SEQ ID NO: 203:5′-AAACAAAAAGA-3′SEQ ID NO: 204:5′-TAAAAATCA-3′ are also included as embodiments. B. Analysis of the Role of Conserved Motifs on Structure within the Aptamer Nas.R-4: The analysis of the role of conserved motifs on structure within aptamer Nas.R-4 was performed in a manner identical to that described for Nas.R-1.FIG.18provides a summary of the motif analysis for aptamer Nas.R-4. There is a thirteen-nucleotide motif present at a frequency that was more than two standard deviations from the overall average motif frequency in the selected libraries, SEQ ID NO 205:5′-AUAAAAAUUUAAA-3′. Sequences containing this motif are also expected to bind to ICAM-1 and are included as embodiments of the present invention. Any sequences containing the corresponding deoxyribonucleotide motif: SEQ ID NO 206:5′-ATAAAAATTTAAA-3′. are also expected to bind to ICAM-1 and are included as embodiments of the present invention. C. Analysis of the Role of Conserved Motifs on Structure within the Aptamer Nas.R-8: The analysis of the role of conserved motifs on structure within aptamer Nas.R-8 was performed in a manner identical to that described for Nas.R-1 and Nas.R-4.FIG.19provides a summary of the motif analysis for aptamer Nas.R-8. There is a twelve-nucleotide motif present at a frequency that was more than two standard deviations from the overall average motif frequency in the selected libraries, SEQ ID NO: 207:5′-GUAAAAAUUAAA-3′ Sequences containing this motif are also expected to bind to ICAM-1 and are included as embodiments of the present invention. Furthermore, any sequences containing the corresponding deoxyribonucleotide motif: SEQ ID NO 208:5′-GTAAAAATTAAA-3′ are also expected to bind to ICAM-1 and are included as embodiments. D. Analysis of Common Motifs within Aptamer Library: A search for common motifs within the top 100 sequences in terms of frequency was performed (seeFIG.20). The lead motifs identified in terms of significant deviation from random distribution were SEQ ID NO: 209 and SEQ IP NO: 210. SEQ ID NO: 209:5′-GUAAAAAAA-3′SEQ ID NO: 210:5′-UNAGCANUUU-3′ Oligonucleotides comprising the motifs SEQ ID NO: 209, SEQ ID NO: 210, or both are included as an embodiment of the current invention. Similarly, any sequences containing the corresponding deoxyribonucleotide motifs SEQ ID NO: 211:5′-GTAAAAAAA-3′SEQ ID NO: 212:5′-TNAGCANTTT-3′ are also expected to bind to ICAM-1 and are included as embodiments of the present invention. TABLE 3List of top sequences from selectionexperiment. All the pyrimidine nucleotidesare fluorinated at the 2′ position of thepentose group.SEQIDNONameSequence1Nas.R-1GGGUGCAUCGUUUACGCGAUUAGUCUGAUAAACAAAAAGAUUUCGCUAAAAAUCAAUCUGCUGCUGAGGAAGGAUAUGAG2Nas.R-2GGGUGCAUCGUUUACGCAGAUAGCAGCAGGAAUCAAGCGGUAGGAGUCUAGCAGAAGCUGCUGCUGAGGAAGGAUAUGAG3Nas.R-3GGGUGCAUCGUUUACGCAUUUUCGUUUUAUUUCAGUUUAAUUGCGUUUAGUAUCUGGCUGCUGCUGAGGAAGGAUAUGAG4Nas.R-4GGGUGCAUCGUUUACGCGCAACAUAAAAAUUUAAAGUGCUCAGUUGUCAAUCUAUGACUGCUGCUGAGGAAGGAUAUGAG5Nas.R-5GGGUGCAUCGUUUACGCGUAAAUGGUCCGCUAUUAAAAGAAAAGAAUGAAGUCUCAGCUGCUGCUGAGGAAGGAUAUGAG6Nas.R-6GGGUGCAUCGUUUACGCUAUUUUCAUUUGUUUUUUUAAUUUACUAGUGUAAACAAUCCUGCUGCUGAGGAAGGAUAUGAG7Nas.R-7GGGUGCAUCGUUUACGCGUAAAUAAGUAGAUAAAGUGGCAGUUUGUUUUCCUUGGAACUGCUGCUGAGGAAGGAUAUGAG8Nas.R-8GGGUGCAUCGUUUACGCGUAAAAAUUAAAGAGAUUAAGGUCCUUAAGCAGUUUUGUCCUGCUGCUGAGGAAGGAUAUGAG9Nas.R-9GGGUGCAUCGUUUACGCGUAAAAAAAUCAAAACUUCAGCAAAUUAUUUAUCAACGUCCUGCUGCUGAGGAAGGAUAUGAG10Nas.R-10GGGUGCAUCGUUUACGCGUAAAAUAAAUUAAAAAGAACUUCUUCAGCAAUCAAUAUCCUGCUGCUGAGGAAGGAUAUGAG11Nas.R-11GGGUGCAUCGUUUACGCGUAAAUAAAAAUGAAAAAUUGUCUCUCAGCUUUCAAAGUCCUGCUGCUGAGGAAGGAUAUGAG12Nas.R-12GGGUGCAUCGUUUACGCGUAAAAAAAAAAUAUCUUCGGAGAAUUCAGCAAUUUUAUCCUGCUGCUGAGGAAGGAUAUGAG13Nas.R-13GGGUGCAUCGUUUACGCGUAAAAAUUUUCAUCUCAGCAAUUAAAUCCAAAGAAUCCACUGCUGCUGAGGAAGGAUAUGAG14Nas.R-14GGGUGCAUCGUUUACGCGUAAAAUAUAUCAGCAAAGUAGUUUAAGCCUCCUCAGUUUCUGCUGCUGAGGAAGGAUAUGAG15Nas.R-15GGGUGCAUCGUUUACGCGUAAAUUAUGAAAAAUACAGCAAGGAUUUAACCUCAGUUUCUGCUGCUGAGGAAGGAUAUGAG16Nas.R-16GGGUGCAUCGUUUACGCGUAAAAUAAAUAAAUCUUCAAAGUACAGACCUCGAUUUUUCUGCUGCUGAGGAAGGAUAUGAG17Nas.R-17GGGUGCAUCGUUUACGCUUAUAGGUAUUAGACAUUUUCAAUUAAAGUGAAUUAGUGUCUGCUGCUGAGGAAGGAUAUGAG18Nas.R-18GGGUGCAUCGUUUACGCGUAAAAUGUGACAGCAGGAUAAUAAAAUAAGUACUCAGUACUGCUGCUGAGGAAGGAUAUGAG19Nas.R-19GGGUGCAUCGUUUACGCGUAAUUAAGAAAAAUAAAAGUACUCUGCAGUUUUUAUCCACUGCUGCUGAGGAAGGAUAUGAG20Nas.R-20GGGUGCAUCGUUUACGCGUAAAAAUAAAAUUUUCCCAGACCAGUUAUCUGCCUUAAACUGCUGCUGAGGAAGGAUAUGAG21Nas.R-21GGGUGCAUCGUUUACGCGUAAAGAAAAAAAUCAGCUUUUAGUCGCCUUCCAUUUUGACUGCUGCUGAGGAAGGAUAUGAG22Nas.R-22GGGUGCAUCGUUUACGCGUAAAUAAAUAAUCAAAAUUACACUCAGUGGCAAUUUCCUCUGCUGCUGAGGAAGGAUAUGAG23Nas.R-23GGGUGCAUCGUUUACGCGUAAAAUACAGGAUACGACAAUAACUCAGCAGAUUUUAUCCUGCUGCUGAGGAAGGAUAUGAG24Nas.R-24GGGUGCAUCGUUUACGCGUUAAAAAUUGUGCACUGAGAUGACGCAGCAUUAACUACACUGCUGCUGAGGAAGGAUAUGAG25Nas.R-25GGGUGCAUCGUUUACGCGUAAAUAAAAAUUAAUCAGCAAUUUUCCACUCAGUUGUACCUGCUGCUGAGGAAGGAUAUGAG26Nas.R-26GGGUGCAUCGUUUACGCGUAAAAAUAAAAAAUCUCGAUCACUGCAGUUUUAUUCCGGCUGCUGCUGAGGAAGGAUAUGAG27Nas.R-27GGGUGCAUCGUUUACGCGUAAACAAAUAUCGAUUAAAAUAAAAUCUCAGCAAGAAUCCUGCUGCUGAGGAAGGAUAUGAG28Nas.R-28GGGUGCAUCGUUUACGCGUAAAAUAAAUAAAAUUAUCCCAGGAGCAAAUUUUCUUCGCUGCUGCUGAGGAAGGAUAUGAG29Nas.R-29GGGUGCAUCGUUUACGCGUAGAAGAAUUAAUAGUGGACAUAUCAAUAGCAGUUUAUCCUGCUGCUGAGGAAGGAUAUGAG30Nas.R-30GGGUGCAUCGUUUACGCGUAAACAUAUUCAGCAGUUAAAAUUUAGUAGGUUCAGUAGCUGCUGCUGAGGAAGGAUAUGAG31Nas.R-31GGGUGCAUCGUUUACGCGUAAAAAAGAUAAAACUUAGUUGCAGAAUUUGCCUUCAUUCUGCUGCUGAGGAAGGAUAUGAG32Nas.R-32GGGUGCAUCGUUUACGCGUAAAAAGUUUGAUGGAAGCAGAUUAGUUUAGUCAAAUUUCUGCUGCUGAGGAAGGAUAUGAG33Nas.R-33GGGUGCAUCGUUUACGCGUAAAAUGAAAUAAGGAAUCCUUCAGCAGUAUUUAUCCUUCUGCUGCUGAGGAAGGAUAUGAG34Nas.R-34GGGUGCAUCGUUUACGCGUAAAGAAUAAAAAUGACAAAAUUCUCAGCUUUUGUCAACCUGCUGCUGAGGAAGGAUAUGAG35Nas.R-35GGGUGCAUCGUUUACGCGUAAAAAAUGAAAUGAAAAAAUUCUCAGCUGUCUAUCUUCCUGCUGCUGAGGAAGGAUAUGAG36Nas.R-36GGGUGCAUCGUUUACGCGUAAAUAAGUAAAAAACUCAGUUUUCAGUUAAGUAUCCAACUGCUGCUGAGGAAGGAUAUGAG37Nas.R-37GGGUGCAUCGUUUACGCGUAAAUUUCAGCAGAGUAAUAAUAACACUUCUUCAGUUUGCUGCUGCUGAGGAAGGAUAUGAG38Nas.R-38GGGUGCAUCGUUUACGCGUAAAAUUAAGAAGUAUUAUCAGUUAGCUUUUUCUUCCAACUGCUGCUGAGGAAGGAUAUGAG39Nas.R-39GGGUGCAUCGUUUACGCGUAAAAUAAAAAGUUUUCCUAUCAGCAAACUCACAAAUUCCUGCUGCUGAGGAAGGAUAUGAG40Nas.R-40GGGUGCAUCGUUUACGCGUAAAAUGAAAUGUAAAAGAAUUGAACUUGGCAGAUUUUCCUGCUGCUGAGGAAGGAUAUGAG41Nas.R-41GGGUGCAUCGUUUACGCGUAAAUUAAAGUAGCAGUAAUUUCAGCAGUUUUUACCUCUCUGCUGCUGAGGAAGGAUAUGAG42Nas.R-42GGGUGCAUCGUUUACGCGUAAAUAAAGGAUAAAAUAAUUUCAGGGCAGUUUCUCAUCCUGCUGCUGAGGAAGGAUAUGAG43Nas.R-43GGGUGCAUCGUUUACGCAGGAUCGUUUUAAGUAAAAUAAAAGAUUUCCUUGGUAAUCCUGCUGCUGAGGAAGGAUAUGAG44Nas.R-44GGGUGCAUCGUUUACGCGUAAAAUAAAGAUCAAUUAAAGGCUUUGAUCGAUUUUCCUCUGCUGCUGAGGAAGGAUAUGAG45Nas.R-45GGGUGCAUCGUUUACGCGUAAAAAUUAGAGAUUAAAAUAGUUCCUUUCAGUUUUGUCCUGCUGCUGAGGAAGGAUAUGAG46Nas.R-46GGGUGCAUCGUUUACGCGUAAAAUUGACAAUGUGAAAAGCAGACAGCAAAUAUUCCUCUGCUGCUGAGGAAGGAUAUGAG47Nas.R-47GGGUGCAUCGUUUACGCGUAAAUAACCAGUUAUACAGAAAGAUCUCAGCAAUUUAUCCUGCUGCUGAGGAAGGAUAUGAG48Nas.R-48GGGUGCAUCGUUUACGCUUACAGAAGGAUUGCACCACAUGCGUACUCGAUGAAACACCUGCUGCUGAGGAAGGAUAUGAG49Nas.R-49GGGUGCAUCGUUUACGCGUAAAAUAAUAAUUAAACUCAGCAAAUUCAAUCCAACUUUCUGCUGCUGAGGAAGGAUAUGAG50Nas.R-50GGGUGCAUCGUUUACGCGUAAACAAGAAUAAAUUCAGCAGUGGUUUUGAUCCUUUGACUGCUGCUGAGGAAGGAUAUGAG51Nas.R-51GGGUGCAUCGUUUACGCGUAAAUUAAUCAGAUUGAACAAAAGUUUUCCCUCAGUUUUCUGCUGCUGAGGAAGGAUAUGAG52Nas.R-52GGGUGCAUCGUUUACGCGUAAAGAAAAACAUCAGAGCAGUUAUAAUAGUCCUUUUUCCUGCUGCUGAGGAAGGAUAUGAG53Nas.R-53GGGUGCAUCGUUUACGCGUAAAGAAAAUAAACUUGAUCAAACUUAGCAGUUUUUAUCCUGCUGCUGAGGAAGGAUAUGAG54Nas.R-54GGGUGCAUCGUUUACGCAUUUUCGUUAUAUUUCUGGUUUUUAUGCGUGAGAAUCCUGCUGCUGCUGAGGAAGGAUAUGAG55Nas.R-55GGGUGCAUCGUUUACGCGUAAAAAUAAGAUCUCACAGCGACAAAUUUUUCUUCCAGUCUGCUGCUGAGGAAGGAUAUGAG56Nas.R-56GGGUGCAUCGUUUACGCGUAAAUUUAAGACAUGACAGCAGACAUUUUAUCUUCAGACCUGCUGCUGAGGAAGGAUAUGAG57Nas.R-57GGGUGCAUCGUUUACGCGUAAUAACAGAAAUAUAACUCAGCUGAAUUAAUUUUUCCGCUGCUGCUGAGGAAGGAUAUGAG58Nas.R-58GGGUGCAUCGUUUACGCGUAAAAAUAAAUUCCAAAAUAUUCAGCAGAAAUCCUCGAACUGCUGCUGAGGAAGGAUAUGAG59Nas.R-59GGGUGCAUCGUUUACGCGUAAAAAUAAUAGGUUCCAAUCAAGCAGUACAAAAUUCCUCUGCUGCUGAGGAAGGAUAUGAG60Nas.R-60GGGUGCAUCGUUUACGCGUAAAAAAUCUAAAAAGAUAUCAGCAGGCAAAUUUUCCUUCUGCUGCUGAGGAAGGAUAUGAG61Nas.R-61GGGUGCAUCGUUUACGCGUAAAAUAAAGAGGAUAACUACAAUCAUCAGCAAUCAUAUCUGCUGCUGAGGAAGGAUAUGAG62Nas.R-62GGGUGCAUCGUUUACGCGUAAAUUUAGUAGAAAGGAAAGACGAAGUUUCCUCAGUUUCUGCUGCUGAGGAAGGAUAUGAG63Nas.R-63GGGUGCAUCGUUUACGCGUAAAAAUAAUAGAUCUCAGAAUAUGAAAGCAGUUCUUUCCUGCUGCUGAGGAAGGAUAUGAG64Nas.R-64GGGUGCAUCGUUUACGCGUAACAAGAUAUUCACAGCAGAUUUUAAAAAAUUCCUCGUCUGCUGCUGAGGAAGGAUAUGAG65Nas.R-65GGGUGCAUCGUUUACGCGUAAAAAGUUGACAAUUAAUAAAAUCUUCUUAGCAUUUUCCUGCUGCUGAGGAAGGAUAUGAG66Nas.R-66GGGUGCAUCGUUUACGCGUAAAACAAAAUGAAACUUAUAGCUCAGCAUAUUUUGAUCCUGCUGCUGAGGAAGGAUAUGAG67Nas.R-67GGGUGCAUCGUUUACGCGUAAAUUAUCAAAAAAGCAGAUUUAAGUAUACCUCAGUUACUGCUGCUGAGGAAGGAUAUGAG68Nas.R-68GGGUGCAUCGUUUACGCGUAAAUAAAAUAGCUCAGCAAGGAAGUUUUUUUCCUCAAACUGCUGCUGAGGAAGGAUAUGAG69Nas.R-69GGGUGCAUCGUUUACGCGUAAAUUUGAGAAAAGAACAGCAGACUCAAAUCUUUUUAACUGCUGCUGAGGAAGGAUAUGAG70Nas.R-70GGGUGCAUCGUUUACGCGUAACAGAAAAUUAAGCUCAGCAAUAGUAAUUAUCCUAGUCUGCUGCUGAGGAAGGAUAUGAG71Nas.R-71GGGUGCAUCGUUUACGCGUAAUGAAAAUAAAUCAGUCUCACAGCAUUUUAAAACUUCCUGCUGCUGAGGAAGGAUAUGAG72Nas.R-72GGGUGCAUCGUUUACGCGUAUUUACAAGCAACAAAGUUACAAUCAGCAGAAUUUAUCCUGCUGCUGAGGAAGGAUAUGAG73Nas.R-73GGGUGCAUCGUUUACGCGUAAAAAAUUGUCUAUAGCACUUUUAGAUUCCCAAACUAACUGCUGCUGAGGAAGGAUAUGAG74Nas.R-74GGGUGCAUCGUUUACGCGUAAAAAAAUCAGCAAAAUCGAAAACUCAUGCAGUUUGUCCUGCUGCUGAGGAAGGAUAUGAG75Nas.R-75GGGUGCAUCGUUUACGCGUAAAAAAUUCCUUAAAAAUUUAACUAACUGGAUAGGUCUCUGCUGCUGAGGAAGGAUAUGAG76Nas.R-76GGGUGCAUCGUUUACGCGUAAAACAAAAUUUCUGACAGCAAUUCCUUCGUUAAAAAUCUGCUGCUGAGGAAGGAUAUGAG77Nas.R-77GGGUGCAUCGUUUACGCGUAAAUUAUUAAAAAAAUCAGCAAAGUUUAUUUCCCACGGCUGCUGCUGAGGAAGGAUAUGAG78Nas.R-78GGGUGCAUCGUUUACGCGUAAUUAAUCAAACAAUAGCAGCAAAUCUCAGCAAUUUUCCUGCUGCUGAGGAAGGAUAUGAG79Nas.R-79GGGUGCAUCGUUUACGCGUAAUUUGAAAGUCUCAUAAAUUUUUUUUUUUUUUUCAAUCUGCUGCUGAGGAAGGAUAUGAG80Nas.R-80GGGUGCAUCGUUUACGCGUAAAAAUUCAGCAUGAUUUCAAUUACUCCUUUCAUUGAUCUGCUGCUGAGGAAGGAUAUGAG81Nas.R-81GGGUGCAUCGUUUACGCGUAAAAUAAAUAAAAAUCAGUAGCAAUCUUUCUCACAGUGCUGCUGCUGAGGAAGGAUAUGAG82Nas.R-82GGGUGCAUCGUUUACGCGUAAAUAAAAAGCAGAUCUCAGCAAAACUCGUAAAUUCAACUGCUGCUGAGGAAGGAUAUGAG83Nas.R-83GGGUGCAUCGUUUACGCGUAAAUAAUGAAGGACUCAGACAGUUAAAAGAUGCAUUAACUGCUGCUGAGGAAGGAUAUGAG84Nas.R-84GGGUGCAUCGUUUACGCGUAAAAAAGAUCAAUAUGAAAAUCAGCAGUUAAUAUCUUCCUGCUGCUGAGGAAGGAUAUGAG85Nas.R-85GGGUGCAUCGUUUACGCGUAAAAAUAACAAACUUCUCAGCUGUUUAAUAUCUCCUGACUGCUGCUGAGGAAGGAUAUGAG86Nas.R-86GGGUGCAUCGUUUACGCGUAAAAUUAAACAAAUAGCUCAGCACGAAAAUUUGCGUAACUGCUGCUGAGGAAGGAUAUGAG87Nas.R-87GGGUGCAUCGUUUACGCGUAAUUAAAAAACCUUCACACAGAAAACAUUCCUCAAUUUCUGCUGCUGAGGAAGGAUAUGAG88Nas.R-88GGGUGCAUCGUUUACGCAUUUUCGUUUUAUUUUAGUUUAAUUGCGUUUAGUAUCUGGCUGCUGCUGAGGAAGGAUAUGAG89Nas.R-89GGGUGCAUCGUUUACGCGUAAAAAGUAUAAAGGUUAGAAAUUCAGCAGUUUGAUAUCCUGCUGCUGAGGAAGGAUAUGAG90Nas.R-90GGGUGCAUCGUUUACGCGUAAAAAGGAGAAUUAGUACUCACCAGUCGUUUAAAAUUUCUGCUGCUGAGGAAGGAUAUGAG91Nas.R-91GGGUGCAUCGUUUACGCGUAAAAAUAAAUAACUACGAGAUCUCAGCAGAUCAUUAUCCUGCUGCUGAGGAAGGAUAUGAG92Nas.R-92GGGUGCAUCGUUUACGCGUAAAAUGGUUUUUCAGCAGUUAACAUAAUGCCUCAGUUUCUGCUGCUGAGGAAGGAUAUGAG93Nas.R-93GGGUGCAUCGUUUACGCGUAAAUAACAAAAAUCUCAGCUUUUGCAGAAUUUAUCCACCUGCUGCUGAGGAAGGAUAUGAG94Nas.R-94GGGUGCAUCGUUUACGCGUAAAUAAACUCACAGCAGAAAAAAUUCCUUCAACUUGUACUGCUGCUGAGGAAGGAUAUGAG95Nas.R-95GGGUGCAUCGUUUACGCAGUAGUUAAUAACAAAUAGUCAGCAGUUUUGUCCUUCAUUCUGCUGCUGAGGAAGGAUAUGAG96Nas.R-96GGGUGCAUCGUUUACGCGUAAAAAUAGCAGUAGAUAGCGGCAGUUUUGUAUUUGUUACUGCUGCUGAGGAAGGAUAUGAG97Nas.R-97GGGUGCAUCGUUUACGCGUAAAAAUUUAAAUAACUCAGCAAUCAUAGAUCCGACUGACUGCUGCUGAGGAAGGAUAUGAG98Nas.R-98GGGUGCAUCGUUUACGCGUAAAGAACAGCUGACAAGAAAUUCAAACCUUCAGAUUUUCUGCUGCUGAGGAAGGAUAUGAG99Nas.R-99GGGUGCAUCGUUUACGCGUAAAGAUAAUAAGCAGUAUUCAGCAGAUUUGUAAGGUUUCUGCUGCUGAGGAAGGAUAUGAG100Nas.R-100GGGUGCAUCGUUUACGCGUAAAUAAGAGGCAGACAGUAUUACAAAUAUCCUAAAAUACUGCUGCUGAGGAAGGAUAUGAG TABLE 4List of deoxyribonucleotides aptamersbased on the top sequences fromselection experiments.SEQID NONameSequence101Nas.D-1GGGTGCATCGTTTACGCGATTAGTCTGATAAACAAAAAGATTTCGCTAAAAATCAATCTGCTGCTGAGGAAGGATATGAG102Nas.D-2GGGTGCATCGTTTACGCAGATAGCAGCAGGAATCAAGCGGTAGGAGTCTAGCAGAAGCTGCTGCTGAGGAAGGATATGAG103Nas.D-3GGGTGCATCGTTTACGCATTTTCGTTTTATTTCAGTTTAATTGCGTTTAGTATCTGGCTGCTGCTGAGGAAGGATATGAG104Nas.D-4GGGTGCATCGTTTACGCGCAACATAAAAATTTAAAGTGCTCAGTTGTCAATCTATGACTGCTGCTGAGGAAGGATATGAG105Nas.D-5GGGTGCATCGTTTACGCGTAAATGGTCCGCTATTAAAAGAAAAGAATGAAGTCTCAGCTGCTGCTGAGGAAGGATATGAG106Nas.D-6GGGTGCATCGTTTACGCTATTTTCATTTGTTTTTTTAATTTACTAGTGTAAACAATCCTGCTGCTGAGGAAGGATATGAG107Nas.D-7GGGTGCATCGTTTACGCGTAAATAAGTAGATAAAGTGGCAGTTTGTTTTCCTTGGAACTGCTGCTGAGGAAGGATATGAG108Nas.D-8GGGTGCATCGTTTACGCGTAAAAATTAAAGAGATTAAGGTCCTTAAGCAGTTTTGTCCTGCTGCTGAGGAAGGATATGAG109Nas.D-9GGGTGCATCGTTTACGCGTAAAAAAATCAAAACTTCAGCAAATTATTTATCAACGTCCTGCTGCTGAGGAAGGATATGAG110Nas.D-10GGGTGCATCGTTTACGCGTAAAATAAATTAAAAAGAACTTCTTCAGCAATCAATATCCTGCTGCTGAGGAAGGATATGAG111Nas.D-11GGGTGCATCGTTTACGCGTAAATAAAAATGAAAAATTGTCTCTCAGCTTTCAAAGTCCTGCTGCTGAGGAAGGATATGAG112Nas.D-12GGGTGCATCGTTTACGCGTAAAAAAAAAATATCTTCGGAGAATTCAGCAATTTTATCCTGCTGCTGAGGAAGGATATGAG113Nas.D-13GGGTGCATCGTTTACGCGTAAAAATTTTCATCTCAGCAATTAAATCCAAAGAATCCACTGCTGCTGAGGAAGGATATGAG114Nas.D-14GGGTGCATCGTTTACGCGTAAAATATATCAGCAAAGTAGTTTAAGCCTCCTCAGTTTCTGCTGCTGAGGAAGGATATGAG115Nas.D-15GGGTGCATCGTTTACGCGTAAATTATGAAAAATACAGCAAGGATTTAACCTCAGTTTCTGCTGCTGAGGAAGGATATGAG116Nas.D-16GGGTGCATCGTTTACGCGTAAAATAAATAAATCTTCAAAGTACAGACCTCGATTTTTCTGCTGCTGAGGAAGGATATGAG117Nas.D-17GGGTGCATCGTTTACGCTTATAGGTATTAGACATTTTCAATTAAAGTGAATTAGTGTCTGCTGCTGAGGAAGGATATGAG118Nas.D-18GGGTGCATCGTTTACGCGTAAAATGTGACAGCAGGATAATAAAATAAGTACTCAGTACTGCTGCTGAGGAAGGATATGAG119Nas.D-19GGGTGCATCGTTTACGCGTAATTAAGAAAAATAAAAGTACTCTGCAGTTTTTATCCACTGCTGCTGAGGAAGGATATGAG120Nas.D-20GGGTGCATCGTTTACGCGTAAAAATAAAATTTTCCCAGACCAGTTATCTGCCTTAAACTGCTGCTGAGGAAGGATATGAG121Nas.D-21GGGTGCATCGTTTACGCGTAAAGAAAAAAATCAGCTTTTAGTCGCCTTCCATTTTGACTGCTGCTGAGGAAGGATATGAG122Nas.D-22GGGTGCATCGTTTACGCGTAAATAAATAATCAAAATTACACTCAGTGGCAATTTCCTCTGCTGCTGAGGAAGGATATGAG123Nas.D-23GGGTGCATCGTTTACGCGTAAAATACAGGATACGACAATAACTCAGCAGATTTTATCCTGCTGCTGAGGAAGGATATGAG124Nas.D-24GGGTGCATCGTTTACGCGTTAAAAATTGTGCACTGAGATGACGCAGCATTAACTACACTGCTGCTGAGGAAGGATATGAG125Nas.D-25GGGTGCATCGTTTACGCGTAAATAAAAATTAATCAGCAATTTTCCACTCAGTTGTACCTGCTGCTGAGGAAGGATATGAG126Nas.D-26GGGTGCATCGTTTACGCGTAAAAATAAAAAATCTCGATCACTGCAGTTTTATTCCGGCTGCTGCTGAGGAAGGATATGAG127Nas.D-27GGGTGCATCGTTTACGCGTAAACAAATATCGATTAAAATAAAATCTCAGCAAGAATCCTGCTGCTGAGGAAGGATATGAG128Nas.D-28GGGTGCATCGTTTACGCGTAAAATAAATAAAATTATCCCAGGAGCAAATTTTCTTCGCTGCTGCTGAGGAAGGATATGAG129Nas.D-29GGGTGCATCGTTTACGCGTAGAAGAATTAATAGTGGACATATCAATAGCAGTTTATCCTGCTGCTGAGGAAGGATATGAG130Nas.D-30GGGTGCATCGTTTACGCGTAAACATATTCAGCAGTTAAAATTTAGTAGGTTCAGTAGCTGCTGCTGAGGAAGGATATGAG131Nas.D-31GGGTGCATCGTTTACGCGTAAAAAAGATAAAACTTAGTTGCAGAATTTGCCTTCATTCTGCTGCTGAGGAAGGATATGAG132Nas.D-32GGGTGCATCGTTTACGCGTAAAAAGTTTGATGGAAGCAGATTAGTTTAGTCAAATTTCTGCTGCTGAGGAAGGATATGAG133Nas.D-33GGGTGCATCGTTTACGCGTAAAATGAAATAAGGAATCCTTCAGCAGTATTTATCCTTCTGCTGCTGAGGAAGGATATGAG134Nas.D-34GGGTGCATCGTTTACGCGTAAAGAATAAAAATGACAAAATTCTCAGCTTTTGTCAACCTGCTGCTGAGGAAGGATATGAG135Nas.D-35GGGTGCATCGTTTACGCGTAAAAAATGAAATGAAAAAATTCTCAGCTGTCTATCTTCCTGCTGCTGAGGAAGGATATGAG136Nas.D-36GGGTGCATCGTTTACGCGTAAATAAGTAAAAAACTCAGTTTTCAGTTAAGTATCCAACTGCTGCTGAGGAAGGATATGAG137Nas.D-37GGGTGCATCGTTTACGCGTAAATTTCAGCAGAGTAATAATAACACTTCTTCAGTTTGCTGCTGCTGAGGAAGGATATGAG138Nas.D-38GGGTGCATCGTTTACGCGTAAAATTAAGAAGTATTATCAGTTAGCTTTTTCTTCCAACTGCTGCTGAGGAAGGATATGAG139Nas.D-39GGGTGCATCGTTTACGCGTAAAATAAAAAGTTTTCCTATCAGCAAACTCACAAATTCCTGCTGCTGAGGAAGGATATGAG140Nas.D-40GGGTGCATCGTTTACGCGTAAAATGAAATGTAAAAGAATTGAACTTGGCAGATTTTCCTGCTGCTGAGGAAGGATATGAG141Nas.D-41GGGTGCATCGTTTACGCGTAAATTAAAGTAGCAGTAATTTCAGCAGTTTTTACCTCTCTGCTGCTGAGGAAGGATATGAG142Nas.D-42GGGTGCATCGTTTACGCGTAAATAAAGGATAAAATAATTTCAGGGCAGTTTCTCATCCTGCTGCTGAGGAAGGATATGAG143Nas.D-43GGGTGCATCGTTTACGCAGGATCGTTTTAAGTAAAATAAAAGATTTCCTTGGTAATCCTGCTGCTGAGGAAGGATATGAG144Nas.D-44GGGTGCATCGTTTACGCGTAAAATAAAGATCAATTAAAGGCTTTGATCGATTTTCCTCTGCTGCTGAGGAAGGATATGAG145Nas.D-45GGGTGCATCGTTTACGCGTAAAAATTAGAGATTAAAATAGTTCCTTTCAGTTTTGTCCTGCTGCTGAGGAAGGATATGAG146Nas.D-46GGGTGCATCGTTTACGCGTAAAATTGACAATGTGAAAAGCAGACAGCAAATATTCCTCTGCTGCTGAGGAAGGATATGAG147Nas.D-47GGGTGCATCGTTTACGCGTAAATAACCAGTTATACAGAAAGATCTCAGCAATTTATCCTGCTGCTGAGGAAGGATATGAG148Nas.D-48GGGTGCATCGTTTACGCTTACAGAAGGATTGCACCACATGCGTACTCGATGAAACACCTGCTGCTGAGGAAGGATATGAG149Nas.D-49GGGTGCATCGTTTACGCGTAAAATAATAATTAAACTCAGCAAATTCAATCCAACTTTCTGCTGCTGAGGAAGGATATGAG150Nas.D-50GGGTGCATCGTTTACGCGTAAACAAGAATAAATTCAGCAGTGGTTTTGATCCTTTGACTGCTGCTGAGGAAGGATATGAG151Nas.D-51GGGTGCATCGTTTACGCGTAAATTAATCAGATTGAACAAAAGTTTTCCCTCAGTTTTCTGCTGCTGAGGAAGGATATGAG152Nas.D-52GGGTGCATCGTTTACGCGTAAAGAAAAACATCAGAGCAGTTATAATAGTCCTTTTTCCTGCTGCTGAGGAAGGATATGAG153Nas.D-53GGGTGCATCGTTTACGCGTAAAGAAAATAAACTTGATCAAACTTAGCAGTTTTTATCCTGCTGCTGAGGAAGGATATGAG154Nas.D-54GGGTGCATCGTTTACGCATTTTCGTTATATTTCTGGTTTTTATGCGTGAGAATCCTGCTGCTGCTGAGGAAGGATATGAG155Nas.D-55GGGTGCATCGTTTACGCGTAAAAATAAGATCTCACAGCGACAAATTTTTCTTCCAGTCTGCTGCTGAGGAAGGATATGAG156Nas.D-56GGGTGCATCGTTTACGCGTAAATTTAAGACATGACAGCAGACATTTTATCTTCAGACCTGCTGCTGAGGAAGGATATGAG157Nas.D-57GGGTGCATCGTTTACGCGTAATAACAGAAATATAACTCAGCTGAATTAATTTTTCCGCTGCTGCTGAGGAAGGATATGAG158Nas.D-58GGGTGCATCGTTTACGCGTAAAAATAAATTCCAAAATATTCAGCAGAAATCCTCGAACTGCTGCTGAGGAAGGATATGAG159Nas.D-59GGGTGCATCGTTTACGCGTAAAAATAATAGGTTCCAATCAAGCAGTACAAAATTCCTCTGCTGCTGAGGAAGGATATGAG160Nas.D-60GGGTGCATCGTTTACGCGTAAAAAATCTAAAAAGATATCAGCAGGCAAATTTTCCTTCTGCTGCTGAGGAAGGATATGAG161Nas.D-61GGGTGCATCGTTTACGCGTAAAATAAAGAGGATAACTACAATCATCAGCAATCATATCTGCTGCTGAGGAAGGATATGAG162Nas.D-62GGGTGCATCGTTTACGCGTAAATTTAGTAGAAAGGAAAGACGAAGTTTCCTCAGTTTCTGCTGCTGAGGAAGGATATGAG163Nas.D-63GGGTGCATCGTTTACGCGTAAAAATAATAGATCTCAGAATATGAAAGCAGTTCTTTCCTGCTGCTGAGGAAGGATATGAG164Nas.D-64GGGTGCATCGTTTACGCGTAACAAGATATTCACAGCAGATTTTAAAAAATTCCTCGTCTGCTGCTGAGGAAGGATATGAG165Nas.D-65GGGTGCATCGTTTACGCGTAAAAAGTTGACAATTAATAAAATCTTCTTAGCATTTTCCTGCTGCTGAGGAAGGATATGAG166Nas.D-66GGGTGCATCGTTTACGCGTAAAACAAAATGAAACTTATAGCTCAGCATATTTTGATCCTGCTGCTGAGGAAGGATATGAG167Nas.D-67GGGTGCATCGTTTACGCGTAAATTATCAAAAAAGCAGATTTAAGTATACCTCAGTTACTGCTGCTGAGGAAGGATATGAG168Nas.D-68GGGTGCATCGTTTACGCGTAAATAAAATAGCTCAGCAAGGAAGTTTTTTTCCTCAAACTGCTGCTGAGGAAGGATATGAG169Nas.D-69GGGTGCATCGTTTACGCGTAAATTTGAGAAAAGAACAGCAGACTCAAATCTTTTTAACTGCTGCTGAGGAAGGATATGAG170Nas.D-70GGGTGCATCGTTTACGCGTAACAGAAAATTAAGCTCAGCAATAGTAATTATCCTAGTCTGCTGCTGAGGAAGGATATGAG171Nas.D-71GGGTGCATCGTTTACGCGTAATGAAAATAAATCAGTCTCACAGCATTTTAAAACTTCCTGCTGCTGAGGAAGGATATGAG172Nas.D-72GGGTGCATCGTTTACGCGTATTTACAAGCAACAAAGTTACAATCAGCAGAATTTATCCTGCTGCTGAGGAAGGATATGAG173Nas.D-73GGGTGCATCGTTTACGCGTAAAAAATTGTCTATAGCACTTTTAGATTCCCAAACTAACTGCTGCTGAGGAAGGATATGAG174Nas.D-74GGGTGCATCGTTTACGCGTAAAAAAATCAGCAAAATCGAAAACTCATGCAGTTTGTCCTGCTGCTGAGGAAGGATATGAG175Nas.D-75GGGTGCATCGTTTACGCGTAAAAAATTCCTTAAAAATTTAACTAACTGGATAGGTCTCTGCTGCTGAGGAAGGATATGAG176Nas.D-76GGGTGCATCGTTTACGCGTAAAACAAAATTTCTGACAGCAATTCCTTCGTTAAAAATCTGCTGCTGAGGAAGGATATGAG177Nas.D-77GGGTGCATCGTTTACGCGTAAATTATTAAAAAAATCAGCAAAGTTTATTTCCCACGGCTGCTGCTGAGGAAGGATATGAG178Nas.D-78GGGTGCATCGTTTACGCGTAATTAATCAAACAATAGCAGCAAATCTCAGCAATTTTCCTGCTGCTGAGGAAGGATATGAG179Nas.D-79GGGTGCATCGTTTACGCGTAATTTGAAAGTCTCATAAATTTTTTTTTTTTTTTCAATCTGCTGCTGAGGAAGGATATGAG180Nas.D-80GGGTGCATCGTTTACGCGTAAAAATTCAGCATGATTTCAATTACTCCTTTCATTGATCTGCTGCTGAGGAAGGATATGAG181Nas.D-81GGGTGCATCGTTTACGCGTAAAATAAATAAAAATCAGTAGCAATCTTTCTCACAGTGCTGCTGCTGAGGAAGGATATGAG182Nas.D-82GGGTGCATCGTTTACGCGTAAATAAAAAGCAGATCTCAGCAAAACTCGTAAATTCAACTGCTGCTGAGGAAGGATATGAG183Nas.D-83GGGTGCATCGTTTACGCGTAAATAATGAAGGACTCAGACAGTTAAAAGATGCATTAACTGCTGCTGAGGAAGGATATGAG184Nas.D-84GGGTGCATCGTTTACGCGTAAAAAAGATCAATATGAAAATCAGCAGTTAATATCTTCCTGCTGCTGAGGAAGGATATGAG185Nas.D-85GGGTGCATCGTTTACGCGTAAAAATAACAAACTTCTCAGCTGTTTAATATCTCCTGACTGCTGCTGAGGAAGGATATGAG186Nas.D-86GGGTGCATCGTTTACGCGTAAAATTAAACAAATAGCTCAGCACGAAAATTTGCGTAACTGCTGCTGAGGAAGGATATGAG187Nas.D-87GGGTGCATCGTTTACGCGTAATTAAAAAACCTTCACACAGAAAACATTCCTCAATTTCTGCTGCTGAGGAAGGATATGAG188Nas.D-88GGGTGCATCGTTTACGCATTTTCGTTTTATTTTAGTTTAATTGCGTTTAGTATCTGGCTGCTGCTGAGGAAGGATATGAG189Nas.D-89GGGTGCATCGTTTACGCGTAAAAAGTATAAAGGTTAGAAATTCAGCAGTTTGATATCCTGCTGCTGAGGAAGGATATGAG190Nas.D-90GGGTGCATCGTTTACGCGTAAAAAGGAGAATTAGTACTCACCAGTCGTTTAAAATTTCTGCTGCTGAGGAAGGATATGAG191Nas.D-91GGGTGCATCGTTTACGCGTAAAAATAAATAACTACGAGATCTCAGCAGATCATTATCCTGCTGCTGAGGAAGGATATGAG192Nas.D-92GGGTGCATCGTTTACGCGTAAAATGGTTTTTCAGCAGTTAACATAATGCCTCAGTTTCTGCTGCTGAGGAAGGATATGAG193Nas.D-93GGGTGCATCGTTTACGCGTAAATAACAAAAATCTCAGCTTTTGCAGAATTTATCCACCTGCTGCTGAGGAAGGATATGAG194Nas.D-94GGGTGCATCGTTTACGCGTAAATAAACTCACAGCAGAAAAAATTCCTTCAACTTGTACTGCTGCTGAGGAAGGATATGAG195Nas.D-95GGGTGCATCGTTTACGCAGTAGTTAATAACAAATAGTCAGCAGTTTTGTCCTTCATTCTGCTGCTGAGGAAGGATATGAG196Nas.D-96GGGTGCATCGTTTACGCGTAAAAATAGCAGTAGATAGCGGCAGTTTTGTATTTGTTACTGCTGCTGAGGAAGGATATGAG197Nas.D-97GGGTGCATCGTTTACGCGTAAAAATTTAAATAACTCAGCAATCATAGATCCGACTGACTGCTGCTGAGGAAGGATATGAG198Nas.D-98GGGTGCATCGTTTACGCGTAAAGAACAGCTGACAAGAAATTCAAACCTTCAGATTTTCTGCTGCTGAGGAAGGATATGAG199Nas.D-99GGGTGCATCGTTTACGCGTAAAGATAATAAGCAGTATTCAGCAGATTTGTAAGGTTTCTGCTGCTGAGGAAGGATATGAG200Nas.D-GGGTGCATCGTTTACGCGTA100AATAAGAGGCAGACAGTATTACAAATATCCTAAAATACTGCTGCTGAGGAAGGATATGAG TABLE 5List of conserved motifs.SEQ ID NOSequence201AAACAAAAAGA202UAAAAAUCA203AAACAAAAAGA204TAAAAATCA205AUAAAAAUUUAAA206ATAAAAATTTAAA207GUAAAAAUUAAA208GTAAAAATTAAA209GUAAAAAAA210UNAGCANUUU211GTAAAAAAA212TNAGCANTTT TABLE 6List of protein sequencesSEQIDNODescriptionSequence213ICAM-1MAPSSPRPALPALLVLLGALFPGPGNAQTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQVEGGAPRANLTVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPRVLEVDTQGTVVCSLDGLFPVSEAQVHLALGDQRLNPTVTYGNDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQETLQTVTIYSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATLEVAGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYEIVIITVVAAAVIMGTAGLSTYLYNRQRKIKKYRLQQAQKGTPMKPNTQATPP214ExtracellularQTSVSPSKVILPRGGSVLVTdomain ofCSTSCDQPKLLGIETPLPKKICAM-1ELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQVEGGAPRANLTVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPRVLEVDTQGTVVCSLDGLFPVSEAQVHLALGDQRLNPTVTYGNDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQETLQTVTIYSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATLEVAGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYE215Ig-like C2-GGSVLVTCSTSCDQPKLLGItype 1ETPLPKKELLLPGNNRKVYEdomainLSNVQEDSQPMCYSNCPDGQSTA216Ig-like C2-GKNLTLRCQVEGGAPRANLTtype 2VVLLRGEKELKREPAVGEPAdomainEVTTTVLVRRDHHGANFSCRTELDLR217Ig-like C2-DTQGTVVCSLDGLFPVSEAQtype 3VHLALGDQRLNPTVTYGNDSdomainFSAKASVSVTAEDEGTQRLTCAVILGNQ218Ig-like C2-GTEVTVKCEAHPRAKVTLNGtype 4VPAQPLGPRAQLLLKATPEDdomainNGRSFSCSATLEVA219Ig-like C2-NSQQTPMCQAWGNPLPELKCtype 5LKDGTFPLPIGESVTVTRDLdomainEGTYLCRARSTQG220Fragment ofQTSVSPSKVILPRICAM-1221Fragment ofSCDQPKLLGIICAM-2222Fragment ofPKKELLLPGNNRKVYEICAM-3223Fragment ofYSNCPDGQSTAKTFLICAM-4231ICAM-3MATMVPSVLWPRACWTLLVCCLLTPGVQGQEFLLRVEPQNPVLSAGGSLFVNCSTDCPSSEKIALETSLSKELVASGMGWAAFNLSNVTGNSRILCSVYCNGSQITGSSNITVYRLPERVELAPLPPWQPVGQNFTLRCQVEDGSPRTSLTVVLLRWEEELSRQPAVEEPAEVTATVLASRDDHGAPFSCRTELDMQPQGLGLFVNTSAPRQLRTFVLPVTPPRLVAPRFLEVETSWPVDCTLDGLFPASEAQVYLALGDQMLNATVMNHGDTLTATATATARADQEGAREIVCNVTLGGERREARENLTVFSFLGPIVNLSEPTAHEGSTVTVSCMAGARVQVTLDGVPAAAPGQPAQLQLNATESDDGRSFFCSATLEVDGEFLHRNSSVQLRVLYGPKIDRATCPQHLKWKDKTRHVLQCQARGNPYPELRCLKEGSSREVPVGIPFFVNVTHNGTYQCQASSSRGKYTLVVVMDIEAGSSHFVPVFVAVLLTLGVVTIVLALMYVFREHQRSGSYHVREESTYLPLTSMQPTEAMGEEPSRAE232ExtracellularQEFLLRVEPQNPVLSAGGSLdomain ofFVNCSTDCPSSEKIALETSLICAM-3SKELVASGMGWAAFNLSNVTGNSRILCSVYCNGSQITGSSNITVYRLPERVELAPLPPWQPVGQNFTLRCQVEDGSPRTSLTVVLLRWEEELSRQPAVEEPAEVTATVLASRDDHGAPFSCRTELDMQPQGLGLFVNTSAPRQLRTFVLPVTPPRLVAPRFLEVETSWPVDCTLDGLFPASEAQVYLALGDQMLNATVMNHGDTLTATATATARADQEGAREIVCNVTLGGERREARENLTVFSFLGPIVNLSEPTAHEGSTVTVSCMAGARVQVTLDGVPAAAPGQPAQLQLNATESDDGRSFFCSATLEVDGEFLHRNSSVQLRVLYGPKIDRATCPQHLKWKDKTRHVLQCQARGNPYPELRCLKEGSSREVPVGIPFFVNVTHNGTYQCQASSSRGKYTLVVVMDIEAGSSH233ICAM-5MPGPSPGLRRALLGLWAALGLGLFGLSAVSQEPFWADLQPRVAFVERGGSLWLNCSTNCPRPERGGLETSLRRNGTQRGLRWLARQLVDIREPETQPVCFFRCARRTLQARGLIRFQRPDRVELMPLPPWQPVGENFTLSCRVPGAGPRASLTLTLLRGAQELIRRSFAGEPPRARGAVLTATVLARREDHGANFSCRAELDLRPHGLGLFENSSAPRELRTFSLSPDAPRLAAPRLLEVGSERPVSCTLDGLFPASEARVYLALGDQNLSPDVTLEGDAFVATATATASAEQEGARQLVCNVTLGGENRETRENVTIYSFPAPLLTLSEPSVSEGQMVTVTCAAGAQALVTLEGVPAAVPGQPAQLQLNATENDDRRSFFCDATLDVDGETLIKNRSAELRVLYAPRLDDSDCPRSWTWPEGPEQTLRCEARGNPEPSVHCARSDGGAVLALGLLGPVTRALSGTYRCKAANDQGEAVKDVTLTVEYAPALDSVGCPERITWLEGTEASLSCVAHGVPPPDVICVRSGELGAVIEGLLRVAREHAGTYRCEATNPRGSAAKNVAVTVEYGPRFEEPSCPSNWTWVEGSGRLFSCEVDGKPQPSVKCVGSGGATEGVLLPLAPPDPSPRAPRIPRVLAPGIYVCNATNRHGSVAKTVVVSAESPPEMDESTCPSHQTWLEGAEASALACAARGRPSPGVRCSREGIPWPEQQRVSREDAGTYHCVATNAHGTDSRTVTVGVEYRPVVAELAASPPGGVRPGGNFTLTCRAEAWPPAQISWRAPPGALNIGLSSNNSTLSVAGAMGSHGGEYECAATNAHGRHARRITVRVAGPWLWVAVGGAAGGAALLAAGAGLAFYVQSTACKKGEYNVQEAESSGEAVCLNGAGGGAGGAAGAEGGPEAAGGAAESPAEGEVFAIQLTSA234ExtracellularEPFWADLQPRVAFVERGGSLdomain ofWLNCSTNCPRPERGGLETSLICAM-5RRNGTQRGLRWLARQLVDIREPETQPVCFFRCARRTLQARGLIRTFQRPDRVELMPLPPWQPVGENFTLSCRVPGAGPRASLTLTLLRGAQELIRRSFAGEPPRARGAVLTATVLARREDHGANFSCRAELDLRPHGLGLFENSSAPRELRTFSLSPDAPRLAAPRLLEVGSERPVSCTLDGLFPASEARVYLALGDQNLSPDVTLEGDAFVATATATASAEQEGARQLVCNVTLGGENRETRENVTIYSFPAPLLTLSEPSVSEGQMVTVTCAAGAQALVTLEGVPAAVPGQPAQLQLNATENDDRRSFFCDATLDVDGETLIKNRSAELRVLYAPRLDDSDCPRSWTWPEGPEQTLRCEARGNPEPSVHCARSDGGAVLALGLLGPVTRALSGTYRCKAANDQGEAVKDVTLTVEYAPALDSVGCPERITWLEGTEASLSCVAHGVPPPDVICVRSGELGAVIEGLLRVAREHAGTYRCEATNPRGSAAKNVAVTVEYGPRFEEPSCPSNWTWVEGSGRLFSCEVDGKPQPSVKCVGSGGATEGVLLPLAPPDPSPRAPRIPRVLAPGIYVCNATNRHGSVAKTVVVSAESPPEMDESTCPSHQTWLEGAEASALACAARGRPSPGVRCSREGIPWPEQQRVSREDAGTYHCVATNAHGTDSRTVTVGVEYRPVVAELAASPPGGVRPGGNFTLTCRAEAWPPAQISWRAPPGALNIGLSSNNSTLSVAGAMGSHGGEYECAATNAHGRHARRITVRVAGPW CombinationsA. An aptamer composition comprising at least one oligonucleotide consisting of: deoxyribonucleotides, ribonucleotides, derivatives of deoxyribonucleotides, derivatives of ribonucleotides, and mixtures thereof; wherein the aptamer composition has a binding affinity for intercellular adhesion molecule 1 (ICAM-1); and wherein the aptamer is configured to reduce the binding of one or more human rhinoviruses to the intercellular adhesion molecule 1 (ICAM-1) and wherein the aptamer composition comprisesi. at least one oligonucleotide selected from the group consisting of oligonucleotides with at least 80% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 200; and/or.ii. at least one oligonucleotide comprising one or more motifs selected from the group consisting of SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, and SEQ ID NO: 212.B. The aptamer composition according to Paragraph A, wherein the at least one oligonucleotide is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8.C. The aptamer composition according to Paragraph A-B, wherein the at least one oligonucleotide shows at least 90%, or 95%, or 96%, or 97%, or 98% or 99% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 200, or wherein the at least one oligonucleotide shows at least 90%, or 95%, or 96%, or 97%, or 98% or 99% nucleotide sequence identity to sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8.D. The aptamer composition according to Paragraph A-C, comprising at least one oligonucleotide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 200.E. The aptamer composition according to Paragraph A-D, comprising at least one oligonucleotide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8.F. The aptamer composition according to Paragraph A-E, wherein the at least one oligonucleotide comprises natural or non-natural nucleobases; preferably wherein the non-natural nucleobases are selected from the group comprising hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-5-methylcytosine, 5-hydroxymethylcytosine, thiouracil, 1-methylhypoxanthine, 6-methylisoquinoline-1-thione-2-yl, 3-methoxy-2-naphthyl, 5-propynyluracil-1-yl, 5-methylcytosin-1-yl, 2-aminoadenin-9-yl, 7-deaza-7-iodoadenin-9-yl, 7-deaza-7-propynyl-2-aminoadenin-9-yl, phenoxazinyl, phenoxazinyl-G-clam, and mixtures thereof.G. The aptamer composition according to Paragraph A-F, wherein the nucleosides of the at least one oligonucleotide are linked by a chemical motif selected from the group comprising natural phosphate diester, chiral phosphorothionate, chiral methyl phosphonate, chiral phosphoramidate, chiral phosphate chiral triester, chiral boranophosphate, chiral phosphoroselenoate, phosphorodithioate, phosphorothionate amidate, methylenemethylimino, 3′-amide, 3′ achiral phosphoramidate, 3′ achiral methylene phosphonates, thioformacetal, thioethyl ether, and mixtures thereof.H. The aptamer composition according to Paragraph A-G, where the derivatives of ribonucleotides or the derivatives of deoxyribonucleotides are selected from the group comprising locked oligonucleotides, peptide oligonucleotides, glycol oligonucleotides, threose oligonucleotides, hexitol oligonucleotides, altritol oligonucleotides, butyl oligonucleotides, L-ribonucleotides, arabino oligonucleotides, 2′-fluoroarabino oligonucleotides, cyclohexene oligonucleotides, phosphorodiamidate morpholino oligonucleotides, and mixtures thereof.I. The aptamer composition according to Paragraph A-H, further comprising at least one polymeric material, wherein the at least one polymeric material is covalently linked to the at least one oligonucleotide; preferably wherein the at least one polymeric material is polyethylene glycol.J. The aptamer composition according to Paragraph A-I wherein the nucleotides at the 5′- and 3′-ends of the at least one oligonucleotide are inverted.K. The aptamer composition according to Paragraph A-J, wherein at least one nucleotide of the at least one oligonucleotide is fluorinated at the 2′ position of the pentose group; preferably wherein the pyrimidine nucleotides of the at least one oligonucleotide are fluorinated at the 2′ position of the pentose group.L. The aptamer composition according to Paragraph A-K, wherein the at least one oligonucleotide is covalently or non-covalently attached to one or more active ingredients, wherein the one or more active ingredients are selected from the group consisting of: respiratory illness treatment agents, cold-treatment agents, flu-treatment agents, antiviral agents, antimicrobial agents, cooling agents, malodor absorbing agents, natural extracts, peptides, enzymes, pharmaceutical active ingredients, metal compounds, and combinations thereof.M. An aptamer composition comprising at least one peptide or protein, wherein the peptide or protein is translated from at least one of the oligonucleotides of anyone of paragraphs A-L.N. The aptamer composition according to Paragraph A-M wherein the aptamer has a binding affinity for the Ig-like C2-type 1 domain (SEQ ID NO: 215) of the intercellular adhesion molecule 1 (ICAM-1), any post-translationally modified versions of said domain, and mixtures thereof.O. The aptamer composition according to Paragraph A-M, wherein the at least one oligonucleotide is covalently or non-covalently attached to one or more nanomaterials comprising one or more active ingredients.P. A personal health care composition comprising the at least one aptamer composition according to paragraph A-O.Q. The personal health care composition according to paragraph P, wherein the at least one nucleic acid aptamer is covalently or non-covalently attached to one or more active ingredients, wherein said one or more active ingredients are selected from the group comprising: respiratory illness treatment agents, cold-treatment agents, flu-treatment agents, antiviral agents, antimicrobial agents, cooling agents, malodor absorbing agents, natural extracts, peptides, enzymes, pharmaceutical active ingredients, metal compounds, and mixtures thereof.R. The aptamer composition according to paragraph A-O or the personal health care composition according to paragraph P or Q for inhibiting human rhinovirus infection by inhibiting binding to the intercellular adhesion molecule 1 (ICAM-1) and thereby inhibiting entering into cells within the nasal cavity and throat and/or for preventing and treating symptoms associated with respiratory tract viral infections, preferably by delivering the composition to the upper respiratory tract.S. A method for delivering a personal health care composition to the upper respiratory tract comprising administering to a subject in need thereof a personal health care composition comprising at least one nucleic acid aptamer, wherein the aptamer has a binding affinity for intercellular adhesion molecule 1 (ICAM-1) and wherein the aptamer is configured to reduce the binding of one or more human rhinoviruses to the intercellular adhesion molecule 1 (ICAM-1). The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm.” Values disclosed herein as ends of ranges are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each numerical range is intended to mean both the recited values and any real numbers including integers within the range. For example, a range disclosed as “1 to 10” is intended to mean “1, 2, 3, 4, 5, 6, 7, 8, 9, and 10” and a range disclosed as “1 to 2” is intended to mean “1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2. Every document cited herein, including any cross referenced or related patent or application and any patent application or patent to which this application claims priority or benefit thereof, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern. While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention. | 160,725 |
11859188 | DETAILED DESCRIPTION OF THE INVENTION To facilitate understanding of the object, characteristics and effects of this present disclosure, embodiments together with the attached drawings for the detailed description of the present disclosure are provided. Magnetic-Assisted Rapid Aptamer Selection (MARAS) According to the previous studies, targeting TXNDC5 could be a novel therapeutic approach against multiple fibrosis-related diseases, such as cardiac fibrosis, heart failure, liver fibrosis, renal fibrosis, chronic kidney diseases and pulmonary fibrosis. A strategy of targeting TXNDC5 is providing DNA aptamer(s) capable of binding TXNDC5 with high specificity and affinity and inhibiting the catalytic ability of TXNDC5. To this end, magnetic-assisted rapid aptamer selection, a novel magnetic-based aptamer screening method, is used to identify multiple TXNDC5-targeting aptamers. The method of selecting TXNDC5-targeting aptamers via MARAS is set forth as following. First, wild-type TXNDC5, catalytic-death TXDNC5 and human serum are biotinylated by EZ-Link NHS-SS-Biotin Kit (Thermo Scientific) according to the manufactures' instructions. A total 100 μs of each biotinylated protein (including the above biotinylated wild-type TXNDC5, catalytic-death TXDNC5 and human serum) is mixed with 50 μl of streptavidin-coated magnetic beads (SA-MNPs) at 4° C. overnight. The mixture of each biotinylated protein and the magnetic beads (MNPs) correspondingly reacting with the biotinylated protein is then subjected to magnetic separation by magnetic stand to remove the unbound biotinylated proteins. The proteins conjugated MNPs (TXNDC5-MNPs) are washed with the binding buffer (the binding buffer contains 20 mM Tris-Cl (pH 7.6), 150 mM NaCl, 50 mM KCl, 2 mM MgCl2, 1 mM CaCl2) and 0.05% Tween-20, the following terms “binding buffer” refers to the binding buffer containing the same components described herein) and stored in the same binding buffer at 4° C. or processed for following experiments. Further, random 50 nucleotides of ssDNA library (aptamers library) are chemically synthesized in 250 nM scale at Integrated DNA Technologies (MedClub Scientific, Taiwan) as starting library. Each aptamer (ssDNA) is composed of the central 20 randomized oligonucleotides, which are flanked with two fixed stem-loop sequences at both ends (5′-AGCAGCACAGAGGTC-N20-GCGTGCTACCGTGAA-3′) for PCR amplification and sequencing (Tsao, S. M et al. Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection. Sci Rep 7, 45478, doi:10.1038/srep45478 (2017)). Two set of primers: 5′-AGCAGCACAGAGGTC-3′ (SEQ ID NO: 15) and 5′-TTCACGGTAGCACGC-3′ (SEQ ID NO: 16) are utilized. After the above ssDNA library has been established, 0.5 μl of randomized oligonucleotide solution (the initial concentration: 100 μM) is used as the starting library and diluted to 10 μl by adding 9.5 μl of the binding buffer (the final concentration of the randomized oligonucleotide solution is 5 The above solution is heated to 95° C. for 5 min and then quickly snapped to 4° C. to make ssDNA form secondary structures. After ssDNA in the above solution has formed into secondary structures, the above solution is stayed at room temperature for 30 min. The first positive selection round is performed by incubating TXNDC5-MNPs (mouse wild type TXNDC5) and the folding oligonucleotide (ssDNA) in the binding buffer for 30 min at room temperature, thereby TXNDC5-MNPs and the folding oligonucleotide are bound together to form aptamer-TXNDC5-MNPs complexes. The aptamer-TXNDC5-MNPs complexes are placed inside the MARAS platform and subjected to a rotating magnetic field with 40-50 KHz and strength of 14 gauss for 10 min. After the above treatment of the rotating magnetic field, the aptamer-TXNDC5-MNPs complexes are stirred by pipetting every 2.5 min to avoid agglomeration. The aptamer-TXNDC5-MNPs complexes are retained and washed three times with 200 μl of the binding buffer. The aptamer-TXNDC5-MNPs complexes are re-suspended into 100 μl of the binding buffer and processed for next positive selection round (human wild type TXNDC5) in identical procedures for refining the aptamers that are attracted by both species of TXNDC5 (human and mouse). Subsequently, for negative selection, a library (ssDNA) from the previous 2 round positive selections is incubated with negative serum-MNPs at room temperature for 30 min. After magnetic separation, aptamers bound with negative serum-MNPs are removed. The collected supernatants are continuously processed to other negative-MNPs (2 rounds of serum-MNPs and 1 round of catalytic-death TXNDC5-MNPs) as the aforementioned negative-selection procedures. The final supernatant containing the aptamer-TXNDC5-MNPs complexes which are not capable of binding to serum and enzymatic-death TXNDC5 is collected. The target-bound aptamers are amplified by PCR and the amplicons of the target-bound aptamers are purified by PCR purification Kit (MinElute PCR purification kit (QIAGEN)) following the manufacturer's instructions. The purified amplicons of the target-bound aptamers are then subcloned into a pGEM-T Easy vector and transformed into DH5a competent cells. The randomly chosen colonies are purified using a High-Speed Plasmid Mini Kit (Geneaid, Taipei, Taiwan) and subjected to sequencing (Genomics, Taiwan). The detail sequences of 14 TXNDC5-bound aptamers are listed in Table 1. TABLE 1List of TXNDC5-hit aptamersNumberSequencesAptamer-1AGC AGC ACA GAG GTC TAG ATG TAA AGG TAC(SEQ IDCTC AGG CGT GCT ACC GTG AANO: 1)Aptamer-2AGC AGC ACA GAG GTC CCT TTA AGG CTT TTG(SEQ IDGTC CGG CGT GCT ACC GTG AANO: 2)Aptamer-3AGC AGC ACA GAG GTC AAT GTA ATC TTT ATC(SEQ IDTAT CGG CGT GCT ACC GTG AANO: 3)Aptamer-4AGC AGC ACA GAG GTC TCG TTT TAC TCT CGT(SEQ IDGTT TGG CGT GCT ACC GTG AANO: 4)Aptamer-5AGC AGC ACA GAG GTC ATC ATC TGG ACT CGG(SEQ IDAAT CGG CGT GCT ACC GTG AANO: 5)Aptamer-6AGC AGC ACA GAG GTC GGT GTA TGA CTT TAT(SEQ IDTTC CGG CGT GCT ACC GTG AANO: 6)Aptamer-7AGC AGC ACA GAG GTC AGG AAC CTT ATG CCT(SEQ IDATG TAG CGT GCT ACC GTG AANO: 7)Aptamer-8AGC AGC ACA GAG GTC CCT ATC AAC CAC ACC(SEQ IDATC TTG CGT GCT ACC GTG AANO: 8)Aptamer-9AGC AGC ACA GAG GTC TAT TGT GAA CTT TTT(SEQ IDCAG CGG CGT GCT ACC GTG AANO: 9)Aptamer-AGC AGC ACA GAG GTC CCT CTC CGG TAT GCT10 (SEQTAT TTG CGT GCT ACC GTG AAID NO:10)Aptamer-AGC AGC ACA GAG GTC TCT TAT TAC TCT CCC11 (SEQGTA CCG CGT GCT ACC GTG AAID NO:11)Aptamer-AGC AGC ACA GAG GTC GAC TCT TGA TTT CCT12 (SEQTGC ATG CGT GCT ACC GTG AAID NO:12)Aptamer-AGC AGC ACA GAG GTC GAC TCT TGA TTT CCT13 (SEQTGC ATG CGT GCT ACC GTG AAID NO:13)Aptamer-AGC AGC ACA GAG GTC ATT CGA TTG TTT TAC14 (SEQAAT TTG CGT GCT ACC GTG AAID NO:14) For further investigating the inhibitory effects of aptamers on TXNDC5 catalytic function, aptamer-3 (Ap-3), aptamer-7 (Ap-7) and aptamer-11 (Ap-11) are representatively selected in the following experiments. Reverse Validation of Isolated Aptamers The 100 nM of aptamer-3, aptamer-7 and aptamer-11 are folded in the binding buffer via the procedures as mentioned above. 20 nM of Aptamer-3, aptamer-7 and aptamer-11 are taken individually and hybridized with positive-(mouse and human TXNDC5, 5 μl) and negative-(serum and catalytic-death TXNDC5, 5 μl) MNPs, and the hybridized aptamers-MNPs are processed according to the above MARAS procedures. Aptamer-3, aptamer-7 and aptamer-11 bound with MNPs are collected and eluted by heating to 95° C. for 5 min in 100 μl ddH2O. The amounts of aptamer-3, aptamer-7 and aptamer-11 are detected by q-PCR and the relative expression levels are utilized as outcomes. The process of detecting the amounts of aptamer-3, aptamer-7 and aptamer-11 by q-PCR is set forth as following. To measure the relative expression of aptamer-3, aptamer-7 and aptamer-11, the q-PCR is performed in 96-well plates with BioRad CFX Connect system. The fivefold-diluted eluted aptamer-3, aptamer-7 and aptamer-11 are individually dissolved in test tubes filled with 100 μl of RNase-free water. The relative expression of aptamer-3, aptamer-7 and aptamer-11 is performed by q-PCR in 96-well plates using BioRad CFX Connect system. A volume of 10 μl q-PCR mixture in the presence of 5 μl 2×_SYBR Green PCR master mix (BioRad), 1 μl of forward aptamer primer (1 Integrated DNA Technologies, IDT), 1 μl of reverse aptamer primer (1 Integrated DNA Technologies, IDT) and 3 μl of eluted aptamers is utilized for q-PCR reaction. The parameters for q-PCR are 95° C. for 3 min; 40 cycles at 94° C. for 30 sec, 60° C. for 30 sec, and 72° C. for 30 sec. As show inFIG.1, aptamer-3, aptamer-7 and aptamer-11 having strong binding with the positive controls and neglectable binding for negative controls are observed. Aptamer-3 reveals the specific binding affinity toward the human-TXNDC5 and the mouse-TXNDC5. Aptamer-11 reveals the specific and strong binding affinity toward the mouse-TXNDC5. Aptamer Structure and Aptamer/TXNDC5 Docking Site Prediction To determine if the TXNDC5-targeting aptamers identified above interact with the catalytic domains of TXNDC5, the 3D structure of aptamer-3, aptamer-7 and aptamer-11 and plausible protein docking sites were predicted using computational modeling. Aptamer structure and aptamer/TXNDC5 docking site prediction is performed in the following procedures. First, the secondary structures of aptamer-3, aptamer-7 and aptamer-11 are predicted by M-fold DNA folding web software (unafold.rna.albany.edu), based on free energy minimization techniques. For setting the parameters of M-fold DNA folding form, the initial sequences of aptamer-3, aptamer-7 and aptamer-11 are set as a linear at a temperature of 25° C. and ionic concentration of 150 mM Na+, 2 mM of Mg2+. The computing is executed in the case that only fold configurations within 5% from the minimum free energy, and it is considered that a maximum number of folds is no limited to the maximum distance between paired bases. Next, the isolated ssDNA aptamer-3, aptamer-7 and aptamer-1 are modified as RNA aptamers and predicted via RNAComposer (rnacomposer.cs.put.poznan.pl) based on the secondary structures. Finally, the docking sites of individual aptamer and structure of wild type human TXNDC5 (I-TASSER, zhanglab.ccmb.med.umich.edu) are predicted through the PatchDock server (bioinfo3d.cs.tau.ac.il). The protein data bank (PDB) code for both aptamer (ligand molecule) and TXNDC5 (receptor molecule) is selected and evaluated at low valve of root mean square deviation (RMSD, 4 Å) with enzyme-inhibitor type. The results of prediction are shown inFIGS.2A,2B,3A,3B,4A,4B, it is observed that all three individual aptamers exhibit single (Ap-11) or double stem-loop at its 3′ and 5′ ends (Ap-3 and -7) structures with free energy values (Ap-3, ΔG=−4.29 kcal/mol, Ap-7, −2.66 kcal/mol, Ap-11, −7.20 kcal/mol). The result of predictions of 3D structures and docking sites demonstrate that aptamer-3, aptamer-7 and aptamer-11 anchor robustly on the wild type human TXNDC5 (FIG.2A-4B), and the binding positions for aptamer-3, aptamer-7 and aptamer-11 may dock approximately to the catalytic thioredoxin (CGHC) domains, it further reveals the potential of TXNDC5-targeting aptamers as potent antagonists against the disulfide isomerase activity of TXNDC5. Dissociation Constant (Kd) of Aptamer-3 and Aptamer-7 For further determining the binding efficiency of TXNDC5-targeting aptamers, the Kdvalues, as the indicator of aptamer and target molecule interaction, of aptamer-3 and aptamer-7 are measured by q-PCR, and the results were fitted with nonlinear regression. The method of measuring the Kdvalues of aptamer-3 and aptamer-7 is illustrated as following. Aptamer-3 and aptamer-7 are isolated and progressively diluted in series from 100 to 3.125 nM, aptamer-3 and aptamer-7 are folded in the binding buffer as the procedures mentioned above. Equal amounts of either mouse-MNPs or humane-MNPs are mixed with aptamer-3 and aptamer-7 and followed the MARAS and q-PCR procedure as described above to specifically bind to aptamer-3 and aptamer-7. The Kdvalues of aptamer-3 and aptamer-7 are measured by fitting the results of each Ct value from q-PCR and the concentrations in a nonlinear regression through PRISM 8 (Graphpad). Each result is performed in duplicate to reduce the errors. As shown inFIGS.5A and5B, the Kdvalue of Ap-3 for mouse TXNDC5 is 7.504 nM and the Kdvalue of Ap-7 for human TXNDC5 is 16.9 nM, it is demonstrated that the TXNDC5-targeting aptamers are capable of binding with TXNDC5 protein sensitively. Insulin Reduction Assay For determining the potency of individual aptamer on inhibiting the disulfide isomerase activity of TXNDC5, an insulin turbidimetric assay is performed. For performing the insulin turbidimetric assay, two isolated thioredoxin domains of TXNDC5 (Trx1 and Trx2) and TXNDC5 protein are prepared as following. TXNDC5 individual domains, Trx1 and Trx2, which have relative highest catalytic functions, are synthesized at the Yao-Hong Biotechnology Inc (Taiwan) with a purity grade of >85% validating through high performance liquid chromatography (HPLC). The purified Trx1 and Trx2 peptides which represent in the powder form are resuspended in 1:3 Acetonitrile/H2O mixture at 1 mg/ml concentration. TXNDC5 Trx1 sequences: skhlytadm fthgiqsaah fvmffapwcg hcqrlqptwn dlgkynsme dakvyvakvd ctahsdvcsa qgvrgyptlk lfkpgqeavk yqgprdfqtl enwmlqtlne (SEQ ID NO: 17); TXNDC5 Trx2 sequences: g lyelsanfe lhvaqgdhfi kffapwcghc kalaptweql alglehsetv kigkvdctqh yelcsgnqvr gyptllwfrd gkkvdqykgk rdleslreyv esqlqrte (SEQ ID NO: 18). High purity of wild type human-TXNDC5, mouse-TXNDC5 and catalytic mutated human-TXNDC5 are generated through baculovirus expression vector system at Sino Biological (Biotools, Taiwan). First, the flanking selected restriction fragments of TXNDC5 cDNA are added by PCR (provided by Sino Biological Inc), then shuttled to the baculovirus vector, the baculovirus vector containing TXNDC5 cDNA are transfected into multiple insect cells for encoding desired entire TXNDC5 protein. Various recombinant TXNDC5 proteins are purified from the soluble fractions of the cell lysates using Ni-purification column. The fractions containing desired entire TXNDC5 are enriched and further dissolved in the formulation buffer (20 mM PBS, 300 mM NaCl, 10% glycerol, pH 7.5). Purified TXNDC5 proteins are aliquoted and stored at −80° C. or processed for assays. Before performing the insulin reduction tubidometric assay for measuring the effects of aptamer titration toward TXNDC5, the optimization of the reductase concentrations of TXNDC5 Trx1 and Trx2 or entire TXNDC5 protein for insulin turbidimetric assay is established according to the method of Smith, A. M. et al. (Smith, A. M. et al. A high-throughput turbidometric assay for screening inhibitors of protein disulfide isomerase activity.J. Biomol. Screen.9, 614-620, doi:10.1177/1087057104265292 (2004)) to achieve a significant signal-to-noise ratio (SNR). The assays are carried out in 384-well plate (Greiner) and a volume of 30 μl of the solution in the presence of final concentrations of 0.16 mM insulin (Sigma-Aldrich) and the reductase. Entire TXNDC5 protein (human or mouse) (33, 1 and 0.02 μg/ml,FIG.6A) or TXNDC5 peptide (Trx1 or Trx2) (5.6, 2.8, 1.12 and 0.28FIG.6C) varies concentrations in an assay buffer (100 mM potassium phosphate and 0.2 mM EDTA, pH 7.0). The 5 μl of 3.5 mM DTT in the final concentration 0.5 mM is added to initiate the reaction, and the reaction is monitored at 650 nm on a Synergy HTX Multi-mode reader (BioTek) for 90 mins at 37° C. The accumulated OD650nmand lag time are presented for validating the optimized concentration of reductases. When a 3-fold to 6-fold SNR is obtained, the indicated concentration of the reductases is decided for subsequent insulin reduction tubidometric assays. The results of the optimization of the reductase concentrations of TXNDC5 Trx1 and Trx2 or entire TXNDC5 protein for insulin turbidimetric assay are shown inFIGS.6A-6D, highest dose of TXNDC5 (33 μg/ml) exhibits about 5%, 20% and 40% increase in the end-point turbidities comparing to 5.5 μg/ml, 1 μg/ml and 0.02 μg/ml of TXNDC5, respectively (FIG.6A). The isomerase reduction reactions were accelerated in a dose-dependent manner as the dramatic turbidity, the isomerase reduction reactions were obtained within 10 mins after addition of dithiothreitol (DTT) at highest amount of TXNDC5. The onset times of chemical reduction of insulin are markedly delayed approximately up to 2250 secs with diluted dose of TXNDC5 (FIG.6B), and the onset times of chemical reduction of insulin are markedly delayed approximately up to 3700 secs with diluted dose of Trx1 and Trx2 (FIG.6D). For individual Trx domains of TXNDC5, Trx1 and Trx2, both domains display relative longer reaction time and weaker kinetic reactions when they compare with full-length TXNDC5 protein (FIG.6A-6D), as entire TXNDC5 protein could catalyze reductive reaction faster (thereby with shorter onset time) in a dose-dependent manner (FIGS.6B and6D). To validate that the formation of precipitated insulin chains were resulted from catalytic functions of TXNDC5, TXNDC5 Trx1 and Trx2 or entire TXNDC5 protein reacts with hydrogen peroxide (H2O2) or protein disulfide isomerase inhibitor 16F16 (50 μm), separately. The reductase activities of TXNDC5 Trx1 and Trx2 and entire TXNDC5 protein are assayed by insulin reduction assay. The assays are carried out in 384-well plate (Greiner), and a volume of 30 μl of the solution in the presence of final concentrations of 0.16 mM insulin (Sigma-Aldrich) and the reductase is prepared. 5.6 μM TXNDC5 peptides (Trx1 and Trx 2,FIG.7A), μg/ml entire TXNDC5 protein (FIG.7B) or 10 μs protein from each cell lysates (human hepatic stellate cell LX2 with wild-type or enzymatic-death mutant TXNDC5 (AAA),FIG.7C) varies concentrations in assay buffer (100 mM potassium phosphate and 0.2 mM EDTA, pH 7.0). To further validate that the formation of precipitated insulin chain are resulted from catalytic functions of TXNDC5, the 5 μl of H2O2in the final concentration 125 mM or 5 μl of 16F16 in the final concentration 50 μM (protein disulfide isomerase inhibitor) is added into 25 μl of reductases/insulin/assay buffer mixture. The 5 μl of 3.5 mM DTT (the final concentration is 0.5 mM) is added into the above mixture to initiate the reaction, and the above reaction is monitored at 650 nm on a Synergy HTX Multi-mode reader (BioTek) for 90 mins at 37° C. The absorbance at 650 nm (OD650nm) is measured in 5-min increments throughout 90 mins at 37° C. The accumulated OD650nmis presented as the enzyme kinetic ability. The LX2 is obtained from Dr. Tung-Hung Su at National Taiwan University Hospital, Taiwan. This cell line is settled in the DMEM containing 10% FBS and 1% penicillin/streptomycin and then is incubated in an incubator with well-controlled of 95% O2and 5% CO2circulation at 37° C. Hydrogen peroxide (H2O2) is able to halt the reaction by depletion of the reductase. Protein disulfide isomerase inhibitor 16F16 is able to diminish the catalytic activity of TXNDC5. Meanwhile, further evaluating the influence of TXNDC5 catalytic domain architectures, the fractions of liver cells (Human hepatic stellate cell LX2), transduced with wild-type or enzymatic-death mutant TXNDC5 (AAA) expressed by Lentiviral transduction system, were subjected to the kinetic reduction assay. The kinetic reduction assay is performed referring to the insulin reduction assay described above. Lentiviral transduction system is employed for ectopic expressing control (pLAS2w.pPuro), human wild type (pLAS2w.pPuro-TXNDC5) or enzymatic-death (pLAS2w.pPuro-TXNDC5-AAA) TXNDC5 within interested cell lines with multiplicity of infection (MOI) of 15, and the interested cell lines are harvested for 24 hrs in Polybrene-contained (8 μg/ml), serum-free DMEM media for boosting the desired protein production. After carefully aspirating the old DMEM media and replacing with the fresh DMEM media, the puromycin (0.5 ng/ml) is used to refine the transduced cells for coming processes. Cells are homogenized using 1× Cell Lysis Buffer (Cell Signaling Technology, MA, USA) supplemented with protease inhibitor cocktail and HALT phosphatase inhibitors (Thermo Fisher Scientific, MA, USA), the cells processed as above are then centrifuged at 4° C. for 10 min at 10,000×g, and the supernatant is collected. The concentration of protein lysate is determined by BCA protein assay. A total of 10 μs of each protein sample is diluted in ddH2O (final volume, 5 μl) for the insulin reduction assay. The results of the above reaction of TXNDC5 Trx1 and Trx2 or entire TXNDC5 protein with H2O2or 16F16 are shown inFIGS.7A-7C. The kinetic reactions of both entire TXNDC5 proteins and isolated Trx1 and Trx2 domains with H2O2were completely and effectively terminated (FIGS.7Aand B). As shown inFIG.7B, the plateau of the end-point kinetic absorbance is declined following addition of 16F16, it is demonstrated that the catalytic ability is required for the reductive cleavage of the interchain disulfide bonds in the insulin. As shown inFIG.7C, cells with ectopic TXNDC5 expression exhibit higher rate of precipitation of insulin. However, such accelerated reactions were obliterated in the cell lysates containing enzyme-dead mutant TXNDC5 or with the addition of H2O2. The procedure for measuring the effects of aptamers (aptamer-3, aptamer-7 and aptamer-11) titration toward TXNDC5 is set forth as following. Step 1 Each of aptamer-3, aptamer-7 and aptamer-11 (60 μl) are mixed in the binding buffer (20 μl) separately, the mixture of aptamer and the binding buffer is heated to 95° C. for 5 min, snap cooled at 4° C. to form 2ndstructures, and maintained at 25° C. for 30 min. Step 2 A half-folded serial dilution of each aptamer mixture is made using 40 μl of the binding buffer as the diluent. Each of aptamer-3, aptamer-7 and aptamer-11 is prepared as various diluents with different concentrations, 7.8125 μM, 15.625 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM. Step 3 Each of 40 μl diluents (with different concentrations, 7.8125 μM, 15.625 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM) of aptamer-3, 7 and 11 is incubated with 5 μl human wild type TXNDC5 (0.4 μl human wild type TXNDC5 (3.125 μg/ml) is diluted in 5 μl) for 30 min with vibrating separately. Step 4 An insulin/assay buffer is prepared by adding 1.6 mM insulin (which is prepared by dissolving insulin in 0.1 N HCl) with the assay buffer consisted of 100 mM potassium phosphate and 0.2 mM EDTA (pH 7). The volume of each insulin/assay buffer is 15 μl, wherein the volume of insulin is 6 μl and the volume of assay buffer is 9 μl. Step 5 Each of the above mixtures of aptamers (Ap-3, Ap7 and Ap-11) with different concentrations and human wild type TXNDC5 is added with a corresponding insulin/assay buffer separately. That is, aptamer-3, aptamer-7 and aptamer-11 have seven experimental group samples (with different concentrations, 7.8125 μM, 15.625 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM) separately. An insulin/assay buffer which is not added with the above mixture of aptamers and human wild type TXNDC5 is prepared as the control group sample. Step 6 All of the experimental group samples and the control group sample are added at 30 μl per well into a 384-well assay plate. 5 μl of 3.5 mM DTT is subsequently added into each of the experimental group samples and the control group sample and quickly mixed to initiate a reaction. Step 7 The enzyme reactions of the experimental group samples and the control group sample are monitored at 650 nm on a Synergy HTX Multi-Mode Reader (BioTek). The results of the enzyme reactions are shown inFIGS.8A-8F; all three aptamers aptamer-3, aptamer-7 and aptamer-11 inhibited the human TXNDC5 activity in a dose-dependent manner with IC50 values of 213.7 μM for aptamer-3 (B), 241.0 μM for aptamer-7 (D) and 263.2 μM for aptamer-11 (F). It is demonstrated that TXNDC5-targeting aptamers are capable of inhibiting the disulfide isomerase activity of TXNDC5. In Vitro Bioactivity Assay For determining the bioactivity of the TXNDC5-targeting DNA aptamers to TXNDC5 in fibroblasts cells, an in vitro bioactivity assay is performed as following. First, 3T3 cells (ATCC, CRL-1658) are cultured with the cell density of 5,000 cells/cm 2 in the cell culture dish, the 3T3 cells are then suspended in 6,400 μl of Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), the 3T3 cells are inoculated in each well of four 8 wells chamber slides (iBidi, 80841), each well contains 200 μl of the above DMEM and 5,000 cells, and the 8 wells chamber slides are placed in an incubator under 37° C. overnight. On the next day, the medium in each well of each chamber slide is completely suck, and then 100 μl of FBS-free DMEM medium is added into each well of each chamber slide. Afterwards, the 8 wells chamber slides are placed in the incubator under 37° C. for 3 hours. After the 8 wells chamber slides are placed in the incubator under 37° C. for 3 hours, FAM_apt7_dT (the fluorescent protein FAM is conjugated to 5′ end of aptamer 7, and 3′ end of aptamer 7 is modified with dT) is added into each well of three chamber slide. Those chamber slides where FAM_apt7_dT has been added into are designated as the first experimental group, the second experimental group and the third experimental group. The chamber slide where FAM_apt7_dT has not been added into is designated as the control group. After FAM_apt7_dT has been added into the first experimental group for 6 hours, the medium in each well of the first experimental group is completely suck, and the cell fractions in each well of the first experimental group are washed out for 5 minutes by PBS, 4% Paraformaldehyde (PFA) is added into each well of the washed first experimental group for 15 minutes to fix 3T3 cells in each well of the washed first experimental group. After 4% Paraformaldehyde has been added into the first experimental group for 15 minute, each well of the first experimental group is washed out for 5 minutes by PBS with repeated 3 times and tenfold-diluted permeabilization buffer (Abcam, ab219801) is added into each well of the washed first experimental group for 20 minutes. After tenfold-diluted permeabilization buffer has been added into the first experimental group for 20 minute, each well of the first experimental group is washed out for 5 minutes by PBS with repeated 3 times and 200 μl/well of 5% bovine serum albumin (BioFroxx) blocking buffer is added into each well of the washed first experimental group for 1 hour at room temperature. After 200 μl/well of 5% bovine serum albumin (BioFroxx) blocking buffer has been added into the first experimental group for 1 hour, each well of the first experimental group is washed out for 5 minutes by PBS with repeated 3 times and primary antibody, TXNDC5 polyclonal antibody (Proteintech, 19834-1-AP) (diluted with the blocking buffer in a 1:100 dilution) is added into each well of the washed first experimental group (the final concentration of TXNDC5 polyclonal antibody is 6.5 μg/ml) overnight at 4° C. After TXNDC5 polyclonal antibody has been added into the first experimental group overnight, each well of the first experimental group is washed out for 5 minutes by PBS with repeated 3 times and secondary antibody, donkey anti-rabbit, Alexa Flour 555 (Invitrogen, A-31572) (diluted with the blocking buffer in a 1:500 dilution) is added into each well of the washed first experimental group (the final concentration of secondary antibody is 4 μg/ml) for 2 hours at room temperature, thereby TXNDC5 in the first experimental group is labeled. After the secondary antibody has been added into the first experimental group for 2 hours, each well of the first experimental group is washed out for 5 minutes by PBS with repeated 3 times, the liquid left over in each well of the washed first experimental group (main liquid is PBS) is further removed. After the liquid left over in the first experimental group is removed, the mounting medium (Southern Biotech, 0100-20) which contains 4′,6-diaminndino-2-phenylinndole (DAPI) is used for labeling the nucleus of 3T3 cells in the first experimental group. The image of the stained 3T3 cells is taken by the fluorescence microscope EVOS M7000 (Invitrogen). The above in vitro bioactivity assay is repeated two times. The second experimental group, the third experimental group and the control repeat the process procedure as the first experimental group as mentioned above. The only different experimental condition between the first experimental group, the second experimental group, the third experimental group and the control group is the reaction time when FAM_apt7_dT has been added into the first experimental group, the second experimental group or the third experimental group. FAM_apt7_dT has been added into the second experimental group for 24 hours, FAM_apt7_dT has been added into the third experimental group for 48 hours, and FAM_apt7_dT has not been added into the control group. The results of the in vitro bioactivity assay are shown inFIGS.9A-9D. FAM′ labeled DNA aptamers (green-colored) were uptaken by 3T3 cells from 6 hr, colocalized with TXNDC5 (red-colored) and remained detectable in the cytoplasm at 48 hr. The results ofFIGS.9A-9Dreveal that these DNA aptamers can be freely uptaken by fibroblasts and interact with TXNDC5. Meanwhile, the intracellular stability of these DNA aptamers could be maintained for at least 48 hours The above results also suggest that DNA aptamers could directly interact with TXNDC5 without any adjuvant. Anti-Fibrotic Effects Assay For determining the anti-fibrotic effects of the TXNDC5-targeting DNA aptamers in fibroblasts cells, an anti-fibrotic effects assay is performed as following. First, 3T3 cells (ATCC, CRL-1658) are cultured with the cell density of cells/cm 2 in the cell culture dish, the 3T3 cells are then suspended in 3,200 μl of Dulbecco's Modified Eagle Medium(DMEM) containing 10% fetal bovine serum (FBS), the 3T3 cells are inoculated in each well of two 8 wells chamber slides (iBidi, 80841), each well contains 200 μl of the above DMEM and 5,000 cells, and the 8 wells chamber slides are placed in an incubator under 37° C. overnight. One of the chamber slides is designated as the 24-hrs group, every two wells of the 24-hrs group are designated as the first experimental group, the second experimental group, the third experimental group and the control group separately. Another chamber slide is designated as the 48-hrs group, the 48-hrs group also has the first experimental group, the second experimental group, the third experimental group and the control group as the 24-hrs group. On the next day, the medium in each well of the 24-hrs group and the 48-hrs group is completely suck, and then 50 μl of FBS-free DMEM medium is added into each well of the 24-hrs group and the 48-hrs group. Afterwards, the 24-hrs group and the 48-hrs group are placed in the incubator under 37° C. for 3 hours. After the 24-hrs group and the 48-hrs group are placed in the incubator under 37° C. for 3 hours, TGFβ (final dose: 10 μg/ml) is added into each well of the first experimental group of the 24-hrs group and the 48-hrs group; TGFβ (final dose: 10 μg/ml) and non-targeting aptamer (Spt_dT) (final dose: 5 μg/ml) are added together into each well of the second experimental group of the 24-hrs group and the 48-hrs group; TGFβ (final dose: 10 μg/ml) and FAM_apt7_dT aforementioned (final dose: 5 μg/ml) are added together into each well of the third experimental group of the 24-hrs group and the 48-hrs group; nothing is added into each well of the control group of the 24-hrs group and the 48-hrs group. After the first experimental group, the second experimental group, the third experimental group and the control group of the 24-hrs group is treated as mentioned above for 24 hours, the medium in each well of the 24-hrs group is completely suck, and the cell fractions in each well of the 24-hrs group are washed out for 5 minutes by PBS, 4% PFA is added into each well of the washed 24-hrs group for 15 minutes to fix 3T3 cells in each well of the washed 24-hrs group. After 4% PFA has been added into the 24-hrs group for 15 minute, each well of the 24-hrs group is washed out for 5 minutes by PBS with repeated 3 times and tenfold-diluted permeabilization buffer (Abcam, ab219801) is added into each well of the washed 24-hrs group for 20 minutes. After tenfold-diluted permeabilization buffer has been added into the 24-hrs group for 20 minute, each well of the 24-hrs group is washed out for 5 minutes by PBS with repeated 3 times and 200 μl/well of 5% bovine serum albumin (BioFroxx) blocking buffer is added into each well of the washed 24-hrs group for 1 hour at room temperature. After 200 μl/well of 5% bovine serum albumin (BioFroxx) blocking buffer has been added into the 24-hrs group for 1 hour, each well of the 24-hrs group is washed out for 5 minutes by PBS with repeated 3 times and primary antibody, αsmooth actin (αSMA) polyclonal antibody (Abcam, ab5694) (diluted with the blocking buffer in a 1:100 dilution) is added into each well of the washed 24-hrs group (the final concentration of αSMA polyclonal antibody is 2 μg/ml) overnight at 4° C. After αSMA polyclonal antibody has been added into the 24-hrs group overnight, each well of the 24-hrs group is washed out for 5 minutes by PBS with repeated 3 times and secondary antibody, donkey anti-rabbit, Alexa Flour 555 (Invitrogen, A-31572) (diluted with the blocking buffer in a 1:500 dilution) is added into each well of the washed 24-hrs group (the final concentration of secondary antibody is 4 μg/ml) for 2 hours at room temperature, thereby αSMA in the 24-hrs group is labeled. After the secondary antibody has been added into the 24-hrs group for 2 hours, each well of the 24-hrs group is washed out for 5 minutes by PBS with repeated 3 times, the liquid left over in each well of the washed 24-hrs group (main liquid is PBS) is further removed. After the liquid left over in the 24-hrs group is removed, the mounting medium (Southern Biotech, 0100-20) which contains DAPI is used for labeling the nucleus of 3T3 cells in the 24-hrs group. The image of the stained 3T3 cells in the 24-hrs group is taken by the fluorescence microscope EVOS M7000 (Invitrogen). The above anti-fibrotic effects assay in the 24-hrs group is repeated three times. The 48-hrs group repeats the process procedure as the 24-hrs group as mentioned above. The only different experimental condition between the 24-hrs group and the 48-hrs group is the reaction when TGFβ, FAM_apt7_dT, Spt_dT or their combination have been added into the 24-hrs group and the 48-hrs group. The reaction time of the 24-hrs group is 24 hours, and the reaction time of the 48-hrs group is 48 hours. The results of the anti-fibrotic effects assay are shown inFIGS.10A and10B.FIG.10Ashows that aptamer 7 inhibits the expression of αSMA which is a marker for fibroblast activation and myofibroblast transdifferentiation in 3T3 fibroblast cells treated with TGFβ stimulation. Compared with the control group, the first experimental group (TGFβ-only group) and the second experimental group (Spt_dT group) significantly express αSMA in 3T3 cells at both 24th and 48th hour, but the third experimental group (FAM_Ap7_dT group) significantly suppresses αSMA in 3T3 cells at both 24th and 48th hour.FIG.10Bshows the αSMA expression rates of the first experimental group, the second experimental group and the third experimental group, the symbol “ns” inFIG.10Bmeans that the data is non-significant, the asterisk inFIG.10Bmeans P value (* P<0.05, ** P<0.01). The treatment of TXNDC5-targeting DNA aptamer (FAM_Apt_dT), but not non-targeting aptamer (Spt_dT), markedly repressed αSMA expression in 3T3 cells in response to TGFβ stimulation, both at 24 and 48 hr. In-Cell Western Assay For determining the inhibitory effect of TXNDC5-targeting DNA aptamer on the cellular expression levels of fibronectin, an in-cell Western assay is performed as following. First, 3T3 cells (ATCC, CRL-1658) are cultured with the cell density of 5,000 cells/cm 2 in the cell culture dish, the 3T3 cells are then suspended in 4,800 μl of Dulbecco's Modified Eagle Medium(DMEM) containing 10% fetal bovine serum (FBS), the 3T3 cells are inoculated in 64 wells of a 96 wells plate, each well contains 100 μl of the above DMEM and 1,600 cells, and the 96 wells plate is placed in an incubator under 37° C. overnight. At the same time, the aptamer for the present in-cell Western assay is prepared. The preparing process of the aptamer is performing as following. 15.75 μl of FAM_apt_dT stock (100 μM), 1 μl of Tris-Cl (400 mM), 2 μl of NaCl (1.5 μM), 1 μl of KCl (1 μM), 0.04 μl of MgCl2(1 μM), 0.2 μl of CaCl2(100 mM) and 0.01 μl of Tween-20 are mixed. The final volume of the above mixture is 20 μl, and the concentration of FAM_apt_dT in the mixture is 78.75 μM. The mixture is then heated to 95° C. for 5 minutes. After the mixture is heated for 5 minutes, the mixture is cooled down at 4° C. for 30 sec and then placed under 25° C. for 30 minutes to make the aptamer folded. FAM_apt_dT stock at a concentration of 78.75 μM is prepared according to the above process. For obtaining the FAM_apt_dT solution at different concentrations, 70 μl of FAM_apt_dT stock (78.75 μM) is mixed with 70 μl of the binding solution to prepare FAM_apt_dT solution at a concentration of 39.375 μM, the FAM_apt_dT solution at the concentration of 39.375 μM is then sequentially diluted according to the previous manner to prepare FAM_apt_dT solution at a concentration of 19.68 μM, 9.84 μM, 4.92 μM, 2.4604 and 1.23 μM separately. On the next day, the medium in the 64 wells of the above 96 wells plate is completely suck, and then 50 μl of FBS-free DMEM medium is added into each of the above 64 wells. Afterwards, the above 96 wells plate is placed in the incubator under 37° C. for 3 hours. After the above 96 wells plate has been placed in the incubator under 37° C. for 3 hours, the 64 wells of the above 96 wells plate are treated with different conditions. 32 wells of the above 96 wells plate are designated as the control group, the 3T3 cells of the control group are not treat with TGFβ. The other 32 wells of the 96 wells plate are designated as the experimental group, the 3T3 cells of the experimental group are treat with 10 μg/ml of TGFβ. Both the experimental group and the control group (which contains 50 μl of FBS-free DMEM medium) are treated with FAM_apt_dT as following. 70 μl of FAM_apt_dT at the concentration of 78.75 μM is added into each of the first four wells of the experimental group/the control group contain (the final concentration of FAM_apt_dT is 20 μM); 70 μl of FAM_apt_dT at the concentration of 39.375 μM is added into each of the second four wells of the experimental group/the control group (the final concentration of FAM_apt_dT is 10 μM); 70 μl of FAM_apt_dT at the concentration of 19.68 μM is added into each of the third four wells of the experimental group/the control group (the final concentration of FAM_apt_dT is 5 μM); 70 μl of FAM_apt_dT at the concentration of 9.84 μM is added into each of the fourth four wells of the experimental group/the control group (the final concentration of FAM_apt_dT is 2.5 μM); 70 μl of FAM_apt_dT at the concentration of 4.92 μM is added into each of the fifth four wells of the experimental group/the control group (the final concentration of FAM_apt_dT is 1.25 μM); 70 μl of FAM_apt_dT at the concentration of 2.46 μM is added into each of the six four wells of the experimental group/the control group (the final concentration of FAM_apt_dT is 0.625 μM); 70 μl of FAM_apt_dT at the concentration of 1.23 μM is added into each of the seventh four wells of the experimental group/the control group (the final concentration of FAM_apt_dT is 0.3125 μM); each of the eighth four wells of the experimental group/the control group does not added with FAM_apt_dT. After the experimental group and the control group have been treated as above, the experimental group and the control group are then placed in the incubator under 37° C. for 72 hours. After the experimental group and the control group have been placed in the incubator for 72 hours, the medium in each well of the experimental group and the control group is completely suck, the cell fractions in each well of the experimental group and the control group are washed out for 5 minutes by PBS with repeated 3 times, and then 4% PFA is added into each well of the washed experimental group and the washed control group for 20 minutes to fix 3T3 cells in each well of the experimental group and the control group. After 4% PFA has been added into the experimental group and the control group for 20 minute, each well of the experimental group and the control group is washed out for 5 minutes by PBS with repeated 3 times, and tenfold-diluted permeabilization buffer (Abcam, ab219801) is added into each well of the washed experimental group and the washed control group for 20 minutes. After tenfold-diluted permeabilization buffer has been added into the experimental group and the control group for 20 minute, each well of the experimental group and the control group is washed out for 5 minutes by PBS with repeated 3 times and 150 μl/well of LI-COR blocking buffer is added into each well of the experimental group and the control group for 1 hour at room temperature. After 150 μl/well of LI-COR blocking buffer has been added into the experimental group and the control group for 1 hour, each well of the experimental group and the control group is washed out for 5 minutes by PBS with repeated 3 times and primary antibody, fibronectin monoclonal antibody (BD Biosciences, 610077) (diluted with the LI-COR blocking buffer in a 1:100 dilution) is added into each well of the experimental group and the control group (the final concentration of fibronectin monoclonal antibody is 2.5 μg/ml) overnight at 4° C. After fibronectin monoclonal antibody has been added into the experimental group and the control group overnight, each well of the experimental group and the control group overnight is washed out for 5 minutes by PBS with repeated 3 times. Afterward, secondary antibody, goat-anti Mouse IRDye 800CW (diluted with the LI-COR blocking buffer in a 1:1000 dilution) and Celltag 700 (diluted with the LI-COR blocking buffer in a 1:500 dilution) are together added into each well of the experimental group and the control group (the final concentration of secondary antibody is 1 μg/ml, the final concentration of Celltag 700 is 0.2 μg/ml) for 2 hours at room temperature, thereby fibronectin and the number of 3T3 cells in the experimental group and the control group is detected. The experimental group and the control group are scanned and analyzed by Odyssey CLx infrared imaging system. IC50of the experimental group and the control group are calculated by Prism. The results of the in-cell Western assay are shown inFIGS.11A and11B.FIGS.11A and11Bshows that a strong inhibitory effect of TXNDC5-targeting DNA aptamer on the cellular expression levels of fibronectin, a critical ECM protein during fibrogenesis, either without TGFβ (IC505.389 μM) or with TGFβ (IC504.12 μM) stimulation. As disclosed above, TXNDC5-targeting aptamers are capable of binding with TXNDC5 protein and inhibiting the catalytic ability of TXNDC5. The previous studies also identified that targeting TXNDC5 is an effective therapeutic approach to prevent or treat organ fibrosis (such as cardiac fibrosis, liver fibrosis, renal fibrosis and pulmonary fibrosi), heart failure and chronic kidney diseases. Therefore, a pharmaceutical composition comprising the TXNDC5-targeting aptamers as an active ingredient is able to be utilized to prevent or treat organ fibrosis, heart failure and chronic kidney diseases. The above pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, such as normal saline, nanoparticles or any known carrier suitable for aptamers. Meanwhile, a method for preventing or treating organ fibrosis can be provided, the method comprises administering an effective amount of the TXNDC5-targeting aptamer to a subject in need thereof. While the present disclosure has been described by means of specific embodiments, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope and spirit of the present disclosure set forth in the claims. | 44,865 |
11859189 | DETAILED DESCRIPTION The present disclosure provides recombinant bacterial cells that have been engineered with optimized genetic circuitry which allow the recombinant bacterial cells to turn on and off an engineered metabolic pathway by sensing a patient's internal environment or by chemical induction during, for example, manufacturing. When turned on, the recombinant bacterial cells complete all of the steps in a metabolic pathway to achieve a therapeutic effect in a host subject and are designed to drive therapeutic effects throughout the body of a host from a point of origin of the microbiome. Specifically, the present disclosure provides recombinant bacterial cells, pharmaceutical compositions thereof, and methods of modulating and treating diseases associated with amino acid metabolism, such as homocystinuria. Specifically, the recombinant bacteria disclosed herein have been constructed to comprise genetic circuits composed of, for example, a methionine decarboxylase to treat disease, as well as other circuitry in order to guarantee the safety and non-colonization of the subject that is administered the recombinant bacteria, such as auxotrophies, etc. These recombinant bacteria are safe and well tolerated and augment the innate activities of the subject's microbiome to achieve a therapeutic effect. In some embodiments, a bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene sequence encoding one or more methionine decarboxylases and is capable of processing (e.g., metabolizing) and reducing levels of methionine. In some embodiments, a bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene sequence encoding one or more methionine decarboxylases and is capable of processing and reducing levels of methionine in low-oxygen environments, e.g., the gut. Thus, the genetically engineered bacterial cells and pharmaceutical compositions comprising the bacterial cells disclosed herein may be used to convert excess methionine into non-toxic molecules in order to treat and/or prevent diseases associated with amino acid metabolism, such as homocystinuria, cystinuria, primary and secondary hypermethioninemia, cancer, and metabolic syndromes/diseases. In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description. As used herein, the term “recombinant bacterial cell” or “recombinant bacteria” (also referred to herein as a “genetically engineered bacterial cell”) refers to a bacterial cell or bacteria that have been genetically modified from their native state. Similarly, “recombinant microorganism” (or genetically engineered microorganism), or “recombinant host cell” (or genetically engineered host cell), refers to a microorganism or host cell that has been genetically modified from their native state. For instance, a recombinant bacterial cell, microorganism, or host cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria, bacterial cell, microorganism, or host cell or on a plasmid in the bacteria, bacterial cell, microorganism, or host cell. Recombinant bacterial cells, microorganisms, or host cells of the disclosure may comprise exogenous or heterologous nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells, microorganisms, or host cells may comprise exogenous or heterologous nucleotide sequences stably incorporated into their chromosome(s). As used herein, the term “gene” refers to a nucleic acid fragment that encodes a protein or fragment thereof, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. In one embodiment, a “gene” does not include regulatory sequences preceding and following the coding sequence. A “native gene” refers to a gene as found in nature, optionally with its own regulatory sequences preceding and following the coding sequence. A “chimeric gene” refers to any gene that is not a native gene, optionally comprising regulatory sequences preceding and following the coding sequence, wherein the coding sequences and/or the regulatory sequences, in whole or in part, are not found together in nature. Thus, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source, but arranged differently than is found in nature. As used herein the term “gene” is also meant to include a codon-optimized gene sequence, which is modified from a native gene sequence, e.g., to reflect the typical codon usage of the host organism, without altering the polypeptide encoded by a gene or nucleic acid molecule. As used herein, the term “gene” may also refer to a gene sequence which encodes a polypeptide that is not naturally occurring. For example, a gene may encode a polypeptide which is derived from a library of engineered, non-naturally occurring polypeptides. As used herein, the term “gene sequence” is meant to refer to a genetic sequence, e.g., a nucleic acid sequence. The gene sequence or genetic sequence is meant to include a complete gene sequence or a partial gene sequence. The gene sequence or genetic sequence is meant to include sequence that encodes a protein or polypeptide and is also meant to include genetic sequence that does not encode a protein or polypeptide, e.g., a regulatory sequence, leader sequence, signal sequence, or other non-protein coding sequence. As used herein, a “heterologous gene” or “heterologous sequence” refers to a nucleotide sequence that is not normally found in a given cell in nature. As used herein, a heterologous sequence encompasses a nucleic acid sequence that is exogenously introduced into a given cell. “Heterologous gene” includes a native gene, or fragment thereof, that has been introduced into the host cell in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene that is reintroduced into the host cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non-native host cell. Alternatively, a heterologous gene may also include a native gene, or fragment thereof, which has been edited within a host cell. Thus, a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and/or the polypeptide which it encodes; and/or two or more nucleic acid sequences that are not found in the same relationship to each other in nature. As used herein, the term “endogenous gene” refers to a native gene in its natural location in the genome of an organism. As used herein, the term “transgene” refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome. As used herein, the term “bacteriostatic” or “cytostatic” refers to a molecule or protein which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of a recombinant bacterial cell of the disclosure. As used herein, the term “bactericidal” refers to a molecule or protein which is capable of killing the recombinant bacterial cell of the disclosure. As used herein, the term “toxin” refers to a protein, enzyme, or polypeptide fragment thereof, or other molecule which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of the recombinant bacterial cell of the disclosure, or which is capable of killing the recombinant bacterial cell of the disclosure. The term “toxin” is intended to include bacteriostatic proteins and bactericidal proteins. The term “toxin” is intended to include, but not limited to, lytic proteins, bacteriocins (e.g., microcins and colicins), gyrase inhibitors, polymerase inhibitors, transcription inhibitors, translation inhibitors, DNases, and RNases. The term “anti-toxin” or “antitoxin,” as used herein, refers to a protein or enzyme which is capable of inhibiting the activity of a toxin. The term anti-toxin is intended to include, but not limited to, immunity modulators, and inhibitors of toxin expression. Examples of toxins and antitoxins are known in the art and described in more detail infra. As used herein, the term “coding region” refers to a nucleotide sequence that codes for a specific amino acid sequence. The term “regulatory sequence” refers to a nucleotide sequence located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing, RNA stability, or translation of the associated coding sequence. Examples of regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, and stem-loop structures. In one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter. “Operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. A regulatory element is operably linked with a coding sequence when it is capable of affecting the expression of the gene coding sequence, regardless of the distance between the regulatory element and the coding sequence. More specifically, operably linked refers to a nucleic acid sequence, e.g., a gene encoding at least one methionine decarboxylase, that is joined to a regulatory sequence in a manner which allows expression of the nucleic acid sequence, e.g., the gene(s) encoding the methionine decarboxylase. In other words, the regulatory sequence acts in cis. In one embodiment, a gene may be “directly linked” to a regulatory sequence in a manner which allows expression of the gene. In another embodiment, a gene may be “indirectly linked” to a regulatory sequence in a manner which allows expression of the gene. In one embodiment, two or more genes may be directly or indirectly linked to a regulatory sequence in a manner which allows expression of the two or more genes. A regulatory region or sequence is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5′ and 3′ untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns. A “promoter” as used herein, refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5′ of the sequence that they regulate. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from promoters found in nature, and/or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may regulate expression of a coding sequence or gene in response to a particular stimulus, e.g., in a cell- or tissue-specific manner, in response to different environmental or physiological conditions, or in response to specific compounds. Prokaryotic promoters are typically classified into two classes: inducible and constitutive. An “inducible promoter” refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region. An “inducible promoter” refers to a promoter that initiates increased levels of transcription of the coding sequence or gene under its control in response to a stimulus or an exogenous environmental condition. A “directly inducible promoter” refers to a regulatory region, wherein the regulatory region is operably linked to a gene encoding a protein or polypeptide, where, in the presence of an inducer of said regulatory region, the protein or polypeptide is expressed. An “indirectly inducible promoter” refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a first gene encoding a first protein, polypeptide, or factor, e.g., a transcriptional regulator, which is capable of regulating a second regulatory region that is operably linked to a second gene, the second regulatory region may be activated or repressed, thereby activating or repressing expression of the second gene. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by “inducible promoter.” Examples of inducible promoters include, but are not limited to, an FNR promoter, a ParaCpromoter, a ParaBADpromoter, a propionate promoter, and a PTetRpromoter, each of which are described in more detail herein. Examples of other inducible promoters are provided herein below. As used herein, a “stably maintained” or “stable” host cell, such as a bacterium, is used to refer to a host cell, such as a bacterial host cell, carrying non-native genetic material, e.g., a methionine decarboxylase, that is incorporated into the host genome or propagated on a self-replicating extra-chromosomal plasmid, such that the non-native genetic material is retained, expressed, and propagated. The stable host cell, such as a stable bacterium, is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. For example, the stable host cell, such as a stable bacterium, may be a genetically engineered host cell, such as a bacterium, comprising an amino acid catabolism gene, in which the plasmid or chromosome carrying the amino acid catabolism gene is stably maintained in the host cell, such as a bacterium, such that the methionine decarboxylase can be expressed in the host cell, such as a bacterium, and the host cell, such as a bacterium, is capable of survival and/or growth in vitro and/or in vivo. In some embodiments, copy number affects the stability of expression of the non-native genetic material. In some embodiments, copy number affects the level of expression of the non-native genetic material. As used herein, the term “expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide As used herein, the term “plasmid” or “vector” refers to an extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated into the genome of a host cell, such as a bacterial host cell. Plasmids are usually circular and capable of autonomous replication. Plasmids may be low-copy, medium-copy, or high-copy, as is well known in the art. Plasmids may optionally comprise a selectable marker, such as an antibiotic resistance gene, which helps select for host cells, such as bacterial host cells, containing the plasmid and which ensures that the plasmid is retained in the host cell, such as a bacterial host cell. A plasmid disclosed herein may comprise a nucleic acid sequence encoding a heterologous gene, e.g., a gene encoding at least one methionine decarboxylase. As used herein, the term “transform” or “transformation” refers to the transfer of a nucleic acid fragment into a host cell, such as a host bacterial cell, resulting in genetically-stable inheritance. Host cells, such as host bacterial cells, comprising the transformed nucleic acid fragment are referred to as “recombinant” or “transgenic” or “transformed” cells or organisms. In some instances where one or more nucleic acid fragments are introduced into a host cell, such as on a plasmid or vector, one or more of the nucleic acid fragments may be retained in the cell, such as by integration into the genome of the cell, while the plasmid or vector itself may be removed from the cell. In such instances, the host cell is considered to be transformed with the nucleic acid fragments that were introduced into the cell regardless of whether the plasmid or vector is retained in the cell or not. The term “genetic modification,” as used herein, refers to any genetic change. Exemplary genetic modifications include those that increase, decrease, or abolish the expression of a gene, including, for example, modifications of native chromosomal or extrachromosomal genetic material. Exemplary genetic modifications also include the introduction of at least one plasmid, modification, mutation, base deletion, base addition, and/or codon modification of chromosomal or extrachromosomal genetic sequence(s), gene over-expression, gene amplification, gene suppression, promoter modification or substitution, gene addition (either single or multi-copy), antisense expression or suppression, or any other change to the genetic elements of a host cell, whether the change produces a change in phenotype or not. Genetic modification can include the introduction of a plasmid, e.g., a plasmid comprising at least one methionine decarboxylase operably linked to a promoter, into a host cell, such as a bacterial host cell. Genetic modification can also involve a targeted replacement in the chromosome, e.g., to replace a native gene promoter with an inducible promoter, regulated promoter, strong promoter, or constitutive promoter. Genetic modification can also involve gene amplification, e.g., introduction of at least one additional copy of a native gene into the chromosome of the cell. Alternatively, chromosomal genetic modification can involve a genetic mutation. As used herein, the term “genetic mutation” refers to a change or changes in a nucleotide sequence of a gene or related regulatory region that alters the nucleotide sequence as compared to its native or wild-type sequence. Mutations include, for example, substitutions, additions, and deletions, in whole or in part, within the wild-type sequence. Such substitutions, additions, or deletions can be single nucleotide changes (e.g., one or more point mutations), or can be two or more nucleotide changes, which may result in substantial changes to the sequence. Mutations can occur within the coding region of the gene as well as within the non-coding and regulatory sequence of the gene. The term “genetic mutation” is intended to include silent and conservative mutations within a coding region as well as changes which alter the amino acid sequence of the polypeptide encoded by the gene. A genetic mutation in a gene coding sequence may, for example, increase, decrease, or otherwise alter the activity (e.g., enzymatic activity) of the gene's polypeptide product. A genetic mutation in a regulatory sequence may increase, decrease, or otherwise alter the expression of sequences operably linked to the altered regulatory sequence. It is routine for one of ordinary skill in the art to make mutations in a gene of interest. Mutations include substitutions, insertions, deletions, and/or truncations of one or more specific amino acid residues or of one or more specific nucleotides or codons in the polypeptide or polynucleotide of the exporter of an asparagine. Mutagenesis and directed evolution methods are well known in the art for creating variants. See, e.g., U.S. Pat. Nos. 7,783,428; 6,586,182; 6,117,679; and Ling, et al., 1999, “Approaches to DNA mutagenesis: an overview,”Anal. Biochem.,254(2):157-78; Smith, 1985, “In vitro mutagenesis,”Ann. Rev. Genet.,19:423-462; Carter, 1986, “Site-directed mutagenesis,”Biochem. J.,237:1-7; and Minshull, et al., 1999, “Protein evolution by molecular breeding,”Current Opinion in Chemical Biology,3:284-290. For example, the lambda red system can be used to knock-out genes inE. coli(see, for example, Datta et al.,Gene,379:109-115 (2006)). The term “inactivated” as applied to a gene refers to any genetic modification that decreases or eliminates the expression of the gene and/or the functional activity of the corresponding gene product (mRNA and/or protein). The term “inactivated” encompasses complete or partial inactivation, suppression, deletion, interruption, blockage, promoter alterations, antisense RNA, dsRNA, or down-regulation of a gene. This can be accomplished, for example, by gene “knockout,” inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or by use of inhibitory RNAs (e.g., sense, antisense, or RNAi technology). A deletion may encompass all or part of a gene's coding sequence. The term “knockout” refers to the deletion of most (at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) or all (100%) of the coding sequence of a gene. In some embodiments, any number of nucleotides can be deleted, from a single base to an entire piece of a chromosome. “Exogenous environmental condition(s)” or “environmental conditions” refer to settings or circumstances under which the promoter described herein is directly or indirectly induced. The phrase is meant to refer to the environmental conditions external to the engineered microorganism, but endogenous or native to the host subject environment. Thus, “exogenous” and “endogenous” may be used interchangeably to refer to environmental conditions in which the environmental conditions are endogenous to a mammalian body, but external or exogenous to an intact microorganism cell. In some embodiments, the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the small intestine of a mammal. In some embodiments, the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut. In some embodiments, exogenous environmental conditions refer to the presence of molecules or metabolites that are specific to the mammalian gut in a healthy or disease-state, e.g., propionate. In some embodiments, the exogenous environmental condition is a tissue-specific or disease-specific metabolite or molecule(s). In some embodiments, the exogenous environmental condition is a low-pH environment. In some embodiments, the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter. In some embodiments, the genetically engineered microorganism of the disclosure comprises an oxygen level-dependent promoter. In some aspects, bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics. As used herein, “exogenous environmental conditions” or “environmental conditions” also refers to settings or circumstances or environmental conditions external to the engineered microorganism, which relate to in vitro culture conditions of the microorganism. “Exogenous environmental conditions” may also refer to the conditions during growth, production, and manufacture of the organism. Such conditions include aerobic culture conditions, anaerobic culture conditions, low oxygen culture conditions and other conditions under set oxygen concentrations. Such conditions also include the presence of a chemical and/or nutritional inducer, such as tetracycline, arabinose, IPTG, rhamnose, and the like in the culture medium. Such conditions also include the temperatures at which the microorganisms are grown prior to in vivo administration. For example, using certain promoter systems, certain temperatures are permissive to expression of a payload, while other temperatures are non-permissive. Oxygen levels, temperature and media composition influence such exogenous environmental conditions. Such conditions affect proliferation rate, rate of induction of the payload or gene of interest, e.g., amino acid catabolism gene, other regulators (e.g., FNRS24Y), and overall viability and metabolic activity of the strain during strain production. In some embodiments, the exogenous environmental condition(s) and/or signal(s) stimulates the activity of an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) that serves to activate the inducible promoter is not naturally present within the gut of a mammal. In some embodiments, the inducible promoter is stimulated by a molecule or metabolite that is administered in combination with the pharmaceutical composition of the disclosure, for example, tetracycline, arabinose, or any biological molecule that serves to activate an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising a recombinant bacterial cell of the disclosure. In some embodiments, the exogenous environmental condition that serves to activate the inducible promoter is naturally present within the gut of a mammal (for example, low oxygen or anaerobic conditions, or biological molecules involved in an inflammatory response). In some embodiments, the loss of exposure to an exogenous environmental condition (for example, in vivo) inhibits the activity of an inducible promoter, as the exogenous environmental condition is not present to induce the promoter (for example, an aerobic environment outside the gut). An “oxygen level-dependent promoter” or “oxygen level-dependent regulatory region” refers to a nucleic acid sequence to which one or more oxygen level-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression. Examples of oxygen level-dependent transcription factors include, but are not limited to, FNR, ANR, and DNR. Corresponding FNR-responsive promoters, ANR-responsive promoters, and DNR-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003). Non-limiting examples are shown in Table 1. In a non-limiting example, a promoter (PfnrS) was derived from theE. coliNissle fumarate and nitrate reductase gene S (fnrS) that is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz, 2010; Boysen et al, 2010). The PfnrS promoter is activated under anaerobic and/or low oxygen conditions by the global transcriptional regulator FNR that is naturally found in Nissle. Under anaerobic and/or low oxygen conditions, FNR forms a dimer and binds to specific sequences in the promoters of specific genes under its control, thereby activating their expression. However, under aerobic conditions, oxygen reacts with iron-sulfur clusters in FNR dimers and converts them to an inactive form. In this way, the PfnrS inducible promoter is adopted to modulate the expression of proteins or RNA. PfnrS is used interchangeably in this application as FNRS, fnrS, FNR, P-FNRS promoter and other such related designations to indicate the promoter PfnrS. TABLE 1Examples of transcription factors andresponsive genes and regulatory regionsTranscriptionExamples of responsive genes,factorpromoters, and/or regulatory regions:FNRnirB, ydfZ, pdhR, focA, ndH, hlyE,narK, narX, narG, yfiD, tdcDDNRnorb, norC As used herein, a “non-native” nucleic acid sequence refers to a nucleic acid sequence not normally present in a host cell, such as a bacterium, e.g., an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al., 2013). The non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in a gene cassette. In some embodiments, “non-native” refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non-native nucleic acid sequence may be present on a plasmid or chromosome. In addition, multiple copies of any regulatory region, promoter, gene, and/or gene cassette may be present in the host cell, such as a bacterium, wherein one or more copies of the regulatory region, promoter, gene, and/or gene cassette may be mutated or otherwise altered as described herein. In some embodiments, the genetically engineered host cell, such as genetically engineered bacteria, are engineered to comprise multiple copies of the same regulatory region, promoter, gene, and/or gene cassette in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions. In some embodiments, the genetically engineered host cell, such as genetically engineered bacteria, of the invention comprise a gene encoding a phenylalanine-metabolizing enzyme that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g., an FNR promoter operably linked to a gene encoding an amino acid metabolism gene. “Constitutive promoter” refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked. Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutiveEscherichia coli σSpromoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutiveEscherichia coli σ32promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutiveEscherichia coli σ70promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015),E. coliCreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_K119000; BBa_K119001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), a constitutiveBacillus subtilis σApromoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PliaG(BBa_K823000), PlepA(BBa_K823002), Pveg(BBa_K823003)), a constitutiveBacillus subtilis σBpromoter (e.g., promoter ctc (BBa_K143010), promoter gsiB (BBa_K143011)), aSalmonellapromoter (e.g., Pspv2 fromSalmonella(BBa_K112706), Pspv fromSalmonella(BBa_K112707)), a bacteriophage T7 promoter (e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa_K113010; BBa_K113011; BBa_K113012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)), and functional fragments thereof. “Gut” refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine. The gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas. The upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine. The lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal. Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines. In some embodiments, the genetically engineered bacteria are active in the gut. In some embodiments, the genetically engineered bacteria are active in the large intestine. In some embodiments, the genetically engineered bacteria are active in the small intestine. In some embodiments, the genetically engineered bacteria are active in the small intestine and in the large intestine. In some embodiments, the genetically engineered bacteria transit through the small intestine. In some embodiments, the genetically engineered bacteria have increased residence time in the small intestine. In some embodiments, the genetically engineered bacteria colonize the small intestine. In some embodiments, the genetically engineered bacteria do not colonize the small intestine. In some embodiments, the genetically engineered bacteria have increased residence time in the gut. In some embodiments, the genetically engineered bacteria colonize the small intestine. In some embodiments, the genetically engineered bacteria do not colonize the gut. As used herein, the term “low oxygen” is meant to refer to a level, amount, or concentration of oxygen (O2) that is lower than the level, amount, or concentration of oxygen that is present in the atmosphere (e.g., <21% O2; <160 torr O2)). Thus, the term “low oxygen condition or conditions” or “low oxygen environment” refers to conditions or environments containing lower levels of oxygen than are present in the atmosphere. In some embodiments, the term “low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O2) found in a mammalian gut, e.g., lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon, rectum, and anal canal. In some embodiments, the term “low oxygen” is meant to refer to a level, amount, or concentration of O2that is 0-60 mmHg O2(0-60 torr O2) (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mmHg O2), including any and all incremental fraction(s) thereof (e.g., 0.2 mmHg, 0.5 mmHg O2, 0.75 mmHg O2, 1.25 mmHg O2, 2.175 mmHg O2, 3.45 mmHg O2, 3.75 mmHg O2, 4.5 mmHg O2, 6.8 mmHg 02, 11.35 mmHg 02, 46.3 mmHg 02, 58.75 mmHg, etc., which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way). In some embodiments, “low oxygen” refers to about 60 mmHg O2or less (e.g., 0 to about 60 mmHg 02). The term “low oxygen” may also refer to a range of O2levels, amounts, or concentrations between 0-60 mmHg 02 (inclusive), e.g., 0-5 mmHg O2, <1.5 mmHg O2, 6-10 mmHg, <8 mmHg, 47-60 mmHg, etc. which listed exemplary ranges are listed here for illustrative purposes and not meant to be limiting in any way. See, for example, Albenberg et al., Gastroenterology, 147(5): 1055-1063 (2014); Bergofsky et al., J Clin. Invest., 41(11): 1971-1980 (1962); Crompton et al., J Exp. Biol., 43: 473-478 (1965); He et al., PNAS (USA), 96: 4586-4591 (1999); McKeown, Br. J. Radiol., 87:20130676 (2014) (doi: 10.1259/brj.20130676), each of which discusses the oxygen levels found in the mammalian gut of various species and each of which are incorporated by reference herewith in their entireties. In some embodiments, the term “low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O2) found in a mammalian organ or tissue other than the gut, e.g., urogenital tract, tumor tissue, etc. in which oxygen is present at a reduced level, e.g., at a hypoxic or anoxic level. In some embodiments, “low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O2) present in partially aerobic, semi aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic, and/or anaerobic conditions. For example, Table 2 summarizes the amount of oxygen present in various organs and tissues. In some embodiments, the level, amount, or concentration of oxygen (O2) is expressed as the amount of dissolved oxygen (“DO”) which refers to the level of free, non-compound oxygen (O2) present in liquids and is typically reported in milligrams per liter (mg/L), parts per million (ppm; 1 mg/L=1 ppm), or in micromoles (μmole) (1 μmole O2=0.022391 mg/L O2). Fondriest Environmental, Inc., “Dissolved Oxygen”, Fundamentals of Environmental Measurements, 19 Nov. 2013. In some embodiments, the term “low oxygen” is meant to refer to a level, amount, or concentration of oxygen (O2) that is about 6.0 mg/L DO or less, e.g., 6.0 mg/L, 5.0 mg/L, 4.0 mg/L, 3.0 mg/L, 2.0 mg/L, 1.0 mg/L, or 0 mg/L, and any fraction therein, e.g., 3.25 mg/L, 2.5 mg/L, 1.75 mg/L, 1.5 mg/L, 1.25 mg/L, 0.9 mg/L, 0.8 mg/L, 0.7 mg/L, 0.6 mg/L, 0.5 mg/L, 0.4 mg/L, 0.3 mg/L, 0.2 mg/L and 0.1 mg/L DO, which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way. The level of oxygen in a liquid or solution may also be reported as a percentage of air saturation or as a percentage of oxygen saturation (the ratio of the concentration of dissolved (O2) in the solution to the maximum amount of oxygen that will dissolve in the solution at a certain temperature, pressure, and salinity under stable equilibrium). Well-aerated solutions (e.g., solutions subjected to mixing and/or stirring) without oxygen producers or consumers are 100% air saturated. In some embodiments, the term “low oxygen” is meant to refer to 40% air saturation or less, e.g., 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% air saturation, including any and all incremental fraction(s) thereof (e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of air saturation levels between 0-40%, inclusive (e.g., 0-5%, 0.05-0.1%, 0.1-0.2%, 0.1-0.5%, 0.5-2.0%, 0-10%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way. In some embodiments, the term “low oxygen” is meant to refer to 9% O2saturation or less, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0%, 02 saturation, including any and all incremental fraction(s) thereof (e.g., 6.5%, 5.0%, 2.2%, 1.7%, 1.4%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of O2saturation levels between 0-9%, inclusive (e.g., 0-5%, 0.05-0.1%, 0.1-0.2%, 0.1-0.5%, 0.5-2.0%, 0-8%, 5-7%, 0.3-4.2% 02, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way. TABLE 2CompartmentOxygen Tensionstomach~60 torr (e.g., 58 +/− 15 torr)duodenum and first~30 torr (e.g., 32 +/− 8 torr);part of jejunum~20% oxygen in ambient airIleum (mid- small~10 torr; ~6% oxygen in ambientintestine)air (e.g., 11 +/− 3 torr)Distal sigmoid colon~3 torr (e.g., 3 +/− 1 torr)colon<2 torrLumen of cecum<1 torrtumor<32 torr (most tumors are <15 torr) “Microorganism” refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microorganisms include bacteria, yeast, viruses, parasites, fungi, certain algae, and protozoa. In some aspects, the microorganism is engineered (“engineered microorganism”) to produce one or more therapeutic molecules or proteins of interest. In certain aspects, the microorganism is engineered to take up and catabolize certain metabolites or other compounds from its environment, e.g., the gut. In certain aspects, the microorganism is engineered to synthesize certain beneficial metabolites or other compounds (synthetic or naturally occurring) and release them into its environment. In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered microorganism is an engineered virus. In certain embodiments, the engineered microorganism is an engineered yeast. When referring to bacteria, engineered bacteria or recombinant bacteria, the embodiments also contemplate other types of microorganisms. “Host cell” refers to a cell that can be used to express a polynucleotide, such as a polynucleotide that encodes a methionine catabolism enzyme, such as a methionine decarboxylase, and/or a methionine importer. “Non-pathogenic bacteria” refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, non-pathogenic bacteria are commensal bacteria, which are present in the indigenous microbiota of the gut. Examples of non-pathogenic bacteria include, but are not limited to,Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia, Lactobacillus, Lactococcus, Saccharomyces, andStaphylococcus, e.g.,Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Escherichia coli, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, andSaccharomyces boulardii(Sonnenborn et al., 2009; Dinleyici et al., 2014; U.S. Pat. Nos. 6,835,376; 6,203,797; 5,589,168; 7,731,976). Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity. “Probiotic” is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic. Examples of probiotic bacteria include, but are not limited to, Bifidobacteria,Escherichia, Lactobacillus, andSaccharomyces, e.g.,Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli, Escherichia colistrain Nissle,Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, andSaccharomyces boulardii(Dinleyici et al., 2014; U.S. Pat. Nos. 5,589,168; 6,203,797; 6,835,376). The probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006). Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties. As used herein, the term “auxotroph” or “auxotrophic” refers to an organism that requires a specific factor, e.g., an amino acid, a sugar, or other nutrient, to support its growth. An “auxotrophic modification” is a genetic modification that causes the organism to die in the absence of an exogenously added nutrient essential for survival or growth because it is unable to produce said nutrient. As used herein, the term “essential gene” refers to a gene which is necessary to for cell growth and/or survival. Essential genes are described in more detail infra and include, but are not limited to, DNA synthesis genes (such as thyA), cell wall synthesis genes (such as dapA), and amino acid genes (such as serA and metA). As used herein, the terms “modulate” and “treat” and their cognates refer to an amelioration of a disease, disorder, and/or condition, or at least one discernible symptom thereof. In another embodiment, “modulate” and “treat” refer to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, “modulate” and “treat” refer to inhibiting the progression of a disease, disorder, and/or condition, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment, “modulate” and “treat” refer to slowing the progression or reversing the progression of a disease, disorder, and/or condition. As used herein, “prevent” and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease, disorder and/or condition or a symptom associated with such disease, disorder, and/or condition. Those in need of treatment may include individuals already having a particular medical disease, as well as those at risk of having, or who may ultimately acquire the disease. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disease, the presence or progression of a disease, or likely receptiveness to treatment of a subject having the disease. Disorders associated with or involved with amino acid metabolism, e.g., homocystinuria or cystinuria, may be caused by inborn genetic mutations for which there are no known cures. Diseases can also be secondary to other conditions, e.g., an intestinal disorder or a bacterial infection. Treating diseases associated with amino acid metabolism may encompass reducing normal levels of one or more amino acids, reducing excess levels of one or more amino acids, or eliminating one or more amino acids, and does not necessarily encompass the elimination of the underlying disease. As used herein the terms “disease associated with amino acid metabolism” or a “disorder associated with amino acid metabolism” is a disease or disorder involving the abnormal, e.g., increased, levels of one or more amino acids, e.g., methionine, in a subject. In one embodiment, a disease or disorder associated with amino acid metabolism, e.g., methionine metabolism, is homocystinuria. In another embodiment, a disease or disorder associated with amino acid metabolism, e.g., methionine metabolism, is cancer. In another embodiment, a disease or disorder associated with amino acid metabolism, e.g., methionine metabolism, is a metabolic disease or a metabolic syndrome. As used herein, the term “amino acid” refers to a class of organic compounds that contain at least one amino group and one carboxyl group. Amino acids include leucine, isoleucine, valine, arginine, lysine, asparagine, serine, glycine, glutamine, tryptophan, methionine, threonine, cysteine, tyrosine, phenylalanine, glutamic acid, aspartic acid, alanine, histidine, and proline. As used herein, the term “amino acid catabolism” or “amino acid metabolism” refers to the processing, breakdown and/or degradation of an amino acid molecule (e.g., methionine, asparagine, lysine or arginine) into other compounds that are not associated with the disease associated with amino acid metabolism, such as homocystinuria, or other compounds which can be utilized by the bacterial cell. In another embodiment, the term “methionine catabolism” refers to the processing, breakdown, and/or degradation of methionine into 3-methylthiopropylamine. In yet another embodiment, the term “methionine catabolism” refers to the processing, breakdown, and/or degradation of methionine to sulfate. In one embodiment, the term “methionine catabolism” refers to the processing, breakdown, and/or degradation of methionine into methanethiol and 2-aminobut-2-enoate. In another embodiment, the term “methionine catabolism” refers to the processing, breakdown, and/or degradation of methionine into 3-methylthio-2-oxobutyric acid. As used herein, the term “importer” is meant to refer to a mechanism, e.g., protein, proteins, or protein complex, for importing a molecule, e.g., amino acid, peptide (di-peptide, tripeptide, polypeptide, etc), toxin, metabolite, substrate, as well as other biomolecules into the microorganism from the extracellular milieu. For example, a methionine importer such as MetP imports methionine into the microorganism. As used herein, “payload” refers to one or more molecules of interest to be produced by a genetically engineered microorganism, such as bacteria or a virus. In some embodiments, the payload is a therapeutic payload, e.g., an amino acid catabolic enzyme or an amino acid importer polypeptide. In some embodiments, the payload is a regulatory molecule, e.g., a transcriptional regulator such as FNR. In some embodiments, the payload comprises a regulatory element, such as a promoter or a repressor. In some embodiments, the payload comprises an inducible promoter, such as from FNRS. In some embodiments the payload comprises a repressor element, such as a kill switch. In some embodiments, the payload is encoded by a gene or multiple genes or an operon. In alternate embodiments, the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism. In some embodiments, the genetically engineered microorganism comprises two or more payloads. The term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. The terms “therapeutically effective dose” and “therapeutically effective amount” are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition. A therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disease or condition associated with excess amino acid levels. A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below. As used herein, the term “polypeptide” includes “polypeptide” as well as “polypeptides,” and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (i.e., peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, “peptides,” “dipeptides,” “tripeptides, “oligopeptides,” “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “dipeptide” refers to a peptide of two linked amino acids. The term “tripeptide” refers to a peptide of three linked amino acids. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology. A polypeptide may be a naturally occurring polypeptide or alternatively may be a polypeptide not naturally occurring, such as a polypeptide identified from a library of engineered polypeptides. In other embodiments, the polypeptide is produced by the genetically engineered bacteria or virus of the current invention. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides, which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, are referred to as unfolded. The term “peptide” or “polypeptide” may refer to an amino acid sequence that corresponds to a protein or a portion of a protein or may refer to an amino acid sequence that corresponds with non-protein sequence, e.g., a sequence selected from a regulatory peptide sequence, leader peptide sequence, signal peptide sequence, linker peptide sequence, and other peptide sequence. An “isolated” polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells, including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e. produced from cells, microbial or mammalian, transformed by an exogenous recombinant DNA expression construct encoding the polypeptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycan. Fragments, derivatives, analogs or variants of the foregoing polypeptides, and any combination thereof are also included as polypeptides. The terms “fragment,” “variant,” “derivative” and “analog” include polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the original peptide and include any polypeptides, which retain at least one or more properties of the corresponding original polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments. Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Polypeptides also include fusion proteins. As used herein, the term “variant” includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide. As used herein, the term “fusion protein” refers to a chimeric protein comprising amino acid sequences of two or more different proteins. Typically, fusion proteins result from well known in vitro recombination techniques. Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion proteins. “Derivatives” include but are not limited to peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. “Similarity” between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M. O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: Ala, Pro, Gly, Gln, Asn, Ser, Thr, Cys, Ser, Tyr, Thr, Val, Ile, Leu, Met, Ala, Phe, Lys, Arg, His, Phe, Tyr, Trp, His, Asp, and Glu. As used herein, the term “sufficiently similar” means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar. Preferably, variants will be sufficiently similar to the amino acid sequence of the peptides of the invention. Such variants generally retain the functional activity of the peptides of the present invention. Variants include peptides that differ in amino acid sequence from the native and wild-type peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or substitution(s). These may be naturally occurring variants as well as artificially designed ones. As used herein, the term “percent identity” refers to a quantitative measurement of the similarity between two sequences (e.g., nucleic acid or amino acid sequence). In some embodiments, the “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.,J. Mol. Biol.215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al.,Nucleic Acids Res.25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. For example, a first nucleic acid sequence may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least about 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of a second nucleic acid. In another example, a first polypeptide may comprise an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least about 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the amino acid sequence of a second polypeptide. As used herein, the term “linker”, “linker peptide” or “peptide linkers” or “linker” refers to synthetic or non-native or non-naturally-occurring amino acid sequences that connect or link two polypeptide sequences, e.g., that link two polypeptide domains. As used herein the term “synthetic” refers to amino acid sequences that are not naturally occurring. Exemplary linkers are described herein. Additional exemplary linkers are provided in US 20140079701, the contents of which are herein incorporated by reference in its entirety. As used herein, the term “codon-optimized sequence” refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism. The term “codon-optimized” refers to the modification of codons in the gene or coding regions of a nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the nucleic acid molecule. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of the host organism. A “codon-optimized sequence” refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. In some embodiments, the improvement of transcription and/or translation involves increasing the level of transcription and/or translation. In some embodiments, the improvement of transcription and/or translation involves decreasing the level of transcription and/or translation. In some embodiments, codon optimization is used to fine-tune the levels of expression from a construct of interest. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism. Many organisms display a bias or preference for use of particular codons to code for insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, is allowed by the degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent, inter alia, on the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. The terms “phage” and “bacteriophage” are used interchangeably herein. Both terms refer to a virus that infects and replicates within a bacterium. As used herein “phage” or bacteriophage” collectively refers to prophage, lysogenic, dormant, temperate, intact, defective, cryptic, and satellite phage, phage tail bacteriocins, tailiocins, and gene transfer agents. As used therein the term “prophage” refers to the genomic material of a bacteriophage, which is integrated into a replicon of the host cell and replicates along with the host. The prophage may be able to produce phages if specifically activated. In some cases, the prophage is not able to produce phages or has never done so (i.e., defective or cryptic prophages). In some cases, prophage also refers to satellite phages. The terms “prophage” and “endogenous phage” are used interchangeably herein. “Endogenous phage” or “endogenous prophage” also refers to a phage that is present in the natural state of a bacterium (and its parental strain). As used herein the term “phage knockout” or “inactivated phage” refers to a phage which has been modified so that it can either no longer produce and/or package phage particles or it produces fewer phage particles than the wild type phage sequence. In some embodiments, the inactivated phage or phage knockout refers to the inactivation of a temperate phage in its lysogenic state, i.e., to a prophage. Such a modification refers to a mutation in the phage; such mutations include insertions, deletions (partial or complete deletion of phage genome), substitutions, inversions, at one or more positions within the phage genome, e.g., within one or more genes within the phage genome. As used herein the adjectives “phage-free”, “phage free” and “phageless” are used interchangeably to characterize a bacterium or strain which contains one or more prophages, one or more of which have been modified. The modification can result in a loss of the ability of the prophage to be induced or release phage particles. Alternatively, the modification can result in less efficient or less frequent induction or less efficient or less frequent phage release as compared to the isogenic strain without the modification. Ability to induce and release phage can be measured using a plaque assay as described herein. As used herein phage induction refers to the part of the life cycle of a lysogenic prophage, in which the lytic phage genes are activated, phage particles are produced and lysis occurs. As used herein a “pharmaceutical composition” refers to a preparation of bacterial cells disclosed herein with other components such as a physiologically suitable carrier and/or excipient. The phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these phrases. The articles “a” and “an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary. For example, as used herein, “a heterologous gene encoding a methionine decarboxylase” should be understood to mean “at least one heterologous gene encoding at least one methionine decarboxylase.” Similarly, as used herein, “a heterologous gene encoding an amino acid importer” should be understood to mean “at least one heterologous gene encoding at least one amino acid importer.” The phrase “and/or,” when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, “A, B, and/or C” indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase “and/or” may be used interchangeably with “at least one of” or “one or more of” the elements in a list. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Host Cells Any suitable host cell may be used to express any of the enzymes disclosed herein, such as methionine catabolism enzymes (e.g., methionine decarboxylases) and methionine importers. Suitable host cells include, but are not limited to, bacterial cells (e.g.,E. colicells), fungal cells (e.g., yeast cells), algal cells, plant cells, insect cells, and animal cells, including mammalian cells. Suitable yeast host cells include, but are not limited to:Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, andYarrowia. In some embodiments, the yeast cell isHansenula polymorpha, Saccharomyces cerevisiae, Saccaromyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, orYarrowia lipolytica. In some embodiments, the yeast strain is an industrial polyploid yeast strain. Other non-limiting examples of fungal cells include cells obtained fromAspergillusspp.,Penicilliumspp.,Fusariumspp.,Rhizopusspp.,Acremoniumspp.,Neurosporaspp., Sordaria spp.,Magnaporthespp.,Allomycesspp.,Ustilagospp.,Botrytisspp., andTrichodermaspp. In certain embodiments, the host cell is an algal cell such asChlamydomonas(e.g., C.Reinhardtii) andPhormidium(P. sp. ATCC29409). In some embodiments, the host cell is an animal cell. In some embodiments, the host cell is a mammalian cell, including, for example, a human cell (e.g., 293, HeLa, W138, PER.C6 or Bowes melanoma cells), a mouse cell (e.g., 3T3, NS0, NS1 or Sp2/0), a hamster cell (e.g., CHO or BHK), or a monkey cell (e.g., COS, FRhL or Vero). In some embodiments, the cell is a hybridoma cell line. In some embodiments, the host cell is a bacterial cell, e.g., a recombinant bacterial cell. The disclosure provides a bacterial cell that comprises a heterologous gene encoding a methionine catabolism enzyme. In some embodiments, the bacterial cell is a non-pathogenic bacterial cell. In some embodiments, the bacterial cell is a commensal bacterial cell. In some embodiments, the bacterial cell is a probiotic bacterial cell. In certain embodiments, the bacterial cell is selected from the group consisting of aBacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Clostridium scindens, Escherichia coli, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, Lactococcus lactis, andOxalobacter formigenesbacterial cell. In one embodiment, the bacterial cell is aBacteroides fragilisbacterial cell. In one embodiment, the bacterial cell is aBacteroides thetaiotaomicronbacterial cell. In one embodiment, the bacterial cell is aBacteroides subtilisbacterial cell. In one embodiment, the bacterial cell is aBifidobacterium animalisbacterial cell. In one embodiment, the bacterial cell is aBifidobacterium bifidumbacterial cell. In one embodiment, the bacterial cell is aBifidobacterium infantisbacterial cell. In one embodiment, the bacterial cell is aBifidobacterium lactisbacterial cell. In one embodiment, the bacterial cell is aClostridium butyricumbacterial cell. In one embodiment, the bacterial cell is aClostridium scindensbacterial cell. In one embodiment, the bacterial cell is anEscherichia colibacterial cell. In one embodiment, the bacterial cell is aLactobacillus acidophilusbacterial cell. In one embodiment, the bacterial cell is aLactobacillus plantarumbacterial cell. In one embodiment, the bacterial cell is aLactobacillus reuteribacterial cell. In one embodiment, the bacterial cell is aLactococcus lactisbacterial cell. In one embodiment, the bacterial cell is aOxalobacter formigenesbacterial cell. In another embodiment, the bacterial cell does not includeOxalobacter formigenes. In one embodiment, the bacterial cell is a Gram positive bacterial cell. In another embodiment, the bacterial cell is a Gram negative bacterial cell. In some embodiments, the bacterial cell isEscherichia colistrain Nissle 1917 (E. coliNissle), a Gram-positive bacterium of the Enterobacteriaceae family that “has evolved into one of the best characterized probiotics” (Ukena et al., 2007). The strain is characterized by its “complete harmlessness” (Schultz, 2008), and “has GRAS (generally recognized as safe) status” (Reister et al., 2014, emphasis added). Genomic sequencing confirmed thatE. coliNissle “lacks prominent virulence factors (e.g.,E. coliα-hemolysin, P-fimbrial adhesins)” (Schultz, 2008), andE. coliNissle “does not carry pathogenic adhesion factors and does not produce any enterotoxins or cytotoxins, it is not invasive, not uropathogenic” (Sonnenborn et al., 2009). As early as in 1917,E. coliNissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use.E. coliNissle has since been used to treat ulcerative colitis in humans in vivo (Rembacken et al., 1999), to treat inflammatory bowel disease, Crohn's disease, and pouchitis in humans in vivo (Schultz, 2008), and to inhibit enteroinvasiveSalmonella, Legionella, Yersinia, andShigellain vitro (Altenhoefer et al., 2004). It is commonly accepted thatE. coliNissle's “therapeutic efficacy and safety have convincingly been proven” (Ukena et al., 2007). In one embodiment, the recombinant bacterial cell does not colonize the subject. One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be adapted for other species, strains, and subtypes of bacteria. Furthermore, genes from one or more different species can be introduced into one another, e.g., an amino acid catabolism gene fromKlebsiella quasipneumoniaecan be expressed inEscherichia coli. In some embodiments, the bacterial cell is a genetically engineered bacterial cell. In another embodiment, the bacterial cell is a recombinant bacterial cell. In some embodiments, the disclosure comprises a colony of bacterial cells. In another aspect, the disclosure provides a recombinant bacterial culture which comprises bacterial cells disclosed herein. In one aspect, the disclosure provides a recombinant bacterial culture which reduces levels of an amino acid, e.g., methionine, in the media of the culture. In one embodiment, the levels of an amino acid are reduced by about 50%, about 75%, or about 100% in the media of the cell culture. In another embodiment, the levels of an amino acid are reduced by about two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold in the media of the cell culture. In one embodiment, the levels of an amino acid, e.g., methionine, are reduced below the limit of detection in the media of the cell culture. In some embodiments of the above described genetically engineered bacteria, the gene encoding a methionine decarboxylase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene encoding a methionine decarboxylase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments of the above described genetically engineered bacteria, the gene encoding a methionine decarboxylase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced. In other embodiments, the gene encoding a methionine decarboxylase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced. In some embodiments of the above described genetically engineered bacteria, the gene encoding a methionine importer is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced. In other embodiments, the gene encoding a methionine importer is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced. In some embodiments of the above described genetically engineered bacteria, the gene encoding a methionine decarboxylase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced and the gene encoding a methionine importer is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced. In other embodiments, the gene encoding a methionine decarboxylase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced and the gene encoding a methionine importer is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced. In some embodiments of the above described genetically engineered bacteria, the gene encoding a methionine decarboxylase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced and the gene encoding a methionine importer is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced. In other embodiments, the gene encoding a methionine decarboxylase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced and the gene encoding a methionine importer is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced. In some embodiments, the genetically engineered bacteria is an auxotroph. In one embodiment, the genetically engineered bacteria is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1 auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a ΔthyA and ΔdapA auxotroph. In some embodiments of the above described genetically engineered bacteria, the gene encoding a methionine decarboxylase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene encoding a methionine decarboxylase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions. A. Methionine Catabolism Enzymes Methionine catabolism enzymes may be expressed or modified in host cells, such as the bacteria disclosed herein, in order to enhance catabolism of methionine. For example, the genetically engineered bacteria comprising at least one heterologous gene encoding a methionine catabolism enzyme can catabolize methionine to treat a disease associated with methionine, including, but not limited to homocystinuria, cystinuria, primary and secondary hypermethioninemia, cystathionine β-synthase (CBS) deficiency, or cancer, e.g., lymphoblastic leukemia. As used herein, the term “methionine catabolism enzyme” refers to an enzyme involved in the catabolism of methionine. Specifically, when a methionine catabolism enzyme is expressed in a recombinant bacterial cell, the bacterial cell hydrolyzes more methionine into 3-methylthiopropylamine (3-MTP) when the catabolism enzyme is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In some embodiments, methionine importers may also be expressed or modified in the recombinant bacteria to enhance methionine import into the cell in order to increase the catabolism of methionine by the methionine catabolism enzyme. In other embodiments, methionine exporters may be knocked-out in the recombinant bacteria to decrease export of methionine and/or increase cytoplasmic concentration of methionine. In one embodiment, the methionine catabolism enzyme is a methionine decarboxylase (MetDC). In another embodiment, the methionine catabolism enzyme is a leucine decarboxylase (LeuDC) which has been modified to further comprise methionine catabolism activity, e.g., methionine decarboxylase activity. For example, SEQ ID NO: 1053 is a leucine decarboxylase which has been modified as compared to a wild-type leucine decarboxylase sequence at positions N2S, V14A, E16G, H17S, R19W, A30E, K41Q, I45D, R48H, A51P, R65Q, L68C, L90R, N147S, L154V, R156Q, R160G, L170Q, H179S, H185S, E218Q, Y220F, C235R, H240D, K254E, V263M, T269A, V304A, S308V, D310E, A318M, V328S, T372E, S394T, I406P, and D411C as compared to a wild type polypeptide. In one embodiment, the leucine decarboxylase gene encodes a polypeptide that has point mutations N2S, V14A, E16G, H17S, R19W, A30E, K41Q, I45D, R48H, A51P, R65Q, L68C, L90R, N147S, L154V, R156Q, R160G, L170Q, H179S, H185S, E218Q, Y220F, C235R, H240D, K254E, V263M, T269A, V304A, S308V, D310E, A318M, V328S, T372E, S394T, 1406P, and D411C and comprises methionine decarboxylase activity. Accordingly, in some embodiments herein, a leucine decarboxylase enzyme that has been modified to have methionine decarboxylase activity may be referred to as a “methionine decarboxylase (MetDC)” which is encoded by a “methionine decarboxylase (metDC) gene.” In one embodiment, the methionine catabolism enzyme increases the rate of methionine catabolism in the cell. In one embodiment, the methionine catabolism enzyme decreases the level of methionine in the cell. In another embodiment, the methionine catabolism enzyme increases the level of 3-methylthiopropylamine in the cell. In one embodiment, 3-methylthiopropylamine is not toxic to the cell. Methionine catabolism enzymes are well known to those of skill in the art (see, e.g., Huang et al.,Mar. Drugs,13(8):5492-5507, 2015). For example, the adenosylmethionine synthase pathway has been identified inAnabaena cylindrica. In the adenosylmethionine synthase pathway, methionine is catabolized into S-adenosyl-L-homocysteine by an S-adenosylmethionine synthase enzyme, followed by conversion of the S-adenosyl-L-homocysteine intoL-homocysteine by an adenosylhomocysteinase enzyme. As another example, two methionine aminotransferase enzymes (including Aro8 and Aro9), and one decarboxylase gene (Aro10) have been identified inSaccharomyces cerevisiaewhich catabolize methionine (Yin et al. (2015) FEMS Microbiol. Lett. 362(5) pii: fnu043). Methionine aminotransferase enzymes catabolize methionine and 2-oxo carboxylate into 2-oxo-4-methylthiobutanoate and an L-amino acid. In some embodiments, a methionine catabolism enzyme is encoded by a gene encoding a methionine catabolism enzyme derived from a bacterial species. In some embodiments, a methionine catabolism enzyme is encoded by a gene encoding a methionine catabolism enzyme derived from a non-bacterial species. In some embodiments, a methionine catabolism enzyme is encoded by a gene derived from a eukaryotic species, e.g., a yeast species or a plant species. In one embodiment, the gene encoding the methionine catabolism enzyme is derived from an organism of the genus or species that includes, but is not limited to,Klebsiella quasipneumoniae, Bacillus subtilis, Caenorhabditis elegans, Entamoeba histolytica, Bacillus halodurans, Methylobacterium aquaticum, Saccharomyces cerevisiae, Escherichia coli, andAnabaena cylindrica. In one embodiment, the methionine catabolism enzyme is a methionine decarboxylase (MDC). In one embodiment, the methionine decarboxylase gene is a MDC gene fromStreptomycessp. 590. On example of such a MDC gene is described, for example, in Misono et al.,Bull. Inst. Chem. Res., Kyoto Univ.,58(3):323-333, 1980. In one embodiment, the methionine decarboxylase gene is a metDC fromStanieriasp. NIES-3757. In one embodiment, the methionine decarboxylase gene is a metDC fromMus musculus. In one embodiment, the methionine decarboxylase gene is a metDC fromEntamoeba histolytica. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a Q70D mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a N82H mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with Q70D N82H mutations referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a Q70D mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a N82H mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with Q70D N82H mutations referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a V491L mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a A500P mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with V491L A500P mutations referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a V491L mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a A500P mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with V491L A500P mutations referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a R41Q mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a Q70D mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with R41Q Q70D mutations referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a R41Q mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with a Q70D mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with R41Q Q70D mutations referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with T66N mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with A203H mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with H379G mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with T66N mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with A203H mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene encodes a polypeptide with H379G mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene has at least about 80% identity with the sequence of SEQ ID NO: 1003. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1003. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1003. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1003. In another embodiment, the methionine decarboxylase gene comprises the sequence of SEQ ID NO: 1003. In yet another embodiment the methionine decarboxylase gene consists of the sequence of SEQ ID NO: 1003. In one embodiment, the methionine decarboxylase gene has at least about 80% identity with the sequence of SEQ ID NO: 1018. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1018. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1018. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1018. In another embodiment, the methionine decarboxylase gene comprises the sequence of SEQ ID NO:1018. In yet another embodiment the methionine decarboxylase gene consists of the sequence of SEQ ID NO: 1018. In one embodiment, the methionine decarboxylase gene has at least about 80% identity with the sequence of SEQ ID NO: 1034. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1034. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1034. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1034. In another embodiment, the methionine decarboxylase gene comprises the sequence of SEQ ID NO: 1034. In yet another embodiment the methionine decarboxylase gene consists of the sequence of SEQ ID NO: 1034. In one embodiment, the methionine decarboxylase gene has at least about 80% identity with the sequence of SEQ ID NO: 1035. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1035. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1035. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1035. In another embodiment, the methionine decarboxylase gene comprises the sequence of SEQ ID NO: 1035. In yet another embodiment the methionine decarboxylase gene consists of the sequence of SEQ ID NO: 1035. In one embodiment, the methionine decarboxylase gene has at least about 80% identity with the sequence of SEQ ID NO: 1036. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1036. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1036. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1036. In another embodiment, the methionine decarboxylase gene comprises the sequence of SEQ ID NO: 1036. In yet another embodiment the methionine decarboxylase gene consists of the sequence of SEQ ID NO: 1036. In one embodiment, the methionine decarboxylase gene has at least about 80% identity with the sequence of SEQ ID NO: 1037. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1037. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1037. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1037. In another embodiment, the methionine decarboxylase gene comprises the sequence of SEQ ID NO: 1037. In yet another embodiment the methionine decarboxylase gene consists of the sequence of SEQ ID NO: 1037. In one embodiment, the methionine decarboxylase gene has at least about 80% identity with the sequence of SEQ ID NO: 1039. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1039. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1039. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1039. In another embodiment, the methionine decarboxylase gene comprises the sequence of SEQ ID NO: 1039. In yet another embodiment the methionine decarboxylase gene consists of the sequence of SEQ ID NO: 1039. In one embodiment, the methionine decarboxylase gene has at least about 80% identity with the sequence of SEQ ID NO: 1040. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1040. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1040. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1040. In another embodiment, the methionine decarboxylase gene comprises the sequence of SEQ ID NO: 1040. In yet another embodiment the methionine decarboxylase gene consists of the sequence of SEQ ID NO: 1040. In one embodiment, the methionine decarboxylase gene has at least about 80% identity with the sequence of SEQ ID NO: 1123. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1123. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1123. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1123. In another embodiment, the methionine decarboxylase gene comprises the sequence of SEQ ID NO: 1123. In yet another embodiment the methionine decarboxylase gene consists of the sequence of SEQ ID NO: 1123. In one embodiment, the methionine decarboxylase gene has at least about 80% identity with the sequence of SEQ ID NO: 1125. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1125. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1125. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1125. In another embodiment, the methionine decarboxylase gene comprises the sequence of SEQ ID NO: 1125. In yet another embodiment the methionine decarboxylase gene consists of the sequence of SEQ ID NO: 1125. In one embodiment, the methionine decarboxylase gene has at least about 80% identity with the sequence of SEQ ID NO: 1127. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1127. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1127. Accordingly, in one embodiment, the methionine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1127. In another embodiment, the methionine decarboxylase gene comprises the sequence of SEQ ID NO: 1127. In yet another embodiment the methionine decarboxylase gene consists of the sequence of SEQ ID NO: 1127. In one embodiment, the recombinant bacteria comprise a gene sequence encoding a methionine catabolism enzyme, wherein the methionine catabolism enzyme is a leucine decarboxylase. In some cases, the leucine decarboxylase may have been modified from a wild-type leucine catabolism enzyme to also catabolize methionine, as described herein. Indeed, in some embodiments disclosed herein, a leucine catabolism enzyme that can metabolize methionine is referred to as a “methionine catabolism enzyme” or a “methionine decarboxylase.” In some embodiment, the leucine decarboxylase gene has at least about 80% with the sequence of SEQ ID NO: 1038. Accordingly, in one embodiment, the leucine decarboxylase gene has at least about 90% identity with the sequence of SEQ ID NO: 1038. Accordingly, in one embodiment, the leucine decarboxylase gene has at least about 95% identity with the sequence of SEQ ID NO: 1038. Accordingly, in one embodiment, the leucine decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1038. In another embodiment, the leucine decarboxylase gene comprises the sequence of SEQ ID NO: 1038. In yet another embodiment the leucine decarboxylase gene consists of the sequence of SEQ ID NO: 1038. In one embodiment, the leucine decarboxylase gene encodes a polypeptide has at least about 80% identity with SEQ ID NO: 1053. Accordingly, in one embodiment, the leucine decarboxylase gene encodes a polypeptide has at least about 90% identity with SEQ ID NO: 1053. Accordingly, in one embodiment, the leucine decarboxylase gene encodes a polypeptide has at least about 95% identity with SEQ ID NO: 1053. Accordingly, in one embodiment, the leucine decarboxylase gene encodes a polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1053. In another embodiment, the leucine decarboxylase gene encodes a polypeptide comprises SEQ ID NO: 1053. In yet another embodiment the leucine decarboxylase gene encodes a polypeptide consists of SEQ ID NO: 1053. In one embodiment, the leucine decarboxylase gene encodes a polypeptide that has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 point mutations as compared to a wild type leucine decarboxylase polypeptide. In one embodiment, the leucine decarboxylase gene encodes a polypeptide that has one or more point mutations selected from N2S, V14A, E16G, H17S, R19W, A30E, K41Q, I45D, R48H, A51P, R65Q, L68C, L90R, N147S, L154V, R156Q, R160G, L170Q, H179S, H185S, E218Q, Y220F, C235R, H240D, K254E, V263M, T269A, V304A, S308V, D310E, A318M, V328S, T372E, S394T, 1406P, and D411C as compared to a wild type polypeptide. In one embodiment, the leucine decarboxylase gene encodes a polypeptide that has point mutations N2S, V14A, E16G, H17S, R19W, A30E, K41Q, I45D, R48H, A51P, R65Q, L68C, L90R, N147S, L154V, R156Q, R160G, L170Q, H179S, H185S, E218Q, Y220F, C235R, H240D, K254E, V263M, T269A, V304A, S308V, D310E, A318M, V328S, T372E, S394T, I406P, and D411C as compared to a wild type polypeptide. In some embodiments, the sequence of a methionine decarboxylase associated with the disclosure comprises one or more amino acid substitutions relative to SEQ ID NO: 1049. In some embodiments, the one or more amino acid substitutions are at a position corresponding to position 41, 66, 70, 82, 203, 379, 491 and/or 500 in SEQ ID NO: 1049. In some embodiments, a methionine decarboxylase comprises: a glutamine (Q) at a position corresponding to position 41 in the sequence of SEQ ID NO: 1049; an asparagine (N) at a position corresponding to position 66 in SEQ ID NO: 1049; an aspartic acid (D) at a position corresponding to position 70 in the sequence of SEQ ID NO: 1049; a histidine (H) at a position corresponding to position 82 in SEQ ID NO: 1049; a histidine (H) at a position corresponding to position 203 in SEQ ID NO: 1049; a glycine (G) at a position corresponding to position 379 in SEQ ID NO: 1049; a leucine (L) at a position corresponding to position 491 in the sequence of SEQ ID NO: 1049; and/or a proline (P) at a position corresponding to position 500 in the sequence of SEQ ID NO: 1049. In some embodiments, the sequence of a methionine decarboxylase associated with the disclosure comprises substitutions at a position corresponding to: position 66; position 203; position 379; positions 70 and 82; positions 491 and 500; or positions 41 and 70 in the sequence of SEQ ID NO: 1049. In some embodiments, the sequence of a methionine decarboxylase comprises the following amino acid substitutions relative to the sequence of SEQ ID NO: 1049: T66N; A203H; H379G; Q70D and N82H; V491L and A500P; or R41Q and Q70D. In one embodiment, the methionine decarboxylase polypeptide has at least about 80% identity with the sequence of any one of SEQ ID NOs: 1048, 1049, 1050, 1051, 1052, 1054, 1055, 1124, 1126, or 1128. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 90% identity with the sequence of any one of SEQ ID NOs: 1048, 1049, 1050, 1051, 1052, 1054, 1055, 1124, 1126, or 1128. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 95% identity with the sequence of any one of SEQ ID NOs: 1048, 1049, 1050, 1051, 1052, 1054, 1055, 1124, 1126, or 1128. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of any one of SEQ ID NOs: 1048, 1049, 1050, 1051, 1052, 1054, 1055, 1124, 1126, or 1128. In another embodiment, the methionine decarboxylase polypeptide comprises the sequence of any one of SEQ ID NOs: 1048, 1049, 1050, 1051, 1052, 1054, 1055, 1124, 1126, or 1128. In yet another embodiment the methionine decarboxylase polypeptide consists of the sequence of any one of SEQ ID NOs: 1048, 1049, 1050, 1051, 1052, 1054, 1055, 1124, 1126, or 1128. In one embodiment, the methionine decarboxylase polypeptide has at least about 80% identity with SEQ ID NO: 1048. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 90% identity with SEQ ID NO: 1048. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 95% identity with SEQ ID NO: 1048. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1048. In another embodiment, the methionine decarboxylase polypeptide comprises SEQ ID NO: 1048. In yet another embodiment the methionine decarboxylase polypeptide consists of SEQ ID NO: 1048. In one embodiment, the methionine decarboxylase polypeptide has at least about 80% identity with SEQ ID NO: 1049. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 90% identity with SEQ ID NO: 1049. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 95% identity with SEQ ID NO: 1049. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1049. In another embodiment, the methionine decarboxylase polypeptide comprises SEQ ID NO: 1049. In yet another embodiment the methionine decarboxylase polypeptide consists of SEQ ID NO: 1049. In one embodiment, the methionine decarboxylase polypeptide has at least about 80% identity with SEQ ID NO: 1050. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 90% identity with SEQ ID NO: 1050. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 95% identity with SEQ ID NO: 1050. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1050. In another embodiment, the methionine decarboxylase polypeptide comprises SEQ ID NO: 1050. In yet another embodiment the methionine decarboxylase polypeptide consists of SEQ ID NO: 1050. In one embodiment, the methionine decarboxylase polypeptide has at least about 80% identity with SEQ ID NO: 1051. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 90% identity with SEQ ID NO: 1051. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 95% identity with SEQ ID NO: 1051. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1051. In another embodiment, the methionine decarboxylase polypeptide comprises SEQ ID NO: 1051. In yet another embodiment the methionine decarboxylase polypeptide consists of SEQ ID NO: 1051. In one embodiment, the methionine decarboxylase polypeptide has at least about 80% identity with SEQ ID NO: 1052. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 90% identity with SEQ ID NO: 1052. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 95% identity SEQ ID NO: 1052. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1052. In another embodiment, the methionine decarboxylase polypeptide comprises SEQ ID NO: 1052. In yet another embodiment the methionine decarboxylase polypeptide consists of SEQ ID NO: 1052. In one embodiment, the methionine decarboxylase polypeptide has at least about 80% identity with SEQ ID NO: 1054. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 90% identity with SEQ ID NO: 1054. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 95% identity with SEQ ID NO: 1054. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1054. In another embodiment, the methionine decarboxylase polypeptide comprises SEQ ID NO: 1054. In yet another embodiment the methionine decarboxylase polypeptide consists of SEQ ID NO: 1054. In one embodiment, the methionine decarboxylase polypeptide has at least 1 or 2 point mutations as compared to a wild type polypeptide. In one embodiment, the methionine decarboxylase polypeptide has point mutations H179S and/or V304A as compared to a wild type polypeptide. For example, SEQ ID NO: 1054 (and SEQ ID NO: 1039 encoding SEQ ID NO: 1054) is a methionine decarboxylase with two amino acid substitutions relative to a wild-type sequence. In one embodiment, the methionine decarboxylase polypeptide has at least about 80% identity with SEQ ID NO: 1055. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 90% identity with SEQ ID NO: 1055. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 95% identity with SEQ ID NO: 1055. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1055. In another embodiment, the methionine decarboxylase polypeptide comprises SEQ ID NO: 1055. In yet another embodiment the methionine decarboxylase polypeptide consists of SEQ ID NO: 1055. In one embodiment, the methionine decarboxylase polypeptide has at least about 80% identity with SEQ ID NO: 1124. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 90% identity with SEQ ID NO: 1124. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 95% identity with SEQ ID NO: 1124. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1124. In another embodiment, the methionine decarboxylase polypeptide comprises SEQ ID NO: 1124. In yet another embodiment the methionine decarboxylase polypeptide consists of SEQ ID NO: 1124. In one embodiment, the methionine decarboxylase polypeptide has at least about 80% identity with SEQ ID NO: 1126. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 90% identity with SEQ ID NO: 1126. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 95% identity with SEQ ID NO: 1126. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1126. In another embodiment, the methionine decarboxylase polypeptide comprises SEQ ID NO: 1126. In yet another embodiment the methionine decarboxylase polypeptide consists of SEQ ID NO: 1126. In one embodiment, the methionine decarboxylase polypeptide has at least about 80% identity with SEQ ID NO: 1128. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 90% identity with SEQ ID NO: 1128. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 95% identity with SEQ ID NO: 1128. Accordingly, in one embodiment, the methionine decarboxylase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1128. In another embodiment, the methionine decarboxylase polypeptide comprises SEQ ID NO: 1128. In yet another embodiment the methionine decarboxylase polypeptide consists of SEQ ID NO: 1128. The present disclosure further comprises genes encoding functional fragments of a methionine decarboxylase enzyme. Assays for testing the activity of a methionine catabolism enzyme, a methionine catabolism enzyme functional variant, or a methionine catabolism enzyme functional fragment are well known to one of ordinary skill in the art. For example, methionine catabolism can be assessed by expressing the protein, functional variant, or fragment thereof, in a recombinant bacterial cell that lacks endogenous methionine catabolism enzyme activity. Other methods are also well known to one of ordinary skill in the art (see, e.g., Dolzan et al.,FEBS Letters,574:141-146, 2004, the entire contents of which are incorporated by reference). Additional methods, i.e., for measuring methionine decarboxylase activity in vitro or in vivo are described in the examples herein. In some embodiments, the genetically engineered host cell, such as genetically engineered bacteria, comprise a stably maintained plasmid or chromosome carrying a gene for producing a methionine decarboxylase, such that the methionine decarboxylase can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo. In some embodiments, a host cell, such as a bacterium, may comprise multiple copies of the gene encoding the methionine decarboxylase. In some embodiments, the gene encoding the methionine decarboxylase is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, the gene encoding the methionine decarboxylase is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the methionine decarboxylase. In some embodiments, the gene encoding the methionine decarboxylase is expressed on a chromosome. In some embodiments, the host cells, such as bacteria host cells are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions. For example, the genetically engineered host cells, such as genetically engineered bacteria, may include four copies of the gene encoding a particular methionine decarboxylase inserted at four different insertion sites. Alternatively, the genetically engineered host cells, such as genetically engineered bacteria, may include three copies of the gene encoding a particular methionine decarboxylase inserted at three different insertion sites and three copies of the gene encoding a different methionine decarboxylase inserted at three different insertion sites. In some embodiments, under conditions where the methionine decarboxylase is expressed, the genetically engineered host cells, such as genetically engineered bacteria, of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the methionine decarboxylase, and/or transcript of the gene(s) in the operon as compared to unmodified host cells, such as unmodified bacteria, of the same subtype under the same conditions. In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the methionine decarboxylase gene(s). Primers specific for methionine decarboxylase the gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain methionine decarboxylase mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C., 60-70° C., and 30-50° C. for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C., 55-65° C., and 35-45° C. for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the methionine decarboxylase gene(s). In one embodiment, the host cell, such as a bacterial host cell, comprises a heterologous gene encoding a methionine catabolism enzyme. In one embodiment, the host cell, such as a bacterial host cell, comprises a heterologous gene encoding an importer of methionine. In one embodiment, the host cell, such as a bacterial host cell, comprises a heterologous gene encoding an importer of methionine and a heterologous gene encoding a methionine catabolism enzyme. In one embodiment, the host cell, such as a bacterial host cell, comprises a heterologous gene encoding a methionine catabolism enzyme and a genetic modification that reduces export of methionine. In one embodiment, the host cell, such as a bacterial host cell, comprises a heterologous gene encoding an importer of methionine, a heterologous gene encoding a methionine catabolism enzyme, and a genetic modification that reduces export of methionine. Importers and exporters are described in more detail in the subsections, below. B. Importers of Methionine Methionine importers may be expressed or modified in the recombinant host cells, such as recombinant bacteria, described herein in order to enhance methionine import into the cell. Specifically, when the importer of methionine is expressed in the recombinant host cells, such as recombinant bacterial cells, described herein, the bacterial cells import more methionine into the cell when the importer is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding importer of methionine which may be used to import methionine into the bacteria so that any gene encoding a methionine catabolism enzyme expressed in the organism can catabolize the methionine to treat a disease associated with methionine, such as homocystinuria. The uptake of methionine into bacterial cells is mediated by proteins well known to those of skill in the art. For example, a methionine importer operon has been identified inCorynebacterium glutamicum(Trotschel et al.,J. Bacteriology,187(11):3786-3794, 2005). In addition, the high affinity MetD ABC importer system has been characterized inEscherichia coli(Kadaba et al. (2008) Science 5886: 250-253; Kadner and Watson (1974)J. Bacteriol.119: 401-9). The MetD importer system is capable of mediating the translocation of several substrates across the bacterial membrane, including methionine. The MetD system ofEscherichia coliconsists of MetN (encoded by metN), which comprises the ATPase domain, MetI (encoded by metI), which comprises the transmembrane domain, and MetQ (encoded by metQ), the cognate binding protein which is located in the periplasm. Orthologues of the genes encoding theE. coliMetD importer system have been identified in multiple organisms including, e.g.,Yersinia pestis, Vibrio cholerae, Pasteurella multocida, Haemophilus influenza, Agrobacterium tumefaciens, Sinorhizobium meliloti, Brucella meliloti, andMesorhizobium loti(Merlin et al. (2002)J. Bacteriol.184: 5513-7). In one embodiment, the at least one gene encoding an importer of methionine is a metN gene, a metI gene, and/or a metQ gene fromCorynebacterium glutamicum, Escherichia coli, andBacillus subtilis(Trotschel et al.,J. Bacteriology,187(11):3786-3794, 2005). In one embodiment, the metN gene has at least about 80% identity with the sequence of SEQ ID NO: 1004. Accordingly, in one embodiment, the metN gene has at least about 90% identity with the sequence of SEQ ID NO: 1004. Accordingly, in one embodiment, the metN gene has at least about 95% identity with the sequence of SEQ ID NO: 1004. Accordingly, in one embodiment, the metN gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1004. In another embodiment, the metN gene comprises the sequence of SEQ ID NO: 1004. In yet another embodiment the metN gene consists of the sequence of SEQ ID NO: 1004. In one embodiment, the metI gene has at least about 80% identity with the sequence of SEQ ID NO: 1005. Accordingly, in one embodiment, the metI gene has at least about 90% identity with the sequence of SEQ ID NO: 1005. Accordingly, in one embodiment, the metI gene has at least about 95% identity with the sequence of SEQ ID NO: 1005. Accordingly, in one embodiment, the metI gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1005. In another embodiment, the metI gene comprises the sequence of SEQ ID NO: 1005. In yet another embodiment the metI gene consists of the sequence of SEQ ID NO: 1005. In one embodiment, the metQ gene has at least about 80% identity with the sequence of SEQ ID NO: 1006. Accordingly, in one embodiment, the metQ gene has at least about 90% identity with the sequence of SEQ ID NO: 1006. Accordingly, in one embodiment, the metQ gene has at least about 95% identity with the sequence of SEQ ID NO: 1006. Accordingly, in one embodiment, the metQ gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1006. In another embodiment, the metQ gene comprises the sequence of SEQ ID NO: 1006. In yet another embodiment the metQ gene consists of the sequence of SEQ ID NO: 1006. In one embodiment, the metNIQ gene has at least about 80% identity with the sequence of SEQ ID NO: 1043, 1045, 1046, or 1047. Accordingly, in one embodiment, the metNIQ gene has at least about 90% identity with the sequence of SEQ ID NO: 1043, 1045, 1046, or 1047. Accordingly, in one embodiment, the metNIQ gene has at least about 95% identity with the sequence of SEQ ID NO: 1043, 1045, 1046, or 1047. Accordingly, in one embodiment, the metNIQ gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1043, 1045, 1046, or 1047. In another embodiment, the metNIQ gene comprises the sequence of SEQ ID NO: 1043, 1045, 1046, or 1047. In yet another embodiment the metNIQ gene consists of the sequence of SEQ ID NO: 1043, 1045, 1046, or 1047. In one embodiment, the metNIQ gene encodes a polypeptide with a P281G mutation in the MetN polypeptide referenced by the MetN polypeptide encoded the gene sequence having the sequence of SEQ ID NO: 1004. In one embodiment, the metNIQ gene encodes a polypeptide with a P281S mutation in the MetN polypeptide referenced by the MetN polypeptide encoded the gene sequence having the sequence of SEQ ID NO: 1004. In one embodiment, at least one gene encoding an importer of methionine is a metP gene. In one embodiment, the metP gene is fromFlavobacterium segetis. In one embodiment, the metP gene is fromFlavobacterium frigoris. In one embodiment, the metP gene is fromBacillus subtilis. In one embodiment, the metP gene is fromSporomusa termitida. In one embodiment, the metP gene is from Bacteroidetes bacterium 43-16. In one embodiment, the metP gene has at least about 80% identity with the sequence of SEQ ID NO: 1041. Accordingly, in one embodiment, the metP gene has at least about 90% identity with the sequence of SEQ ID NO: 1041. Accordingly, in one embodiment, the metP gene has at least about 95% identity with the sequence of SEQ ID NO: 1041. Accordingly, in one embodiment, the metP gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1041. In another embodiment, the metP gene comprises the sequence of SEQ ID NO: 1041. In yet another embodiment the metP gene consists of the sequence of SEQ ID NO: 1041. In one embodiment, the metP gene has at least about 80% identity with the sequence of SEQ ID NO: 1042. Accordingly, in one embodiment, the metP gene has at least about 90% identity with the sequence of SEQ ID NO: 1042. Accordingly, in one embodiment, the metP gene has at least about 95% identity with the sequence of SEQ ID NO: 1042. Accordingly, in one embodiment, the metP gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1042. In another embodiment, the metP gene comprises the sequence of SEQ ID NO: 1042. In yet another embodiment the metP gene consists of the sequence of SEQ ID NO: 1042. In one embodiment, the metP gene has at least about 80% identity with the sequence of SEQ ID NO: 1044. Accordingly, in one embodiment, the metP gene has at least about 90% identity with the sequence of SEQ ID NO: 1044. Accordingly, in one embodiment, the metP gene has at least about 95% identity with the sequence of SEQ ID NO: 1044. Accordingly, in one embodiment, the metP gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1044. In another embodiment, the metP gene comprises the sequence of SEQ ID NO: 1044. In yet another embodiment the metP gene consists of the sequence of SEQ ID NO: 1044. In one embodiment, the metP gene has at least about 80% identity with the sequence of SEQ ID NO: 1129. Accordingly, in one embodiment, the metP gene has at least about 90% identity with the sequence of SEQ ID NO: 1129. Accordingly, in one embodiment, the metP gene has at least about 95% identity with the sequence of SEQ ID NO: 1129. Accordingly, in one embodiment, the metP gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1129. In another embodiment, the metP gene comprises the sequence of SEQ ID NO: 1129. In yet another embodiment the metP gene consists of the sequence of SEQ ID NO: 1129. In one embodiment, the metP gene has at least about 80% identity with the sequence of SEQ ID NO: 1131. Accordingly, in one embodiment, the metP gene has at least about 90% identity with the sequence of SEQ ID NO: 1131. Accordingly, in one embodiment, the metP gene has at least about 95% identity with the sequence of SEQ ID NO: 1131. Accordingly, in one embodiment, the metP gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1131. In another embodiment, the metP gene comprises the sequence of SEQ ID NO: 1131. In yet another embodiment the metP gene consists of the sequence of SEQ ID NO: 1131. In one embodiment, the MetP is fromFlavobacterium segetis. In one embodiment, the MetP is fromFlavobacterium frigoris. In one embodiment, MetP gene is fromBacillus subtilis. In one embodiment, MetP gene is fromSporomusa termitida. In one embodiment, MetP gene is from Bacteroidetes bacterium 43-16. In one embodiment, the MetP polypeptide has at least about 80% identity with the sequence of any one of SEQ ID NOs: 1056, 1057, 1061, 1130, or 1132. Accordingly, in one embodiment, the MetP polypeptide has at least about 90% identity with the sequence of any one of SEQ ID NOs: 1056, 1057, 1061, 1130, or 1132. Accordingly, in one embodiment, the MetP polypeptide has at least about 95% identity with the sequence of any one of SEQ ID NOs: 1056, 1057, 1061, 1130, or 1132. Accordingly, in one embodiment, the MetP polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of any one of SEQ ID NOs: 1056, 1057, 1061, 1130, or 1132. In another embodiment, the MetP polypeptide comprises the sequence of any one of SEQ ID NOs: 1056, 1057, 1061, 1130, or 1132. In yet another embodiment the MetP polypeptide consists of the sequence of any one of SEQ ID NOs: 1056, 1057, 1061, 1130, or 1132. In one embodiment, the MetP polypeptide has at least about 80% identity with the sequence of SEQ ID NO: 1056. Accordingly, in one embodiment, the MetP polypeptide has at least about 90% identity with the sequence of SEQ ID NO: 1056. Accordingly, in one embodiment, the MetP polypeptide has at least about 95% identity with the sequence of SEQ ID NO: 1056. Accordingly, in one embodiment, the MetP polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1056. In another embodiment, the MetP polypeptide comprises the sequence of SEQ ID NO: 1056. In yet another embodiment the MetP polypeptide consists of the sequence of SEQ ID NO: 1056. In one embodiment, the MetP polypeptide has at least about 80% identity with the sequence of SEQ ID NO: 1057. Accordingly, in one embodiment, the MetP polypeptide has at least about 90% identity with the sequence of SEQ ID NO: 1057. Accordingly, in one embodiment, the MetP polypeptide has at least about 95% identity with the sequence of SEQ ID NO: 1057. Accordingly, in one embodiment, the MetP polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1057. In another embodiment, the MetP polypeptide comprises the sequence of SEQ ID NO: 1057. In yet another embodiment the MetP polypeptide consists of the sequence of SEQ ID NO: 1057. In one embodiment, the MetP polypeptide has at least about 80% identity with the sequence of SEQ ID NO: 1061. Accordingly, in one embodiment, the MetP polypeptide has at least about 90% identity with the sequence of SEQ ID NO: 1061. Accordingly, in one embodiment, the MetP polypeptide has at least about 95% identity with the sequence of SEQ ID NO: 1061. Accordingly, in one embodiment, the MetP polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1061. In another embodiment, the MetP polypeptide comprises the sequence of SEQ ID NO: 1061. In yet another embodiment the MetP polypeptide consists of the sequence of SEQ ID NO: 1061. In one embodiment, the MetP polypeptide has at least about 80% identity with the sequence of SEQ ID NO: 1130. Accordingly, in one embodiment, the MetP polypeptide has at least about 90% identity with the sequence of SEQ ID NO: 1130. Accordingly, in one embodiment, the MetP polypeptide has at least about 95% identity with the sequence of SEQ ID NO: 1130. Accordingly, in one embodiment, the MetP polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1130. In another embodiment, the MetP polypeptide comprises the sequence of SEQ ID NO: 1130. In yet another embodiment the MetP polypeptide consists of the sequence of SEQ ID NO: 1130. In one embodiment, the MetP polypeptide has at least about 80% identity with the sequence of SEQ ID NO: 1132. Accordingly, in one embodiment, the MetP polypeptide has at least about 90% identity with the sequence of SEQ ID NO: 1132. Accordingly, in one embodiment, the MetP polypeptide has at least about 95% identity with the sequence of SEQ ID NO: 1132. Accordingly, in one embodiment, the MetP polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1132. In another embodiment, the MetP polypeptide comprises the sequence of SEQ ID NO: 1132. In yet another embodiment the MetP polypeptide consists of the sequence of SEQ ID NO: 1132. In one embodiment, the MetN polypeptide has at least about 80% identity with the sequence of SEQ ID NO: 1058. Accordingly, in one embodiment, the MetN polypeptide has at least about 90% identity with the sequence of SEQ ID NO: 1058. Accordingly, in one embodiment, the MetN polypeptide has at least about 95% identity with the sequence of SEQ ID NO: 1058. Accordingly, in one embodiment, the MetN polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1058. In another embodiment, the MetN polypeptide comprises the sequence of SEQ ID NO: 1058. In yet another embodiment the MetN polypeptide consists of the sequence of SEQ ID NO: 1058. In one embodiment, the MetN polypeptide has at least about 80% identity with the sequence of SEQ ID NO: 1062. Accordingly, in one embodiment, the MetN polypeptide has at least about 90% identity with the sequence of SEQ ID NO: 1062. Accordingly, in one embodiment, the MetN polypeptide has at least about 95% identity with the sequence of SEQ ID NO: 1062. Accordingly, in one embodiment, the MetN polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1062. In another embodiment, the MetN polypeptide comprises the sequence of SEQ ID NO: 1062. In yet another embodiment the MetN polypeptide consists of the sequence of SEQ ID NO: 1062. In one embodiment, the MetN polypeptide has at least about 80% identity with the sequence of SEQ ID NO: 1063. Accordingly, in one embodiment, the MetN polypeptide has at least about 90% identity with the sequence of SEQ ID NO: 1063. Accordingly, in one embodiment, the MetN polypeptide has at least about 95% identity with the sequence of SEQ ID NO: 1063. Accordingly, in one embodiment, the MetN polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1063. In another embodiment, the MetN polypeptide comprises the sequence of SEQ ID NO: 1063. In yet another embodiment the MetN polypeptide consists of the sequence of SEQ ID NO: 1063. In one embodiment, the MetI polypeptide has at least about 80% identity with the sequence of SEQ ID NO: 1059. Accordingly, in one embodiment, the MetI polypeptide has at least about 90% identity with the sequence of SEQ ID NO: 1059. Accordingly, in one embodiment, the MetI polypeptide has at least about 95% identity with the sequence of SEQ ID NO: 1059. Accordingly, in one embodiment, the MetI polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1059. In another embodiment, the MetI polypeptide comprises the sequence of SEQ ID NO: 1059. In yet another embodiment the MetI polypeptide consists of the sequence of SEQ ID NO: 1059. In one embodiment, the MetQ polypeptide has at least about 80% identity with the sequence of SEQ ID NO: 1060. Accordingly, in one embodiment, the MetQ polypeptide has at least about 90% identity with the sequence of SEQ ID NO: 1060. Accordingly, in one embodiment, the MetQ polypeptide has at least about 95% identity with the sequence of SEQ ID NO: 1060. Accordingly, in one embodiment, the MetQ polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1060. In another embodiment, the MetQ polypeptide comprises the sequence of SEQ ID NO: 1060. In yet another embodiment the MetQ polypeptide consists of the sequence of SEQ ID NO: 1060. In some embodiments, the importer of methionine is encoded by an importer of methionine gene derived from a bacterial genus or species, including but not limited to,Corynebacterium glutamicum, Escherichia coli, andBacillus subtilis. In some embodiments, the bacterial species isEscherichia colistrain Nissle. Assays for testing the activity of an importer of methionine, a functional variant of an importer of methionine, or a functional fragment of importer of methionine are well known to one of ordinary skill in the art. For example, import of methionine may be determined using the methods as described in Trotschel et al.,J. Bacteriology,187(11):3786-3794, 2005, the entire contents of which are expressly incorporated by reference herein. In one embodiment, when the importer of a methionine is expressed in the recombinant bacterial cells described herein, the bacterial cells import 10% more methionine into the bacterial cell when the importer is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when the importer of methionine is expressed in the recombinant bacterial cells described herein, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more methionine into the bacterial cell when the importer is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the importer of methionine is expressed in the recombinant bacterial cells described herein, the bacterial cells import two-fold more methionine into the cell when the importer is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the importer of methionine is expressed in the recombinant bacterial cells described herein, the bacterial cells import three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold more methionine into the cell when the importer is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. C. Exporters of Methionine Methionine exporters may be modified in the recombinant bacteria described herein in order to reduce methionine export from the cell. Specifically, when the recombinant bacterial cells described herein comprise a genetic modification that reduces export of methionine, the bacterial cells retain more methionine in the bacterial cell than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the recombinant bacteria comprising a genetic modification that reduces export of methionine may be used to retain more methionine in the bacterial cell so that any methionine catabolism enzyme expressed in the organism, e.g., co-expressed methionine catabolism enzyme, can catabolize the methionine. Exporters of methionine are well known to one of ordinary skill in the art. For example, the MetE methionine exporter fromBacillus atrophaeus, and the BrnFE methionine exporter fromCorynebacterium glutamicumhave been described (Trotschel et al.,J. Bacteriology,187(11):3786-3794, 2005). The YjeH methionine exporter fromE. colihas also been described (Liu et al., 2015: Applied and Environmental Microbiology,81(22):7753-7766). In one embodiment, the methionine exporter is yjeH. In one embodiment, the yjeH gene has at least about 80% identity with the sequence of SEQ ID NO: 1014. Accordingly, in one embodiment, the yjeH gene has at least about 90% identity with the sequence of SEQ ID NO:1014. Accordingly, in one embodiment, the yjeH gene has at least about 95% identity with the sequence of SEQ ID NO:1014. Accordingly, in one embodiment, the yjeH gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:1014. In another embodiment, the yjeH gene comprises the sequence of SEQ ID NO:1014. In yet another embodiment the yjeH gene consists of the sequence of SEQ ID NO:1014. In one embodiment, the yjeH gene is deleted. In another embodiment, a point mutation in the yjeH gene prevents export of methionine from the cell. In one embodiment, the genetic modification is a mutation in an endogenous gene encoding an exporter of methionine. In another embodiment, the genetic mutation results in an exporter having reduced activity as compared to a wild-type exporter protein. In one embodiment, the activity of the exporter is reduced at least 50%, at least 75%, or at least 100%. In another embodiment, the activity of the exporter is reduced at least two-fold, three-fold, four-fold, or five-fold. In another embodiment, the genetic mutation results in an exporter having no activity and which cannot export methionine from the bacterial cell. In another embodiment, the genetic modification is a mutation in a promoter of an endogenous gene encoding an exporter of methionine. In yet another embodiment, the genetic modification is an overexpression of a repressor of an exporter of methionine. In one embodiment, the overexpression of the repressor of the exporter is caused by a mutation which renders the promoter of the repressor constitutively active. In another embodiment, the overexpression of the repressor of the exporter is caused by the insertion of an inducible promoter in front of the repressor so that the expression of the repressor can be induced. Inducible promoters are described in more detail herein. D. Inducible Promoters In some embodiments, the host cell, such as a bacterial host cell, comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding the methionine decarboxylase(s) and/or the methionine importers, such that the methionine decarboxylase(s) and/or the methionine importers can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, the host cell, such as a bacterial host cell, comprises two or more distinct methionine decarboxylase and/or the methionine importer genes or operons. In some embodiments, the host cell, such as a bacterial host cell, comprises three or more distinct methionine decarboxylase and/or methionine importer genes or operons. In some embodiments, the host cell, such as a bacterial host cell, comprises 4, 5, 6, 7, 8, 9, 10, or more distinct methionine decarboxylase and/or methionine importer genes or operons. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacteria, comprise multiple copies of the same methionine decarboxylase gene(s) or methionine importer genes. In some embodiments, the gene encoding the methionine decarboxylase or methionine importer genes is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the methionine decarboxylase or methionine importer genes is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the methionine decarboxylase or methionine importer genes is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the methionine decarboxylase or methionine importer genes is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the methionine decarboxylase or methionine importer genes is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline, arabinose or Isopropyl β-D-1-thiogalactopyranoside (IPTG). In some embodiments, the inducible promoter is a IPTG inducible promoter, e.g., Ptac. In one embodiment, the IPTG inducible promoter comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 1108. In some embodiments, the recombinant bacterium further comprises a gene sequence encoding a gene sequence encoding a transcriptional regulator, e.g., a repressor IPTG inducible promoter. In some embodiments, the gene sequence encoding a repressor comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 1105. In some embodiments, the repressor comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 1106. TABLE 3IPTG inducible promoter and LacI sequencesDescriptionSEQ ID NOSequencesLacI in reverseTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAorientationATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCSEQ ID NO:CAGGGTGGTTTTTCTTTTCACCAGTGAGACTGGCAACAGCTGATTGC1105CCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTATCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCCTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCATLacIMKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNSEQ ID NO:YIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVV1106VSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQPlacI (promoterCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGfor lacI inGreverseorientation)SEQ ID NO:1113PlacI-RBS - lacITCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTA(reverseATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCorientation)CAGGGTGGTTTTTCTTTTCACCAGTGAGACTGGCAACAGCTGATTGCSEQ ID NO:CCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTG1114GTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTATCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCCTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCATATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGPtac (minimalttgacaattaatcatcggctcgtataatgpromoter forgene expression- includes −10and −35 region)SEQ ID NO:1115ExemplaryCGCGCCGCTTCGTCAGGCCACATAGCTTTCTTGTTCTGATCGGAACGspacer regionATCGTTGGCTGtgSEQ ID NO:1116Exemplary LacaattgtgagcgctcacaattoperatorSEQ ID NO:1107Exemplary pTacttgacaattaatcatcggctcgtataatgtgtggaattgtgagcgctcacaattagctgtpromotercomprising −10and −35 regionsand Lac operonSEQ ID NO:1108ExemplarytaacaccgtgcgtgttgOperator 1SEQ ID NO:miExemplaryTacctctggcggtgataOperator 2SEQ ID NO:1112Exemplary LacIATTCACCACCCTGAATTGACTCTCTTRBS (reverseorientation)SEQ ID NO:1117 In some embodiments, the bacterial cells comprise endogenous gene(s) encoding the IPTG sensing transcriptional regulator, LacI. In some embodiments, the lacI gene is heterologous. In some embodiments, the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI, is present on a plasmid. In some embodiments, the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI, and the gene encoding the methionine decarboxylase/methionine importer are present on different plasmids. In some embodiments, the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI, and the gene encoding the methionine decarboxylase or methionine importer are present on the same plasmid. In some embodiments, the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI, is present on a chromosome. In some embodiments, the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI, and the gene encoding the methionine decarboxylase or methionine importer are present on different chromosomes. In some embodiments, the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI, and the gene encoding the methionine decarboxylase or methionine importer are present on the same chromosome, either at the same or a different insertion site. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the methionine decarboxylase or methionine importer, e.g., a constitutive promoter. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the methionine decarboxylase or methionine importer. In some embodiments, the transcriptional regulator and the methionine decarboxylase or methionine importer are divergently transcribed from a promoter region. In some embodiments, the promoter that is operably linked to the gene encoding the methionine decarboxylase or methionine importer is directly induced by exogenous environmental conditions. In some embodiments, the promoter that is operably linked to the gene encoding the methionine decarboxylase or methionine importer is indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell. In one embodiment, the inducible promoter is an anhydrotetracycline (ATC)-inducible promoter. In one embodiment, the inducible promoter is an IPTG promoter. In one embodiment, the IPTG promoter is Ptac. As used herein the term “pTac” or “pTac promoter” includes the minimal promoter having −35 and −10 regions and at least the lac operator region. As used herein in certain instances, the term “pTac” or “pTac promoter” may also include an RBS in addition the minimal promoter and the Lac operator region. Non-limiting examples of suitable RBSs are listed herein. In a non-limiting example, pTac promoter sequence comprises SEQ ID NO: 1108. In some instances an RBS may be included at the 3′ end of SEQ ID NO: 1108. In a non-limiting example, the RBS comprises SEQ ID NO: 1107. In certain embodiments, the bacterial cell comprises a gene encoding a methionine decarboxylase and/or a methionine importer expressed under the control of a fumarate and nitrate reductase regulator (FNR) responsive promoter. InE. coli, FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et al., 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive. FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed in the chart, below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. TABLE 4FNR Re-sponsivePromoterSequenceSEQ IDGTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGNO: 1GGCGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCCTTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAASEQ IDATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGNO: 2ACTTATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGTTACGTGGGCTTCGACTGTAAATCAGAAAGGAGAAAACACCTSEQ IDGTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGNO: 3GGCGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCCTTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATSEQ IDCATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCNO: 4GACTTATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATSEQ IDAGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTANO: 5GTAAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGTAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTCTTGGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT In one embodiment, the FNR responsive promoter comprises SEQ ID NO: 1. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 2. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 3. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 4. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 5. In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria. In alternate embodiments, the genetically engineered bacteria comprise a gene encoding a methionine decarboxylase and/or a gene encoding a methionine importer expressed under the control of an alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al., 2010) or ANR (Ray et al., 1997). In these embodiments, expression of the methionine decarboxylase gene or methionine importer gene is particularly activated in a low-oxygen or anaerobic environment, such as in the gut. In one embodiment, the mammalian gut is a human mammalian gut. In some embodiments, the bacterial cell comprises an oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter from a different bacterial species. The heterologous oxygen-level dependent transcriptional regulator and promoter increase the transcription of genes operably linked to said promoter, e.g., the gene encoding the methionine decarboxylase or the gene encoding a methionine importer, in a low-oxygen or anaerobic environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions. In certain embodiments, the non-native oxygen-level dependent transcriptional regulator is an FNR protein fromN. gonorrhoeae(see, e.g., Isabella et al., 2011). In some embodiments, the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity. In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that is mutated relative to the wild-type promoter from bacteria of the same subtype. The mutated promoter enhances binding to the wild-type transcriptional regulator and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the methionine decarboxylase or the methionine importer, in a low-oxygen or anaerobic environment, as compared to the wild-type promoter under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent promoter, e.g., FNR, ANR, or DNR promoter, and corresponding transcriptional regulator that is mutated relative to the wild-type transcriptional regulator from bacteria of the same subtype. The mutated transcriptional regulator enhances binding to the wild-type promoter and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the methionine decarboxylase or the gene encoding the methionine importer, in a low-oxygen or anaerobic environment, as compared to the wild-type transcriptional regulator under the same conditions. In certain embodiments, the mutant oxygen-level dependent transcriptional regulator is an FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et al., (2006). In some embodiments, the bacterial cells comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator, e.g., the FNR gene. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the methionine decarboxylase or methionine importer are present on different plasmids. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the methionine decarboxylase or methionine importer are present on the same plasmid. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the methionine decarboxylase or methionine importer are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the methionine decarboxylase or methionine importer are present on the same chromosome. In some instances, it may be advantageous to express the oxygen level-sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the methionine decarboxylase or methionine importer. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the methionine decarboxylase or methionine importer. In some embodiments, the transcriptional regulator and the methionine decarboxylase or methionine importer are divergently transcribed from a promoter region. In some embodiments, any of the gene(s) of the present disclosure may be integrated into the chromosome of a host cell, such as a bacterial chromosome, at one or more integration sites. For example, one or more copies of one or more gene(s) encoding a methionine decarboxylase or methionine importer may be integrated into the chromosome of a host cell, such as a bacterial chromosome. Having multiple copies of the gene or gene(s) integrated into the chromosome allows for greater production of the methionine decarboxylase(s) and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the secretion or exporter circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the chromosome of a host cell, such as a bacterial chromosome, at one or more different integration sites to perform multiple different functions. E. Temperature Dependent Regulation In some instances, thermoregulators may be advantageous because of strong transcriptional control without the use of external chemicals or specialized media. Thermoregulated protein expression using the mutant cI857 repressor and the pL and/or pR phage λ promoters have been used to engineer recombinant bacterial strains. For example, a gene of interest cloned downstream of the a promoters can be efficiently regulated by the mutant thermolabile cI857 repressor of bacteriophage L. At temperatures below 37° C., cI857 binds to the oL or oR regions of the pR promoter and inhibits transcription by RNA polymerase. At higher temperatures, the functional cI857 dimer is destabilized, binding to the oL or oR DNA sequences is abrogated, and mRNA transcription is initiated. In certain instances, it may be advantageous to reduce, diminish, or shut off production of one or more protein(s) of interest. This can be done in a thermoregulated system by growing a bacterial strain at temperatures at which the temperature regulated system is not optimally active. Temperature regulated expression can then be induced as desired by changing the temperature to a temperature where the system is more active or optimally active. For example, a thermoregulated promoter may be induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. Bacteria comprising gene sequences or gene cassettes either indirectly or directly operably linked to a temperature sensitive system or promoter may, for example, could be induced by temperatures between 37° C. and 42° C. In some instances, the cultures may be grown aerobically. Alternatively, the cultures are grown anaerobically. In some embodiments, the host cell, such as a bacterial host cell, described herein comprise one or more gene sequence(s) or gene cassette(s) which are directly or indirectly operably linked to a temperature regulated promoter. In some embodiments, the gene sequence(s) or gene cassette(s) are induced in vitro during growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the gene sequence(s) are induced upon or during in vivo administration. In some embodiments, the gene sequence(s) are induced during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration and upon or during in vivo administration. In some embodiments, the genetically engineered host cell, such as genetically engineered bacteria, further comprise gene sequence (s) encoding a transcription factor which is capable of binding to the temperature sensitive promoter. In some embodiments, the transcription factor is a repressor of transcription. In one embodiment, the thermoregulated promoter is operably linked to a construct having gene sequence(s) or gene cassette(s) encoding one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the thermoregulated promoter is induced under a first set of exogenous conditions, and the second promoter is induced under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., thermoregulation and arabinose or IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., permissive temperature, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, one or more thermoregulated promoters drive expression of one or more protein(s) of interest in combination with an oxygen regulated promoter, e.g., FNR, driving the expression of the same gene sequence(s). In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the host cell chromosome, such as a bacterial chromosome. Exemplary insertion sites are described herein. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 309. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 313. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 316. In some embodiments, the thermoregulated construct further comprises a gene encoding mutant cI857 repressor, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 310. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 312. In some embodiments, the thermoregulated construct further comprises a gene encoding mutant cI38 repressor, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 314. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 315. SEQ ID NOs: 309, 310, and 312-316 are shown in Table 5. TABLE 5Inducible promoter construct sequences and related elementsDescriptionSEQ ID NORegion comprising TemperatureSEQ ID NO: 309sensitive promotermutant cI857 repressorSEQ ID NO: 310nucleotide sequencemutant cI857 repressor polypeptideSEQ ID NO: 312sequencePr/Pl promoterSEQ ID NO: 313mutant cI38 repressor nucleotideSEQ ID NO: 314sequencemutant cI38 repressor polypeptideSEQ ID NO: 315sequenceTemperature sensitive promoterSEQ ID NO: 316 In some embodiments, the bacterial cells comprise gene(s) encoding a temperature sensing transcriptional regulator/repressor described herein, e.g., cI857 or a mutant thereof. In some embodiments, the gene encoding the temperature sensing transcriptional regulator, is present on a plasmid. In some embodiments, the gene encoding the temperature sensing transcriptional regulator, and the gene encoding the methionine decarboxylase/methionine importer are present on different plasmids. In some embodiments, the gene encoding the temperature sensing transcriptional regulator, and the gene encoding the methionine decarboxylase or methionine importer are present on the same plasmid. In some embodiments, the gene encoding the temperature sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the temperature sensing transcriptional regulator, and the gene encoding the methionine decarboxylase or methionine importer are present on different chromosomes. In some embodiments, the gene encoding the temperature sensing transcriptional regulator, and the gene encoding the methionine decarboxylase or methionine importer are present on the same chromosome, either at the same or at different insertion sites. In some embodiments, expression of temperature sensing transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the methionine decarboxylase or methionine importer, e.g., a constitutive promoter. In some embodiments, expression of the temperature sensing transcriptional regulator is controlled by the same promoter that controls expression of the methionine decarboxylase or methionine importer. In some embodiments, the temperature sensing transcriptional regulator and the methionine decarboxylase or methionine importer are divergently transcribed from a promoter region. In any of these embodiments, gene expression may be further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability. F. Phage Deletion In some embodiments, the genetically engineered bacteria comprise one or moreE. coliNissle bacteriophage, e.g., Phage 1, Phage 2, and Phage 3. In some embodiments, the genetically engineered bacteria comprise one or mutations in Phage 3. Such mutations include deletions, insertions, substitutions and inversions and are located in or encompass one or more Phage 3 genes. In some embodiments, the one or more insertions comprise an antibiotic cassette. In some embodiments, the mutation is a deletion. In some embodiments, the genetically engineered bacteria comprise one or more deletions, which are located in or comprise one or more genes selected from ECOLIN_09965, ECOLIN_09970, ECOLIN_09975, ECOLIN_09980, ECOLIN_09985, ECOLIN_09990, ECOLIN_09995, ECOLIN_10000, ECOLIN_10005, ECOLIN_10010, ECOLIN_10015, ECOLIN_10020, ECOLIN_10025, ECOLIN_10030, ECOLIN_10035, ECOLIN_10040, ECOLIN_10045, ECOLIN_10050, ECOLIN_10055, ECOLIN_10065, ECOLIN_10070, ECOLIN_10075, ECOLIN_10080, ECOLIN_10085, ECOLIN_10090, ECOLIN_10095, ECOLIN_10100, ECOLIN_10105, ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, ECOLIN_10170, ECOLIN_10175, ECOLIN_10180, ECOLIN_10185, ECOLIN_10190, ECOLIN_10195, ECOLIN_10200, ECOLIN_10205, ECOLIN_10210, ECOLIN_10220, ECOLIN_10225, ECOLIN_10230, ECOLIN_10235, ECOLIN_10240, ECOLIN_10245, ECOLIN_10250, ECOLIN_10255, ECOLIN_10260, ECOLIN_10265, ECOLIN_10270, ECOLIN_10275, ECOLIN_10280, ECOLIN_10290, ECOLIN_10295, ECOLIN_10300, ECOLIN_10305, ECOLIN_10310, ECOLIN_10315, ECOLIN_10320, ECOLIN_10325, ECOLIN_10330, ECOLIN_10335, ECOLIN_10340, and ECOLIN_10345. In one embodiment, the genetically engineered bacteria comprise a complete or partial deletion of one or more of ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, ECOLIN_10170, and ECOLIN_10175. In one specific embodiment, the deletion is a complete deletion of ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, and ECOLIN_10170, and a partial deletion of ECOLIN_10175. In one embodiment, the sequence of SEQ ID NO: 1064 is deleted from the Phage 3 genome. In one embodiment, a sequence comprising SEQ ID NO: 1064 is deleted from the Phage 3 genome. G. Colibactin Island (Also Known as pks Island) In some embodiments, the engineered bacterium further comprises a modified pks island (colibactin island). Non-limiting examples are described in PCT/US2021/061579, the contents of which are herein incorporated by reference in their entirety. Colibactin is a cyclomodulin that is synthetized by enzymes encoded by the pks genomic island. See Fais 2018. The pks genomic island is “highly conserved” in Enterobacteriaceae. Id. InEscherichia coli, a 54-kilobase pks genomic island contains 19 genes, clbA to clbS, and encodes various enzymes that have been described as an “assembly line responsible for colibactin synthesis.” Id. The pks genomic island assembly line for colibactin synthesis includes three polyketide synthases (ClbC, ClbI, ClbO), three non-ribosomal peptide synthases (ClbH, ClbJ, ClbN), two hybrid non-ribosomal peptide/polyketide synthases (ClbB, ClbK), and nine accessory, tailoring, and editing proteins. The polyketide synthases, non-ribosomal peptide synthases, and hybrid enzymes “are usually organized in mega-complexes as an assembly line, in which the synthesized compound is transferred from one enzymatic module to the following one.” Id. Colibactin undergoes a prodrug activation mechanism that incorporates an N-terminal structural motif, which is removed during the final stage of biosynthesis. In some embodiments, the engineered microorganism, e.g., engineered bacterium, comprises a modified pks island (colibactin island). In some embodiments, the engineered microorganism, e.g., engineered bacterium, comprises a modified clb sequence selected from one or more of the clbA, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbL, clbJ, clbK, clbL, clbM, clbN, cibO, clbP, clbQ, clbR, and clbS gene sequences, as compared to a suitable control, e.g., the native pks island in an unmodified bacterium of the same strain and/or subtype. In some embodiments, the modified clb sequence is an insertion, a substitution, and/or a deletion as compared to the control. In some embodiments, the modified clb sequence is a deletion of the clb island, e.g., clbA, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbI, clbJ, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, clbR, and clbS. In one embodiment, the colibactin deletion is the whole island except for the clbS gene, e.g., a deletion of clbA, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbI, clbJ, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, and clbR. In some embodiments, the modified endogenous colibactin island comprises one or more modified clb sequences selected from clbA (SEQ ID NO: 1065), clbB (SEQ ID NO: 1066), clbC (SEQ ID NO: 1067), clbD (SEQ ID NO: 1068), clbE (SEQ ID NO: 1069), clbF (SEQ ID NO: 1070), clbG (SEQ ID NO: 1071), clbH (SEQ ID NO: 1072), clbI (SEQ ID NO: 1073), clbJ (SEQ ID NO: 1074), clbK (SEQ ID NO: 1075), clbL (SEQ ID NO: 1076), clbM (SEQ ID NO: 1077), clbN (SEQ ID NO: 1078), clbO (SEQ ID NO: 1079), clbP (SEQ ID NO: 1080), clbQ (SEQ ID NO: 1081), clbR (SEQ ID NO: 1082), or clbS (SEQ ID NO: 1803) gene. In some embodiments, the modified endogenous colibactin island comprises a deletion of clbA (SEQ ID NO: 1065), clbB (SEQ ID NO: 1066), clbC (SEQ ID NO: 1067), clbD (SEQ ID NO: 1068), clbE (SEQ ID NO: 1069), clbF (SEQ ID NO: 1070), clbG (SEQ ID NO: 1071), clbH (SEQ ID NO: 1072), clbI (SEQ ID NO: 1073), clbJ (SEQ ID NO: 1074), clbK (SEQ ID NO: 1075), clbL (SEQ ID NO: 1076), clbM (SEQ ID NO: 1077), clbN (SEQ ID NO: 1078), clbO (SEQ ID NO: 1079), clbP (SEQ ID NO: 1080), clbQ (SEQ ID NO: 1081), and clbR (SEQ ID NO: 1082). Essential Genes and Auxotrophs As used herein, the term “essential gene” refers to a gene which is necessary to for cell growth and/or survival. Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, for example, Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes,Nucl. Acids Res.,37: D455-D458 and Gerdes et al., Essential genes on metabolic maps,Curr. Opin. Biotechnol.,17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference). An “essential gene” may be dependent on the circumstances and environment in which an organism lives. For example, a mutation of, modification of, or excision of an essential gene may result in the recombinant bacteria of the disclosure becoming an auxotroph. An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In some embodiments, any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth. In one embodiment, the essential gene is an oligonucleotide synthesis gene, for example, thyA. In another embodiment, the essential gene is a cell wall synthesis gene, for example, dapA. In yet another embodiment, the essential gene is an amino acid gene, for example, serA or metA. Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1, as long as the corresponding wild-type gene product is not produced in the bacteria. For example, thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death. The thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003). In some embodiments, the bacterial cell of the disclosure is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene. A thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo. In some embodiments, the bacterial cell of the disclosure is auxotrophic in a gene that is complemented when the bacterium is present in the mammalian gut. Without sufficient amounts of thymine, the thyA auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut). Diaminopimelic acid (DAP) is an amino acid synthetized within the lysine biosynthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971). In some embodiments, any of the genetically engineered bacteria described herein is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene. A dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g., by adding DAP to growth media in vitro. Without sufficient amounts of DAP, the dapD auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut). In other embodiments, the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene. The uraA gene codes for UraA, a membrane-bound importer that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et al., 1995). A uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g., by adding uracil to growth media in vitro. Without sufficient amounts of uracil, the uraA auxotroph dies. In some embodiments, auxotrophic modifications are used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product (e.g., outside of the gut). In complex communities, it is possible for bacteria to share DNA. In very rare circumstances, an auxotrophic bacterial strain may receive DNA from a non-auxotrophic strain, which repairs the genomic deletion and permanently rescues the auxotroph. Therefore, engineering a bacterial strain with more than one auxotroph may greatly decrease the probability that DNA transfer will occur enough times to rescue the auxotrophy. In some embodiments, the genetically engineered bacteria comprise a deletion or mutation in two or more genes required for cell survival and/or growth. Other examples of essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, lpxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, rnc, ftsB, eno, pyrG, chpR, lgt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, lspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsL, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, lpxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, infB, nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsL, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def fnt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB, csrA, ispF, ispD, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, lpxD, fabZ, lpxA, lpxB, dnaE, accA, tilS, proS, yafF, tsf pyrH, olA, rlpB, leuS, lnt, ginS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, me, yceQ, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, ribA, fabI, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, and gpsA. Other essential genes are known to those of ordinary skill in the art. In some embodiments, the genetically engineered bacterium of the present disclosure is a synthetic ligand-dependent essential gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic auxotrophs with a mutation in one or more essential genes that only grow in the presence of a particular ligand (see Lopez and Anderson “Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3 Biosafety Strain, “ACS Synthetic Biology (2015) DOI: 10.1021/acssynbio.5b00085, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the SLiDE bacterial cell comprises a mutation in an essential gene. In some embodiments, the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG and adk. In some embodiments, the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is dnaN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS comprising one or more of the following mutations: L36V, C38A and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L5I and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L5I and L6G. In some embodiments, the genetically engineered bacterium is complemented by a ligand. In some embodiments, the ligand is selected from the group consisting of benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid, and L-histidine methyl ester. For example, bacterial cells comprising mutations in metG (E45Q, N47R, I49G, and A51C) are complemented by benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid or L-histidine methyl ester. Bacterial cells comprising mutations in dnaN (H191N, R240C, I317S, F319V, L340T, V347I, and S345C) are complemented by benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells comprising mutations in pheS (F125G, P183T, P184A, R186A, and I188L) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in adk (I4L, L5I and L6G) are complemented by benzothiazole or indole. In some embodiments, the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand. In some embodiments, the bacterial cell comprises mutations in two essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other embodiments, the bacterial cell comprises mutations in three essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G), metG (E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A, and I188L). In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system described herein. In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein. For example, the recombinant bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s) (such as the recombinase systems described herein). Other embodiments are described in Wright et al., “GeneGuard: A Modular Plasmid System Designed for Biosafety,” ACS Synthetic Biology (2015) 4: 307-16, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein, as well as another biosecurity system, such a conditional origin of replication (see Wright et al., supra). Isolated Plasmids In other embodiments, the disclosure provides an isolated plasmid comprising a first nucleic acid encoding a methionine decarboxylase or methionine importer operably linked to a first inducible promoter. In another embodiment, the disclosure provides an isolated plasmid comprising a second nucleic acid encoding at least one additional methionine decarboxylase or methionine importer. In one embodiment, the first nucleic acid and the second nucleic acid are operably linked to the first promoter. In another embodiment, the second nucleic acid is operably linked to a second inducible promoter. In one embodiment, the first inducible promoter and the second inducible promoter are separate copies of the same inducible promoter. In another embodiment, the first inducible promoter and the second inducible promoter are different inducible promoters. In one embodiment, the first promoter, the second promoter, or the first promoter and the second promoter, are each directly or indirectly induced by low-oxygen or anaerobic conditions. In another embodiment, the first promoter, the second promoter, or the first promoter and the second promoter, are each IPTG inducible. In another embodiment, the first promoter, the second promoter, or the first promoter and second promoter are each regulated by changes in temperature. In any of the above-described embodiments, the plasmid is a high-copy plasmid. In another embodiment, the plasmid is a low-copy plasmid. In another aspect, the disclosure provides a recombinant host cell, such as a recombinant bacterial cell, comprising an isolated plasmid described herein. In another embodiment, the disclosure provides a pharmaceutical composition comprising the recombinant bacterial cell. Integration In some embodiments, any of the gene(s) or gene cassette(s) of the present disclosure may be integrated into the host cell chromosome, such as a bacterial chromosome, at one or more integration sites. One or more copies of the gene (for example, an amino acid catabolism gene) or gene cassette (for example, a gene cassette comprising an amino acid catabolism gene and an amino acid importer gene) may be integrated into the host cell chromosome, such as a bacterial chromosome. Having multiple copies of the gene or gene cassette integrated into the chromosome allows for greater production of the methionine decarboxylase, and other enzymes of the gene cassette, and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the kill-switch circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the host cell chromosome, such as a bacterial chromosome, at one or more different integration sites to perform multiple different functions. In one non-limiting example, gene sequences encoding MetP and MetDC are integrated to facilitate Met import and metabolism. In one embodiment, metP is derived fromFlavobacterium segetisand facilitates the uptake of Met into the cell. In one embodiment, MetDC is derived fromStreptomycessp. 590 and includes two modifications (Q70D and N82H). In one embodiment, MetP is derived fromFlavobacterium segetis. In one embodiment, both genes or gene sequences are operably linked to a chemically inducible promoter. In some embodiments, the promoter is induced by the compound Isopropyl β-D-1-thiogalactopyranoside (IPTG) e.g., PTac promoter (see e.g.,FIG.14A). In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprises a single integrated copy of metDC. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprises two or more integrated copies of metDC. In some embodiments, two or more copies of the metDC gene are present at the same integration site, arranged in a cassette, and operably linked to the same promoter. In some embodiments, two or more copies of the metDC gene are present at the same integration site, arranged in a cassette, and one or more copies of the metDC gene are operably linked to different copies of same promoter or different promoters. Alternatively, in some embodiments, a genetically engineered host cell, such as a genetically engineered bacterium, may comprise two or more copies of the metDC gene and each copy of the metDC gene may be integrated at distinct sites. In some embodiments, each copy of the metDC gene is linked to a separate promoter at each integration site. In some embodiments, each copy of the metDC gene is operably linked to a different copy of the same promoter. In some embodiments, the promoters are different between two or more copies of the metDC gene. In some embodiments, the promoter is an inducible promoter, e.g., an IPTG inducible promoter. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprises a gene cassette comprising a metDC gene operatively linked to an IPTG inducible promoter, e.g., a pTac promoter. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprise a Ptac-metDC cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprises a single integrated copy of metP. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprises two or more integrated copies of metP. In some embodiments, two or more copies of the metP gene are present at the same integration site, arranged in a cassette, and operably linked to the same promoter. In some embodiments, multiple copies of the metP gene are present at the same integration site, arranged in a cassette, and one or more copies of the metP gene are operably linked to different copies of same promoter or different promoters. Alternatively, in some embodiments, a genetically engineered host cell, such as a genetically engineered bacterium, may comprise two or more copies of the metP gene and each copy of the metP gene may be integrated at distinct sites. In some embodiments, each copy of the metP gene is linked to a separate promoter at each integration site. In some embodiments, each copy of the metP gene is operably linked to a different copy of the same promoter. In some embodiments, the promoters are different between two or more copies of the metP gene. In some embodiments, the promoter is an inducible promoter, e.g., an IPTG inducible promoter. In some embodiments, the genetically engineered bacterium comprises a gene cassette comprising a metP gene operatively linked to an IPTG inducible promoter, e.g., Ptac. In some embodiments, the genetically engineered bacteria comprise a Ptac-metP cassette. In one embodiment, both metDC and metP genes are arranged in a cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprises a single integrated copy of metDC and/or metP. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprises one or more integrated copies of metDC and one or more integrated copies of metP. In some embodiments, the metDC gene and the metP gene are present at the same integration site, arranged in a cassette, and operably linked to the same promoter. In some embodiments, the metDC gene and the metP gene are present at the same integration site, arranged in a cassette, and the metDC gene and the metP gene are operably linked to different copies of same promoter or different promoters. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprises a single integrated copy of a gene cassette comprising metDC and metP. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprises two or more integrated copies a gene cassette comprising metDC and metP. In some embodiments, multiple copies of a gene cassette comprising metDC and metP are present at the same integration site, and operably linked to the same promoter. In some embodiments, multiple copies of the gene cassette comprising metDC and metP are present at the same integration site, and one or more copies of the cassette comprising metDC and metP are operably linked to different copies of same promoter or different promoters. In any of these embodiments, the metDC gene and the metP within the gene cassette may be operably linked to the same promoter. Alternatively, in any of these embodiments, the metDC gene and the metP may be each operably linked to a different copy of the same promoter. In another alternative, in any of these embodiments, the metDC gene and the metP may each be operably linked to a different promoter. Alternatively, in some embodiments, a genetically engineered host cell, such as a genetically engineered bacterium or microorganism, may comprise two or more copies of the gene cassette comprising metDC and metP and each copy of the gene cassette comprising metDC and metP may be integrated at distinct sites. In some embodiments, each copy of the gene cassette comprising metDC and metP is linked to one or more separate promoters at each integration site. In some embodiments, each copy of the gene cassette comprising metDC and metP is operably linked to one or more different copies of the same promoter. In some embodiments, the promoters are different between two or more copies of gene cassette comprising metDC and metP gene. In any of these embodiments, the metDC gene and the metP within the gene cassette may be operably linked to the same promoter. Alternatively, in any of these embodiments, the metDC gene and the metP may be each operably linked to a different copy of the same promoter. In another alternative, in any of these embodiments, the metDC gene and the metP may each be operably linked to a different promoter. In some embodiments, the promoter is an inducible promoter, e.g., an IPTG inducible promoter, e.g., a Ptac promoter. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprises gene cassette comprising metDC and metP, wherein metDC and metP are operably linked to the same IPTG inducible promoter. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprises gene cassette comprising metDC and metP, wherein metDC and metP are each operably linked to a different copy of an IPTG inducible promoter. In some embodiments metDC and metP are each operably linked to a different copy of a Ptac promoter. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprise a Ptac-metP-metDC cassette or a Ptac-metDC-metP-cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a Ptac-metP-Ptac-metDC cassette or a Ptac-metDC-Ptac-metP-cassette. Alternatively, in some embodiments, a genetically engineered host cell, such as a genetically engineered bacterium, may comprise one or more copies of the metDC gene and one or more copies of the metP gene and the metDC gene and the metP gene may be integrated at distinct sites. In some embodiments, the metP gene and the metDC gene are linked to a separate promoter at each integration site. In some embodiments, the metP gene and the metDC gene are operably linked to a different copy of the same promoter. In some embodiments, the promoters are different between the metP gene and the metDC gene. In some embodiments, the promoter is an inducible promoter, e.g., an IPTG inducible promoter. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprises a metDC gene operatively linked to an IPTG inducible promoter, e.g., a pTac promoter and a metP gene operatively linked to a different IPTG inducible promoter, e.g., a pTac promoter. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprise a cassette comprising Ptac-metDC cassette and a cassette comprising Ptac-metP. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, further comprise a LacI gene. In some embodiments, the lacI gene is non-native or heterologous. In some embodiments, the lacI gene is native. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprises a single integrated copy of lac. In some embodiments, the genetically engineered bacterium comprises two or more integrated copies of lacI. In some embodiments, two or more copies of the lacI gene are present at the same integration site, arranged in a cassette, and operably linked to the same promoter. In some embodiments, two or more copies of the lacI gene are present at the same integration site, arranged in a cassette, and one or more copies of the lacI gene are operably linked to different copies of same promoter or different promoters. Alternatively, in some embodiments, a genetically engineered host cell, such as a genetically engineered bacterium or microorganism, may comprise two or more copies of the lacI gene and each copy of the lacI gene may be integrated at distinct sites. In some embodiments, each copy of the lacI gene is linked to a separate promoter at each integration site. In some embodiments, each copy of the lacI gene is operably linked to a different copy of the same promoter. In some embodiments, the promoters are different between two or more copies of the lacI gene. In some embodiments, the promoter is a native lacI promoter. In some embodiments, the promoter is a non-native promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the genetically engineered bacterium comprises a gene cassette comprising a lacI gene operatively linked to a constitutive promoter, e.g., a Plac promoter. In some embodiments, the genetically engineered bacteria or microorganism comprise a Plac-lacI cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a gene cassette having a lacI gene and a metDC and/or metP gene. In some embodiments, the PlacI promoter and the lacI gene sequences are located upstream of a metDC gene, a metP gene or a gene cassette comprising metDC and metP. In some embodiments, the lacI gene is in reverse orientation relative to the metDC gene, metP gene or gene cassette comprising metDC and metP, i.e., LacI is divergently transcribed from a promoter region relative to the metDC gene, metP gene or gene cassette comprising metDC and met. Accordingly, in some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a lacI-PlacI cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a lacI-PlacI-Ptac-metDC cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a lacI-PlacI-Ptac-metP cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a lacI-PlacI-Ptac-metP-metDC cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a lacI-PlacI-Ptac-metDC-metP cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a lacI-PlacI-Ptac-metP-Ptac-metDC cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a lacI-PlacI-Ptac-metDC-Ptac-metP-cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a PlacI-lacI-Ptac-metDC cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a PlacI-lacI-Ptac-metP cassette. In some embodiments, the genetically engineered bacteria or microorganism comprise a PlacI-lacI-Ptac-metP-metDC cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a PlacI-lacI-Ptac-metDC-metP cassette. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a PlacI-lacI-Ptac-metP-Ptac-metDC cassette In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprise a PlacI-lacI-Ptac-metDC-Ptac-metP-cassette. In more specific embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprises three integrated copies of a metDC gene. In some embodiments, each of the three copies are integrated at separate integration sites. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium, comprises a single integrated copy of metP. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprises three integrated copies of metDC and one integrated copy of metP. In some embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprises a single integrated copy of a gene cassette comprising metDC and metP and further comprises a two integrated copies of a metDC gene. In some embodiments, each copy of metDC and metP are operably linked to different copies of the same promoter. In some embodiments, the promoter is an IPTG inducible promoter, e.g., Ptac. In some embodiments, the metDC gene encodes metDC fromStreptomycessp. 590, having Q70D and N82H mutations. In some embodiments, the metP gene encodes MetP fromFlavobacterium segetis. In some embodiments, the gene cassette comprising metDC and metP further comprises lacI. In some embodiments, one copy of a metDC gene is present in a gene cassette which further comprises lac. In some embodiments, a second copy of a metDC gene is not in a gene cassette further comprising lac. In some embodiments, the lacI promoter is a constitutive promoter, e.g., Plac. In one embodiment, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprises three integrated copies of a metDC gene, integrated at three separate integration sites, wherein one of the three metDC gene copies is present in a cassette further comprising a metP gene. In some embodiments, each of the three copies of the metDC genes and the metP genes, are linked to different copies of the same promoter. In some embodiments, the promoter is an inducible promoter, such as an IPTG inducible promoter, e.g. pTac. In some embodiments, the metDC gene encodes MetDC fromStreptomycessp. 590, having Q70D and N82H mutations and the metP gene encodes MetP fromFlavobacterium segetis. In any of these embodiments, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, may further comprise one or more of (1) a deletion in yjeH gene that encodes a Met/branched chain amino acid exporter (2) a deletion of the dapA gene that encodes for dihydrodipicolinate synthase (3) a deletion in the pks island which encodes colibactin and (4) an endogenous Nissle prophage gene deletion. In one specific embodiment, the host cell, such as a genetically engineered bacterium or microorganism, comprises three copies of a metDC gene derived fromStreptomycessp. 590 and comprising two modifications (Q70D and N82H), each integrated at separate integration sites, wherein one of the three metDC gene copies is present in a cassette further comprising metP derived fromFlavobacterium segetis, wherein the three copies of the metDC gene and the metP gene are each operably linked to separate copies of the same IPTG inducible promoter, wherein the host cell, such as a genetically engineered bacterium or microorganism, further comprises two non-native copies of the lacI gene each operably linked to separate copies of the same constitutive Plac promoter, wherein a first copy is of the lacI gene is present in reverse orientation upstream of the metP-metDC gene cassette and the second copy is present in reverse orientation upstream of a-metDC gene, and wherein the host cell, such as a genetically engineered bacterium or microorganism, further comprises a deletion in yjeH gene, a deletion of the dapA gene, a deletion in the pks island, and an endogenous Nissle prophage gene deletion. In one embodiment, the genetically engineered host cell, such as a genetically engineered bacterium or microorganism, comprises lacI-PlacI-Ptac-metDC, Ptac-metDC, and lacI-PlacI-Ptac-metP-Ptac-metDC, wherein the metDC genes encode MetDC fromStreptomycessp. 590, having Q70D and N82H mutations and metP gene encoding MetP fromFlavobacterium segetis, and wherein the bacterium further comprises a deletion in yjeH gene, a deletion of the dapA gene, a deletion in the pks island, and an endogenous Nissle prophage gene deletion. In Vivo Models The recombinant host cells, such as recombinant bacteria, may be evaluated in vivo, e.g., in an animal model. Any suitable animal model of a disease or condition associated with amino acid metabolism, such as homocystinuria, may be used. Pharmaceutical Compositions and Formulations Pharmaceutical compositions comprising the genetically engineered bacteria described herein may be used to treat, manage, ameliorate, and/or prevent a disorder associated with amino acid catabolism, e.g., homocystinuria. Pharmaceutical compositions comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided. Pharmaceutical compositions comprising the genetically engineered microorganisms of the invention may be used to treat, manage, ameliorate, and/or prevent a disorder associated with amino acid catabolism or symptom(s) associated with diseases or disorders associated with amino acid catabolism. Pharmaceutical compositions of the invention comprising one or more genetically engineered bacteria, and/or one or more genetically engineered virus, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided. In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic modifications described herein, e.g., to express a methionine decarboxylase. In alternate embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria that are each engineered to comprise the genetic modifications described herein, e.g., to express a methionine decarboxylase. The pharmaceutical compositions of the invention described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration. The genetically engineered microorganisms may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, injectable, intravenous, sub-cutaneous, immediate-release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 104 to 1012 bacteria. The composition may be administered once or more daily, weekly, or monthly. The composition may be administered before, during, or following a meal. In one embodiment, the pharmaceutical composition is administered before the subject eats a meal. In one embodiment, the pharmaceutical composition is administered currently with a meal. In on embodiment, the pharmaceutical composition is administered after the subject eats a meal The genetically engineered bacteria may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. In some embodiments, the genetically engineered bacteria of the invention may be formulated in a solution of sodium bicarbonate, e.g., 1 molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example). The genetically engineered bacteria may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. The genetically engineered microorganisms may be administered intravenously, e.g., by infusion or injection. The genetically engineered microorganisms of the disclosure may be administered intrathecally. In some embodiments, the genetically engineered microorganisms of the invention may be administered orally. The genetically engineered microorganisms disclosed herein may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well known to one of skill in the art. See, e.g., “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA. In an embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. In one embodiment, the pharmaceutical composition comprising the host cell may be formulated as a hygiene product. For example, the hygiene product may be an antibacterial formulation, or a fermentation product such as a fermentation broth. Hygiene products may be, for example, shampoos, conditioners, creams, pastes, lotions, and lip balms. The host cells disclosed herein may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate-polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,N-dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co-glycolides), carrageenan, starch poly-anhydrides, starch polymethacrylates, polyamino acids, and enteric coating polymers. In some embodiments, the genetically engineered microorganisms are enterically coated for release into the gut or a particular region of the gut, for example, the large intestine. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels. Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered microorganisms described herein. In one embodiment, the genetically engineered microorganisms of the disclosure may be formulated in a composition suitable for administration to adult subjects or pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et al., Pediatrics, 134(2):361-372, 2014). Moreover, pediatric formulation acceptability and preferences, such as route of administration and taste attributes, are critical for achieving acceptable pediatric compliance. Thus, in one embodiment, the composition suitable for administration to pediatric subjects may include easy-to-swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers. In one embodiment, a composition suitable for administration to pediatric subjects may also be suitable for administration to adults. In one embodiment, the composition suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In one embodiment, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors. In one embodiment, the composition suitable for administration to pediatric subjects may include a flavor. As used herein, “flavor” is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry. In certain embodiments, the genetically engineered microorganisms may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. In another embodiment, the pharmaceutical composition comprising the recombinant bacteria of the invention may be a comestible product, for example, a food product. In one embodiment, the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria-fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional food products, animal feeds, or dietary supplements. In one embodiment, the food product is a fermented food, such as a fermented dairy product. In one embodiment, the fermented dairy product is yogurt. In another embodiment, the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir. In another embodiment, the recombinant bacteria of the invention are combined in a preparation containing other live bacterial cells intended to serve as probiotics. In another embodiment, the food product is a beverage. In one embodiment, the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts. In another embodiment, the food product is a jelly or a pudding. Other food products suitable for administration of the recombinant bacteria of the invention are well known in the art. For example, see U.S. 2015/0359894 and US 2015/0238545, the entire contents of each of which are expressly incorporated herein by reference. In yet another embodiment, the pharmaceutical composition of the invention is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese. In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents. The genetically engineered microorganisms described herein may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The genetically engineered microorganisms may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection, including intravenous injection, subcutaneous injection, local injection, direct injection, or infusion. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt). In some embodiments, disclosed herein are pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g., by infusion. Single dosage forms of the pharmaceutical composition may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient. In other embodiments, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., U.S. Pat. No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used. Dosage regimens may be adjusted to provide a therapeutic response. Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD50, ED50, EC50, and IC50 may be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans. The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C. and 8° C. and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase. In some embodiments, the genetically engineered viruses are prepared for delivery, taking into consideration the need for efficient delivery and for overcoming the host antiviral immune response. Approaches to evade antiviral response include the administration of different viral serotypes as part of the treatment regimen (serotype switching), formulation, such as polymer coating to mask the virus from antibody recognition and the use of cells as delivery vehicles. In another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., U.S. Pat. No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used. The genetically engineered bacteria of the invention may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Methods of Treatment Further disclosed herein are methods of treating diseases associated with methionine metabolism. In some embodiments, disclosed herein are methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases or disorders. As used herein the terms “disease associated with amino acid metabolism” or a “disorder associated with amino acid metabolism” is a disease or disorder involving the abnormal, e.g., increased, levels of one or more amino acids in a subject. In one embodiment, a disease or disorder associated with amino acid metabolism is homocystinuria, cancer, or a metabolic syndrome/disease. For example, for metabolic indications, a methionine-restricted diet has been shown to increase lifespan, reduce adiposity, decrease systemic inflammation, and improve insulin sensitivity in rodent and some large animal models (see, for example, Dong et al.,EClinicalMedicine,2019). For indications in immune-oncology and cancer, there is preclinical data supporting a link between tumoral methionine restriction and antitumor activity (see, for example, Gay et al.,Cancer Medicine,2017, 6(6):1437-1452). In some embodiments, a disease or disorder associated with amino acid metabolism is cystinuria. Cystinuria is a condition in which stones made from cysteine dimers (known as cystine) form in the kidney, ureter, and bladder. The condition is inherited in an autosomal recessive manner. Normally, most cystine dissolves and returns to the bloodstream after entering the kidneys. Subjects with cystinuria have a genetic defect in SLC3A1 or SLC7A9. As a result, cystine builds up in the urine and forms crystals or stones (˜10× increase vs healthy subjects). The goal of current treatments is to relieve symptoms and prevent more stones from forming. Treatment involves drinking plenty of water, use drugs that make urine more alkaline, dietary salt/animal protein restriction, kidney/bladder surgery. A large proportion of patients fail to achieve therapeutic success even with adherence to current SOC. Cystinuria is a lifelong condition. Stones often return. In some embodiments, the disclosure provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases. The method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. In some embodiments, the genetically engineered bacteria disclosed herein are administered orally, e.g., in a liquid suspension. In some embodiments, the genetically engineered bacteria are lyophilized in a gel cap and administered orally. In some embodiments, the genetically engineered bacteria are administered via a feeding tube or gastric shunt. In some embodiments, the genetically engineered bacteria are administered rectally, e.g., by enema. In some embodiments, the genetically engineered bacteria are administered topically, intraintestinally, intrajejunally, intraduodenally, intraileally, and/or intracolically. In one embodiment, the genetically engineered bacteria are injected directly into a tumor. In certain embodiments, administering the pharmaceutical composition to the subject reduces the level of an amino acid, e.g., methionine, homocysteine, cysteine or cystine in a subject. In some embodiments, the methods of the present disclosure may reduce the level of an amino acid, e.g., methionine, homocysteine, cysteine or cystine in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, reduction is measured by comparing the amino acid concentration in a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating a disease or disorder allows one or more symptoms of the condition or disorder to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more as compared to levels in an untreated or control subject, or as compared to levels in the subject prior to administration. Amino acid levels may be measured by methods known in the art (see methionine decarboxylase section, supra). Before, during, and after the administration of the pharmaceutical composition, methionine concentrations in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions to reduce amino acid, e.g., methionine concentrations in a subject to undetectable levels, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject's amino acid concentration(s) prior to treatment. Before, during, and after the administration of the pharmaceutical composition, homocysteine concentrations in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions to reduce amino acid, e.g., homocysteine concentrations in a subject to undetectable levels, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject's amino acid concentration(s) prior to treatment. Before, during, and after the administration of the pharmaceutical composition, cysteine concentrations in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions to reduce amino acid, e.g., cysteine concentrations in a subject to undetectable levels, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject's amino acid concentration(s) prior to treatment. Before, during, and after the administration of the pharmaceutical composition, cystine concentrations in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions to reduce amino acid, e.g., cystine concentrations in a subject to undetectable levels, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject's amino acid concentration(s) prior to treatment. The methods disclosed herein may further comprise isolating a sample from the subject prior to administration of a composition and determining the level of the amino acid(s) in the sample. In some embodiments, the methods may further comprise isolating a sample from the subject after to administration of a composition and determining the level of amino acid(s) in the sample. In certain embodiments, administering the pharmaceutical composition to the subject prevents or reduces formation, occurrence, or presence of stones in a subject. In some embodiments, the stones are present in the kidney, bladder or urether. In some embodiments, the stones are cystine stones. In some embodiments, the methods of the present disclosure may reduce or reduce/prevent an increase in the formation, occurrence, or presence of a stone, e.g., a cystine stone, in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, levels of formation, occurrence, or presence of a stone, e.g., a cystine stone, is measured by comparing stone formation, occurrence or presence, respectively, in a subject before and after administration of the pharmaceutical composition, e.g., within a certain time span. In some embodiments, the formation, occurrence, or presence of a stone, e.g., a cystine stone, in a subject may be prevented completely, or completely within a certain time span. In some embodiments, the methods of the present disclosure may reduce or reduce or prevent an increase in cystine stone number, stone volume, stone area or stone weight, e.g., over time, in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, levels of cystine stone number, volume, area or weight over time are measured by comparing cystine stone number, stone volume, stone area or stone weight, respectively, in a subject before and after administration of the pharmaceutical composition, e.g., within a certain time span. In some embodiments, the methods of the present disclosure may prevent a change, e.g., an increase, in number of cystine stones, stone volume, stone area or stone weight, e.g., over time, in a subject completely, or completely within a certain time span. In some embodiments, the method of treating or ameliorating a disease or disorder allows the symptom of stone formation, e.g., cystine stone formation, e.g., in a subject having cystinuria, to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more as compared to levels in an untreated or control subject, or as compared to levels in the subject prior to administration. Presence of stones and stone attributes, e.g. cystine stone number, volume, area and weight may be measured by methods known in the art, e.g., CT scan ultrasound or MRI. In certain embodiments, the genetically engineered bacteria comprising a methionine decarboxylase isE. coliNissle. The genetically engineered bacteria may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009), or by activation of a kill switch, several hours or days after administration. Thus, the pharmaceutical composition comprising the methionine decarboxylase may be re-administered at a therapeutically effective dose and frequency. In alternate embodiments, the genetically engineered bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut. The methods disclosed herein may comprise administration of a composition alone or in combination with one or more additional therapies. The pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents, including but not limited to, sodium phenylbutyrate, sodium benzoate, and glycerol phenylbutyrate. The methods may also comprise following an amino acid, e.g., methionine, restricted diet, and/or administration of betaine, pyridoxine, and/or other enzyme replacement-based therapies such as OT-58 or AGLE-177. OT-58 represents a therapeutic approach incorporating the use of a modified version of the native human CBS enzyme. The goal of this treatment is to introduce the CBS enzyme into circulation, resulting in reduced Hey levels, increased crystalthionine levels, and normalized cysteine levels. AGLE-177 is an engineered human enzyme designed to degrade both homocysteine and homocysteine (two homocysteine molecules bound together) to lower abnormally high levels of homocysteine in the blood. Methionine abundance in natural sources of protein ranges from 1-2% (or 1-2 g/100 g protein intake). Assuming the average human subject needs to degrade about 1.0 g methionine per day with meals, and assuming the recombinant bacteria provides 3 hours of activity per dose, that leaves 3× doses per day at 5×1011dose and 1.0 g methionine per day (0.33 g/dose). 0.33 g methionine/dose=2230 μmol methionine. 2230 μmol/3 hours/5×1011cells leads to 1.49 μmol/hr/1×109cells. The target dose is 5×1011live recombinant bacterial cells/mL. For human subjects on a low protein diet eating 10 g protein/day, the subject needs to degrade about 0.1-1 g, e.g. 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g or 1 g, methionine per day with meals. Assuming the recombinant bacteria provides 3 hours of activity per dose, that leaves 3× doses per day at 5×1011dose and 0.1 g per day (0.033 g/dose). 0.033 g methionine/dose=223 μmol methionine. 223 μmol/3 hours/5×1011cells leads to 0.15 μmol/hr/1×109cells. The target dose is 5×1011live recombinant bacterial cells/mL. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.1 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.15 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.2 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.25 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.3 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.4 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.5 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.6 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.7 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.8 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.9 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 1.0 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 1.10 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 1.30 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 1.30 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 1.40 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 1.45 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 1.50 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.1 μmol/hr/1×109cells to about 1.5 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.2 μmol/hr/1×109cells to about 1.5 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.1 μmol/hr/1×109cells to about 1.4 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.4 μmol/hr/1×109cells to about 1.1 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.1 μmol/hr/1×109cells to about 1.0 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.5 μmol/hr/1×109cells to about 1.5 μmol/hr/1×109cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a methionine degradation activity of about 0.75 μmol/hr/1×109cells to about 1.25 μmol/hr/1×109cells. In one embodiment, about 0.1 g to about 1.0 g of methionine are degraded per day. In one embodiment, about 0.01 to about 1.5 g of methionine are degraded per day. In one embodiment, about 0.1 g of methionine are degraded per day. In one embodiment, about 0.2 g of methionine are degraded per day. In one embodiment, about 0.3 g of methionine are degraded per day. In one embodiment, about 0.4 g of methionine are degraded per day. In one embodiment, about 0.5 g of methionine are degraded per day. In one embodiment, about 0.6 g of methionine are degraded per day. In one embodiment, about 0.7 g of methionine are degraded per day. In one embodiment, about 0.8 g of methionine are degraded per day. In one embodiment, about 0.9 g of methionine are degraded per day. In one embodiment, about 1.0 g of methionine are degraded per day. In one embodiment, about 1.1 g of methionine are degraded per day. In one embodiment, about 1.2 g of methionine are degraded per day. In one embodiment, about 1.3 g of methionine are degraded per day. In one embodiment, about 1.4 g of methionine are degraded per day. In one embodiment, about 1.5 g of methionine are degraded per day. An important consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the genetically engineered bacteria disclosed herein, e.g., the agent(s) must not kill the bacteria. In some embodiments, the pharmaceutical composition is administered with food. In alternate embodiments, the pharmaceutical composition is administered before or after eating food. The pharmaceutical composition may be administered in combination with one or more dietary modifications, e.g., low-protein diet or amino acid supplementation. The dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician. EXAMPLES The present disclosure is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references, including literature references, issued patents, and published patent applications, as cited throughout this application are hereby expressly incorporated herein by reference. It should further be understood that the contents of all the figures and tables attached hereto are also expressly incorporated herein by reference. Example 1: Strain Development and Testing All strains in this example utilize medium copy plasmids. These plasmids contain either Methionine gamma lyase (MGL) or Methionine decarboxylase (MDC) under the control of an anhydrotetracycline (ATC)-inducible promoter. Plasmids were constructed through TypeIIS cloning of synthesized gBlock fragments (IDT, Coralville, IA) containing these genes, followed by Sanger sequencing for sequence verification. Plasmids were used to transformE. coliNissle (EcN). EcN strains harboring either MGL or MDC plasmids were grown to early log phase and induced for expression with 200 ng/mL ATC. Induction was allowed to proceed for 4 h, at which time cells were harvested by centrifugation and biomass stored in PBS containing 15% glycerol at −80° C. For testing of Methionine degradation activity, frozen biomass was thawed on ice and brought to an OD600=1 in M9 minimal media containing 0.5% glucose and 10 mM methionine and incubated at 37° C. statically. Supernatant samples were removed at 0, 30, 60, and 120 mins to determine the concentration of Methionine remaining over time. For activated biomass, 2 mL cultures were grown overnight in LB media. Overnight cultures were back-diluted 1:100 in 10-20 mL fresh LB media in 50 mL baffled flasks and grown for 2 hours at 37° C. with shaking at 250 rpm. After 2 hours of growth, induction with 2× anhydrotetracycline (ATC) occurred, and cells were grown an additional four hours at 37° C. with shaking at 250 rpm. After 6 hours of total growth, bacterial cells were pelleted by centrifugation at 8000 rpm for five minutes. The supernatant was removed, cells were placed on ice, and cells were resuspended in PBS buffer. The cells were either frozen at −80° C. or the consumption assay was run. For the methionine consumption assay, cells were thawed on ice and OD600was measured. The volume of cells equivalent to an OD of 1 were added to 1 mL of M9 minimal media containing 5% glucose in an 1.7 mL tube. The tube was vortexed briefly to evenly distribute the cells, and the tubes were placed at 37° C. with no shaking. 150 μL of cell/media suspension was removed at 0.5, 1.0, 1.5, 2.0 and 4.0 hour time points, spun at high speed for about 1 minute to pellet cells, and 100 μL was added to the well of a 96-well plate (avoiding pellet). The amount of L-methionine was measured using HPLC. FIG.3is a graph depicting methionine disappearance from minimal media inE. coliNissle harboring methionine gamma lyase (MGL) or methionine decarboxylase (MDC also referred to herein as MetDC) under the control of an anhydrotetracycline (ATC)-inducible promoter. EcN control is wild-typeE. coliNissle with no methionine catabolism enzymes. BA CGL/MGL is MGL fromBrevibacterium aurantiacum(DOI 10.1124/jpet.119.256537). CF MGL is MGL fromCitrobacter freundii. PG MGL is MGL from Poprhyromonasgingivalis. EcN-MetDC is MDC fromStreptomycessp. 590. These data demonstrate increased disappearance of methionine in the strains comprising a methionine catabolism enzyme as compared to EcN control. FIG.4is a graph depicting L-Met consumption over time.E. coliNissle, SYN7344, containing a medium copy plasmid (p15A ori) encoding an anhydrotetracycline-inducible MetDC were grown in LB to early log phase followed by induction of MetDC expression for 4 hours. Activated cells were harvested and frozen in PBS buffer containing 15% glycerol at −80° C. On the day of testing, activated biomass was thawed and resuspended to an OD600=1 in M9 minimal media containing 0.5% glucose and 10 mM Met. Supernatant samples were removed over 2 hours to quantify Met disappearance. These data demonstrate increased consumption of methionine in theE. coliNissle strains comprising MetDC as compared toE. coliNissle control strains. The recombinant bacteria may be further modified by knocking out methionine exporters, such as yjeH, an efflux pump known to import methionine out of the cell. Such a knockout will increase the cytoplasmic concentration of methionine to assist in driving methionine degradation reactions. In addition,E. colicontains an ABC importer, encoded in the metNIQ operon, known to import methionine into the cell. This importer may also be expressed or over expressed to increase availability of methionine to the recombinant bacteria. FIG.5is a graph depicting L-Met consumption over time. Strains SYN094 (control), SYN7328 (metNIQ (SEQ ID NOs: 1058, 1059, and 1060)), SYN7344 (SpmetDC (SEQ ID NO: 1049)), SYN7345 (ΔyjeH), SYN7346 (ΔyjeH, SpmetDC (SEQ ID NO: 1049), SYN7347 (ΔyjeH, metNIQ (SEQ ID NOs: 1058, 1059, and 1060)), SYN7348 (SpmetDC (SEQ ID NO: 1049), metNIQ (SEQ ID NOs: 1058, 1059, and 1060)), and SYN7349 (ΔyjeH, SpmetDC (SEQ ID NO: 1049), metNIQ (SEQ ID NOs: 1058, 1059, and 1060)). The strains used are shown in Table 6 and results in Table 7. EcN containing a medium copy plasmid (p15A ori) encoding an anhydrotetracycline-inducible MetDC and/or a low copy plasmid (pSC101 ori) encoding an anhydrotetracycline-inducible MetNIQ were grown in LB to early log phase followed by induction of MetDC and/or MetNIQ expression for 4 hours. Activated cells were harvested and frozen in formulation buffer containing 15% glycerol at −80° C. On the day of testing, activated biomass was resuspended to an OD600=1 in M9 minimal media containing 0.5% glucose and 10 mM Met. Supernatant samples were removed over 2 hours to quantify Met disappearance. Deletion of yjeH and/or addition of metNIQ show an additive effect when tested in combination with metDC. Expression of the Met importer, metNIQ, increased Met consumption. Similarly, deletion of the Met exporter, yjeH, increased Met consumption. Combining expression of metNIQ and deletion of yjeH with the expression of MetDC lead to an additive effect and greater Met consumption. Increasing internal Met concentration by increasing uptake and decreasing release of Met surprisingly increases whole cell activity. TABLE 6E. coliStrainsStrainAntibioticNo.Background/genotyperesistanceSYN7328SYN001 (WT EcN); Logic2375(pSC101;carbeni -Ptet:metNIQ (SEQ ID NOs: 1058,cillin1059, and 1060)(carb)SYN7344SYN001; Logic2279(p15a; Ptet:metDCkanamycin(SEQ ID NO: 1049))(kan)SYN7345SYN001; ΔyjeHchloram-phenicol(cam)SYN7346SYN001; ΔyjeH; Logic2279(p15a;cam, kanPtet:metDC (SEQ ID NO: 1049))SYN7347SYN001; ΔyjeH; Logic2375(pSC101;cam, carbPtet: metNIQ (SEQ ID NOs: 1058,1059, and 1060))SYN7348SYN001; Logic2279(p15a; Ptet: metDCkan, carb(SEQ ID NO: 1049)); Logic2375(pSC101;Ptet: metNIQ (SEQ ID NOs: 1058,1059, and 1060))SYN7349SYN001; ΔyjeH; Logic2279(p15a; Ptet:cam, carb,metDC (SEQ ID NO: 1049));kanLogic2375(pSC101; Ptet: metNIQ (SEQID NOs: 1058, 1059, and 1060)) TABLE 7Met ConsumptionTime (min)SYN094 - EcN ControlSYN7346 - EcN + ΔyjeH + MetDC010.438457110.3859836510.400650810.438457110.3859836510.40065083010.323979210.1582280510.049256959.836327359.792908359.717648756010.310529910.2443247510.174113059.53544789.53608329.599746759010.2097660510.0717077510.1042729.272727559.12985089.082389951209.661062859.94305699.65274978.56593338.591437558.62101895SYN7328 - EcN + MetNIQSYN7347 - EcN + ΔyjeH + MetNIQ010.438457110.3859836510.400650810.438457110.3859836510.40065083010.0767556510.129546810.07169019.952746759.94157439.999519256010.2405310.1500737510.251790710.06275929.993571210.02763579010.156621910.04330899.9164769.20440449.96723749.966301951209.921576859.97786279.86073739.78318329.73708149.76205615SYN7344 - EcN + MetDCSYN7348 - EcN + MetDC + MetNIQ010.438457110.3859836510.400650810.438457110.3859836510.4006508309.945510259.96000099.857754459.32714259.80319839.72354385609.594239959.754713759.731662859.50328959.42153479.482286909.336549959.27804029.30165599.02066799.07181769.027392551208.7251018.82079938.8221768.48989718.583142058.7146875SYN7345 - EcN + ΔyjeHSYN7349 - EcN + ΔyjeH + MetDC + MetNIQ010.438457110.3859836510.400650810.438457110.3859836510.40065083010.128240710.147761610.10494279.95597679.896443259.921647456010.158139810.123016310.2387659.30278559.34055659.377462659010.126369810.1596400510.11129678.779763058.88486888.94632611209.8561139.900396859.915752358.318780358.31082028.2659539 Example 2: Strain Activity Calculation Methionine abundance in natural sources of protein ranges from 1-2% (or 1-2 g/100 g protein intake). Assuming the average human subject needs to degrade about 1.0 g methionine per day with meals, and assuming the recombinant bacteria provides 3 hours of activity per dose, that leaves 3× doses per day at 5×1011dose and 1.0 g per day (0.33 g/dose). 0.33 g methionine/dose=2230 μmol methionine. 2230 μmol/3 hours/5×1011cells leads to 1.49 μmol/hr/1×109cells. The target dose is 5×1011live recombinant bacterial cells/mL. For human subjects on a low protein diet eating 10 g protein/day, the subject needs to degrade about 0.1 g methionine per day with meals. Assuming the recombinant bacteria provides 3 hours of activity per dose, that leaves 3× doses per day at 5×1011dose and 0.1 g per day (0.033 g/dose). 0.033 g methionine/dose=223 μmol methionine. 223 μmol/3 hours/5×1011cells leads to 0.15 μmol/hr/1×109cells. The target dose is 5×1011live recombinant bacterial cells/mL. The target performance is about 4.0 μmol Met degraded per hour per 1×109live cells. In one embodiment, the target performance is about 3.5 μmol Met degraded per hour per 1×109live cells. In one embodiment, the target performance is about 4.5 μmol Met degraded per hour per 1×109live cells. In one embodiment, the target performance is about 3.5 to about 4.5 μmol Met degraded per hour per 1×109live cells. In one embodiment, the target performance is about 3.75 to about 4.25 μmol Met degraded per hour per 1×109live cells. This is approximately five-fold what previous bacteria were able to degrade. The Met degradation rate for the SYN7344 strain is 0.81 μmol/hr/1×109cells. The Met degradation rate for the SYN7346 strain is 0.91 μmol/hr/1×109cells. The Met degradation rate for the SYN7348 strain is 0.91 μmol/hr/1×109cells. The Met degradation rate for the SYN7349 strain is 1.05 μmol/hr/1×109cells. Example 3: Production and Formulation Recombinant bacteria are cultured in LB media. 2 mL cultures were grown shaking overnight in 14 mL culture tubes. On the day of biomass preparation, 10 mL of fresh LB in a 50 mL baffled flask was inoculated with overnight culture at a 1:100 back-dilution. Cells were grown for 2 h at 37° C. in a shaking incubator (250 rpm). At 2 h, 200 ng/mL ATC was added for induction of recombinant genes. The induction phase was allowed to continue for 4 h. After induction, cells were spun down in a centrifuge at 5000×g for 10 min, and resuspended in PBS containing 15% glycerol and stored at −80° C. until the day of testing. The current formulation comprises biomass stored in PBS comprising 15% glycerol. Example 4: Design of In Vivo Study with Acute Mouse Model of Homocystinuria An in vivo study was designed to evaluate the activity of the recombinant bacterial strains in a mouse model of acute hypermethionemia. Briefly, mice were fasted overnight and orally gavaged with a dose of 200 mg/kg of D4-Met (labeled methionine) the following day. Blood samples were taken at 20 minutes, 1, 2 and 5 hours and urine samples at 5 hours post administration. Baseline samples were collected prior to the administration of D4-Met. Mice were kept fasting throughout the study. Intestinal effluent samples were collected at the end of the study after euthanization. Samples were analyzed using LC-MS/MS for primarily labelled and unlabeled Met and Homocysteine. As shown inFIG.6A, the plasma level of D4-Met reached a peak at about 20 minutes post administration, and the level dropped back to the baseline within 5 hours. A significant increase in D4-Met and D4-Hcy urinary excretion was observed in mice at 5 hours post administration (FIG.6B). The level of D4-Met in different gastrointestinal segments were also measured. As shown inFIG.6C, the level of D4-Met in the gastrointestinal segments correlated with the expected absorption gradient of methionine, where the upper small intestine had the highest level of D4-Met, followed by the middle small intestine, the lower small intestine and the colon. Similar patterns were observed for the level of endogenous methionine in plasma, urine and intestinal effluent samples (FIGS.6D-6F). However, the level of endogenous methionine in plasma samples reached a peak at about 2 hours post administration. Example 5. Evaluation of the Activity of Strain SYN7349 in HCU Mouse Model The activity of the recombinant bacterial strain, SYN7349 (ΔyjeH; metDC (SEQ ID NO: 1049)); metNIQ (SEQ ID NOs: 1058, 1059, and 1060))), was evaluated in the previously described acute mouse model of HCU based on the oral administration of labeled methionine (D4-Met). Briefly, mice were fasted overnight prior to dose and administered orally using a flexible feeding tube attached to a sterile single use syringe with 100 μL of D4-Met (200 mg/kg) and 200 μl of the recombinant bacterial strains, SYN7349 and/or SYN094 (about 2.8×1010live cells), or the glycerol/PBS vehicle. D4-Met was dosed 10 minutes after administration of the bacterial strains. Urine samples were collected before dose and 5 hours post dose using free catch method. Gastrointestinal samples were collected at the end of the study after euthanization. Samples were flushed with PBS and effluents were collected from small intestines and colon were collected. The small intestine was divided into three equal sections and effluents from the sections were collected into separate tubes. All samples were kept on ice and stored on 96-well plates at −80° C. for quantification. Samples were analyzed using LC-MS/MS for primarily labelled and unlabeled Met, Homocysteine and methylthiopropinoic acid (3MTP). Mice receiving the SYN7349 strain excreted a significant higher level of D4-3-methylthiopropinoic acid (3MTP) in urine samples than mice receiving the SYN094 strain or the vehicle control (FIG.7A). A similar pattern was shown for the elevated endogenous methionine, which is converted into 3MTP (FIG.7B). In addition, a significant increase in the level of 3MTP in the colon sample of mice receiving the SYN7349 strain was also observed at 5 hours (FIG.7C), suggesting that mice receiving the SYN7349 strain had a better capacity to consume methionine and excrete the decarboxylated product. These data demonstrate that the SYN7349 strains are capable of consuming methionine in vivo and are promising therapeutic treatment for metabolic diseases involving dysregulation of methionine metabolism, such as homocystinuria. Example 6. Evaluation of Methionine Consumption and 3-MTP Production Activities of EngineeredE. coliStrains FIG.8is a graph depicting L-Met consumption over time.E. coliNissle strains, SYN7349 (containing a medium copy plasmid (p15A ori) encoding an MetDC (Streptomycessp. 590), a low copy plasmid (pSC101) encoding an endogenous methionine importer (metNIQ), and a knockout of yjeH) and SYN7346 (containing a medium copy plasmid (p15A ori) encoding an MetDC (Streptomycessp. 590), and a knockout of yjeH), were grown in LB to early log phase followed by induction of MetDC expression for 4 hours. Activated cells were harvested and frozen in formulation buffer containing 15% glycerol at −80° C. On the day of testing, activated biomass was resuspended to an OD600=1 in M9 minimal media containing 0.5% glucose and 10 mM Met. Supernatant samples were removed over 2 hours to quantify Met disappearance. These data demonstrate increased consumption of methionine in theE. coliNissle strains comprising MetDC or MetDC/MetNIQ as compared toE. coliNissle control strains (SYN094). A metagenomic and a protein engineered library were screened for MetDC candidate enzymes having improved activity and for proteins that facilitate import of methionine.FIGS.9A and9Cdepict a primary screen (FIG.9A) and a secondary screen (FIG.9C) to identify optimal MetDC candidates. MetDC amino acid decarboxylases were expressed from plasmids inE. coliNissle. These strains were incubated with L-methionine for a set period of time. Samples were taken at the endpoint and analyzed using LCMS for the presence of the decarboxylation product of methionine, 3-methylthiopropylamine (3-MTP), which was used as a measure of activity. Surprisingly, multiple MetDC enzymes identified in the screen showed a multiple fold increase in activity relative to baseline. Screens were also conducted to identify optimal Met importer (MetP or MetNIQ) candidates are shown inFIG.10A. MetP and MetNIQ amino acid importers were expressed from plasmids in WTE. coliNissle. These strains were subsequently incubated with serial dilutions of norleucine, a toxic methionine analog, in liquid medium. These plates were used to calculate minimum inhibitory concentration of norleucine, under the hypothesis that lower MICs (higher sensitivity to toxin) would correspond to more active methionine importers. FIG.10Bis a graph depicting 3-MTP production over time when methionine importers were added to MetDC expressing strains. Genes encoding MetP (SYN7818 or SYN7819) or MetNIQ (SYN7815, SYN7816, or SYN7817) were added to strain expressing MetDC (SYN7346). Both MetP and MetNIQ increased 3-MTP production in comparison to strain containing only MetDC. Assays were performed as described herein. In vitro Simulation (IVS) assays were performed with SYN094 (control), SYN7349 (ΔyjeH, metDC, metNIQ), SYN7818 (ΔyjeH, metDC, metP((F. frigoris)), and SYN7819 (ΔyjeH, metDC, metP((F. segetis)) (FIG.10C). 3-MTP production was increased in SYN7349, SYN7818, and SYN7819 compared to the control strain. Strains with added MetP genes (SYN7818 and SYN7819) showed an increase of 3-MTP production in comparison to SYN7349 with only MetDC. SYN7819 showed approximately a 2-fold increase of 3-MTP production when compared to SYN7349. Briefly, frozen aliquots were thawed at room temperature, mixed, and placed on ice. Each strain was prepared in 1 mL aliquot at 5×109live cells/mL in 0.077 M sodium bicarbonate buffer, pH 7, and 400 uL were aliquoted into 3 wells in a 96-well plate. Samples were incubated in an Anoxic chamber set at 4% O2. 400 uL of Simulated Intestinal Fluid (SIF), with 10 mM Methionine, was added to each well. The plate was incubated for 2 hours at 37° C. with shaking with a breathable plate seal. 100 uL samples were collected at time points 0, 30, 60, 120, and 180 minutes. Each sample was centrifuged at 400 rpm for 5 mins and 90 uL supernatant was collected. The supernatants were stored at −80° C. until LC-MS/MS analysis. TABLE 8SIF Experimental MixtureVolume forOne SampleComponentConcentration[uL]Simulated Gastric Chyme1×500Simulated Intestinal Fluid1.25×275Pancreatin Solution800 Trypsin U/125mL in SIFBile Salts160 mM in SIF62.5CaCl2 Stock Solution0.3 M1HCl Stock Solution1 M7.5Water29Total Volume1000 FIG.11Ais a graph depicting Met-d4 consumption andFIG.11Bis a graph depicting 3-MTP-d4 production by strains containing the identified MetDC proteins from the MetDC screen. SYN094 (control), SYN7346 (ΔyjeH, metDC SEQ ID NO: 1049), SYN7640 (metDC (SEQ ID NO: 1049), ΔyjeH), SYN7641 (metDC (V491L A500P; SEQ ID NO: 1050; engineered library), ΔyjeH), SYN7642 (metDC (Q70D N82H; SEQ ID NO: 1048; engineered library), ΔyjeH), SYN7643 (metDC (R41Q Q70D; SEQ ID NO: 1051; engineered library), ΔyjeH), SYN7644 (metDC (Stanieriasp. NIES-3757; metagenomic library), ΔyjeH), SYN7689 (engineered metDC (Mus musculus; SEQ ID NO: 1054; metagenomic library), ΔyjeH), SYN7690 (engineered leuDC (Mus musculus; SEQ ID NO: 1053; metagenomic library), ΔyjeH), and SYN7691 (metDC (Entamoeba histolytica; SEQ ID NO: 1055; metagenomics library), ΔyjeH). MetDC (Q70D N82H) fromStreptomycessp. 590 (SYN7642) showed both increased Met-d4 consumption and 3-MTP-d4 production when compared to the control strain and/or prototype strain. Example 7. Gene Integration intoE. coliNissle Genome Genes encoding MetP and MetDC were integrated to facilitate methionine import and metabolism. The importer metP is derived fromFlavobacterium segetisand facilitates the uptake of methionine into the cell. MetDC is derived fromStreptomycessp. 590 and includes two modifications (Q70D and N82H) to improve its activity at converting methionine to 3-MTP and CO2. Both genes are under the regulatory control of a chemically inducible promoter (Ptac), which is induced by IPTG. To prevent release of methionine from the bacteria in the GI tract once it enters the cell, the yjeH gene that encodes a methionine/branched chain amino acid exporter was deleted. To control growth in vivo and in the environment, strains were engineered to be an auxotrophic strain through deletion of the dapA gene that encodes for dihydrodipicolinate synthase, which is essential for the cell wall. This deletion renders SYNB1353 unable to synthesize DAP, thereby preventing the proper formation of bacterial cell wall unless the strain is supplemented with DAP exogenously. IVS assays for 3-MTP production by SYN094 (control), SYN7642 (metDC (Q70D N82H; SEQ ID NO: 1048; engineered library), SYN7970 (2 copies metDC (Q70D N82H; SEQ ID NO: 1048; engineered library), metP (metagenomics library;F. segetis; SEQ ID NO: 1056), ΔyjeH, Δdap, Δϕ), SYN8002 (3 copies metDC (Q70D N82H; SEQ ID NO: 1048; engineered library), metP (metagenomics library;F. segetis; SEQ ID NO: 1056), ΔyjeH, Δdap, Δϕ), and SYN8003 (3 copies metDC (Q70D N82H; SEQ ID NO: 1048; engineered library), metP (metagenomics library;F. segetis; SEQ ID NO: 1056), ΔyjeH, Δdap, Δϕ) showed 3-MTP production (FIG.12). SYN8003 has three copies of MetDC and one copy of MetP and was shown produce approximately 3-fold more 3-MTP than the control strain. Lyophilization of SYN8003 decreased 3-MTP production by approximately by 1.2-fold. (Note: SYN8002 differs from SYN8003 by the RBS for MetDC. SYN8003 has a stronger RBS). The pks island (aka colibactin island or clb island), and an endogenous Nissle prophage gene, have also been deleted from the genome.FIG.13depicts Met consumption byE. colistrains: SYN094 (control), SYN7349 (ΔyjeH, metDC (SEQ ID NO: 1049), metNIQ (endogenous)), SYN8003 (3 copies metDC (Q70D N82H; SEQ ID NO: 1048; engineered library), metP (metagenomics library;F. segetis; SEQ ID NO: 1056), ΔyjeH, Δdap, Δϕ), and SYN8070 (3 copies metDC (Q70D N82H; SEQ ID NO: 1048; engineered library), metP (metagenomics library;F. segetis; SEQ ID NO: 1056), ΔyjeH, Δdap, Δϕ, Δpks). Both SYN8003 and SYN8070 (aka SYNB1353) showed approximately 1.4 fold to about 1.5-fold decrease of methionine compared to the control strain (SYN094). Deletion of the endogenous clb (colibactin) island (also referred to as pks island) in SYN8070 did not impact methionine consumption in comparison to SYN8003 with an intact clb gene. Strain names SYN8070 and SYNB1353 are used interchangeably herein. Additionally, SYNB1353 was designed as a DAP auxotroph strain by deleting the dapA gene that encodes 4-hydroxy-tetrahydropicolinate synthase, which is essential for bacterial growth. This deletion renders SYNB1353 unable to synthesize DAP, thereby preventing the proper formation of bacterial cell wall unless the strain is supplemented with DAP exogenously. There are no antibiotic resistance genes in SYNB1353, and whole genome sequencing (PacBio) confirmed that all insertions and deletions in SYNB1353 were created in the proper chromosomal locations and contained the expected sequence identities. Plating on selective media confirmed that SYNB1353 did not grow in the presence of any of the antibiotics used during strain construction and did not grow without exogenously supplied diaminopimelic acid. Example 8. Analysis of Methionine Degradation and 3-MTP Production with SYNB1353 In Vitro SYNB1353 comprises a metP gene, metDC gene, and deletion of the yjeH gene, as shown inFIG.14A. The ability of SYNB1353 to degrade methionine to 3-MTP and CO2by its engineered pathway was measured. SYNB1353 and SYN094 were grown and activated in a bioreactor following optimized processes intended to be used for the scale-up of drug product. Activated cell batches were resuspended to the specified live cell count in assay media, and cells were statically incubated at 37° C. Supernatants were collected at defined timepoints, and the quantity of each analyte (methionine and 3-MTP) in each sample was determined by liquid chromatography mass spectrometry (LC-MS/MS). As observed inFIG.14B, SYNB1353 degraded methionine and produced 3-MTP de novo, as designed. The control strain, SYN094, consumed methionine at a low rate and did not produce any 3-MTP. In vitro Met consumption assays, as described above, show consumption of methionine and production of 3-MTP by SYNB1353 and not the EcN control (FIG.14B). In vitro, SYNB1353 consumed methionine at a rate of 1.3±0.13 μmol/h/1×109live cells and concomitantly produced 3-MTP at a rate of 1.3±0.087 μmol/h/1×109live cells. Example 9. Dose-Response of SYNB1353 in Healthy Mice Receiving a Bolus of D4-Methionine The ability of SYNB1353 to metabolize dietary and gastrointestinal methionine to produce 3-MTP in healthy mice was assessed. Two identical studies were performed, and the data from both studies were combined and shown inFIG.15D. For each study, male C57BL/6J mice of approximately 8 weeks of age were acclimated for at least 4 days before being placed on study. Mice were fasted overnight and orally administered a single dose of SYN094 (3.5×10{circumflex over ( )}10 live cells, n=18/group) or SYNB1353 (3.0×10{circumflex over ( )}9, 1.0×10{circumflex over ( )}10, 3.5×10{circumflex over ( )}10 live cells, n=18/group). Thirty (30) minutes later, mice received a bolus of 200 mg of D4-methionine (PO) and were immediately placed in metabolic cages (3 per cage) without access to food for a total of 5 hours. Cumulative urine was collected for 3-MTP and creatinine measurements. The effects of SYNB1353 on urinary recovery of 3-MTP and D4-3-MTP are shown inFIG.15D. Urinary 3-MTP, D4-3-MTP, and creatinine were quantified by LC-MS/MS, and the ratio of metabolites to creatinine determined. Overall, the urinary recovery of 3-MTP in healthy mice was low but detectable, and only mice receiving SYNB1353 at 3.5×10{circumflex over ( )}10 live cells registered 3-MTP concentrations above the lower limit of quantitation (LLOQ) of the assay (0.16 μg/mL). SYNB1353 dose-dependently increased the recovery of both 3-MTP and D4-3-MTP, indicating conversion of both endogenous and orally administered methionine by SYNB1353 in the gut. At the highest dose tested, SYNB1353 resulted in a statistically significant 25- and 61-fold increase in urinary recovery of 3-MTP and D4-3-MTP, respectively, as compared to SYN094. In conclusion, this study indicates that SYNB1353 can dose-dependently convert both endogenous and dietary methionine into 3-MTP. Example 10. Development of an Acute Model of Homocystinuria in Healthy Nonhuman Primates The objective of this study was to develop an acute model of homocystinuria in nonhuman primates. Male cynomolgus monkeys of approximately 2-5 years of age (average weight of 3.4 kg) were fasted overnight and orally administered a methionine load at 100 or 300 mg/kg, and plasma was collected at 0-, 0.5-, 1-, 2-, 4-, 6-, and 24-hours post-dose for methionine and total homocysteine measurements by LC-MS/MS. Oral administration of methionine (100 or 300 mg/kg) resulted in a dose-dependent increase in plasma methionine levels, with peak concentration recorded at 30 minutes and 1 hour post dose for 100 mg/kg and 300 mg/kg, respectively (FIG.16A). Plasma methionine concentrations gradually decreased over time and reached pre-dose levels by 24 hours. The oral methionine load also resulted in a significant elevation in total plasma homocysteine by 30 minutes post dose, but no statistically significant difference between groups was noted (FIG.16B). By 24 hours, total homocysteine levels had returned to baseline values for both groups. In conclusion, this study indicates that oral administration of a methionine load to nonhuman primates leads to acute homocystinuria. Example 11. Activity of SYNB1353 on Plasma Methionine and Plasma Homocysteine Levels in Nonhuman Primates Receiving Different Methionine Loads The objective of this study was to assess the ability of SYNB1353 to metabolize methionine in an acute model of homocystinuria in nonhuman primates. SYNB1353 activity was assessed following a single dose of 1×10 live cells compared to vehicle in order to determine the impact of the combined EcN chassis and methionine engineering in this model. Male cynomolgus monkeys were fasted overnight and orally administered a methionine load (100 or 300 mg/kg) followed by sodium bicarbonate (1.8 mmol), and formulation buffer (vehicle), or SYNB1353 at 1×10 live cells. Plasma was collected at 0, 0.5, 1, 2, 4, and 6-hours post-dose for methionine and total homocysteine measurements and cumulative urine was collected at 6 hours for 3-MTP recovery (and normalized to creatinine levels to account for differences in urinary volumes). Metabolites were measured using LC-MS/MS. Administration of SYNB1353 resulted in a significant and treatment-related elevation in urinary 3-MTP levels as compared to vehicle, suggesting conversion of methionine by the strain in the gut (FIG.17B). As shown inFIG.17CandFIG.17D, SYNB1353 significantly blunted the plasma appearance of methionine and total homocysteine at both methionine loads, with a 42% and 55% reduction in plasma total homocysteine area under the curve (AUC) compared to vehicle. In conclusion, these data indicate that SYNB1353 is capable of consuming methionine in the gut of nonhuman primates with acute homocystinuria. Example 12. Dose-Response of SYNB1353 in Nonhuman Primates Receiving a Methionine Load The objective of this study was to evaluate the dose-response of SYNB1353 and assess the specific effect of methionine engineering against a control strain (SYN094) in an acute model of homocystinuria in nonhuman primates. Male cynomolgus monkeys were fasted overnight and orally administered a methionine load (100 mg/kg) followed by sodium bicarbonate (1.8 mmol), and formulation buffer (vehicle), SYN094, or SYNB1353 at 5×10{circumflex over ( )}11 or 1×10{circumflex over ( )}12 live cells. Two sets of 3 studies were conducted in a cross-over manner so that all 12 colony animals received each treatment. Plasma was collected at 0-, 0.5-, 1-, 2-, 4-, and 6-hours post-dose for methionine and total homocysteine measurements and cumulative urine was collected at 6 hours for 3-MTP recovery (and normalized to creatinine levels to account for differences in urinary volumes). Each set of 3 studies was normalized to its respective vehicle for data representation. Administration of SYNB1353 resulted in a dose-dependent elevation in urinary 3-MTP levels as compared to SYN094, suggesting that the methionine-metabolizing pathway engineered in SYNB1353, and not the bacterial chassis itself, was responsible for methionine metabolism in the gut (FIG.18B). As shown inFIG.18CandFIG.18D, SYNB1353 dose-dependently blunted the plasma appearance of methionine and total homocysteine in this model as compared to SYN094. In conclusion, these data indicate that the engineering of SYNB1353 is responsible for methionine consumption in the gut of nonhuman primates with acute homocystinuria and results in a dose-dependent blunting of plasma total homocysteine appearance in the blood. Example 13. In Vivo Evaluation of Methionine Consumption of EngineeredE. coliStrains after Systemic Administration of Methionine The objectives of this study were to determine (1) whether enterorecirculation of methionine occurs, and (2) whether orally-administered SYNB1353 can consume peripherally administered (IP) labeled methionine in mice. In a first study, healthy male C57BL/6 mice (n=3/group) were fasted overnight and received a single IP dose of D4-methionine (100 mg/kg). Blood and gut effluents (SI, cecum or colon) were collected at 0, 0.5, 1, or 2 hours post dosing for D4-methionine measurements. Results shown inFIGS.19A-19Dindicate that there is enterorecirculation of methionine from the plasma into the gut. In a second study, healthy male C57BL/6 mice (n=10-18/group) were fasted overnight and received a single IP dose of D4-Met (100 mg/kg) followed by 2 doses of SYNB1353 PO 0.5 and 1.5 hours later. Blood and urine were collected for D4-Met, D4-tHcy and D4-3-MTP measurements. Results are shown inFIGS.20A-20Cand illustrate that SYNB1353 is capable of consuming peripherally-administered labeled methionine and blunts plasma labeled methionine and labeled homocysteine levels. Example 14. Effect of Methionine-Restricted Diet in a Mouse Model of Cystinuria Cystinuria is a genetic disorder of amino acid import in the kidney characterized by excessive excretion of cystine, and dibasic amino acids (ornitihine, lysine, and arginine) in the urine, and cystine stone formation in the urinary tract. The potential of a methionine consuming strain described herein to treat, prevent, or reduce cystinuria was evaluated by analyzing the effect of a methionine restricted diet in a Slc3a1 knockout (KO) mouse model for cystinuria. Slc3a1 KO mice were subjected to a reduction in the methionine content of diet from the standard 0.62% to 0.12% for eight weeks, and cysteine as well as cystine levels in urine and plasma, and stone formation in the bladder were evaluated according to a scheme shown inFIG.23. Cystine stone formation was not observed in any of the twelve mice on the low-methionine diet. In contrast, bladder stones were observed in nine out of twelve mice (75%) on the 0.62% diet. Time of stone formation ranged from 2-8 weeks following diet treatment. These data suggest that a treatment resulting in a reduction in plasma or urinary methionine, e.g., administration of a methionine-consuming strain described herein, is a promising approach for the treatment of cystinuria. Methods Mouse Breeding and Diet Modification KO mice were generated using Slc−/−×Slc−/− matings and one-half of the male mice from a given litter were used for the 0.12% methionine study and the other half for the 0.62% study. Mice were identified using ear tags. One group of KO mice (N=12, age six weeks) was placed on a diet containing 0.12% methionine and another group on a diet containing 0.62% methionine. A third group of WT mice was placed on the 0.62% methionine diet. Both diets contained 10 kcal % fat. The 0.62% methionine diet is equivalent to the regular mouse diet with respect to methionine content, whereas the 0.12% diet is approximately 20% of regular methionine content. Computed Tomography KO mice were scanned by computed tomography (CT) on the Albira PET/CT system (Bruker Corporation) and only mice showing no evidence of stone at the age of six weeks were used for the diet studies. KO mice were then scanned every two weeks and bladder stone volume determined using the VivoQuant software (from Invicro) installed on the CT scanner. WT mice were not scanned. Body Weight, Food Consumption, and Urine Collection Mice were weighed and urine collected at baseline. The amount of food added per cage was also weighed. Mice were maintained on the above diets for eight weeks and the amount of food in each cage weighed weekly. There were three or four mice per cage. From these data, the average food consumption per mouse per day was calculated. Water was provided ad libitum, but water consumption was not measured. Mice were then weighed weekly and urine collected every two weeks. Urine samples were stored at −80° C. Mouse Sacrifice, Blood Collection, and Plasma Separation Mice were sacrificed using CO2 exposure at the end of the 8-week treatment period and blood was collected by cardiac puncture into heparin (green top tubes) from each of the low- and high-methionine groups and from nine WT mice. Plasma was separated by centrifugation and then stored at −80° C. A couple of the KO mice on 0.12% methionine diet had lost 20% of their body weights and were therefore euthanized at the recommendations of veterinary staff. Tissue Fixation and Storage Kidneys, bladder, and a small portion of the liver were removed from each mouse and placed in 10% formalin. Bladder Dissection and Stone Enumeration After three days in formalin, bladders were dissected, and any stones removed and weighed. The dissected bladders were also weighed, photographed and then returned to formalin. Stone number and size distribution (based on surface area of the stone image) were determined using NIH Image J software. Data Analysis Where appropriate, data were analyzed using standard statistical techniques and a p value of <0.05 was considered significant. Results Body Weight The average body weight in the two KO groups at the start of the study was 19.24 g, but there was a rapid decline in body weight to 14.73 g after one-week on the 0.12% diet (a loss of 23.4% relative to baseline). This was followed by a gradual decline over time, and the weight at eight weeks of treatment was 11.92 g (a loss of 38.0% relative to baseline). As indicated in the Methods section, two mice in this group were sacrificed at 3- and 8-weeks of treatment, respectively. By comparison, body weight of KO mice on the 0.62% diet increased over time, reaching 20.55 g after eight weeks of treatment (an increase of 6.81%). Results are shown inFIG.24A. Body weight in the WT mice on the 0.62% diet increased in a linear manner over time (black line), going from 20.48 g at baseline to 25.17 g after eight weeks (an increase of 22.9%). Food Consumption Three cages (with four mice per cage) were set-up for each of the 0.12% and 0.62% diets for the KO mice and four cages (with three mice per cage) were set-up for the 0.62% diet for WT mice. The average food consumption in the three groups was 1.68, 2.57, and 3.00 g/day/mouse, respectively. This may be related to differences in body weight among the three groups. Results normalized to body weight are shown inFIG.24B. CT Scanning The KO mice on the 0.12 and 0.62% methionine diets were CT scanned every two weeks. Bladder stones were not detected in any mice on the 0.12% diet. Bladder stones were detected by CT in 7 of the 12 KO mice on the 0.62% diet and the onset of stone detection ranged from two weeks to eight weeks of dietary treatment. In two mice, stones were not detected by CT, but small amounts of stone material were present when the bladders were dissected. Therefore, nine of the 12 KO mice on the 0.62% diet demonstrated evidence of stone presence. Results are showing inFIG.24Cand graphs of stone volume versus treatment period were almost parallel, indicating that, once stone formation has started, the rate of stone volume increase was comparable in all seven mice in which stones were detected by CT. Bladder and Stone Weight As indicated above, stones were identified in bladders from nine KO mice on the 0.62% methionine diet (7 by CT and two following bladder dissection). In the absence of stones, bladder weight in these mice was typically 16 mg. In the presence of stones, bladders were enlarged, with bladder and stone weights ranging from 19.6-82.1 and 20.2-83.3 mg, respectively, in the seven mice with CT-verified stones. Bladder weight in the 11 KO mice on the 0.12% diet was in the range 5.7-11.0 g (mean=8.38, SD=1.45). Bladder weights normalized to body weight are shown inFIG.24D. Bladder weight in the 12 WT mice on the 0.62% diet was in the range 13.5-23.5 g, but Grubbs' test using GraphPad software (GraphPad.com/quickcalcs/) indicated that the bladder of mouse #2728 (23.5 g) was an outlier (P<0.05). It was therefore removed from the data, giving an adjusted mean and SD of 16.36 and 1.70, respectively. The difference in bladder weight of this mouse compared with the others is evident in the photographs of the bladders from KO mice on the 012% and 0.62% methionine diets (FIG.25). The difference in bladder weight between the KO mice on the 0.12% diet and WT mice on the 0.62% diet was statistically significant using the unpaired t-test (P<0.0001). This is due to the decrease in body weight in the KO mice on the 0.12% diet versus WT mice. As shown inFIG.24D, when the bladder weight was normalized to total body weight, the difference in bladder weight between KO on 0.12% diet and WT was no longer significant. Bladder Stone Enumeration Stones from each of the nine mice were enumerated and the surface area of the stone image determined using NIH Image J software. Stone number, average stone area, and stone weight are summarized inFIGS.26A-26C. The number of stones per mouse was in the range 4-99 and the average surface area was in the range 0.077-3.574 mm2. Of the 260 stones identified, 137 occupied a surface area within the range 0.00-0.50 mm2 and one stone was at the extreme end (20.01-25.00 mm2). Conclusion A diet containing 0.62% methionine promoted stone formation in nine out of 12 KO mice (75%), whereas stones were not observed in any of the 12 KO mice that were on a diet containing 0.12% methionine for 8 weeks. These results indicate that a reduction in methionine levels as a result of methionine restriction leads to a reduction in urolithiasis and provides a rationale for using a methionine consuming strain described herein as a treatment for the reduction of stone formation in a subject having cystinuria. Example 15. Metabolite Levels in a Mouse Model of Homocystinuria Urine and plasma samples collected from several mice in each of the three groups in the study described in the previous example, and urinary cystine, cysteine, and methionine levels as well as plasma cysteine levels were measured. In brief, cysteine, cystine, ornithine, lysine, and arginine were quantitated in mouse plasma and urine by LC-MS/MS. Samples were deprotonated with sulfosalicylic acid then diluted with acetonitrile containing heavy-isotope internal standards for each analyte. To measure total cysteine, separate sample aliquots were first reduced with DL-dithiothreitol. Analytes were separated using hydrophilic interaction chromatography and detected using selected reaction monitoring of compound specific ions. Peaks were integrated and analyte/internal standard area ratios were used to calculate unknown concentrations relative to a standard curve. Results of the urine analysis are shown inFIGS.27A-27C, and results of the plasma analysis is shown inFIG.28and demonstrate that methionine restriction in the mouse model of cystinuria lowered urinary cysteine, cystine and methionine levels. Low-Methionine group was also associated with decreased plasma cysteine. These results further support the conclusion that reducing methionine levels in subjects with cystinuria, e.g., by using a methionine consuming strain described herein, can reduce elevated cysteine and cystine in urine and/or plasma and consequently can reduce or prevent cystine stone formation in kidney or bladder in these subjects. Example 16. Mouse Model of Homocystinuria Methods Generation of an Inducible Mouse Model of Homocystinuria To develop an inducible model of homocystinuria, short hairpin RNA (shRNA) targeting cystathionine β-synthase (CBS) packaged in adeno-associated virus (AAV) particles were purchased from Vector Biolabs (Malvern, PA) and used to inject 6-week-old C57BL/6J male mice. For this experiment, 18 male C57BL/6J mice were group housed and assigned to groups (n=9/group) based on average cage body weight. Mice received a single intravenous dose (tail vein) of vehicle (PBS) or AAV CBS-shRNA (1×1012genomic copies [GC]) and left unmanipulated for 72 hours. Blood was collected 21- or 28-days post IV dosing for tHcy analysis. Animals were euthanized by CO2 asphyxiation on day 35 and livers were snap-frozen in liquid nitrogen for future analysis. Liver samples were added to bead-bug tubes and homogenized for 30 seconds in 1 mL of lysis buffer (TPER) with a cocktail of protease inhibitors, followed by a 3-minutes centrifugation at 25,200 g. Protein concentration was determined on the supernatants by the Bradford method, 50 μg of liver homogenate was loaded onto a 4-12% Bis-Tris gel and proteins transferred onto PVDF membranes. Membranes were incubated overnight with a primary rabbit monoclonal antibody against CBS (D8F2P, Cell Signaling Technologies Cat. 14782S) at 1:333 dilution. Membranes were then washed and incubated with anti-rabbit IgG HRP-linked secondary antibody (Cell Signaling Technologies Cat. 70745) at 1:1000 dilution for 60 minutes at room temperature. Protein signal was revealed using ECL Reagent and developed using SignalFire™ ECL Reagent #6883 (Cell Signaling Technologies Cat. 6883S). Assessment of SYNB1353 Activity in AAV-CBS Mice ShRNA targeting CBS packaged in AAV particles was administered to 8-week-old C57BL/6J male mice by IV injection (1×1012genomic copies (GC) of AAV). Six weeks post-AAV injection, mice were orally administered a bolus of labeled methionine (50 mg/kg) with EcN (2.7×1010live cells, n=8/group) or SYNB1353 (2.7×1010live cells, n=8/group). One hour later, mice received another dose of bacteria (EcN or SYNB1353 at 2.7×1010live cells) and blood was collected 0, 0.5, or 2 hours post-labeled methionine. Results To assess the activity of SYNB1353 on plasma total homocysteine (tHcy) levels in mice, we developed a new model by delivering short hairpin RNA (shRNA) targeting CBS packaged in adeno-associated virus (AAV) particles by intravenous (IV) injection to 6-week-old C57BL/6J male mice. Mice were administered vehicle or 1×1012genomic copies (GC) of AAV by tail vein injection. On days 21 and 28 post AAV injection, blood was collected for total homocysteine determination and livers were harvested on day 35 to assess CBS expression. AAV-targeted delivery of CBS shRNA resulted in substantial lowering of hepatic CBS protein (61 kDa) expression by Western Blot compared to vehicle treated animals. Because of residual CBS protein in one AAV CBS-shRNA, suggesting incomplete CBS knockdown, and lack of housekeeping protein expression (GAPDH) in another, these 2 animals were removed from further analysis. Total homocysteine levels remained low in animals with intact CBS (average 5.1 μM), while downregulation of hepatic CBS expression resulted in a significant 10-12-fold elevation in plasma tHcy. Short hairpin RNA (shRNA) targeting cystathionine β-synthase (CBS) packaged in adeno-associated virus (AAV) particles was administered to 8-week-old C57BL/6J male mice by intravenous (IV) injection (1×1012genomic copies (GC) of AAV). Six weeks post-AAV injection, mice were orally administered a bolus of labeled methionine (50 mg/kg) with EcN (2.7×1010live cells, n=8/group) or SYNB1353 (2.7×1010live cells, n=8/group). One hour later, mice received another dose of bacteria (EcN or SYNB1353 at 2.7×1010live cells) and blood was collected 0, 0.5, or 2 hours post-labeled methionine. The bolus of labeled methionine resulted in significant elevations in plasma labeled methionine and labeled homocysteine in AAV mice, and SYNB1353 significantly blunted the appearance of both amino acids in plasma as demonstrated by a significant reduction in the area under the curve (AUC) with 35% and 23% for methionine and homocysteine, respectively. TABLE 9E. coli StrainsAntibioticStrain No.Background/genotyperesistanceSYN094wt Nissle, strepRStrepSYN7349Logic2279, which is a medium-copy p15a plasmid (pTET, atccam, carb,induction) expressing a methionine decarboxylase fromkanStreptomycessp. 590 (SEQ ID NO: 1003) to convert methionineinto 3-methylthiopropylamine and Logic2375, which is a low-copy pSC101 plasmid (pTET, atc induction) expressing anendogenous methionine importer (metNIQ); yjeHSYN7815ΔyjeH, containing Logic2279. Also containing logic2501 with anCam, kan,engineered MetNIQ importer (MetN P281S) (SEQ ID NO: 1047spec(metN(P281S)IQ)); (pLacO, IPTG induction)SYN7816Logic2279, methionine decarboxylase fromStreptomycessp. 590Cam, kan,(SEQ ID NO: 1003). Also containing plasmid logic2502 with anspecengineered MetNIQ importer (MetN P281G) (SEQ ID NO: 1045(metN(P281G)IQ)); (pLacO, IPTG induction); ΔyjeHSYN7817Logic2279, methionine decarboxylase fromStreptomycessp. 590Cam, kan,(SEQ ID NO: 1003). Also containing logic2503 with a recodedspecMetNIQ importer (SEQ ID NO: 1046); (pLacO, IPTGinduction); ΔyjeHSYN7818Logic2279, methionine decarboxylase fromStreptomycessp. 590Cam, kan,(SEQ ID NO: 1003). Also containing logic2534 with the MetPspecimporter (F. frigoris) (SEQ ID NO: 1042); (pLacO I, IPTGinduction); ΔyjeHSYN7819Logic2279, methionine decarboxylase fromStreptomycessp. 590Cam, kan,(SEQ ID NO: 1003). Also containing logic2535 with the MetPspecimporter (F. segetis) (SEQ ID NO: 1041); (pLacO, IPTGinduction); ΔyjeHSYN7346Logic2279, methionine decarboxylase fromStreptomycessp. 590cam, kan(SEQ ID NO: 1003); ΔyjeHSYN7640recodedStreptomycesMetDC (SEQ ID NO: 1003), pSC101Cam, kan(pTET, atc induction); ΔyjeHSYN7641protein engineered MetDC (V491L A500P) (SEQ ID NO: 1035)Cam, kanwith high 3MTP production, pSC101 (pTET, atc induction);ΔyjeHSYN7642protein engineered MetDC (Q70D N82H) (SEQ ID NO: 1034)Cam, kanwith high 3MTP production, pSC101 (pTET, atc induction);ΔyjeHSYN7643protein engineered MetDC (R41Q Q70D) (SEQ ID NO: 1036)Cam, kanwith high 3MTP production, pSC101 (pTET, atc induction);ΔyjeHSYN7644Stanieriasp. NIES-3757 Methionine decarboxylase (SEQ IDCam kanNO: 1037) from MetDC metagenomic library screen with high3MTP production, pSC101 (pTET, atc induction); ΔyjeH,SYN7689SYN7345 containing Logic2491, which expresses an engineeredCam kanMetDC fromMus musculus(SEQ ID NO: 1039). pSC101(pTET, atc induction); ΔyjeHSYN7690SYN7345 containing Logic2492, which expresses an engineeredCam kanLeuDC fromMus musculus(SEQ ID NO: 1038). pSC101(pTET, atc induction); ΔyjeHSYN7691SYN7345 containing Logic2493, which expresses a MetDC fromCam kanEntamoeba histolytica(SEQ ID NO: 1040). pSC101 (pTET, atcinduction); ΔyjeHSYN7345EcN with endogenous methionine and branched-chain a.a.camexporter (yjeH) knocked out.SYN7970(lacI-Ptac, IPTG) 2× MetDC (Q70D N82H) (SEQ ID NO: 1034);none1× MetP (F. segetis) (SEQ ID NO: 1041); Δdap; ΔyjeH, ΔϕSYN8002(lacI-Ptac, IPTG) 3× MetDC (Q70D N82H) (SEQ ID NO: 1034);None1× MetP (F. segetis) (SEQ ID NO: 1041); Δdap; ΔyjeH; ΔϕSYN8003(lacI-Ptac, IPTG) 3× MetDC (Q70D N82H) (SEQ ID NO: 1034);none1× MetP (F. segetis) (SEQ ID NO: 1041); Δdap; ΔyjeH; Δϕ(stronger RBS than SYN8002)SYN8070(lacI-Ptac, IPTG) 3× MetDC (Q70D N82H) (SEQ ID NO: 1034);None1× MetP (F. segetis) (SEQ ID NO: 1041); Δϕ; Δdap; ΔyjeH; Δpks(integration: thiC/rsd::attB2-lacI-Ptac-metDC; glmS/pstS::attB5-lacI-Ptac-metP-Ptac-metDC; hypothetical protein/yfjJ::attB7-Ptac-metDC)SYNB1353(lacI-Ptac, IPTG) 3× MetDC (Q70D N82H) (SEQ ID NO: 1034);None1× MetP (F. segetis) (SEQ ID NO: 1041); Δϕ; Δdap; ΔyjeH;Δpks (integration: thiC/rsd::attB2-lacI-Ptac-metDC;glmS/pstS::attB5-lacI-Ptac-metP-Ptac-metDC; hypotheticalprotein/yfjJ::attB7-Ptac-metDC) TABLE 10Exemplary Methionine Decarboxylase Nucleotide SequencesDescriptionSEQ ID NO:Nucleotide SequenceMetDC Q70DATGtccccgacggcgtttccagcggccgaaacagctactgcccctgcaactgccgtcgatcctgggccagaacN82HtggacggcggagatttcgcccttccagagggcgggctggatgacgatcgtcgcttacgtgcattggacgcagttgaSEQ ID NO:cgagtatttgacccgcaagcgcaagcatttggttgggtaccaagctacccaggatatggacggaacggccttggat1034ttagcccgtttcatgccccacaacatcaacaacctgggagatcctttccagtcgggtgggtataaaccaaatacgaaagtcgttgagcgtgccgtactggactactatgcaaaattgtggcacgcagaacgtccacacgacccagctgacccagaaagctactggggttacatgttatcgatgggctcaactgagggcaacatgtacgccctgtggaatgcacgtgactacctgtcgggtaaggctttgattcagcctcccacggcaccatttgacgctgttcgctacgtgaaggctgaccccgatcgccgcaatcctaacgcacaccacccagtcgcattctactcggaggatacccactattcttttgctaaagccgttgcggtgctgggtgtcgaaactttccacgctgtgggtctggagaaatacgctgacgagtgccccttggtggatccagtaaccggccttcgtacctggccgaccgaagttccatcgcgcccggggccgtcgggtttaagctgggacggccctggtgagattgatgttgatgcgcttgcagtactggtcgagttcttcgcagcgaagggtcaccccgtcttcgtcaaccttaacttggggtctacatttaaaggagcacatgatgacgtacgtgcggtatgtgaacgcttattaccaatcttcgagcgccatggcttagtacaacgtgaagttgtatatgggagctgtccccaaaccggccgccctttagtggatgtacgtcgcggattttggatccacgtagatggggcacttggggcggggtatgccccttttctgcgtcttgccgccgaagacccggaaggttatggttggacccctgaggcagaattacctgagttcgacttcggcttacgtttgccgacggcggggcatggagaagttgatatggttagcagcatcgccatgagtggacataagtgggcaggcgcgccgtggccatgcggcatctatatgacgaaagtgaaatatcagattagtccaccgtcacagcccgattatattggtgctcctgacacaacatttgccggttcccgtaacggcttttcgccgttaattttgtgggatcatttatcgcgctactcgtaccgcgaccaggtagagcgcatccgcgaagcacaggagcttgcagcatatttggaacgccgccttaccgctatggagcgcgagctgggagtggaactttggccagcccgcacaccgggtgctgtaaccgtacgttttcgcaaaccctctgctgagctggttgcgaagtggtccttgtcgtcgcaggatgttttaatggtgccgggtgatgaaactacgcgtcgtagttacgttcatgtgttcgtgatgccttctgttgatcgtgcaaagttagatgcgttgctggcagaattggccgaagatcccgtcatcttgggtgcgccttaaMetDCATGTCCCCGACGGCGTTTCCAGCGGCCGAAACAGCTACTGCCCCTGC(Streptomyces)AACTGCCGTCGATCCTGGGCCAGAACTGGACGGCGGAGATTTCGCCCSEQ ID NO:TTCCAGAGGGCGGGCTGGATGACGATCGTCGCTTACGTGCATTGGAC1003GCAGTTGACGAGTATTTGACCCGCAAGCGCAAGCATTTGGTTGGGTACCAAGCTACCCAGGATATGCAGGGAACGGCCTTGGATTTAGCCCGTTTCATGCCCAACAACATCAACAACCTGGGAGATCCTTTCCAGTCGGGTGGGTATAAACCAAATACGAAAGTCGTTGAGCGTGCCGTACTGGACTACTATGCAAAATTGTGGCACGCAGAACGTCCACACGACCCAGCTGACCCAGAAAGCTACTGGGGTTACATGTTATCGATGGGCTCAACTGAGGGCAACATGTACGCCCTGTGGAATGCACGTGACTACCTGTCGGGTAAGGCTTTGATTCAGCCTCCCACGGCACCATTTGACGCTGTTCGCTACGTGAAGGCTGACCCCGATCGCCGCAATCCTAACGCACACCACCCAGTCGCATTCTACTCGGAGGATACCCACTATTCTTTTGCTAAAGCCGTTGCGGTGCTGGGTGTCGAAACTTTCCACGCTGTGGGTCTGGAGAAATACGCTGACGAGTGCCCCTTGGTGGATCCAGTAACCGGCCTTCGTACCTGGCCGACCGAAGTTCCATCGCGCCCGGGGCCGTCGGGTTTAAGCTGGGACGGCCCTGGTGAGATTGATGTTGATGCGCTTGCAGTACTGGTCGAGTTCTTCGCAGCGAAGGGTCACCCCGTCTTCGTCAACCTTAACTTGGGGTCTACATTTAAAGGAGCACATGATGACGTACGTGCGGTATGTGAACGCTTATTACCAATCTTCGAGCGCCATGGCTTAGTACAACGTGAAGTTGTATATGGGAGCTGTCCCCAAACCGGCCGCCCTTTAGTGGATGTACGTCGCGGATTTTGGATCCACGTAGATGGGGCACTTGGGGCGGGGTATGCCCCTTTTCTGCGTCTTGCCGCCGAAGACCCGGAAGGTTATGGTTGGACCCCTGAGGCAGAATTACCTGAGTTCGACTTCGGCTTACGTTTGCCGACGGCGGGGCATGGAGAAGTTGATATGGTTAGCAGCATCGCCATGAGTGGACATAAGTGGGCAGGCGCGCCGTGGCCATGCGGCATCTATATGACGAAAGTGAAATATCAGATTAGTCCACCGTCACAGCCCGATTATATTGGTGCTCCTGACACAACATTTGCCGGTTCCCGTAACGGCTTTTCGCCGTTAATTTTGTGGGATCATTTATCGCGCTACTCGTACCGCGACCAGGTAGAGCGCATCCGCGAAGCACAGGAGCTTGCAGCATATTTGGAACGCCGCCTTACCGCTATGGAGCGCGAGCTGGGAGTGGAACTTTGGCCAGCCCGCACACCGGGTGCTGTAACCGTACGTTTTCGCAAACCCTCTGCTGAGCTGGTTGCGAAGTGGTCCTTGTCGTCGCAGGATGTTTTAATGGTGCCGGGTGATGAAACTACGCGTCGTAGTTACGTTCATGTGTTCGTGATGCCTTCTGTTGATCGTGCAAAGTTAGATGCGTTGCTGGCAGAATTGGCCGAAGATCCCGTCATCTTGGGTGCGCCTtaaMetDCatgagcccgaccgccttccccgccgccgagaccgcgaccgcgcccgcgaccgccgtcgatcccggtccggag(Streptomyces)ctggacggcggtgacttcgccctccccgagggcggcctggacgacgaccggcggctgcgcgcgctcgacgccSEQ ID NO:gtggacgagtacctgacccgcaagcgcaagcacctggtcggctaccaggccacccaggacatgcagggcaccg1018cactggacctcgcccggttcatgccgaacaacatcaacaacctcggcgacccgttccagagcggcggatacaagcccaacaccaaggtcgtcgagcgggccgtgctcgactactacgcgaagctctggcacgccgagcgcccgcacgacccggccgacccggagtcgtactggggctacatgctgtccatgggctcgaccgagggcaacatgtacgccctctggaacgccagggactacctgagcggcaaggcgctgatccagccgccgaccgcccccttcgacgcggtgcgctacgtcaaggccgaccccgaccgacggaacccgaacgcccaccacccggtggccttctactccgaggacacccactactccttcgccaaggccgtggccgtcctcggcgtggagaccttccacgccgtcggcctggagaagtacgccgacgagtgcccgctggtcgacccggtgaccgggctgcgcacctggcccaccgaggtgccctcccgcccgggtccgtccggcctgtcctgggacggccccggcgagatagacgtcgacgccctcgccgtactcgtcgagttcttcgccgccaagggtcacccggtcttcgtcaacctcaacctcggcagcaccttcaagggcgcccacgacgacgtccgcgccgtctgcgagcgcttgctgccgatcttcgagcggcacgggctcgtccagcgcgaggtggtctacggcagctgcccgcagaccggccggccgctggtggacgtgcgccgcggcttctggatccacgtggacggcgcgctcggcgccggctacgcgccgttcctgcggctggccgccgaggacccggaagggtacggctggacgcccgaggcggagctgcccgagttcgacttcggcctgcggctgcccaccgccgggcacggcgaggtggacatggtctcctcgatcgcgatgagcggccacaagtgggccggcgcgccgtggccgtgcggcatctacatgaccaaggtgaagtaccagatctcgccgccgtcccagccggactacatcggcgccccggacaccaccttcgccggctcccgcaacggcttctccccgctgatcctctgggaccacctgtcccggtactcctaccgggaccaggtggagcggatccgcgaggcccaggagctggccgcctacctggagcggcggctgaccgccatggagcgcgaactcggcgtcgagctctggccggcccgtaccccgggcgccgtcaccgtacggttccgcaagccgagcgccgagctggtggccaagtggtcgctgtcctcccaggacgtgctgatggtcccgggcgacgagaccacccggcgcagctacgtgcacgtcttcgtgatgccctcggtcgaccgggccaagctggacgcgctgctcgccgaactcgccgaggacccggtgatcctgggcgcaccgtagMetDCatgtccccgacggcgtttccagcggccgaaacagctactgcccctgcaactgccgtcgatcctgggccagaactg(V491LgacggcggagatttcgcccttccagagggcgggctggatgacgatcgtcgcttacgtgcattggacgcagttgacA500P)gagtatttgacccgcaagcgcaagcatttggttgggtaccaagctacccaggatatgcagggaacggccttggatttSEQ ID NO:agcccgtttcatgcccaacaacatcaacaacctgggagatcctttccagtcgggtgggtataaaccaaatacgaaa1035gtcgttgagcgtgccgtactggactactatgcaaaattgtggcacgcagaacgtccacacgacccagctgacccagaaagctactggggttacatgttatcgatgggctcaactgagggcaacatgtacgccctgtggaatgcacgtgactacctgtcgggtaaggctttgattcagcctcccacggcaccatttgacgctgttcgctacgtgaaggctgaccccgatcgccgcaatcctaacgcacaccacccagtcgcattctactcggaggatacccactattcttttgctaaagccgttgcggtgctgggtgtcgaaactttccacgctgtgggtctggagaaatacgctgacgagtgccccttggtggatccagtaaccggccttcgtacctggccgaccgaagttccatcgcgcccggggccgtcgggtttaagctgggacggccctggtgagattgatgttgatgcgcttgcagtactggtcgagttcttcgcagcgaagggtcaccccgtcttcgtcaaccttaacttggggtctacatttaaaggagcacatgatgacgtacgtgcggtatgtgaacgcttattaccaatcttcgagcgccatggcttagtacaacgtgaagttgtatatgggagctgtccccaaaccggccgccctttagtggatgtacgtcgcggattttggatccacgtagatggggcacttggggcggggtatgccccttttctgcgtcttgccgccgaagacccggaaggttatggttggacccctgaggcagaattacctgagttcgacttcggcttacgtttgccgacggcggggcatggagaagttgatatggttagcagcatcgccatgagtggacataagtgggcaggcgcgccgtggccatgcggcatctatatgacgaaagtgaaatatcagattagtccaccgtcacagcccgattatattggtgctcctgacacaacatttgccggttcccgtaacggcttttcgccgttaattttgtgggatcatttatcgcgctactcgtaccgcgaccaggtagagcgcatccgcgaagcacaggagcttgcagcatatttggaacgccgccttaccgctatggagcgcgagctgggagtggaactttggccagcccgcacaccgggtgctctgaccgtacgttttcgcaaaccctctccggagctggttgcgaagtggtccttgtcgtcgcaggatgttttaatggtgccgggtgatgaaactacgcgtcgtagttacgttcatgtgttcgtgatgccttctgttgatcgtgcaaagttagatgcgttgctggcagaattggccgaagatcccgtcatcttgggtgcgccttaaMetDC (R41QatgtccccgacggcgtttccagcggccgaaacagctactgcccctgcaactgccgtcgatcctgggccagaactgQ70D)gacggcggagatttcgcccttccagagggcgggctggatgacgatcagcgcttacgtgcattggacgcagttgacSEQ ID NO:gagtatttgacccgcaagcgcaagcatttggttgggtaccaagctacccaggatatggacggaacggccttggattt1036agcccgtttcatgcccaacaacatcaacaacctgggagatcctttccagtcgggtgggtataaaccaaatacgaaagtcgttgagcgtgccgtactggactactatgcaaaattgtggcacgcagaacgtccacacgacccagctgacccagaaagctactggggttacatgttatcgatgggctcaactgagggcaacatgtacgccctgtggaatgcacgtgactacctgtcgggtaaggctttgattcagcctcccacggcaccatttgacgctgttcgctacgtgaaggctgaccccgatcgccgcaatcctaacgcacaccacccagtcgcattctactcggaggatacccactattcttttgctaaagccgttgcggtgctgggtgtcgaaactttccacgctgtgggtctggagaaatacgctgacgagtgccccttggtggatccagtaaccggccttcgtacctggccgaccgaagttccatcgcgcccggggccgtcgggtttaagctgggacggccctggtgagattgatgttgatgcgcttgcagtactggtcgagttcttcgcagcgaagggtcaccccgtcttcgtcaaccttaacttggggtctacatttaaaggagcacatgatgacgtacgtgcggtatgtgaacgcttattaccaatcttcgagcgccatggcttagtacaacgtgaagttgtatatgggagctgtccccaaaccggccgccctttagtggatgtacgtcgcggattttggatccacgtagatggggcacttggggcggggtatgccccttttctgcgtcttgccgccgaagacccggaaggttatggttggacccctgaggcagaattacctgagttcgacttcggcttacgtttgccgacggcggggcatggagaagttgatatggttagcagcatcgccatgagtggacataagtgggcaggcgcgccgtggccatgcggcatctatatgacgaaagtgaaatatcagattagtccaccgtcacagcccgattatattggtgctcctgacacaacatttgccggttcccgtaacggcttttcgccgttaattttgtgggatcatttatcgcgctactcgtaccgcgaccaggtagagcgcatccgcgaagcacaggagcttgcagcatatttggaacgccgccttaccgctatggagcgcgagctgggagtggaactttggccagcccgcacaccgggtgctgtaaccgtacgttttcgcaaaccctctgctgagctggttgcgaagtggtccttgtcgtcgcaggatgttttaatggtgccgggtgatgaaactacgcgtcgtagttacgttcatgtgttcgtgatgccttctgttgatcgtgcaaagttagatgcgttgctggcagaattggccgaagatcccgtcatcttgggtgcgccttaaMetDCatggggttccagttactgtctaaacataagctgtcagccgaggatcaacagaaacttgaccgcttttatcgtgatattc(Stanieriasp.agacagaagcagaacgattcctgggttacccatgtaacgaactctttgactactcccccttgttccggttcctgcaataNIES-3757)tccgctgaataacgtcggcgacccgtacctgccgagtaactaccacctgaacacgcacaactttgagtgcgaagtaSEQ ID NO:ctggaaatcttccgtaccctgaccgaggctactgaaggttcgacttggggctacgtgaccaacggcggtacggaa1037ggtaatcattatggtcttttcctggcgagagagctgctgcctgaaggccttgtttactattctcaggatgcgcactactcgatcgataaaatcctgaggtgcctcaacctccgtagcataatgattcgcagccacgacgacggacgcatggacctggatgatctgcgtgaaactctgcgtatccatcgcgacttgccgccgatcgtttgcgctaccattggtactacaatgaagggcgctgtagatgacatcgcaggcattaaaaagatcttcaaagatctggcaatacaccgtcactatatccatgctgacgcggccctaggtggcatgattttaccgttcctggataactccccaccgtggaattttaaagctggaatcgactctatcgctatctccggtcacaaaatggtgggcagtcctatcccgtgtggggttgtcctggctaaaaagtcgaacgttgaacgtattgcacagagcgtggaatacattggtactctggataccaccctgtctggctcccgtaacgccttgactccgttatttctgtggtacgcgttccacaccgttggtatcgaaggtttcaaacgtatcatcccggcatgcttaaaaatggcggactatgccatcgctcagctgaacaaaattaaccgcaatgcgtggcgctacccttacagcaacacggtagtcttcgatcgcccaagccccgaagtgactcgttattggcagctggcttgtcagggcaacctgagccacctaatcaccatgccacacgttacatctactcaaattgatcatctggttgctgacatcatcgcttctgagccgataccgccgctgccgaccctgtcagttactccggcatgcgaactgctgacttctaccccggaccaggatattacgctgatcggcaccgctaatcataatctgctctccgaagtatctaccgccctggctgccgagggtctgtcaattgaaaacctggctgctgtggcggtagaaagcgaggacgttgaagttgtaaggctccgcgttaacaaccgtgagcgtgcactgcaaatcctgaaccagaacctggatatcggtcgttgctacggtcaggctcgaccctttggcaacgaagaagcgacgcaggtactgtcccagctggaatatcaaagcgtgggggaggatgcactactggtccagcttgacgattgccctggcagcctggcggagctgttgaaggattgccgcaacgaagcggtaaaaatccgtaatatccgactgctttggcgtgggcacggtaagggcgtcgtagcaattgctaccacttctccagatgcgctgaaaacgctgctgaaagaccgtattcttttgagctaaLeuDCATGtccacacctagtgaagtaaagaaggatttgctgggtgcagcagggtcattatggccgtcggagcccattac(Musmusculus)gctgggtccaggtgaaagtgcttggcagctggtattgaagaagatccaagagttgagtgacagcggtcatcaagacSEQ ID NO:ccgttcatggttgcagaccttgatgtccttgtgtctcgtcatcagacgttctgtcaagcactgcctagagtacaaccctt1038ctatgcagtaaagtgcaatagtaacccatgggtgttacgggtgttggcagctcttggcacgggatttgattgtgcttctcagggagaattggagcaagttttgggcttgggtgtagcgccgtcacggataatcttcgcaaatccctgtaaagcagtcagccacattcagtttgcagctcggtgcggtgtgcaattgttgacattcgacagcgaagaggagttaatcaaggttgcgcagtaccatccaggcgcacggttggtgcttcggattcaaacccaggactcacaatcaacgttcccactttccaccaagttcggtgcttctttagaagcatgtggacaccttctgcaggttgccagagagctgggtcttgccgtggtaggtgctagctttcatgtaggaagcgactgccacacacctcagagttttcgtcaggccatcgcagattgtcatcgtgtgttcgagatgggccgtaaggcaggtcatgatatgtcgcttcttgatttgggtggagggttcccaggtgtggaaggttccgaggcgaagtttgaggagatggcaagagtaatcaatgccgctcttgctcagtactttccggaagagactggcatcgaggtgatcgcggaacctggtcgtttctacgctgggtcggtgtgcactgcagctgtgaacatcatcgcgaagaagtctgtcttggaaccaggtggtcatcgtaagcttatgtactaccttaatgaaggacattacggttctttcagattgttcttgcgtgatccagtgcctcgtattcccatcgtggtgaaagagttcccatccgaaccaccactgtttccttgcactttgtacggtcccacatgtgacgcctatgatcggttgttttccgaagaggtacaattgccagagctggatgttggagattggttgatcttcccagatatgggtgcctatacctcctcaatgtcctcgaccttcaacggatttccaccggccaccgtgtattgcgcaatgtcaccgcagttacgctccctgttggagactgtaccataaMetDCatgaacacacctagtgaagtaaagaaggatttgctgggtgttgcagaacatttacgtccgtcggagcccattacgct(Musmusculus)gggtccaggtgcgagtgcttggcagctggtattgaagaagatcaaggagttgagtattagcggtcgtcaagacgctSEQ ID NO:ttcatggttgcagaccttgatgtccttgtgtctcgtcatcggacgttcttacaagcactgcctagagtacaacccttctat1039gcagtaaagtgcaatagtaacccatgggtgttacttgtgttggcagctcttggcacgggatttgattgtgcttctcagggagaattggagcaagttttgggcttgggtgtagcgccgtcacggataatcttcgcaaatccctgtaaagcagtcagccacattcagtttgcagctcggtgcggtgtgcaattgttgacattcgacaatgaagaggagttaatcaagttagcgcgttaccatccacgtgcacggttggtgcttcggattcaaaccctggactcacaatcaacgttcccacttagcaccaagttcggtgctcacttagaagcatgtggacaccttctgcaggttgccagagagctgggtcttgccgtggtaggtgctagctttcatgtaggaagcgactgccacacacctgagagttaccgtcaggccatcgcagattgtcatcgtgtgttcgagatgggctgtaaggcaggtcatcacatgtcgcttcttgatttgggtggagggttcccaggtgtgaaaggttccgaggcgaagtttgaggaggttgcaagagtaatcaataccgctcttgctcagtactttccggaagagactggcatcgaggtgatcgcggaacctggtcgtttctacgctgggtcggtgtgcactgcagctgtgaacatcatcgccaagaagtctagtttggacccaggtggtcatcgtaagcttgcttactaccttaatgaaggacattacggtgtattcagattgttcttgcgtgatccagtgcctcgtattcccatcgtggtgaaagagttcccatccgaaccaccactgtttccttgcactttgtacggtcccacatgtgacgcctatgatcggttgttttccaccgaggtacaattgccagagctggatgttggagattggttgatcttcccagatatgggtgcctattcgtcctcaatgtcctcgaccttcaacggatttccaatagccaccgtgtatgatgcaatgtcaccgcagttacgctccctgttggagactgtaccataaMetDCatgaaacaaacgtcccttgaggtgaaggaatttgccttgaatctcatttctcagttcgaaccagaaaaccagcctctg(Entamoebaggtttctggatattcgacaccgaaggcgttgagaaagcggtagaacgctggaaaaagaacatgccgactgtccgtchistolytica)cctgttttgcagttaaatgcaacccggagccgcacctggtgaaattactgggggaactgggttgcggcttcgattgcSEQ ID NO:gctagcctgaacgaaatcaaagaggtactggacttgggttttaatccggaagatatcacttatagtcagaccttcaaa1040ccgtacaaccagttaattgaagcttcgcatctgggcatcaaccacacgatcgttgattcaatcgacgaagttcaaaaaattgctaaatacgcgcctaagatgggtatcatgattcggatcatggaaaatgacacaagcgcaggccacgtctttggagagaaattcggtctgcatgatgatgaagttgagatcgtactgaaggaaattaaagacaaaggtctgaacctggacggcgttcatttccacgttggctctgattcccacaacagcgaagtgtttactaaggcactgaccaaagctcgtaacactgtaaccctggccgaacagttcggcatgaaaccgtacctgatcgacattggtggcgggttctctcaggttgcgccgttcgaagaatttgctgctaccatcgaaaaaactataaaggaactggaatttccagagcgaactcgtttcattgcagagccgggtcgctatatggcatcaaatgcctttcaccttgtctcttcgctgcatggtaaaagggtgcgcatccagaacggtaagaaacagatcgaatacaccagcggcgatgggctgcacggctccttcggctgttgcatctggttcgaaaaacagaagtcttgcgaatgtataacacaaaaagtaaacgagaacaccaaaatgtatgaaagcatcatctacggcccatcttgcaacggttcggacaaagtggccacgcaggagttgccggaaatggagccgggtaaagattggctgctgttccccaatatgggtgcttacactatttccatggcgaccaactttaacggcttcgaagaacgtaaccatgtaatctatacgttaccactcaaaagtactaaaataattcagatccctaaaagcattgaatgcaactccgttccgtctttaaacggaatcccacactacgcgtaaSpMetDCatgtccccgacggcgtttccagcggccgaaacagctactgcccctgcaactgccgtcgatcctgggccagaactgT66NgacggcggagatttcgcccttccagagggcgggctggatgacgatcgtcgcttacgtgcattggacgcagttgacCodon-gagtatttgacccgcaagcgcaagcatttggttgggtaccaagctaatcaggatatgcagggaacggccttggatttoptimizedagcccgtttcatgcccaacaacatcaacaacctgggagatcctttccagtcgggtgggtataaaccaaatacgaaasequencegtcgttgagcgtgccgtactggactactatgcaaaattgtggcacgcagaacgtccacacgacccagctgacccagSEQ ID NO:aaagctactggggttacatgttatcgatgggctcaactgagggcaacatgtacgccctgtggaatgcacgtgactac1123ctgtcgggtaaggctttgattcagcctcccacggcaccatttgacgctgttcgctacgtgaaggctgaccccgatcgccgcaatcctaacgcacaccacccagtcgcattctactcggaggatacccactattcttttgctaaagccgttgcggtgctgggtgtcgaaactttccacgctgtgggtctggagaaatacgctgacgagtgccccttggtggatccagtaaccggccttcgtacctggccgaccgaagttccatcgcgcccggggccgtcgggtttaagctgggacggccctggtgagattgatgttgatgcgcttgcagtactggtcgagttcttcgcagcgaagggtcaccccgtcttcgtcaaccttaacttggggtctacatttaaaggagcacatgatgacgtacgtgcggtatgtgaacgcttattaccaatcttcgagcgccatggcttagtacaacgtgaagttgtatatgggagctgtccccaaaccggccgccctttagtggatgtacgtcgcggattttggatccacgtagatggggcacttggggcggggtatgccccttttctgcgtcttgccgccgaagacccggaaggttatggttggacccctgaggcagaattacctgagttcgacttcggcttacgtttgccgacggcggggcatggagaagttgatatggttagcagcatcgccatgagtggacataagtgggcaggcgcgccgtggccatgcggcatctatatgacgaaagtgaaatatcagattagtccaccgtcacagcccgattatattggtgctcctgacacaacatttgccggttcccgtaacggcttttcgccgttaattttgtgggatcatttatcgcgctactcgtaccgcgaccaggtagagcgcatccgcgaagcacaggagcttgcagcatatttggaacgccgccttaccgctatggagcgcgagctgggagtggaactttggccagcccgcacaccgggtgctgtaaccgtacgttttcgcaaaccctctgctgagctggttgcgaagtggtccttgtcgtcgcaggatgttttaatggtgccgggtgatgaaactacgcgtcgtagttacgttcatgtgttcgtgatgccttctgttgatcgtgcaaagttagatgcgttgctggcagaattggccgaagatcccgtcatcttgggtgcgccttaaSpMetDCatgtccccgacggcgtttccagcggccgaaacagctactgcccctgcaactgccgtcgatcctgggccagaactgA203HgacggcggagatttcgcccttccagagggcgggctggatgacgatcgtcgcttacgtgcattggacgcagttgacCodon-gagtatttgacccgcaagcgcaagcatttggttgggtaccaagctacccaggatatgcagggaacggccttggatttoptimizedagcccgtttcatgcccaacaacatcaacaacctgggagatcctttccagtcgggtgggtataaaccaaatacgaaasequencegtcgttgagcgtgccgtactggactactatgcaaaattgtggcacgcagaacgtccacacgacccagctgacccagSEQ ID NO:aaagctactggggttacatgttatcgatgggctcaactgagggcaacatgtacgccctgtggaatgcacgtgactac1125ctgtcgggtaaggctttgattcagcctcccacggcaccatttgacgctgttcgctacgtgaaggctgaccccgatcgccgcaatcctaacgcacaccacccagtcgcattctactcggaggatacccactattcttttgctaaagccgttcatgtgctgggtgtcgaaactttccacgctgtgggtctggagaaatacgctgacgagtgccccttggtggatccagtaaccggccttcgtacctggccgaccgaagttccatcgcgcccggggccgtcgggtttaagctgggacggccctggtgagattgatgttgatgcgcttgcagtactggtcgagttcttcgcagcgaagggtcaccccgtcttcgtcaaccttaacttggggtctacatttaaaggagcacatgatgacgtacgtgcggtatgtgaacgcttattaccaatcttcgagcgccatggcttagtacaacgtgaagttgtatatgggagctgtccccaaaccggccgccctttagtggatgtacgtcgcggattttggatccacgtagatggggcacttggggcggggtatgccccttttctgcgtcttgccgccgaagacccggaaggttatggttggacccctgaggcagaattacctgagttcgacttcggcttacgtttgccgacggcggggcatggagaagttgatatggttagcagcatcgccatgagtggacataagtgggcaggcgcgccgtggccatgcggcatctatatgacgaaagtgaaatatcagattagtccaccgtcacagcccgattatattggtgctcctgacacaacatttgccggttcccgtaacggcttttcgccgttaattttgtgggatcatttatcgcgctactcgtaccgcgaccaggtagagcgcatccgcgaagcacaggagcttgcagcatatttggaacgccgccttaccgctatggagcgcgagctgggagtggaactttggccagcccgcacaccgggtgctgtaaccgtacgttttcgcaaaccctctgctgagctggttgcgaagtggtccttgtcgtcgcaggatgttttaatggtgccgggtgatgaaactacgcgtcgtagttacgttcatgtgttcgtgatgccttctgttgatcgtgcaaagttagatgcgttgctggcagaattggccgaagatcccgtcatcttgggtgcgccttaaSpMetDCatgtccccgacggcgtttccagcggccgaaacagctactgcccctgcaactgccgtcgatcctgggccagaactgH379GgacggcggagatttcgcccttccagagggcgggctggatgacgatcgtcgcttacgtgcattggacgcagttgacCodon-gagtatttgacccgcaagcgcaagcatttggttgggtaccaagctacccaggatatgcagggaacggccttggatttoptimizedagcccgtttcatgcccaacaacatcaacaacctgggagatcctttccagtcgggtgggtataaaccaaatacgaaasequencegtcgttgagcgtgccgtactggactactatgcaaaattgtggcacgcagaacgtccacacgacccagctgacccagSEQ ID NO:aaagctactggggttacatgttatcgatgggctcaactgagggcaacatgtacgccctgtggaatgcacgtgactac1127ctgtcgggtaaggctttgattcagcctcccacggcaccatttgacgctgttcgctacgtgaaggctgaccccgatcgccgcaatcctaacgcacaccacccagtcgcattctactcggaggatacccactattcttttgctaaagccgttgcggtgctgggtgtcgaaactttccacgctgtgggtctggagaaatacgctgacgagtgccccttggtggatccagtaaccggccttcgtacctggccgaccgaagttccatcgcgcccggggccgtcgggtttaagctgggacggccctggtgagattgatgttgatgcgcttgcagtactggtcgagttcttcgcagcgaagggtcaccccgtcttcgtcaaccttaacttggggtctacatttaaaggagcacatgatgacgtacgtgcggtatgtgaacgcttattaccaatcttcgagcgccatggcttagtacaacgtgaagttgtatatgggagctgtccccaaaccggccgccctttagtggatgtacgtcgcggattttggatccacgtagatggggcacttggggcggggtatgccccttttctgcgtcttgccgccgaagacccggaaggttatggttggacccctgaggcagaattacctgagttcgacttcggcttacgtttgccgacggcggggggcggagaagttgatatggttagcagcatcgccatgagtggacataagtgggcaggcgcgccgtggccatgcggcatctatatgacgaaagtgaaatatcagattagtccaccgtcacagcccgattatattggtgctcctgacacaacatttgccggttcccgtaacggcttttcgccgttaattttgtgggatcatttatcgcgctactcgtaccgcgaccaggtagagcgcatccgcgaagcacaggagcttgcagcatatttggaacgccgccttaccgctatggagcgcgagctgggagtggaactttggccagcccgcacaccgggtgctgtaaccgtacgttttcgcaaaccctctgctgagctggttgcgaagtggtccttgtcgtcgcaggatgttttaatggtgccgggtgatgaaactacgcgtcgtagttacgttcatgtgttcgtgatgccttctgttgatcgtgcaaagttagatgcgttgctggcagaattggccgaagatcccgtcatcttgggtgcgccttaa TABLE 11Exemplary Importer Nucleotide SequencesDescriptionSEQ ID NO:Nucleotide SequenceMetPatggggaccattaacacgaagatctataaatacatgagcatctggaaaacaaaacctctgtccgtgctcttgtctgaa(F. segetis)gcaactgaggatgaaaaaggcctgaagcgcactctgtcggcccgttcacttgttgcgctgggtgtcggtgctattatSEQ ID NO:cggcgctggtttattctctctgaccggcatagctgcggcagacaatgctggaccggcagtaaccctgagctttatcct1041ggcctccgttggttgcgcgttcgctggcctgtgttacgcagaatttgcttctatgattccagttgcgggtagcgcctacacttatagttatgctaccatgggcgagttcgtggcgtggatcatcggttgggatctggtactcgaatacgcattgggcgcagctactgttgccgttagctggtcccagtacgtggacaaattcttgcaaaactacggcatccatattccgaactctatcctccacgggccgtgggataccacccccggtattatcaatttaccgtcgatatttatcatctgcctgctgagcgtgctgctgattcgtggtactaaagaatctgctctgatcaacaacattctggtaatcctgaaagtcacggttgtcatcgtgttcattggcctgggctgggggttcatgaactccgcaaaccacacgccctttatcccggttaacgaaggtgaggctctactgtcttctggtgaaatgagtttcctcaactttttcagcagtgactactttggacactacggatggtccggtattcttcgcggcgctggtgtagtattcttcgcatttatcggcttcgacgcggtgagcactgcggcacaggaggccaaggatccgcagaaaggcatgccaatcggtattctgggctcactgatcatttgcaccgttctgtacgtgcttttcgctttcgttctgaccggtctggaaaactatctaaacttcaaaggtgacgcttctcctgtcaccactgcatttgccaaaacaggctatactttcctgaatagcggtctgacgatcgctatcatagcgggctacacatccgttatgctggtaatgttgatgggtcagtcccgtgtcttttatagtatgtctgtggatggcctgcttccgaagtttttctcgaccctgcataccaaaaacaggactccgtacaaaactaatttgctgttcatggttttcgtaagcctgttcgctggctttgttccggtcagcgacctgggccatatggtatccatcggtaccctcttcgctttctgcctggtgtgtatcggcgttatcgttatgcgaaaaaccaacccagacgccgttcgcggttttcgtgttccttttgtaccggttttcccgattatcggtgtagttatttgtctggttctaatggcgggcctgccgattgaatcttgggaacgtctggcgatctggatgattctgggtgtcgtgatctacttcttctactctaaaaagaactctaaactgaataaccccgaataaMetPatggggacgatcaatactaagaccaacaaatatatgagcatttggaaaaccaaaccgttgtctgtactgttaaacgag(F. frigoris)gcctcagaagatgaaaagggcctgaaaaggactctgtcctctcgttccctcgtggctctgggtgtcggtgcgatcatSEQ ID NO:tggcgcaggtctgtttagcctaacaggcatcgcagctgcggaacatgctggtccagcggttactctgagtttcatact1042ggccgctgttggttgtgctttcgcaggcctgtgctacgcggagtttgcgtcgatgatccctgtggctgggtctgcttacacctatagctacgcaaccatgggcgaatttatggcgtggatcattggctgggaccttgtactggaatacgctctgggtgcagcgactgttggtgtatcctggtcccgttacttactggaattgctgaacaaatatggtgttcacctgaacccgaaattcatctgctctccgtgggagacacttaccctgggcgacggcactattatcgatggcgggtacatcaatctgccggcaattctgatcgtgagcgccctcagcttgctgctgattagaggtacccaggaatctgcttctattaacaacatcctggttgtgctgaaagtaatagtcgtgatcatgttcatcgttttaggatgggactatatcgatcccgcaaattactcaccttacatcccggaaaacaccggcgtaaagggccaattcggttggtcgggtatcgctgcgggtgctggtacggttttctttgccttcattggtttcgacgccgtttccactgcggctcaggaggctaaaaacccgcagaaaggcatgccaattggcatcctggggtctttggtaatttgtacgatcctgtacgtcctttttgcccacgttatgacgggcctggtgccgtattataagttcgctggagatgctaaacccgctgcgacagcattcgcagtcaccggttacagttttctgcaaactggactgattgttgcgatcctggctggctatactagcgttatgctggtcatgctgatggggcagagtcgtgttttctacaccatgagcaaagacggtctgctaccaccgctgttcggtcagatccattcgaaatttcgcactccgtacaagactaacctgttctttatggtattcgtttctttattcgcgggtttcgtgccggttagcgacctcggccacatggtcagcatcggtaccctcctggcgtttgttcttgtgtgcataggtgtgctggtgatgcgaaaaaagatgccagatgctccgcgttctttcaaaaccccgttcgttccgtatgtacccatcgcaggcgtcctggtgtgcacttacctgatgtactccctcccttacgaatcctggattcgcttagtgctttggatggctatcggcgtagccctgtacttcgtgtatggaaaaaagcactcaaaactgaacaatccggataaMetNIQatgataaaactttcgaatatcaccaaagtgttccaccagggcacccgcaccatccaggcgttgaacaacgtcagcctSEQ ID NO:gcatgtgccagctggacaaatttatggcgttatcggtgcctcaggcgcgggtaagagtacgcttatacgttgtgtaaa1043cctgctggagcgcccaaccgagggtagcgtgctggtcgatggccaggaactgaccacgctgtcagaatccgagttgaccaaagctcgtcgccagattggtatgattttccagcattttaacctgctctcttcgcgtactgtttttggcaacgtggctctgccgctggagctggacaacacaccgaaagacgagatcaaacgtcgcgtgacggaattgctgtcattggttggtcttggcgataagcatgatagctacccgtcgaatctttccggtgggcagaaacaacgtgtggcgattgcccgtgcattagccagcaatcccaaagtattgctgtgtgatgaagccaccagcgcgctggacccggcaacgacacgttctattctcgaactgctgaaagacatcaaccgccgtctgggtttgacgattctgttgatcactcacgaaatggacgttgtgaagcgcatttgtgattgcgtggcggtcatcagcaatggcgaactgatcgagcaggacacggtaagtgaagtgttctcgcatccgaaaacgccgctggcgcagaagtttattcagtcgaccctgcatctggatatcccggaagattaccaggaacgtctgcaagcggagccatttactgactgcgtgccgatgctgcgtctggagtttaccggtcaatcggtcgatgccccactgctttctgaaaccgcgcgtcgtttcaacgtcaacaacaacattattagcgcgcagatggattacgccggtggcgttaagttcggcatcatgctgactgaaatgcacggcacacaacaagatacgcaagccgccattgcctggctgcaggaacaccatgtaaaagtagaggtactgggttatgtctgagccgatgatgtggctgctggttcgtggcgtatgggaaacgctggcaatgaccttcgtatccggtttttttggctttgtgattggtctgccggttggcgttctgctttatgtcacgcgtccggggcaaattattgctaacgcgaagttgtatcgtaccatttctgcgattgtgaacattttccgttccatcccgttcattatcttgctggtatggatgattccgtttacccgcgttattgtcggtacatcgattggattgcaggcagcgattgttccgttaaccgttggtgcagcaccgtttattgcccgtatggtcgagaacgctctgctggagatcccaaccgggttaattgaagcttcccgcgcaatgggggccacgccaatgcagatcgtccgtaaagtgctgttaccggaagcgttgccgggtctggtgaatgcggcaactatcaccctgattaccctggttggttattccgcgatgggtggtgcagtcggtgccggtggtttaggtcagattggctatcagtatggctacatcggctacaacgcgacggtgatgaatacggtactggtattgctggtcattctggtttatttaattcagttcgcaggcgaccgcatcgtccgggctgtcactcgcaagtaacgttcaacacaacataaataattgaagaaggaataaggtatggcgttcaaattcaaaacctttgcggcagtgggagccctgattggatcactggcactggtaggctgcggtcaggatgaaaaagatccaaaccacattaaagtcggcgtgattgttggtgccgaacagcaggttgcagaagtcgcgcagaaagttgcgaaagacaaatatggcctggacgttgagctggtaaccttcaacgactatgttctgccaaacgaagcattgagcaaaggcgatatcgacgccaacgccttccagcataaaccgtaccttgatcagcaactgaaagatcgtggctacaaactggtcgcagtaggcaacacatttgtttatccgattgctggttactccaagaaaatcaaatcactggatgaactgcaggatggttcgcaggttgccgtgccaaacgacccaactaaccttggtcgttcactgctgctgctgcaaaaagtgggcttgatcaaactgaaagatggcgttggcctgctgccgaccgttcttgatgttgttgagaacccaaaaaatctgaaaattgttgaactggaagcaccgcagctaccgcgctctctggacgacgcgcaaatcgctctggcagttatcaataccacctatgccagccagattggcctgactccagcgaaagacggtatctttgtcgaagataaagagtccccgtacgtaaacctgatcgtaacgcgtgaagacaacaaagacgccgaaaacgtgaagaaattcgttcaggcttatcagtctgacgaagtttacgaagcagcaaacaaagtgtttaacggcggcgctgttaaaggctggtaaMethionineatgtttgaga agtattttcc aaatgttgac ttgaccgagt tatggaatgc cacatatgaaactctgtataimport systemtgacattgat ttccttactg tttgccttcg taatcggcgt catcctgggattgctgttat tcttaacatcpermeasetaaggggtct ctttggcaaa ataaagcagt aaattccgttatcgcagccg ttgtcaacat ctttcgttcaprotein MetPattcccttcc ttattttaat catcctgcttcttggtttca ctaaattctt agtgggaaca attttgggac(Bacilluscaaatgcggc tcttcccgcgttagtcatcg gtagtgctcc cttttatgct cgtctggtcg aaatcgcactsubtilis)tcgtgaagtggacaaaggag tgattgaggc ggcgaaatcg atgggggcta agacgagcac tattatttttSEQ ID NO:aaggttctta tccccgagtc catgcccgcg ctgatttccg gaattacagt gactgcgatt gcattgatcg1044ggtcaaccgc catcgcagga gctattggtt ctggtggatt gggaaacttagcatacgttg aaggctatcaatcgaataat gcggatgtga ccttcgtggc cacagttttcatcctgatta ttgttttcat cattcagatcattggtgacc ttattaccaa catcatcgataaacgcMetNIQatgataaaactttcgaatatcaccaaagtgttccaccagggcacccgcaccatccaggcgttgaacaacgtcagcct(P281G)gcatgtgccagctggacaaatttatggcgttatcggtgcctcaggcgcgggtaagagtacgcttatacgttgtgtaaaSEQ ID NO:cctgctggagcgcccaaccgagggtagcgtgctggtcgatggccaggaactgaccacgctgtcagaatccgagtt1045gaccaaagctcgtcgccagattggtatgattttccagcattttaacctgctctcttcgcgtactgtttttggcaacgtggctctgccgctggagctggacaacacaccgaaagacgagatcaaacgtcgcgtgacggaattgctgtcattggttggtcttggcgataagcatgatagctacccgtcgaatctttccggtgggcagaaacaacgtgtggcgattgcccgtgcattagccagcaatcccaaagtattgctgtgtgatgaagccaccagcgcgctggacccggcaacgacacgttctattctcgaactgctgaaagacatcaaccgccgtctgggtttgacgattctgttgatcactcacgaaatggacgttgtgaagcgcatttgtgattgcgtggcggtcatcagcaatggcgaactgatcgagcaggacacggtaagtgaagtgttctcgcatccgaaaacgccgctggcgcagaagtttattcagtcgaccctgcatctggatatcccggaagattaccaggaacgtctgcaagcggagccatttactgactgcgtgccgatgctgcgtctggagtttaccggtcaatcggtcgatgccggcctgctttctgaaaccgcgcgtcgtttcaacgtcaacaacaacattattagcgcgcagatggattacgccggtggcgttaagttcggcatcatgctgactgaaatgcacggcacacaacaagatacgcaagccgccattgcctggctgcaagaacaccatgtaaaagtagaggtactgggttatgtctgagccgatgatgtggctgctggttcgtggcgtatgggaaacgctggcaatgaccttcgtatccggtttttttggctttgtgattggtctgccggttggcgttctgctttatgtcacgcgtccggggcaaattattgctaacgcgaagttgtatcgtaccatttctgcgattgtgaacattttccgttccatcccgttcattatcttgctggtatggatgattccgtttacccgcgttattgtcggtacatcgattggattgcaggcagcgattgttccgttaaccgttggtgcagcaccgtttattgcccgtatggtcgagaacgctctgctggagatcccaaccgggttaattgaagcttcccgcgcaatgggggccacgccaatgcagatcgtccgtaaagtgctgttaccggaagcgttgccgggtctggtgaatgcggcaactatcaccctgattaccctggttggttattccgcgatgggtggtgcagtcggtgccggtggtttaggtcagattggctatcagtatggctacatcggctacaacgcgacggtgatgaatacggtactggtattgctggtcattctggtttatttaattcagttcgcaggcgaccgcatcgtccgggctgtcactcgcaagtaacgttcaacacaacataaataattgaagaaggaataaggtatggcgttcaaattcaaaacctttgcggcagtgggagccctgattggatcactggcactggtaggctgcggtcaggatgaaaaagatccaaaccacattaaagtcggcgtgattgttggtgccgaacagcaggttgcagaagtcgcgcagaaagttgcgaaagacaaatatggcctggacgttgagctggtaaccttcaacgactatgttctgccaaacgaagcattgagcaaaggcgatatcgacgccaacgccttccagcataaaccgtaccttgatcagcaactgaaagatcgtggctacaaactggtcgcagtaggcaacacatttgtttatccgattgctggttactccaagaaaatcaaatcactggatgaactgcaagatggttcgcaggttgccgtgccaaacgacccaactaaccttggtcgttcactgctgctgctgcaaaaagtgggcttgatcaaactgaaagatggcgttggcctgctgccgaccgttcttgatgttgttgagaacccaaaaaatctgaaaattgttgaactggaagcaccgcagctaccgcgctctctggacgacgcgcaaatcgctctggcagttatcaataccacctatgccagccagattggcctgactccagcgaaagacggtatctttgtcgaagataaagagtccccgtacgtaaacctgatcgtaacgcgtgaagacaacaaagacgccgaaaacgtgaagaaattcgttcaggcttatcagtctgacgaagtttacgaagcagcaaacaaagtgtttaacggcggcgctgttaaaggctggtaaRecodedatgataaaactttcgaatatcaccaaagtgttccaccagggcacccgcaccatccaggcgttgaacaacgtcagcctMetNIQgcatgtgccagctggacaaatttatggcgttatcggtgcctcaggcgcgggtaagagtacgcttatacgttgtgtaaaSEQ ID NO:cctgctggagcgcccaaccgagggtagcgtgctggtcgatggccaggaactgaccacgctgtcagaatccgagtt1046gaccaaagctcgtcgccagattggtatgattttccagcattttaacctgctctcttcgcgtactgtttttggcaacgtggctctgccgctggagctggacaacacaccgaaagacgagatcaaacgtcgcgtgacggaattgctgtcattggttggtcttggcgataagcatgatagctacccgtcgaatctttccggtgggcagaaacaacgtgtggcgattgcccgtgcattagccagcaatcccaaagtattgctgtgtgatgaagccaccagcgcgctggacccggcaacgacacgttctattctcgaactgctgaaagacatcaaccgccgtctgggtttgacgattctgttgatcactcacgaaatggacgttgtgaagcgcatttgtgattgcgtggcggtcatcagcaatggcgaactgatcgagcaggacacggtaagtgaagtgttctcgcatccgaaaacgccgctggcgcagaagtttattcagtcgaccctgcatctggatatcccggaagattaccaggaacgtctgcaagcggagccatttactgactgcgtgccgatgctgcgtctggagtttaccggtcaatcggtcgatgccccactgctttctgaaaccgcgcgtcgtttcaacgtcaacaacaacattattagcgcgcagatggattacgccggtggcgttaagttcggcatcatgctgactgaaatgcacggcacacaacaagatacgcaagccgccattgcctggctgcaagaacaccatgtaaaagtagaggtactgggttatgtctgagccgatgatgtggctgctggttcgtggcgtatgggaaacgctggcaatgaccttcgtatccggtttttttggctttgtgattggtctgccggttggcgttctgctttatgtcacgcgtccggggcaaattattgctaacgcgaagttgtatcgtaccatttctgcgattgtgaacattttccgttccatcccgttcattatcttgctggtatggatgattccgtttacccgcgttattgtcggtacatcgattggattgcaggcagcgattgttccgttaaccgttggtgcagcaccgtttattgcccgtatggtcgagaacgctctgctggagatcccaaccgggttaattgaagcttcccgcgcaatgggggccacgccaatgcagatcgtccgtaaagtgctgttaccggaagcgttgccgggtctggtgaatgcggcaactatcaccctgattaccctggttggttattccgcgatgggtggtgcagtcggtgccggtggtttaggtcagattggctatcagtatggctacatcggctacaacgcgacggtgatgaatacggtactggtattgctggtcattctggtttatttaattcagttcgcaggcgaccgcatcgtccgggctgtcactcgcaagtaacgttcaacacaacataaataattgaagaaggaataaggtatggcgttcaaattcaaaacctttgcggcagtgggagccctgattggatcactggcactggtaggctgcggtcaggatgaaaaagatccaaaccacattaaagtcggcgtgattgttggtgccgaacagcaggttgcagaagtcgcgcagaaagttgcgaaagacaaatatggcctggacgttgagctggtaaccttcaacgactatgttctgccaaacgaagcattgagcaaaggcgatatcgacgccaacgccttccagcataaaccgtaccttgatcagcaactgaaagatcgtggctacaaactggtcgcagtaggcaacacatttgtttatccgattgctggttactccaagaaaatcaaatcactggatgaactgcaagatggttcgcaggttgccgtgccaaacgacccaactaaccttggtcgttcactgctgctgctgcaaaaagtgggcttgatcaaactgaaagatggcgttggcctgctgccgaccgttcttgatgttgttgagaacccaaaaaatctgaaaattgttgaactggaagcaccgcagctaccgcgctctctggacgacgcgcaaatcgctctggcagttatcaataccacctatgccagccagattggcctgactccagcgaaagacggtatctttgtcgaagataaagagtccccgtacgtaaacctgatcgtaacgcgtgaagacaacaaagacgccgaaaacgtgaagaaattcgttcaggcttatcagtctgacgaagtttacgaagcagcaaacaaagtgtttaacggcggcgctgttaaaggctggtaaMetNIQatgataaaactttcgaatatcaccaaagtgttccaccagggcacccgcaccatccaggcgttgaacaacgtcagcct(P281S)gcatgtgccagctggacaaatttatggcgttatcggtgcctcaggcgcgggtaagagtacgcttatacgttgtgtaaaSEQ ID NO:cctgctggagcgcccaaccgagggtagcgtgctggtcgatggccaggaactgaccacgctgtcagaatccgagtt1047gaccaaagctcgtcgccagattggtatgattttccagcattttaacctgctctcttcgcgtactgtttttggcaacgtggctctgccgctggagctggacaacacaccgaaagacgagatcaaacgtcgcgtgacggaattgctgtcattggttggtcttggcgataagcatgatagctacccgtcgaatctttccggtgggcagaaacaacgtgtggcgattgcccgtgcattagccagcaatcccaaagtattgctgtgtgatgaagccaccagcgcgctggacccggcaacgacacgttctattctcgaactgctgaaagacatcaaccgccgtctgggtttgacgattctgttgatcactcacgaaatggacgttgtgaagcgcatttgtgattgcgtggcggtcatcagcaatggcgaactgatcgagcaggacacggtaagtgaagtgttctcgcatccgaaaacgccgctggcgcagaagtttattcagtcgaccctgcatctggatatcccggaagattaccaggaacgtctgcaagcggagccatttactgactgcgtgccgatgctgcgtctggagtttaccggtcaatcggtcgatgcctccctgctttctgaaaccgcgcgtcgtttcaacgtcaacaacaacattattagcgcgcagatggattacgccggtggcgttaagttcggcatcatgctgactgaaatgcacggcacacaacaagatacgcaagccgccattgcctggctgcaagaacaccatgtaaaagtagaggtactgggttatgtctgagccgatgatgtggctgctggttcgtggcgtatgggaaacgctggcaatgaccttcgtatccggtttttttggctttgtgattggtctgccggttggcgttctgctttatgtcacgcgtccggggcaaattattgctaacgcgaagttgtatcgtaccatttctgcgattgtgaacattttccgttccatcccgttcattatcttgctggtatggatgattccgtttacccgcgttattgtcggtacatcgattggattgcaggcagcgattgttccgttaaccgttggtgcagcaccgtttattgcccgtatggtcgagaacgctctgctggagatcccaaccgggttaattgaagcttcccgcgcaatgggggccacgccaatgcagatcgtccgtaaagtgctgttaccggaagcgttgccgggtctggtgaatgcggcaactatcaccctgattaccctggttggttattccgcgatgggtggtgcagtcggtgccggtggtttaggtcagattggctatcagtatggctacatcggctacaacgcgacggtgatgaatacggtactggtattgctggtcattctggtttatttaattcagttcgcaggcgaccgcatcgtccgggctgtcactcgcaagtaacgttcaacacaacataaataattgaagaaggaataaggtatggcgttcaaattcaaaacctttgcggcagtgggagccctgattggatcactggcactggtaggctgcggtcaggatgaaaaagatccaaaccacattaaagtcggcgtgattgttggtgccgaacagcaggttgcagaagtcgcgcagaaagttgcgaaagacaaatatggcctggacgttgagctggtaaccttcaacgactatgttctgccaaacgaagcattgagcaaaggcgatatcgacgccaacgccttccagcataaaccgtaccttgatcagcaactgaaagatcgtggctacaaactggtcgcagtaggcaacacatttgtttatccgattgctggttactccaagaaaatcaaatcactggatgaactgcaagatggttcgcaggttgccgtgccaaacgacccaactaaccttggtcgttcactgctgctgctgcaaaaagtgggcttgatcaaactgaaagatggcgttggcctgctgccgaccgttcttgatgttgttgagaacccaaaaaatctgaaaattgttgaactggaagcaccgcagctaccgcgctctctggacgacgcgcaaatcgctctggcagttatcaataccacctatgccagccagattggcctgactccagcgaaagacggtatctttgtcgaagataaagagtccccgtacgtaaacctgatcgtaacgcgtgaagacaacaaagacgccgaaaacgtgaagaaattcgttcaggcttatcagtctgacgaagtttacgaagcagcaaacaaagtgtttaacggcggcgctgttaaaggctggtaaSporomusaatgaactttttccgcactaaatgtattgacaagttaaaagaaggcgcagagcagcaaggtttgaaaaagagtctgggtermitidaMetPggctaccgatctgatccttctgggtatcggatgcatcattggcacaggcatcttcgttctgacgggcgtcgcggctgc- Codon-aaattatgccggtccgggtataatgctgtccttcgtgatctcgggtctggcgtgcgcttttgcagctctggcctacgcgoptimizedgaactagctgctatggttccaattgctggcagcgcgtacacttattcttacgccgcgttaggcgaaatcgtagcatggsequenceattgtaggttggaacctgatcctcgagtacagcgttgggtcttcagctgtggccgcgggttggtccggctacatggtaSEQ ID NO:ggcctgctgaaaagcggtggtatcgaactgcctaaagctttcaccgcagttccggctgatggtggtttggtgaacttg1129ccggccatgttaattgctctgctgctgtcggttctgctagtccgtggcaccaaagaatctgtgactctgaataaggtcctggttgtgattaaactggcagcggttttcatctttctggctctggcgggccccaaagtaaacccggctaactggtcccctctgatgccgtatgggttctctggtgtagcggcgggtgcagccattatctttttcgcttacatcggcttcgacgcagtagctaccgctgctgaagagtgccggaacccgaaacgagacttaccagcaggtatcatcgggtcactggttatctgcactatactgtatattgttgttgcgggcgtcctgactggcgtcgttccgtaccagcagctcaacaacgctgaaccggttgcatacgctctgagagcgatcggctacaatttcgggtctgctctcgttggtaccggagctatcgccggcattacgacagtgctgcttgtcctgatgtatggtcagacccgcatcttctttgcaatgagccgtgatggcctgatcccggctcgtatctgtaaagtacatccacgttatggaactcctcacataattaccatggcagcgggtatcgcagttgctctgattgcaggttttacacctatcggcattatcgcggaactgactaacatcggcaccttgttcgcgttcgtggtagccgccatcggtgtactggtgctcaggtacacccgcccggacatcccgcgtagctttaagtgcccggctgtcaaagtgattgctccgctggctgttctgtcctgcggatacctgatggccaatctgccagcagagacttggatccgcttcggtatctggtccgccattggcttcgtcgtttactttgtttattcttatcaccatagcgttctgaacaaagcggaagtggctgggaaggaataaBacteroidetesatgggtatctttgcgaaaaagcagctgaatcaattgattgccgaggcttccgaatctgaaaaaggcttaaaaaagactbacterium43-ctgtcagctggagcactcgtgagtctagggatcggtgccataatcggcgcgggcctgttctctcttacgggcatggc16 MetP -tgctgccgacaacgcaggtccggcggttgtattcagctttatcctggcagctgtcggttgcgggttcgctggtctgtgCodon-ttacgcagaatttgcgagcatgattcctgttgctggctccgcatatacatactcttatgctaccatgggcgaactgattgoptimizedcttggatcatcggttgggatctggtactggagtacgcgctgggtgccgcaaccgttgctgtgtcgtggagccagtacsequencegttaacaaattccttcactccgtcggcatcgacctgccacagtatctattgcatggtccgtgggatgaagtgaacggcSEQ ID NO:gttgcgatgaacggtattattaacctgcccgcgatcatcattgtatgcctgctgtctctgctgctgatacgcggcacta1131aagagagtgcgctattgaataacatcctggtgatcctgaaagtcgttgtagttttggtcttcatctgtattggctggtctttcatcaacccggctaatcacgaaccgtttattccggttaacgctggtgaagagatggtaaaaagcggtaccatgtctttttggagcttcttcacttccgaaagcttcggatcttacggtatctcgggcatcctgcgtggcgctggtgtggttttctttgcatttatcggattcgatgccgttagcaccgctgctcaagaagccaagaacccgcagaaaggcatgccaatcgggatcattggtagtctggttgtgtgcactatcctgtatgtgctcttcgcatacgtactgactggcctggagaactacataaactttaaaggtaatgcgtccccggttaccaccgcgttcgcacacaccggttatacgtttttaaactctttccttactatggcgattatcgcaggttacacctcagttatgcttgtaatgctgatgggccagtcccgtgtgttctatagtatgtcggtggacggactgctgcctaaaatgttttctgacctgcataagaaaaacaggacaccgtacaagactaacctgatcttcatggtgtttgtctcactgttcgcaggcttcgttccggtagcagatctggggcacatggtcagcatcggtacattattcgcattcgctctggtgtgcattggcgttatagtgatgcgcaaaactaatcccgatgccgttagagggttccgtactccgttcgtcccagttctccctatcctgggtgtcttagtatgtgtagtactgatgctgggcctgccgaaagaatcctgggaacgtttggccatctggcttggtttgggcctgattatctactttgcttacagcaagaaaaactctaaaattggaaacaaataa TABLE 12Exemplary Methionine Decarboxylase Amino Acid SequencesDescriptionSEQ ID NO:Amino Acid SequenceMetDC Q70DMSPTAFPAAETATAPATAVDPGPELDGGDFALPEGGLDDDRRLRALDAVN82HDEYLTRKRKHLVGYQATQDMDGTALDLARFMPHNINNLGDPFQSGGYSEQ ID NO:KPNTKVVERAVLDYYAKLWHAERPHDPADPESYWGYMLSMGSTEGN1048MYALWNARDYLSGKALIQPPTAPFDAVRYVKADPDRRNPNAHHPVAFYSEDTHYSFAKAVAVLGVETFHAVGLEKYADECPLVDPVTGLRTWPTEVPSRPGPSGLSWDGPGEIDVDALAVLVEFFAAKGHPVFVNLNLGSTFKGAHDDVRAVCERLLPIFERHGLVQREVVYGSCPQTGRPLVDVRRGFWIHVDGALGAGYAPFLRLAAEDPEGYGWTPEAELPEFDFGLRLPTAGHGEVDMVSSIAMSGHKWAGAPWPCGIYMTKVKYQISPPSQPDYIGAPDTTFAGSRNGFSPLILWDHLSRYSYRDQVERIREAQELAAYLERRLTAMERELGVELWPARTPGAVTVRFRKPSAELVAKWSLSSQDVLMVPGDETTRRSYVHVFVMPSVDRAKLDALLAELAEDPVILGAPMetDCMSPTAFPAAETATAPATAVDPGPELDGGDFALPEGGLDDDRRLRALDAV(Streptomyces)DEYLTRKRKHLVGYQATQDMQGTALDLARFMPNNINNLGDPFQSGGYSEQ ID NO:KPNTKVVERAVLDYYAKLWHAERPHDPADPESYWGYMLSMGSTEGN1049MYALWNARDYLSGKALIQPPTAPFDAVRYVKADPDRRNPNAHHPVAFYSEDTHYSFAKAVAVLGVETFHAVGLEKYADECPLVDPVTGLRTWPTEVPSRPGPSGLSWDGPGEIDVDALAVLVEFFAAKGHPVFVNLNLGSTFKGAHDDVRAVCERLLPIFERHGLVQREVVYGSCPQTGRPLVDVRRGFWIHVDGALGAGYAPFLRLAAEDPEGYGWTPEAELPEFDFGLRLPTAGHGEVDMVSSIAMSGHKWAGAPWPCGIYMTKVKYQISPPSQPDYIGAPDTTFAGSRNGFSPLILWDHLSRYSYRDQVERIREAQELAAYLERRLTAMERELGVELWPARTPGAVTVRFRKPSAELVAKWSLSSQDVLMVPGDETTRRSYVHVFVMPSVDRAKLDALLAELAEDPVILGAPMetDCMSPTAFPAAETATAPATAVDPGPELDGGDFALPEGGLDDDRRLRALDAV(V491LDEYLTRKRKHLVGYQATQDMQGTALDLARFMPNNINNLGDPFQSGGYA500P)KPNTKVVERAVLDYYAKLWHAERPHDPADPESYWGYMLSMGSTEGNSEQ ID NO:MYALWNARDYLSGKALIQPPTAPFDAVRYVKADPDRRNPNAHHPVAFY1050SEDTHYSFAKAVAVLGVETFHAVGLEKYADECPLVDPVTGLRTWPTEVPSRPGPSGLSWDGPGEIDVDALAVLVEFFAAKGHPVFVNLNLGSTFKGAHDDVRAVCERLLPIFERHGLVQREVVYGSCPQTGRPLVDVRRGFWIHVDGALGAGYAPFLRLAAEDPEGYGWTPEAELPEFDFGLRLPTAGHGEVDMVSSIAMSGHKWAGAPWPCGIYMTKVKYQISPPSQPDYIGAPDTTFAGSRNGFSPLILWDHLSRYSYRDQVERIREAQELAAYLERRLTAMERELGVELWPARTPGALTVRFRKPSPELVAKWSLSSQDVLMVPGDETTRRSYVHVFVMPSVDRAKLDALLAELAEDPVILGAPMetDC (R41QMSPTAFPAAETATAPATAVDPGPELDGGDFALPEGGLDDDQRLRALDAQ70D)VDEYLTRKRKHLVGYQATQDMDGTALDLARFMPNNINNLGDPFQSGGSEQ ID NO:YKPNTKVVERAVLDYYAKLWHAERPHDPADPESYWGYMLSMGSTEGN1051MYALWNARDYLSGKALIQPPTAPFDAVRYVKADPDRRNPNAHHPVAFYSEDTHYSFAKAVAVLGVETFHAVGLEKYADECPLVDPVTGLRTWPTEVPSRPGPSGLSWDGPGEIDVDALAVLVEFFAAKGHPVFVNLNLGSTFKGAHDDVRAVCERLLPIFERHGLVQREVVYGSCPQTGRPLVDVRRGFWIHVDGALGAGYAPFLRLAAEDPEGYGWTPEAELPEFDFGLRLPTAGHGEVDMVSSIAMSGHKWAGAPWPCGIYMTKVKYQISPPSQPDYIGAPDTTFAGSRNGFSPLILWDHLSRYSYRDQVERIREAQELAAYLERRLTAMERELGVELWPARTPGAVTVRFRKPSAELVAKWSLSSQDVLMVPGDETTRRSYVHVFVMPSVDRAKLDALLAELAEDPVILGAPMetDCMGFQLLSKHKLSAEDQQKLDRFYRDIQTEAERFLGYPCNELFDYSPLFRF(Stanieriasp.LQYPLNNVGDPYLPSNYHLNTHNFECEVLEIFRTLTEATEGSTWGYVTNNIES-3757)GGTEGNHYGLFLARELLPEGLVYYSQDAHYSIDKILRCLNLRSIMIRSHDSEQ ID NO:DGRMDLDDLRETLRIHRDLPPIVCATIGTTMKGAVDDIAGIKKIFKDLAIH1052RHYIHADAALGGMILPFLDNSPPWNFKAGIDSIAISGHKMVGSPIPCGVVLAKKSNVERIAQSVEYIGTLDTTLSGSRNALTPLFLWYAFHTVGIEGFKRIIPACLKMADYAIAQLNKINRNAWRYPYSNTVVFDRPSPEVTRYWQLACQGNLSHLITMPHVTSTQIDHLVADIIASEPIPPLPTLSVTPACELLTSTPDQDITLIGTANHNLLSEVSTALAAEGLSIENLAAVAVESEDVEVVRLRVNNRERALQILNQNLDIGRCYGQARPFGNEEATQVLSQLEYQSVGEDALLVQLDDCPGSLAELLKDCRNEAVKIRNIRLLWRGHGKGVVAIATTSPDALKTLLKDRILLSLeuDCMSTPSEVKKDLLGAAGSLWPSEPITLGPGESAWQLVLKKIQELSDSGHQ(Musmusculus)DPFMVADLDVLVSRHQTFCQALPRVQPFYAVKCNSNPWVLRVLAALGTSEQ ID NO:GFDCASQGELEQVLGLGVAPSRIIFANPCKAVSHIQFAARCGVQLLTFDS1053EEELIKVAQYHPGARLVLRIQTQDSQSTFPLSTKFGASLEACGHLLQVARELGLAVVGASFHVGSDCHTPQSFRQAIADCHRVFEMGRKAGHDMSLLDLGGGFPGVEGSEAKFEEMARVINAALAQYFPEETGIEVIAEPGRFYAGSVCTAAVNIIAKKSVLEPGGHRKLMYYLNEGHYGSFRLFLRDPVPRIPIVVKEFPSEPPLFPCTLYGPTCDAYDRLFSEEVQLPELDVGDWLIFPDMGAYTSSMSSTFNGFPPATVYCAMSPQLRSLLETVPMetDCMNTPSEVKKDLLGVAEHLRPSEPITLGPGASAWQLVLKKIKELSISGRQD(Musmusculus)AFMVADLDVLVSRHRTFLQALPRVQPFYAVKCNSNPWVLLVLAALGTGSEQ ID NO:FDCASQGELEQVLGLGVAPSRIIFANPCKAVSHIQFAARCGVQLLTFDNE1054EELIKLARYHPRARLVLRIQTLDSQSTFPLSTKFGAHLEACGHLLQVARELGLAVVGASFHVGSDCHTPESYRQAIADCHRVFEMGCKAGHHMSLLDLGGGFPGVKGSEAKFEEVARVINTALAQYFPEETGIEVIAEPGRFYAGSVCTAAVNIIAKKSSLDPGGHRKLAYYLNEGHYGVFRLFLRDPVPRIPIVVKEFPSEPPLFPCTLYGPTCDAYDRLFSTEVQLPELDVGDWLIFPDMGAYSSSMSSTFNGFPIATVYDAMSPQLRSLLETVPMetDCMKQTSLEVKEFALNLISQFEPENQPLGFWIFDTEGVEKAVERWKKNMPT(EntamoebaVRPCFAVKCNPEPHLVKLLGELGCGFDCASLNEIKEVLDLGFNPEDITYShistolytica)QTFKPYNQLIEASHLGINHTIVDSIDEVQKIAKYAPKMGIMIRIMENDTSASEQ ID NO:GHVFGEKFGLHDDEVEIVLKEIKDKGLNLDGVHFHVGSDSHNSEVFTKA1055LTKARNTVTLAEQFGMKPYLIDIGGGFSQVAPFEEFAATIEKTIKELEFPERTRFIAEPGRYMASNAFHLVSSLHGKRVRIQNGKKQIEYTSGDGLHGSFGCCIWFEKQKSCECITQKVNENTKMYESIIYGPSCNGSDKVATQELPEMEPGKDWLLFPNMGAYTISMATNFNGFEERNHVIYTLPLKSTKIIQIPKSIECNSVPSLNGIPHYASpMetDCMSPTAFPAAETATAPATAVDPGPELDGGDFALPEGGLDDDRRLRALDAVT66NDEYLTRKRKHLVGYQANQDMQGTALDLARFMPNNINNLGDPFQSGGYSEQ ID NO:KPNTKVVERAVLDYYAKLWHAERPHDPADPESYWGYMLSMGSTEGN1124MYALWNARDYLSGKALIQPPTAPFDAVRYVKADPDRRNPNAHHPVAFYSEDTHYSFAKAVAVLGVETFHAVGLEKYADECPLVDPVTGLRTWPTEVPSRPGPSGLSWDGPGEIDVDALAVLVEFFAAKGHPVFVNLNLGSTFKGAHDDVRAVCERLLPIFERHGLVQREVVYGSCPQTGRPLVDVRRGFWIHVDGALGAGYAPFLRLAAEDPEGYGWTPEAELPEFDFGLRLPTAGHGEVDMVSSIAMSGHKWAGAPWPCGIYMTKVKYQISPPSQPDYIGAPDTTFAGSRNGFSPLILWDHLSRYSYRDQVERIREAQELAAYLERRLTAMERELGVELWPARTPGAVTVRFRKPSAELVAKWSLSSQDVLMVPGDETTRRSYVHVFVMPSVDRAKLDALLAELAEDPVILGAPSpMetDCMSPTAFPAAETATAPATAVDPGPELDGGDFALPEGGLDDDRRLRALDAVA203HDEYLTRKRKHLVGYQATQDMQGTALDLARFMPNNINNLGDPFQSGGYSEQ ID NO:KPNTKVVERAVLDYYAKLWHAERPHDPADPESYWGYMLSMGSTEGN1126MYALWNARDYLSGKALIQPPTAPFDAVRYVKADPDRRNPNAHHPVAFYSEDTHYSFAKAVHVLGVETFHAVGLEKYADECPLVDPVTGLRTWPTEVPSRPGPSGLSWDGPGEIDVDALAVLVEFFAAKGHPVFVNLNLGSTFKGAHDDVRAVCERLLPIFERHGLVQREVVYGSCPQTGRPLVDVRRGFWIHVDGALGAGYAPFLRLAAEDPEGYGWTPEAELPEFDFGLRLPTAGHGEVDMVSSIAMSGHKWAGAPWPCGIYMTKVKYQISPPSQPDYIGAPDTTFAGSRNGFSPLILWDHLSRYSYRDQVERIREAQELAAYLERRLTAMERELGVELWPARTPGAVTVRFRKPSAELVAKWSLSSQDVLMVPGDETTRRSYVHVFVMPSVDRAKLDALLAELAEDPVILGAPSpMetDCMSPTAFPAAETATAPATAVDPGPELDGGDFALPEGGLDDDRRLRALDAVH379GDEYLTRKRKHLVGYQATQDMQGTALDLARFMPNNINNLGDPFQSGGYSEQ ID NO:KPNTKVVERAVLDYYAKLWHAERPHDPADPESYWGYMLSMGSTEGN1128MYALWNARDYLSGKALIQPPTAPFDAVRYVKADPDRRNPNAHHPVAFYSEDTHYSFAKAVAVLGVETFHAVGLEKYADECPLVDPVTGLRTWPTEVPSRPGPSGLSWDGPGEIDVDALAVLVEFFAAKGHPVFVNLNLGSTFKGAHDDVRAVCERLLPIFERHGLVQREVVYGSCPQTGRPLVDVRRGFWIHVDGALGAGYAPFLRLAAEDPEGYGWTPEAELPEFDFGLRLPTAGGGEVDMVSSIAMSGHKWAGAPWPCGIYMTKVKYQISPPSQPDYIGAPDTTFAGSRNGFSPLILWDHLSRYSYRDQVERIREAQELAAYLERRLTAMERELGVELWPARTPGAVTVRFRKPSAELVAKWSLSSQDVLMVPGDETTRRSYVHVFVMPSVDRAKLDALLAELAEDPVILGAP TABLE 13Exemplary Importer Amino Acid SequencesDescriptionSEQ ID NO:Amino Acid SequenceMetPMGTINTKIYKYMSIWKTKPLSVLLSEATEDEKGLKRTLSARSLVALGVG(F. segetis)AIIGAGLFSLTGIAAADNAGPAVTLSFILASVGCAFAGLCYAEFASMIPVASEQ ID NO:GSAYTYSYATMGEFVAWIIGWDLVLEYALGAATVAVSWSQYVDKFLQ1056NYGIHIPNSILHGPWDTTPGIINLPSIFIICLLSVLLIRGTKESALINNILVILKVTVVIVFIGLGWGFMNSANHTPFIPVNEGEALLSSGEMSFLNFFSSDYFGHYGWSGILRGAGVVFFAFIGFDAVSTAAQEAKDPQKGMPIGILGSLIICTVLYVLFAFVLTGLENYLNFKGDASPVTTAFAKTGYTFLNSGLTIAIIAGYTSVMLVMLMGQSRVFYSMSVDGLLPKFFSTLHTKNRTPYKTNLLFMVFVSLFAGFVPVSDLGHMVSIGTLFAFCLVCIGVIVMRKTNPDAVRGFRVPFVPVFPIIGVVICLVLMAGLPIESWERLAIWMILGVVIYFFYSKKNSKLNNPEMetPMGTINTKTNKYMSIWKTKPLSVLLNEASEDEKGLKRTLSSRSLVALGVG(F. frigoris)AIIGAGLFSLTGIAAAEHAGPAVTLSFILAAVGCAFAGLCYAEFASMIPVASEQ ID NO:GSAYTYSYATMGEFMAWIIGWDLVLEYALGAATVGVSWSRYLLELLNK1057YGVHLNPKFICSPWETLTLGDGTIIDGGYINLPAILIVSALSLLLIRGTQESASINNILVVLKVIVVIMFIVLGWDYIDPANYSPYIPENTGVKGQFGWSGIAAGAGTVFFAFIGFDAVSTAAQEAKNPQKGMPIGILGSLVICTILYVLFAHVMTGLVPYYKFAGDAKPAATAFAVTGYSFLQTGLIVAILAGYTSVMLVMLMGQSRVFYTMSKDGLLPPLFGQIHSKFRTPYKTNLFFMVFVSLFAGFVPVSDLGHMVSIGTLLAFVLVCIGVLVMRKKMPDAPRSFKTPFVPYVPIAGVLVCTYLMYSLPYESWIRLVLWMAIGVALYFVYGKKHSKLNNPDMetNMIKLSNITKVFHQGTRTIQALNNVSLHVPAGQIYGVIGASGAGKSTLIRCSEQ ID NO:VNLLERPTEGSVLVDGQELTTLSESELTKARRQIGMIFQHFNLLSSRTVFG1058NVALPLELDNTPKDEIKRRVTELLSLVGLGDKHDSYPSNLSGGQKQRVAIARALASNPKVLLCDEATSALDPATTRSILELLKDINRRLGLTILLITHEMDVVKRICDCVAVISNGELIEQDTVSEVFSHPKTPLAQKFIQSTLHLDIPEDYQERLQAEPFTDCVPMLRLEFTGQSVDAPLLSETARRFNVNNNIISAQMDYAGGVKFGIMLTEMHGTQQDTQAAIAWLQEHHVKVEVLGYVMetIMSEPMMWLLVRGVWETLAMTFVSGFFGFVIGLPVGVLLYVTRPGQIIANSEQ ID NO:AKLYRTISAIVNIFRSIPFIILLVWMIPFTRVIVGTSIGLQAAIVPLTVGAAPF1059IARMVENALLEIPTGLIEASRAMGATPMQIVRKVLLPEALPGLVNAATITLITLVGYSAMGGAVGAGGLGQIGYQYGYIGYNATVMNTVLVLLVILVYLIQFAGDRIVRAVTRKMetQMAFKFKTFAAVGALIGSLALVGCGQDEKDPNHIKVGVIVGAEQQVAEVSEQ ID NO:AQKVAKDKYGLDVELVTFNDYVLPNEALSKGDIDANAFQHKPYLDQQL1060KDRGYKLVAVGNTFVYPIAGYSKKIKSLDELQDGSQVAVPNDPTNLGRSLLLLQKVGLIKLKDGVGLLPTVLDVVENPKNLKIVELEAPQLPRSLDDAQIALAVINTTYASQIGLTPAKDGIFVEDKESPYVNLIVTREDNKDAENVKKFVQAYQSDEVYEAANKVFNGGAVKGWMethionineMFEKYFPNVDLTELWNATYETLYMTLISLLFAFVIGVILGLLLFLTSKGSLimport systemWQNKAVNSVIAAVVNIFRSIPFLILIILLLGFTKFLVGTILGPNAALPALVIpermeaseGSAPFYARLVEIALREVDKGVIEAAKSMGAKTSTIIFKVLIPESMPALISGIprotein MetPTVTAIALIGSTAIAGAIGSGGLGNLAYVEGYQSNNADVTFVATVFILIIVFI(BacillusIQIIGDLITNIIDKRsubtilis)SEQ ID NO:1061MetN (P281G)MIKLSNITKVFHQGTRTIQALNNVSLHVPAGQIYGVIGASGAGKSTLIRCSEQ ID NO:VNLLERPTEGSVLVDGQELTTLSESELTKARRQIGMIFQHFNLLSSRTVFG1062NVALPLELDNTPKDEIKRRVTELLSLVGLGDKHDSYPSNLSGGQKQRVAIARALASNPKVLLCDEATSALDPATTRSILELLKDINRRLGLTILLITHEMDVVKRICDCVAVISNGELIEQDTVSEVFSHPKTPLAQKFIQSTLHLDIPEDYQERLQAEPFTDCVPMLRLEFTGQSVDAGLLSETARRFNVNNNIISAQMDYAGGVKFGIMLTEMHGTQQDTQAAIAWLQEHHVKVEVLGYVMetN (P281S)MIKLSNITKVFHQGTRTIQALNNVSLHVPAGQIYGVIGASGAGKSTLIRCSEQ ID NO:VNLLERPTEGSVLVDGQELTTLSESELTKARRQIGMIFQHFNLLSSRTVFG1063NVALPLELDNTPKDEIKRRVTELLSLVGLGDKHDSYPSNLSGGQKQRVAIARALASNPKVLLCDEATSALDPATTRSILELLKDINRRLGLTILLITHEMDVVKRICDCVAVISNGELIEQDTVSEVFSHPKTPLAQKFIQSTLHLDIPEDYQERLQAEPFTDCVPMLRLEFTGQSVDASLLSETARRFNVNNNIISAQMDYAGGVKFGIMLTEMHGTQQDTQAAIAWLQEHHVKVEVLGYVSporomusaMNFFRTKCIDKLKEGAEQQGLKKSLGATDLILLGIGCIIGTGIFVLTGVAAtermitidaMetPANYAGPGIMLSFVISGLACAFAALAYAELAAMVPIAGSAYTYSYAALGESEQ ID NO:IVAWIVGWNLILEYSVGSSAVAAGWSGYMVGLLKSGGIELPKAFTAVPA1130DGGLVNLPAMLIALLLSVLLVRGTKESVTLNKVLVVIKLAAVFIFLALAGPKVNPANWSPLMPYGFSGVAAGAAIIFFAYIGFDAVATAAEECRNPKRDLPAGIIGSLVICTILYIVVAGVLTGVVPYQQLNNAEPVAYALRAIGYNFGSALVGTGAIAGITTVLLVLMYGQTRIFFAMSRDGLIPARICKVHPRYGTPHIITMAAGIAVALIAGFTPIGIIAELTNIGTLFAFVVAAIGVLVLRYTRPDIPRSFKCPAVKVIAPLAVLSCGYLMANLPAETWIRFGIWSAIGFVVYFVYSYHHSVLNKAEVAGKEBacteroidetesMGIFAKKQLNQLIAEASESEKGLKKTLSAGALVSLGIGAIIGAGLFSLTGbacterium43-MAAADNAGPAVVFSFILAAVGCGFAGLCYAEFASMIPVAGSAYTYSYA16 MetPTMGELIAWIIGWDLVLEYALGAATVAVSWSQYVNKFLHSVGIDLPQYLLSEQ ID NO:HGPWDEVNGVAMNGIINLPAIIIVCLLSLLLIRGTKESALLNNILVILKVV1132VVLVFICIGWSFINPANHEPFIPVNAGEEMVKSGTMSFWSFFTSESFGSYGISGILRGAGVVFFAFIGFDAVSTAAQEAKNPQKGMPIGIIGSLVVCTILYVLFAYVLTGLENYINFKGNASPVTTAFAHTGYTFLNSFLTMAIIAGYTSVMLVMLMGQSRVFYSMSVDGLLPKMFSDLHKKNRTPYKTNLIFMVFVSLFAGFVPVADLGHMVSIGTLFAFALVCIGVIVMRKTNPDAVRGFRTPFVPVLPILGVLVCVVLMLGLPKESWERLAIWLGLGLIIYFAYSKKNSKIGNK TABLE 14Phage Nucleotide SequenceDescriptionSEQ ID NO:Phage 3SEQ ID NO: 1064 TABLE 15Colibactin Nucleotide SequencesDescriptionSEQ ID NO:clbASEQ ID NO: 1065clbBSEQ ID NO: 1066clbCSEQ ID NO: 1067clbDSEQ ID NO: 1068clbESEQ ID NO: 1069clbFSEQ ID NO: 1070clbGSEQ ID NO: 1071clbHSEQ ID NO: 1072clbISEQ ID NO: 1073clbJSEQ ID NO: 1074clbKSEQ ID NO: 1075clbLSEQ ID NO: 1076clbMSEQ ID NO: 1077clbNSEQ ID NO: 1078clbOSEQ ID NO: 1079clbPSEQ ID NO: 1080clbQSEQ ID NO: 1081clbRSEQ ID NO: 1082clbSSEQ ID NO: 1083 TABLE 16Colibactin Amino Acid SequencesDescriptionSEQ ID NO:Amino Acid SequenceclbASEQ ID NO: 1084clbBSEQ ID NO: 1085clbCSEQ ID NO: 1086clbDSEQ ID NO: 1087clbESEQ ID NO: 1088clbFSEQ ID NO: 1089clbGSEQ ID NO: 1090clbHSEQ ID NO: 1091clbISEQ ID NO: 1092clbJSEQ ID NO: 1093clbKSEQ ID NO: 1094clbLSEQ ID NO: 1095clbMSEQ ID NO: 1096clbNSEQ ID NO: 1097clbOSEQ ID NO: 1098clbPSEQ ID NO: 1099clbQSEQ ID NO: 1100clbRSEQ ID NO: 1101clbSSEQ ID NO: 1102 TABLE 17Other Exemplary SequencesDescriptionSequencePtac-metPttgacaattaatcatcggctcgtataatgtgtggaattgtgagcgctcacaattagctgtgaccagaggtaaggaggtaacSEQ ID NO:aaccatgcgagtgttgaagaaacatcttaatcatgctgcggagggtttctaatggggaccattaacacgaagatctataaa1118tacatgagcatctggaaaacaaaacctctgtccgtgctcttgtctgaagcaactgaggatgaaaaaggcctgaagcgcactctgtcggcccgttcacttgttgcgctgggtgtcggtgctattatcggcgctggtttattctctctgaccggcatagctgcggcagacaatgctggaccggcagtaaccctgagctttatcctggcctccgttggttgcgcgttcgctggcctgtgttacgcagaatttgcttctatgattccagttgcgggtagcgcctacacttatagttatgctaccatgggcgagttcgtggcgtggatcatcggttgggatctggtactcgaatacgcattgggcgcagctactgttgccgttagctggtcccagtacgtggacaaattcttgcaaaactacggcatccatattccgaactctatcctccacgggccgtgggataccacccccggtattatcaatttaccgtcgatatttatcatctgcctgctgagcgtgctgctgattcgtggtactaaagaatctgctctgatcaacaacattctggtaatcctgaaagtcacggttgtcatcgtgttcattggcctgggctgggggttcatgaactccgcaaaccacacgccctttatcccggttaacgaaggtgaggctctactgtcttctggtgaaatgagtttcctcaactttttcagcagtgactactttggacactacggatggtccggtattcttcgcggcgctggtgtagtattcttcgcatttatcggcttcgacgcggtgagcactgcggcacaggaggccaaggatccgcagaaaggcatgccaatcggtattctgggctcactgatcatttgcaccgttctgtacgtgcttttcgctttcgttctgaccggtctggaaaactatctaaacttcaaaggtgacgcttctcctgtcaccactgcatttgccaaaacaggctatactttcctgaatagcggtctgacgatcgctatcatagcgggctacacatccgttatgctggtaatgttgatgggtcagtcccgtgtcttttatagtatgtctgtggatggcctgcttccgaagtttttctcgaccctgcataccaaaaacaggactccgtacaaaactaatttgctgttcatggttttcgtaagcctgttcgctggctttgttccggtcagcgacctgggccatatggtatccatcggtaccctcttcgctttctgcctggtgtgtatcggcgttatcgttatgcgaaaaaccaacccagacgccgttcgcggttttcgtgttccttttgtaccggttttcccgattatcggtgtagttatttgtctggttctaatggcgggcctgccgattgaatcttgggaacgtctggcgatctggatgattctgggtgtcgtgatctacttcttctactctaaaaagaactctaaactgaataaccccgaataaPtac-metDCttgacaattaatcatcggctcgtataatgtgtggaattgtgagcgctcacaattaagtgaATTGCCAATAACASEQ ID NO:ATTACTAAGGAGGTTTTTTATGtccccgacggcgtttccagcggccgaaacagctactgcccctgc1119aactgccgtcgatcctgggccagaactggacggcggagatttcgcccttccagagggcgggctggatgacgatcgtcgcttacgtgcattggacgcagttgacgagtatttgacccgcaagcgcaagcatttggttgggtaccaagctacccaggatatggacggaacggccttggatttagcccgtttcatgccccacaacatcaacaacctgggagatcctttccagtcgggtgggtataaaccaaatacgaaagtcgttgagcgtgccgtactggactactatgcaaaattgtggcacgcagaacgtccacacgacccagctgacccagaaagctactggggttacatgttatcgatgggctcaactgagggcaacatgtacgccctgtggaatgcacgtgactacctgtcgggtaaggctttgattcagcctcccacggcaccatttgacgctgttcgctacgtgaaggctgaccccgatcgccgcaatcctaacgcacaccacccagtcgcattctactcggaggatacccactattcttttgctaaagccgttgcggtgctgggtgtcgaaactttccacgctgtgggtctggagaaatacgctgacgagtgccccttggtggatccagtaaccggccttcgtacctggccgaccgaagttccatcgcgcccggggccgtcgggtttaagctgggacggccctggtgagattgatgttgatgcgcttgcagtactggtcgagttcttcgcagcgaagggtcaccccgtcttcgtcaaccttaacttggggtctacatttaaaggagcacatgatgacgtacgtgcggtatgtgaacgcttattaccaatcttcgagcgccatggcttagtacaacgtgaagttgtatatgggagctgtccccaaaccggccgccctttagtggatgtacgtcgcggattttggatccacgtagatggggcacttggggcggggtatgccccttttctgcgtcttgccgccgaagacccggaaggttatggttggacccctgaggcagaattacctgagttcgacttcggcttacgtttgccgacggcggggcatggagaagttgatatggttagcagcatcgccatgagtggacataagtgggcaggcgcgccgtggccatgcggcatctatatgacgaaagtgaaatatcagattagtccaccgtcacagcccgattatattggtgctcctgacacaacatttgccggttcccgtaacggcttttcgccgttaattttgtgggatcatttatcgcgctactcgtaccgcgaccaggtagagcgcatccgcgaagcacaggagcttgcagcatatttggaacgccgccttaccgctatggagcgcgagctgggagtggaactttggccagcccgcacaccgggtgctgtaaccgtacgttttcgcaaaccctctgctgagctggttgcgaagtggtccttgtcgtcgcaggatgttttaatggtgccgggtgatgaaactacgcgtcgtagttacgttcatgtgttcgtgatgccttctgttgatcgtgcaaagttagatgcgttgctggcagaattggccgaagatcccgtcatcttgggtgcgccttaayjeHatgagtggactcaaacaagaactggggctggcccagggcatcggcctactatcgacgtcattattaggcactggcgtgtSEQ ID NO:ttgccgttcctgcgttagctgcgctagtagcaggcaataacagcctgtgggcgtggcccgttttgattatcttagtgttccc1014gattgcgattgtgtttgcgattctgggtcgccactatcccagcgcaggcggcgtcgcacacttcgtcggtatggcgtttggttcgcggcttgagcgagtcaccggctggttgtttttatcggtcattcccgtgggtttgcctgccgcgctacaaattgctgccggattcggccaggcaatgtttggctggcatagcgggcaactgttgttggcagaactcggtacgctggcgctggtgtggtatatcggtactcgaggtgccagttccagtgctaatctacaaacagttattgccgggcttatcgtcgcactgattgtcgctatctggtgggcgggcgatatcaaacctgcgaatatccccttccctgcgccaggaaatatcgaacttaccgggttattcgctgcgttatcagtgatgttctggtgttttgtcggtctggaagcatttgcccatcttgcctcggaatttaaaaatccagagcgtgattttcctcgtgctttgatgattggcctgctgctggcaggattagtctattggggctgtacggtagtcgtcttacacttcgacgcctatggtgaacaaatggcggcggcagcatcgcttcccaaaattgtagtgcagttattcggtgtaggagcgttatggattgcctgcgtaattggctatctggcctgctttgccagtctcaacatttatatacagagcttcgcccgcctggtctggtcgcaggcgcaacataatcctgaccattacctggcacgcctctcttctcgccatattccgaataatgccctcaatgcggtgctcggctgctgcgtggtgagcacgttggtgattcatgctttagagatcaatctggacgctcttattatttatgccaatggcatctttattatgatttatctgttatgcatgctggcaggctgtaaattattgcaaggacgttatcgactactggcagtggttggcgggctattatgcgttctgttactggcaatggtcggctggaaaagtctctacgcgctgatcatgctggcggggttatggctgtttctgccaaaacgaaaaacgccggaaaatggcataaccacataayjeH K/Oaatgtgaatggcacgattatgcgggatacttacaccaccgacggaatatgaaaatcaatattatcgacggctcagaagtg(100 bp uptctagattatccgtggcgatCTGACATGGGAATTAGCCATGGTCCATATGAATATCCTandCCttAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGdownstreamAAGCAGCTCCAGCCTACACAATCGCTCAAGACGTGTAATtccggcgtttcgacattand the scaraatcctggcgatcgtctttatgatcaaggcggtcgcggtcatcatcctttcgctggtactcaccatcaaaagtattaccgccsite inabetween)SEQ ID NO:1120YjeH AminoMSGLKQELGLAQGIGLLSTSLLGTGVFAVPALAALVAGNNSLWAWPVLIILacidVFPIAIVFAILGRHYPSAGGVAHFVGMAFGSRLERVTGWLFLSVIPVGLPAAsequenceLQIAAGFGQAMFGWHSGQLLLAELGTLALVWYIGTRGASSSANLQTVIAGLSEQ ID NO:IVALIVAIWWAGDIKPANIPFPAPGNIELTGLFAALSVMFWCFVGLEAFAHL1121ASEFKNPERDFPRALMIGLLLAGLVYWGCTVVVLHFDAYGEQMAAAASLPKIVVQLFGVGALWIACVIGYLACFASLNIYIQSFARLVWSQAQHNPDHYLARLSSRHIPNNALNAVLGCCVVSTLVIHALEINLDALIIYANGIFIMIYLLCMLAGCKLLQGRYRLLAVVGGLLCVLLLAMVGWKSLYALIMLAGLWLFLPKRKTPENGITTExemplarygaccagaggtaaggaggtaacaaccatgcgagtgttgaagaaacatcttaatcatgctgcggagggtttctaRBS forMetPSEQ ID NO:1109ExemplaryATTGCCAATAACAATTACTAAGGAGGTTTTTTRBS forMetDCSEQ ID NO:1122 | 360,403 |
11859190 | DETAILED DESCRIPTION Disclosed herein are compositions and methods for modifying an endogenous albumin gene, for example, for expressing a transgene in a secretory tissue. In some embodiments, the transgene is inserted into an endogenous albumin gene to allow for very high expression levels that are moreover limited to hepatic tissue. The transgene can encode any protein or peptide including those providing therapeutic benefit. Thus, the methods and compositions of the invention can be used to express therapeutically beneficial proteins (from a transgene) from highly expressed loci in secretory tissues. For example, the transgene can encode a protein involved in disorders of the blood, for example, clotting disorders, and a variety of other monogenic diseases. In some embodiments, the transgene can be inserted into the endogenous albumin locus such that expression of the transgene is controlled by the albumin expressional control elements, resulting in liver-specific expression of the transgene encoded protein at high concentrations. Proteins that may be expressed may include clotting factors such as Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XIII, vWF and the like, antibodies, proteins relevant to lysosomal storage, insulin, alpha 1-antitrypsin, and indeed any peptide or protein that when so expressed provides benefit. In addition, any transgene can be introduced into patient derived cells, e.g., patient derived induced pluripotent stem cells (iPSCs) or other types of stem cells (embryonic, hematopoietic, neural, or mesenchymal as a non-limiting set) for use in eventual implantation into secretory tissues. The transgene can be introduced into any region of interest in these cells, including, but not limited to, into an albumin gene or a safe harbor gene. These altered stem cells can be differentiated for example, into hepatocytes and implanted into the liver. Alternately, the transgene can be directed to the secretory tissue as desired through the use of viral or other delivery systems that target specific tissues. For example, use of the liver-trophic adenovirus associated virus (AAV) vector AAV8 as a delivery vehicle can result in the integration of the transgene at the desired locus when specific nucleases are co-delivered with the transgene. General Practice of the methods, as well as preparation and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields as are within the skill of the art. These techniques are fully explained in the literature. See, for example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) Humana Press, Totowa, 1999. Definitions The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T. The terms “polypeptide,” “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally-occurring amino acids. “Binding” refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (Kd) of 10−6M−1or lower. “Affinity” refers to the strength of binding: increased binding affinity being correlated with a lower Kd. A “binding protein” is a protein that is able to bind non-covalently to another molecule. A binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein). In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity. A “zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. See, e.g., U.S. Patent Publication No. 2011/0301073, incorporated by reference herein in its entirety. Zinc finger and TALE binding domains can be “engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein. Therefore, engineered DNA binding proteins (zinc fingers or TALEs) are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering DNA-binding proteins are design and selection. A designed DNA binding protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also International Patent Publication Nos. WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Patent Publication No. 2011/0301073. A “selected” zinc finger protein or TALE is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; International Patent Publication Nos. WO 95/19431; O 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; WO 02/099084 and U.S. Patent Publication No. 2011/0301073. “Recombination” refers to a process of exchange of genetic information between two polynucleotides. For the purposes of this disclosure, “homologous recombination (HR)” refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells via homology-directed repair mechanisms. This process requires nucleotide sequence homology, uses a “donor” molecule to template repair of a “target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or “synthesis-dependent strand annealing,” in which the donor is used to re-synthesize genetic information that will become part of the target, and/or related processes. Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide. In the methods of the disclosure, one or more targeted nucleases as described herein create a double-stranded break in the target sequence (e.g., cellular chromatin) at a predetermined site, and a “donor” polynucleotide, having homology to the nucleotide sequence in the region of the break, can be introduced into the cell. The presence of the double-stranded break has been shown to facilitate integration of the donor sequence. The donor sequence may be physically integrated or, alternatively, the donor polynucleotide is used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence as in the donor into the cellular chromatin. Thus, a first sequence in cellular chromatin can be altered and, in certain embodiments, can be converted into a sequence present in a donor polynucleotide. Thus, the use of the terms “replace” or “replacement” can be understood to represent replacement of one nucleotide sequence by another, (i.e., replacement of a sequence in the informational sense), and does not necessarily require physical or chemical replacement of one polynucleotide by another. In any of the methods described herein, additional pairs of zinc-finger or TALEN proteins can be used for additional double-stranded cleavage of additional target sites within the cell. In certain embodiments of methods for targeted recombination and/or replacement and/or alteration of a sequence in a region of interest in cellular chromatin, a chromosomal sequence is altered by homologous recombination with an exogenous “donor” nucleotide sequence. Such homologous recombination is stimulated by the presence of a double-stranded break in cellular chromatin, if sequences homologous to the region of the break are present. In any of the methods described herein, the first nucleotide sequence (the “donor sequence”) can contain sequences that are homologous, but not identical, to genomic sequences in the region of interest, thereby stimulating homologous recombination to insert a non-identical sequence in the region of interest. Thus, in certain embodiments, portions of the donor sequence that are homologous to sequences in the region of interest exhibit between about 80 to 99% (or any value therebetween) sequence identity to the genomic sequence that is replaced. In other embodiments, the homology between the donor and genomic sequence is higher than 99%, for example if only 1 nucleotide differs as between donor and genomic sequences of over 101 contiguous base pairs. In certain cases, a non-homologous portion of the donor sequence can contain sequences not present in the region of interest, such that new sequences are introduced into the region of interest. In these instances, the non-homologous sequence is generally flanked by sequences of 50-1,000 base pairs (or any integral value therebetween) or any number of base pairs greater than 1,000, that are homologous or identical to sequences in the region of interest. In other embodiments, the donor sequence is non-homologous to the first sequence, and is inserted into the genome by non-homologous recombination mechanisms. Any of the methods described herein can be used for partial or complete inactivation of one or more target sequences in a cell by targeted integration of donor sequence that disrupts expression of the gene(s) of interest. Cell lines with partially or completely inactivated genes are also provided. Furthermore, the methods of targeted integration as described herein can also be used to integrate one or more exogenous sequences. The exogenous nucleic acid sequence can comprise, for example, one or more genes or cDNA molecules, or any type of coding or non-coding sequence, as well as one or more control elements (e.g., promoters). In addition, the exogenous nucleic acid sequence may produce one or more RNA molecules (e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs (miRNAs), etc.). “Cleavage” refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage. A “cleavage half-domain” is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity). The terms “first and second cleavage half-domains;” “+ and − cleavage half-domains” and “right and left cleavage half-domains” are used interchangeably to refer to pairs of cleavage half-domains that dimerize. An “engineered cleavage half-domain” is a cleavage half-domain that has been modified so as to form obligate heterodimers with another cleavage half-domain (e.g., another engineered cleavage half-domain). See, also, U.S. Patent Publication Nos. 2005/0064474, 2007/0218528, 2008/0131962 and 2011/0201055, incorporated herein by reference in their entireties. The term “sequence” refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded. The term “donor sequence” refers to a nucleotide sequence that is inserted into a genome. A donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value therebetween or thereabove), preferably between about 100 and 1,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides in length. “Chromatin” is the nucleoprotein structure comprising the cellular genome. Cellular chromatin comprises nucleic acid, primarily DNA, and protein, including histones and non-histone chromosomal proteins. The majority of eukaryotic cellular chromatin exists in the form of nucleosomes, wherein a nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and linker DNA (of variable length depending on the organism) extends between nucleosome cores. A molecule of histone H1 is generally associated with the linker DNA. For the purposes of the present disclosure, the term “chromatin” is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic. Cellular chromatin includes both chromosomal and episomal chromatin. A “chromosome” is a chromatin complex comprising all or a portion of the genome of a cell. The genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome of the cell. The genome of a cell can comprise one or more chromosomes. An “episome” is a replicating nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part of the chromosomal karyotype of a cell. Examples of episomes include plasmids and certain viral genomes. A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist. An “exogenous” molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. “Normal presence in the cell” is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat-shocked cell. An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally-functioning endogenous molecule. An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases. An exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid. For example, an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer. An exogenous molecule can also be the same type of molecule as an endogenous molecule but derived from a different species than the cell is derived from. For example, a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster. By contrast, an “endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid. Additional endogenous molecules can include proteins, for example, transcription factors and enzymes. A “fusion” molecule is a molecule in which two or more subunit molecules are linked, preferably covalently. The subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules. Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a ZFP or TALE DNA-binding domain and one or more activation domains) and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein describedsupra). Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex-forming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid. Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein. Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure. A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product (see infra), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. “Gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation. “Modulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Genome editing (e.g., cleavage, alteration, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to any reduction in gene expression as compared to a cell that does not include a ZFP or TALEN as described herein. Thus, gene inactivation may be partial or complete. A “region of interest” is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and/or targeted recombination. A region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example. A region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region. A region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs. “Eukaryotic” cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells (e.g., T-cells). “Secretory tissues” are those tissues that secrete products. Examples of secretory tissues that are localized to the gastrointestinal tract include the cells that line the gut, the pancreas, and the gallbladder. Other secretory tissues include the liver, tissues associated with the eye and mucous membranes such as salivary glands, mammary glands, the prostate gland, the pituitary gland and other members of the endocrine system. Additionally, secretory tissues include individual cells of a tissue type which are capable of secretion. The terms “operative linkage” and “operatively linked” (or “operably linked”) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. By way of illustration, a transcriptional regulatory sequence, such as a promoter, is operatively linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous. With respect to fusion polypeptides, the term “operatively linked” can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked. For example, with respect to a fusion polypeptide in which a ZFP or TALE DNA-binding domain is fused to an activation domain, the ZFP or TALE DNA-binding domain and the activation domain are in operative linkage if, in the fusion polypeptide, the ZFP or TALE DNA-binding domain portion is able to bind its target site and/or its binding site, while the activation domain is able to up-regulate gene expression. When a fusion polypeptide in which a ZFP or TALE DNA-binding domain is fused to a cleavage domain, the ZFP or TALE DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the ZFP or TALE DNA-binding domain portion is able to bind its target site and/or its binding site, while the cleavage domain is able to cleave DNA in the vicinity of the target site. A “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function are well-known. For example, the DNA-binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility-shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See Ausubel et al.,supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, both genetic and biochemical. See, for example, Fields et al. (1989)Nature340:245-246; U.S. Pat. No. 5,585,245 and International Patent Publication No. WO 98/44350. A “vector” is capable of transferring gene sequences to target cells. Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors. A “reporter gene” or “reporter sequence” refers to any sequence that produces a protein product that is easily measured, preferably although not necessarily in a routine assay. Suitable reporter genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins which mediate enhanced cell growth and/or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA or any detectable amino acid sequence. “Expression tags” include sequences that encode reporters that may be operably linked to a desired gene sequence in order to monitor expression of the gene of interest. Nucleases Described herein are compositions, particularly nucleases, which are useful targeting a gene for the insertion of a transgene, for example, nucleases that are specific for albumin. In certain embodiments, the nuclease is naturally occurring. In other embodiments, the nuclease is non-naturally occurring, i.e., engineered in the DNA-binding domain and/or cleavage domain. For example, the DNA-binding domain of a naturally-occurring nuclease may be altered to bind to a selected target site (e.g., a meganuclease that has been engineered to bind to site different than the cognate binding site). In other embodiments, the nuclease comprises heterologous DNA-binding and cleavage domains (e.g., zinc finger nucleases; TAL-effector nucleases; meganuclease DNA-binding domains with heterologous cleavage domains). A. DNA-Binding Domains In certain embodiments, the nuclease is a meganuclease (homing endonuclease). Naturally-occurring meganucleases recognize 15-40 base-pair cleavage sites and are commonly grouped into four families: the LAGLIDADG (SEQ ID NO: 162) family, the GIY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Their recognition sequences are known. See also U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort et al. (1997)Nucleic Acids Res.25:3379-3388; Dujon et al. (1989)Gene82:115-118; Perler et al. (1994)Nucleic Acids Res.22, 1125-1127; Jasin (1996)Trends Genet.12:224-228; Gimble et al. (1996)J Mol. Biol.263:163-180; Argast et al. (1998)J. Mol. Biol.280:345-353 and the New England Biolabs catalogue. In certain embodiments, the nuclease comprises an engineered (non-naturally occurring) homing endonuclease (meganuclease). The recognition sequences of homing endonucleases and meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. See also U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort et al. (1997)Nucleic Acids Res.25:3379-3388; Dujon et al. (1989)Gene82:115-118; Perler et al. (1994)Nucleic Acids Res.22, 1125-1127; Jasin (1996)Trends Genet.12:224-228; Gimble et al. (1996)J. Mol. Biol.263:163-180; Argast et al. (1998)J. Mol. Biol.280:345-353 and the New England Biolabs catalogue. In addition, the DNA-binding specificity of homing endonucleases and meganucleases can be engineered to bind non-natural target sites. See, for example, Chevalier et al. (2002)Molec. Cell10:895-905; Epinat et al. (2003)Nucleic Acids Res.31:2952-2962; Ashworth et al. (2006)Nature441:656-659; Paques et al. (2007)Current Gene Therapy7:49-66; U.S. Patent Publication No. 2007/0117128. The DNA-binding domains of the homing endonucleases and meganucleases may be altered in the context of the nuclease as a whole (i.e., such that the nuclease includes the cognate cleavage domain) or may be fused to a heterologous cleavage domain. In other embodiments, the DNA-binding domain comprises a naturally occurring or engineered (non-naturally occurring) TALE DNA binding domain. See, e.g., U.S. Patent Publication No. 2011/0301073, incorporated by reference in its entirety herein. The plant pathogenic bacteria of the genusXanthomonasare known to cause many diseases in important crop plants. Pathogenicity ofXanthomonasdepends on a conserved type III secretion (T3 S) system which injects more than 25 different effector proteins into the plant cell. Among these injected proteins are transcription activator-like effectors (TALE) which mimic plant transcriptional activators and manipulate the plant transcriptome (see Kay et al. (2007)Science318:648-651). These proteins contain a DNA binding domain and a transcriptional activation domain. One of the most well characterized TALEs is AvrBs3 fromXanthomonas campestgrispv.Vesicatoria(see Bonas et al. (1989)Mol Gen Genet218: 127-136 and International Patent Publicaiton No. WO 2010/079430). TALEs contain a centralized domain of tandem repeats, each repeat containing approximately 34 amino acids, which are key to the DNA binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcriptional activation domain (for a review see Schornack S, et al. (2006)J Plant Physiol163(3):256-272). In addition, in the phytopathogenic bacteriaRalstonia solanacearumtwo genes, designated brg11 and hpx17 have been found that are homologous to the AvrBs3 family ofXanthomonasin theR. solanacearumbiovar 1 strain GMI1000 and in the biovar 4 strain RS1000 (See Heuer et al. (2007)Appl and Envir Micro73(13):4379-4384). These genes are 98.9% identical in nucleotide sequence to each other but differ by a deletion of 1,575 bp in the repeat domain of hpx17. However, both gene products have less than 40% sequence identity with AvrBs3 family proteins ofXanthomonas. Thus, in some embodiments, the DNA binding domain that binds to a target site in a target locus (e.g., albumin or safe harbor) is an engineered domain from a TALE similar to those derived from the plant pathogensXanthomonas(see Boch et al. (2009)Science326:1509-1512 and Moscou and Bogdanove (2009)Science326:1501) andRalstonia(see Heuer et al. (2007)Applied and Environmental Microbiology73(13):4379-4384); U.S. Patent Publication No. 2011/0301073 and U.S. Patent Publication No. 2011/0145940. In certain embodiments, the DNA binding domain comprises a zinc finger protein (e.g., a zinc finger protein that binds to a target site in an albumin or safe-harbor gene). Preferably, the zinc finger protein is non-naturally occurring in that it is engineered to bind to a target site of choice. See, for example, See, for example, Beerli et al. (2002)Nature Biotechnol.20:135-141; Pabo et al. (2001)Ann. Rev. Biochem.70:313-340; Isalan et al. (2001)Nature Biotechnol.19:656-660; Segal et al. (2001)Curr. Opin. Biotechnol.12:632-637; Choo et al. (2000)Curr. Opin. Struct. Biol.10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated herein by reference in their entireties. An engineered zinc finger binding or TALE domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference herein in their entireties. Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02/077227. In addition, as disclosed in these and other references, DNA-binding domains (e.g., multi-finger zinc finger proteins or TALE domains) may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The DNA binding proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02/077227. Selection of target sites; DNA-binding domains and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Pat. Nos. 6,140,815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; 6,200,759; International Patent Publication Nos. WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Patent Publication No. 2011/0301073. In addition, as disclosed in these and other references, DNA-binding domains (e.g., multi-finger zinc finger proteins) may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. B. Cleavage Domains Any suitable cleavage domain can be operatively linked to a DNA-binding domain to form a nuclease. For example, ZFP DNA-binding domains have been fused to nuclease domains to create ZFNs—a functional entity that is able to recognize its intended nucleic acid target through its engineered (ZFP) DNA binding domain and cause the DNA to be cut near the ZFP binding site via the nuclease activity. See, e.g., Kim et al. (1996)Proc Nat'l Acad Sci USA93(3):1156-1160. More recently, ZFNs have been used for genome modification in a variety of organisms. See, for example, U.S. Patent Publication Nos. 2003/0232410; 2005/0208489; 2005/0026157; 2005/0064474; 2006/0188987; 2006/0063231; and International Patent Publication No. WO 07/014275. Likewise, TALE DNA-binding domains have been fused to nuclease domains to create TALENs. See, e.g., U.S. Patent Publication No. 2011/0301073. As noted above, the cleavage domain may be heterologous to the DNA-binding domain, for example a zinc finger DNA-binding domain and a cleavage domain from a nuclease or a TALEN DNA-binding domain and a cleavage domain, or meganuclease DNA-binding domain and cleavage domain from a different nuclease. Heterologous cleavage domains can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, MA; and Belfort et al. (1997)Nucleic Acids Res.25:3379-3388. Additional enzymes which cleave DNA are known (e.g., Si Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains. Similarly, a cleavage half-domain can be derived from any nuclease or portion thereof, as set forth above, that requires dimerization for cleavage activity. In general, two fusion proteins are required for cleavage if the fusion proteins comprise cleavage half-domains. Alternatively, a single protein comprising two cleavage half-domains can be used. The two cleavage half-domains can be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragments thereof). In addition, the target sites for the two fusion proteins are preferably disposed, with respect to each other, such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation to each other that allows the cleavage half-domains to form a functional cleavage domain, e.g., by dimerizing. Thus, in certain embodiments, the near edges of the target sites are separated by 5-8 nucleotides or by 15-18 nucleotides. However any integral {why are we always using the qualifiers “integral” and “integer”—are these really necessary? They just seem restrictive and their use would seem to open us up to workarounds}. number of nucleotides or nucleotide pairs can intervene between two target sites (e.g., from 2 to 50 nucleotide pairs or more). In general, the site of cleavage lies between the target sites. Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme FokI catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992)Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993)Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a)Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b)J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is FokI. This particular enzyme is active as a dimer. Bitinaite et al. (1998)Proc. Natl. Acad. Sci. USA 95:10,570-10,575. Accordingly, for the purposes of the present disclosure, the portion of the FokI enzyme used in the disclosed fusion proteins is considered a cleavage half-domain. Thus, for targeted double-stranded cleavage and/or targeted replacement of cellular sequences using zinc finger-FokI fusions, two fusion proteins, each comprising a FokI cleavage half-domain, can be used to reconstitute a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a DNA binding domain and two FokI cleavage half-domains can also be used. A cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity, or that retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain. Exemplary Type IIS restriction enzymes are described in International Patent Publication No. WO 07/014275, incorporated herein in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains, and these are contemplated by the present disclosure. See, for example, Roberts et al. (2003)Nucleic Acids Res.31:418-420. In certain embodiments, the cleavage domain comprises one or more engineered cleavage half-domain (also referred to as dimerization domain mutants) that minimize or prevent homodimerization, as described, for example, in U.S. Patent Publication Nos. 2005/0064474; 2006/0188987; 2008/0131962 and 2011/0201055, the disclosures of all of which are incorporated by reference in their entireties herein. Amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of FokI are all targets for influencing dimerization of the FokI cleavage half-domains. Exemplary engineered cleavage half-domains of FokI that form obligate heterodimers include a pair in which a first cleavage half-domain includes mutations at amino acid residues at positions 490 and 538 of FokI and a second cleavage half-domain includes mutations at amino acid residues 486 and 499. Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys (K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at 486 replaced Gln (Q) with Glu (E); and the mutation at position 499 replaces Iso (I) with Lys (K). Specifically, the engineered cleavage half-domains described herein were prepared by mutating positions 490 (E→K) and 538 (I→K) in one cleavage half-domain to produce an engineered cleavage half-domain designated “E490K:1538K” and by mutating positions 486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce an engineered cleavage half-domain designated “Q486E:1499L”. The engineered cleavage half-domains described herein are obligate heterodimer mutants in which aberrant cleavage is minimized or abolished. See, e.g., U.S. Patent Publication No. 2008/0131962, the disclosure of which is incorporated by reference in its entirety for all purposes. In certain embodiments, the engineered cleavage half-domain comprises mutations at positions 486, 499 and 496 (numbered relative to wild-type FokI), for instance mutations that replace the wild type Gln (Q) residue at position 486 with a Glu (E) residue, the wild type Iso (I) residue at position 499 with a Leu (L) residue and the wild-type Asn (N) residue at position 496 with an Asp (D) or Glu (E) residue (also referred to as a “ELD” and “ELE” domains, respectively). In other embodiments, the engineered cleavage half-domain comprises mutations at positions 490, 538 and 537 (numbered relative to wild-type FokI), for instance mutations that replace the wild type Glu (E) residue at position 490 with a Lys (K) residue, the wild type Iso (I) residue at position 538 with a Lys (K) residue, and the wild-type His (H) residue at position 537 with a Lys (K) residue or a Arg (R) residue (also referred to as “KKK” and “KKR” domains, respectively). In other embodiments, the engineered cleavage half-domain comprises mutations at positions 490 and 537 (numbered relative to wild-type FokI), for instance mutations that replace the wild type Glu (E) residue at position 490 with a Lys (K) residue and the wild-type His (H) residue at position 537 with a Lys (K) residue or a Arg (R) residue (also referred to as “KIK” and “KIR” domains, respectively). (See U.S. Patent Publication No. 2011/0201055). Engineered cleavage half-domains described herein can be prepared using any suitable method, for example, by site-directed mutagenesis of wild-type cleavage half-domains (FokI) as described in U.S. Patent Publication Nos. 2005/0064474; 2008/0131962 and 2011/0201055. Alternatively, nucleases may be assembled in vivo at the nucleic acid target site using so-called “split-enzyme” technology (see e.g., U.S. Patent Publication No. 2009/0068164). Components of such split enzymes may be expressed either on separate expression constructs, or can be linked in one open reading frame where the individual components are separated, for example, by a self-cleaving 2A peptide or IRES sequence. Components may be individual zinc finger binding domains or domains of a meganuclease nucleic acid binding domain. Nucleases can be screened for activity prior to use, for example in a yeast-based chromosomal system as described in International Patent Publication No. WO 2009/042163 and U.S. Patent Publication No. 2009/0068164. Nuclease expression constructs can be readily designed using methods known in the art. See, e.g., U.S. Patent Publication Nos. 2003/0232410; 2005/0208489; 2005/0026157; 2005/0064474; 2006/0188987; 2006/0063231; and International Patent Publication No. WO 07/014275. Expression of the nuclease may be under the control of a constitutive promoter or an inducible promoter, for example the galactokinase promoter which is activated (de-repressed) in the presence of raffinose and/or galactose and repressed in presence of glucose. Target Sites As described in detail above, DNA domains can be engineered to bind to any sequence of choice in a locus, for example an albumin or safe-harbor gene. An engineered DNA-binding domain can have a novel binding specificity, compared to a naturally-occurring DNA-binding domain. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual (e.g., zinc finger) amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of DNA binding domain which bind the particular triplet or quadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference herein in their entireties. Rational design of TAL-effector domains can also be performed. See, e.g., U.S. Patent Publication No. 2011/0301073. Exemplary selection methods applicable to DNA-binding domains, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878 and WO 01/88197 and GB 2,338,237. Selection of target sites; nucleases and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Patent Publication Nos. 2005/0064474 and 2006/0188987, incorporated by reference in their entireties herein. In addition, as disclosed in these and other references, DNA-binding domains (e.g., multi-finger zinc finger proteins) may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids. See, e.g., U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual DNA-binding domains of the protein. See, also, U.S. Patent Publication No. 2011/0301073. As noted above, insertion of an exogenous sequence (also called a “donor sequence” or “donor”), for example for correction of a mutant gene or for increased expression of a wild-type gene. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence where it is placed. A donor sequence can contain a non-homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest. Additionally, donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin. A donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest. The donor polynucleotide can be DNA or RNA, single-stranded or double-stranded and can be introduced into a cell in linear or circular form. See, e.g., U.S. Patent Publication Nos. 2010/0047805 and 2011/0207221. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987)Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al. (1996)Science272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. A polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)). The donor is generally inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the albumin gene. However, it will be apparent that the donor may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific promoter. The donor molecule may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed. For example, a transgene as described herein may be inserted into an albumin locus such that some or none of the endogenous albumin sequences are expressed, for example as a fusion with the transgene. In other embodiments, the transgene (e.g., with or without albumin encoding sequences) is integrated into any endogenous locus, for example a safe-harbor locus. See, e.g., U.S. Patent Publication Nos. 2008/0299580; 2008/0159996 and 2010/00218264. When albumin sequences (endogenous or part of the transgene) are expressed with the transgene, the albumin sequences may be full-length sequences (wild-type or mutant) or partial sequences. Preferably the albumin sequences are functional. Non-limiting examples of the function of these full length or partial albumin sequences include increasing the serum half-life of the polypeptide expressed by the transgene (e.g., therapeutic gene) and/or acting as a carrier. Furthermore, although not required for expression, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals. Delivery The nucleases, polynucleotides encoding these nucleases, donor polynucleotides and compositions comprising the proteins and/or polynucleotides described herein may be delivered in vivo or ex vivo by any suitable means. Methods of delivering nucleases as described herein are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Nucleases and/or donor constructs as described herein may also be delivered using vectors containing sequences encoding one or more of the zinc finger or TALEN protein(s). Any vector systems may be used including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, incorporated by reference herein in their entireties. Furthermore, it will be apparent that any of these vectors may comprise one or more of the sequences needed for treatment. Thus, when one or more nucleases and a donor construct are introduced into the cell, the nucleases and/or donor polynucleotide may be carried on the same vector or on different vectors. When multiple vectors are used, each vector may comprise a sequence encoding one or multiple nucleases and/or donor constructs. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding nucleases and donor constructs in cells (e.g., mammalian cells) and target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson,Science256:808-813 (1992); Nabel & Felgner,TIBTECH11:211-217 (1993); Mitani & Caskey,TIBTECH11:162-166 (1993); Dillon,TIBTECH11:167-175 (1993); Miller,Nature357:455-460 (1992); Van Brunt,Biotechnology6(10):1149-1154 (1988); Vigne,Restorative Neurology and Neuroscience8:35-36 (1995); Kremer & Perricaudet,British Medical Bulletin51(1):31-44 (1995); Haddada et al., inCurrent Topics in Microbiology and ImmunologyDoerfler and Böhm (eds.) (1995); and Yu et al.,Gene Therapy1:13-26 (1994). Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), BTX Molecular Delivery Systems (Holliston, MA) and Copernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336). Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, International Patent Publication Nos. WO 91/17424, WO 91/16024. The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal,Science270:404-410 (1995); Blaese et al.,Cancer Gene Ther.2:291-297 (1995); Behr et al.,Bioconjugate Chem.5:382-389 (1994); Remy et al.,Bioconjugate Chem.5:647-654 (1994); Gao et al.,Gene Therapy2:710-722 (1995); Ahmad et al.,Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787). Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis. Once in the cell, the contents are released (see MacDiarmid et al. (2009)Nature Biotechnology27(7):643). The use of RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered ZFPs take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of ZFPs include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al.,J. Virol.66:2731-2739 (1992); Johann et al.,J. Virol.66:1635-1640 (1992); Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al.,J. Virol.63:2374-2378 (1989); Miller et al.,J. Virol.65:2220-2224 (1991); International Patent Publication No. WO 94/26877). In applications in which transient expression is preferred, adenoviral based systems can be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al.,Virology160:38-47 (1987); U.S. Pat. No. 4,797,368; International Patent Publication No. WO 93/24641; Kotin,Human Gene Therapy5:793-801 (1994); Muzyczka,J. Clin. Invest.94:1351 (1994). Construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al.,Mol. Cell. Biol.5:3251-3260 (1985); Tratschin, et al.,Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al.,J. Virol.63:03822-3828 (1989). At least six viral vector approaches are currently available for gene transfer in clinical trials, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent. pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn et al.,Nat. Med.1:1017-102 (1995); Malech et al.,PNAS94:22 12133-12138 (1997)). PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al.,Science270:475-480 (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al.,Immunol Immunother44(1):10-20 (1997); Dranoff et al.,Hum. Gene Ther.1:111-2 (1997). Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al.,Gene Ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9, AAV rh10 and pseudotyped AAV such as AAV2/8, AAV2/5 and AAV2/6 can also be used in accordance with the present invention. Replication-deficient recombinant adenoviral vectors (Ad) can be produced at high titer and readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for anti-tumor immunization with intramuscular injection (Sterman et al.,Hum. Gene Ther.7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al.,Infection24:1 5-10 (1996); Sterman et al.,Hum. Gene Ther.9:7 1083-1089 (1998); Welsh et al.,Hum. Gene Ther.2:205-18 (1995); Alvarez et al.,Hum. Gene Ther.5:597-613 (1997); Topf et al.,Gene Ther.5:507-513 (1998); Sterman et al.,Hum. Gene Ther.7:1083-1089 (1998). Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and w2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. Accordingly, a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al.,Proc. Natl. Acad. Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell-surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences which favor uptake by specific target cells. Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector. Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing nucleases and/or donor constructs can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. Vectors suitable for introduction of polynucleotides described herein include non-integrating lentivirus vectors (IDLV). See, for example, Ory et al. (1996)Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al. (1998)J. Virol.72:8463-8471; Zuffery et al. (1998)J. Virol.72:9873-9880; Follenzi et al. (2000)Nature Genetics25:217-222; U.S. Patent Publication No. 2009/054985. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989). It will be apparent that the nuclease-encoding sequences and donor constructs can be delivered using the same or different systems. For example, a donor polynucleotide can be carried by a plasmid, while the one or more nucleases can be carried by a AAV vector. Furthermore, the different vectors can be administered by the same or different routes (intramuscular injection, tail vein injection, other intravenous injection, intraperitoneal administration and/or intramuscular injection. The vectors can be delivered simultaneously or in any sequential order. Formulations for both ex vivo and in vivo administrations include suspensions in liquid or emulsified liquids. The active ingredients often are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, stabilizing agents or other reagents that enhance the effectiveness of the pharmaceutical composition. Applications The methods and compositions of the invention can be used in any circumstance wherein it is desired to supply a transgene encoding one or more proteins such that the protein(s) is(are) secreted from the targeted cell. Thus, this technology is of use in a condition where a patient is deficient in some protein due to problems (e.g., problems in expression level or problems with the protein expressed as sub- or non-functioning). Particularly useful with this invention is the expression of transgenes to correct or restore functionality in clotting disorders. Additionally, A1AT-deficiency disorders such as COPD or liver damage, or other disorders, conditions or diseases that can be mitigated by the supply of exogenous proteins by a secretory organ may be successfully treated by the methods and compositions of this invention. Lysosomal storage diseases can be treated by the methods and compositions of the invention, as are metabolic diseases such as diabetes. Proteins that are useful therapeutically and that are typically delivered by injection or infusion are also useful with the methods and compositions of the invention. By way of non-limiting examples, production of a C-peptide (e.g., Ersatta™ by Cebix) or insulin for use in diabetic therapy. A further application includes treatment of Epidermolysis Bullosa via production of collagen VII. Expression of IGF-1 in secretory tissue as described herein can be used to increase levels of this protein in patients with liver cirrhosis and lipoprotein lipase deficiency by expression of lipoprotein lipase. Antibodies may also be secreted for therapeutic benefit, for example, for the treatment of cancers, autoimmune and other diseases. Other proteins related to clotting could be produced in secretory tissue, include fibrinogen, prothrombin, tissue factor, Factor V, Factor XI, Factor XII (Hageman factor), Factor XIII (fibrin-stabilizing factor), von Willebrand factor, prekallikrein, high molecular weight kininogen (Fitzgerald factor), fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen, alpha 2-antiplasmin, tissue plasminogen activator, urokinase, plasminogen activator inhibitor-1, and plasminogen activator inhibitor-2. The following Examples relate to exemplary embodiments of the present disclosure in which the nuclease comprises a zinc finger nuclease (ZFN) or TALEN. It will be appreciated that this is for purposes of exemplification only and that other nucleases can be used, for instance homing endonucleases (meganucleases) with engineered DNA-binding domains and/or fusions of naturally occurring of engineered homing endonucleases (meganucleases) DNA-binding domains and heterologous cleavage domains. EXAMPLES Example 1: Design, Construction and Characterization of Zinc Finger Protein Nucleases (ZFN) Targeted to the Mouse Albumin Gene Zinc finger proteins were designed to target cleavage sites within introns 1, 12 and 13 of the mouse albumin gene. Corresponding expression constructs were assembled and incorporated into plasmids, AAV or adenoviral vectors essentially as described in Urnov et al. (2005)Nature435(7042):646-651, Perez et al. (2008)Nature Biotechnology26(7):808-816, and as described in U.S. Pat. No. 6,534,261. Table 1 shows the recognition helices within the DNA binding domain of exemplary mouse albumin specific ZFPs while Table 2 shows the target sites for these ZFPs. Nucleotides in the target site that are contacted by the ZFP recognition helices are indicated in uppercase letters; non-contacted nucleotides indicated in lowercase. TABLE 1Murine albumin-specific zinc finger nucleases helix designsTargetDesignSBS #F1F2F3F4F5F6IntronTSGSLTRRSDALSTQSATRTKTSGHLSRQSGNLARNA1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30724NO: 1)NO: 2)NO: 3)NO: 4)NO: 5)IntronRSDHLSATKSNRTKDRSNLSRWRSSLRADSSDRKKNA1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30725NO: 6)NO: 7)NO: 8)NO: 9)NO: 10)IntronTSGNLTRDRSTRRQTSGSLTRERGTLARTSANLSRNA1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30732NO: 11)NO: 12)NO: 1)NO: 13)NO: 14)IntronDRSALARRSDHLSEHRSDRTRQSGALARQSGHLSRNS1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30733NO: 15)NO: 16)NO: 17)NO: 18)NO: 19)IntronRSDNLSTDRSALARDRSNLSRDGRNLRHRSDNLARQSNALNR13(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30759NO: 20)NO: 15)NO: 8)NO: 21)NO: 22)NO: 23)IntronDRSNLSRLKQVLVRQSGNLARQSTPLFAQSGALARNA13(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30761NO: 8)NO: 24)NO: 5)NO: 25)NO: 18)IntronDRSNLSRDGRNLRHRSDNLARQSNALNRNANA13(SEQ ID(SEQ ID(SEQ ID(SEQ ID30760NO: 8)NO: 21)NO: 22)NO: 23)IntronRSDNLSVHSNARKTRSDSLSAQSGNLARRSDSLSVQSGHLSR13(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30767NO: 26)NO: 27)NO: 28)NO: 5)NO: 29)NO: 19)IntronRSDNLSEERANRNSQSANRTKERGTLARRSDALTQNA13(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30768NO: 30)NO: 31)NO: 32)NO: 13)NO: 33)IntronTSGSLTRDRSNLSRDGRNLRHERGTLARRSDALTQNA13(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30769NO: 1)NO: 8)NO: 21)NO: 13)NO: 33)IntronQSGHLARRSDHLTQRSDHLSQWRSSLVARSDVLSERNQHRKT12(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30872NO: 34)NO: 35)NO: 36)NO: 37)NO: 38)NO: 39)IntronQSGDLTRRSDALARQSGDLTRRRDPLINRSDNLSVIRSTLRD12(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30873NO: 40)NO: 41)NO: 40)NO: 42)NO: 26)NO: 43)IntronRSDNLSVYSSTRNSRSDHLSASYWSRTVQSSDLSRRTDALRG12(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30876NO: 26)NO: 44)NO: 6)NO: 45)NO: 46)NO: 47)IntronRSDNLSTQKSPLNTTSGNLTRQAENLKSQSSDLSRRTDALRG12(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30877NO: 20)NO: 48)NO: 11)NO: 49)NO: 46)NO: 47)IntronRSDNLSVRRAHLNQTSGNLTRSDTNRFKRSDNLSTQSGHLSR12(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30882NO: 26)NO: 50)NO: 11)NO: 51)NO: 20)NO: 19)IntronDSSDRKKDRSALSRTSSNRKTQSGALARRSDHLSRNA12(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID30883NO: 10)NO: 52)NO: 53)NO: 18)NO: 54) TABLE 2Target sites of murine albumin-specific ZFNsTargetSBS #Target siteIntron 130724ctGAAGGTgGCAATGGTTcctctctgct_ (SEQ ID NO: 55)Intron 130725ttTCCTGTAACGATCGGgaactggcatc_ (SEQ ID NO: 56)Intron 130732aaGATGCCaGTTCCCGATcgttacagga_ (SEQ ID NO: 57)Intron 130733agGGAGTAGCTTAGGTCagtgaagagaa_ (SEQ ID NO: 58)Intron 1330759acGTAGAGAACAACATCTAGattggtgg_ (SEQ ID NO: 59)Intron 1330761ctGTAATAGAAACTGACttacgtagctt_ (SEQ ID NO: 60)Intron 1330760acGTAGAGAACAACatctagattggtgg_ (SEQ ID NO: 59)Intron 1330767agGGAATGtGAAATGATTCAGatatata_ (SEQ ID NO: 61)Intron 1330768ccATGGCCTAACAACAGtttatcttctt_ (SEQ ID NO: 62)Intron 1330769ccATGGCCtAACAACaGTTtatcttctt_ (SEQ ID NO: 62)Intron 1230872ctTGGCTGTGTAGGAGGGGAgtagcagt_ (SEQ ID NO: 63)Intron 1230873ttCCTAAGTTGGCAGTGGCAtgcttaat_ (SEQ ID NO: 64)Intron 1230876ctTTGGCTTTGAGGATTAAGcatgccac_ (SEQ ID NO: 65)Intron 1230877acTTGGCTcCAAGATTTATAGccttaaa_ (SEQ ID NO: 66)Intron 1230882caGGAAAGTAAGATAGGAAGgaatgtga_ (SEQ ID NO: 67)Intron 1230883ctGGGGTAAATGTCTCCttgctcttctt_ (SEQ ID NO: 68) Example 2: Activity of Murine Albumin-Specific ZFNs The ZFNs in Table 1 were tested for the ability to cleave their endogenous target sequences in mouse cells. To accomplish this, constructs expressing the ZFNs in Table 1 were transfected into Neuro2A cells in the pairings indicated inFIG.1. Cells were then maintained at 37° C. for 3 days or subjected to a hypothermic shock (30° C., see co-owned U.S. Patent Publication No. 2011/0041195). Genomic DNA was then isolated from Neuro2A cells using the DNeasy kit (Qiagen) and subjected to the Cel-I assay (Surveyor™, Transgenomics) as described in Perez et al. (2008)Nat. Biotechnol.26: 808-816 and Guschin et al., (2010)Methods Mol Biol.649:247-56), in order to quantify chromosomal modifications induced by ZFN-cleavage. In this assay, PCR is used to amplify a DNA fragment bearing the ZFN target site, and then the resultant amplicon is digested with the mismatch-specific nuclease Cel-I (Yang et al. (2000)Biochemistry39:3533-3541), followed by resolution of intact and cleaved amplicon on an agarose gel. By quantifying the degree of amplicon cleavage, one may calculate the fraction of mutated alleles in amplicon and therefore in the original cellular pool. In these experiments, all ZFN pairs were ELD/KKR FokI mutation pairs (described above). Results from the Cel-I assay are shown inFIG.1, and demonstrate that the ZFNs are capable of inducing cleavage and consequent mutations at their respective target sites. The “percent indel” value shown beneath each lane indicates the fraction of ZFN targets that were successfully cleaved and subsequently mutated during cellular repair of the double stranded break via NHEJ. The data also demonstrate increased activity when the transduction procedure incorporates the hypothermic shock. Example 3: Canine Albumin-Specific ZFNs A pair of ZFNs targeting the canine albumin locus was constructed for use in in vivo models. The pair was constructed as described in Example 1, and is shown below in Table 3. The target for each ZFN is provided in Table 4. TABLE 3Canine albumin-specific zinc finger nucleases helix designsTargetSBS #F1F2F3F4FSIntron33115QRSNLDSQSSDLSRYHWYLKKRSDDLSVTSSNRTK1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ IDNO: 83)NO: 46)NO: 84)NO: 85)NO: 86)Intron34077QSGNLARQYTHLVARSDHLSTRSDARTTDRSALAR1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ IDNO: 5)NO: 87)NO: 88)NO: 89)NO: 15) TABLE 4Target sites of canine albumin-specific ZFNsTargetSBS #Target siteIntron 133115agTATTCGTTTGCTcCAAaatatttgcc(SEQ ID NO: 90)Intron 134077aaGTCATGTGGAGAGAAacacaaagagt(SEQ ID NO: 91) The canine specific ZFNs were tested in vitro for activity essentially as described in example 2, except that the canine cell line D17 was used. As shown inFIG.2, the ZFNs were shown to generate ˜30% indels in this study. Example 4: Non-Human Primate Albumin Specific ZFNs ZFNs targeting the albumin locus inrhesus macaquemonkeys (Macaca mulatta) were also made. The pairs were constructed as described above and are shown below in Table 5. The targets for the ZFNs are shown in Table 6. As shown below, the human (SEQ ID NO:92) andrhesus macaque(SEQ ID NO:93) sequences for the binding site for SBS #35396 (see below, Table 7 and 8) are perfectly conserved. The differences between the human andrhesussequences are boxed. HUMAN LEADS HUMAN RHESUSGNOTE:G IN SOME INSTANCES Thus, for the development of therhesusalbumin specific pair, 35396 was paired with a series of partners which were designed to replace the human 35364 partner inrhesus. These proteins are shown below (Table 5) along with their target sequences (Table 6). TABLE 5Rhesus albumin-specific zinc finger nucleases helix designsRhesusTargetSBS #F1F2F3F4F5Intron36813QSGNLARHLGNLKTLKHHLTDDRSNLSRRLDNRTA1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ IDNO: 5)NO: 94)NO: 95)NO: 8)NO: 96)Intron36808QSGNLARLMQNRNQLKHHLTDDRSNLSRRSDHLTT1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ IDNO: 5)NO: 97)NO: 95)NO: 8)NO: 98)Intron36820QRSNLVRLRMNLTKLKHHLTDDRSNLSRRSDHLTT1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ IDNO: 99)NO: 100)NO: 95)NO: 8)NO: 98)Intron36819QRSNLVRLRMNLTKLKHHLTDDRSNLSRRSDHLTQ1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ IDNO: 99)NO: 100)NO: 95)NO: 8)NO: 35)Intron36806QSGNLARLMQNRNQLKHHLTDDRSNLSRRSDHLTQ1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ IDNO: 5)NO: 97)NO: 95)NO: 8)NO: 35) TABLE 6Target sites of rhesus albumin-specific ZFNsTargetSBS #Target siteIntron 136813ttAGGGACAGTTATGAAttcaatcttca_ (SEQ ID NO: 101)Intron 136808ttAGGGACAGTTATGAAttcaatcttca_ (SEQ ID NO: 101)Intron 136820ttAGGGACAGTTATGAAttcaatcttca_ (SEQ ID NO: 101)Intron 136819ttAGGGACAGTTATGAAttcaatcttca_ (SEQ ID NO: 101)Intron 136806ttAGGGACAGTTATGAAttcaatcttca_ (SEQ ID NO: 101) Therhesusalbumin specific ZFNs were tested in pairs to determine the pair with the greatest activity. In each pair, SBS #35396 was tested with the potential partners shown in Tables 5 and 6 in therhesuscell line RF/6A using the methods described above. The resultant activity, as determined by percent of mismatch detected using the Cel-I assay is shown in the body of the matrix (Table 7), and demonstrate that the ZFNs pairs have activity against therhesusalbumin locus. TABLE 7Activity at the rhesus macaque albumin locus36813368083682036819368063539621%26%23%30%20.5% Two pairs were examined more extensively, comparing sequence specificity by SELEX analysis and by a titration of each pair for activity in vitro. The results demonstrate that the 35396/36806 pair was the most desirable lead pair (seeFIG.12). Comparison of the sequence of the human albumin locus with the sequences of other non-human primates demonstrates that similar pairs may be developed for work in other primates such as cynologous monkeys (see,FIGS.3A and3B). Example 5: In Vivo Cleavage by ZFNs in Mice To deliver the albumin-specific ZFNs to the liver in vivo, the normal site of albumin production, we generated a hepatotropic adeno-associated virus vector, serotype 8 expressing the albumin-specific ZFNs from a liver-specific enhancer and promoter (Shen et al.,ibidand Miao et al.,ibid). Adult C57BL/6 mice were subjected to genome editing at the albumin gene as follows: adult mice were treated by i.v. (intravenous) injection with 1×1011v.g. (viral genomes)/mouse of either ZFN pair 1 (SBS 30724 and SBS 30725), or ZFN pair 2 (SBS 30872 and SBS 30873) and sacrificed seven days later. The region of the albumin gene encompassing the target site for pair 1 was amplified by PCR for the Cel-I mismatch assay using the following 2 PCR primers: Cel1 F1:(SEQ ID NO: 69)5′ CCTGCTCGACCATGCTATACT 3′Cel1 R1:(SEQ ID NO: 70)5′ CAGGCCTTTGAAATGTTGTTC 3′ The region of the albumin gene encompassing the target site for pair 2 was amplified by PCR for the Cel-I assay using these PCR primers: mAlb set4F4:(SEQ ID NO: 71)5′ AAGTGCAAAGCCTTTCAGGA 3′mAlb set4R4:(SEQ ID NO: 72)5′ GTGTCCTTGTCAGCAGCCTT 3′ As shown inFIG.4, the ZFNs induce indels in up to 17% of their target sites in vivo in this study. The mouse albumin specific ZFNs SBS30724 and SBS30725 which target a sequence in intron 1 were also tested in a second study. Genes for expressing the ZFNs were introduced into an AAV2/8 vector as described previously (Li et al. (2011)Nature475 (7355): 217). To facilitate AAV production in the baculovirus system, a baculovirus containing a chimeric serotype 8.2 capsid gene was used. Serotype 8.2 capsid differs from serotype 8 capsid in that the phopholipase A2 domain in capsid protein VP1 of AAV8 has been replaced by the comparable domain from the AAV2 capsid creating a chimeric capsid. Production of the ZFN containing virus particles was done either by preparation using a HEK293 system or a baculovirus system using standard methods in the art (See Li et al.,ibid, see e.g., U.S. Pat. No. 6,723,551). The virus particles were then administered to normal male mice (n=6) using a single dose of 200 microliter of 1.0el 1 total vector genomes of either AAV2/8 or AAV2/8.2 encoding the mouse albumin-specific ZFN. 14 days post administration of rAAV vectors, mice were sacrificed, livers harvested and processed for DNA or total proteins using standard methods known in the art. Detection of AAV vector genome copies was performed by quantitative PCR. Briefly, qPCR primers were made specific to the bGHpA sequences within the AAV as follows: Oligo200 (Forward)(SEQ ID NO: 102)5′-GTTGCCAGCCATCTGTTGTTT-3′Oligo201 (Reverse)(SEQ ID NO: 103)5′-GACAGTGGGAGTGGCACCTT-3′Oligo202 (Probe)(SEQ ID NO: 104)5′-CTCCCCCGTGCCTTCCTTGACC-3′ Cleavage activity of the ZFN was measured using a Cel-I assay performed using a LC-GX apparatus (Perkin Elmer), according to manufacturer's protocol. Expression of the ZFNs in vivo was measured using a FLAG-Tag system according to standard methods. As shown inFIG.5(for each mouse in the study) the ZFNs were expressed, and cleave the target in the mouse liver gene. The % indels generated in each mouse sample is provided at the bottom of each lane. The type of vector and their contents are shown above the lanes. Mismatch repair following ZFN cleavage (indicated % indels) was detected at nearly 16% in some of the mice. The mouse specific albumin ZFNs were also tested for in vivo activity when delivered via use of a variety of AAV serotypes including AAV2/5, AAV2/6, AAV2/8 and AAV2/8.2. In these AAV vectors, all the ZFN encoding sequence is flanked by the AAV2 ITRs, contain, and then encapsulated using capsid proteins from AAV5, 6, or 8, respectively. The 8.2 designation is the same as described above. The SBS30724 and SBS30725 ZFNs were cloned into the AAV as described previously (Li et al.,ibid), and the viral particles were produced either using baculovirus or a HEK293 transient transfection purification as described above. Dosing was done in normal mice in a volume of 200 μL per mouse via tail injection, at doses from 5e10 to 1e12 vg per dose. Viral genomes per diploid mouse genome were analyzed at days 14, and are analyzed at days 30 and 60. In addition, ZFN directed cleavage of the albumin locus was analyzed by Cel-I assay as described previously at day 14 and is analyzed at days 30 and 60. As shown inFIG.6, cleavage was observed at a level of up to 21% indels. Also included in Figure are the samples from the previous study as a comparison (far right, “mini-mouse” study-D14 and a background band (“unspecific band”). Example 6: In Vivo Co-Delivery of a Donor Nucleic Acid and Albumin ZFNs Insertion of human Factor IX: ZFNs were used to target integration of the gene for the clotting protein Factor IX (F.IX) into the albumin locus in adult wild-type mice. In these experiments, the mice were treated by I.V. injection with either 1×1011v.g./mouse albumin-specific ZFN pair 1 targeting intron 1+donor (“mAlb (intron1)”), 1×1011v.g./mouse albumin-specific ZFN pair 2 targeting intron 12+donor (“mAlb(intron12)”) or a ZFN set that targets a human gene plus donor as a control (“Control”). The ZFN pair #1 was 30724/30725, targeting intron 1, and ZFN pair 2 was 30872/30873, targeting exon 12. In these experiments, the F.IX donor transgene was integrated via end capture following ZFN-induced cleavage. Alternatively, the F.IX transgene was inserted into a donor vector such that the transgene was flanked by arms with homology to the site of cleavage. In either case, the F.IX transgene was the “SA— wild-type hF9 exons 2-8” cassette (see co-owned U.S. Provisional Patent Application No. 61/392,333). Transduced mice were then sampled for serum human F.IX levels, which were elevated (seeFIG.7, showing stabilized expression of human F.IX for at least eight weeks following insertion into intron 1). The expressed human F.IX is also functional, as evidenced by the reduction in clotting time in hemophilic mice with a human F.IX transgene targeted into the albumin locus (seeFIG.8). Notably, within two weeks following transgene insertion, the clotting time is not significantly different than clotting time in a wild type mouse. When the intron 1 specific donor was inserted into the intron 12 locus, correct splicing to result in expression of the huF.IX cannot occur. The lack of signal in this sample verifies that the signal from the intron 1 donor being integrated into the intron 1 site is truly from correct transgene integration, and not from random integration and expression at another non-specific site. Insertion of human alpha galactosidase (huGLa): Similar to the insertion of the human F.IX gene, the gene encoding human alpha galatosidase (deficient in patients with Fabry's disease) was inserted into the mouse albumin locus. The ZFN pair 30724/30725 was used as described above using an alpha galactosidase transgene in place of the F.IX transgene. In this experiment, 3 mice were treated with an AAV2/8 virus containing the ZFN pair at a dose of 3.0e11 viral genomes per mouse and an AAV2/8 virus containing the huGLa donor at 1.5e12 viral genomes per mouse. Control animals were given either the ZFN containing virus alone or the huGLa donor virus alone. Western blots done on liver homogenates showed an increase in alpha galactosidase-specific signal, indicating that the alpha galactosidase gene had been integrated and was being expressed (FIG.13A). In addition, an ELISA was performed on the liver lysate using a human alpha galactosidase assay kit (Sino) according to manufacturer's protocol. The results, shown inFIG.13B, demonstrated an increase in signal in the mice that had been treated with both the ZFNs and the huGLa donor. Example 7: Design of Human Albumin Specific ZFNs To design ZFNs with specificity for the human albumin gene, the DNA sequence of human albumin intron 1 was analyzed using previously described methods to identify target sequences with the best potential for ZFN binding. Regions throughout the intron (loci 1-5) were chosen and several ZFNs were designed to target these regions region (for example, seeFIG.9which shows the binding sites of ZFNs from loci 1-3). In this analysis, five loci were identified to target in the albumin intron1 (seeFIG.3B). The target and helices are shown in Tables 8 and 9. TABLE 8Human albumin-specific zinc finger nucleases helix designsTargetDesignSBS #F1F2F3F4F5F6IntronQSSDLSRLRHNLRADQSNLRARPYTLRLQSSDLSRHRSNLNK1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35393NO: 46)NO: 105)NO: 106)NO: 107)NO: 46)NO: 108)IntronQSSDLSRHRSNLNKDQSNLRARPYTLRLQSSDLSRHRSNLNK1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35394NO: 46)NO: 108)NO: 106)NO: 107)NO: 46)NO: 108)IntronQSSDLSRLKWNLRTDQSNLRARPYTLRLQSSDLSRHRSNLNK1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35396NO: 46)NO: 109)NO: 106)NO: 107)NO: 46)NO: 108)IntronQSSDLSRLRHNLRADQSNLRARPYTLRLQSSDLSRHRSNLNK1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35398NO: 46)NO: 105)NO: 106)NO: 107)NO: 46)NO: 108)IntronQSSDLSRHRSNLNKDQSNLRARPYTLRLQSSDLSRHRSNLNK1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35399NO: 46)NO: 108)NO: 106)NO: 107)NO: 46)NO: 108)IntronQSSDLSRWKWNLRADQSNLRARPYTLRLQSSDLSRHRSNLNK1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35405NO: 46)NO: 110)NO: 106)NO: 107)NO: 46)NO: 108)IntronQSGNLARLMQNRNQLKQHLNETSGNLTRRRYYLRLN/A1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35361NO: 5)NO: 97NO: 111)NO: 11)NO: 112)IntronQSGNLARHLGNLKTLKQHLNETSGNLTRRRDWRRDN/A1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35364NO: 5)NO: 94)NO: 111)NO: 11)NO: 113)IntronQSGNLARLMQNRNQLKQHLNETSGNLTRRRDWRRDNIA1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35370NO: 5)NO: 97)NO: 111)NO: 11)NO: 113)IntronQRSNLVRTSSNRKTLKHHLTDTSGNLTRRRDWRRDN/A1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35379NO: 99)NO: 53)NO: 95)NO: 11)NO: 113)IntronDKSYLRPTSGNLTRHRSARKRQSSDLSRWRSSLKTN/A1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35458NO: 114)NO: 11)NO: 115)NO: 46)NO: 116)IntronTSGNLTRHRSARKRQSGDLTRNRHHLKSN/AN/A1(SEQ ID(SEQ ID(SEQ ID(SEQ ID35480NO: 11)NO: 115)NO: 40)NO: 163)IntronQSGDLTRQSGNLHVQSAHRKNSTAALSYTSGSLSRRSDALAR1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35426NO: 40)NO: 117)NO: 118)NO: 119)NO: 120)NO: 41)IntronQSGDLTRQRSNLNIQSAHRKNSTAALSYDRSALSRRSDALAR1(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID(SEQ ID35428NO: 40)NO: 121)NO: 118)NO: 119)NO: 52)NO: 41) TABLE 9Target sites of Human albumin-specific ZFNsTargetSBS #Target siteIntron 135393ccTATCCATTGCACTATGCTttatttaa(SEQ ID NO: 127)(locus 2)Intron 135394ccTATCCATTGCACTATGCTttatttaa(SEQ ID NO: 127)(locus 2)Intron 135396ccTATCCATTGCACTATGCTttatttaa(SEQ ID NO: 127)(locus 2)Intron 135398ccTATCCATTGCACTATGCTttatttaa(SEQ ID NO: 127)(locus 2)Intron 135399ccTATCCATTGCACTATGCTttatttaa(SEQ ID NO: 127)(locus 2)Intron 135405ccTATCCATTGCACTATGCTttatttaa(SEQ ID NO: 127)(locus 2)Intron 135361ttTGGGATAGTTATGAAttcaatcttca(SEQ ID NO: 128)(locus 2)Intron 135364ttTGGGATAGTTATGAAttcaatcttca(SEQ ID NO: 128)(locus 2)Intron 135370ttTGGGATAGTTATGAAttcaatcttca(SEQ ID NO: 128)(locus 2)Intron 135379ttTGGGATAGTTATGAAttcaatcttca(SEQ ID NO: 128)(locus 2)Intron 135458ccTGTGCTGTTGATCTCataaatagaac(SEQ ID NO: 129)(locus 3)Intron 135480ccTGTGCTGTTGATctcataaatagaac(SEQ ID NO: 129)(locus 3)Intron 135426ttGTGGTTTTTAAAtAAAGCAtagtgca(SEQ ID NO: 130)(locus 3)Intron 135428ttGTGGTTTTTAAAtAAAGCAtagtgca(SEQ ID NO: 130)(locus 3)Intron 134931acCAAGAAGACAGActaaaatgaaaata (SEQ ID NO: 131)(locus 4)Intron 133940ctGTTGATAGACACTAAAAGagtattag (SEQ ID NO: 132)(locus 4) These nucleases were tested in pairs to determine the pair with the highest activity. The resultant matrices of tested pairs are shown in Tables 10 and 11, below where the ZFN used for the right side of the dimer is shown across the top of each matrix, and the ZFN used for the left side of the dimer is listed on the left side of each matrix. The resultant activity, as determined by percent of mismatch detected using the Cel-I assay is shown in the body of both matrices: TABLE 10Activity of Human albumin-specific ZFNs (% mutated targets)353933539435396353983539935405ave.353611819252223212135364n.d.24231921212235370211922n.d.222321353792121n.d.19192120 TABLE 11Activity of Human albumin-specific ZFNs (% mutated targets))3545835480ave.354264.573354284.963.6(note: ‘n.d.’ means the assay on this pair was not done) Thus, highly active nucleases have been developed that recognize target sequences in intron 1 of human albumin. Example 8: Design of Albumin Specific TALENs TALENs were designed to target sequences within human albumin intron 1. Base recognition was achieved using the canonical RVD-base correspondences (the “TALE code”: NI for A, HD for C, NN for G (NK in half repeat), NG for T). TALENs were constructed as previously described (see co-owned U.S. Patent Publication No. 2011/0301073). Targets for a subset of TALENs were conserved in cynomolgus monkey andrhesus macaquealbumin genes (seeFIG.10). The TALENs were constructed in the “+17” and “+63” TALEN backbones as described in U.S. Patent Publication No. 2011/0301073. The targets and numeric identifiers for the TALENs tested are shown below in Table 12. TABLE 12Albumin specific TALENs# ofSEQ IDSBS #siteRVDsNO:102249gtTGAAGATTGAATTCAta15133102250gtTGAAGATTGAATTCATAac17164102251gtGCAATGGATAGGTCTtt15134102252atAGTGCAATGGATAGGtc15135102253atTGAATTCATAACTATcc15136102254atTGAATTCATAACTATCCca17137102255atAAAGCATAGTGCAATGGat17138102256atAAAGCATAGTGCAATgg15139102257ctATGCTTTATTTAAAAac15140102258ctATGCTTTATTTAAAAACca17141102259atTTATGAGATCAACAGCAca17142102260ctATTTATGAGATCAACAGca17158102261ttCATTTTAGTCTGTCTTCtt17143102262atTTTAGTCTGTCTTCTtg15144102263ctAATACTCTTTTAGTGTct16145102264atCTAATACTCTTTTAGTGtc17146102265atAATTGAACATCATCCtg15147102266atAATTGAACATCATCCTGag17148102267atATTGGGCTCTGATTCCTac17149102268atATTGGGCTCTGATTCct15150102269ttTTTCTGTAGGAATCAga15159102270ttTTTCTGTAGGAATCAGag16151102271ttATGCATTTGTTTCAAaa15152102272atTATGCATTTGTTTCAaa15153 The TALENs were then tested in pairs in HepG2 cells for the ability to induce modifications at their endogenous chromosomal targets, and the results showed that many proteins bearing the +17 truncation point were active. Similarly, many TALENs bearing the +63 truncation point were also active (see Table 13 andFIG.11). Note that the pair numbers shown in Table 13 correspond with the pair numbers shown above the lanes inFIG.11. Side by side comparisons with three sets of non-optimized albumin ZFNs showed that the TALENs and ZFNs have activities that are in the same approximate range. TABLE 13TALEN-induced target modification in HepG2-C3a cellsSam-%%plemodification,modification,pairTALEN C17C17TALEN C63C63Gap1102251:10224915102251:1022490122102251:1022500102251:1022500103102252:1022490102252:1022498.3154102252:10225032102252:1022508.0135102255:10225338102255:10225321136102255:10225443102255:1022540117102256:1022530102256:10225323158102256:10225428102256:10225416139102259:10225718102259:102257151310102259:10225815102259:10225801111102260:10225715102260:102257131512102260:10225824102260:102258111313102263:1022610102263:102261161714102263:1022620102263:102262151615102264:1022610102264:102261221816102264:1022620102264:102262171720102267:10226547102267:1022659.81321102267:1022664.7102267:10226601122102268:1022654.2102268:1022657.91523102268:10226610102268:10226601344102271:10226914102271:10226901225102271:1022700102271:10227001126102272:1022690102272:10226901327102272:1022700102272:102270012ZFNs1735361:353963135361:353962961835426:354581035426:35458761934931:339407.334931:3394076 As noted previously (see co-owned U.S. Patent Publication No. 20110301073), the C17 TALENs have greater activity when the gap size between the two TALEN target sites is approximately 11-15 bp, while the C63 TALENs sustain activity at gap sizes up to 18 bp (seeFIG.10,11Cand Table 13). All patents, patent applications and publications mentioned herein are hereby incorporated by reference in their entirety. Although disclosure has been provided in some detail by way of illustration and example for the purposes of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be practiced without departing from the spirit or scope of the disclosure. Accordingly, the foregoing descriptions and examples should not be construed as limiting. | 100,489 |
11859191 | DETAILED DESCRIPTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Where a term is provided in the singular, the inventors also contemplate aspects of the disclosure described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the art in which they are used. The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, plant breeding, and biotechnology, which are within the skill of the art. See, e.g., Green and Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, 4th edition (2012); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)); Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL; ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)); RECOMBINANT PROTEIN PURIFICATION: PRINCIPLES AND METHODS, 18-1142-75, GE Healthcare Life Sciences; C. N. Stewart, A. Touraev, V. Citovsky, T. Tzfira eds. (2011) PLANT TRANSFORMATION TECHNOLOGIES (Wiley-Blackwell); and R. H. Smith (2013) PLANT TISSUE CULTURE. TECHNIQUES AND EXPERIMENTS (Academic Press, Inc.). The inventors do not intend to be limited to a mechanism or mode of action. Reference thereto is provided for illustrative purposes only. Any references cited herein are incorporated by reference in their entireties. As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof. Thus, for example, reference to “plant,” “the plant,” or “a plant” also includes a plurality of plants; also, depending on the context, use of the term “plant” can also include genetically similar or identical progeny of that plant; use of the term “a nucleic acid” optionally includes, as a practical matter, many copies of that nucleic acid molecule. As used herein, the term “about” indicates that a value includes the inherent variation of error for the method being employed to determine a value, or the variation that exists among experiments. As used herein, “encoding” refers either to a polynucleotide (DNA or RNA) encoding for the amino acids of a polypeptide or a DNA encoding for the nucleotides of an RNA. As used herein, “coding sequence” and “coding region” are used interchangeably and refer to a polynucleotide that encodes a polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5′ end and a translation stop codon at its 3′ end. As used herein, the term “identity” when used in relation to nucleic acids, describes the degree of similarity between two or more nucleotide sequences. The percentage of “sequence identity” between two sequences can be determined by comparing two optimally aligned sequences over a comparison window, such that the portion of the sequence in the comparison window may comprise additions or deletions (gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. A sequence that is identical at every position in comparison to a reference sequence is said to be identical to the reference sequence and vice-versa. An alignment of two or more sequences may be performed using any suitable computer program. For example, a widely used and accepted computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson, et al. (1994) Nucl. Acids Res., 22: 4673-4680). As used herein, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide are used interchangeably and refer to deoxyribonuclotides (DNA), ribonucleotides (RNA), and functional analogues thereof, such as complementary DNA (cDNA) in linear or circular conformation. Nucleic acid molecules provided herein can be single stranded or double stranded. Nucleic acid molecules comprise the nucleotide bases adenine (A), guanine (G), thymine (T), cytosine (C). Uracil (U) replaces thymine in RNA molecules. Analogues of the natural nucleotide bases, as well as nucleotide bases that are modified in the base, sugar, and/or phosphate moieties are also provided herein. The symbol “N” can be used to represent any nucleotide base (e.g., A, G, C, T, or U). The symbol “Y” can be used to represent thymine or cytosine bases. The symbol “V” can be used to represent the nucleotide bases A, C or G. As used herein, “complementary” in reference to a nucleic acid molecule or nucleotide bases refers to A being complementary to T (or U), and G being complementary to C. Two complementary nucleic acid molecules are capable of hybridizing with each other under appropriate conditions. In an aspect of the present disclosure, two nucleic acid sequences are homologous if they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with each other. As used herein, the term “plant” refers to any photosynthetic, eukaryotic, unicellular or multicellular organism of the kingdom Plantae and includes a whole plant or a cell or tissue culture derived from a plant, comprising any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, protoplasts and/or progeny of the same. A progeny plant can be from any filial generation, e.g., F1, F2, F3, F4, F5, F6, F7, etc. A “plant cell” is a biological cell of a plant, taken from a plant or derived through culture from a cell taken from a plant. The term plant encompasses monocotyledonous and dicotyledonous plants. The methods, systems, and compositions described herein are useful across a broad range of plants. Suitable plants in which the methods, systems, and compositions disclosed herein can be used include, but are not limited to, cereals and forage grasses (e.g., alfalfa, rice, maize, wheat, barley, oat, sorghum, pearl millet, finger millet, cool-season forage grasses, and bahiagrass), oilseed crops (e.g., soybean, oilseed brassicas including canola and oilseed rape, sunflower, peanut, flax, sesame, and safflower), legume grains and forages (e.g., common bean, cowpea, pea, faba bean, lentil, tepary bean, Asiatic beans, pigeonpea, vetch, chickpea, lupine, alfalfa, and clovers), temperate fruits and nuts (e.g., apple, pear, peach, plums, berry crops, cherries, grapes, olive, almond, and Persian walnut), tropical and subtropical fruits and nuts (e.g., citrus including limes, oranges, and grapefruit; banana and plantain, pineapple, papaya, mango, avocado, kiwifruit, passionfruit, and persimmon), vegetable crops (e.g., solanaceous plants including tomato, eggplant, and peppers; vegetable brassicas; radish, carrot, cucurbits, alliums, asparagus, and leafy vegetables), sugar cane, tubers (e.g., beets, parsnips, potatoes, turnips, sweet potatoes), and fiber crops (sugarcane, sugar beet, stevia, potato, sweet potato, cassava, and cotton), plantation crops, ornamentals, and turf grasses (tobacco, coffee, cocoa, tea, rubber tree, medicinal plants, ornamentals, and turf grasses), and forest tree species. As used herein, “plant genome” refers to a nuclear genome, a mitochondrial genome, or a plastid (e.g., chloroplast) genome of a plant cell. In some embodiments, a plant genome may comprise a parental genome contributed by the male and a parental genome contributed by the female. In some embodiments, a plant genome may comprise only one parental genome. As used herein, “polynucleotide” refers to a nucleic acid molecule containing multiple nucleotides and generally refers both to “oligonucleotides” (a polynucleotide molecule of 18-25 nucleotides in length) and polynucleotides of 26 or more nucleotides. Aspects of this disclosure include compositions including oligonucleotides having a length of 18-25 nucleotides (e. g., 18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), or medium-length polynucleotides having a length of 26 or more nucleotides (e. g., polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides), or long polynucleotides having a length greater than about 300 nucleotides (e. g., polynucleotides of between about 300 to about 400 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 900 to about 1000 nucleotides, between about 300 to about 500 nucleotides, between about 300 to about 600 nucleotides, between about 300 to about 700 nucleotides, between about 300 to about 800 nucleotides, between about 300 to about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, for example up to the entire length of a target gene including coding or non-coding or both coding and non-coding portions of the target gene). Where a polynucleotide is double-stranded, its length can be similarly described in terms of base pairs. As used herein, terms “polypeptide”, “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally-occurring amino acids. As used herein, “protoplast” refers to a plant cell that has had its protective cell wall completely or partially removed using, for example, mechanical or enzymatic means resulting in an intact biochemical competent unit of living plant that can reform their cell wall, proliferate and regenerate grow into a whole plant under proper growing conditions. As used herein, a “recombinant nucleic acid” refers to a nucleic acid molecule (DNA or RNA) having a coding and/or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. In some aspects, a recombinant nucleic acid provided herein is used in any composition, system or method provided herein. In some aspects, a recombinant nucleic acid may encode any CRISPR enzyme provided herein can be used in any composition, system or method provided herein. In some aspects, a recombinant nucleic acid may comprise or encode any guide RNA provided herein can be used in any composition, system or method provided herein. In an aspect, a vector provided herein comprises any recombinant nucleic acid provided herein. In another aspect, a cell provided herein comprises a recombinant nucleic acid provided herein. In another aspect, a cell provided herein comprises a vector provided herein. As used herein, the term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as meristem, or particular cell types (e.g., pollen). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); and SV40 enhancer. As used herein, the terms “target sequence” or “target site” refer to a nucleotide sequence against which a guide RNA is capable of hybridizing. A target sequence may be genic or non-genic. In some aspects, a target sequence provided herein comprises a genic region. In other aspects, a target sequence provided herein comprises an intergenic region. In yet another aspect, a target sequence provided herein comprises both a genic region and an intergenic region. In an aspect, a target sequence provided herein comprises a coding nucleic acid sequence. In another aspect, a target sequence provided herein comprises a non-coding nucleic acid sequence. In an aspect, a target sequence provided herein is located in a promoter. In another aspect, a target sequence provided herein comprises an enhancer sequence. In yet another aspect, a target sequence provided herein comprises both a coding nucleic acid sequence and a non-coding nucleic acid sequence. In one aspect, a target sequence provided herein is recognized and cleaved by a double-strand break inducing agent, such as a system comprising a Cpf1 enzyme and a guide RNA. As used herein, the term “donor” or “donor DNA” means a single stranded or double stranded DNA that comprises a polynucleotide sequence to be inserted at or near the target site of a Cpf1 enzyme and guide system. In some embodiments, the donor DNA comprises a transgene for insertion into the plant cell genome. In some embodiments, the donor DNA comprises a first and a second region of homology that flank the transgene, where the first and second regions of homology share homology to a first and a second genomic region present in or flanking the target site. A region of homology can be of any length that is sufficient to promote homologous recombination at the target site. For example, a region of homology can comprise at least 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1,000, 1,000-1,150, 1,150-1,200, 1,200-1,250, 1,250-1,300, 1,300-1,350, 1,350-1,400, 1,400-1,450, 1,450-1,500, 1,500-1,550, 1,550-1,600, 1,600-1,650, 1,650-1,700, 1,700-1,750, 1,750-1,800, 1,800-1,850, 1,850-1,900, 1,900-1,950, 1,950-2,000, or more bases in length. In some embodiments, the donor DNA comprises a polynucleotide sequence that comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide modifications compared to the target site. In some embodiments, the donor DNA comprises a polynucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to a polynucleotide sequence at or adjacent to the target site. In some embodiments, the donor DNA is 20, 25, 26, 27, 28, 29, 30, 31, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1,000, 1,000-1,150, 1,150-1,200, 1,200-1,250, 1,250-1,300, 1,300-1,350, 1,350-1,400, 1,400-1,450, 1,450-1,500, 1,500-1,550, 1,550-1,600, 1,600-1,650, 1,650-1,700, 1,700-1,750, 1,750-1,800, 1,800-1,850, 1,850-1,900, 1,900-1,950, 1,950-2,000, 2,000-2,100, 2,000-2,200, 2,000-2,300, 2,000-2,400, 2,000-2,500, 2,000-2,600, 2,000-2,700, 2,000-2,800, 2,000-2,900, 2,000-3,000, 3,000-3,100, 3,000-3,200, 3,000-3,300, 3,000-3,400, 3,000-3,500, 3,000-3,600, 3,000-3,700, 3,000-3,800, 3,000-3,900, 3,000-4,000, 4,000-4,100, 4,000-4,200, 4,000-4,300, 4,000-4,400, 4,000-4,500, 4,000-4,600, 4,000-4,700, 4,000-4,800, 4,000-4,900, 4,000-5,000, or more nucleotides in length. In an aspect, a Cpf1 nuclease provided herein is a Lachnospiraceae bacterium Cpf1 (LbCpf1) nuclease. In another aspect, a Cpf1 nuclease provided herein is aFrancisella novicidaCpf1 (FnCpf1) nuclease. A prerequisite for cleavage of the target site by a CRISPR ribonucleoprotein is the presence of a conserved Protospacer Adjacent Motif (PAM) near the target site. Depending on the CRISPR nuclease, cleavage can occur within a certain number of nucleotides (e.g., between 18-23 nucleotides for Cpf1) from the PAM site. PAM sites are only required for type I and type II CRISPR associated proteins, and different CRISPR endonucleases recognize different PAM sites. Without being limiting, the Cpf1 from Lachnospiraceae bacterium can recognize at least the following PAM sites: TTTN, and YTN; (where T is thymine; Y is thymine or cytosine; and N is thymine, cytosine, guanine, or adenine). Without being limiting, the Cpf1 fromFrancisella novicidacan recognize at least the following PAM sites: TTN (where T is thymine; and N is thymine, cytosine, guanine, or adenine). In certain embodiments, the LbCpf1 protein disclosed here has been modified to recognize a non-natural PAM. LbCpf1 variants comprising one or more amino acid substitutions resulting in altered PAM sequence specificities have been disclosed in the art (for example see Gao et. al., Nature Biotech., 2017 August; 35(8):789-792). Gao et. al. have disclosed two LbCpf1 variants: SEQ ID NO: 39 comprising the amino acid substitutions G532R/K595R that can recognize TYCV PAM (where T is thymine; Y is thymine or cytosine; C is cytosine and V is cytosine, guanine, or adenine) and SEQ ID NO: 76 comprising the amino acid substitutions G532R/K538V/Y542R that can recognize the TATV PAM (where T is thymine; A is adenine; and V is cytosine, guanine, or adenine). As used herein, LbCpf1(TYC) variant refers to an LbCpf1 nuclease comprising the amino acid substitutions G532R/K595R. As used herein, LbCpf1(TAT) variant (SEQ ID NO: 76) refers to an LbCpf1 nuclease comprising the mutations G532R/K538V/Y542R. The instant disclosure provides a recombinant nucleic acid encoding the Cpf1 nuclease of SEQ ID NO 2, 39, 43, 76 or a fragment thereof, wherein the recombinant nucleic acid is optimized for expression in a plant cell. A sequence can be optimized for expression in a plant cell by modifying a nucleotide sequence encoding a protein such as, for example, the nucleic acid sequence encoding the Cpf1 nuclease of SEQ ID NO 2, 39, 43 or a fragment thereof, using one or more plant-preferred codons for improved expression. In some embodiments, the plant-optimized recombinant nucleic acid encoding the Cpf1 nuclease of SEQ ID NO 2, or a fragment thereof, comprises a sequence having at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99%, or 100% identity to a sequence selected from SEQ ID NOs: 1 and 10, or a fragment thereof. In some embodiments, the plant-optimized recombinant nucleic acid encoding the LbCpf1(TYC) nuclease (SEQ ID NO: 39), or a fragment thereof, comprises a sequence having at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99%, or 100% identity to a sequence selected from SEQ ID NOs: 38, or a fragment thereof. In some embodiments, the plant-optimized recombinant nucleic acid encoding the LbCpf1 (TAT) nuclease (SEQ ID NO: 76) or a fragment thereof, comprises a sequence having at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99%, or 100% identity to a sequence selected from SEQ ID NOs: 75, or a fragment thereof. In some embodiments, the plant-optimized recombinant nucleic acid encoding the FnCpf1 nuclease (SEQ ID NO 43), or a fragment thereof, comprises a sequence having at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99%, or 100% identity to a sequence selected from SEQ ID NOs: 45-48, 50, 51 or a fragment thereof. In some embodiments, the plant-optimized recombinant nucleic acid is operably linked to a heterologous promoter. In one aspect, a recombinant nucleic acid provided herein comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more heterologous promoters operably linked to one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more plant-optimized recombinant nucleic acids encoding a Cpf1 nuclease. In some embodiments, a plant-optimized recombinant nucleic acids encoding a Cpf1 nuclease provided herein is provided to a plant cell in combination with one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more guide polynucleotides. As used herein, the term “guide polynucleotide” refers to a polynucleotide sequence that can form a complex with a Cpf1 endonuclease and enables the Cpf1 endonuclease to bind to, and optionally cleave, a target site. The guide polynucleotide sequence can be a RNA sequence, a DNA sequence, or any combination thereof (e.g., a RNA-DNA hybrid sequence). In one aspect, a guide polynucleotide provided herein comprises a CRISPR repeat sequence and a spacer sequence that is complementary to a target site. In one aspect, a guide polynucleotide provided herein comprises one or more repeats of a CRISPR repeat sequence, a spacer sequence, and a CRISPR repeat sequence. In some embodiments, the guide polynucleotide comprises two or more spacer sequences that are complementary to different target sites. In some embodiments, the guide polynucleotide comprises one or more CRISPR repeat sequences selected from a pre-crRNA and a mature cr-RNA. In some embodiments, the guide polynucleotide is operably linked to a promoter. In certain embodiments, recombinant nucleic acids encoding guide polynucleotides may be designed in an array format such that multiple guide polynucleotides can be simultaneously released. In some embodiments, expression of one or more guide polynucleotides is U6-driven. In some embodiments, Cpf1 enzymes complex with multiple guide polynucleotides to mediate genome editing and at multiple target sequences. Some embodiments relate to expression of singly or in tandem array format from 1 up to 4 or more different guide sequences; e.g. up to about 20 or about 30 guides sequences. Each individual guide sequence may target a different target sequence. Such may be processed from, e.g. one chimeric pol3 transcript. Pol3 promoters such as U6 or H1 promoters may be used. In some embodiments, a plant-optimized recombinant nucleic acid as disclosed herein is expressed or delivered in a vector. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is anAgrobacteriumT-DNA. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, Tobacco mosaic virus (TMV), Potato virus X (PVX) and Cowpea mosaic virus (CPMV), tobamovirus, Gemini viruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. In some embodiments, a viral vector may be delivered to a plant usingAgrobacterium. Certain vectors are capable of autonomous replication in a host cell into which they are introduced. Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors”. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.). In some embodiments, an expression vector can comprise a plant-optimized recombinant nucleic acid in a form suitable for expression of the plant-optimized recombinant nucleic acid in a plant cell, which means that the expression vector comprises one or more regulatory elements that are operatively-linked to the plant-optimized recombinant nucleic acid to be expressed. Regulatory elements may include enhancers, termination sequences, introns, etc. In certain embodiments, the plant-optimized recombinant nucleic acid may be operably linked to a nucleic acid sequence encoding one or more nuclear localization signal (NLS), nuclear export signal (NES), functional domains, and flexible linkers. The one or more of the NLS, the NES or the functional domain may be conditionally activated or inactivated. In particular embodiments it can be of interest to target the Cpf1 encoded by the plant-optimized recombinant nucleic acid to the chloroplast. In many cases, this targeting may be achieved by the operably linking the plant-optimized recombinant nucleic acid encoding Cpf1 to a nucleic acid encoding a chloroplast transit peptide (CTP) or plastid transit peptide. Other options for targeting to the chloroplast which have been described are the maize cab-m7 signal sequence (U.S. Pat. No. 7,022,896, WO 97/41228, incorporated by reference herein) a pea glutathione reductase signal sequence (WO 97/41228, incorporated by reference herein) and the CTP described in US2009029861, incorporated by reference herein. Several embodiments relate to a method for modifying a target sequence in the genome of a plant cell, the method comprising: introducing a recombinant nucleic acid optimized for expression in a plant cell comprising one or more of SEQ ID NOs: 1, 4, 6, 10, 12, 14, 15, 26, 31, 36, 38, 40, 41, 45, 46, 47, 48, 49, 50, 51, 63, 65, 66, 67, 68, 68, 70, 71, 72, 73, and 75 and a guide polynucleotide comprising a targeting domain that is complementary to a target sequence into the plant cell, where the recombinant nucleic acid expresses Cpf1 endonuclease in the plant cell and the Cpf1 endonuclease and the guide polynucleotide are capable of forming a complex that can recognize, bind to, and optionally nick or cleave the target sequence. In some embodiments, the guide polynucleotide and/or the recombinant nucleic acid are introduced into the plant cell by biolistic delivery. Several embodiments relate to a method for modifying a target sequence in the genome of a plant cell, the method comprising: introducing a guide polynucleotide comprising a targeting domain that is complementary to a target sequence in the plant genome into a plant cell comprising a recombinant nucleic acid optimized for expression in a plant cell, wherein the recombinant nucleic acid comprises one or more of SEQ ID NOs: 11, 4, 6, 10, 12, 14, 15, 26, 31, 36, 38, 40, 41, 45, 46, 47, 48, 49, 50, 51, 63, 65, 66, 67, 68, 68, 70, 71, 72, 73, and 75 where the recombinant nucleic acid expresses Cpf1 endonuclease in the plant cell and the Cpf1 endonuclease and the guide polynucleotide are capable of forming a complex that can recognize, bind to, and optionally nick or cleave the target sequence. In some embodiments, the guide polynucleotide is introduced into the plant cell by biolistic delivery. In some embodiments, the method further comprises incubating the plant cell at temperatures between 24° C. and 25° C., 25° C. and 26° C., 26° C. and 27° C., 27° C. and 28° C., 28° C. and 29° C., 29° C. and 30° C., 30° C. and 31° C., 31° C. and 32° C., 32° C. and 33° C., 33° C. and 34° C., 34° C. and 35° C., 35° C. and 36° C., 36° C. and 37° C., 37° C. and 38° C., 38° C. and 39° C., 39° C. and 40° C., for a period of at least about 10 min., 15 min., 20 min., 25 min., 30 min., 35 min., 40 min., 45 min., 50 min., 55 min., 1 hr., 2 hrs., 3 hr., 4 hrs., 5 hrs., 6 hrs., 7 hrs., 8 hrs., 9 hrs., 10 hrs., 11 hrs., 12 hrs., 13 hrs., 14 hrs., 15 hrs., 16 hrs., 17 hrs., 18 hrs., 19 hrs., 20 hrs. 21 hrs., 22 hrs., 23 hrs., 24 hrs., 25 hrs., 26 hrs., 27 hrs., 28 hrs., 29 hrs., 30 hrs., 31 hrs., 32 hrs., 33 hrs., 34 hrs., 35 hrs., 36 hrs., 37 hrs., 38 hrs., 39 hrs., 40 hrs., 41 hrs., 42 hrs., 43 hrs. 44 hrs., 45 hrs., 46 hrs., 47 hrs., 48 hrs., 3 days, 4 days, 5 days, 6 days, or 7 days. In some embodiments, the methods described herein can further comprise identifying at least one plant cell, plant or progeny plant that has a modification at the target sequence, where the modification at the target sequence is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii). The method can further provide a donor DNA to the plant cell, where the donor DNA comprises a polynucleotide sequence of interest. This can produce a plant cell or plant having a detectable targeted genome modification. Several embodiments relate to a method for modifying a target sequence in the genome of a plant cell, method comprising: obtaining a plant cell comprising in its genome a recombinant nucleic acid comprising a sequence selected from the group consisting of: SEQ ID NOs 1, 4, 6, 10, 12, 14, 15, 26, 31, 36, 38, 40, 41, 45, 46, 47, 48, 49, 50, 51, 63, 65, 66, 67, 68, 68, 70, 71, 72, 73, and 75 and introducing into the plant cell a guide polynucleotide comprising a targeting domain that is complementary to a target sequence in the plant genome or a recombinant nucleic acid encoding the guide polynucleotide, where the guide polynucleotide and Cpf1 endonuclease encoded by the recombinant nucleic acid are capable of forming a complex that can bind to, and modify the target sequence. In some embodiments, the guide polynucleotide is introduced into the plant cell by biolistic delivery. In some embodiments, the method further comprises incubating the plant cell at temperatures between 24° C. and 25° C., 25° C. and 26° C., 26° C. and 27° C., 27° C. and 28° C., 28° C. and 29° C., 29° C. and 30° C., 30° C. and 31° C., 31° C. and 32° C., 32° C. and 33° C., 33° C. and 34° C., 34° C. and 35° C., 35° C. and 36° C., 36° C. and 37° C., 37° C. and 38° C., 38° C. and 39° C., 39° C. and 40° C., for a period of at least about 10 min., 15 min., 20 min., 25 min., 30 min., 35 min., 40 min., 45 min., 50 min., 55 min., 1 hr., 2 hrs., 3 hr., 4 hrs., 5 hrs., 6 hrs., 7 hrs., 8 hrs., 9 hrs., 10 hrs., 11 hrs., 12 hrs., 13 hrs., 14 hrs., 15 hrs., 16 hrs., 17 hrs., 18 hrs., 19 hrs., 20 hrs. 21 hrs., 22 hrs., 23 hrs., 24 hrs., 25 hrs., 26 hrs., 27 hrs., 28 hrs., 29 hrs., 30 hrs., 31 hrs., 32 hrs., 33 hrs., 34 hrs., 35 hrs., 36 hrs., 37 hrs., 38 hrs., 39 hrs., 40 hrs., 41 hrs., 42 hrs., 43 hrs. 44 hrs., 45 hrs., 46 hrs., 47 hrs., 48 hrs., 3 days, 4 days, 5 days, 6 days, or 7 days. In some embodiments, the methods described herein can further comprise identifying at least one plant cell, plant or progeny plant that has a modification at the target sequence, where the modification at the target sequence is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii). The method can further provide a donor DNA to the plant cell, where the donor DNA comprises a polynucleotide sequence of interest. This can produce a plant cell or plant having a detectable targeted genome modification. The plant cell may be of a monocot or dicot. In some embodiments, the plant cell may be from or of a crop or grain plant such as cassava, corn, sorghum, alfalfa, cotton, soybean, canola, wheat, oat or rice. The plant cell may also be of an algae, tree or production plant, fruit or vegetable (e.g., trees such as citrus trees, e.g., orange, grapefruit or lemon trees; peach or nectarine trees; apple or pear trees; nut trees such as almond or walnut or pistachio trees; nightshade plants; plants of the genusBrassica; plants of the genusLactuca; plants of the genusSpinacia; plants of the genusCapsicum; cotton, tobacco, asparagus, avocado,papaya, cassava, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, potato, squash, melon, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc). The methods for genome editing using the recombinant nucleic acid molecules as described herein can be used to confer desired traits on essentially any plant. A wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation methods mentioned above. EXAMPLES The following examples are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); ss, single stranded; ds, double stranded and the like. Example 1 Design and Analysis of LbCpf1-CO1, an Engineered Polynucleotide Optimized for Expression in Plant Cells This example describes the creation and testing of a synthetic polynucleotide encoding Lachnospiraceae bacterium ND2006 (LbCpf1) nuclease that is optimized for expression in plant cells. A nucleotide sequence of Cpf1 from Lachnospiraceae bacterium ND2006 (LbCpf1) that was codon optimized for expression in human cells has been described by Zetsche et. al, (Cell 2015, 163, 759-771). The human codon optimized sequence disclosed by Zetsche et. al., was modified through algorithmic methods, partly based on corn codon preference, to design LbCpf1-CO1(Coding sequence Optimized version 1) (SEQ ID NO: 1) to optimize the sequence for expression of the LbCpf1 protein (SEQ ID NO: 2) in plant cells. The plant-optimized LbCpf1-CO1 sequence was then incorporated into six different expression vectors to test its activity in corn cells. Three of the expression vectors were designed with an expression cassette (SEQ ID NO: 3) comprising the LbCpf1-CO1 nuclease and a nucleotide sequence encoding the Nuclear Localization Sequence (NLS) from the heat stress transcription factor 1 (HSFA1) gene fromSolanum lycopersicum(SEQ ID NO:4) on the 5′ and 3′ ends. Three of the expression vectors were designed with an expression cassette (SEQ ID NO:5) comprising a processable potato LS1 intron sequence (SEQ ID NO: 6) introduced into the NLS-LbCpf1-CO1-NLS sequence to eliminate expression of the LbCpf1 protein inAgrobacterium. The NLS-LbCpf1-CO1-NLS expression cassettes also comprised aZea maysUbiquitin M1 promoter leader and intron sequence (SEQ ID NOs:7) operably linked to the NLS-LbCpf1-CO1-NLS nuclease and a transcription terminator sequence from a rice Lipid transfer protein (LTP) gene (SEQ ID NO:8). Each plant vector also comprised a gRNA expression array comprising either 2 or 4 guide RNA sequences (mature crRNA+spacer) positioned in tandem and targeting 2 or 4 sites in a corn chromosome. The first crRNA sequence was 35 nt while the remaining ones were 20 nt and the spacer sequence was 30 nt. The gRNA arrays were operably linked to the maize U6 Pol III promoter (SEQ ID NO:9) and a poly T terminator sequence. All the expression vectors also included a third expression cassette containing the selectable marker gene CP4 that provides resistance to the herbicide glyphosate. See Table 1. TABLE 1SUMMARY OF RESULTS OF FRAGMENT LENGTH ANALYSIS (FLA)GENERATED FROM CORN PLANTS STABLY TRANSFORMED WITHLBCPF1-CO1 AND GRNAS TARGETING 8 UNIQUE GENOMIC TARGET SITES.Intronin Cpf1-Plants# EditedMutationCO1Genomic sitesPlantsreturningsamples byefficiencyVectorcassettetargeted (TS)testeddataFLA(%)1NoZmTS1, ZmTS24734002NoZmTS3, ZmTS4,555000ZmTS5, ZmTS63NoZmTS7, ZmTS84544004YesZmTS7, ZmTS8383712.635YesZmTS1, Zm TS26564006YesZmTS3, ZmTS4,352900ZmTS5, ZmTS6Total2852581 Corn 01DKD2 cultivar embryos were transformed withAgrobacteriumcontaining the plant expression vectors described in Table 1. Transformed plants were selected on glyphosate, leaf samples from regenerated plantlets were harvested after 2 weeks and genomic DNA was extracted for Fragment Length Analysis (FLA). FLA is a PCR-based molecular assay that can be used to identify indel (insertion or deletion) mutations introduced at the target site by NHEJ-mediated (Non Homologous End Joining) DNA repair following dsDNA cleavage by the Cpf1-guide complex. Genomic DNA was subjected to a PCR reaction with primers flanking the target site to generate amplicons. The amplicons fragment length was then compared to a wild type amplicon to identify mutants. PCR reactions were carried out using 5′ FAM-labeled primer, a standard primer and Phusion™ polymerase (New England Biolabs, MA) according to manufactures instructions to generate 200 to 500 bp PCR fragments. 1 ul PCR product was combined with 0.5 ul GeneScan 1200 LIZ Size Standard (Thermo Fisher, MA), 8.5 ul formamide and run on ABI sequencer (Thermo Fisher, MA). Two FLA reactions were multiplexed and subsequently analyzed for fragment length variation to identify plants with mutations at the target sites. As shown in Table 1, 258 plants returned high quality FLA data, out of which only 1 plant was identified as having mutations at one of the target sites. Example 2 Design and Analysis of LbCpf1-CO2, an Engineered Polynucleotide Optimized for Expression in Plant Cells This example describes the design and expression analysis of Lachnospiraceae bacterium ND2006 (LbCpf1) nuclease that is optimized for expression in plant cells. The LbCpf1-CO1 nucleotide sequence described in Example 1 was manually analyzed for the presence of deleterious motifs that could potentially reduce gene expression. These deleterious motifs were given a higher priority for removal/replacement by nucleotide sequences coding for synonymous codons. Additionally, a monocot-specific codon frequency table was used for optimization of the nucleotide sequence for expression in monocots. Based on these criteria, a second optimized LbCpf1 (referred to as LbCpf1-CO2) nucleotide sequence was generated (SEQ ID NO: 10) for expression of the LbCpf1 protein (SEQ ID NO: 2) in planta. When compared to LbCpf1-CO1, the LbCpf1-CO2 sequence was determined to have a threefold reduction in the presence of deleterious motifs within its coding sequence. The full length LbCpf1-CO2 nucleotide sequence shows only 85.6% sequence identity with the human codon optimized LbCpf1 nucleotide sequence disclosed by Zetsche et. al., (Cell 2015, 163, 759-771), only 77.5% sequence identity with LbCpf1-CO1 and only 69.4% sequence identity with the native bacterial LbCpf1 sequence. Three expression cassettes (Prom35S::HIStag:NLS:LbCpf1-CO2:mOrange:NLS::TermNOS; Prom35S::HIStag:NLS:LbCpf1-Os:mOrange:NLS::TermNOS; and Prom35S::HIStag:NLS:mOrange:NLS::TermNOS) were generated by standard cloning techniques and as described below: (1) Prom35S::HIS tag:NLS:LbCpf1-CO2:mOrange:NLS::TermNOS The LbCpf1-CO2 coding sequence was fused 5′ to the coding sequence of mOrange (mOr) fromEntacmaea quadricolor(SEQ ID NO: 52). The LbCpf1-CO2:mOrange fusion gene was then flanked at the 5′ and 3′ ends with the NLS sequence from tomato HSFA1 gene (SEQ ID NO:3) and a nucleotide sequence encoding a HIS tag (MGSS7H) (SEQ ID NO: 54) was introduced to the 5′ end. The nucleotide sequence was operably linked to the Cauliflower mosaic virus 35S promoter and anAgrobacteriumNOS terminator. (2) Prom35S::HIS Tag:NLS:LbCpf1-Os:mOrange:NLS::TermNOS The rice codon-optimized Cpf1 (LbCpf1-Os) nucleotide sequence described by Xu et. al. (Plant Biotechnology Journal, 2017, 15, 713-717) (SEQ ID NO:11) was used as a control to compare in planta expression. The LbCpf1-Os coding sequence was fused 5′ to the coding sequence of mOrange (mOr) fromEntacmaea quadricolor(SEQ ID NO: 52). The LbCpf1-Os:mOrange fusion gene was then flanked at the 5′ and 3′ ends with the NLS sequence from tomato HSFA1 gene (SEQ ID NO:3) and a nucleotide sequence encoding a HIS tag (MGSS7H) (SEQ ID NO:54) was introduced to the 5′ end. The nucleotide sequence was operably linked to the Cauliflower mosaic virus 35S promoter and anAgrobacteriumNOS terminator. (3) Prom35S::HIS tag:NLS:mOrange:NLS::TermNOS The coding sequence of mOrange (mOr) gene (SEQ ID NO:52) fromEntacmaea quadricolorwas flanked at the 5′ and 3′ ends with the NLS sequence from tomato HSFA1 gene (SEQ ID NO:3) and a nucleotide sequence encoding a HIS tag (MGSS7H) (SEQ ID NO:54) was introduced to the 5′ end. The nucleotide sequence was operably linked to the Cauliflower mosaic virus 35S promoter and anAgrobacteriumNOS terminator. The expression cassettes described above were cloned into plant expression constructs. Corn leaf protoplasts were transfected with either the LbCpf1-CO2-mOr construct, the LbCpf1-Os-mOr construct, or the control mOr construct to evaluate expression levels (Table 2). Since mOrange was fused to LbCpf1-CO2 and LbCpf1-Os, the relative mOrange fluorescence levels reflects LbCpf-CO2 and LbCpf1-Os expression levels. Transformations were carried out using standard polyethylene glycol (PEG) based transfection methods. To quantify transformation frequency, an expression vector comprising the luciferase gene was co-transfected. Following transformation, the protoplasts were incubated in the dark in incubation buffer and harvested after 48 hours. Transformation efficiency was calculated by quantifying luciferase expression. The average mOrange expression from 3 technical replicates was determined using Operetta™ (Perkin Elmer) analysis software. As shown inFIG.1and Table 2, mOrange intensity was significantly higher in protoplasts expressing LbCpf1-CO2-mOrange than in cells expressing LbCpf1-Os-mOrange. TABLE 2EXPRESSION ANALYSIS OF LBCPF1-CO2-MOR AND LBCPF1-OS-MOR FLUORESCENT PROTEINS IN CORN PROTOPLASTSFoldincrease inexpressionFluo-comparedrescenceto Cpf1-Expression ConstructdetectedOs-mOrProm35S::HIStag:NLS:LbCpf1-Yes14CO2:mOrange:NLS::TermNOSProm35S::HIStag:NLS:LbCpf1-Yes1Os:mOrange:NLS::TermNOSProm35S:: HIS tag:NLS:mOrange:NLS::TermNOSYes135 Example 3 Analysis of LbCpf1-CO2 Activity in Corn Plants This example describes testing the LbCpf1-CO2 nucleotide sequence for activity at multiple genomic sites in corn plants using multiplexed guide RNAs. AnAgrobacteriumLbCpf1-CO2 T-DNA vector comprising: an expression cassette for a selectable marker conferring resistance to the herbicide glyphosate; an expression cassette (SEQ ID NO:15) comprising NLS-LbCpf1-CO2-NLS (SEQ ID NO: 12) linked to a 5′ Kozak sequence (SEQ ID NO:13) resulting in Koz-NLS-LbCpf1-CO2-NLS (SEQ ID NO: 14), which was operably linked to aZea maysUbiquitin M1 promoter cassette (SEQ ID NOs:7) and the transcription terminator sequence from rice LTP (SEQ ID NO:8); and an expression cassette comprising theZea maysU6 promoter (SEQ ID NO:9) and a polyT terminator operably linked to gRNA expression array comprising three gRNAs positioned in tandem and targeting the three genomic sites, was created. Each gRNA comprised a 21 bp crRNA sequence linked to a 23 bp spacer sequence that was complementary to target site ZmTS9, ZmTS10 or ZmTS11 in the corn genome. As a control, anAgrobacteriumLbCpf1-Os T-DNA vector comprising: an expression cassette for a selectable marker conferring resistance to the herbicide glyphosate; an expression cassette (SEQ ID NO:18) comprising a Kozak sequence immediately upstream of the coding sequence of LbCpf1-Os (SEQ ID NO: 11) fused to the tomato HSFA NLS (SEQ ID NO:3) at the 5′ end and the 3′ end which was operably linked to theZea maysUbiquitin M1 promoter cassette (SEQ ID NO: 7) and to the rice LTP terminator (SEQ ID NO: 8); and an expression cassette comprising theZea maysU6 promoter (SEQ ID NO:9) operably linked to gRNA expression array comprising three gRNAs positioned in tandem and targeting the three genomic sites, was created. Each gRNA comprised a 21 bp crRNA sequence linked to a 23 bp spacer sequence that was complementary to target site ZmTS9, ZmTS10 or ZmTS11 in the corn genome. Corn 01DKD2 cultivar embryos were transformed with either the LbCpf1-CO2 or LbCpf1-Os T-DNA vectors described above byAgrobacterium-mediated transformation. Transformed plants were selected on glyphosate, leaf samples from regenerated plantlets were harvested after 2 weeks and genomic DNA was extracted for Fragment Length Analysis (FLA) to determine genome mutation rates, as described in Example 1. Table 3 summarizes the results and shows the mutation rate detected at each site in stably transformed corn plants. TABLE 3SUMMARY OF RESULTS OF FLA GENERATED FROM CORNPLANTS STABLY TRANSFORMED WITH EITHER LBCPF1-CO2OR LBCPF1-OS AND GRNA ARRAY TARGETING 3 UNIQUEGENOMIC TARGET SITEST-DNAPlants# Edited plantsMutationVectorTarget siteassayedby FLA analysisfrequencyLbCpf1-CO2ZmTS9482041.6%ZmTS1047612.7%ZmTS114624.3%LbCpf1-OsZmTS94948%ZmTS104912%ZmTS114724.3% As shown in Table 3, all three sites targeted for cleavage with the guide/LbCpf1-CO2 system described above exhibited the presence of mutations which is indicative of DNA cleavage and repair. The frequency of mutations at the three sites ranged from 4.3% at ZmTS11, 12.7% for ZmTS10 to almost 42% at ZmTS9. 20 plants identified as having mutations in ZmTS9 were further analyzed to confirm the presence of mutations at the target site. PCR primers flanking the target site were used to generate amplicons which were cloned via Zero blunt-end Topo™ cloning (LifeTechnologies), sequenced and compared to the reference sequence. The presence of mutations was confirmed in all 20 events. For the guide/LbCpf1-Os system, mutations were identified at all three sites and the frequency of mutations at the three sites ranged from 2% at TS10, 4.3% for TS11 to almost 9% at TS1. Taken together, the data shows that the plant coding sequence optimized LbCpf1-CO2 is properly transcribed and translated in the corn host cell, is functional and can successfully promote gRNA directed chromosomal cleavage at target sites. Example 4 Analysis of LbCpf1-CO2 Activity in Combination with a Single gRNA Expression System in Corn Plants This example describes the testing the LbCpf1-CO2 nucleotide sequence for the ability to induce cleavage and subsequent edits at a genomic target site in corn plants utilizing a single gRNA expression cassette. AnAgrobacteriumT-DNA vector comprising: an expression cassette for a selectable marker gene that conferred resistance to the herbicide glyphosate; an expression cassette (SEQ ID NO:15) comprising a Kozak sequence introduced 5′ to the NLS-LbCpf1-CO2-NLS nucleotide sequence and operably linked to aZea maysUbiquitin M1 promoter cassette and the transcription terminator sequence from rice LTP; and an expression cassette comprising theZea maysU6 Pol III promoter (SEQ ID NO: 9) and a poly T terminator operably linked to a single guide RNA (gRNA) comprising a crRNA sequence linked to a 23 bp spacer sequence complementary to a unique target site (ZmTS12) in the corn chromosome. Corn 01DKD2 cultivar embryos were transformed withAgrobacteriumcontaining the T-DNA vector and stably transformed plants were selected on glyphosate. Leaf samples from regenerated plantlets were harvested after 2 weeks and genomic DNA was extracted for Fragment Length Analysis (FLA) to determine genome mutation rates, as described in Example 1. TABLE 4FLA RESULTS GENERATED FROM CORN PLANTSSTABLY TRANSFORMED WITH LBCPF1-CO2 ANDGRNA TARGETING ZMTS12 GENOMIC TARGET SITE.# EditedNucleaseplants by FLAMutationsequenceTarget sitePlants assayedanalysisfrequencyLbCpf1-CO2ZmTS1224715864% As shown in Table 4, mutations were identified at the target site in 64% of corn plants stably transformed with a vector comprising the LbCp1-CO2 nucleotide sequence and a single guide RNA. Example 5 Analysis of the Effect of the Addition of a Kozak Fragment Upstream of the LbCpf1-Os Nucleotide Sequence on Nuclease Activity in Plant Cells This example describes testing the addition of the Kozak sequence (SEQ ID NO:15) upstream of the LbCpf1-Os nucleotide sequence for the ability to enhance nuclease activity in corn plants. AnAgrobacteriumLbCpf1-Os (Kozak minus) T-DNA vector comprising: an expression cassette for a selectable marker conferring resistance to the herbicide glyphosate; an expression cassette (SEQ ID NO:19) comprising NLS-LbCpf1-Os-NLS (SEQ ID NO:16), with an ATG sequence incorporated immediately 5′ to SEQ ID NO:16 and operably linked to aZea maysUbiquitin M1 promoter cassette (SEQ ID NOs:7) and the transcription terminator sequence from rice LTP (SEQ ID NO:8); and an expression cassette comprising theZea maysU6 promoter (SEQ ID NO:9) operably linked to gRNA expression array comprising three gRNAs positioned in tandem and targeting the three genomic sites, was created. Each gRNA comprised a 21 bp crRNA sequence linked to a 23 bp spacer sequence that was complementary to target site ZmTS9, ZmTS10 or ZmTS11 in the corn genome. Corn plants were transformed withAgrobacteriumcontaining either the T-DNA vector described above comprising the LbCpf1-Os (Kozak minus) expression cassette (SEQ ID NO:19) or the T-DNA vector described in Example 3 comprising a Kozak sequence immediately upstream of the coding sequence of LbCpf1-Os (SEQ ID NO:18). Transformed plants were selected on glyphosate, leaf samples from regenerated plantlets were harvested after 2 weeks and genomic DNA was extracted for Fragment Length Analysis (FLA) to determine genome mutation rates, as described in Example 1. Table 5 summarizes the results and shows the mutation rate for each site in stably transformed corn plants. TABLE 5SUMMARY OF FLA RESULTS GENERATED FROM CORNPLANTS STABLY TRANSFORMED WITH LBCPF1-OS ANDGRNA ARRAY TARGETING 3 UNIQUE GENOMIC TARGET SITES# Editedplants byKozakNucleasePlantsFLAMutationsequencesequenceTarget siteassayedanalysisfrequency+LbCpf1-OsZmTS94948%ZmTS104912%ZmTS114724.3%−LbCpf1-OsZmTS93300%ZmTS103500%ZmTS113500% Plants transformed with the LbCpf1-Os comprising a Kozak sequence upstream of the nuclease coding sequence exhibited mutations at all three target sites at frequency ranging from 2% at ZmTS10, 4.3% for ZmTS11 to almost 8% at ZmTS9. No mutants were identified at any of the three target sites in plants transformed with the LbCpf1-Os expression cassette lacking the Kozak sequence. Example 6 Analysis of LbCpf1-CO2 Activity in Soybean Plants This example describes testing the LbCpf1-CO2 nucleotide sequence for activity in soybean plants by assaying the ability of the nuclease to target cleavage at multiple unique genomic sites using multiplexed guides. AnAgrobacteriumLbCpf1-CO2 T-DNA vector was created comprising: an expression cassette for a selectable marker conferring resistance to the antibiotic spectinomycin; an expression cassette (SEQ ID NO: 20) comprising NLS-LbCpf1-CO2-NLS (SEQ ID NO:12) with ATGGCG fused in frame 5′ to SEQ ID NO 12 as the translational start site, which was operably linked to a promoter sequence (SEQ ID NO:37) and a transcriptional terminator sequence fromMedicago truncatula(disclosed in US20140283200); and an expression cassette comprising theGlycine maxU6 Pol III promoter (disclosed in US20170166912) and a polyT terminator operably linked to a gRNA array comprising three gRNAs arranged in tandem and a transcriptional terminator sequence. Each gRNA comprised a 21 bp mature crRNA sequence linked to a 23 bp spacer sequence that was complementary to either the GmFAD2-1A-TS, GmPDS-TS1 or GmPDS-TS2 target site. AnAgrobacteriumLbCpf1-Os T-DNA control vector was created comprising: an expression cassette for a selectable marker conferring resistance to the antibiotic spectinomycin; an expression cassette (SEQ ID NO: 21) comprising NLS-LbCpf1-Os-NLS with ATGGCG fused in frame 5′ as the translational start site, which was operably linked to a promoter sequence (SEQ ID NO:37) and a transcriptional terminator sequence fromMedicago truncatula(disclosed in US20140283200); and an expression cassette comprising theGlycine maxU6 Pol III promoter (disclosed in US20170166912) and polyT terminator operably linked to a gRNA array comprising three gRNAs arranged in tandem and a transcriptional terminator sequence. Each gRNA comprised a 21 bp mature crRNA sequence linked to a 23 bp spacer sequence that was complementary to either the GmFAD2-1A-TS, GmPDS-TS1 or GmPDS-TS2 target site. Excised embryos from A3555 soybean plants were co-cultured with theAgrobacteriumcontaining either the LbCpf1-CO2 T-DNA vector or the LbCpf1-Os T-DNA control vector described above. Transformed plants were selected on spectinomycin, leaf samples from regenerated plantlets were harvested after 4 weeks, and genomic DNA was extracted for Fragment Length Analysis (FLA) to determine genome mutation rates, as described in Example 1. A summary of FLA results generated from soy plants stably transformed with either LbCpf1-CO2 or LbCpf1-Os and gRNA array targeting 3 unique genomic target sites is provided in Table 6. The plants were also scored for the albino phenotype typically associated with reduction/loss of PDS gene function (Table 7). PDS catalyzes a rate-limiting step in the biosynthesis of carotenoids in plants (Misawa, et. al.,The Plant Journal,1993, 4; 833-840). Reducing the endogenous PDS gene expression will therefore result in plants with a bleached phenotype and lowered chlorophyll content. Presence of an albino phenotype is therefore indicative of mutations at the PDS locus. TABLE 6SUMMARY OF FLA RESULTS GENERATED FROM SOYPLANTS STABLY TRANSFORMED WITH EITHER LBCPF1-CO2OR LBCPF1-OS AND GRNA ARRAY TARGETING 3 UNIQUEGENOMIC TARGET SITES# EditedNuclease seqplants byMutationvariantTarget sitesPlants assayedFLArateLbCpf1-CO2GmFAD2-1A622236%GmPDS-TS16200%GmPDS-TS2622845%LbCpf1-OsGmFAD2-1A882022%GmPDS-TS18800%GmPDS-TS2883742% TABLE 7SUMMARY OF PLANTS SCORED FOR PDS GENEMUTATIONS INDICATED BY AN ALBINO PHENOTYPE.Albino frequencyNucleasePlants assayedAlbino plantsrateLbCpf1-CO2625284%LbCpf1-Os886068% As summarized in Table 6, of the 3 sites targeted by LbCpf1-Os and LbCpf1-CO2, soybean plants were recovered where mutations were identified at FAD2 and PDS1-TS2 sites. The mutations at the PDS locus was further confirmed by scoring for the albino phenotype (see Table 7). Example 7 Plant Expression Vectors with Unique Cpf1-CO2 Expression Cassettes PromMt.Ubiq::NLS:LbCpf1-CO2:NLS::TermMt: An expression cassette (SEQ ID NO: 26) for the expression of a Cpf1-CO2 endonuclease was created comprising: a promoter (SEQ ID NO:22), leader (SEQ ID NO:23) and intron (SEQ ID NO:24) derived fromMedicago truncatulaUbiquitin operably linked 5′ to the NLS-LbCpf1-CO2-NLS coding sequence (SEQ ID NO: 12) wherein ATGGCG sequence was fused in frame 5′ to SEQ ID NO 12 and served as the translational start site. The resulting sequence was in turn operably linked 5′ to a UTR sequence from a gene fromMedicago truncatula(SEQ ID NO:25). The expression cassette was introduced into anAgrobacteriumvector that also comprised a gRNA cassette designed to guide LbCpf1 to a unique GmTS1 target site on the soy chromosome. The gRNA comprised a 21 bp crRNA sequence linked to a 23 bp spacer sequence that was complementary to GmTS1 within the soy genome. The gRNA was operably linked toGlycine maxU6 Pol III promoter (disclosed in US20170166912, incorporated by reference herein) and a poly T terminator. The vector also comprised an expression cassette for a selectable marker conferring resistance to the antibiotic spectinomycin. PromEF1a::NLS:LbCpf1-CO2:NLS::TermMt: An expression cassette (SEQ ID NO: 31) for the expression of a Cpf1-CO2 endonuclease was created comprising: a promoter (SEQ ID NO:27), leader 5′ (SEQ ID NO:28), intron (SEQ ID NO:29), leader 3′ (SEQ ID NO:30) derived fromCucumis meloEIF1alpha gene operably linked 5′ to the NLS-LbCpf1-CO2-NLS coding sequence (SEQ ID NO: 12) wherein ATGGCG sequence was fused in frame 5′ to SEQ ID NO 12 and served as the translational start site. The resulting sequence was operably linked to a UTR sequence from a gene fromMedicago truncatula(SEQ ID NO:25). The expression cassette was introduced into anAgrobacteriumvector that also comprised a gRNA cassette designed to guide LbCpf1 to a unique GmTS2 target site on the soy chromosome. The gRNA comprised a 21 bp crRNA sequence linked to a 23 bp spacer sequence that was complementary to GmTS2 within the soy genome. The gRNA was operably linked toGlycine maxU6 Pol III promoter (disclosed in US20170166912) and a poly T terminator. The vector also comprised an expression cassette for a selectable marker conferring resistance to the antibiotic spectinomycin. PromAt.Ubiq::NLS:LbCpf1-CO2:NLS::TermGb: An expression cassette (SEQ ID NO: 36) for the expression of a Cpf1-CO2 endonuclease was created comprising a promoter (SEQ ID NO:32), leader (SEQ ID NO:33) and intron (SEQ ID NO:34) derived fromArabidopsisUbiquitin 10 gene operably linked 5′ to the NLS-LbCpf1-CO2-NLS coding sequence (SEQ ID NO: 12) where ATGGCG sequence was fused in frame 5′ to SEQ ID NO 12 and served as the translational start site. The resulting sequence was operably linked to a UTR sequence from a gene fromGossypium barbadense(SEQ ID NO: 35). The expression cassette was introduced into anAgrobacteriumvector that also comprised a gRNA cassette designed to guide LbCpf1 to a unique GmTS3 target site on the soy chromosome. The gRNA comprised a 21 bp crRNA sequence linked to a 23 bp spacer sequence that was complementary to GmTS3 within the soy genome. The gRNA was operably linked toGlycine maxU6 Pol III promoter (disclosed in US20170166912) and a poly T terminator. The vector also comprised an expression cassette for a selectable marker conferring resistance to the antibiotic spectinomycin. Example 8 Testing the Activity of LbCpf1-CO2 Expression Cassettes TheAgrobacteriumT-DNA vectors described in Example 7, were introduced intoA. tumefaciens. Excised embryos from A3555 Soybean plants were co-cultured with theAgrobacteriumcontaining the vectors by standard methods known in the art and grown on spectinomycin to select for transformed plants. Leaf samples from regenerated plantlets were harvested after 2 weeks and genomic DNA was extracted for Fragment Length Analysis (FLA) to determine genome mutation rates at the target sites GmTS1, GmTS2 and GmTS3 as described in Example 1. A summary of FLA results generated from soy plants stably transformed with the three LbCpf1-CO2 expression cassettes and gRNAs targeting the unique soy genomic target sites is provided in Table 8. TABLE 8SUMMARY OF FLA RESULTS GENERATED FROM SOYPLANTS STABLY TRANSFORMED WITH LBCPF1-CO2EXPRESSION CASSETTES AND GRNATARGETING 3 UNIQUE GENOMIC TARGET SITESGenomic# of editedExpression vector withtargetPlantsplants byTarget siteLbCpf1 cassettesiteassayedFLAmutationPromMt.Ubiq::NLS:LbCpf1-GmTS1846881%CO2:NLS::TermMtPromEFIa::NLS:LbCpf1-GmTS2845869%CO2:NLS::TermMtPromAt.Ubiq::NLS:LbCpf1-GmTS3847286%CO2:NLS::TermGb As shown in Table 8, all three sites targeted for cleavage with the guide/LbCpf1-CO2 expression systems described above exhibited the presence of mutations which is indicative of DNA cleavage and repair. Example 9 Analysis of LbCpf1(TYC)-CO2 Variant Activity in Corn Plants This example describes the testing of a recombinant polynucleotide encoding Lachnospiraceae LbCpf1(TYC) PAM variant nuclease that is optimized for expression in plant cells. LbCpf1 variants comprising amino acid mutations resulting in altered PAM sequence specificities have been described by Gao et. al. (see Nature Biotech., 2017 August; 35(8):789-792). For example, Gao et. al. have described an LbCpf1(TYC) variant comprising the mutations G532R/K595R that can be engineered to recognize TYCV PAM. Two nucleotide substitutions were introduced into the LbCpf1-CO2 sequence (SEQ ID NO:10) resulting in LbCpf1(TYC)-CO2 (SEQ ID NO:38) encoding the LbCpf1(TYC) protein (SEQ ID NO:39) comprising the mutations G532R/K595R. To test the activity of LbCpf1(TYC), anAgrobacteriumT-DNA vector was generated. The vector comprised a Cpf1 expression cassette (SEQ ID NO:40) comprising the maize ubiquitin promoter (SEQ ID NO: 7) operably linked to a sequence (SEQ ID NO: 41) encoding LbCpf1(TYC)-CO2 comprising two nuclear localization signals (SEQ ID NOs: 42 and 3). The NLS-LbCpf1(TYC)-CO2-NLS was operably linked to a transcription terminator sequence from a rice Lipid transfer protein (LTP) gene (disclosed in US201801058230-0175, incorporated herein by reference). The vector also comprised a gRNA expression cassette encoding gRNAs designed to target two unique target sites in the corn genome, ZmTS13 and ZMTS14. The ZmTS13 and ZMTS14 sites were chosen since the TYCV PAM was present immediately upstream to each site. The 5′PAM for ZmTS13 was the sequence TTCA. The 5′PAM for ZmTS14 was the sequence TCCA. The gRNA expression cassette comprised theZea maysU6 Pol III promoter (SEQ ID NO: 9) operably linked to two guide RNAs positioned in tandem and targeting the ZmTS13 and ZmTS14 sites. The expression vector also included a third expression cassette containing the selectable marker gene that provides resistance to the herbicide glyphosate. Corn 01DKD2 cultivar embryos were transformed with the LbCpf1(TYC)-CO2 vector described above byAgrobacterium-mediated transformation. Transformed plants were selected on glyphosate, leaf samples from regenerated plantlets were harvested and genomic DNA was extracted for Fragment Length Analysis (FLA) to determine genome mutation rates specifically at ZmTS13 and ZmTS14 sites, as described in Example 1. ZmTS13 and ZmTS14 are arrayed in antisense orientation relative to each other in the genome and overlap by 8 nts, thus individual editing rates at each gRNA target site were not able to be ascertained. Table 9 summarizes the results and shows the cumulative mutation rate detected at or near the two sites in stably transformed corn plants. As shown in Table 9, 48% (40 of the 83) plants tested exhibited the presence of mutations at the expected region which is indicative of DNA cleavage by LbCpf1(TYC) and subsequent repair. TABLE 9FLA RESULTS GENERATED FROM CORN PLANTS STABLYTRANSFORMED WITH LBCPF1(TYC)-CO2 EXPRESSIONCASSETTE AND GRNA TARGETING 2 UNIQUE GENOMICTARGET SITES.# Edited plantsCumulativeTarget sitesPlantsby FLAMutationT-DNA VectortestedassayedanalysisfrequencyLbCpf1(TYC)-ZmTS13834048%CO2ZmTS14 Example 10 Analysis of FnCpf1 Engineered Polynucleotides Optimized for Expression in Plant Cells This example describes the design and expression analysis of polynucleotide sequences encodingFrancisella novicida(FnCpf1) nuclease that are optimized for expression in plant cells. A nucleotide sequence of Cpf1 fromFrancisella novicida(FnCpf1) that was codon optimized for expression in human cells has been described by Zetsche et. al, (Cell 2015, 163, 759-771). To optimize the expression of the FnCpf1 protein (SEQ ID NO:43) in plant cells, the human codon optimized sequence disclosed by Zetsche et. al., (SEQ ID NO:44), described here as FnCpf1-Hs was modified through algorithmic methods, partly based on plant codon frequency tables, to design seven FnCpf1 CO (Codon optimized) sequences (see Table 10). TABLE 10CODON OPTIMIZED FNCPF1 AND THE CODON FREQUENCYTABLES USED TO DESIGN EACH SEQUENCE.Codon optimized FnCpf1Codon frequency tableSEQ ID NO:FnCpf1-CO1Glycine max45FnCpf1-CO2Monocot46FnCpf1-CO3Glycine max47FnCpf1-CO4Monocot48FnCpf1-CO5Oryza sativa49FnCpf1-CO6Oryza sativa50FnCpf1-CO7Zea mays51 Expression Analysis of FnCpf1-CO Variants Via Quantification of FnCpf1-mOr Intensity Three expression cassettes (Prom35S::HIStag:NLS:FnCpf1-CO1:mOrange:NLS::TermNOS; Prom35S::HIStag:NLS:FnCpf1-CO2:mOrange:NLS::TermNOS; and Prom35S::HIStag:NLS:FnCpf1-Hs:mOrange:NLS::TermNOS) were generated by standard cloning techniques and are described below: (1) Prom35S::HIS tag:NLS:FnCpf1-CO1:mOrange:NLS::TermNOS The FnCpf1-CO1 coding sequence (SEQ ID NO: 45) was fused 5′ to the coding sequence of mOrange (mOr) fromEntacmaea quadricolor(SEQ ID NO:52) The FnCpf1-CO1:mOrange fusion gene was flanked at the 5′ end with an NLS sequence from potato (SEQ ID NO: 42) and at the 3′ end with an NLS sequence from tomato HSFA1 gene (SEQ ID NO:3) resulting in NLS:FnCpf1-CO1:mOrange:NLS (SEQ ID NO: 53). A nucleotide sequence encoding a HIS tag (MGSS7H) (SEQ ID NO: 54) was introduced at the 5′ end of SEQ ID NO:53. A ‘TAG’ termination codon was introduced to the 3′ end of the resulting nucleotide sequence (SEQ ID NO: 55) which was then operably linked to the Cauliflower mosaic virus 35S promoter (disclosed in U.S. Pat. No. 9,938,535-0047, incorporated herein by reference) and anAgrobacteriumNOS terminator (MK078637). The expression cassette (SEQ ID NO: 56) was cloned into a plant expression vector. (2) Prom35S::HIS tag:NLS:FnCpf1-CO2:mOrange:NLS::TermNOS The FnCpf1-CO2 coding sequence (SEQ ID NO: 46) was fused 5′ to the coding sequence of mOrange (mOr) fromEntacmaea quadricolor. (SEQ ID NO:52). The FnCpf1-CO2:mOrange fusion gene was flanked at the 5′ end with an NLS sequence from potato (SEQ ID NO: 42) and at the 3′ end with an NLS sequence from tomato HSFA1 gene (SEQ ID NO:3) resulting in NLS-FnCpf1-CO2-NLS(SEQ ID NO:57). A nucleotide sequence encoding a HIS tag (MGSS7H) (SEQ ID NO: 54) was introduced at the 5′ end of SEQ ID NO:57. A ‘TAG’ termination codon was introduced to the 3′ end of the resulting nucleotide sequence(SEQ ID NO:58) which was then operably linked to the Cauliflower mosaic virus 35S promoter and anAgrobacteriumNOS terminator (MK078637). The expression cassette (SEQ ID NO:59) was cloned into a plant expression vector. (3) Prom35S::HIS tag:NLS:FnCpf1-Hs:mOrange:NLS::TermNOS The human codon-optimized Cpf1 (FnCpf1-Hs) nucleotide sequence described by Zetsche et. al, (Cell 2015, 163, 759-771) (SEQ ID NO:44) was fused 5′ to the coding sequence of mOrange (mOr) fromEntacmaea quadricolor. (SEQ ID NO:52). The FnCpf1-Hs:mOrange fusion gene was then flanked at the 5′ end with an NLS sequence from potato (SEQ ID NO: 42) and at the 3′ end with an NLS sequence from tomato HSFA1 gene (SEQ ID NO:3) resulting in NLS-FnCpf1-Hs:mOrange-NLS(SEQ ID NO: 60). A nucleotide sequence encoding a HIS tag (MGSS7H) (SEQ ID NO: 54) was introduced at the 5′ end of SEQ ID NO:60. A ‘TAG’ termination codon was introduced to the 3′ end of the resulting nucleotide sequence (SEQ ID NO:61) which was then operably linked to the Cauliflower mosaic virus 35S promoter and anAgrobacteriumNOS terminator (MK078637). The expression cassette (SEQ ID NO:62) was cloned into a plant expression vector. To evaluate and quantify the expression of the fusion proteins, corn leaf protoplasts were transfected with expression vectors comprising either of the three expression cassettes described above. Since mOrange was fused to FnCpf1-CO1, FnCpf1-CO2 and FnCpf1-Hs, the relative mOrange fluorescence levels reflects FnCpf1 expression levels. Transformations were carried out using standard polyethylene glycol (PEG) based transfection methods. To quantify transformation frequency, an expression vector comprising the luciferase gene was co-transfected. Following transformation, the protoplasts were incubated in the dark in incubation buffer and harvested after 48 hours. Transformation efficiency was calculated by quantifying luciferase expression. The average mOrange expression from 5 technical replicates was determined using Operetta™ (Perkin Elmer) analysis software. As shown inFIG.2, mOrange fluorescence was detected from all three samples. The observed intensity was the highest in protoplasts expressing the FnCpf1-CO2-mOrange expression construct. Expression Analysis FnCpf1-CO Variants Via Qualitative Western Blots: In addition to the three expression constructs described above, five expression constructs were generated and are described below: (4) PromUbiq::NLS:FnCpf1-CO3:NLS::TermOs: The FnCpf1-CO3 sequence (SEQ ID NO:47) was flanked at the 5′ end with an NLS sequence from potato (SEQ ID NO: 42) and at the 3′ end with an NLS sequence from tomato HSFA1 gene (SEQ ID NO:3) resulting in NLS-FnCpf1-CO3-NLS (SEQ ID NO:63). An ATG sequence encoding the translation initiation codon was added 5′ to the potato NLS sequence and a TGA termination codon sequence was introduced 3′ to the tomato NLS sequence. The resulting sequence was operably linked to the maize ubiquitin promoter (SEQ ID NO: 7) and a transcription terminator sequence from a rice (SEQ ID NO:64). The FnCpf1-CO3 expression cassette sequence is set forth as SEQ ID NO:65. The expression cassette was cloned into a plant expression vector. (5) PromUbiq::NLS:FnCpf1-CO4:NLS::TermOs: The FnCpf1-CO4 sequence (SEQ ID NO:48) was flanked at the 5′ end with an NLS sequence from potato (SEQ ID NO: 42) and at the 3′ end with an NLS sequence from tomato HSFA1 gene (SEQ ID NO:3) resulting in NLS-FnCpf1-CO4-NLS(SEQ ID NO:66). An ATG sequence encoding the translation initiation codon was added 5′ to the potato NLS sequence and a TAG termination codon sequence was introduced 3′ to the tomato NLS sequence. The resulting sequence was operably linked to the maize ubiquitin promoter (SEQ ID NO: 7) and a transcription terminator sequence from a rice (SEQ ID NO: 64). The FnCpf1-CO4 expression cassette sequence is set forth as SEQ ID NO:67. The expression cassette was cloned into a plant expression vector. (6) PromUbiq::NLS:FnCpf1-CO5:NLS::TermOs: The FnCpf1-CO5 sequence (SEQ ID NO:49) was flanked at the 5′ end with an NLS sequence from potato (SEQ ID NO: 42) and at the 3′ end with an NLS sequence from tomato HSFA1 gene (SEQ ID NO:3) resulting in NLS-FnCpf1-CO5-NLS (SEQ ID NO:68). An ATG sequence encoding the translation initiation codon was added 5′ to the potato NLS sequence and a TGA termination codon sequence was introduced 3′ to the tomato NLS sequence. The resulting sequence was operably linked to the maize ubiquitin promoter (SEQ ID NO: 7) and a transcription terminator sequence from a rice (SEQ ID NO: 64). The FnCpf1-CO5 expression cassette sequence is set forth as SEQ ID NO:69. The expression cassette was cloned into a plant expression vector. (7) PromUbiq::NLS:FnCpf1-CO6:NLS::TermOs: The FnCpf1-CO6 sequence (SEQ ID NO:50) was flanked at the 5′ end with an NLS sequence from potato (SEQ ID NO: 42) and at the 3′ end with an NLS sequence from tomato HSFA1 gene (SEQ ID NO:3) resulting in NLS-FnCpf1-CO6-NLS (SEQ ID NO:70). An ATG sequence encoding the translation initiation codon was added 5′ to the potato NLS sequence and a TGA termination codon sequence was introduced 3′ to the tomato NLS sequence. The resulting sequence was operably linked to the maize ubiquitin promoter (SEQ ID NO: 7) and a transcription terminator sequence from a rice (SEQ ID NO: 64). The FnCpf1-CO6 expression cassette sequence is set forth as SEQ ID NO:71. The expression cassette was cloned into a plant expression vector. (8) PromUbiq::NLS:FnCpf1-CO7:NLS::TermOs: The FnCpf1-CO7 sequence (SEQ ID NO:51) was flanked at the 5′ end with an NLS sequence from potato (SEQ ID NO: 42) and at the 3′ end with an NLS sequence from tomato HSFA1 gene (SEQ ID NO:3) resulting in NLS-FnCpf1-CO7-NLS (SEQ ID NO:72). An ATG sequence encoding the translation initiation codon was added 5′ to the potato NLS sequence and a TGA termination codon sequence was introduced 3′ to the tomato NLS sequence. The resulting sequence was operably linked to the maize ubiquitin promoter (SEQ ID NO: 7) and a transcription terminator sequence from a rice (SEQ ID NO: 64). The FnCpf1-CO7 expression cassette sequence is set forth as SEQ ID NO:73. The expression cassette was cloned into a plant expression vector. Corn protoplast cells were transformed with the eight plant expression vectors described above and in Table 11. As a negative control, cells were transformed with an expression vector for GFP. Transformations were carried out using standard polyethylene glycol (PEG) based transfection methods. Following transformation, the protoplasts were incubated in the dark in incubation buffer and harvested after 48 hours. 32*104cells from each transformation were lysed using 50 uL of lysis buffer. Total protein was extracted from each of the lysed samples and 30 ug protein per sample was resolved on an SDS-PAGE gel and electro-blotted onto nitrocellulose membranes by standard methods. 5 ng, 1 ng and 500 pg of purified FnCpf1 protein were loaded as positive controls. Western blots using anti-FnCpf1 antibody (Cell Signaling Technology, Danvers, MA) were performed to detect the presence of FnCpf1 proteins using standard methods. As noted in Table 11, a band corresponding to the FnCpf1-mOr was visually observed in the lanes containing protein extract from protoplasts expressing FnCpf1-CO2-mOr (Sample 3). Similarly, bands corresponding to FnCpf1 were visually observed in the lanes containing protein extract from protoplasts expressing FnCpf1-CO3 and FnCpf1-CO4 (Samples 4 and 5). TABLE 11EXPRESSION ANALYSIS FNCPF1-CODON OPTIMIZED VARIANTS VIAQUALITATIVE WESTERN BLOTSProtein bandSampleExpression cassetteobserved1Prom35S::HIStag:NLS:FnCpf1Hs:mOrange:NLS::TermNOSNo2Prom35S:: HIS tag:NLS:FnCpf1-CO1:mOrange:NLS::TermNOSNo3Prom35S:: HIS tag:NLS:FnCpf1-CO2:mOrange:NLS::TermNOSYes4PromUbiq::NLS:FnCpf1-CO3:NLS::TermOsYes5PromUbiq::NLS:FnCpf1-CO4:NLS::TermOsYes6PromUbiq::NLS:FnCpf1-CO5:NLS::TermOsNo7PromUbiq::NLS:FnCpf1-CO6:NLS::TermOsNo8PromUbiq::NLS:FnCpf1-CO7:NLS::TermOsNo95 ng purified FnCpf1 protein (Positive control)Yes101 ng purified FnCpf1 protein (Positive control)Yes11500 pg purified FnCpf1 protein (Positive control)Yes12Prom35S::GFP::TermNOS(Negative control)No Example 11 Analysis of FnCpf1 Activity in Corn Protoplasts The assay used to evaluate FnCpf1 activity in corn protoplasts was integration of a blunt-end, double-stranded DNA (dsDNA) fragment into the DSB (Double stranded break) created by FnCpf1 protein at a specific target site. The blunt-end dsDNA fragment (disclosed in WO2019084148-021, incorporated herein by reference) was prepared by pre-annealing complementary ssDNA oligonucleotides. The ZmTS9 target site was chosen as the insertion site and a gRNA expression cassette targeting TS9 was designed. The expression cassette comprised a synthetic U6 promoter operably linked to a 21 bp crRNA sequence linked to a 23 bp spacer sequence that was complementary to ZmTS9 in the corn genome. The gRNA expression cassette was introduced into a plant expression vector. The gRNA vector and the eight plant vectors described in Example 11, each containing an expression cassette for a codon optimized FnCpf1 variant were co-transformed into isolated corn leaf protoplasts along with the double-stranded DNA (dsDNA) fragment essentially as described in patent application publication WO2015131101 (incorporated herein by reference), with minor modifications. Approximately 3.2×105protoplasts were transformed using PEG with a total of 12 μg of plasmid DNA and 50 pmoles of the dsDNA fragment (assays 2-9 in Table 12). Protoplast samples lacking the nuclease expressing plasmids served as a negative control (see assay 10 in Table 12). Additionally, protoplast samples transformed with nuclease vectors and gRNA cassettes lacking the spacer sequence were used as negative controls (see assays 11-19 in Table 12). As a positive control (assay 1 in Table 12), protoplasts were transformed with the gRNA cassette and a vector comprising an expression cassette (SEQ ID NO:74) for LbCpf1-CO2 that has been shown to be active in corn (see Examples 3-4). The expression cassette (SEQ ID NO: 20) comprised NLS-LbCpf1-CO2-NLS (SEQ ID NO:12) with ATGGCG fused in frame 5′ to SEQ ID NO 12 as the translational start site, and TGA termination codon fused 3′ to SEQ ID NO:12. The resulting sequence was operably linked to the maize ubiquitin promoter (SEQ ID NO: 7) and a transcription terminator sequence from a rice (SEQ ID NO:64). To determine transformation efficiency, 3 ug of GFP internal control plasmid was transformed along with test constructs. Following transformation, the corn protoplasts were incubated in the dark and harvested after 48 hours. Genomic DNA was extracted and assayed for integration of the dsDNA fragment. Integration of the dsDNA fragment into the genomic DNA was detected by standard PCR and agarose gel electrophoresis to assess PCR amplicons. The dsDNA fragment may have integrated in either a 5′ or 3′ orientation with respect to the 5′- and 3′-ends of the DSB. Therefore, two PCR primer sets were run for the target site where the primer sets contained a primer specific to the dsDNA fragment and a primer specific to either the 5′ side or the 3′ side of the DSB at TS11. The PCR amplicons were separated using standard agarose gel electrophoresis and the size of the amplicon was confirmed by comparison to a molecular weight marker. The presence of a band of expected size was indicative of site-directed integration of the donor oligo at the ZmTS9 site following FnCpf1 mediated dsDNA cleavage. As shown in Table 12, expected bands were amplified from protoplasts expressing LbCpf1-CO2, FnCpf1-CO1, FnCpf1-CO2, FnCpf1-CO3, FnCpf1-CO4, FnCpf1-CO6, FnCpf1-CO7 along with the cognate gRNA cassette and ds DNA. Expected bands were not amplified from protoplasts expressing FnCpf1-CO5 or any of the negative controls. TABLE 12FNCPF1 MEDIATED SITE DIRECTED INTEGRATION OF DSDNA OLIGO AT ZMTS9TARGET SITE.gRNAExpectedtargetingbandAssayNuclease Expression cassetteZmTS9amplified1PromUbiq::NLS:LbCpf1-CO2:NLS::TermOs(Positive control)+Yes2Prom35S::HIStag:NLS:FnCpf1Hs:mOrange:NLS::TermNOS+No3Prom35S:: HIS tag:NLS:FnCpf1-CO1:mOrange:NLS::TermNOS+Yes4Prom35S:: HIS tag:NLS:FnCpf1-CO2:mOrange:NLS::TermNOS+Yes5PromUbiq::NLS:FnCpf1-CO3:NLS::TermOs+Yes6PromUbiq::NLS:FnCpf1-CO4:NLS::TermOs+Yes7PromUbiq::NLS:FnCpf1-CO5:NLS::TermOs+No8PromUbiq::NLS:FnCpf1-CO6:NLS::TermOs+Yes9PromUbiq::NLS:FnCpf1-CO7:NLS::TermOs+Yes10None+No11PromUbiq::NLS:LbCpf1-CO2:NLS::TermOs−No12Prom35S::HIStag:NLS:FnCpf1Hs:mOrange:NLS::TermNOS−No13Prom35S:: HIS tag:NLS:FnCpf1-CO1:mOrange:NLS::TermNOS−No14Prom35S:: HIS tag:NLS:FnCpf1-CO2:mOrange:NLS::TermNOS−No15PromUbiq::NLS:FnCpf1-CO3:NLS::TermOs−No16PromUbiq::NLS:FnCpf1-CO4:NLS::TermOs−No17PromUbiq::NLS:FnCpf1-CO5:NLS::TermOs−No18PromUbiq::NLS:FnCpf1-CO6:NLS::TermOs−No19PromUbiq::NLS:FnCpf1-CO7:NLS::TermOs−No | 82,109 |
11859192 | BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID Nos: 1 to 11 set forth sequences of exemplary promoters for topping responsive root specific or preferred expression. SEQ ID Nos: 11 to 21 set forth sequences of exemplary promoters for topping responsive leaf specific or preferred expression. SEQ ID No: 22 sets forth a sequence of an exemplary DNA construct encoding a non-coding RNA suppressing an ornithine decarboxylase (ODC). SEQ ID Nos: 23 to 28 set forth cDNA sequences of exemplary tobacco ODC genes. SEQ ID Nos: 29 to 34 set forth amino acid sequences encoded by exemplary ODC genes. SEQ ID Nos: 35 and 36 set forth two miRNA sequences targeting an ODC gene in accordance with the present disclosure. Various sequences include “N” in nucleotide sequences or “X” in amino acid sequences. “N” can be any nucleotide, e.g., A, T, G, C, or a deletion or insertion of one or more nucleotides. In some instant, a string of “N” are shown. The number of “N” does not necessarily correlate with the actual number of undetermined nucleotides at that position. The actual nucleotide sequences can be longer or shorter than the shown segment of “N”. Similarly, “X” can be any amino acid residue or a deletion or insertion of one or more amino acids. Again, the number of “X” does not necessarily correlate with the actual number of undetermined amino acids at that position. The actual amino acid sequences can be longer or shorter than the shown segment of “X”. Notwithstanding the use of A, T, G, C (compared to A, U, G, C) in describing any SEQ ID in the sequence listing, that SEQ ID can also refer to a RNA sequence, depending on the context in which the SEQ ID is mentioned. DETAILED DESCRIPTION Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. One skilled in the art will recognize many methods can be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described. For purposes of the present disclosure, the following terms are defined below. Any references cited herein, including, e.g., all patents and publications are incorporated by reference in their entirety. As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof. The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. To avoid any doubt, used herein, terms or phrases such as “about”, “at least”, “at least about”, “at most”, “less than”, “greater than”, “within” or alike, when followed by a series of list of numbers of percentages, such terms or phrases are deemed to modify each and every number of percentage in the series or list. As used herein, a tobacco plant can be from any plant from theNicotianagenus including, but not limited toNicotiana tabacum, Nicotiana amplexicaulisPI 271989;Nicotiana benthamianaPI 555478;Nicotiana bigeloviiPI 555485;Nicotiana debneyi; Nicotiana excelsiorPI 224063;Nicotiana glutinosaPI 555507;Nicotiana goodspeediiPI 241012;Nicotiana gosseiPI 230953;Nicotiana hesperisPI 271991;Nicotiana knightianaPI 555527;Nicotiana maritimaPI 555535;Nicotiana megalosiphonPI 555536;Nicotiana nudicaulisPI 555540;Nicotiana paniculataPI 555545;Nicotiana plumbaginifoliaPI 555548;Nicotiana repandaPI 555552;Nicotiana rustica; Nicotiana suaveolensPI 230960;Nicotiana sylvestrisPI 555569;Nicotiana tomentosaPI 266379;Nicotiana tomentosiformis; andNicotiana trigonophyllaPI 555572. In an aspect, the present disclosure provides tobacco plants, or part thereof, comprising an inducible promoter operably linked to a transcribable DNA sequence encoding a non-coding RNA for suppression of an ornithine decarboxylase (ODC) gene. In one aspect, tobacco plants comprise a mutation or a transgene conferring a reduced level of nicotine. In an aspect, tobacco plants are low-alkaloid tobacco plants. In one aspect, tobacco plants of the present disclosure comprise a nic1 mutation, a nic2 mutation, or both. In an aspect, tobacco plants comprise nicotine at a level below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, or below 80% of the nicotine level of a control plant when grown in similar growth conditions, where the control plant shares an essentially identical genetic background with the tobacco plant except a low-nicotine conferring mutation or transgene. In another aspect, tobacco plants comprise nicotine or total alkaloids at a level below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, or below 80% of the nicotine or total alkaloids level of the control plant when grown in similar growth conditions. In another aspect, tobacco plants comprise a total alkaloid level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05% of the nicotine level of a control plant when grown in similar growth conditions, where the control plant shares an essentially identical genetic background with the tobacco plant except a low-nicotine conferring mutation or transgene. In another aspect, tobacco plants comprise a nicotine or total alkaloid level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05% of the nicotine or total alkaloids level of the control plant when grown in similar growth conditions. In an aspect, tobacco plants comprise a transgene or mutation directly suppressing the expression or activity of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, or all twenty-one genes or loci encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBB1, BBL, MYC2, nic1, nic2, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter. See Dewey and Xie, Molecular genetics of alkaloid biosynthesis inNicotiana tabacum, Phytochemistry 94 (2013) 10-27. In an aspect, tobacco plants further comprise one or more mutations in one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten genes selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. In one aspect, tobacco plants further comprise one or more mutations in ERF189, ERF115, or both. In an aspect, tobacco plants further comprise one or more transgenes targeting and suppressing a gene encoding one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten proteins selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. In an aspect, tobacco plants are capable of producing leaves, when cured, having a USDA grade index value selected from the group consisting of 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more. In another aspect, tobacco plants are capable of producing leaves, when cured, having a USDA grade index value comparable to that of a control plant when grown and cured in similar conditions, where the control plant shares an essentially identical genetic background with the tobacco plant except a low-nicotine conferring mutation or transgene. In a further aspect, tobacco plants are capable of producing leaves, when cured, having a USDA grade index value of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% of the USDA grade index value of a control plant when grown in similar conditions, where the control plant shares an essentially identical genetic background with the tobacco plant except a low-nicotine conferring mutation or transgene. In a further aspect, tobacco plants are capable of producing leaves, when cured, having a USDA grade index value of between 65% and 130%, between 70% and 130%, between 75% and 130%, between 80% and 130%, between 85% and 130%, between 90% and 130%, between 95% and 130%, between 100% and 130%, between 105% and 130%, between 110% and 130%, between 115% and 130%, or between 120% and 130% of the USDA grade index value of the control plant. In a further aspect, tobacco plants are capable of producing leaves, when cured, having a USDA grade index value of between 70% and 125%, between 75% and 120%, between 80% and 115%, between 85% and 110%, or between 90% and 100% of the USDA grade index value of the control plant. In another aspect, tobacco plants are capable of producing leaves, when cured, having a USDA grade index value selected from the group consisting of 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more. In another aspect, tobacco plants are capable of producing leaves, when cured, having a USDA grade index value selected from the group consisting of between 50 and 95, between 55 and 95, between 60 and 95, between 65 and 95, between 70 and 95, between 75 and 95, between 80 and 95, between 85 and 95, between 90 and 95, between 55 and 90, between 60 and 85, between 65 and 80, between 70 and 75, between 50 and 55, between 55 and 60, between 60 and 65, between 65 and 70, between 70 and 75, between 75 and 80, between 80 and 85, between 85 and 90, and between 90 and 95. In a further aspect, tobacco plants are capable of producing leaves, when cured, having a USDA grade index value of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% of the USDA grade index value of the control plant. In a further aspect, tobacco plants are capable of producing leaves, when cured, having a USDA grade index value of between 65% and 130%, between 70% and 130%, between 75% and 130%, between 80% and 130%, between 85% and 130%, between 90% and 130%, between 95% and 130%, between 100% and 130%, between 105% and 130%, between 110% and 130%, between 115% and 130%, or between 120% and 130% of the USDA grade index value of the control plant. In a further aspect, tobacco plants are capable of producing leaves, when cured, having a USDA grade index value of between 70% and 125%, between 75% and 120%, between 80% and 115%, between 85% and 110%, or between 90% and 100% of the USDA grade index value of the control plant. In an aspect, the present disclosure also provides a tobacco variety, cultivar, or line comprising a mutation selected from the group consisting of a nic1 mutation, a nic2 mutation, and a combination thereof, where the tobacco variety, cultivar, or line has a leaf grade comparable to the leaf grade of a control tobacco variety, cultivar, or line when grown in similar growth conditions, where the control tobacco variety shares an essentially identical genetic background with the tobacco variety, cultivar, or line except the mutation. In an aspect, the present disclosure further provides non-transgenic tobacco plants, or part thereof, comprising a nicotine or total alkaloid level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%, where the tobacco plants are capable of producing leaves, when cured, having a USDA grade index value of 50 or more 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more. In another aspect, such non-transgenic tobacco plants comprise a nicotine level of less than 2.0% and are capable of producing leaves, when cured, having a USDA grade index value of 70 or more. In a further aspect, such non-transgenic tobacco plants comprise a nicotine level of less than 1.0% and are capable of producing leaves, when cured, having a USDA grade index value of 70 or more. In an aspect, the present disclosure also provides a tobacco plant, or part thereof, comprising a non-transgenic mutation, where the non-transgenic mutation reduces the nicotine or total alkaloid level of the tobacco plant to below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, or below 80% of the nicotine level of a control plant when grown in similar growth conditions, where the tobacco plant is capable of producing leaves, when cured, having a USDA grade index value comparable to the USDA grade index value of the control plant, and where the control plant shares an essentially identical genetic background with the tobacco plant except the non-transgenic mutation. In an aspect, the present disclosure provides tobacco plants, or part thereof, comprising a mutation in a gene or locus, where the mutation is absent from LA Burley 21. In an aspect, tobacco plants provided herein comprise a shorter chromosomal introgression at a locus of interest compared to LA Burley 21. In another aspect, tobacco plants provided herein comprise no deletion of a complete gene or a complete genic coding sequence in the locus of interest. In an aspect, tobacco plants provided herein are homozygous at the locus of interest. In another aspect, tobacco plants provided herein are heterozygous at the locus of interest. In an aspect, tobacco plants provided herein comprise a mutation selected from the group consisting of a point mutation, a deletion, an insertion, a duplication, and an inversion at the gene or locus of interest. In an aspect, mutations in the tobacco plants provided herein are introduced by an approach selected from the group consisting of random mutagenesis and targeted mutagenesis. In another aspect, mutations in the tobacco plants provided herein are introduced by a targeted mutagenesis approach selected from the group consisting of meganuclease, zinc finger nuclease, TALEN, and CRISPR. As used herein, a mutation refers to an inheritable genetic modification introduced into a gene to alter the expression or activity of a product encoded by the gene. Such a modification can be in any sequence region of a gene, for example, in a promoter, 5′ UTR, exon, intron, 3′ UTR, or terminator region. In an aspect, a mutation reduces, inhibits, or eliminates the expression or activity of a gene product. In another aspect, a mutation increases, elevates, strengthens, or augments the expression or activity of a gene product. In an aspect, mutations are not natural polymorphisms that exist in a particular tobacco variety or cultivar. As used herein, a “mutant allele” refers to an allele from a locus where the allele comprises a mutation. As used herein, “mutagenic” refers to generating a mutation without involving a transgene or with no mutation-related transgene remaining in an eventual mutant. In an aspect, mutagenic is cisgenic. In another aspect, mutagenic is via gene or genome editing. In a further aspect, mutagenic is via random mutagenesis, for example, chemical (e.g., EMS) or physical (r-irradiation) mutagenesis. In an aspect, tobacco plants provided herein comprise one or more mutations within one or more genes comprising a coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 23 to 28, and fragments thereof. In an aspect, one or more mutations reduce the expression or activity of one or more genes comprising a coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 23 to 28, and fragments thereof. In an aspect, tobacco plants provided herein comprise one or more mutations within one or more genes encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 29 to 34, and fragments thereof. In an aspect, one or more mutations reduce the expression or activity of one or more genes encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 29 to 34, and fragments thereof. LA Burley 21 (also referenced as LA BU21) is a low total alkaloid tobacco line produced by incorporation of a low alkaloid gene(s) from a Cuban cigar variety into Burley 21 through several backcrosses (Legg et al. 1970). It has approximately 0.2% total alkaloids (dry weight) compared to the about 3.5% (dry weight) of its parent, Burley 21. LA BU21 has a leaf grade well below commercially acceptable standards. LA BU21 also exhibits other unfavorable leaf phenotypes characterized by lower yields, delayed ripening and senescence, higher susceptibility to insect herbivory, and poor end-product quality after curing (Chaplin and Weeks, 1976; Legg et al. 1970; Chaplin and Burk 1983). LA BU21 leaves further exhibit traits such as higher polyamine content, higher chlorophyll content and more mesophyll cells per unit leaf area. In plants, polyamines are reportedly involved in developmental, physiological and metabolic processes such as cell growth and division, stress tolerance, vascular differentiation, lignin polymerization, pathogen defense, senescence and ripening (Fariduddin Q, Varshney P, Yusuf M, Ahmad A (2013) Polyamines: potent modulators of plant responses to stress. J. Plant Interac. 8: 1-16; Kusano T, Suzuki H (2015). Polyamines a universal molecularnexusfor growth, survival and specialized metabolism. Tokyo: Springer). Several studies have linked polyamines to the regulation of plant cell senescence (Sobieszczuk-Nowicka, E., Kubala, S., Zmienko, A., Malecka, A., Legocka, J. 2016. From accumulation to degradation: Reprograming polyamine metabolism facilitates dark-induced senescence in Barley leaf cells. Front. Plant Sci. doi: 10.3389/fpls.2015.01198). In fruit and vegetative tissues, polyamines act as anti-senescence and anti-ripening regulators that prevent the decay of chloroplast photosystem complexes and changes in cell wall/membrane composition (Lester G E (2000). Polyamines and their cellular anti-senescence properties in honey dew musk melon fruit. Plant Sci. 160: 105-112; Mattoo A K, Handa A K (2008). Higher polyamines restore and enhance metabolic memory in ripening fruit. Plant Sci. 174: 386-393; Serafini-Fracassini D, Di Sandro A, Del Duca S (2010). Spermine delays leaf senescence inLactuca sativaand prevents the decay of chloroplast photosystems. Plant Physiol. Biochem. 48: 602-611). Higher levels of polyamines increase the longevity of tomato vines (Mehta R A, Cassol T, Li N, Ali N, Handa A K, Mattoo A K (2002). Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality and vine life. Nat. Biotechnol. 20: 613-618), and delayed ripening and leaf senescence was observed in transgenic tomato plants overexpressing a yeast spermidine synthase (Nambeesan S, Datsenka T, Ferruzzi M G, Malladi A, Mattoo A K, Handa A K (2010). Overexpression of yeast spermidine synthase impacts ripening, senescence and decay symptoms in tomato. Plant J. 63: 836-847). Polyamines may act directly by stabilizing cell walls or through crosstalk with phytohormones such as ethylene, abscisic acid, cytokinins and gibberellins (Kussano and Suzuki 2015). In most plants, putrescine can be synthesized either directly from ornithine by ornithine decarboxylase (ODC) or from arginine via three enzymatic steps, initiated by arginine decarboxylase (ADC) (Michael A J, Furze J M, Rhodes M J, Burtin D (1996). Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA. Biochem. J. 314: 241-248; Piotrowski M, Janowitz T, Kneifel H (2003). Plant C—N hydrolases and the identification of a plant N-carbamoylputrescine amidohydrolase involved in polyamine biosynthesis J. Biol. Chem. 278: 1708-1712; Illingworth C, Mayer M J, Elliot K, Hanfrey C, Walton N J, Michael A J (2003). The diverse bacterial origins of theArabidopsispolyamine biosynthetic pathway FEBS Letters 549: 26-30). Previous studies have stated that the ADC route to putrescine has only a minor effect on the alkaloid profile of tobacco whereas the ODC pathway plays the major role in nicotine biosynthesis (Chintapakorn Y, Hamill J D (2007). Antisense-mediated reduction in ADC activity causes minor alterations in the alkaloid profile of cultured hairy root and regenerated transgenic plants ofNicotiana tabacum. Phytochem. 68: 2465-2479; DeBoer K D, Dalton H L, Edward F J, Hamill J D (2011). RNAi-mediated down-regulation of ornithine decarboxylase (ODC) leads to reduced nicotine and increased anatabine levels in transgenicNicotiana tabacumL. Phytochem. 72: 344-355; DeBoer K D, Dalton H L, Edward F J, Ryan S M, Hamill J D (2013). RNAi-mediated down-regulation of ornithine decarboxylase (ODC) impedes wound-stress stimulation of anabasine synthesis inNicotiana glauca. Phytochem. 86: 21-28; Dalton H L, Blomstedt C K, Neale A D, Gleadow R, DeBoer K D, Hamill J D (2016). Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth inNicotiana tabacumL. J. Exp. Bot. 67: 3367-3381). Putrescine is converted to spermidine and then spermine by the successive addition of aminopropyl groups derived from decarboxylated S-adenosylmethionine (SAM), in reactions catalyzed by the enzymes spermidine synthase and spermine synthase, respectively. SAM is also a substrate for the biosynthesis of ethylene (Tiburcio A F, Altabella T, Bitrián M, Alcazar R (2014). The roles of polyamines during the lifespan of plants: from development to stress. Planta 240: 1-18), which regulates senescence and fruit ripening (Fluhr R, Mattoo A K (1996). Ethylene—biosynthesis and perception. Crit. Rev. Plant Sci. 15:479-523). The polyamine and ethylene biosynthesis pathways compete for the common precursor SAM but have opposing developmental effects, particularly during the developmental switch from vegetative growth to ripening/senescence (Nambeesan S, Handa A K, Mattoo A K (2008). Polyamines and regulation of ripening and senescence. In: Paliyath G, Murr D P, Handa A K, Lurie S (eds) Postharvest biology and technology of fruits, vegetables and flowers. Willey-Blackwell Publ, Ames. pp 319-340, Harpaz-Saad S, Yoon G M, Mattoo A K, Kieber J J (2012). The formation of ACC and competition between polyamines and ethylene for SAM. Annu. Plant Reviews. 44: 53-81, Gupta A, Pal R K, Rajam M V (2013). Delayed ripening and improved fruit processing quality in tomato by RNAi-mediated silencing of three homologs of 1-aminopropane-1-carboxylate synthase gene. J. Plant Physiol. 170: 987-995). Polyamine levels decrease and ethylene levels increase during the onset of fruit ripening in tomato (Saftner R A, Baldi B G (1990). Polyamine levels and tomato fruit development: possible interaction with ethylene. Plant Physiol. 92: 547-550; Morilla A, Garcia J M, Albi M A (1996). Free polyamine contents and decarboxylase activities during tomato development and ripening. J. Agri. Food Chem. 44: 2608-2611) and avocado (Kushad M M, Yelenosky G, Knight R (1988). Interrelationship of polyamine and ethylene biosynthesis during avocado fruit development and ripening. Plant Physiol. 87:463-467), which reflects the mutually antagonistic effect of ethylene on polyamine biosynthesis and vice versa (Harpaz-Saad et al. 2012; Anwar R, Mattoo A, Handa A (2015). Polyamine interactions with plant hormones: crosstalk at several levels in Kusano T, Suzuki H (eds). Polyamines a Universal MolecularNexusfor Growth, Survival and Specialized Metabolism. Tokyo: Springer. pp 267-303). However, transgenic tomato plants expressing yeast S-adenosylmethionine decarboxylase (SAMDC) under the control of the ripening-specific E8 promoter produced higher levels of ethylene and polyamines simultaneously during fruit ripening, indicating the absence of any competition for SAM in this system (Mehta R A, Cassol T, Li N, Ali N, Handa A K, Mattoo A K (2002). Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality and vine life. Nat. Biotechnol. 20: 613-618). Without being bound to any scientific theory, the suppression of nicotine biosynthesis in LA tobacco plants can affect crosstalk between the nicotine, polyamine and ethylene pathways, resulting in the accumulation of putrescine. This would in turn increase metabolic flux towards the higher polyamines spermidine and spermine while inhibiting ethylene biosynthesis, causing a dramatic effect on leaf ripening and senescence. In an aspect, the present disclosure provides tobacco plants, or part thereof, comprising a low nicotine or low alkaloid-conferring mutation or transgene and capable of producing a leaf comprising a comparable level of one or more polyamines relative to a comparable leaf of a control plant not comprising the same mutation or transgene. In one aspect, a comparable level of one or more polyamines is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In an aspect, a comparable level of one or more polyamines is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In a further aspect, a comparable level of one or more polyamines is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In an aspect, the present disclosure provides tobacco plants, or part thereof, comprising a low nicotine or low alkaloid-conferring mutation or transgene and capable of producing a leaf comprising a comparable chlorophyll level relative to a comparable leaf of a control plant not comprising the same mutation or transgene. In one aspect, a comparable chlorophyll level is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In an aspect, a comparable chlorophyll level is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In a further aspect, a comparable chlorophyll level is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In an aspect, the present disclosure provides tobacco plants, or part thereof, comprising a low nicotine or low alkaloid-conferring mutation or transgene and capable of producing a leaf comprising a comparable number of mesophyll cell per unit of leaf area relative to a comparable leaf of a control plant not comprising the same mutation or transgene. In one aspect, a comparable number of mesophyll cell per unit of leaf area is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In an aspect, a comparable number of mesophyll cell per unit of leaf area is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In a further aspect, a comparable number of mesophyll cell per unit of leaf area is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In an aspect, the present disclosure provides tobacco plants, or part thereof, comprising a low nicotine or low alkaloid-conferring mutation or transgene and capable of producing a leaf comprising a comparable epidermal cell size relative to a comparable leaf of a control plant not comprising the same mutation or transgene. In one aspect, a comparable epidermal cell size is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In an aspect, a comparable epidermal cell size is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In a further aspect, a comparable epidermal cell size is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In an aspect, the present disclosure provides tobacco plants, or part thereof, comprising a low nicotine or low alkaloid-conferring mutation or transgene and capable of producing a leaf comprising a comparable leaf yield relative to a comparable leaf of a control plant not comprising the same mutation or transgene. In one aspect, a comparable leaf yield is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In an aspect, a comparable leaf yield is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In a further aspect, a comparable leaf yield is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In an aspect, the present disclosure provides tobacco plants, or part thereof, comprising a low nicotine or low alkaloid-conferring mutation or transgene and exhibiting a comparable insect herbivory susceptibility relative to a comparable leaf of a control plant not comprising the same mutation or transgene. In one aspect, a comparable insect herbivory susceptibility is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In an aspect, a comparable insect herbivory susceptibility is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. In a further aspect, a comparable insect herbivory susceptibility is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene. Insect herbivory susceptibility level can be assayed by methods known in the art, for example, in an insect feeding assay. In short, a quarter inch layer of 0.7% agar in water is added to a 100 mm Petri dish and allowed to solidify. Leaf discs are cut from the petri dish lid, placed in the plates and pushed gently into the agar. Leaf discs are taken from plants at the 4-5 leaf stage. Discs were taken from lamina only to exclude major midribs. A single disc is taken from each of the four largest leaves of the plant generating 4 replicates per plant. Four plants are sampled for a total of 16 biological replicates test line. A single budworm at the second instar stage is added to the leaf and allowed to feed for 48 hours at ambient temperature. After 48 hours the budworm larvae are weighed and final larval weights are recorded. Unless specified otherwise, measurements of alkaloid, polyamine, or nicotine levels (or another leaf chemistry or property characterization) or leaf grade index values mentioned herein for a tobacco plant, variety, cultivar, or line can refer to average measurements, including, for example, depending on the context, an average of multiple leaves of a single plant or an average measurement from a population of tobacco plants from a single variety, cultivar, or line. In an aspect, the nicotine, alkaloid, or polyamine level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a pooled leaf sample collected from leaf number 3, 4, and 5 after topping. In another aspect, the nicotine, alkaloid, or polyamine level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a leaf having the highest level of nicotine, alkaloid, or polyamine (or another leaf chemistry or property characterization). In an aspect, the nicotine, alkaloid, or polyamine level of a tobacco plant is measured after topping in leaf number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In another aspect, the nicotine, alkaloid, or polyamine level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a pool of two or more leaves with consecutive leaf numbers selected from the group consisting of leaf number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In another aspect, the nicotine, alkaloid, or polyamine level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a leaf with a leaf number selected from the group consisting of between 1 and 5, between 6 and 10, between 11 and 15, between 16 and 20, between 21 and 25, and between 26 and 30. In another aspect, the nicotine, alkaloid, or polyamine level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a pool of two or more leaves with leaf numbers selected from the group consisting of between 1 and 5, between 6 and 10, between 11 and 15, between 16 and 20, between 21 and 25, and between 26 and 30. In another aspect, the nicotine, alkaloid, or polyamine level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a pool of three or more leaves with leaf numbers selected from the group consisting of between 1 and 5, between 6 and 10, between 11 and 15, between 16 and 20, between 21 and 25, and between 26 and 30. Alkaloid levels can be assayed by methods known in the art, for example by quantification based on gas-liquid chromatography, high performance liquid chromatography, radio-immunoassays, and enzyme-linked immunosorbent assays. As used herein, leaf numbering is based on the leaf position on a tobacco stalk with leaf number 1 being the oldest leaf (at the base) after topping and the highest leaf number assigned to the youngest leaf (at the tip). A population of tobacco plants or a collection of tobacco leaves for determining an average measurement (e.g., alkaloid or nicotine level or leaf grading) can be of any size, for example, 5, 10, 15, 20, 25, 30, 35, 40, or 50. Industry-accepted standard protocols are followed for determining average measurements or grad index values. As used herein, “topping” refers to the removal of the stalk apex, including the SAM, flowers, and up to several adjacent leaves, when a tobacco plant is near vegetative maturity and around the start of reproductive growth. Typically, tobacco plants are topped in the button stage (soon after the flower begins to appear). For example, greenhouse or field-grown tobacco plants can be topped when 50% of the plants have at least one open flower. Topping a tobacco plant results in the loss of apical dominance and also induce increased alkaloid production. Typically, the nicotine, alkaloid, or polyamine level (or another leaf chemistry or property characterization) of a tobacco plant is measured about 2 weeks after topping. Other time points can also be used. In an aspect, the nicotine, alkaloid, or polyamine level (or another leaf chemistry or property characterization) of a tobacco plant is measured about 1, 2, 3, 4, or 5 weeks after topping. In another aspect, the nicotine, alkaloid, or polyamine level (or another leaf chemistry or property characterization) of a tobacco plant is measured about 3, 5, 7, 10, 12, 14, 17, 19, or 21 days after topping. As used herein, “similar growth conditions” refer to similar environmental conditions and/or agronomic practices for growing and making meaningful comparisons between two or more plant genotypes so that neither environmental conditions nor agronomic practices would contribute to or explain any difference observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water (humidity), and nutrition (e.g., nitrogen and phosphorus). Agronomic practices include, for example, seeding, clipping, undercutting, transplanting, topping, and suckering. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp 70-103. As used herein, “comparable leaves” refer to leaves having similar size, shape, age, and/or stalk position. In an aspect, the present disclosure provides tobacco plants, or part thereof, comprising an inducible promoter operably linked to a transcribable DNA sequence encoding a non-coding RNA for suppression of an ornithine decarboxylase (ODC) gene. In one aspect, an inducible promoter is a topping-inducible promoter. In an aspect, an inducible promoter is also a tissue-specific or tissue-preferred promoter. In one aspect, a tissue-specific or tissue-preferred promoter is specific or preferred for one or more tissues or organs selected from the group consisting of shoot, root, leaf, stem, flower, sucker, root tip, mesophyll cells, epidermal cells, and vasculature. In a further aspect, a topping inducible promoter comprises a promoter sequence from a tobacco nicotine demethylase gene, for example, CYP82E4, CYP82E5, or CYP82E10. Various types of promoters can be used here, which are classified according to a variety of criteria relating to the pattern of expression of a coding sequence or gene (including a transgene) operably linked to the promoter, such as constitutive, developmental, tissue-specific, tissue-preferred, inducible, etc. Promoters that initiate transcription in all or most tissues of the plant are referred to as “constitutive” promoters. Promoters that initiate transcription during certain periods or stages of development are referred to as “developmental” promoters. Promoters whose expression is enhanced in certain tissues of the plant relative to other plant tissues are referred to as “tissue-enhanced” or “tissue-preferred” promoters. Thus, a “tissue-preferred” promoter causes relatively higher or preferential expression in a specific tissue(s) of the plant, but with lower levels of expression in other tissue(s) of the plant. Promoters that express within a specific tissue(s) of the plant, with little or no expression in other plant tissues, are referred to as “tissue-specific” promoters. A promoter that expresses in a certain cell type of the plant is referred to as a “cell type specific” promoter. An “inducible” promoter is a promoter that initiates transcription in response to an environmental stimulus such as cold, drought, heat or light, or other stimuli, such as wounding or chemical application. A promoter may also be classified in terms of its origin, such as being heterologous, homologous, chimeric, synthetic, etc. A “heterologous” promoter is a promoter sequence having a different origin relative to its associated transcribable sequence, coding sequence, or gene (or transgene), and/or not naturally occurring in the plant species to be transformed. The term “heterologous” more broadly includes a combination of two or more DNA molecules or sequences when such a combination is not normally found in nature. For example, two or more DNA molecules or sequences would be heterologous with respect to each other if they are normally found in different genomes or at different loci in the same genome, or if they are not identically combined in nature. In an aspect, an inducible promoter provides root specific or preferred expression. In one aspect, a root specific or preferred inducible promoter comprises a sequence selected from the group consisting of SEQ ID Nos: 1-11 and a functional fragment thereof. Table 1 provides a comparison of estimated leaf versus root specific expression level driven by SEQ ID Nos: 1-11. In an aspect, an inducible promoter provides leaf specific or preferred expression. In one aspect, a leaf specific or preferred inducible promoter comprises a sequence selected from the group consisting of SEQ ID Nos: 12-21 and a functional fragment thereof. Table 2 provides a comparison of estimated leaf versus root specific expression level driven by SEQ ID Nos: 12-21. TABLE 1Exemplary inducible promoters for topping-responsive root specific or preferred expressionRootLeaf3 days after3 days4 wks3 daystoppingGeneLaybyBeforeafterafterLaybyFloweringafter(nitrogenSEQ IdIdstageToppingToppingToppingSenescencestagetimeToppingdeficient)SEQ IDg786550.00.00.00.01.11.71.0146.0251.0NO: 1SEQ IDg720210.00.01.70.73.34.36.9501.7468.7NO: 2SEQ IDg782523.20.00.01.50.00.93.077.3167.8NO: 3SEQ IDg657200.00.01.70.03.80.90.033.56.0NO: 4SEQ IDg741083.23.30.00.73.62.64.081.6142.9NO: 5SEQ IDg4746612.731.60.069.643.20.01.022.310.6NO: 6SEQ IDg1028683.23.30.00.00.20.91.036.923.4NO: 7SEQ IDg230570.00.01.70.08.72.613.8247.4287.2NO: 8SEQ IDg3468412.741.643.067.4104.021.5105.71947.52177.0NO: 9SEQ IDg10594815.83.30.08.811.916.314.878.2768.7NO: 10SEQ IDg81261145.668.372.390.273.144.7115.62206.92112.0NO: 11 TABLE 2Exemplary inducible promoters for topping-responsiveleaf specific or preferred expression1 day3 days1 week2 weeks3 weeksBeforeafterafterafterafterafterHarvestSEQ IdGeneToppingToppingToppingToppingToppingToppingtimeSEQ IDg2237201703443057064321049NO: 12SEQ IDg3114241611774801671530892NO: 13SEQ IDg75488419306534472297138NO: 14SEQ IDg94193119128846165037743371546NO: 15SEQ IDg3475615861137322183611NO: 16SEQ IDg104299481101665761381521994NO: 17SEQ IDg4481021199980492140521NO: 18SEQ IDg7167115265821040348299NO: 19SEQ IDg294273434395314474591339NO: 20SEQ IDg490242928229230280382595NO: 21 In an aspect, an inducible promoter is a heterologous to the operably linked transcribable DNA sequence. In one aspect, a transcribable DNA sequence encodes a non-coding RNA selected from the group consisting of microRNA (miRNA), anti-sense RNA, small interfering RNA (siRNA), a trans-acting siRNA (ta-siRNA), and hairpin RNA (hpRNA). In an aspect, a non-coding RNA comprises a nucleotide sequence having at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% identity to a sequence selected from the group consisting of SEQ ID Nos: 35 and 36, and any portions thereof. In one aspect, a non-coding RNA is provided in an ODC RNAi construct comprising a nucleotide sequence having at least 99%, at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, or at least 75% identity to SEQ ID No: 22. “Alkaloids” are complex, nitrogen-containing compounds that naturally occur in plants, and have pharmacological effects in humans and animals. “Nicotine” is the primary natural alkaloid in commercialized cigarette tobacco and accounts for about 90 percent of the alkaloid content inNicotiana tabacum. Other major alkaloids in tobacco include cotinine, nornicotine, myosmine, nicotyrine, anabasine and anatabine. Minor tobacco alkaloids include nicotine-n-oxide, N-methyl anatabine, N-methyl anabasine, pseudooxynicotine, 2,3 dipyridyl and others. In an aspect, tobacco plants provided herein comprise a lower level of total alkaloid or an individual alkaloid compared to a control tobacco plant without a nic1 mutation and/or a nic2 mutation when grown in similar growth conditions. In another aspect, tobacco plants provided herein comprise a lower level of one or more alkaloids selected from the group consisting of cotinine, nornicotine, myosmine, nicotyrine, anabasine and anatabine, compared to a control tobacco plant when grown in similar growth conditions. In an aspect, a lower alkaloid or nicotine level refers to an alkaloid or nicotine level of below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, or below 80% of the alkaloid or nicotine level of a control tobacco plant. In another aspect, a lower alkaloid or nicotine level refers to an alkaloid or nicotine level of about between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, between 19% and 20%, between 21% and 22%, between 22% and 23%, between 23% and 24%, between 24% and 25%, between 25% and 26%, between 26% and 27%, between 27% and 28%, between 28% and 29%, or between 29% and 30% of the alkaloid or nicotine level of a control tobacco plant. In a further aspect, a lower alkaloid or nicotine level refers to an alkaloid or nicotine level of about between 0.5% and 5%, between 5% and 10%, between 10% and 20%, between 20% and 30% of the alkaloid or nicotine level of a control tobacco plant. Alkaloid levels can be assayed by methods known in the art, for example by quantification based on gas-liquid chromatography, high performance liquid chromatography, radio-immunoassays, and enzyme-linked immunosorbent assays. For example, nicotinic alkaloid levels can be measured by a GC-FID method based on CORESTA Recommended Method No. 7, 1987 and ISO Standards (ISO TC 126N 394 E. See also Hibi et al.,Plant Physiology100: 826-35 (1992) for a method using gas-liquid chromatography equipped with a capillary column and an FID detector. Unless specified otherwise, all alkaloid levels described here are measured using a method in accordance with CORESTA Method No 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatographic Analysis, February 2005, and those defined in the Centers for Disease Control and Prevention's Protocol for Analysis of Nicotine, Total Moisture and pH in Smokeless Tobacco Products, as published in the Federal Register Vol. 64, No. 55 Mar. 23, 1999 (and as amended in Vol. 74, No. 4, Jan. 7, 2009). Alternatively, tobacco total alkaloids can be measured using a segmented-flow colorimetric method developed for analysis of tobacco samples as adapted by Skalar Instrument Co (West Chester, Pa.) and described by Collins et al.,Tobacco Science13:79-81 (1969). In short, samples of tobacco are dried, ground, and extracted prior to analysis of total alkaloids and reducing sugars. The method then employs an acetic acid/methanol/water extraction and charcoal for decolorization. Determination of total alkaloids was based on the reaction of cyanogen chloride with nicotine alkaloids in the presence of an aromatic amine to form a colored complex which is measured at 460 nm. Unless specified otherwise, total alkaloid levels or nicotine levels shown herein are on a dry weight basis (e.g., percent total alkaloid or percent nicotine). In an aspect, tobacco plants provided herein comprise a lower level of nicotine compared to a control tobacco plant without a nic1 mutation and/or a nic2 mutation when grown in similar growth conditions. In an aspect, a lower nicotine level refers to an average nicotine level of below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, or below 80% of the average nicotine level of a control tobacco plant. In another aspect, a lower nicotine level refers to an average nicotine level of about between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, between 19% and 20%, between 21% and 22%, between 22% and 23%, between 23% and 24%, between 24% and 25%, between 25% and 26%, between 26% and 27%, between 27% and 28%, between 28% and 29%, or between 29% and 30% of the average nicotine level of a control tobacco plant. In a further aspect, a lower nicotine level refers to an average nicotine level of about between 0.5% and 5%, between 5% and 10%, between 10% and 20%, between 20% and 30% of the average nicotine level of a control tobacco plant. In an aspect, tobacco plants provided herein comprise an average nicotine or total alkaloid level selected from the group consisting of about 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, and 9% on a dry weight basis. In another aspect, tobacco plants provided herein comprise an average nicotine or total alkaloid level selected from the group consisting of about between 0.01% and 0.02%, between 0.02% and 0.05%, between 0.05% and 0.75%, between 0.75% and 0.1%, between 0.1% and 0.15%, between 0.15% and 0.2%, between 0.2% and 0.3%, between 0.3% and 0.35%, between 0.35% and 0.4%, between 0.4% and 0.5%, between 0.5% and 0.6%, between 0.6% and 0.7%, between 0.7% and 0.8%, between 0.8% and 0.9%, between 0.9% and 1%, between 1% and 1.1%, between 1.1% and 1.2%, between 1.2% and 1.3%, between 1.3% and 1.4%, between 1.4% and 1.5%, between 1.5% and 1.6%, between 1.6% and 1.7%, between 1.7% and 1.8%, between 1.8% and 1.9%, between 1.9% and 2%, between 2% and 2.1%, between 2.1% and 2.2%, between 2.2% and 2.3%, between 2.3% and 2.4%, between 2.4% and 2.5%, between 2.5% and 2.6%, between 2.6% and 2.7%, between 2.7% and 2.8%, between 2.8% and 2.9%, between 2.9% and 3%, between 3% and 3.1%, between 3.1% and 3.2%, between 3.2% and 3.3%, between 3.3% and 3.4%, between 3.4% and 3.5%, and between 3.5% and 3.6% on a dry weight basis. In a further aspect, tobacco plants provided herein comprise an average nicotine or total alkaloid level selected from the group consisting of about between 0.01% and 0.1%, between 0.02% and 0.2%, between 0.03% and 0.3%, between 0.04% and 0.4%, between 0.05% and 0.5%, between 0.75% and 1%, between 0.1% and 1.5%, between 0.15% and 2%, between 0.2% and 3%, and between 0.3% and 3.5% on a dry weight basis. The present disclosure also provides tobacco plants having altered nicotine levels without negative impacts over other tobacco traits, e.g., leaf grade index value. In an aspect, a low-nicotine or nicotine-free tobacco variety provides cured tobacco of commercially acceptable grade. Tobacco grades are evaluated based on factors including, but not limited to, the leaf stalk position, leaf size, leaf color, leaf uniformity and integrity, ripeness, texture, elasticity, sheen (related to the intensity and the depth of coloration of the leaf as well as the shine), hygroscopicity (the faculty of the tobacco leaves to absorb and to retain the ambient moisture), and green nuance or cast. Leaf grade can be determined, for example, using an Official Standard Grade published by the Agricultural Marketing Service of the US Department of Agriculture (7 U.S.C. § 511). See, e.g., Official Standard Grades for Burley Tobacco (U.S. Type 31 and Foreign Type 93), effective Nov. 5, 1990 (55 F.R. 40645); Official Standard Grades for Flue-Cured Tobacco (U.S. Types 11, 12, 13, 14 and Foreign Type 92), effective Mar. 27, 1989 (54 F.R. 7925); Official Standard Grades for Pennsylvania Seedleaf Tobacco (U.S. Type 41), effective Jan. 8, 1965 (29 F.R. 16854); Official Standard Grades for Ohio Cigar-Leaf Tobacco (U.S. Types 42, 43, and 44), effective Dec. 8, 1963 (28 F.R. 11719 and 28 F.R. 11926); Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55), effective Nov. 20, 1969 (34 F.R. 17061); Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55), effective Nov. 20, 1969 (34 F.R. 17061); Official Standard Grades for Georgia and Florida Shade-Grown Cigar-Wrapper Tobacco (U.S. Type 62), Effective April 1971. A USDA grade index value can be determined according to an industry accepted grade index. See, e.g., Bowman et al,Tobacco Science,32:39-40(1988); Legacy Tobacco Document Library (Bates Document #523267826-523267833, Jul. 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index); and Miller et al., 1990, Tobacco Intern.,192:55-57 (all foregoing references are incorporated by inference in their entirety). In an aspect, a USDA grade index is a 0-100 numerical representation of federal grade received and is a weighted average of all stalk positions. A higher grade index indicates higher quality. Alternatively, leaf grade can be determined via hyper-spectral imaging. See e.g., WO 2011/027315 (published on Mar. 10, 2011, and incorporated by inference in its entirety). A comparable leaf grade index indicates a leaf grade index that does not vary more than 30% above or below an appropriate control or comparator when comparing leaves from similar stalk positions. In an aspect, a comparable leaf grade index does not vary more than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% above or below an appropriate control or comparator when comparing leaves from similar stalk positions. In an aspect, tobacco plants provided herein comprise a similar level of one or more tobacco aroma compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, a labdenoid, a cembrenoid, a sugar ester, and a reducing sugar, compared to control tobacco plants when grown in similar growth conditions. In another aspect, tobacco plants provided herein comprise a nic1 mutation, a nic2 mutation, or a combination thereof having no impact over the level of one or more tobacco aroma compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, a labdenoid, a cembrenoid, a sugar ester, and a reducing sugar. As used herein, tobacco aroma compounds are compounds associated with the flavor and aroma of tobacco smoke. These compounds include, but are not limited to, 3-methylvaleric acid, valeric acid, isovaleric acid, cembrenoid and labdenoid diterpenes, and sugar esters. Concentrations of tobacco aroma compounds can be measured by any known metabolite profiling methods in the art including, without limitation, gas chromatography mass spectrometry (GC-MS), Nuclear Magnetic Resonance Spectroscopy, liquid chromatography-linked mass spectrometry. See The Handbook of Plant Metabolomics, edited by Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013). As used herein, “reducing sugar(s)” are any sugar (monosaccharide or polysaccharide) that has a free or potentially free aldehdye or ketone group. Glucose and fructose act as nicotine buffers in cigarette smoke by reducing smoke pH and effectively reducing the amount of “free” unprotonated nicotine. Reducing sugars balances smoke flavor, for example, by modifying the sensory impact of nicotine and other tobacco alkaloids. An inverse relationship between sugar content and alkaloid content has been reported across tobacco varieties, within the same variety, and within the same plant line caused by planting conditions. Reducing sugar levels can be measured using a segmented-flow colorimetric method developed for analysis of tobacco samples as adapted by Skalar Instrument Co (West Chester, Pa.) and described by Davis,Tobacco Science20:139-144 (1976). For example, a sample is dialyzed against a sodium carbonate solution. Copper neocuproin is added to the sample and the solution is heated. The copper neocuproin chelate is reduced in the presence of sugars resulting in a colored complex which is measured at 460 nm. In an aspect, tobacco plants provided herein comprise one or more non-naturally existing mutant alleles at nic1 and/or nic2 locus which reduce or eliminate one or more gene activity from nic1 and/or nic2 locus. In an aspect, these mutant alleles result in lower nicotine levels. Mutant nic1 and/or nic2 alleles can be introduced by any method known in the art including random or targeted mutagenesis approaches. Such mutagenesis methods include, without limitation, treatment of seeds with ethyl methylsulfate (EMS) (Hildering and Verkerk, In, The use of induced mutations in plant breeding. Pergamon press, pp 317-320, 1965) or UV-irradiation, X-rays, and fast neutron irradiation (see, for example, Verkerk,Neth. J. Agric. Sci.19:197-203, 1971; and Poehlman, Breeding Field Crops, Van Nostrand Reinhold, N.Y. (3.sup.rd ed), 1987), transposon tagging (Fedoroff et al., 1984; U.S. Pat. Nos. 4,732,856 and 5,013,658), as well as T-DNA insertion methodologies (Hoekema et al., 1983; U.S. Pat. No. 5,149,645). EMS-induced mutagenesis consists of chemically inducing random point mutations over the length of the genome. Fast neutron mutagenesis consists of exposing seeds to neutron bombardment which causes large deletions through double stranded DNA breakage. Transposon tagging comprises inserting a transposon within an endogenous gene to reduce or eliminate expression of the gene. The types of mutations that may be present in a tobacco gene include, for example, point mutations, deletions, insertions, duplications, and inversions. Such mutations desirably are present in the coding region of a tobacco gene; however mutations in the promoter region, and intron, or an untranslated region of a tobacco gene may also be desirable. In addition, a fast and automatable method for screening for chemically induced mutations, TILLING (Targeting Induced Local Lesions In Genomes), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the present disclosure. See, McCallum et al. (2000)Nat. Biotechnol.18:455-457. Mutations that impact gene expression or that interfere with the function of genes can be determined using methods that are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues can be particularly effective in inhibiting the function of a protein. In an aspect, tobacco plants comprise a nonsense (e.g., stop codon) mutation in one or more NCG genes described in U.S. Provisional Application Nos. 62/616,959 and 62/625,878, both of which are incorporated by reference in their entirety. In an aspect, the present disclosure also provides tobacco lines with altered nicotine levels while maintaining commercially acceptable leaf quality. These lines can be produced by introducing mutations into one or more genes at nic1 and/or nic2 locus via precise genome engineering technologies, for example, Transcription activator-like effector nucleases (TALENs), meganuclease, zinc finger nuclease, and a clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 system, a CRISPR/Cpf1 system, a CRISPR/Csm1 system, and a combination thereof (see, for example, U.S. Patent Application publication 2017/0233756). See, e.g., Gaj et al.,Trends in Biotechnology,31(7):397-405 (2013). The screening and selection of mutagenized tobacco plants can be through any methodologies known to those having ordinary skill in the art. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for detection of a polynucleotide, Northern blots, RNase protection, primer-extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, Next Generation sequencing technologies (e.g., Illumina, PacBio, Ion Torrent, 454), enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are known. In an aspect, a tobacco plant or plant genome provided herein is mutated or edited by a nuclease selected from the group consisting of a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas9 nuclease, a CRISPR/Cpf1 nuclease, or a CRISPR/Csm1 nuclease. In an aspect, tobacco plants provided herein comprising a nic1 mutation, a nic2 mutation, or both, further comprises a transgene or mutation providing an early-senescence trait. In one aspect, a mutation providing an early-senescence trait is yellow burley1 (−yb1). In an aspect, a mutation providing an early-senescence trait is yellow burley2 (−yb2). In one aspect, a mutation providing an early-senescence trait is pale yellow (PY). In an aspect, a tobacco plant, or part thereof, comprises relative to a control tobacco plant: a first genome modification providing a lower level of nicotine or total alkaloid, and a second genome modification providing a comparable level of one or more traits selected from the group consisting of total leaf polyamine level, total root polyamine level, total leaf chlorophyll level, mesophyll cell number per leaf area unit, and leaf epidermal cell size; and where the control plant does not have both the first and the second genome modifications. In one aspect, a tobacco plant, or part thereof, comprises relative to a control tobacco plant: a first genome modification providing a lower level of nicotine or total alkaloid, and a second genome modification providing a comparable level of total leaf polyamine level, where the control plant does not have both the first and the second genome modifications. In an aspect, a tobacco plant, or part thereof, comprises relative to a control tobacco plant: a first genome modification providing a lower level of nicotine or total alkaloid, and a second genome modification providing a comparable level of total root polyamine level, where the control plant does not have both the first and the second genome modifications. In one aspect, a tobacco plant, or part thereof, comprises relative to a control tobacco plant: a first genome modification providing a lower level of nicotine or total alkaloid, and a second genome modification providing a comparable level of total leaf chlorophyll level, where the control plant does not have both the first and the second genome modifications. In an aspect, a tobacco plant, or part thereof, comprises relative to a control tobacco plant: a first genome modification providing a lower level of nicotine or total alkaloid, and a second genome modification providing a comparable level of mesophyll cell number per leaf area unit, where the control plant does not have both the first and the second genome modifications. In one aspect, a tobacco plant, or part thereof, comprises relative to a control tobacco plant: a first genome modification providing a lower level of nicotine or total alkaloid, and a second genome modification providing a comparable level of leaf epidermal cell size, where the control plant does not have both the first and the second genome modifications. In an aspect, a first genome modification, a second genome modification, or both comprise a transgene, a mutation, or both. In one aspect, a genome modification, a second genome modification, or both comprise a transgene. In an aspect, a first genome modification, a second genome modification, or both comprise a mutation. In one aspect, a first genome modification, a second genome modification, or both are not transgene-based. In an aspect, a first genome modification, a second genome modification, or both are not mutation-based. In an aspect, tobacco plants provided herein comprise a first genome modification providing a lower level of nicotine compared to a control tobacco plant. In one aspect, tobacco plants provided herein comprise a first genome modification comprising a nic1 mutation, a nic2 mutation, or both. In an aspect, tobacco plants provided herein comprise a transgene targeting the Nic1 locus, a transgene targeting the Nic2 locus, or both. In an aspect, tobacco plants provided herein comprise a first genome modification comprising a mutation in a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), and S-adenosyl-methionine synthetase (SAMS), A622, NBB1, BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter. In one aspect, tobacco plants provided herein comprise a first genome modification comprises a transgene targeting and suppressing a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), and S-adenosyl-methionine synthetase (SAMS), A622, NBB1, BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter. In an aspect, tobacco plants provided herein comprise a first genome modification comprising a mutation in a gene or locus encoding a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. In one aspect, tobacco plants provided herein comprise a first genome modification comprises a transgene targeting and suppressing a gene or locus encoding a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have one or more mutations in one or more NCG genes selected from the group consisting of NCG1 to NCG35, where the one or more mutations reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have one or more mutations in one or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where the one or more mutations reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have one or more mutations in one or more NCG genes selected from the group consisting of NCG1 to NCG21, where the one or more mutations reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have one or more mutations in one or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG15, NCG16, and NCG17, where the one or more mutations reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have one or more mutations in one or more NCG genes selected from the group consisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where the one or more mutations reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have one or more mutations in one or more NCG genes selected from the group consisting of NCG12, NCG15, NCG16, and NCG17, where the one or more mutations reduce or eliminate the activity or expression of the one or more NCG genes. In another aspect, an edited or mutated tobacco plant having one or more, two or more, three or more, four or more, or five or more NCG mutations further comprises one or more mutations in one or more, two or more, three or more, four or more, or five or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the one or more mutations reduce or eliminate the activity or expression of the one or more ERF genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have two or more mutations in two or more NCG genes selected from the group consisting of NCG1 to NCG35, where the two or more mutations reduce or eliminate the activity or expression of the two or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have two or more mutations in two or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where the two or more mutations reduce or eliminate the activity or expression of the two or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have two or more mutations in two or more NCG genes selected from the group consisting of NCG1 to NCG21, where the two or more mutations reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have two or more mutations in two or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG15, NCG16, and NCG17, where the two or more mutations reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have two or more mutations in two or more NCG genes selected from the group consisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where the two or more mutations reduce or eliminate the activity or expression of the two or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have two or more mutations in two or more NCG genes selected from the group consisting of NCG12, NCG15, NCG16, and NCG17, where the two or more mutations reduce or eliminate the activity or expression of the two or more NCG genes. In another aspect, an edited or mutated tobacco plant having two or more NCG mutations further comprises two or more mutations in two or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the two or more mutations reduce or eliminate the activity or expression of the two or more ERF genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have three or more mutations in three or more NCG genes selected from the group consisting of NCG1 to NCG35, where the three or more mutations reduce or eliminate the activity or expression of the three or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have three or more mutations in three or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where the three or more mutations reduce or eliminate the activity or expression of the three or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have three or more mutations in three or more NCG genes selected from the group consisting of NCG1 to NCG21, where the three or more mutations reduce or eliminate the activity or expression of the three or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have three or more mutations in three or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG15, NCG16, and NCG17, where the three or more mutations reduce or eliminate the activity or expression of the three or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have three or more mutations in three or more NCG genes selected from the group consisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where the three or more mutations reduce or eliminate the activity or expression of the three or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have three or more mutations in three or more NCG genes selected from the group consisting of NCG12, NCG15, NCG16, and NCG17, where the three or more mutations reduce or eliminate the activity or expression of the three or more NCG genes. In another aspect, an edited or mutated tobacco plant having three or more NCG mutations further comprises three or more mutations in three or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the three or more mutations reduce or eliminate the activity or expression of the three or more ERF genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have four or more mutations in four or more NCG genes selected from the group consisting of NCG1 to NCG35, where the four or more mutations reduce or eliminate the activity or expression of the four or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have four or more mutations in four or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where the four or more mutations reduce or eliminate the activity or expression of the four or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have four or more mutations in four or more NCG genes selected from the group consisting of NCG1 to NCG21, where the four or more mutations reduce or eliminate the activity or expression of the four or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have four or more mutations in four or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG15, NCG16, and NCG17, where the four or more mutations reduce or eliminate the activity or expression of the four or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have four or more mutations in four or more NCG genes selected from the group consisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where the four or more mutations reduce or eliminate the activity or expression of the four or more NCG genes. In an aspect, a tobacco plant or plant genome provided is mutated or edited to have four or more mutations in four or more NCG genes selected from the group consisting of NCG12, NCG15, NCG16, and NCG17, where the four or more mutations reduce or eliminate the activity or expression of the four or more NCG genes. In another aspect, an edited or mutated tobacco plant having four or more NCG mutations further comprises four or more mutations in four or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the four or more mutations reduce or eliminate the activity or expression of the four or more ERF genes. In an aspect, a tobacco plant or plant genome provided comprises one or more transgenes targeting one or more NCG genes selected from the group consisting of NCG1 to NCG35, where the one or more transgenes reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises one or more transgenes targeting one or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where the one or more transgenes reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises one or more transgenes targeting one or more NCG genes selected from the group consisting of NCG1 to NCG21, where the one or more transgenes reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises one or more transgenes targeting one or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG15, NCG16, and NCG17, where the one or more transgenes reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises one or more transgenes targeting one or more NCG genes selected from the group consisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where the one or more transgenes reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises one or more transgenes targeting one or more NCG genes selected from the group consisting of NCG12, NCG15, NCG16, and NCG17, where the one or more transgenes reduce or eliminate the activity or expression of the one or more NCG genes. In another aspect, a tobacco plant having one or more, two or more, three or more, four or more, or five or more NCG-targeting transgenes further comprises one or more mutations in one or more, two or more, three or more, four or more, or five or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the one or more mutations reduce or eliminate the activity or expression of the one or more ERF genes. In another aspect, a tobacco plant having one or more, two or more, three or more, four or more, or five or more NCG-targeting transgenes further comprises one or more transgenes targeting one or more, two or more, three or more, four or more, or five or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the one or more ERF-targeting transgenes reduce or eliminate the activity or expression of the one or more ERF genes. In an aspect, a tobacco plant or plant genome provided comprises two or more transgenes targeting two or more NCG genes selected from the group consisting of NCG1 to NCG35, where the two or more transgenes reduce or eliminate the activity or expression of the two or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises two or more transgenes targeting two or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where the two or more transgenes reduce or eliminate the activity or expression of the two or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises two or more transgenes targeting two or more NCG genes selected from the group consisting of NCG1 to NCG21, where the two or more transgenes reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises two or more transgenes targeting two or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG15, NCG16, and NCG17, where the two or more transgenes reduce or eliminate the activity or expression of the one or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises two or more transgenes targeting two or more NCG genes selected from the group consisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where the two or more transgenes reduce or eliminate the activity or expression of the two or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises two or more transgenes targeting two or more NCG genes selected from the group consisting of NCG12, NCG15, NCG16, and NCG17, where the two or more transgenes reduce or eliminate the activity or expression of the two or more NCG genes. In another aspect, a tobacco plant having two or more NCG-targeting transgenes further comprises two or more mutations in two or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the two or more mutations reduce or eliminate the activity or expression of the two or more ERF genes. In another aspect, a tobacco plant having two or more NCG-targeting transgenes further comprises two or more transgenes targeting two or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the two or more ERF-targeting transgenes reduce or eliminate the activity or expression of the two or more ERF genes. In an aspect, a tobacco plant or plant genome provided comprises three or more transgenes targeting three or more NCG genes selected from the group consisting of NCG1 to NCG35, where the three or more transgenes reduce or eliminate the activity or expression of the three or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises three or more transgenes targeting three or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where the three or more transgenes reduce or eliminate the activity or expression of the three or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises three or more transgenes targeting three or more NCG genes selected from the group consisting of NCG1 to NCG21, where the three or more transgenes reduce or eliminate the activity or expression of the three or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises three or more transgenes targeting three or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG15, NCG16, and NCG17, where the three or more transgenes reduce or eliminate the activity or expression of the three or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises three or more transgenes targeting three or more NCG genes selected from the group consisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where the three or more transgenes reduce or eliminate the activity or expression of the three or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises three or more transgenes targeting three or more NCG genes selected from the group consisting of NCG12, NCG15, NCG16, and NCG17, where the three or more transgenes reduce or eliminate the activity or expression of the three or more NCG genes. In another aspect, a tobacco plant having three or more NCG-targeting transgenes further comprises three or more mutations in three or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the three or more mutations reduce or eliminate the activity or expression of the three or more ERF genes. In another aspect, a tobacco plant having three or more NCG-targeting transgenes further comprises three or more transgenes targeting three or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the three or more ERF-targeting transgenes reduce or eliminate the activity or expression of the three or more ERF genes. In an aspect, a tobacco plant or plant genome provided comprises four or more transgenes targeting four or more NCG genes selected from the group consisting of NCG1 to NCG35, where the four or more transgenes reduce or eliminate the activity or expression of the four or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises four or more transgenes targeting four or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where the four or more transgenes reduce or eliminate the activity or expression of the four or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises four or more transgenes targeting four or more NCG genes selected from the group consisting of NCG1 to NCG21, where the four or more transgenes reduce or eliminate the activity or expression of the four or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises four or more transgenes targeting four or more NCG genes selected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG15, NCG16, and NCG17, where the four or more transgenes reduce or eliminate the activity or expression of the four or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises four or more transgenes targeting four or more NCG genes selected from the group consisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where the four or more transgenes reduce or eliminate the activity or expression of the four or more NCG genes. In an aspect, a tobacco plant or plant genome provided comprises four or more transgenes targeting four or more NCG genes selected from the group consisting of NCG12, NCG15, NCG16, and NCG17, where the four or more transgenes reduce or eliminate the activity or expression of the four or more NCG genes. In another aspect, a tobacco plant having four or more NCG-targeting transgenes further comprises four or more mutations in four or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the four or more mutations reduce or eliminate the activity or expression of the four or more ERF genes. In another aspect, a tobacco plant having four or more NCG-targeting transgenes further comprises four or more transgenes targeting four or more ERF genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the four or more ERF-targeting transgenes reduce or eliminate the activity or expression of the four or more ERF genes. In an aspect, tobacco plants provided herein comprise second genome modification comprising an inducible promoter operably linked to a transcribable DNA sequence encoding a non-coding RNA for suppression of an ornithine decarboxylase (ODC) gene. In another aspect, a tobacco plant is provided having suppressed MYB8 activity via either transgene suppression, mutagenesis, or targeted genome editing. For simplicity, every instance here mentioning ODC suppression (e.g., operably linked to any particular type of promoters) is equally applicable to MYB8 suppression. In an aspect, tobacco plants provided herein comprise a reduced amount of total conjugated polyamines in leaves relative to the control tobacco plant. In one aspect, tobacco plants provided herein comprise a reduced amount of total conjugated polyamines in roots relative to the control tobacco plant. Used here, conjugated polyamines include, but are not limited to, soluble conjugated polyamines such as phenolamides containing a backbone consisting of a free polyamine (e.g., putrescine, spermine, and/or spermidine) conjugated with one or more phenylpropanoids such as ferulic, caffeic and courmaric acids. Conjugated polyamines also include, but are not limited to, insoluble conjugated polyamines incorporated into structural polymers such as lignin. In an aspect, tobacco plants provided herein comprise a reduced amount of total free polyamines (e.g., putrescine, spermine, and spermidine) in leaves relative to the control tobacco plant. In one aspect, tobacco plants provided herein comprise a reduced amount of total conjugated polyamines in roots relative to the control tobacco plant. In an aspect, tobacco plants provided herein comprise a reduced amount of total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in leaves relative to the control tobacco plant. In one aspect, tobacco plants provided herein comprise a reduced amount of total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in roots relative to the control tobacco plant. In an aspect, tobacco plants provided herein comprise a reduced amount of total free form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in leaves relative to the control tobacco plant. In one aspect, tobacco plants provided herein comprise a reduced amount of total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in roots relative to the control tobacco plant. In an aspect, a characteristic or a trait of a tobacco plant described here are measured at a time selected from the group consisting of immediately before flowering, at topping, 1 week-post-topping (WPT), 2 WPT, 3 WPT, 4 WPT, 5 WPT, 6 WPT, 7 WPT, 8 WPT, and at harvest. In one aspect, tobacco plants provided herein comprising a first and a second genome modification are capable of producing a leaf with a leaf grade comparable to that of a leaf from a control plant. In an aspect, tobacco plants provided herein comprising a first and a second genome modification have a total leaf yield comparable to a control plant. As used herein, “editing” or “genome editing” refers to targeted mutagenesis of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an endogenous plant genome nucleic acid sequence, or removal or replacement of an endogenous plant genome nucleic acid sequence. In an aspect, an edited nucleic acid sequence provided has at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity with an endogenous nucleic acid sequence. In an aspect, an edited nucleic acid sequence provided has at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity with SEQ ID Nos: 23-28, and fragments thereof. In another aspect, an edited nucleic acid sequence provided has at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity with a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs:29-34. Meganucleases, ZFNs, TALENs, CRISPR/Cas9, CRISPR/Csm1 and CRISPR/Cpf1 induce a double-strand DNA break at a target site of a genomic sequence that is then repaired by the natural processes of homologous recombination (HR) or non-homologous end-joining (NHEJ). Sequence modifications then occur at the cleaved sites, which can include deletions or insertions that result in gene disruption in the case of NHEJ, or integration of donor nucleic acid sequences by HR. In an aspect, a method provided comprises editing a plant genome with a nuclease provided to mutate at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more than 10 nucleotides in the plant genome via HR with a donor polynucleotide. In an aspect, a mutation provided is caused by genome editing using a nuclease. In another aspect, a mutation provided is caused by non-homologous end-joining or homologous recombination. In an aspect, a mutation provided here provides a dominant mutant that activates the expression or activity of a gene of interest, e.g., a gene selected from the group consisting of a biosynthetic enzyme, a regulatory transcription factor, a transporter, a catabolic enzyme, or a combination thereof, for one or more antioxidants. Meganucleases, which are commonly identified in microbes, are unique enzymes with high activity and long recognition sequences (>14 bp) resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 bp). The engineering of meganucleases can be more challenging than that of ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity. ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to the cleavage domain of the FokI restriction endonuclease. ZFNs can be designed to cleave almost any long stretch of double-stranded DNA for modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of a non-specific DNA cleavage domain of FokI endonuclease fused to a zinc finger array engineered to bind a target DNA sequence. The DNA-binding domain of a ZFN is typically composed of 3-4 zinc-finger arrays. The amino acids at positions −1, +2, +3, and +6 relative to the start of the zinc finger ∞-helix, which contribute to site-specific binding to the target DNA, can be changed and customized to fit specific target sequences. The other amino acids form the consensus backbone to generate ZFNs with different sequence specificities. Rules for selecting target sequences for ZFNs are known in the art. The FokI nuclease domain requires dimerization to cleave DNA and therefore two ZFNs with their C-terminal regions are needed to bind opposite DNA strands of the cleavage site (separated by 5-7 bp). The ZFN monomer can cute the target site if the two-ZF-binding sites are palindromic. The term ZFN, as used herein, is broad and includes a monomeric ZFN that can cleave double stranded DNA without assistance from another ZFN. The term ZFN is also used to refer to one or both members of a pair of ZFNs that are engineered to work together to cleave DNA at the same site. Without being limited by any scientific theory, because the DNA-binding specificities of zinc finger domains can in principle be re-engineered using one of various methods, customized ZFNs can theoretically be constructed to target nearly any gene sequence. Publicly available methods for engineering zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly. TALENs are artificial restriction enzymes generated by fusing the transcription activator-like effector (TALE) DNA binding domain to a FokI nuclease domain. When each member of a TALEN pair binds to the DNA sites flanking a target site, the FokI monomers dimerize and cause a double-stranded DNA break at the target site. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that work together to cleave DNA at the same site. Transcription activator-like effectors (TALEs) can be engineered to bind practically any DNA sequence. TALE proteins are DNA-binding domains derived from various plant bacterial pathogens of the genusXanthomonas. TheXanthomonaspathogens secrete TALEs into the host plant cell during infection. The TALE moves to the nucleus, where it recognizes and binds to a specific DNA sequence in the promoter region of a specific DNA sequence in the promoter region of a specific gene in the host genome. TALE has a central DNA-binding domain composed of 13-28 repeat monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat-variable diresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine/adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs. Besides the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity. A relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. Software programs such as DNA Works can be used to design TALE constructs. Other methods of designing TALE constructs are known to those of skill in the art. See Doyle et al,Nucleic Acids Research(2012) 40: W117-122; Cermak et al.,Nucleic Acids Research(2011). 39:e82; and tale-nt.cac.cornell.edu/about. A CRISPR/Cas9 system, CRISPR/Csm1, or a CRISPR/Cpf1 system are alternatives to the FokI-based methods ZFN and TALEN. The CRISPR systems are based on RNA-guided engineered nucleases that use complementary base pairing to recognize DNA sequences at target sites. CRISPR/Cas9, CRISPR/Csm1, and a CRISPR/Cpf1 systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids such as viruses by cleaving the foreign DNA in a sequence-dependent manner. The immunity is acquired by the integration of short fragments of the invading DNA known as spacers between two adjacent repeats at the proximal end of a CRISPR locus. The CRISPR arrays, including the spacers, are transcribed during subsequent encounters with invasive DNA and are processed into small interfering CRISPR RNAs (crRNAs) approximately 40 nt in length, which combine with the trans-activating CRISPR RNA (tracrRNA) to activate and guide the Cas9 nuclease. This cleaves homologous double-stranded DNA sequences known as protospacers in the invading DNA. A prerequisite for cleavage is the presence of a conserved protospacer-adjacent motif (PAM) downstream of the target DNA, which usually has the sequence 5-NGG-3 but less frequently NAG. Specificity is provided by the so-called “seed sequence” approximately 12 bases upstream of the PAM, which must match between the RNA and target DNA. Cpf1 and Csm1 act in a similar manner to Cas9, but Cpf1 and Csm1 do not require a tracrRNA. In still another aspect, a tobacco plant provided further comprises one or more mutations in one or more loci encoding a nicotine demethylase (e.g., CYP82E4, CYP82E5, CYP82E10) that confer reduced amounts of nornicotine (See U.S. Pat. Nos. 8,319,011; 8,124,851; 9,187,759; 9,228,194; 9,228,195; 9,247,706) compared to control plant lacking one or more mutations in one or more loci encoding a nicotine demethylase. In an aspect, a modified tobacco plant described further comprises reduced nicotine demethylase activity compared to a control plant when grown and cured under comparable conditions. In a further aspect, a tobacco plant provided further comprises one or more mutations or transgenes providing an elevated level of one or more antioxidants (See U.S. patent application Ser. No. 15/727,523 and PCT Application No. PCT/US2017/055618). In another aspect, a tobacco plant provided further comprises one or more mutations or transgenes providing a reduced level of one or more TSNAs (such as N′-nitrosonornicotine (NNN), 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanatabine (NAT) N′-nitrosoanabasine (NAB)). The present disclosure also provides compositions and methods for inhibiting the expression or function of one or more genes involved in polyamine biosynthesis or regulation thereof, in a plant, particularly plants of theNicotianagenus, including tobacco plants of the various commercial varieties. In an aspect, the present disclosure provides tobacco plants, or part thereof, comprising a heterologous expression cassette comprising an ODC inhibitory sequence. In another aspect, tobacco plants, or part thereof, comprise a heterologous expression cassette comprising an inhibitory sequence of a gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 23-28, and fragments thereof, where the inhibitory sequence is operably linked to a promoter that is functional in a plant cell, and where the inhibitory sequence has at least 90% sequence identity to a fragment of at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides of the sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 23-28, and fragments thereof. As used herein, the terms “inhibit,” “inhibition,” and “inhibiting” are defined as any method known in the art or described herein that decreases the expression or function of a gene product of interest (e.g., a target gene product). “Inhibition” can be in the context of a comparison between two plants, for example, a genetically altered plant versus a wild-type plant. Alternatively, inhibition of expression or function of a target gene product can be in the context of a comparison between plant cells, organelles, organs, tissues, or plant parts within the same plant or between different plants, and includes comparisons between developmental or temporal stages within the same plant or plant part or between plants or plant parts. “Inhibition” includes any relative decrement of function or production of a gene product of interest, up to and including complete elimination of function or production of that gene product. The term “inhibition” encompasses any method or composition that down-regulates translation and/or transcription of the target gene product or functional activity of the target gene product. In an aspect, the mRNA or protein level of one or more genes in a modified plant is less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the mRNA or protein level of the same gene in a plant that is not a mutant or that has not been genetically modified to inhibit the expression of that gene. The term “inhibitory sequence” encompasses any polynucleotide or polypeptide sequence capable of inhibiting the expression or function of a gene involved in nicotine biosynthesis regulation from Nic1b locus in a plant, such as full-length polynucleotide or polypeptide sequences, truncated polynucleotide or polypeptide sequences, fragments of polynucleotide or polypeptide sequences, variants of polynucleotide or polypeptide sequences, sense-oriented nucleotide sequences, antisense-oriented nucleotide sequences, the complement of a sense- or antisense-oriented nucleotide sequence, inverted regions of nucleotide sequences, hairpins of nucleotide sequences, double-stranded nucleotide sequences, single-stranded nucleotide sequences, combinations thereof, and the like. The term “polynucleotide sequence” includes sequences of RNA, DNA, chemically modified nucleic acids, nucleic acid analogs, combinations thereof, and the like. Inhibitory sequences are designated by the name of the target gene product. Thus, a “ODC inhibitory sequence” refers to an inhibitory sequence that is capable of inhibiting the expression of an ODC gene involved in polyamine biosynthesis regulation in a plant, for example, at the level of transcription and/or translation, or which is capable of inhibiting the function of a gene product. When the phrase “capable of inhibiting” is used in the context of a polynucleotide inhibitory sequence, it is intended to mean that the inhibitory sequence itself exerts the inhibitory effect; or, where the inhibitory sequence encodes an inhibitory nucleotide molecule (for example, hairpin RNA, miRNA, or double-stranded RNA polynucleotides), or encodes an inhibitory polypeptide (e.g., a polypeptide that inhibits expression or function of the target gene product), following its transcription (for example, in the case of an inhibitory sequence encoding a hairpin RNA, miRNA, or double-stranded RNA polynucleotide) or its transcription and translation (in the case of an inhibitory sequence encoding an inhibitory polypeptide), the transcribed or translated product, respectively, exerts the inhibitory effect on the target gene product (e.g., inhibits expression or function of the target gene product). A ODC inhibitory sequence disclosed can be a sequence triggering gene silencing via any silencing pathway or mechanism known in the art, including, but not limited to, sense suppression/cosuppression, antisense suppression, double-stranded RNA (dsRNA) interference, hairpin RNA interference and intron-containing hairpin RNA interference, amplicon-mediated interference, ribozymes, small interfering RNA, artificial or synthetic microRNA, and artificial trans-acting siRNA. An ODC inhibitory sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 70 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, and up to the full-length polynucleotide encoding the proteins of the present disclosure, depending upon the desired outcome. In an aspect, a ODC inhibitory sequence can be a fragment of between about 50 and about 400 nucleotides, between about 70 and about 350 nucleotides, between about 90 and about 325 nucleotides, between about 90 and about 300 nucleotides, between about 90 and about 275 nucleotides, between about 100 and about 400 nucleotides, between about 100 and about 350 nucleotides, between about 100 and about 325 nucleotides, between about 100 and about 300 nucleotides, between about 125 and about 300 nucleotides, or between about 125 and about 275 nucleotides in length. The use of the term “polynucleotide” is not intended to limit the present disclosure to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the present disclosure also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. In an aspect, the present disclosure provides recombinant DNA constructs comprising a promoter that is functional in a tobacco cell and operably linked to a polynucleotide that encodes an RNA molecule capable of binding to an RNA encoding a polypeptide having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 28, and fragments thereof, and where the RNA molecule suppresses the expression of the polypeptide. In an aspect, the RNA molecule is selected from the group consisting of a microRNA, an siRNA, and a trans-acting siRNA. In another aspect, the recombinant DNA construct encodes a double stranded RNA. Also provided are transgenic tobacco plants or part thereof, cured tobacco material, or tobacco products comprising these recombinant DNA constructs. In an aspect, these transgenic plants, cured tobacco material, or tobacco products comprise a lower level of nicotine compared to a control tobacco plant without the recombinant DNA construct. Further provided are methods of reducing the nicotine level of a tobacco plant, the method comprising transforming a tobacco plant with any of these recombinant DNA constructs. As used herein, “operably linked” refers to a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. As used herein and when used in reference to a sequence, “heterologous” refers to a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic location by deliberate human intervention. The term also is applicable to nucleic acid constructs, also referred to herein as “polynucleotide constructs” or “nucleotide constructs.” In this manner, a “heterologous” nucleic acid construct is intended to mean a construct that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic location by deliberate human intervention. Heterologous nucleic acid constructs include, but are not limited to, recombinant nucleotide constructs that have been introduced into a plant or plant part thereof, for example, via transformation methods or subsequent breeding of a transgenic plant with another plant of interest. In an aspect, a promoter used is heterologous to the sequence driven by the promoter. In another aspect, a promoter used is heterologous to tobacco. In a further aspect, a promoter used is native to tobacco. In an aspect, a modified tobacco plant described is a cisgenic plant. As used herein, “cisgenesis” or “cisgenic” refers to genetic modification of a plant, plant cell, or plant genome in which all components (e.g., promoter, donor nucleic acid, selection gene) have only plant origins (i.e., no non-plant origin components are used). In an aspect, a modified plant, plant cell, or plant genome provided is cisgenic. Cisgenic plants, plant cells, and plant genomes provided can lead to ready-to-use tobacco lines. In another aspect, a modified tobacco plant provided comprises no non-tobacco genetic material or sequences. As used herein, “gene expression” refers to the biosynthesis or production of a gene product, including the transcription and/or translation of the gene product. In an aspect, recombinant DNA constructs or expression cassettes can also comprise a selectable marker gene for the selection of transgenic cells. Selectable marker genes include, but are not limited to, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP). In an aspect, recombinant DNA constructs or expression cassettes comprise a promoter selected from the group consisting of a constitutive promoter, an inducible promoter, and a tissue-preferred promoter (for example, a leaf-specific or root-specific promoter). Exemplary constitutive promoters include the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Exemplary chemical-inducible promoters include the tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-inducible promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156). Additional exemplary promoters that can be used are those responsible for heat-regulated gene expression, light-regulated gene expression (for example, the pea rbcS-3A; the maize rbcS promoter; the chlorophyll alb-binding protein gene found in pea; or the Arabssu promoter), hormone-regulated gene expression (for example, the abscisic acid (ABA) responsive sequences from the Em gene of wheat; the ABA-inducible HVA1 and HVA22, and rd29A promoters of barley andArabidopsis; and wound-induced gene expression (for example, of wunl), organ specific gene expression (for example, of the tuber-specific storage protein gene; the 23-kDa zein gene from maize described by; or the French bean (β-phaseolin gene), or pathogen-inducible promoters (for example, the PR-1, prp-1, or (β-1,3 glucanase promoters, the fungal-inducible wir1a promoter of wheat, and the nematode-inducible promoters, TobRB7-5A and Hmg-1, of tobacco arid parsley, respectively). In an aspect, a tobacco plant provided further comprises increased or reduced expression of activity of genes involved in nicotine biosynthesis or transport. Genes involved in nicotine biosynthesis include, but are not limited to, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), and S-adenosyl-methionine synthetase (SAMS). Nicotine Synthase, which catalyzes the condensation step between a nicotinic acid derivative and methylpyrrolinium cation, has not been elucidated although two candidate genes (A622 and NBB1) have been proposed. See US 2007/0240728 A1 and US 2008/0120737A1. A622 encodes an isoflavone reductase-like protein. In addition, several transporters may be involved in the translocation of nicotine. A transporter gene, named MATE, has been cloned and characterized (Morita et al., PNAS 106:2447-52 (2009)). In an aspect, a tobacco plant provided further comprises an increased or reduced level of mRNA, protein, or both of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1, compared to a control tobacco plant. In another aspect, a tobacco plants provided further comprises a transgene directly suppressing the expression of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1. In another aspect, a tobacco plant provided further comprises a transgene or mutation suppressing the expression or activity of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1. In another aspect, a tobacco plant provided further comprises a transgene overexpressing one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1. Also disclosed are the transformation of tobacco plants with recombinant constructs or expression cassettes described using any suitable transformation methods known in the art. Methods for introducing polynucleotide sequences into tobacco plants are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. “Stable transformation” refers to transformation where the nucleotide construct of interest introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” is intended to mean that a sequence is introduced into the plant and is only temporally expressed or is only transiently present in the plant. Suitable methods of introducing polynucleotides into plant cells of the present disclosure include microinjection (Crossway et al. (1986)Biotechniques4:320-334), electroporation (Shillito et al. (1987)Meth. Enzymol.153:313-336; Riggs et al. (1986)Proc. Natl. Acad. Sci. USA83:5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,104,310, 5,149,645, 5,177,010, 5,231,019, 5,463,174, 5,464,763, 5,469,976, 4,762,785, 5,004,863, 5,159,135, 5,563,055, and 5,981,840), direct gene transfer (Paszkowski et al. (1984)EMBO J.3:2717-2722), and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050, 5,141,131, 5,886,244, 5,879,918, and 5,932,782; Tomes et al. (1995) inPlant Cell, Tissue, and Organ Culture Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988)Biotechnology6:923-926). Also see Weissinger et al. (1988)Ann. Rev. Genet.22:421-477; Christou et al. (1988)Plant Physiol.87:671-674 (soybean); McCabe et al. (1988)Bio/Technology6:923-926 (soybean); Finer and McMullen (1991)In Vitro Cell Dev. Biol.27P: 175-182 (soybean); Singh et al. (1998)Theor. Appl. Genet.96:319-324 (soybean); De Wet et al. (1985) inThe Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports9:415-418 and Kaeppler et al. (1992)Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell4:1495-1505 (electroporation). In another aspect, recombinant constructs or expression cassettes may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating an expression cassette of the present disclosure within a viral DNA or RNA molecule. It is recognized that promoters for use in expression cassettes also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996)Molecular Biotechnology5:209-221. Any plant tissue that can be subsequently propagated using clonal methods, whether by organogenesis or embryogenesis, may be transformed with a recombinant construct or an expression cassette. By “organogenesis” in intended the process by which shoots and roots are developed sequentially from meristematic centers. By “embryogenesis” is intended the process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. Exemplary tissues that are suitable for various transformation protocols described include, but are not limited to, callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems) and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem), hypocotyls, cotyledons, leaf disks, pollen, embryos, and the like. In an aspect, a tobacco plant provided is from a tobacco type selected from the group consisting of flue-cured tobacco, air-cured tobacco, dark air-cured tobacco, dark fire-cured tobacco, Galpao tobacco, and Oriental tobacco. In another aspect, a tobacco plant provided is from a tobacco type selected from the group consisting of Burley tobacco, Maryland tobacco, and dark tobacco. In an aspect, a tobacco plant provided is in a flue-cured tobacco background or exhibits one or more flue-cured tobacco characteristic described here. Flue-cured tobaccos (also called Virginia or bright tobaccos) amount to approximately 40% of world tobacco production. Flue-cured tobaccos are often also referred to as “bright tobacco” because of the golden-yellow to deep-orange color it reaches during curing. Flue-cured tobaccos have a light, bright aroma and taste. Flue-cured tobaccos are generally high in sugar and low in oils. Major flue-cured tobacco growing countries are Argentina, Brazil, China, India, Tanzania and the U.S. In an aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in a flue-cured tobacco background selected from the group consisting of CC 13, CC 27, CC 33, CC 37, CC 65, CC 67, CC 700, GF 318, GL 338, GL 368, GL 939, K 346, K 399, K326, NC 102, NC 196, NC 291, NC 297, NC 299, NC 471, NC 55, NC 606, NC 71, NC 72, NC 92, PVH 1118, PVH 1452, PVH 2110, SPEIGHT 168, SPEIGHT 220, SPEIGHT 225, SPEIGHT 227, SPEIGHT 236, and any variety essentially derived from any one of the foregoing varieties. In another aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in a flue-cured tobacco background selected from the group consisting of Coker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL 939, K 149, K326, K 340, K 346, K 358, K 394, K 399, K 730, NC 27NF, NC 37NF, NC 55, NC 60, NC 71, NC 72, NC 82, NC 95, NC 297, NC 606, NC 729, NC 2326, McNair 373, McNair 944, Ox 207, Ox 414 NF, Reams 126, Reams 713, Reams 744, RG 8, RG 11, RG 13, RG 17, RG 22, RG 81, RG H4, RG H51, Speight H-20, Speight G-28, Speight G-58, Speight G-70, Speight G-108, Speight G-111, Speight G-117, Speight 168, Speight 179, Speight NF-3, Va 116, Va 182, and any variety essentially derived from any one of the foregoing varieties. See WO 2004/041006 A1. In a further aspect, low-alkaloid or low-nicotine tobacco plants, seeds, hybrids, varieties, or lines are in any flue cured background selected from the group consisting of K326, K346, and NC196. In an aspect, a tobacco plant provided is in an air-cured tobacco background or exhibits one or more air-cured tobacco characteristic described here. Air-cured tobaccos include Burley, Md., and dark tobaccos. The common factor is that curing is primarily without artificial sources of heat and humidity. Burley tobaccos are light to dark brown in color, high in oil, and low in sugar. Burley tobaccos are air-cured in barns. Major Burley growing countries are Argentina, Brazil, Italy, Malawi, and the U.S. Maryland tobaccos are extremely fluffy, have good burning properties, low nicotine and a neutral aroma. Major Maryland growing countries include the U.S. and Italy. In an aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in a Burley tobacco background selected from the group consisting of Clay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN 86, TN 90, TN 97, R 610, R 630, R 711, R 712, NCBH 129, Bu 21×Ky 10, HBO4P, Ky 14×L 8, Kt 200, Newton 98, Pedigo 561, Pf561 and Va 509. In a further aspect, low-alkaloid or low-nicotine tobacco plants, seeds, hybrids, varieties, or lines are in any Burley background selected from the group consisting of TN 90, KT 209, KT 206, KT212, and HB 4488. In another aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in a Maryland tobacco background selected from the group consisting of Md 10, Md 40, Md 201, Md 609, Md 872 and Md 341. In an aspect, a tobacco plant provided is in a dark air-cured tobacco background or exhibits one or more dark air-cured tobacco characteristic described here. Dark air-cured tobaccos are distinguished from other types primarily by its curing process which gives dark air-cured tobacco its medium- to dark-brown color and distinct aroma. Dark air-cured tobaccos are mainly used in the production of chewing tobacco and snuff. In an aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in a dark air-cured tobacco background selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One sucker, Green River, Va. sun-cured, and Paraguan Passado. In an aspect, a tobacco plant provided is in a dark fire-cured tobacco background or exhibits one or more dark fire-cured tobacco characteristic described here. Dark fire-cured tobaccos are generally cured with low-burning wood fires on the floors of closed curing barns. Their leaves have low sugar content but high nicotine content. Dark fire-cured tobaccos are used for making pipe blends, cigarettes, chewing tobacco, snuff and strong-tasting cigars. Major growing regions for dark fire-cured tobaccos are Tennessee, Kentucky, and Virginia, USA. In an aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in a dark fire-cured tobacco background selected from the group consisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stalk Black Mammoth, DT 508, DT 518, DT 592, KY 171, DF 911, DF 485, TN D94, TN D950, VA 309, and VA 359. In an aspect, a tobacco plant provided is in an Oriental tobacco background or exhibits one or more Oriental tobacco characteristic described here. Oriental tobaccos are also referred to as Greek, aroma and Turkish tobaccos due to the fact that they are typically grown in eastern Mediterranean regions such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, and Romania. The small plant and leaf size, characteristic of today's Oriental varieties, as well as its unique aroma properties are a result of the plant's adaptation to the poor soil and stressful climatic conditions in which it develop over many past centuries. In an aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in an Oriental tobacco background selected from the group consisting of Izmir, Katerini, Samsun, Basma and Krumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Semdinli, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra, Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir, and any variety essentially derived from any one of the foregoing varieties. In an aspect, low-alkaloid or low-nicotine tobacco plants, seeds, hybrids, varieties, or lines are essentially derived from or in the genetic background of BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14×L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’ tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, or VA359, Maryland 609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, ShireyLC, or any commercial tobacco variety according to standard tobacco breeding techniques known in the art. All foregoing mentioned specific varieties of dark air-cured, Burley, Md., dark fire-cured, or Oriental type are listed only for exemplary purposes. Any additional dark air-cured, Burley, Md., dark fire-cured, Oriental varieties are also contemplated in the present application. Also provided are populations of tobacco plants described. In an aspect, a population of tobacco plants has a planting density of between about 5,000 and about 8000, between about 5,000 and about 7,600, between about 5,000 and about 7,200, between about 5,000 and about 6,800, between about 5,000 and about 6,400, between about 5,000 and about 6,000, between about 5,000 and about 5,600, between about 5,000 and about 5,200, between about 5,200 and about 8,000, between about 5,600 and about 8,000, between about 6,000 and about 8,000, between about 6,400 and about 8,000, between about 6,800 and about 8,000, between about 7,200 and about 8,000, or between about 7,600 and about 8,000 plants per acre. In another aspect, a population of tobacco plants is in a soil type with low to medium fertility. Also provided are containers of seeds from tobacco plants described. A container of tobacco seeds of the present disclosure may contain any number, weight, or volume of seeds. For example, a container can contain at least, or greater than, about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, the container can contain at least, or greater than, about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more seeds. Containers of tobacco seeds may be any container available in the art. By way of non-limiting example, a container may be a box, a bag, a packet, a pouch, a tape roll, a tube, or a bottle. Also provided is cured tobacco material made from a low-alkaloid or low-nicotine tobacco plant described. Further provided is cured tobacco material made from a tobacco plant described with higher levels of total alkaloid or nicotine. “Curing” is the aging process that reduces moisture and brings about the destruction of chlorophyll giving tobacco leaves a golden color and by which starch is converted to sugar. Cured tobacco therefore has a higher reducing sugar content and a lower starch content compared to harvested green leaf. In an aspect, green leaf tobacco provided can be cured using conventional means, e.g., flue-cured, barn-cured, fire-cured, air-cured or sun-cured. See, for example, Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford) for a description of different types of curing methods. Cured tobacco is usually aged in a wooden drum (e.g., a hogshead) or cardboard cartons in compressed conditions for several years (e.g., two to five years), at a moisture content ranging from 10% to about 25%. See, U.S. Pat. Nos. 4,516,590 and 5,372,149. Cured and aged tobacco then can be further processed. Further processing includes conditioning the tobacco under vacuum with or without the introduction of steam at various temperatures, pasteurization, and fermentation. Fermentation typically is characterized by high initial moisture content, heat generation, and a 10 to 20% loss of dry weight. See, e.g., U.S. Pat. Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S. Publication No. 2005/0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cure, aged, and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, for example, U.S. Pat. Nos. 4,528,993; 4,660,577; and 4,987,907. In an aspect, the cured tobacco material of the present disclosure is sun-cured. In another aspect, the cured tobacco material of the present disclosure is flue-cured, air-cured, or fire-cured. Tobacco material obtained from the tobacco lines, varieties or hybrids of the present disclosure can be used to make tobacco products. As used herein, “tobacco product” is defined as any product made or derived from tobacco that is intended for human use or consumption. Tobacco products provided include, without limitation, cigarette products (e.g., cigarettes and bidi cigarettes), cigar products (e.g., cigar wrapping tobacco and cigarillos), pipe tobacco products, products derived from tobacco, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, tabs, shaped parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, and the like. See, e.g., U.S. Patent Publication No. US 2006/0191548. As used herein, “cigarette” refers a tobacco product having a “rod” and “filler”. The cigarette “rod” includes the cigarette paper, filter, plug wrap (used to contain filtration materials), tipping paper that holds the cigarette paper (including the filler) to the filter, and all glues that hold these components together. The “filler” includes (1) all tobaccos, including but not limited to reconstituted and expanded tobacco, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials and other spices that may accompany tobaccos rolled within the cigarette paper), (3) casings, (4) flavorings, and (5) all other additives (that are mixed into tobaccos and substitutes and rolled into the cigarette). As used herein, “reconstituted tobacco” refers to a part of tobacco filler made from tobacco dust and other tobacco scrap material, processed into sheet form and cut into strips to resemble tobacco. In addition to the cost savings, reconstituted tobacco is very important for its contribution to cigarette taste from processing flavor development using reactions between ammonia and sugars. As used herein, “expanded tobacco” refers to a part of tobacco filler which is processed through expansion of suitable gases so that the tobacco is “puffed” resulting in reduced density and greater filling capacity. It reduces the weight of tobacco used in cigarettes. Tobacco products derived from plants of the present disclosure also include cigarettes and other smoking articles, particularly those smoking articles including filter elements, where the rod of smokable material includes cured tobacco within a tobacco blend. In an aspect, a tobacco product of the present disclosure is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah tobacco, shredded tobacco, and cut tobacco. In another aspect, a tobacco product of the present disclosure is a smokeless tobacco product. Smokeless tobacco products are not combusted and include, but not limited to, chewing tobacco, moist smokeless tobacco, snus, and dry snuff. Chewing tobacco is coarsely divided tobacco leaf that is typically packaged in a large pouch-like package and used in a plug or twist. Moist smokeless tobacco is a moist, more finely divided tobacco that is provided in loose form or in pouch form and is typically packaged in round cans and used as a pinch or in a pouch placed between an adult tobacco consumer's cheek and gum. Snus is a heat treated smokeless tobacco. Dry snuff is finely ground tobacco that is placed in the mouth or used nasally. In a further aspect, a tobacco product of the present disclosure is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. In yet another aspect, a tobacco product of the present disclosure is selected from the group consisting of an electronically heated cigarette, an e-cigarette, an electronic vaporing device. In an aspect, a tobacco product of the present disclosure can be a blended tobacco product. In one aspect, a blended tobacco product comprises cured tobacco materials. In an aspect, a cured tobacco material constitutes about at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of cured tobacco in a tobacco blend by weight. In one aspect, a cured tobacco material constitutes about at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of cured tobacco in a tobacco blend by volume. In an aspect, a tobacco product of the present disclosure can be a low nicotine tobacco product. In a further aspect, a tobacco product of the present disclosure may comprise nornicotine at a level of less than about 3 mg/g. For example, the nornicotine content in such a product can be about 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 pg/g, 500 pg/g, 250 pg/g, 100 pg/g, 75 pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, or undetectable. In an aspect, cured tobacco material or tobacco products provided comprise an average nicotine or total alkaloid level selected from the group consisting of about 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, and 9% on a dry weight basis. In another aspect, cured tobacco material or tobacco products provided comprise an average nicotine or total alkaloid level selected from the group consisting of about between 0.01% and 0.02%, between 0.02% and 0.05%, between 0.05% and 0.75%, between 0.75% and 0.1%, between 0.1% and 0.15%, between 0.15% and 0.2%, between 0.2% and 0.3%, between 0.3% and 0.35%, between 0.35% and 0.4%, between 0.4% and 0.5%, between 0.5% and 0.6%, between 0.6% and 0.7%, between 0.7% and 0.8%, between 0.8% and 0.9%, between 0.9% and 1%, between 1% and 1.1%, between 1.1% and 1.2%, between 1.2% and 1.3%, between 1.3% and 1.4%, between 1.4% and 1.5%, between 1.5% and 1.6%, between 1.6% and 1.7%, between 1.7% and 1.8%, between 1.8% and 1.9%, between 1.9% and 2%, between 2% and 2.1%, between 2.1% and 2.2%, between 2.2% and 2.3%, between 2.3% and 2.4%, between 2.4% and 2.5%, between 2.5% and 2.6%, between 2.6% and 2.7%, between 2.7% and 2.8%, between 2.8% and 2.9%, between 2.9% and 3%, between 3% and 3.1%, between 3.1% and 3.2%, between 3.2% and 3.3%, between 3.3% and 3.4%, between 3.4% and 3.5%, and between 3.5% and 3.6% on a dry weight basis. In a further aspect, cured tobacco material or tobacco products provided comprise an average nicotine or total alkaloid level selected from the group consisting of about between 0.01% and 0.1%, between 0.02% and 0.2%, between 0.03% and 0.3%, between 0.04% and 0.4%, between 0.05% and 0.5%, between 0.75% and 1%, between 0.1% and 1.5%, between 0.15% and 2%, between 0.2% and 3%, and between 0.3% and 3.5% on a dry weight basis. The present disclosure also provides methods for breeding tobacco lines, cultivars, or varieties comprising a desirable level of total alkaloid or nicotine, e.g., low nicotine or nicotine free. Breeding can be carried out via any known procedures. DNA fingerprinting, SNP mapping, haplotype mapping or similar technologies may be used in a marker-assisted selection (MAS) breeding program to transfer or breed a desirable trait or allele into a tobacco plant. For example, a breeder can create segregating populations in a F2or backcross generation using F1 hybrid plants or further crossing the F1 hybrid plants with other donor plants with an agronomically desirable genotype. Plants in the F2or backcross generations can be screened for a desired agronomic trait or a desirable chemical profile using one of the techniques known in the art or listed herein. Depending on the expected inheritance pattern or the MAS technology used, self-pollination of selected plants before each cycle of backcrossing to aid identification of the desired individual plants can be performed. Backcrossing or other breeding procedure can be repeated until the desired phenotype of the recurrent parent is recovered. A recurrent parent in the present disclosure can be a flue-cured variety, a Burley variety, a dark air-cured variety, a dark fire-cured variety, or an Oriental variety. Other breeding techniques can be found, for example, in Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go, Inc., New York, N.Y., incorporated herein by reference in their entirety. Results of a plant breeding program using the tobacco plants described includes useful lines, cultivars, varieties, progeny, inbreds, and hybrids of the present disclosure. As used herein, the term “variety” refers to a population of plants that share constant characteristics which separate them from other plants of the same species. A variety is often, although not always, sold commercially. While possessing one or more distinctive traits, a variety is further characterized by a very small overall variation between individuals within that variety. A “pure line” variety may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. A variety can be essentially derived from another line or variety. As defined by the International Convention for the Protection of New Varieties of Plants (Dec. 2, 1961, as revised at Geneva on Nov. 10, 1972; on Oct. 23, 1978; and on Mar. 19, 1991), a variety is “essentially derived” from an initial variety if: a) it is predominantly derived from the initial variety, or from a variety that is predominantly derived from the initial variety, while retaining the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety; b) it is clearly distinguishable from the initial variety; and c) except for the differences which result from the act of derivation, it conforms to the initial variety in the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety. Essentially derived varieties can be obtained, for example, by the selection of a natural or induced mutant, a somaclonal variant, a variant individual from plants of the initial variety, backcrossing, or transformation. A first tobacco variety and a second tobacco variety from which the first variety is essentially derived, are considered as having essentially identical genetic background. A “line” as distinguished from a variety most often denotes a group of plants used non-commercially, for example in plant research. A line typically displays little overall variation between individuals for one or more traits of interest, although there may be some variation between individuals for other traits. As used herein, “locus” is a chromosome region where a polymorphic nucleic acid, trait determinant, gene, or marker is located. The loci of this disclosure comprise one or more polymorphisms in a population; e.g., alternative alleles are present in some individuals. As used herein, “allele” refers to an alternative nucleic acid sequence at a particular locus. The length of an allele can be as small as 1 nucleotide base, but is typically larger. For example, a first allele can occur on one chromosome, while a second allele occurs on a second homologous chromosome, e.g., as occurs for different chromosomes of a heterozygous individual, or between different homozygous or heterozygous individuals in a population. As used herein, the term “chromosome interval” designates a contiguous linear span of genomic DNA that resides on a single chromosome. As used herein, “introgression” or “introgress” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. As used herein, “crossed” or “cross” means to produce progeny via fertilization (e.g. cells, seeds or plants) and includes crosses between plants (sexual) and self fertilization (selfing). As used herein, “backcross” and “backcrossing” refer to the process whereby a progeny plant is repeatedly crossed back to one of its parents. In a backcrossing scheme, the “donor” parent refers to the parental plant with the desired gene or locus to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. The initial cross gives rise to the F1 generation. The term “BC1” refers to the second use of the recurrent parent, “BC2” refers to the third use of the recurrent parent, and so on. In an aspect, a backcross is performed repeatedly, with a progeny individual of each successive backcross generation being itself backcrossed to the same parental genotype. As used herein, “single gene converted” or “single gene conversion” refers to plants that are developed using a plant breeding technique known as backcrossing, or via genetic engineering, where essentially all of the desired morphological and physiological characteristics of a variety are recovered in addition to the single gene transferred into the variety via the backcrossing technique or via genetic engineering. As used herein, “elite variety” means any variety that has resulted from breeding and selection for superior agronomic performance. As used herein, “selecting” or “selection” in the context of marker-assisted selection or breeding refer to the act of picking or choosing desired individuals, normally from a population, based on certain pre-determined criteria. As used herein, the term “trait” refers to one or more detectable characteristics of a cell or organism which can be influenced by genotype. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, genomic analysis, an assay for a particular disease tolerance, etc. In some cases, a phenotype is directly controlled by a single gene or genetic locus, e.g., a “single gene trait.” In other cases, a phenotype is the result of several genes. It is understood that any tobacco plant of the present disclosure can further comprise additional agronomically desirable traits, for example, by transformation with a genetic construct or transgene using a technique known in the art. Without limitation, an example of a desired trait is herbicide resistance, pest resistance, disease resistance; high yield; high grade index value; curability; curing quality; mechanical harvestability; holding ability; leaf quality; height, plant maturation (e.g., early maturing, early to medium maturing, medium maturing, medium to late maturing, or late maturing); stalk size (e.g., a small, medium, or a large stalk); or leaf number per plant (e.g., a small (e.g., 5-10 leaves), medium (e.g., 11-15 leaves), or large (e.g., 16-21) number of leaves), or any combination. In an aspect, low-nicotine or nicotine-free tobacco plants or seeds disclosed comprise one or more transgenes expressing one or more insecticidal proteins, such as, for example, a crystal protein ofBacillus thuringiensisor a vegetative insecticidal protein fromBacillus cereus, such as VIP3 (see, for example, Estruch et al. (1997)Nat. Biotechnol.15:137). In another aspect, tobacco plants further comprise an introgressed trait conferring resistance to brown stem rot (U.S. Pat. No. 5,689,035) or resistance to cyst nematodes (U.S. Pat. No. 5,491,081). The present disclosure also provides tobacco plants comprising an altered nicotine or total alkaloid level but having a yield comparable to the yield of corresponding initial tobacco plants without such a nicotine level alternation. In an aspect, a low-nicotine or nicotine-free tobacco variety provides a yield selected from the group consisting of about between 1200 and 3500, between 1300 and 3400, between 1400 and 3300, between 1500 and 3200, between 1600 and 3100, between 1700 and 3000, between 1800 and 2900, between 1900 and 2800, between 2000 and 2700, between 2100 and 2600, between 2200 and 2500, and between 2300 and 2400 lbs/acre. In another aspect, a low-nicotine or nicotine-free tobacco variety provides a yield selected from the group consisting of about between 1200 and 3500, between 1300 and 3500, between 1400 and 3500, between 1500 and 3500, between 1600 and 3500, between 1700 and 3500, between 1800 and 3500, between 1900 and 3500, between 2000 and 3500, between 2100 and 3500, between 2200 and 3500, between 2300 and 3500, between 2400 and 3500, between 2500 and 3500, between 2600 and 3500, between 2700 and 3500, between 2800 and 3500, between 2900 and 3500, between 3000 and 3500, and between 3100 and 3500 lbs/acre. In a further aspect, low-nicotine or nicotine-free tobacco plants provide a yield between 65% and 130%, between 70% and 130%, between 75% and 130%, between 80% and 130%, between 85% and 130%, between 90% and 130%, between 95% and 130%, between 100% and 130%, between 105% and 130%, between 110% and 130%, between 115% and 130%, or between 120% and 130% of the yield of a control plant having essentially identical genetic background except a nic1b mutation, a nic2 mutation, a Nic1b transgene, a Nic2 transgene, or combinations thereof. In a further aspect, low-nicotine or nicotine-free tobacco plants provide a yield between 70% and 125%, between 75% and 120%, between 80% and 115%, between 85% and 110%, or between 90% and 100% of the yield of a control plant having essentially identical genetic background except a nic1 mutation, a nic2 mutation, a Nic1 transgene, a Nic2 transgene, or combinations thereof. In an aspect, a tobacco plant (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobacco variety) does not exhibit one or more, two or more, three or more, or all of the LA BU21 traits selected from the group consisting of lower yield, delayed ripening and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing. In an aspect, a tobacco plant disclosed (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobacco variety) does not exhibit two or more of the LA BU21 traits selected from the group consisting of lower yield, delayed ripening and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing. In an aspect, a tobacco plant disclosed (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobacco variety) does not exhibit three or more of the LA BU21 traits selected from the group consisting of lower yield, delayed ripening and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing. In an aspect, a tobacco plant disclosed (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobacco variety) exhibits at a lower level compared to LA BU21, LAFC53, or LN KY171, one or more, two or more, three or more, or all of the LA BU21 traits selected from the group consisting of lower yield, delayed ripening and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing. In an aspect, a tobacco plant disclosed (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobacco variety) exhibits at a lower level compared to LA BU21, LAFC53, or LN KY171, two or more of the LA BU21 traits selected from the group consisting of lower yield, delayed ripening and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing. In an aspect, a tobacco plant disclosed (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobacco variety) exhibits at a lower level compared to LA BU21, LAFC53, or LN KY171, three or more, or all of the LA BU21 traits selected from the group consisting of lower yield, delayed ripening and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing. In an aspect, a modified tobacco plant (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobacco variety) comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) without substantially impacting a trait selected from the group consisting of yield, ripening and senescence, susceptibility to insect herbivory, polyamine content after topping, chlorophyll level, mesophyll cell number per unit leaf area, and end-product quality after curing. In an aspect, a modified tobacco plant comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a trait substantially comparable to an unmodified control plant, where the trait is selected from the group consisting of yield, ripening and senescence, susceptibility to insect herbivory, polyamine content after topping, chlorophyll level, mesophyll cell number per unit leaf area, and end-product quality after curing. In an aspect, a modified tobacco plant comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a yield which is more than 80%, more than 85%, more than 90%, more than 95%, more than 100%, more than 105%, more than 110%, more than 115%, more than 120%, more than 125%, more than 130%, more than 135%, or more than 140% relative to the yield of an unmodified control plant. In an aspect, a modified tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a yield which is between 70% and 140%, between 75% and 135%, between 80% and 130%, between 85% and 125%, between 90% and 120%, between 95% and 115%, or between 100% and 110% relative to the yield of an unmodified control plant. In an aspect, a modified tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a yield which is between 70% and 80%, between 75% and 85%, between 80% and 90%, between 85% and 95%, between 90% and 100%, between 95% and 105%, between 105% and 115%, between 110% and 120%, between 115% to 125%, between 120% and 130%, between 125 and 135%, or between 130% and 140% relative to the yield of an unmodified control plant. In an aspect, a modified tobacco plant comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a polyamine content after topping which is more than 80%, more than 85%, more than 90%, more than 95%, more than 100%, more than 105%, more than 110%, more than 115%, more than 120%, more than 125%, more than 130%, more than 135%, or more than 140% relative to the polyamine content after topping of an unmodified control plant. In an aspect, a modified tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a polyamine content after topping which is between 70% and 140%, between 75% and 135%, between 80% and 130%, between 85% and 125%, between 90% and 120%, between 95% and 115%, or between 100% and 110% relative to the polyamine content after topping of an unmodified control plant. In an aspect, a modified tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a polyamine content after topping which is between 70% and 80%, between 75% and 85%, between 80% and 90%, between 85% and 95%, between 90% and 100%, between 95% and 105%, between 105% and 115%, between 110% and 120%, between 115% to 125%, between 120% and 130%, between 125 and 135%, or between 130% and 140% relative to the polyamine content after topping of an unmodified control plant. In an aspect, a modified tobacco plant comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a chlorophyll level which is more than 80%, more than 85%, more than 90%, more than 95%, more than 100%, more than 105%, more than 110%, more than 115%, more than 120%, more than 125%, more than 130%, more than 135%, or more than 140% relative to the chlorophyll level of an unmodified control plant. In an aspect, a modified tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a chlorophyll level which is between 70% and 140%, between 75% and 135%, between 80% and 130%, between 85% and 125%, between 90% and 120%, between 95% and 115%, or between 100% and 110% relative to the chlorophyll level of an unmodified control plant. In an aspect, a modified tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a chlorophyll level which is between 70% and 80%, between 75% and 85%, between 80% and 90%, between 85% and 95%, between 90% and 100%, between 95% and 105%, between 105% and 115%, between 110% and 120%, between 115% to 125%, between 120% and 130%, between 125 and 135%, or between 130% and 140% relative to the chlorophyll level of an unmodified control plant. In an aspect, a modified tobacco plant comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a mesophyll cell number per unit leaf area which is more than 80%, more than 85%, more than 90%, more than 95%, more than 100%, more than 105%, more than 110%, more than 115%, more than 120%, more than 125%, more than 130%, more than 135%, or more than 140% relative to the mesophyll cell number per unit leaf area of an unmodified control plant. In an aspect, a modified tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a mesophyll cell number per unit leaf area which is between 70% and 140%, between 75% and 135%, between 80% and 130%, between 85% and 125%, between 90% and 120%, between 95% and 115%, or between 100% and 110% relative to the mesophyll cell number per unit leaf area of an unmodified control plant. In an aspect, a modified tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a mesophyll cell number per unit leaf area which is between 70% and 80%, between 75% and 85%, between 80% and 90%, between 85% and 95%, between 90% and 100%, between 95% and 105%, between 105% and 115%, between 110% and 120%, between 115% to 125%, between 120% and 130%, between 125 and 135%, or between 130% and 140% relative to the mesophyll cell number per unit leaf area of an unmodified control plant. In an aspect, a low-nicotine or nicotine-free tobacco variety is adapted for machine harvesting. In another aspect, a low-nicotine or nicotine-free tobacco variety disclosed is harvested mechanically. In an aspect, a method for improving leaf quality in a reduced-alkaloid tobacco plant is provided, the method comprising: growing a tobacco plant; reducing the level of putrescine in the tobacco plant, and harvesting leaves from the tobacco plant. In an aspect, a method for improving leaf quality in a reduced-alkaloid tobacco plant is provided, the method comprising: growing a tobacco plant; suppressing the expression or activity of an ornithine decarboxylase (ODC) gene in the tobacco plant, and harvesting leaves from the tobacco plant. In one aspect, the suppressing step is within 2, 4, 6, or 8 WPT. In an aspect, the suppressing step comprises suppressing a ODC gene both prior to and after topping a tobacco plant. In one aspect, the suppressing step does not include the use of a chemical inhibitor. In an aspect, the suppressing step is accomplished by inducing the expression of a non-coding RNA for suppression of an ornithine decarboxylase (ODC) gene. In one aspect, the suppressing step comprises applying an ODC inhibitor to the tobacco plant. In an aspect, the suppressing is accomplished by applying an ODC inhibitor to a tobacco plant. In one aspect, an ODC inhibitor is DFMO. In an aspect, tobacco plants provided are hybrid plants. Hybrids can be produced by preventing self-pollination of female parent plants (e.g., seed parents) of a first variety, permitting pollen from male parent plants of a second variety to fertilize the female parent plants, and allowing F1 hybrid seeds to form on the female plants. Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation can be prevented on the female parent plants using a form of male sterility. For example, male sterility can be produced by male sterility (MS), or transgenic male sterility where a transgene inhibits microsporogenesis and/or pollen formation, or self-incompatibility. Female parent plants containing MS are particularly useful. In aspects in which the female parent plants are MS, pollen may be harvested from male fertile plants and applied manually to the stigmas of MS female parent plants, and the resulting F1 seed is harvested. Plants can be used to form single-cross tobacco F1 hybrids. Pollen from a male parent plant is manually transferred to an emasculated female parent plant or a female parent plant that is male sterile to form F1 seed. Alternatively, three-way crosses can be carried out where a single-cross F1 hybrid is used as a female parent and is crossed with a different male parent. As another alternative, double-cross hybrids can be created where the F1 progeny of two different single-crosses are themselves crossed. Self-incompatibility can be used to particular advantage to prevent self-pollination of female parents when forming a double-cross hybrid. In an aspect, a low-nicotine or nicotine-free tobacco variety is male sterile. In another aspect, a low-nicotine or nicotine-free tobacco variety is cytoplasmic male sterile. Male sterile tobacco plants may be produced by any method known in the art. Methods of producing male sterile tobacco are described in Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go, Inc., New York, N.Y. 761 pp. In a further aspect, tobacco parts provided include, but are not limited to, a leaf, a stem, a root, a seed, a flower, pollen, an anther, an ovule, a pedicel, a fruit, a meristem, a cotyledon, a hypocotyl, a pod, an embryo, endosperm, an explant, a callus, a tissue culture, a shoot, a cell, and a protoplast. In an aspect, tobacco part provided does not include seed. In an aspect, this disclosure provides tobacco plant cells, tissues, and organs that are not reproductive material and do not mediate the natural reproduction of the plant. In another aspect, this disclosure also provides tobacco plant cells, tissues, and organs that are reproductive material and mediate the natural reproduction of the plant. In another aspect, this disclosure provides tobacco plant cells, tissues, and organs that cannot maintain themselves via photosynthesis. In another aspect, this disclosure provides somatic tobacco plant cells. Somatic cells, contrary to germline cells, do not mediate plant reproduction. The provided cells, tissues and organs may be from seed, fruit, leaf, cotyledon, hypocotyl, meristem, embryos, endosperm, root, shoot, stem, pod, flower, inflorescence, stalk, pedicel, style, stigma, receptacle, petal, sepal, pollen, anther, filament, ovary, ovule, pericarp, phloem, vascular tissue. In another aspect, this disclosure provides a tobacco plant chloroplast. In a further aspect, this disclosure provides epidermal cells, stomata cell, leaf or root hairs, a storage root, or a tuber. In another aspect, this disclosure provides a tobacco protoplast. Skilled artisans understand that tobacco plants naturally reproduce via seeds, not via asexual reproduction or vegetative propagation. In an aspect, this disclosure provides tobacco endosperm. In another aspect, this disclosure provides tobacco endosperm cells. In a further aspect, this disclosure provides a male or female sterile tobacco plant, which cannot reproduce without human intervention. In an aspect, the present disclosure provides a nucleic acid molecule comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 23-28, and fragments thereof. In an aspect, the present disclosure provides a polypeptide or protein comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-34. In another aspect, the present disclosure provides a biologically active variant of a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-34. A biologically active variant of a protein of the present disclosure may differ from that protein by as few as 1-15 amino acid residues, as few as 10, as few as 9, as few as 8, as few as 7, as few as 6, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue. Also provided are orthologous genes or proteins of genes or proteins from the ODC pathway. “Orthologs” are genes derived from a common ancestral gene and which are found in different species as a result of speciation. Orthologs may share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity or similarity at the nucleotide sequence and/or the protein sequence level. Functions of orthologs are often highly conserved among species. As used herein, the term “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are deemed to have “sequence similarity” or “similarity.” Nucleic acid molecules, polypeptides, or proteins provided can be isolated or substantially purified. An “isolated” or “purified” nucleic acid molecule, polypeptide, protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. For example, an isolated or purified polynucleotide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. The present disclosure further provides a method manufacturing a tobacco product comprising tobacco material from tobacco plants disclosed. In an aspect, methods comprise conditioning aged tobacco material made from tobacco plants to increase its moisture content from between about 12.5% and about 13.5% to about 21%, blending the conditioned tobacco material to produce a desirable blend. In an aspect, the method of manufacturing a tobacco product further comprises casing or flavoring the blend. Generally, during the casing process, casing or sauce materials are added to blends to enhance their quality by balancing the chemical composition and to develop certain desired flavor characteristics. Further details for the casing process can be found in Tobacco Production, Chemistry and Technology, Edited by L. Davis and M. Nielsen, Blackwell Science, 1999. Tobacco material provided can be also processed using methods including, but not limited to, heat treatment (e.g., cooking, toasting), flavoring, enzyme treatment, expansion and/or curing. Both fermented and non-fermented tobaccos can be processed using these techniques. Examples of suitable processed tobaccos include dark air-cured, dark fire cured, burley, flue cured, and cigar filler or wrapper, as well as the products from the whole leaf stemming operation. In an aspect, tobacco fibers include up to 70% dark tobacco on a fresh weight basis. For example, tobacco can be conditioned by heating, sweating and/or pasteurizing steps as described in U.S. Publication Nos. 2004/0118422 or 2005/0178398. Tobacco material provided can be subject to fermentation. Fermenting typically is characterized by high initial moisture content, heat generation, and a 10 to 20% loss of dry weight. See, e.g., U.S. Pat. Nos. 4,528,993; 4,660,577; 4,848,373; and 5,372,149. In addition to modifying the aroma of the leaf, fermentation can change either or both the color and texture of a leaf. Also during the fermentation process, evolution gases can be produced, oxygen can be taken up, the pH can change, and the amount of water retained can change. See, for example, U.S. Publication No. 2005/0178398 and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured, or cured and fermented tobacco can be further processed (e.g., cut, expanded, blended, milled or comminuted) prior to incorporation into the oral product. The tobacco, in some cases, is long cut fermented cured moist tobacco having an oven volatiles content of between 48 and 50 weight percent prior to mixing with the copolymer and optionally flavorants and other additives. In an aspect, tobacco material provided can be processed to a desired size. In an aspect, tobacco fibers can be processed to have an average fiber size of less than 200 micrometers. In an aspect, tobacco fibers are between 75 and 125 micrometers. In another aspect, tobacco fibers are processed to have a size of 75 micrometers or less. In an aspect, tobacco fibers include long cut tobacco, which can be cut or shredded into widths of about 10 cuts/inch up to about 110 cuts/inch and lengths of about 0.1 inches up to about 1 inch. Double cut tobacco fibers can have a range of particle sizes such that about 70% of the double cut tobacco fibers falls between the mesh sizes of −20 mesh and 80 mesh. Tobacco material provided can be processed to have a total oven volatiles content of about 10% by weight or greater; about 20% by weight or greater; about 40% by weight or greater; about 15% by weight to about 25% by weight; about 20% by weight to about 30% by weight; about 30% by weight to about 50% by weight; about 45% by weight to about 65% by weight; or about 50% by weight to about 60% by weight. Those of skill in the art will appreciate that “moist” tobacco typically refers to tobacco that has an oven volatiles content of between about 40% by weight and about 60% by weight (e.g., about 45% by weight to about 55% by weight, or about 50% by weight). As used herein, “oven volatiles” are determined by calculating the percentage of weight loss for a sample after drying the sample in a pre-warmed forced draft oven at 110° C. for 3.25 hours. The oral product can have a different overall oven volatiles content than the oven volatiles content of the tobacco fibers used to make the oral product. The processing steps described can reduce or increase the oven volatiles content. Having now generally described the disclosure, the same will be more readily understood through reference to the following examples that are provided by way of illustration, and are not intended to be limiting of the present disclosure, unless specified. The following are exemplary embodiments of the present disclosure. Embodiment 1. A tobacco plant comprising an inducible promoter operably linked to a transcribable DNA sequence encoding a non-coding RNA for suppression of an ornithine decarboxylase (ODC) gene. Embodiment 2. The tobacco plant of Embodiment 1, wherein said tobacco plant comprises a mutation or a transgene conferring a reduced level of nicotine. Embodiment 3. The tobacco plant of Embodiments 1 or 2, wherein said tobacco plant is a low-alkaloid tobacco plant. Embodiment 4. The tobacco plant of any one of Embodiments 1-3, wherein said tobacco plant comprises a nic1 mutation, a nic2 mutation, or both. Embodiment 5. The tobacco plant of any one of Embodiments 1-4, wherein said tobacco plant comprises a mutation in a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBB1, BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter. Embodiment 6. The tobacco plant of any one of Embodiments 1-5, wherein said tobacco plant comprises a mutation in a gene or locus encoding a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. Embodiment 7. The tobacco plant of any one of Embodiments 1-6, wherein said tobacco plant comprises a transgene targeting and suppressing a gene encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBB1, BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter. Embodiment 8. The tobacco plant of any one of Embodiments 1-7, wherein said tobacco plant comprises a transgene targeting and suppressing a gene encoding a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. Embodiment 9. The tobacco plant of any one of Embodiments 1-8, wherein said tobacco plant is capable of producing a leaf comprising a comparable level of one or more polyamines relative to a comparable leaf of a control plant not comprising said mutation or said transgene. Embodiment 10. The tobacco plant of any one of Embodiments 1-9, wherein said comparable level is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in said control. Embodiment 11. The tobacco plant of any one of claims Embodiments 1-10, wherein said tobacco plant is capable of producing a leaf comprising a comparable chlorophyll level relative to a comparable leaf of a control plant not comprising said mutation or said transgene. Embodiment 12. The tobacco plant of any one of Embodiments 1-11, wherein said comparable chlorophyll level is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in said control. Embodiment 13. The tobacco plant of any one of Embodiments 1-12, wherein said tobacco plant is capable of producing a leaf comprising a comparable number of mesophyll cell per unit of leaf area relative to a comparable leaf of a control plant not comprising said mutation or said transgene. Embodiment 14. The tobacco plant of any one of Embodiments 1-13, wherein said comparable mesophyll cell per unit of leaf area is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in said control. Embodiment 15. The tobacco plant of any one of Embodiments 1-14, wherein said tobacco plant is capable of producing a leaf comprising a comparable epidermal cell size relative to a comparable leaf of a control plant not comprising said mutation or said transgene. Embodiment 16. The tobacco plant of any one of Embodiments 1-15, wherein said comparable epidermal cell size is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in said control. Embodiment 17. The tobacco plant of any one of Embodiments 1-16, wherein said tobacco plant comprises a comparable leaf yield relative to a comparable leaf of a control plant not comprising said mutation or said transgene. Embodiment 18. The tobacco plant of any one of Embodiments 1-17, wherein said comparable leaf yield is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in said control. Embodiment 19. The tobacco plant of any one of Embodiments 1-18, wherein said tobacco plant exhibits a comparable insect herbivory susceptibility relative to a comparable leaf of a control plant not comprising said mutation or said transgene. Embodiment 20. The tobacco plant of any one of Embodiments 1-19, wherein said ornithine decarboxylase (ODC) gene encodes a polypeptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID Nos: 29-34. Embodiment 21. The tobacco plant of any one of Embodiments 1-20, wherein said ODC gene comprises a nucleotide sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID Nos: 23-28. Embodiment 22. The tobacco plant of any one of Embodiments 1-21, wherein said inducible promoter is a topping-inducible promoter. Embodiment 23. The tobacco plant of any one of Embodiments 1-22, wherein said inducible promoter is also a tissue-specific or tissue-preferred promoter. Embodiment 24. The tobacco plant of any one of Embodiments 1-23, wherein said tissue-specific or tissue-preferred promoter is specific or preferred for one or more tissues or organs selected from the group consisting of shoot, root, leaf, stem, flower, sucker, root tip, mesophyll cells, epidermal cells, and vasculature. Embodiment 25. The tobacco plant of any one of Embodiments 1-24, wherein said inducible promoter regulates root specific or preferred expression. Embodiment 26. The tobacco plant of any one of Embodiments 1-25, wherein said inducible promoter comprises a sequence selected from the group consisting of SEQ ID Nos: 1-11. Embodiment 27. The tobacco plant of any one of Embodiments 1-26, wherein said inducible promoter regulates leaf specific or preferred expression. Embodiment 28. The tobacco plant of any one of Embodiments 1-27, wherein said inducible promoter comprises a sequence selected from the group consisting of SEQ ID Nos: 12-21. Embodiment 29. The tobacco plant of any one of Embodiments 1-28, wherein said inducible promoter is a heterologous to said transcribable DNA sequence. Embodiment 30. The tobacco plant of any one of Embodiments 1-29, wherein said non-coding RNA is selected from the group consisting of microRNA (miRNA), anti-sense RNA, small interfering RNA (siRNA), a trans-acting siRNA (ta-siRNA), and hairpin RNA (hpRNA). Embodiment 31. The tobacco plant of any one of Embodiments 1-30, wherein said non-coding RNA comprises a nucleotide sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID Nos: 35 and 36. Embodiment 32. The tobacco plant of any one of Embodiments 1-31, wherein said non-coding RNA is provided in an ODC RNAi construct comprising a nucleotide sequence having at least 90% identity to SEQ ID No: 22. Embodiment 33. A tobacco plant, or part thereof, comprising a nic1 mutation, a nic2 mutation, or both, and further comprising a transgene or mutation providing an early-senescence trait. Embodiment 34. The tobacco plant of Embodiment 33, wherein said mutation providing an early-senescence trait is yellow burley1 (−yb1). Embodiment 35. The tobacco plant of Embodiments 33 or 34, wherein said mutation providing an early-senescence trait is yellow burley2 (−yb2). Embodiment 36. The tobacco plant of any one of Embodiments 33-35, wherein said mutation providing an early-senescence trait is pale yellow (PY). Embodiment 37. A tobacco plant, or part thereof, comprising relative to a control tobacco plant:a. a first genome modification providing a lower level of nicotine or total alkaloid, andb. a second genome modification providing a comparable level of one or more traits selected from the group consisting ofi. total leaf polyamine level,ii. total root polyamine level,iii. total leaf chlorophyll level,iv. mesophyll cell number per leaf area unit, andv. leaf epidermal cell size; andwherein said control plant does not have both said first and said second genome modifications. Embodiment 38. A tobacco plant, or part thereof, comprising relative to a control tobacco plant:a. a first genome modification providing a lower level of nicotine or total alkaloid, andb. a second genome modification providing a comparable level of total leaf polyamine level, wherein said control plant does not have both said first and said second genome modifications. Embodiment 39. A tobacco plant, or part thereof, comprising relative to a control tobacco plant:a. a first genome modification providing a lower level of nicotine or total alkaloid, andb. a second genome modification providing a comparable level of total root polyamine level, wherein said control plant does not have both said first and said second genome modifications. Embodiment 40. A tobacco plant, or part thereof, comprising relative to a control tobacco plant:a. a first genome modification providing a lower level of nicotine or total alkaloid, andb. a second genome modification providing a comparable level of total leaf chlorophyll level, wherein said control plant does not have both said first and said second genome modifications. Embodiment 41. A tobacco plant, or part thereof, comprising relative to a control tobacco plant:a. a first genome modification providing a lower level of nicotine or total alkaloid, andb. a second genome modification providing a comparable level of mesophyll cell number per leaf area unit, wherein said control plant does not have both said first and said second genome modifications. Embodiment 42. A tobacco plant, or part thereof, comprising relative to a control tobacco plant:a. a first genome modification providing a lower level of nicotine or total alkaloid, andb. a second genome modification providing a comparable level of leaf epidermal cell size, wherein said control plant does not have both said first and said second genome modifications. Embodiment 43. The tobacco plant, or part thereof, of any one of Embodiments 37-42, wherein said tobacco plant comprises a reduced amount of total conjugated polyamines in leaves relative to said control tobacco plant. Embodiment 44. The tobacco plant, or part thereof, of any one of Embodiments 37-43, wherein said tobacco plant comprises a reduced amount of total conjugated polyamines in roots relative to said control tobacco plant. Embodiment 45. The tobacco plant, or part thereof, of any one of Embodiments 37-44, wherein said tobacco plant comprises a reduced amount of total free polyamines in leaves relative to said control tobacco plant. Embodiment 46. The tobacco plant, or part thereof, of any one of Embodiments 37-45, wherein said tobacco plant comprises a reduced amount of total conjugated polyamines in roots relative to said control tobacco plant. Embodiment 47. The tobacco plant, or part thereof, of any one of Embodiments 37-46, wherein said tobacco plant comprises a reduced amount of total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in leaves relative to said control tobacco plant. Embodiment 48. The tobacco plant, or part thereof, of any one of Embodiments 37-47, wherein said tobacco plant comprises a reduced amount of total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in roots relative to said control tobacco plant. Embodiment 49. The tobacco plant, or part thereof, of any one of Embodiments 37-48, wherein said tobacco plant comprises a reduced amount of total free form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in leaves relative to said control tobacco plant. Embodiment 50. The tobacco plant, or part thereof, of any one of Embodiments 37-49, wherein said tobacco plant comprises a reduced amount of total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in roots relative to said control tobacco plant. Embodiment 51. The tobacco plant, or part thereof, of any one of Embodiments 37-50, wherein said first genome modification provides a lower level of nicotine compared to said control tobacco plant. Embodiment 52. The tobacco plant, or part thereof, of any one of Embodiments 37-51, said first genome modification, said second genome modification, or both comprise a transgene, a mutation, or both. Embodiment 53. The tobacco plant, or part thereof, of any one of Embodiments 37-52, said first genome modification, said second genome modification, or both comprise a transgene. Embodiment 54. The tobacco plant, or part thereof, of any one of Embodiments 37-53, said first genome modification, said second genome modification, or both comprise a mutation. Embodiment 55. The tobacco plant, or part thereof, of any one of Embodiments 37-54, said first genome modification, said second genome modification, or both are not transgene-based. Embodiment 56. The tobacco plant, or part thereof, of any one of Embodiments 37-55, said first genome modification, said second genome modification, or both are not mutation-based. Embodiment 57. The tobacco plant, or part thereof, of any one of Embodiments 37-56, wherein said first genome modification comprises a nic1 mutation, a nic2 mutation, or both. Embodiment 58. The tobacco plant, or part thereof, of any one of Embodiments 37-57, wherein said first genome modification comprises a transgene targeting the Nic1 locus, a transgene targeting the Nic2 locus, or both. Embodiment 59. The tobacco plant, or part thereof, of any one of Embodiments 37-58, wherein said second genome modification comprises a transcribable DNA sequence encoding a non-coding RNA for suppression of an ornithine decarboxylase (ODC) gene, a MYB8 gene, or both. Embodiment 60. The tobacco plant, or part thereof, of any one of Embodiments 37-59, wherein said a transcribable DNA sequence is operably linked to an heterologous promoter selected from the group consisting of a constitutive promoter, a developmental promoter, a tissue-specific promoter, a tissue-preferred promoter, an inducible promoter, and any combination thereof. Embodiment 61. The tobacco plant, or part thereof, of any one of Embodiments 37-60, wherein said second genome modification comprises overexpression of an diamine oxidase, suppression of an arginine decarboxylase, or both. Embodiment 62. The tobacco plant, or part thereof, of any one of Embodiments 37-61, wherein said first genome modification comprises a mutation in a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), and S-adenosyl-methionine synthetase (SAMS), A622, NBB1, BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter. Embodiment 63. The tobacco plant, or part thereof, of any one of Embodiments 37-62, wherein said first genome modification comprises a mutation in a gene or locus encoding a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. Embodiment 64. The tobacco plant, or part thereof, of any one of Embodiments 37-63, wherein said first genome modification comprises a transgene targeting and suppressing a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), and S-adenosyl-methionine synthetase (SAMS), A622, NBB1, BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter. Embodiment 65. The tobacco plant, or part thereof, of any one of Embodiments 37-64, wherein said first genome modification comprises a transgene targeting and suppressing a gene or locus encoding a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. Embodiment 66. The tobacco plant, or part thereof, of any one of Embodiments 37-65, wherein said lower level is measured at a time selected from the group consisting of immediately before flowering, at topping, 1 week-post-topping (WPT), 2 WPT, 3 WPT, 4 WPT, 5 WPT, 6 WPT, 7 WPT, 8 WPT, and at harvest. Embodiment 67. The tobacco plant, or part thereof, of any one of Embodiments 37-66, wherein said comparable level is measured at a time selected from the group consisting of immediately before flowering, at topping, 1 week-post-topping (WPT), 2 WPT, 3 WPT, 4 WPT, 5 WPT, 6 WPT, 7 WPT, 8 WPT, and at harvest. Embodiment 68. The tobacco plant, or part thereof, of any one of Embodiments 37-67, wherein said tobacco plant is capable of producing a leaf with a leaf grade comparable to that of a leaf from said control plant. Embodiment 69. The tobacco plant, or part thereof, of any one of Embodiments 37-68, wherein said tobacco plant has a total leaf yield comparable to said control plant. Embodiment 70. The tobacco plant, or part thereof, of any one of the preceding Embodiments, wherein said tobacco plant comprises a nicotine level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%. Embodiment 71. The tobacco plant, or part thereof, of any one of Embodiments 37-70, wherein said tobacco plant comprises nicotine at a level below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, or below 80% of the nicotine level of said control plant when grown in comparable growth conditions. Embodiment 72. A population of the tobacco plants of any one of the preceding Embodiments. Embodiment 73. Cured tobacco material from the tobacco plant of any one of the preceding Embodiments. Embodiment 74. The cured tobacco material of Embodiment 73, wherein said cured tobacco material is made by a curing process selected from the group consisting of flue curing, air curing, fire curing, and sun curing. Embodiment 75. A tobacco blend comprising said cured tobacco material of Embodiments 73 or 74. Embodiment 76. The tobacco blend of any one of Embodiments 73-75, wherein said cured tobacco material constitutes about at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of cured tobacco in said tobacco blend by weight. Embodiment 77. The tobacco blend of any one of Embodiments 73-76, wherein said cured tobacco material constitutes about at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of cured tobacco in said tobacco blend by volume. Embodiment 78. A tobacco product comprising the cured tobacco material of any one of Embodiments 73-77. Embodiment 79. The tobacco product of Embodiment 78, wherein said tobacco product is selected from the group consisting of a cigarette, a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco. Embodiment 80. The tobacco product of Embodiments 78 or 79, wherein said tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. Embodiment 81. A method for improving leaf quality in a reduced-alkaloid tobacco plant, said method comprising:a. Growing a tobacco plant;b. Reducing the level of putrescine in said tobacco plant,c. Harvesting leaves from said tobacco plant. Embodiment 82. A method for improving leaf quality in a reduced-alkaloid tobacco plant, said method comprising:a. Growing a tobacco plant;b. Suppressing the expression or activity of an ornithine decarboxylase (ODC) gene in said tobacco plant,c. Harvesting leaves from said tobacco plant. Embodiment 83. The method of Embodiment 81 or 82, wherein said suppressing is within 2, 4, 6, or 8 WPT. Embodiment 84. The method of any one of Embodiments 81-83, wherein said suppressing comprises suppressing said ODC gene both prior to and after topping said tobacco plant. Embodiment 85. The method of any one of Embodiments 82-84, wherein said suppressing does not include the use of a chemical inhibitor. Embodiment 86. The method of any one of Embodiments 82-85, wherein said suppressing comprises applying an ODC inhibitor to said tobacco plant. Embodiment 87. The method of any one of Embodiments 82-86, wherein said suppressing is by applying an ODC inhibitor to said tobacco plant. Embodiment 88. The method of any one of Embodiments 82-87, wherein said ODC inhibitor is DFMO. Embodiment 89. The method of any one of Embodiments 82-88, wherein said suppressing is by inducing the expression of a non-coding RNA for suppression of said ornithine decarboxylase (ODC) gene. Embodiment 90. The method of any one of Embodiments 82-89, wherein said method further comprises reducing nitrogen fertilization or reducing nitrate. EXAMPLES Example 1: Plant Material and General Growth Conditions Seeds ofNicotiana tabacumL. cv. Burley 21 wild-type NA, as well as HI (nic2), LI (nic1) and LA (nic1nic2) near-isogenic varieties were obtained from the USNicotianaGermplasm Collection at North Carolina State University and used in all greenhouse experiments. Seeds were germinated in pots under greenhouse conditions at 27/23° C. day/night temperature and a 16-h photoperiod (˜200 mmol s−1m−2; λ=400-700 nm) at 70% relative humidity. Five week-old tobacco plantlets were transferred to 13-L pots with standard substrate (Einheitserde, Fröndenberg, Germany). The plants were attached to a continuous drip irrigation system active every 4 h for ˜5 min and were irrigated with 0.7% (w/v) Ferty 2 Mega containing 16% nitrogen, (Planta Düngemittel, Regenstauf, Germany) for 5 min h−1during the 16-h photoperiod and grown for 4 additional weeks. Greenhouse plants were topped when 50% of the plants had at least one open flower. After topping, plants grew for additional 4-7 weeks until harvest, 30 days post-topping. Tobacco leaves used for polyamine analysis were collected from greenhouse-grown plants at three time points: before flowering (6.5-week-old plants), just before topping, i.e. the removal of the floral apex (9-week-old plants) and at harvest (13-week-old plants, 4 weeks post-topping). Root samples were collected at topping and harvest. LA and NA tobacco plants were grown in the field under 135 units of nitrogen per acre and sampled at 1 week post-topping for polyamine analysis. For treatment with polyamine biosynthesis inhibitors, 5 mM D-Arginine (AKos, Steinen, Germany), 2 mM DFMO (Synchem Ug & Co. KG, Felsberg, Germany) alone or in combination with 0.5 mM Ethephon®, or 0.5 mM Ethephon® alone (Merck KGaA, Darmstadt, Germany), were diluted in the same amount of water used for daily irrigation and applied to LA plants every 4 hours at 9 am, 12 am, 3 pm and 6 pm three times per week instead of the drip irrigation system. The treatment with D-arginine and DFMO started before flowering (˜2.5 weeks before topping when plants were still in the vegetative growth stage) for a period of 6 weeks until harvest, whereas Ethephon® was applied from topping to harvest (4 weeks in total) to avoid early senescence in the LA plants. Twelve plants per inhibitor were treated. NA plants were used as controls and treated in the same way as the LA plants. More description of experimental procedures and data can be found at Nölke G, et al. Polyamines delay leaf maturation in low-alkaloid tobacco varieties.Plant Direct.2018; 2:1-12. Example 2: Chlorophyll Measurements Chlorophyll contents were determined by measuring leaf absorbance in the red and infrared regions using a SPAD-502 Plus device (Minolta Camera Co., Osaka, Japan). Chlorophyll was measured twice in the same day at different positions in all fully expanded (length>15 cm) leaves (leaves 6-26) from six randomly-selected plants from each line at five growth stages: before flowering (6.5-week-old plants and 2.5 weeks before topping), at topping, 1 and 2.5 WPT and at harvest (30 days post topping) before flowering. The total chlorophyll content was calculated as an average of all measured leaf chlorophyll values per plant to minimize the influence of leaf position. Example 3: Leaf Cell Microscopy Four leaf discs (1 cm2) cut form leaf 15 from six biological replicates at different development stages (before flowering, at topping, 1 WPT and at harvest) were mounted on slides and imaged using a Leica DM R microscope (Leica, Wetzlar, Germany) with a 10× air objective. Images were imported into ImageJ and Adobe Photoshop CS5.1 software and the cells per unit area were counted using Count Tool in the Photoshop CS5. A standard area was designated to use for cell counting three times across all images and care was taken to avoid counting any cell twice. Example 4: Determination of ODC and ADC Activities To determine enzymatic activities, 500 mg of tobacco leaf tissue collected from leaf 23 of three biological replicates of was ground in 1 ml HEPES extraction buffer (100 mM HEPES, 2 mM dithiothreitol (DTT), 1 mM EDTA, pH 7.5) and 100 mg of polyvinylpyrrolidone was added during grinding. Following centrifugation (13,000 g, 10 min, 4° C.), the enzyme activities were measured using an isotopic method as described by Capell et al. (1998) by measuring the release of14CO2. L-[1-14C]Arg and L-[1-14C]Orn were used as radioactive substrates. Example 5: Polyamine Extraction and Analysis For polyamine analysis, 150 μg of leaf or root material was harvested from plants grown in the greenhouse at different stages of development: before flowering (leaves 6 and 12, numbered from base), at topping (leaves 19 and 23 and roots) and at harvest (leaves 23 and 24 and roots). Samples were collected after 4 h of illumination from three biological replicates and were flash frozen in liquid nitrogen. For field-grown plants, leaf material was collected from five well-expanded upper leaves from three biological replicates. Plant material was ground in 1.6 ml pre-chilled 10% (v/v) perchloric acid and incubated at 4° C. for 1 h. The extract was vortexed for 10 s and centrifuged (16,000 g, 15 min, 4° C.) before 800 μl of the supernatant was mixed with 100 μl 1 mM hexamethylenediamine. Then, 10 μl of the clear supernatant was transferred to a fresh 2-ml tube and polyamines were extracted with 200 μl of cyclohexane for the dansilation of free polyamines. For the extraction of conjugated polyamines, the pellet was resuspended in 1600 μl 1 M NaOH and 200 μl 1 mM hexamethylenediamine and centrifuged as above. The clear supernatant (200 μl) was transferred to a 2-ml glass ampule containing 12 M HCl, mixed and incubated for 16 h at 110° C. overnight for the hydrolysis of conjugated polyamines. The dansilation of free and conjugated polyamines was carried out with dansyl chloride as described by Flores and Galston (1982). The dansylated polyamines were measured by LC-MS/MS. All experiments were carried out on a 3200 QTRAP™ mass spectrometer (Sciex, Darmstadt, Germany) coupled to an HPLC Agilent 1200 system (Waldbronn, Germany). The mass spectrometer was equipped with an electrospray ionization source. The sample was separated on a reversed-phase Synergi Fusion with 80 Å pore size, 4 μm particle size and dimensions of 50 mm×2.0 mm internal diameter (Phenomenex, Aschaffenburg, Germany) with the corresponding guard column at a flow rate of 800 μl/min. The column oven was heated to 30° C. For elution, solvent A comprised 94.9% (v/v) water, 5% (v/v) acetonitrile, 0.1% (v/v) formic acid and solvent B comprised 94.9% (v/v) acetonitrile, 5% (v/v) water, 0.1% (v/v) formic acid. The elution following elution profiles was used: 1 min, hold at 60% solvent A/40% solvent B; 3 min, linear increase to 100% solvent B, 3 min hold at 100% solvent B; rapid linear decrease to 60% solvent A/40% solvent B in 0.1 min; hold for 1 min. The total run time was 8 min and the sample volume injected in each run was 10 μl. The mass spectrometer was set to unit resolution in Q1 and Q3. All measurements were captured in multiple reaction monitoring mode. For compound optimization, standards were prepared according to the dansylation protocol, diluted in 50:50 (v/v) methanol/water and infused with a flow rate of 10 μl/min with the syringe pump directly connected to the ion source. Declustering potential, collision energy, collision cell entrance potential, collision cell exit potential and entrance potential were optimized for all compounds using automated compound optimization (Table 3). The ion source parameters were set to: capillary voltage=5.5 kV, heater gas temperature=500° C., curtain gas=30 psi, nebulizing gas=70 psi, drying gas=70 psi, and collision gas=medium. For each analyte, one transition was used for quantification and another as a qualifier. The acquired data was processed using Analyst v1.6 (Sciex). The mass calibration of the 3200 QTRAP was achieved using polypropylene glycol standards (Standards Chemical Kit with Low/High Concentration PPGs, Sciex) according to the manufacturer's instructions. TABLE 3Compound parameter for polyamine quantification. DP = declustering potential, CE = collisionenergy, CEP = collision cell entrance potential, CXP = collision cell exit potential, EP = entrance potential.DansylatedQuantifier/ParentProductDPEPCEPCECXPRTCompoundQualifiermass [m/z]mass [m/z][eV][eV][eV][eV][eV][min]SpermineQuantifier1135.39360.38610486544.3SpermineQualifier1135.39170.386104812144.3SpermidineQuantifier845.228360.3969.5345343.9SpermidineQualifier845.228170.3969.5348143.9PutrescineQuantifier555.119170.3617.5244543.2PutrescineQualifier555.119168.3617.5247943.2HexamethyldiamineQuantifier583.14170.3701028.8095043.5HexamethyldiamineQualifier583.14169.2701028.8095043.5 Example 6: Statistical Analysis Differences between the genotypes were determined by applying one-way analysis of variance (ANOVA) followed by post-hoc Bonferroni test using Excel software (Microsoft, Redmond, Wash., USA). Two-tailed t-tests were applied. A p-value<0.05 was considered statistically significant. Example 7: Biochemical and Morphological Differences Among the Four Varieties During Leaf Ripening Progression of senescence in the Burley 21 NA, HI, LI and LA lines was monitored by measuring the loss of chlorophyll a and b in the leaves. The chlorophyll levels had declined significantly (p<0.01) in all genotypes after 1 week post-topping (WPT) (FIG.1A). However, the leaves of the LI and LA plants contained significantly (p<0.001) higher levels of chlorophyll than the NA controls at 2.5 WPT (22% more in both genotypes) and at harvest (36% and 44% more in the LI and LA leaves, respectively), indicating slower chlorophyll degradation compared to NA controls. Loss of chlorophyll was correlated with morphological changes in the leaves of NA plants, i.e. they became wrinkly and leathery with yellow patches, whereas the LA leaves remained smooth, shiny and green (FIG.1B). Given the distinct leaf morphology in the LA and NA lines, the size and shape of the mesophyll cells were investigated at different time points. Before flowering, leaf 15 (numbered from the base) of the LA plants had smaller and more abundant mesophyll cells (more cells per unit leaf area) compared to the NA plants (FIG.1C/D). From that time point until harvest, the number of leaf mesophyll cells per unit area declined at a similar rate in both the NA and LA lines, but the LA plants retained a significantly (p<0.05) greater number of mesophyll cells throughout ripening. The greatest difference in mesophyll cell number per unit area (54% more cells in the LA plants compared to NA controls) was observed at earlier stages of leaf development (before flowering). LI plants also contained more mesophyll cells than the NA plants but not to the degree observed in the LA plants, and there was no significant difference in mesophyll cell number between the HI and NA lines (data not shown). Example 8: LA Plants Accumulate Higher Levels of Polyamines than NA Plants To investigate the impact of the nic1nic2 double mutation on polyamine biosynthesis, the levels of free and conjugated putrescine, spermidine and spermine in the NA and LA plants were compared by liquid chromatography tandem mass spectrometry (LC-MS/MS). First, the polyamine content were analyzed in leaves 16-18 of field-grown plants. At 1 WPT, the total polyamine content was significantly higher (1.9-fold, p<0.001) in the LA plants compared to the NA plants (FIG.2A). Compositional analysis revealed significantly higher levels of free putrescine (1.4-fold, p<0.05), conjugated putrescine (2.3-fold, p<0.005) and conjugated spermidine (1.9-fold, (p<0.005) levels in the leaves of the LA plants, indicating that the polyamine biosynthesis pathway is strongly induced by the nic1nic2 double mutant or that the inability of the substrates to be further processed into nicotine results in a buildup of these materials. In contrast, the level of free spermidine in the LA plants was lower than in the NA plants, although the difference was not statistically significant (p>0.05). To minimize the effect of variable environmental factors on polyamine biosynthesis, further experiments were performed under controlled greenhouse conditions mirroring the average field conditions in terms of temperature, light and humidity (data not shown). The phenotypes of the NA and LA plants in the greenhouse at harvest (30 days post-topping) were similar to their counterparts grown in the field in terms of plant height, leaf number and leaf morphology (data not shown). The impact of wounding on polyamine biosynthesis was minimized by designing the experiments so that each leaf/root sample was collected only once per plant and time point. Time-course monitoring of the total polyamine content in leaves at the same developmental stage—i.e. leaf 12 before flowering, leaf 19 at topping and leaf 24 at harvest—revealed significantly (p<0.05) higher levels of polyamines in the LA plants before flowering (1.5-fold) and at harvest (2.1-fold) compared to the NA controls (FIG.2B). The LA plants also accumulated significantly (p<0.05) higher levels of total polyamines in the roots at topping (2.4-fold) and at harvest (1.4-fold) compared to the NA controls (FIG.2B) Example 9: Effect of the Nic1nic2 Double Mutation on Polyamine Biosynthesis Comparative analysis of the polyamine composition in selected leaves (leaf 6 before flowering, young leaf 23 at topping and mature leaf 23 at harvest) in the four varieties revealed that, before flowering, lines LI and LA contained significantly (p<0.05) higher levels of free putrescine than the NA controls (1.6-fold and 4.2-fold higher, respectively) and even higher levels of conjugated putrescine (2.1-fold and 5-fold higher, respectively) (FIG.3A). The conjugated putrescine and spermidine fractions increased continuously during ripening in all four varieties, but remained significantly higher in LI and LA plants compared to NA controls (FIG.3A). The greatest differential in polyamine content was observed in the LA leaves at harvest, with a 2.1-fold increase in the level of total polyamines compared to NA controls, including a 1.8-fold increase in free putrescine, a 2.9-fold increase in conjugated putrescine and a 2.4-fold increase in conjugated spermidine. However, there was no significant difference between the NA and HI varieties, indicating that the nic2 single mutation had a lower impact on polyamine accumulation. At topping, the roots of the LA plants contained significantly (p<0.05) higher levels of free putrescine, conjugated putrescine and conjugated spermidine than the NA plants (2.6-fold, 2.9-fold and 2.5-fold increases, respectively) and such differences were also observed at harvest (1.6-fold, 1.4-fold and 2.5-fold increases, respectively) (FIG.3B). Example 10: The Polyamine Biosynthesis Pathway is More Active in the LA Plants The relative contribution of ADC and ODC to putrescine biosynthesis was evaluated by measuring the activity of each enzyme in the leaves (leaf 23) and roots of the NA and LA plants at topping and harvest. ADC and ODC activity varied in an organ-specific and developmental stage-specific manner in both lines (FIG.4). Whereas ADC activity was high in the leaves but minimal in the roots of both lines, ODC activity was higher in the younger leaves and roots, indicating that ODC is mainly responsible for putrescine biosynthesis in the roots. ADC activity was significantly higher (1.4-fold, p<0.05) in the leaves of the LA plants compared to the NA controls at topping and harvest (FIG.4A). Similarly, ODC activity was significantly higher (p<0.05) in the LA plants compared to the NA controls in the roots at topping (1.8-fold) and at harvest (1.7-fold), and in young leaves at topping (1.5-fold) (FIG.4B). Example 11: Inhibition of Polyamine Biosynthesis in the LA Variety Given the correlation between the higher polyamine levels in the LA variety and the undesirable leaf morphology, the effect of treating the plants with chemicals that inhibit ADC and ODC was evaluated. Preliminary experiments defined the appropriate inhibitor concentration, application time, treatment intensity and duration (data not shown). The levels of free and conjugated putrescine were significantly higher in the LA plants than the NA controls before flowering and at harvest (FIG.3), so the ADC inhibitor D-arginine and the ODC inhibitor difluoromethylornithine (DFMO) were applied beginning 2.5 weeks before topping and continued the treatment until harvest. In addition, the plant growth regulator Ethephon® was used alone or in combination with DFMO to accelerate ripening via the liberation of ethylene. To avoid the early induction of senescence, Ethephon® was applied from topping until harvest. The DFMO and DFMO/Ethephon® treatments achieved a partial amelioration of the morphological phenotype, such that the leaves of the LA plants took on some of the characteristics of the NA leaves (wrinkling and chlorophyll degradation), whereas treatment with Ethephon® alone reduced the chlorophyll content but did not affect leaf morphology (FIG.5). Starting the DFMO treatment before flowering resulted in growth arrest, which was not observed when the treatment was started at topping (data not shown). The D-arginine treatment had no effect on the chlorophyll level or morphology of the LA plants. The analysis of polyamine levels revealed that the DFMO treatment 2.5 weeks before topping increased the levels of total polyamines in the LA leaves by 2.1-fold, mainly reflecting higher levels of conjugated putrescine and conjugated spermidine (FIG.6A). This higher proportion of conjugated polyamines remained until harvest in the plants treated with DFMO and DFMO/Ethephon®. In contrast, the treatment with Ethephon® alone led to a significant reduction in total polyamine levels at harvest, mainly reflecting the reduction of free and conjugated putrescine. In the roots, the DFMO treatment significantly reduced (p<0.05) the total polyamine content of the LA plants at topping (1.5-fold) and at harvest (1.4-fold) due mainly to reduction of free and conjugated putrescine and conjugated spermidine (FIG.6B). This decrease was not reversed by the addition of Ethephon®. In contrast to the effect in leaves, the application of Ethephon® alone had no effect on the polyamine content of the roots. The D-arginine treatment had no effect on the polyamine content of the LA plants. The loss of polyamines in the roots could therefore reflect the inhibition of ODC activity, the main enzyme responsible for putrescine biosynthesis in roots. Example 12: Alteration of Polyamine Levels by Genetic Engineering Modified tobacco plants are made to suppress ODC activity in a nic1 nic2 mutant background. A topping-responsive promoter (e.g., SED ID Nos: 1 to 21) is used to drive an ODC RNAi cassette (e.g., SEQ ID No: 22) to achieve the suppression of one or more ODC genes (e.g., coding sequences or protein sequences shown in SEQ ID Nos: 23 to 34). Transgenic plants are generated and assessed for leaf phenotypes, including for example, total leaf polyamine level, total root polyamine level, total leaf chlorophyll level, mesophyll cell number per leaf area unit, leaf epidermal cell size, and cured leaf grade. Modified tobacco plants are also made to modulate the expression and activity of a MYB8 gene in a nic1 nic2 mutant Burley background. MYB8 was reported to control inducible phenolamide levels by activating three hydroxycinnamoyl-coenzyme A:polyamine transferases inNicotiana attenuata. See Onkokesung et al., Plant Physiology 158 (1) 389-407 (2012). A constitutive promoter or a topping-responsive promoter (e.g., SED ID Nos: 1 to 21) is used to drive an MYB8 RNAi cassette or an MYB8 cDNA sequence to achieve suppression or overexpression, respectively. Transgenic plants are generated and assessed for leaf phenotypes, including for example, total leaf polyamine level, total root polyamine level, total leaf chlorophyll level, mesophyll cell number per leaf area unit, leaf epidermal cell size, and cured leaf grade. Example 13: A Breeding Population Low-alkaloid tobacco hybrids, varieties, or lines can be made as a Burley type, a dark type, a flue-cured type, a Maryland type or an Oriental type tobacco, or can be essentially derived from BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14×L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’ tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, or VA359, Maryland 609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, ShireyLC, or any commercial tobacco variety according to standard tobacco breeding techniques known in the art. | 222,681 |
11859193 | KEY TO THE SEQUENCE LISTING SEQ ID NO:1Arabidopsis thalianaDGAT1 polypeptide (CAB44774.1) SEQ ID NO:2Arabidopsis thalianaDGAT2 polypeptide (NP 566952.1) SEQ ID NO:3Ricinus communisDGAT2 polypeptide (AAY16324.1) SEQ ID NO:4Vernicia fordiiDGAT2 polypeptide (ABC94474.1) SEQ ID NO:5Mortierella ramannianaDGAT2 polypeptide (AAK84179.1) SEQ ID NO:6Homo sapiensDGAT2 polypeptide (Q96PD7.2) SEQ ID NO:7Homo sapiensDGAT2 polypeptide (Q58HT5.1) SEQ ID NO:8Bos taurusDGAT2 polypeptide (Q70VZ8.1) SEQ ID NO:9Mus musculusDGAT2 polypeptide (AAK84175.1) SEQ ID NO:10 YFP tripeptide—conserved DGAT2 and/or MGAT1/2 sequence motif SEQ ID NO:11 HPHG tetrapeptide—conserved DGAT2 and/or MGAT1/2 sequence motif SEQ ID NO:12 EPHS tetrapeptide—conserved plant DGAT2 sequence motif SEQ ID NO:13 RXGFX(K/R)XAXXXGXXX(L/V)VPXXXFG(E/Q)—long conserved sequence motif of DGAT2 which is part of the putative glycerol phospholipid domain SEQ ID NO:14 FLXLXXXN—conserved sequence motif of mouse DGAT2 and MGAT1/2 which is a putative neutral lipid binding domain SEQ ID NO:15 plsC acyltransferase domain (PF01553) of GPAT SEQ ID NO:16 HAD-like hydrolase (PF12710) superfamily domain of GPAT SEQ ID NO:17 Phosphoserine phosphatase domain (PF00702). GPAT4-8 contain a N-terminal region homologous to this domain SEQ ID NO:18 Conserved GPAT amino acid sequence GDLVICPEGTTCREP SEQ ID NO:19 Conserved GPAT/phosphatase amino acid sequence (Motif I) SEQ ID NO:20 Conserved GPAT/phosphatase amino acid sequence (Motif III) SEQ ID NO:21Arabidopsis thalianaWRI1 polypeptide (A8MS57) SEQ ID NO:22Arabidopsis thalianaWRI1 polypeptide (Q6X5Y6) SEQ ID NO:23Arabidopsis lyratasubsp.lyrataWRI1 polypeptide (XP_002876251.1) SEQ ID NO:24Brassica napusWRI1 polypeptide (ABD16282.1) SEQ ID NO:25Brassica napusWRI1 polypeptide (AD016346.1) SEQ ID NO:26Glycine maxWRI1 polypeptide (XP_003530370.1) SEQ ID NO:27Jatropha curcasWRI1 polypeptide (AE022131.1) SEQ ID NO:28Ricinus communisWRI1 polypeptide (XP_002525305.1) SEQ ID NO:29Populus trichocarpaWRI1 polypeptide (XP_002316459.1) SEQ ID NO:30Vitis viniferaWRI1 polypeptide (CB129147.3) SEQ ID NO:31Brachypodium distachyonWRI1 polypeptide (XP_003578997.1) SEQ ID NO:32Hordeum vulgaresubsp.vulgareWRI1 polypeptide (BAJ86627.1) SEQ ID NO:33Oryza sativaWRI1 polypeptide (EAY79792.1) SEQ ID NO:34Sorghum bicolorWRI1 polypeptide (XP_002450194.1) SEQ ID NO:35Zea maysWRI1 polypeptide (ACG32367.1) SEQ ID NO:36Brachypodium distachyonWRI1 polypeptide (XP_003561189.1) SEQ ID NO:37Brachypodium sylvaticumWRI1 polypeptide (ABL85061.1) SEQ ID NO:38Oryza sativaWRI1 polypeptide (BAD68417.1) SEQ ID NO:39Sorghum bicolorWRI1 polypeptide (XP_002437819.1) SEQ ID NO:40Sorghum bicolorWRI1 polypeptide (XP_002441444.1) SEQ ID NO:41Glycine maxWRI1 polypeptide (XP_003530686.1) SEQ ID NO:42Glycine maxWRI1 polypeptide (XP_003553203.1) SEQ ID NO:43Populus trichocarpaWRI1 polypeptide (XP_002315794.1) SEQ ID NO:44Vitis viniferaWRI1 polypeptide (XP_002270149.1) SEQ ID NO:45Glycine maxWRI1 polypeptide (XP_003533548.1) SEQ ID NO:46Glycine maxWRI1 polypeptide (XP_003551723.1) SEQ ID NO:47Medicago truncatulaWRI1 polypeptide (XP_003621117.1) SEQ ID NO:48Populus trichocarpaWRI1 polypeptide (XP_002323836.1) SEQ ID NO:49Ricinus communisWRI1 polypeptide (XP_002517474.1) SEQ ID NO:50Vitis viniferaWRI1 polypeptide (CAN79925.1) SEQ ID NO:51Brachypodium distachyonWRI1 polypeptide (XP_003572236.1) SEQ ID NO:52Oryza sativaWRI1 polypeptide (BAD10030.1) SEQ ID NO:53Sorghum bicolorWRI1 polypeptide (XP_002444429.1) SEQ ID NO:54Zea maysWRI1 polypeptide (NP 001170359.1) SEQ ID NO:55Arabidopsis lyratasubsp.lyrataWRI1 polypeptide (XP_002889265.1) SEQ ID NO:56Arabidopsis thalianaWRI1 polypeptide (AAF68121.1) SEQ ID NO:57Arabidopsis thalianaWRI1 polypeptide (NP 178088.2) SEQ ID NO:58Arabidopsis lyratasubsp.lyrataWRI1 polypeptide (XP_002890145.1) SEQ ID NO:59Thellungiella halophilaWRI1 polypeptide (BAJ33872.1) SEQ ID NO:60Arabidopsis thalianaWRI1 polypeptide (NP 563990.1) SEQ ID NO:61Glycine maxWRI1 polypeptide (XP_003530350.1) SEQ ID NO:62Brachypodium distachyonWRI1 polypeptide (XP_003578142.1) SEQ ID NO:63Oryza sativaWRI1 polypeptide (EAZ09147.1) SEQ ID NO:64Sorghum bicolorWRI1 polypeptide (XP_002460236.1) SEQ ID NO:65Zea maysWRI1 polypeptide (NP 001146338.1) SEQ ID NO:66Glycine maxWRI1 polypeptide (XP_003519167.1) SEQ ID NO:67Glycine maxWRI1 polypeptide (XP_003550676.1) SEQ ID NO:68Medicago truncatulaWRI1 polypeptide (XP_003610261.1) SEQ ID NO:69Glycine maxWRI1 polypeptide (XP_003524030.1) SEQ ID NO:70Glycine maxWRI1 polypeptide (XP_003525949.1) SEQ ID NO:71Populus trichocarpaWRI1 polypeptide (XP_002325111.1) SEQ ID NO:72Vitis viniferaWRI1 polypeptide (CBI36586.3) SEQ ID NO:73Vitis viniferaWRI1 polypeptide (XP_002273046.2) SEQ ID NO:74Populus trichocarpaWRI1 polypeptide (XP_002303866.1) SEQ ID NO:75Vitis viniferaWRI1 polypeptide (CBI25261.3) SEQ ID NO:76 Sorbi-WRL1 SEQ ID NO: 77 Lupan-WRL1 SEQ ID NO:78 Ricco-WRL1 SEQ ID NO:79Lupin angustifoliusWRI1 polypeptide SEQ ID NO:80Aspergillus fumigatusDGAT1 polypeptide (XP_755172.1) SEQ ID NO:81Ricinus communisDGAT1 polypeptide (AAR11479.1) SEQ ID NO:82Vernicia fordiiDGAT1 polypeptide (ABC94472.1) SEQ ID NO:83Vernonia galamensisDGAT1 polypeptide (ABV21945.1) SEQ ID NO:84Vernonia galamensisDGAT1 polypeptide (ABV21946.1) SEQ ID NO:85Euonymus alatusDGAT1 polypeptide (AAV31083.1) SEQ ID NO:86Caenorhabditis elegansDGAT1 polypeptide (AAF82410.1) SEQ ID NO:87Rattus norvegicusDGAT1 polypeptide (NP_445889.1) SEQ ID NO:88Homo sapiensDGAT1 polypeptide (NP_036211.2) SEQ ID NO:89 WRI1 motif (R G V T/S R H R W T G R) SEQ ID NO:90 WRI1 motif (F/Y E A H L W D K) SEQ ID NO:91 WRI1 motif (D L A A L K Y W G) SEQ ID NO:92 WRI1 motif (S X G F S/A R G X) SEQ ID NO:93 WRI1 motif (H H H/Q N G R/K W E A R I G R/K V) SEQ ID NO:94 WRI1 motif (Q E E A A A X Y D) SEQ ID NO:95Brassica napusoleosin polypeptide (CAA57545.1) SEQ ID NO:96Brassica napusoleosin S1-1 polypeptide (ACG69504.1) SEQ ID NO:97Brassica napusoleosin S2-1 polypeptide (ACG69503.1) SEQ ID NO:98Brassica napusoleosin S3-1 polypeptide (ACG69513.1) SEQ ID NO:99Brassica napusoleosin S4-1 polypeptide (ACG69507.1) SEQ ID NO:100Brassica napusoleosin S5-1 polypeptide (ACG69511.1) SEQ ID NO:101Arachis hypogaeaoleosin 1 polypeptide (AAZ20276.1) SEQ ID NO:102Arachis hypogaeaoleosin 2 polypeptide (AAU21500.1) SEQ ID NO:103Arachis hypogaeaoleosin 3 polypeptide (AAU21501.1) SEQ ID NO:104Arachis hypogaeaoleosin 5 polypeptide (ABC96763.1) SEQ ID NO:105Ricinus communisoleosin 1 polypeptide (EEF40948.1) SEQ ID NO:106Ricinus communisoleosin 2 polypeptide (EEF51616.1) SEQ ID NO:107Glycine maxoleosin isoform a polypeptide (P29530.2) SEQ ID NO:108Glycine maxoleosin isoform b polypeptide (P29531.1) SEQ ID NO:109Linum usitatissimumoleosin low molecular weight isoform polypeptide (ABB01622.1) SEQ ID NO:110 amino acid sequence ofLinum usitatissimumoleosin high molecular weight isoform polypeptide (ABB01624.1) SEQ ID NO:111Helianthus annuusoleosin polypeptide (CAA44224.1) SEQ ID NO:112Zea maysoleosin polypeptide (NP_001105338.1) SEQ ID NO:113Brassica napussteroleosin polypeptide (ABM30178.1) SEQ ID NO:114Brassica napussteroleosin SLO1-1 polypeptide (ACG69522.1) SEQ ID NO:115Brassica napussteroleosin SLO2-1 polypeptide (ACG69525.1) SEQ ID NO:116Sesamum indicumsteroleosin polypeptide (AAL13315.1) SEQ ID NO:117Zea mayssteroleosin polypeptide (NP_001152614.1) SEQ ID NO:118Brassica napuscaleosin CLO-1 polypeptide (ACG69529.1) SEQ ID NO:119Brassica napuscaleosin CLO-3 polypeptide (ACG69527.1) SEQ ID NO:120Sesamum indicumcaleosin polypeptide (AAF13743.1) SEQ ID NO:121Zea mayscaleosin polypeptide (NP_001151906.1) SEQ ID NO:122 pJP3502 TDNA (inserted into genome) sequence SEQ ID NO:123 pJP3507 vector sequence SEQ ID NO:124 Linker sequence SEQ ID NO:125 PartialNicotiana benthamianaCGI-58 sequence selected for hpRNAi silencing (pTV46) SEQ ID NO:126 PartialN. tabacumAGPase sequence selected for hpRNAi silencing (pTV35) SEQ ID NO:127 GXSXG lipase motif SEQ ID NO:128 HX(4)D acyltransferase motif SEQ ID NO:129 VX(3)HGF probable lipid binding motif SEQ ID NO:130Arabidopsis thalianaCGi58 polynucleotide (NM_118548.1) SEQ ID NO:131Brachypodium distachyonCGi58 polynucleotide (XM_003578402.1) SEQ ID NO:132Glycine maxCGi58 polynucleotide (XM_003523590.1) SEQ ID NO:133Zea maysCGi58 polynucleotide (NM_001155541.1) SEQ ID NO:134Sorghum bicolorCGi58 polynucleotide (XM_002460493.1) SEQ ID NO:135Ricinus communisCGi58 polynucleotide (XM_002510439.1) SEQ ID NO:136Medicago truncatulaCGi58 polynucleotide (XM_003603685.1) SEQ ID NO:137Arabidopsis thalianaLEC2 polynucleotide (NM_102595.2) SEQ ID NO:138Medicago truncatulaLEC2 polynucelotide (X60387.1) SEQ ID NO:139Brassica napusLEC2 polynucelotide (HM370539.1) SEQ ID NO:140Arabidopsis thalianaBBM polynucleotide (NM_121749.2) SEQ ID NO:141Medicago truncatulaBBM polynucleotide (AY899909.1) SEQ ID NO:142Arabidopsis thalianaLEC2 polypeptide (NP_564304.1) SEQ ID NO:143Medicago truncatulaLEC2 polypeptide (CAA42938.1) SEQ ID NO:144Brassica napusLEC2 polypeptide (AD016343.1) SEQ ID NO:145Arabidopsis thalianaBBM polypeptide (NP_197245.2) SEQ ID NO:146Medicago truncatulaBBM polypeptide (AAW82334.1) SEQ ID NO:147 InducibleAspergillus nigeralcA promoter SEQ ID NO:148 AlcR inducer that activates the AlcA promotor in the presence of ethanol SEQ ID NO:149Arabidopsis thalianaLEC1; (AAC39488) SEQ ID NO:150Arabidopsis lyrataLEC1 (XP_002862657) SEQ ID NO:151Brassica napusLEC1 (ADF81045) SEQ ID NO:152Ricinus communisLEC1 (XP_002522740) SEQ ID NO:153Glycine maxLEC1 (XP_006582823) SEQ ID NO:154Medicago truncatulaLEC1 (AFK49653) SEQ ID NO:155Zea maysLEC1 (AAK95562) SEQ ID NO:156Arachis hypogaeaLEC1 (ADC33213) SEQ ID NO:157Arabidopsis thalianaLEC1-like (AAN15924) SEQ ID NO:158Brassica napusLEC1-like (AHI94922) SEQ ID NO:159Phaseolus coccineusLEC1-like (AAN01148) SEQ ID NO:160Arabidopsis thalianaFUS3 (AAC35247) SEQ ID NO:161Brassica napusFUS3 SEQ ID NO:162Medicago truncatulaFUS3 SEQ ID NO:163Arabidopsis thalianaSDP1 cDNA sequence, Accession No. NM_120486, 3275 nt SEQ ID NO:164Brassica napusSDP1 cDNA; Accession No. GN078290 SEQ ID NO:165Brachypodium distachyonSDP1 cDNA, 2670 nt SEQ ID NO:166Populus trichocarpaSDP1 cDNA, 3884 nt SEQ ID NO:167Medicago truncatulaSDP1 cDNA; XM_003591377; 2490 nt SEQ ID NO:168Glycine maxSDP1 cDNA XM_003521103; 2783 nt SEQ ID NO:169Sorghum bicolorSDP1 cDNA XM_002458486; 2724 nt SEQ ID NO:170Zea maysSDP1 cDNA, NM_001175206; 2985 nt SEQ ID NO:171Physcomitrella patensSDP1 cDNA, XM_001758117; 1998 nt SEQ ID NO:172Hordeum vulgareSDP1 cDNA, AK372092; 3439 nt SEQ ID NO:173Nicotiana benthamianaSDP1 cDNA, Nbv5tr6404201 SEQ ID NO:174Nicotiana benthamianaSDP1 cDNA region targeted for hpRNAi silencing SEQ ID NO:175 Promoter ofArabidopsis thalianaSDP1 gene, 1.5 kb SEQ ID NO:176 Nucleotide sequence of the complement of the pSSU-Oleosin gene in the T-DNA of pJP3502. In order (complementary sequences):Glycine maxLectin terminator 348 nt, 3′ exon 255 nt, UBQ10 intron 304 nt, 5′ exon 213 nt, SSU promoter 1751 nt SEQ ID NO:177Arabidopsis thalianaplastidial GPAT cDNA, NM_179407 SEQ ID NO:178Arabidopsis thalianaplastidial GPAT polypeptide, NM_179407 SEQ ID NO:179Populus trichocarpaplastidial GPAT cDNA, XP_006368351 SEQ ID NO:180Jatropha curcasplastidial GPAT cDNA, ACR61638 SEQ ID NO:181Ricinus communisplastidial GPAT cDNA, XP_002518993 SEQ ID NO:182Helianthus annuusplastidial GPAT cDNA, ADV16382 SEQ ID NO:183Medicago truncatulaplastidial GPAT cDNA, XP_003612801 SEQ ID NO:184Glycine maxplastidial GPAT cDNA, XP_003516958 SEQ ID NO:185Carthamus tinctoriusplastidial GPAT cDNA, CAHG3PACTR SEQ ID NO:186Solanum tuberosumplastidial GPAT cDNA, XP_006352898 SEQ ID NO:187Oryza sativa Japonicaplastidial GPAT cDNA, NM_001072027 SEQ ID NO:188Sorghum bicolorplastidial GPAT cDNA, XM_002467381 SEQ ID NO:189Zea maysplastidial GPAT cDNA, NM_001158637 SEQ ID NO:190Hordeum vulgareplastidial GPAT cDNA, AK371419 SEQ ID NO:191Physcomitrella patensplastidial GPAT cDNA, XM_001771247 SEQ ID NO:192Chlamydomonas reinhardtiiplastidial GPAT cDNA, XM_001694925 SEQ ID NO:193Arabidopsis thalianaFATA1 SEQ ID NO:194Arabidopsis thalianaFATA2 SEQ ID NO:195Arabidopsis thalianaFATB SEQ ID NO:196Arabidopsis thalianaWRI3 SEQ ID NO:197Arabidopsis thalianaWRI4 SEQ ID NO:198Avena sativaWRI1 SEQ ID NO:199Sorghum bicolorWRI1 SEQ ID NO:200Zea maysWRI1 SEQ ID NO:201Triadica sebiferaWRH SEQ ID NO:202S. tuberosumPatatin B33 promoter sequence SEQ ID NOs 203 to 206 and 236 to 245 Oligonucleotide primers SEQ ID NO:207Z. maysSEE1 promoter region (1970 nt from Accession number AJ494982) SEQ ID NO:208A. littoralisAlSAP promoter sequence, Accession No DQ885219 SEQ ID NO:209A. rhizogenesArRolC promoter sequence, Accession No. DQ160187 SEQ ID NO:210 hpRNAi construct containing a 732 bp fragment ofN. benthamianaplastidial GPAT SEQ ID NO:211Elaeis guineensis(oil palm) DGAT1 SEQ ID NO:212G. maxMYB73, Accession No. ABH02868 SEQ ID NO:213A. thalianabZIP53, Accession No. AAM14360 SEQ ID NO:214A. thalianaAGL15, Accession No NP_196883 SEQ ID NO:215A. thalianaMYB118, Accession No. AAS58517 SEQ ID NO:216A. thalianaMYB115, Accession No. AAS10103 SEQ ID NO:217A. thalianaTANMEI, Accession No. BAE44475 SEQ ID NO:218A. thalianaWUS, Accession No. NP_565429 SEQ ID NO:219B. napusGFR2a1, Accession No. AFB74090 SEQ ID NO:220B. napusGFR2a2, Accession No. AFB74089 SEQ ID NO:221A. thalianaPHR1, Accession No. AAN72198 SEQ ID NO:222N. benthamianaTGD1 fragment SEQ ID NO:223 Potato SDP1 amino acid SEQ ID NO:224 Potato SDP1 nucleotide sequence SEQ ID NO:225 Potato AGPase small subunit SEQ ID NO:226 Potato AGPase small subunit nucleotide sequence: SEQ ID NO:227Sapium sebiferumLDAP-1 nucleotide sequence SEQ ID NO:228Sapium sebiferumLDAP-1 amino acid sequence SEQ ID NO:229Sapium sebiferumLDAP-2 nucleotide sequence SEQ ID NO:230Sapium sebiferumLDAP-2 amino acid sequence SEQ ID NO:231Sapium sebiferumLDAP-3 nucleotide sequence SEQ ID NO:232Sapium sebiferumLDAP-3 amino acid sequence SEQ ID NO:233S. bicolorSDP1 (accession number XM_002463620) SEQ ID NO:234T. aestivumSDP1 nucleotide sequence (Accession number AK334547) SEQ ID NO:235S. bicolorSDP1 hpRNAi fragment. SEQ ID NO:246Saccharumhybrid DIRIGENT (DIR16) promoter sequence SEQ ID NO:247Saccharumhybrid 0-Methyl transferase (OMT) promoter sequence SEQ ID NO:248 Sequence of the A1 promoter allele of theSaccharumhybrid R1MYB1 gene SEQ ID NO:249Saccharumhybrid Loading Stem Gene 5 (LSG5) promoter sequence SEQ ID NO:250 Nucleotide sequence of the protein coding region of the cDNA forSorghum bicolorTGD5 gene, Accession No. XM_002442154; 297 nt SEQ ID NO:251 Amino acid sequence ofSorghum bicolorTGD5 polypeptide, Accession No. XM_002442154; 98aa SEQ ID NO:252 Nucleotide sequence of the protein coding region of the cDNA forZea maysTGD5 gene, Accession No. EU972796.1; 297 nt SEQ ID NO:253 Amino acid sequence ofZea maysTGD5 polypeptide, Accession No. EU972796.1; 98aa SEQ ID NO:254 Nucleotide sequence of the protein coding region of the cDNA forSorghum bicolorgene encoding AGPase small subunit (Accession No. XM_002462095.1); 1533 nt SEQ ID NO:255 Amino acid sequence ofSorghum bicolorAGPase small subunit polypeptide (Accession No. XM_002462095.1); 510aa SEQ ID NO:256 Nucleotide sequence of the protein coding region of the cDNA forZea maysgene encoding AGPase small subunit polypeptide (Accession No. XM_008666513.1); 1554 nt SEQ ID NO:257 Amino acid sequence ofZea maysAGPase small subunit polypeptide (Accession No. XM_008666513.1); 517aa SEQ ID NO:258 Nucleotide sequence of the protein coding region of the cDNA forSorghum bicolorPDAT1 gene (Accession No. XM_002462417.1); SEQ ID NO:259 Amino acid sequence ofSorghum bicolorPDAT1 polypeptide (Accession No. XM_002462417.1); 682aa SEQ ID NO:260 Nucleotide sequence of the protein coding region of the cDNA forZea maysPDAT1 gene (Accession No. NM_001147943); 2037 nt SEQ ID NO:261 Amino acid sequence ofZea maysPDAT1 polypeptide (Accession No. NM_001147943); 678aa SEQ ID NO:262 Nucleotide sequence of the protein coding region of a cDNA forSorghum bicolorPDCT gene (Accession No. XM_002437214); 846 nt SEQ ID NO:263 Amino acid sequence of aSorghum bicolorPDCT polypeptide (Accession No. XM_002437214); 281aa SEQ ID NO:264 Nucleotide sequence of the protein coding region of the cDNA forZea maysPDCT gene (Accession No. EU973573.1); 849 nt SEQ ID NO:265 Amino acid sequence ofZea maysPDCT polypeptide (Accession No. EU973573.1); 282aa SEQ ID NO:266 Nucleotide sequence of the protein coding region of the cDNA forSorghum bicolorTST1 gene (Accession No. XM_002467535.1); 2223 nt SEQ ID NO:267 Amino acid sequence ofSorghum bicolorTST1 polypeptide (Accession No. XM_002467535.1); 740aa SEQ ID NO:268 Nucleotide sequence of the protein coding region of the cDNA forZea maysTST1 gene (Accession No. NM_001158464); 2244 nt SEQ ID NO:269 Amino acid sequence ofZea maysTST1 polypeptide (Accession No. NM_001158464); 747aa SEQ ID NO:270 Nucleotide sequence of the protein coding region of the cDNA forSorghum bicolorTST2 gene (Sb04G008150; Sobic.004G099300; Accession No. KXG29849.1); 2238 nt SEQ ID NO:271 Amino acid sequence ofSorghum bicolorTST2 polypeptide (Accession No. KXG29849.1); 745aa SEQ ID NO:272 Nucleotide sequence of the protein coding region of the cDNA forZea maysTST2 gene (Accession No. XM_008647398.1); 2238 nt SEQ ID NO:273 Amino acid sequence ofZea maysTST2 polypeptide (Accession No. XM_008647398.1); 745aa SEQ ID NO:274 Nucleotide sequence of the protein coding region of the cDNA forSorghum bicolorINV3 gene (Sobic.004G004800; Sb04g000620; Accession No. XM_002451312); 1464 nt SEQ ID NO:275 Amino acid sequence ofSorghum bicolorINV3 polypeptide (Accession No. XM_002451312); 487aa SEQ ID NO:276 Amino acid sequence ofSorghum bicolorINV3 polypeptide; alternative longer splicing form (Accession No. EES04332.2); 638aa SEQ ID NO:277 Nucleotide sequence of the protein coding region of the cDNA forZea maysINV2 gene (maize homolog to Sb INV3) (Accession No. NM_001305860.1); 2022 nt SEQ ID NO:278 Amino acid sequence ofZea maysINV2 polypeptide (maize homolog to Sb INV3) (Accession No. NM_001305860.1); 673aa SEQ ID NO:279 Nucleotide sequence of the protein coding region of the cDNA forSorghum bicolorSUS4 gene (Sobic.001G344500; Sb01g033060; Accession No. XM_002465116.1); 2451 nt SEQ ID NO:280 Amino acid sequence ofSorghum bicolorSUS4 polypeptide (Accession No. XM_002465116.1); 816aa SEQ ID NO:281 Nucleotide sequence of the protein coding region of the cDNA forZea maysSUS1 gene (maize homolog to Sb SUS4) (Accession No. NM_001111853); 2451 nt SEQ ID NO:282 Amino acid sequence ofZea maysSUS1 polypeptide (Accession No. NM_001111853); 816aa SEQ ID NO:283 Nucleotide sequence of the protein coding region of the cDNA forSorghum bicolorbCIN gene (Sobic.004G172700; Sb04g022350; Accession No. XM_002453920.1); SEQ ID NO:284 Amino acid sequence ofSorghum bicolorbCIN polypeptide (Accession No. XM_002453920.1); 559aa SEQ ID NO:285 Nucleotide sequence of the protein coding region of the cDNA forZea mayscytosolic INV gene (homolog of Sb bCIN) (Accession No. NM_001175248.1); 1680 nt SEQ ID NO:286 Amino acid sequence ofZea maysINV polypeptide (Accession No. NM_001175248.1); 559aa SEQ ID NO:287 Nucleotide sequence of the protein coding region of the cDNA forSorghum bicolorSUT4 gene (Sb04g038030; Accession No. XM_002453038.1); 1785 nt SEQ ID NO:288 Amino acid sequence ofSorghum bicolorSUT4 polypeptide (Accession No. XM_002453038.1); 594aa SEQ ID NO:289 Nucleotide sequence of the protein coding region of the cDNA forZea maysSUT2 gene (Accession No. AY581895.1); 1779 nt SEQ ID NO:290 Amino acid sequence ofZea maysSUT2 polypeptide (Accession No. AY581895.1); 592aa SEQ ID NO:291 Nucleotide sequence of the protein coding region of the cDNA forArabidopsis thalianaSWEET16 gene (Accession No. NM_001338249.1); 693 nt SEQ ID NO:292 Amino acid sequence ofArabidopsis thalianaSWEET16 polypeptide (Accession No. NM_001338249.1); 230aa SEQ ID NO:293 Nucleotide sequence of the protein coding region of the cDNA forArabidopsis thalianaMED15-1 gene (Accession No. NM_101446.4); 4008 nt SEQ ID NO:294 Amino acid sequence ofArabidopsis thalianaMED15-1 polypeptide (Accession No. NM_101446.4); 1335aa SEQ ID NO:295 Nucleotide sequence of the protein coding region of the cDNA forZea maysMED15-1 gene (Accession No. NM_001321633.1); 3927 nt SEQ ID NO:296 Amino acid sequence ofZea maysMED15-1 polypeptide (Accession No. NM_001321633.1); 1308aa SEQ ID NO:297 Nucleotide sequence of the protein coding region of the cDNA forArabidopsis thaliana14-3-3K gene (Accession No. AY079350); SEQ ID NO:298 Amino acid sequence ofArabidopsis thaliana14-3-3K polypeptide (Accession No. AY079350); 248aa SEQ ID NO:299 Nucleotide sequence of the protein coding region of the cDNA forSorghum bicolor14-3-3K gene (Accession No. XM_002445734.1); 762 nt SEQ ID NO:300 Amino acid sequence ofSorghum bicolor14-3-3K polypeptide (Accession No. XM_002445734.1); 253aa SEQ ID NO:301 Nucleotide sequence of the protein coding region of the cDNA forArabidopsis thaliana14-3-3λ gene (Accession No. NM_001203346); 777 nt SEQ ID NO:302 Amino acid sequence ofArabidopsis thaliana14-3-3λ polypeptide (Accession No. NM_001203346); 258aa SEQ ID NO:303 Nucleotide sequence of the protein coding region of the cDNA forSorghum bicolor14-3-3λ gene (Accession No. XM_002445734.1); 762 nt SEQ ID NO:304 Amino acid sequence ofSorghum bicolor14-3-3λ polypeptide (Accession No. XM_002445734.1); 253aa SEQ ID NO:305 Amino acid sequence ofSesamum indicumoleosinL polypeptide (Accession No. AF091840) SEQ ID NO:306 Amino acid sequence ofFicus pumilavar.awkeotsangoleosinL ortholog polypeptide (Accession No. ABQ57397.1) SEQ ID NO:307 Amino acid sequence ofCucumis sativusoleosinL ortholog polypeptide (Accession No. XP_004146901.1) SEQ ID NO:308 Amino acid sequence ofLinum usitatissimumoleosinL ortholog polypeptide (Accession No. ABB01618.1) SEQ ID NO:309 Amino acid sequence ofGlycine maxoleosinL ortholog polypeptide (Accession No. XP_003556321.2) SEQ ID NO:310 Amino acid sequence ofAnanas comosusoleosinL ortholog polypeptide (Accession No. OAY72596.1) SEQ ID NO:311 Amino acid sequence ofSetaria italicaoleosinL ortholog polypeptide (Accession No. XP_004956407.1) SEQ ID NO:312 Amino acid sequence ofFragaria vescasubsp.vescaoleosinL ortholog polypeptide (Accession No. XP_004307777.1) SEQ ID NO:313 Amino acid sequence ofBrassica napusoleosinL ortholog polypeptide (Accession No. CDY03377.1) SEQ ID NO:314 Amino acid sequence ofSolanum lycopersicumoleosinL ortholog polypeptide (Accession No. XP_004240765.1) SEQ ID NO: 315. Amino acid sequence of U1 Oleosin fromVanilla planifolia SEQ ID NO: 316. Amino acid sequence of TsLDAP1 fromTriadica sebifera(Chinese tallow) SEQ ID NO: 317. Amino acid sequence of TsLDAP2 fromTriadica sebifera(Chinese tallow) SEQ ID NO: 318. Amino acid sequence of TsLDAP3 fromTriadica sebifera(Chinese tallow) SEQ ID NO: 319. Amino acid sequence of a GPAT9 fromCocos nucifera(Coconut) SEQ ID NO: 320. Amino acid sequence of aZea maysCPT1 (Accession No. NP_001151915.1) SEQ ID NO: 321. Amino acid sequence of aZea maysCPT1 (Accession No. XP_008649199.1) SEQ ID NO: 322. Amino acid sequence of aSorghum bicolorCPT1 (Accession No. XP_002451408.1) SEQ ID NO: 323. Amino acid sequence of aSorghum bicolorCPT1 (Accession No. XP_021305900.1) DETAILED DESCRIPTION OF THE INVENTION General Techniques Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, plant biology, cell biology, protein chemistry, lipid and fatty acid chemistry, animal nutrition, biofeul production, and biochemistry). Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present). Selected Definitions The term “exogenous” in the context of a polynucleotide or polypeptide refers to the polynucleotide or polypeptide when present in a cell or a plant or part thereof which does not naturally comprise the polynucleotide or polypeptide. Such a cell is referred to herein as a “recombinant cell” or a “transgenic cell” and a plant comprising the cell as a “transgenic plant”. In an embodiment, the exogenous polynucleotide or polypeptide is from a different genus to the cell of the plant or part thereof comprising the exogenous polynucleotide or polypeptide. In another embodiment, the exogenous polynucleotide or polypeptide is from a different species. In one embodiment, the exogenous polynucleotide or polypeptide expressed in the plant cell is from a different species or genus. The exogenous polynucleotide or polypeptide may be non-naturally occurring, such as for example, a synthetic DNA molecule which has been produced by recombinant DNA methods. The DNA molecule may, preferably, include a protein coding region which has been codon-optimised for expression in the plant cell, thereby producing a polypeptide which has the same amino acid sequence as a naturally occurring polypeptide, even though the nucleotide sequence of the protein coding region is non-naturally occurring. The exogenous polynucleotide may encode, or the exogenous polypeptide may be, for example: a diacylglycerol acyltransferase (DGAT) such as a DGAT1 or a DGAT2, a Wrinkled 1 (WRI1) transcription factor, on OBC such as an Oleosin or preferably an LDAP, a fatty acid thioesterase such as a FATA or FATB polypeptide, or a silencing suppressor polypeptide. In an embodiment, a cell of the invention is a recombinant cell. As used herein, the term “triacylglycerol (TAG) content” or variations thereof refers to the amount of TAG in the cell, plant or part thereof. TAG content can be calculated using techniques known in the art such as the sum of glycerol and fatty acyl moieties using a relation: % TAG by weight=100×((41×total mol FAME/3)+(total g FAME−(15×total mol FAME)))/g, where 41 and 15 are molecular weights of glycerol moiety and methyl group, respectively (where FAME is fatty acid methyl esters) (see Examples such as Example 1). As used herein, the term “total fatty acid (TFA) content” or variations thereof refers to the total amount of fatty acids in the cell, plant or part thereof on a weight basis, as a percentage of the weight of the cell, plant or part thereof. Unless otherwise specified, the weight of the cell, plant or part thereof is the dry weight of the cell, plant or part thereof. TFA content is measured as described in Example 1 herein. The method involves conversion of the fatty acids in the sample to FAME and measurement of the amount of FAME by GC, using addition of a known amount of a distinctive fatty acid standard such as C17:0 as a quantitation standard in the GC. TFA therefore represents the weight of just the fatty acids, not the weight of the fatty acids and their linked moieties in the plant lipid. As used herein, the“TAG/TFA Quotient” or “TTQ” parameter is calculated as the level of TAG (%) divided by the level of TFA (%), each as a percentage of the dry weight of the plant material. For example, a TAG level of 6% comprised in a TFA level of 10% yields a TTQ of 0.6. The TAG and TFA levels are measured as described herein. It is understood that, in this context, the TFA level refers to the weight of the total fatty acid content and the TAG level refers to the weight of TAG, including the glycerol moiety of TAG. As used herein, the term “soluble protein content” or variations thereof refers to the amount of soluble protein in the plant or part thereof. Soluble protein content can be calculated using techniques known in the art. For instance, fresh tissue can be ground, chlorophyll and soluble sugars extracted by heating to 80° C. in 50-80% (v/v) ethanol in 2.5 mM HEPES buffer at pH 7.5, centriguation, washing pellet in distilled water, resuspending the pellet 0.1 M NaOH and heating to 95° C. for 30 min, and then the Bradford assay (Bradford, 1976) is used determined soluble protein content. Alternatively, fresh tissue can be ground in buffer containing 100 mM Tris-HCl pH 8.0 and 10 mM MgCl2. As used herein, the term “nitrogen content” or variations thereof refers to the amount of nitrogen in the plant or part thereof. Nitrogen content can be calculated using techniques known in the art. For example, freeze-dried tissue can be analysed using a Europa 20-20 isotope ratio mass spectrometer with an ANCA preparation system, comprising a combustion and reduction tube operating at 1000° C. and 600° C., respectively, to determine nitrogen content. As used herein, the term “carbon content” or variations thereof refers to the amount of carbon in the plant or part thereof. Carbon content can be calculated using techniques known in the art. For example, organic carbon levels can be deteremined using the method described by Shaw (1959), or as described in Example 1. As used herein, the term “carbon:nitrogen ratio” or variations thereof refers to the relative amount of carbon in the cell, plant or part thereof when compared to the amount of nitrogen in the cell, plant or part thereof. Carbon and nitrogen contents can be calculated as described above and represented as a ratio. As used herein, the term “photosynthetic gene expression” or variations thereof refers to one or more genes expressing proteins involved in photosynthetic pathways in the plant of part thereof. Examples of photosynthetic genes which may be upregulated in plants or parts thereof of the invention include, but are not limited to, one or more of the genes listed in Table 10. As used herein, the term “photosynthetic capacity” or variations thereof refers to the ability of the plant or part thereof to photosynthesize (convert light energy to chemical energy). Photosynthetic capacity (Amax) is a measure of the maximum rate at which leaves are able to fix carbon during photosynthesis. It is typically measured as the amount of carbon dioxide that is fixed per metre squared per second, for example as μmol m−2sec−1. Photosynthetic capacity can be calculated using techniques known in the art. As used herein, the term “total dietary fibre (TDF) content” or variations thereof refers to the amount of fiber (including soluble and insoluble fibre) in the cell, plant or part thereof. As the skilled person would understand, dietary fiber includes non-starch polysaccharides such as arabinoxylans, cellulose, and many other plant components such as resistant starch, resistant dextrins, inulin, lignin, chitins, pectins, β-glucans, and oligosaccharides. TDF can be calculated using techniques known in the art. For example, using the Prosky method (Prosky et al. 1985), the McCleary method (McCleary et al., 2007) or the rapid integrated total dietary fiber method (McCleary et al., 2015). As used herein, the term “energy content” or variations thereof refers to the amount of food energy in the plant or part thereof. More specifically, the amount of chemical energy that animals (including humans) derive from their food. Energy content can be calculated using techniques known in the art. For example, energy content can be determined based on heats of combustion in a bomb calorimeter and corrections that take into consideration the efficiency of digestion and absorption and the production of urea and other substances in the urine. As another example, energy content can be calculated as described in Example 1. As used herein, the term “extracted lipid” refers to a composition extracted from a cell, plant or part thereof of the invention, such as a transgenic cell, plant or part thereof of the invention, which comprises at least 60% (w/w) lipid. As used herein, the term “non-polar lipid” refers to fatty acids and derivatives thereof which are soluble in organic solvents but insoluble in water. The fatty acids may be free fatty acids and/or in an esterified form. Examples of esterified forms of non-polar lipid include, but are not limited to, triacylglycerol (TAG), diacylyglycerol (DAG), monoacylglycerol (MAG). Non-polar lipids also include sterols, sterol esters and wax esters. Non-polar lipids are also known as “neutral lipids”. Non-polar lipid is typically a liquid at room temperature. Preferably, the non-polar lipid predominantly (>50%) comprises fatty acids that are at least 16 carbons in length. More preferably, at least 50% of the total fatty acids in the non-polar lipid are C18 fatty acids for example, oleic acid. In an embodiment, at least 5% of the total fatty acids in the non-polar lipids are C12 or C14 fatty acids, or both. In an embodiment, at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% of the fatty acids in non-polar lipid of the invention are present as TAG. The non-polar lipid may be further purified or treated, for example by hydrolysis with a strong base to release the free fatty acid, or by fractionation, distillation, or the like. Non-polar lipid may be present in or obtained from plant parts such as seed, leaves, tubers, beets or fruit. Non-polar lipid of the invention may form part of “seedoil” if it is obtained from seed. The free and esterified sterol (for example, sitosterol, campesterol, stigmasterol, brassicasterol, Δ5-avenasterol, sitostanol, campestanol, and cholesterol) concentrations in the extracted lipid may be as described in Phillips et al. (2002). Sterols in plant oils are present as free alcohols, esters with fatty acids (esterified sterols), glycosides and acylated glycosides of sterols. Sterol concentrations in naturally occurring vegetable oils (seedoils) ranges up to a maximum of about 1100 mg/100 g. Hydrogenated palm oil has one of the lowest concentrations of naturally occurring vegetable oils at about 60 mg/100 g. The recovered or extracted seedoils of the invention preferably have between about 100 and about 1000 mg total sterol/100 g of oil. For use as food or feed, it is preferred that sterols are present primarily as free or esterified forms rather than glycosylated forms. In the seedoils of the present invention, preferably at least 50% of the sterols in the oils are present as esterified sterols, except for soybean seedoil which has about 25% of the sterols esterified. The canola seedoil and rapeseed oil of the invention preferably have between about 500 and about 800 mg total sterol/100 g, with sitosterol the main sterol and campesterol the next most abundant. The corn seedoil of the invention preferably has between about 600 and about 800 mg total sterol/100 g, with sitosterol the main sterol. The soybean seedoil of the invention preferably has between about 150 and about 350 mg total sterol/100 g, with sitosterol the main sterol and stigmasterol the next most abundant, and with more free sterol than esterified sterol. The cottonseed oil of the invention preferably has between about 200 and about 350 mg total sterol/100 g, with sitosterol the main sterol. The coconut oil and palm oil of the invention preferably have between about 50 and about 100 mg total sterol/100 g, with sitosterol the main sterol. The safflower seedoil of the invention preferably has between about 150 and about 250 mg total sterol/100 g, with sitosterol the main sterol. The peanut seedoil of the invention preferably has between about 100 and about 200 mg total sterol/100 g, with sitosterol the main sterol. The sesame seedoil of the invention preferably has between about 400 and about 600 mg total sterol/100 g, with sitosterol the main sterol. The sunflower seedoil of the invention preferably has between about 200 and 400 mg total sterol/100 g, with sitosterol the main sterol. Oils obtained from vegetative plant parts according to the invention preferably have less than 200 mg total sterol/100 g, more preferably less than 100 mg total sterol/100 g, and most preferably less than 50 mg total sterols/100 g, with the majority of the sterols being free sterols. As used herein, the term “vegetative oil” refers to a composition obtained from vegetative parts of a plant which comprises at least 60% (w/w) lipid, or obtainable from the vegetative parts if the oil is still present in the vegetative part. That is, vegetative oil of the invention includes oil which is present in the vegetative plant part, as well as oil which has been extracted from the vegetative part (extracted oil). The vegetative oil is preferably extracted vegetative oil. Vegetative oil is typically a liquid at room temperature. Preferably, the total fatty acid (TFA) content in the vegetative oil predominantly (>50%) comprises fatty acids that are at least 16 carbons in length. More preferably, at least 50% of the total fatty acids in the vegetative oil are C18 fatty acids for example, oleic acid. The fatty acids are typically in an esterified form such as for example, TAG, DAG, acyl-CoA, galactolipid or phospholipid. The fatty acids may be free fatty acids and/or in an esterified form. In an embodiment, at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% of the fatty acids in vegetative oil of the invention can be found as TAG. In an embodiment, vegetative oil of the invention is “substantially purified” or “purified” oil that has been separated from one or more other lipids, nucleic acids, polypeptides, or other contaminating molecules with which it is associated in the vegetative plant part or in a crude extract. It is preferred that the substantially purified vegetative oil is at least 60% free, more preferably at least 75% free, and more preferably, at least 90% free from other components with which it is associated in the vegetative plant part or extract. Vegetative oil of the invention may further comprise non-fatty acid molecules such as, but not limited to, sterols. In an embodiment, the vegetative oil is canola oil (Brassicasp. such asBrassica carinata, Brassica juncea, Brassica napobrassica, Brassica napus) mustard oil (Brassica juncea), otherBrassicaoil (e.g.,Brassica napobrassica, Brassica camelina), sunflower oil (Helianthussp. such asHelianthus annuus), linseed oil (Linum usitatissimum), soybean oil (Glycine max), safflower oil (Carthamus tinctorius), corn oil (Zea mays), tobacco oil (Nicotianasp. such asNicotiana tabacumorNicotiana benthamiana), peanut oil (Arachis hypogaea), palm oil (Elaeis guineensis), cotton oil (Gossypium hirsutum), coconut oil (Cocos nucifera), avocado oil (Persea americana), olive oil (Olea europaea), cashew oil (Anacardium occidentale),macadamiaoil (Macadamia intergrifolia), almond oil (Prunus amygdalus), oat oil (Avena sativa), rice oil (Oryzasp. such asOryza sativaandOryza glaberrima),Arabidopsisoil (Arabidopsis thaliana),Aracinis hypogaea(peanut),Beta vulgaris(sugar beet),Camelina sativa(false flax),Crambe abyssinica(Abyssinian kale),Cucumis melo(melon),Hordeum vulgare(barley),Jatropha curcas(physic nut),Joannesia princeps(arara nut-tree),Licania rigida(oiticica),Lupinus angustifolius(lupin),Miscanthussp. such asMiscanthusxgiganteusandMiscanthus sinensis, Panicum virgatum(switchgrass),Pongamia pinnata(Indian beech),Populus trichocarpa, Ricinus communis(castor),Saccharumsp. (sugarcane),Sesamum indicum(sesame),Solanum tuberosum(potato),Sorghumsp. such asSorghum bicolor, Sorghum vulgare, Theobroma grandiforum(cupuassu),Trifoliumsp., andTriticumsp. (wheat) such asTriticum aestivum. Vegetative oil may be extracted from vegetative plant parts by any method known in the art, such as for extracting seedoils. This typically involves extraction with nonpolar solvents such as diethyl ether, petroleum ether, chloroform/methanol or butanol mixtures, generally associated with first crushing of the seeds. Lipids associated with the starch or other polysaccharides may be extracted with water-saturated butanol. The seedoil may be “de-gummed” by methods known in the art to remove polar lipids such as phospholipids or treated in other ways to remove contaminants or improve purity, stability, or colour. The TAGs and other esters in the vegetative oil may be hydrolysed to release free fatty acids, or the oil hydrogenated, treated chemically, or enzymatically as known in the art. As used herein, the term “seedoil” has an analogous meaning except that it refers to a lipid composition obtained from seeds of plants of the invention. As used herein, the term “fatty acid” refers to a carboxylic acid with an aliphatic tail of at least 8 carbon atoms in length, either saturated or unsaturated. Preferred fatty acids have a carbon-carbon bonded chain of at least 12 carbons in length. Most naturally occurring fatty acids have an even number of carbon atoms because their biosynthesis involves acetate which has two carbon atoms. The fatty acids may be in a free state (non-esterified) or in an esterified form such as part of a TAG, DAG, MAG, acyl-CoA (thio-ester) bound, acyl-ACP bound, or other covalently bound form. When covalently bound in an esterified form, the fatty acid is referred to herein as an “acyl” group. The fatty acid may be esterified as a phospholipid such as a phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or diphosphatidylglycerol. Saturated fatty acids do not contain any double bonds or other functional groups along the chain. The term “saturated” refers to hydrogen, in that all carbons (apart from the carboxylic acid [—COOH] group) contain as many hydrogens as possible. In other words, the omega (ω) end contains 3 hydrogens (CH3—) and each carbon within the chain contains 2 hydrogens (—CH2—). Unsaturated fatty acids are of similar form to saturated fatty acids, except that one or more alkene functional groups exist along the chain, with each alkene substituting a singly-bonded “—CH2—CH2—” part of the chain with a doubly-bonded “—CH═CH—” portion (that is, a carbon double bonded to another carbon). The two next carbon atoms in the chain that are bound to either side of the double bond can occur in a cis or trans configuration. As used herein, the terms “monounsaturated fatty acid” or “MUFA” refer to a fatty acid which comprises at least 12 carbon atoms in its carbon chain and only one alkene group (carbon-carbon double bond), which may be in an esterified or non-esterified (free) form. As used herein, the terms “polyunsaturated fatty acid” or “PUFA” refer to a fatty acid which comprises at least 12 carbon atoms in its carbon chain and at least two alkene groups (carbon-carbon double bonds), which may be in an esterified or non-esterified form. “Monoacylglyceride” or “MAG” is glyceride in which the glycerol is esterified with one fatty acid. As used herein, MAG comprises a hydroxyl group at an sn-1/3 (also referred to herein as sn-1 MAG or 1-MAG or 1/3-MAG) or sn-2 position (also referred to herein as 2-MAG), and therefore MAG does not include phosphorylated molecules such as PA or PC. MAG is thus a component of neutral lipids in a plant or part thereof. “Diacylglyceride” or “DAG” is glyceride in which the glycerol is esterified with two fatty acids which may be the same or, preferably, different. As used herein, DAG comprises a hydroxyl group at a sn-1,3 or sn-2 position, and therefore DAG does not include phosphorylated molecules such as PA or PC. DAG is thus a component of neutral lipids in a plant or part thereof. In the Kennedy pathway of DAG synthesis (FIG.1), the precursor sn-glycerol-3-phosphate (G3P) is esterified to two acyl groups, each coming from a fatty acid coenzyme A ester, in a first reaction catalysed by a glycerol-3-phosphate acyltransferase (GPAT) at position sn-1 to form LysoPA, followed by a second acylation at position sn-2 catalysed by a lysophosphatidic acid acyltransferase (LPAAT) to form phosphatidic acid (PA). This intermediate is then de-phosphorylated by PAP to form DAG. DAG may also be formed from TAG by removal of an acyl group by a lipase, or from PC essentially by removal of a choline headgroup by any of the enzymes PDCT, PLC or PLD (FIG.1). “Triacylglyceride” or “TAG” is a glyceride in which the glycerol is esterified with three fatty acids which may be the same (e.g. as in tri-olein) or, more commonly, different. In the Kennedy pathway of TAG synthesis, DAG is formed as described above, and then a third acyl group is esterified to the glycerol backbone by the activity of DGAT. Alternative pathways for formation of TAG include one catalysed by the enzyme PDAT (FIG.1) and the MGAT pathway described herein. As used herein, the term “wild-type” or variations thereof refers to cell, plant or part thereof such as a cell, vegetative plant part, seed, tuber or beet, that has not been genetically modified, such as cells, plants or parts thereof that do not comprise the first and second exogenous polynucleotides, according to this invention. The term “corresponding” refers to a cell, plant or part thereof such as a cell, vegetative plant part, seed, tuber or beet, that has the same or similar genetic background as a cell, plant or part thereof such as a vegetative plant part, seed, tuber or beet of the invention but which has not been modified as described herein (for example, a vegetative plant part or seed which lacks the first and second exogenous polynucleotides). In a preferred embodiment, the corresponding plant or part thereof such as a vegetative plant part is at the same developmental stage as the plant or part thereof such as a vegetative plant part of the invention. For example, if the plant is a flowering plant, then preferably the corresponding plant is also flowering. A corresponding cell, plant or part thereof such as a vegetative plant part, can be used as a control to compare levels of nucleic acid or protein expression, or the extent and nature of trait modification, for example TTQ and/or TAG content, with the cell, plant or part thereof such as a vegetative plant part of the invention which is modified as described herein. A person skilled in the art is readily able to determine an appropriate “corresponding” cell, plant or part thereof such as a vegetative plant part for such a comparison. As used herein, “compared with” or “relative to” refers to comparing levels of, for example, TTQ or triacylglycerol (TAG) content, one or more or all of soluble protein content, nitrogen content, carbon:nitrogen ratio, photosynthetic gene expression, photosynthetic capacity, total dietary fibre (TDF) content, carbon content, and energy content, or non-polar lipid content or composition, total non-polar lipid content, total fatty acid content or other parameter of the cell, plant or part thereof comprising the one or more exogenous polynucleotides, genetic modifications or exogenous polypeptides with a cell, plant or part thereof such as a vegetative plant part lacking the one or more exogenous polynucelotides, genetic modifications or polypeptides. As used herein, “synergism”, “synergistic”, “acting synergistically” and related terms are each a comparative term that means that the effect of a combination of elements present in a plant or part thereof of the invention, for example a combination of elements A and B, is greater than the sum of the effects of the elements separately in corresponding plants or parts thereof, for example the sum of the effect of A and the effect of B. Where more than two elements are present in the plant or part thereof, for example elements A, B and C, it means that the effect of the combination of all of the elements is greater than the sum of the effects of the individual effects of the elements. In a preferred embodiment, it means that the effect of the combination of elements A, B and C is greater than the sum of the effect of elements A and B combined and the effect of element C. In such a case, it can be said that element C acts synergistically with elements A and B. As would be understood, the effects are measured in corresponding cells, plants or parts thereof, for example grown under the same conditions and at the same stage of biological development. As used herein, “germinate at a rate substantially the same as for a corresponding wild-type plant” or similar phrases refers to seed of a plant of the invention being relatively able to germinate when compared to seed of a wild-type plant lacking the defined exogenous polynucleotide(s) and genetic modifications. Germination may be measured in vitro on tissue culture medium or in soil as occurs in the field. In one embodiment, the number of seeds which germinate, for instance when grown under optimal greenhouse conditions for the plant species, is at least 75%, more preferably at least 90%, when compared to corresponding wild-type seed. In another embodiment, the seeds which germinate, for instance when grown under optimal glasshouse conditions for the plant species, produce seedlings which grow at a rate which, on average, is at least 75%, more preferably at least 90%, when compared to corresponding wild-type plants. This is referred to as “seedling vigour”. In an embodiment, the rate of initial root growth and shoot growth of seedlings of the invention is essentially the same compared to a corresponding wild-type seedling grown under the same conditions. In an embodiment, the leaf biomass (dry weight) of the plants of the invention is at least 80%, preferably at least 90%, of the leaf biomass relative to a corresponding wild-type plant grown under the same conditions, preferably in the field. In an embodiment, the height of the plants of the invention is at least 70%, preferably at least 80%, more preferably at least 90%, of the plant height relative to a corresponding wild-type plant grown under the same conditions, preferably in the field and preferably at maturity. As used herein, the term “an exogenous polynucleotide which down-regulates the production and/or activity of an endogenous polypeptide” or variations thereof, refers to a polynucleotide that encodes an RNA molecule, herein termed a “silencing RNA molecule” or variations thereof (for example, encoding an amiRNA or hpRNAi), that down-regulates the production and/or activity, or itself down-regulates the production and/or activity (for example, is an amiRNA or hpRNA which can be delivered directly to, for example, the plant or part thereof) of an endogenous polypeptide. This includes where the initial RNA transcript produced by expression of the exogenous polynucleotide is processed in the cell to form the actual silencing RNA molecule. The endogenous polypeptides whose production or activity are downregulated include, for example, SDP1 TAG lipase, plastidial GPAT, plastidial LPAAT, TGD polypeptide such as TGD5, TST such as TST1 or TST2, AGPase, PDCT, CPT or Δ12 fatty acid desturase (FAD2), or a combination of two or more thereof. Typically, the RNA molecule decreases the expression of an endogenous gene encoding the polypeptide. The extent of down-regulation is typically less than 100%, for example the production or activity is reduced by between 25% and 95% relative to the wild-type. The optimal level of remaining production or activity can be routinely determined. As used herein, the term “on a weight basis” refers to the weight of a substance (for example, TAG, DAG, fatty acid, protein, nitrogen, carbon) as a percentage of the weight of the composition comprising the substance (for example, seed, leaf dry weight). For example, if a transgenic seed has 25 μg total fatty acid per 120 μg seed weight; the percentage of total fatty acid on a weight basis is 20.8%. As used herein, the term “on a relative basis” refers to a parameter such as the amount of a substance in a composition comprising the substance in comparison with the parameter for a corresponding composition, as a percentage. For example, a reduction from 3 units to 2 units is a reduction of 33% on a relative basis. As used herein, “plastids” are organelles in plants, including algae, which are the site of manufacture of carbon-based compounds from photosynthesis including sugars, starch and fatty acids. Plastids include chloroplasts which contain chlorophyll and carry out photosynthesis, etioplasts which are the predecessors of chloroplasts, as well as specialised plastids such as chromoplasts which are coloured plastids for synthesis and storage of pigments, gerontoplasts which control the dismantling of the photosynthetic apparatus during senescence, amyloplasts for starch synthesis and storage, elaioplasts for storage of lipids, and proteinoplasts for storing and modifying proteins. As used herein, the term “biofuel” refers to any type of fuel, typically as used to power machinery such as automobiles, planes, boats, trucks or petroleum powered motors, whose energy is derived from biological carbon fixation. Biofuels include fuels derived from biomass conversion, as well as solid biomass, liquid fuels and biogases. Examples of biofuels include bioalcohols, biodiesel, synthetic diesel, vegetable oil, bioethers, biogas, syngas, solid biofuels, algae-derived fuel, biohydrogen, biomethanol, 2,5-Dimethylfuran (DMF), biodimethyl ether (bioDME), Fischer-Tropsch diesel, biohydrogen diesel, mixed alcohols and wood diesel. As used herein, the term “bioalcohol” refers to biologically produced alcohols, for example, ethanol, propanol and butanol. Bioalcohols are produced by the action of microorganisms and/or enzymes through the fermentation of sugars, hemicellulose or cellulose. As used herein, the term “biodiesel” refers to a composition comprising fatty acid methyl- or ethyl-esters derived from lipids by transesterification, the lipids being from living cells not fossil fuels. As used herein, the term “synthetic diesel” refers to a form of diesel fuel which is derived from renewable feedstock rather than the fossil feedstock used in most diesel fuels. As used herein, the term “vegetable oil” includes a pure plant oil (or straight vegetable oil) or a waste vegetable oil (by product of other industries), including oil produced in either a vegetative plant part or in seed. Vegetable oil includes vegetative oil and seedoil, as defined herein. As used herein, the term “biogas” refers to methane or a flammable mixture of methane and other gases produced by anaerobic digestion of organic material by anaerobes. As used herein, the term “syngas” refers to a gas mixture that contains varying amounts of carbon monoxide and hydrogen and possibly other hydrocarbons, produced by partial combustion of biomass. Syngas may be converted into methanol in the presence of catalyst (usually copper-based), with subsequent methanol dehydration in the presence of a different catalyst (for example, silica-alumina). As used herein, the term “biochar” refers to charcoal made from biomass, for example, by pyrolysis of the biomass. As used herein, the term “feedstock” refers to a material, for example, biomass or a conversion product thereof (for example, syngas) when used to produce a product, for example, a biofuel such as biodiesel or a synthetic diesel. As used herein, the term “industrial product” refers to a hydrocarbon product which is predominantly made of carbon and hydrogen such as, for example, fatty acid methyl- and/or ethyl-esters or alkanes such as methane, mixtures of longer chain alkanes which are typically liquids at ambient temperatures, a biofuel, carbon monoxide and/or hydrogen, or a bioalcohol such as ethanol, propanol, or butanol, or biochar. The term “industrial product” is intended to include intermediary products that can be converted to other industrial products, for example, syngas is itself considered to be an industrial product which can be used to synthesize a hydrocarbon product which is also considered to be an industrial product. The term industrial product as used herein includes both pure forms of the above compounds, or more commonly a mixture of various compounds and components, for example the hydrocarbon product may contain a range of carbon chain lengths, as well understood in the art. As used herein, “progeny” means the immediate and all subsequent generations of offspring produced from a parent, for example a second, third or later generation offspring. As used herein, the term “ancestor” refers to any earlier generation of the plant comprising the first and second exogenous polynucleotides. The ancestor may be the parent plant, grandparent plant, great grandparent plant and so on. As used herein, the term “selecting a plant” means actively selecting the plant on the basis that it has the desired phenotype, such as increased TTQ, increased TAG and protein content when compared to the corresponding wild-type plant. As used herein, phrases such as “comprise a TFA content of about 5% (w/w dry weight)”, or “comprise a total TAG content of about 6% (w/w dry weight)”, or similarly structured phrases, mean that more than the defined level may be present. For instance, the phrase “comprise a TFA content of about 5% (w/w dry weight)” can be used interchangeably with “comprises at least about 5% TFA (w/w dry weight)”. Extending this example further, a vegetative plant part which comprise a TFA content of about 5% (w/w dry weight) may have a 6%, or 7.5% or higher TFA content. As used herein, unless the context indicates otherwise, the term “increased content” when used in reference to a polypeptide, or similar pharses including refrence to specific polypeptide, refers to either an exogenous polypeptide or an endogenous polypeptide. For example, a vegetative plant part of the invention may comprise an increased content of a WRI1 polypeptide, an increased content of a DGAT polypeptide, and a decreased content of a SDP1 polypeptide, each relative to a corresponding wild-type vegetative plant part, wherein each of the WRI1 and DGAT polypeptides is independently either an exogenous polypeptide or an endogenous polypeptide. As another example, a vegetative plant part of the invention may comprise an increased content of a WRI1 polypeptide, an increased content of a DGAT polypeptide, and an increased content of a LEC2 polypeptide, each relative to a corresponding wild-type vegetative plant part, wherein each of the WRI1, DGAT and LEC2 polypeptides is independently either an exogenous polypeptide or an endogenous polypeptide. As a further example, a vegetative plant part of the invention may comprise an increased content of a PDAT or DGAT polypeptide, a decreased content of a TGD polypeptide, and a decreased content of a SDP1 polypeptide, each relative to a corresponding wild-type vegetative plant part wherein the PDAT or DGAT is either an exogenous polypeptide or an endogenous polypeptide, and so on. An exogenous polypepetide may be the result of expression of a transgene encoding the polypeptide in the cell or plant or part thereof of the invention. The endogenous polypeptide may be the result of increased expression of an endogenous gene, such as inducing overexpression and/or providing increased levels of a transcription factor(s) for the gene. Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning. As used herein, the term about, unless stated to the contrary, refers to +/−10%, more preferably +/−5%, more preferably +/−2%, more preferably +/−1%, even more preferably +/−0.5%, of the designated value. Production of Plants with Modified Traits The present invention is based on the finding that plant traits, such two or more of non-polar lipid content, protein content, TTQ, TAG content, nitrogen content, carbon content, in plants or parts thereof can be increased by a combination of modifications selected from those designated herein as: (A). Push, (B). Pull, (C). Protect, (D). Package, (E). Plastidial Export, (F). Plastidial Import and (G). Prokaryotic Pathway. Plants or parts thereof such as a vegetative plant parts of the invention therefore have a number of combinations of exogenous polynucleotides and/or genetic modifications each of which provide for one of the modifications. These exogenous polynucleotides and/or genetic modifications include:(A) an exogenous polynucleotide which encodes a transcription factor polypeptide that increases the expression of one or more glycolytic and/or fatty acid biosynthetic genes in the plant or part thereof such as a vegetative plant part, providing the “Push” modification,(B) an exogenous polynucleotide which encodes a polypeptide involved in the biosynthesis of one or more non-polar lipids in the plant or part thereof such as a vegetative plant part, providing the “Pull” modification,(C) a genetic modification which down-regulates endogenous production and/or activity of a polypeptide involved in the catabolism of triacylglycerols (TAG) in the plant or part thereof such as a vegetative plant part when compared to a corresponding plant or part thereof such as a vegetative plant part lacking the genetic modification, providing the “Protect” modification,(D) an exogenous polynucleotide which encodes an oil body coating (OBC) polypeptide such as a lipid droplet associated polypeptide (LDAP), providing the “Package” modification,(E) an exogenous polynucleotide which encodes a polypeptide which increases the export of fatty acids out of plastids of the plant or part thereof such as a vegetative plant part, when compared to a corresponding plant or part thereof such as a vegetative plant part lacking the exogenous polynucleotide, providing the “Plastidial Export” modification,(F) a genetic modification which down-regulates endogenous production and/or activity of a polypeptide involved in importing fatty acids into plastids of the plant or part thereof such as a vegetative plant part when compared to a corresponding plant or part thereof such as a vegetative plant part lacking the genetic modification, providing the “Plastidial Import” modification, andG) a genetic modification which down-regulates endogenous production and/or activity of a polypeptide involved in diacylglycerol (DAG) production in the plastid of the plant or part thereof such as a vegetative plant part when compared to a corresponding plant or part thereof such as a vegetative plant part lacking the genetic modification, providing the “prokaryotic Pathway” modification. Preferred combinations (also referred to herein as sets) of exogenous polynucleotides and/or genetic modifications of the invention are;1) A, B and optionally one of C, D, E, F or G;2) A, C and optionally one of D, E, F or G;3) A, D and optionally one of E, F or G;4) A, E and optionally F or G;5) A, F and optionally G;6) A and G;7) A, B, C and optionally one of D, E, F or G;8) A, B, D and optionally one of E, F or G;9) A, B, E and optionally F or G;10) A, B, F and optionally G;11) A, B, C, D and optionally one of E, F or G;12) A, B, C, E and optionally F or G;13) A, B, C, F and optionally G;14) A, B, D, E and optionally F or G;15) A, B, D, F and optionally G;16) A, B, E, F and optionally G;17) A, C, D and optionally one of E, F or G;18) A, C, E and optionally F or G;19) A, C, F and optionally G;20) A, C, D, E and optionally F or G;21) A, C, D, F and optionally G;22) A, C, E, F and optionally a fifth modification G;23) A, D, E and optionally F or G;24) A, D, F and optionally G;25) A, D, E, F and optionally G;26) A, E, F and optionally G;27) Six of A, B, C, D, E, F and G omitting one of A, B, C, D, E, F or G, and28) Any one of 1-26 above where there are two or more exogenous polynucleotides encoding two or more different transcription factor polypeptides that increases the expression of one or more glycolytic and/or fatty acid biosynthetic genes in the plant or part thereof, for example one exogenous polynucleotide encoding WRI1 and another exogenous polynucleotide encoding LEC2. In each of the above preferred combinations there may be at least two different exogenous polynucleotides which encode at least two different transcription factor polypeptides that increases the expression of one or more glycolytic and/or fatty acid biosynthetic genes in the plant or part thereof such as a vegetative plant part. These modifications are described more fully as follows: A. The “Push” modification is characterised by an increased synthesis of total fatty acids in the plastids of the plant or part thereof. In an embodiment, this occurs by the increased expression and/or activity of a transcription factor which regulates fatty acid synthesis in the plastids. In one embodiment, this can be achieved by expressing in a transgenic plant or part thereof an exogenous polynucleotide which encodes a transcription factor polypeptide that increases the expression of one or more glycolytic and/or fatty acid biosynthetic genes in the plant or part thereof. In an embodiment, the increased fatty acid synthesis is not caused by the provision to the plant or part thereof of an altered ACCase whose activity is less inhibited by fatty acids, relative to the endogenous ACCase in the plant or part thereof. In an embodiment, the plant or part thereof comprises an exogenous polynucleotide which encodes the transcription factor, preferably under the control of a promoter other than a constitutive promoter. The transcription factor may be selected from the group consisting of WRI1, LEC1, LEC1-like, LEC2, BBM, FUS3, ABI3, ABI4, ABI5, Dof4, Dofl1 or the group consisting of MYB73, bZIP53, AGL15, MYB115, MYB118, TANMEI, WUS, GFR2a1, GFR2a2 and PHR1, and is preferably WRI1, LEC1 or LEC2. In a further embodiment, the increased synthesis of total fatty acids is relative to a corresponding wild-type plant or part thereof. In an embodiment, there are two or more exogenous polynucleotides encoding two or more different transcription factor polypeptides. The “Push” modification may also be achieved by increased expression of polypeptides which modulate activity of WRI1, such as MED15 or 14-3-3 polypeptides. B. The “Pull” modification is characterised by increased expression and/or activity in the plant or part thereof of a fatty acyl acyltransferase which catalyses the synthesis of TAG, DAG or MAG in the plant or part thereof, such as a DGAT, PDAT, LPAAT, GPAT or MGAT, preferably a DGAT or a PDAT. In one embodiment, this can be achieved by expressing in a transgenic plant or part thereof an exogenous polynucleotide which encodes a polypeptide involved in the biosynthesis of one or more non-polar lipids. In an embodiment, the acyltransferase is a membrane-bound acyltransferase that uses an acyl-CoA substrate as the acyl donor in the case of DGAT, LPAAT, GPAT or MGAT, or an acyl group from PC as the acyl donor in the case of PDAT. The Pull modification can be relative to a corresponding wild-type plant or part thereof or, preferably, relative to a corresponding plant or part thereof which has the Push modification. In an embodiment, the plant or part thereof comprises an exogenous polynucleotide which encodes the fatty acyl acyltransferase. The “Pull” modification can also be achieved by increased expression of a PDCT, CPT or phospholipase C or D polypeptide which increases the production of DAG from PC. C. The “Protect” modification is characterised by a reduction in the catabolism of triacylglycerols (TAG) in the plant or part thereof. In an embodiment, this can be achieved through a genetic modification in the plant or part thereof which down-regulates endogenous production and/or activity of a polypeptide involved in the catabolism of triacylglycerols (TAG) in the plant or part thereof when compared to a corresponding plant or part thereof lacking the genetic modification. In an embodiment, the plant or part thereof has a reduced expression and/or activity of an endogenous TAG lipase in the plant or part thereof, preferably an SDP1 lipase, a Cgi58 polypeptide, an acyl-CoA oxidase such as the ACX1 or ACX2, or a polypeptide involved in β-oxidation of fatty acids in the plant or part thereof such as a PXA1 peroxisomal ATP-binding cassette transporter. This may occur by expression in the plant or part thereof of an exogenous polynucleotide which encodes an RNA molecule which reduces the expression of, for example, an endogenous gene encoding the TAG lipase such as the SDP1 lipase, acyl-CoA oxidase or the polypeptide involved in (3-oxidation of fatty acids in the plant or part thereof, or by a mutation in an endogenous gene encoding, for example, the TAG lipase, acyl-CoA oxidase or polypeptide involved in β-oxidation of fatty acids. In an embodiment, the reduced expression and/or activity is relative to a corresponding wild-type plant or part thereof or relative to a corresponding plant or part thereof which has the Push modification. D. The “Package” modification is characterised by an increased expression and/or accumulation of an oil body coating (OBC) polypeptide. In an embodiment, this can be achieved by expressing in a transgenic plant or part thereof an exogenous polynucleotide which encodes an oil body coating (OBC) polypeptide. The OBC polypeptide may be an oleosin, such as for example a polyoleosin, a caoleosin or a steroleosin, or preferably an LDAP. In an embodiment, the level of oleosin that is accumulated in the plant or part thereof is at least 2-fold higher relative to the corresponding plant or part thereof comprising the oleosin gene from the T-DNA of pJP3502. In an embodiment, the increased expression or accumulation of the OBC polypeptide is not caused solely by the Push modification. In an embodiment, the expression and/or accumulation is relative to a corresponding wild-type plant or part thereof or, preferably, relative to a corresponding plant or part thereof which has the Push modification. E. The “Plastidial Export” modification is characterised by an increased rate of export of total fatty acids out of the plastids of the plant or part thereof. In one embodiment, this can be achieved by expressing in a plant or part thereof an exogenous polynucleotide which encodes a polypeptide which increases the export of fatty acids out of plastids of the plant or part thereof when compared to a corresponding plant or part thereof lacking the exogenous polynucleotide. In an embodiment, this occurs by the increased expression and/or activity of a fatty acid thioesterase (TE), a fatty acid transporter polypeptide such as an ABCA9 polypeptide, or a long-chain acyl-CoA synthetase (LACS). In an embodiment, the plant or part thereof comprises an exogenous polynucleotide which encodes the TE, fatty acid transporter polypeptide or LACS. The TE may be a FATB polypeptide or preferably a FATA polypeptide. In an embodiment, the Plastidial Export modification is relative to a corresponding wild-type plant or part thereof or, preferably, relative to a corresponding plant or part thereof which has the Push modification. F. The “Plastidial Import” modification is characterised by a reduced rate of import of fatty acids into the plastids of the plant or part thereof from outside of the plastids. In an embodiment, this can be achieved through a genetic modification in the plant or part thereof which down-regulates endogenous production and/or activity of a polypeptide involved in importing fatty acids into plastids of the plant or part thereof when compared to a corresponding plant or part thereof lacking the genetic modification. For example, this may occur by expression in the plant or part thereof of an exogenous polynucleotide which encodes an RNA molecule which reduces the expression of an endogenous gene encoding an transporter polypeptide such as a TGD polypeptide, for example a TGD1, TGD2, TGD3, TGD4 or preferably a TGD5 polypeptide, or by a mutation in an endogenous gene encoding the TGD polypeptide. In an embodiment, the reduced rate of import is relative to a corresponding wild-type plant or part thereof or relative to a corresponding plant or part thereof which has the Push modification. G. The “Prokaryotic Pathway” modification is characterised by a decreased amount of DAG or rate of production of DAG in the plastids of the plant or part thereof. In an embodiment, this can be achieved through a genetic modification in the plant or part thereof which down-regulates endogenous production and/or activity of a polypeptide involved in diacylglycerol (DAG) production in the plastid when compared to a corresponding plant or part thereof lacking the genetic modification. In an embodiment, the decreased amount or rate of production of DAG occurs by a decreased production of LPA from acyl-ACP and G3P in the plastids. The decreased amount or rate of production of DAG may occur by expression in the plant or part thereof of an exogenous polynucleotide which encodes an RNA molecule which reduces the expression of an endogenous gene encoding a plastidial GPAT, plastidial LPAAT or a plastidial PAP, preferably a plastidial GPAT, or by a mutation in an endogenous gene encoding the plastidial polypeptide. In an embodiment, the decreased amount or rate of production of DAG is relative to a corresponding wild-type plant or part thereof or, preferably, relative to a corresponding plant or part thereof which has the Push modification. The Push modification is highly desirable in the invention, and the Pull modification is preferred. The Protect and Package modifications may be complementary i.e. one of the two may be sufficient. The plant or part thereof may comprise one, two or all three of the Plastidial Export, Plastidial Import and Prokaryotic Pathway modifications. In an embodiment, at least one of the exogenous polynucleotides in the plant or part thereof, preferably at least the exogenous polynucleotide encoding the transcription factor which regulates fatty acid synthesis in the plastids, is expressed under the control of (H) a promoter other than a constitutive promoter such as, for example, a developmentally related promoter, a promoter that is preferentially active in photosynthetic cells, a tissue-specific promoter, a promoter which has been modified by reducing its expression level relative to a corresponding native promoter, or is preferably a senesence-specific promoter. More preferably, at least the exogenous polynucleotide encoding the transcription factor which regulates fatty acid synthesis in the plastids is expressed under the control of a promoter other than a constitutive promoter and the exogenous polynucleotide which encodes an RNA molecule which down-regulates endogenous production and/or activity of a polypeptide involved in the catabolism of triacylglycerols is also expressed under the control of a promoter other than a constitutive promoter, which promoters may be the same or different. Alternatively in monocotyledonous plants, the exogenous polynucleotide encoding the transcription factor which regulates fatty acid synthesis in the plastids is expressed under the control of a constitutive promoter such as, for example, a ubiquitin gene promoter or an actin gene promoter. Plants produce some, but not all, of their membrane lipids such as MGDG in plastids by the so-called prokaryotic pathway (FIG.1). In plants, there is also a eukaryotic pathway for synthesis of galactolipids and glycerolipids which synthesizes FA first of all in the plastid and then assembles the FA into glycerolipids in the ER. MGDG synthesised by the eukaryotic pathway contains C18:3 (ALA) fatty acid esterified at the sn-2 position of MGDG. The DAG backbone including the ALA for the MGDG synthesis by this pathway is assembled in the ER and then imported into the plastid. In contrast, the MGDG synthesized by the prokaryotic pathway contains C16:3 fatty acid esterified at the sn-2 position of MGDG. The ratio of the contribution of the prokaryotic pathway relative to the eukaryotic pathway in producing MGDG (16:3) vs MGDG (18:3) is a characteristic and distinctive feature of different plant species (Mongrand et al. 1998). This distinctive fatty acid composition of MGDG allows all higher plants (angiosperms) to be classified as either so-called 16:3 or 18:3 plants. 16:3 species, exemplified byArabidopsisandBrassica napus, generally have both of the prokaryotic and eukaryotic pathways of MGDG synthesis operating, whereas the 18:3 species exemplified bySorghum bicolor, Zea mays, Nicotiana tabacum, Pisum sativumandGlycine maxgenerally have only (or almost entirely) the eukaryotic pathway of MGDG synthesis, providing little or no C16:3 fatty acid accumulation in the vegetative tissues. As used herein, a “16:3 plant” or “16:3 species” is one which has more than 2% C16:3 fatty acid in the total fatty acid content of its photosynthetic tissues. As used herein, a “18:3 plant” or “18:3 species” is one which has less than 2% C16:3 fatty acid in the total fatty acid content of its photosynthetic tissues. As described herein, a plant can be converted from being a 16:3 plant to an 18:3 plant by suitable genetic modifications. The proportion of flux between the prokaryote and eukaryote pathways is not conserved across different plant species or tissues. In 16:3 species up to 40% of flux in leaves occurs via the prokaryotic pathway (Browse et al., 1986), while in 18:3 species, such as pea and soybean, about 90% of FAs which are synthesized in the plastid are exported out of the plastid to the ER to supply the source of FA for the eukaryotic pathway (Ohlrogge and Browse, 1995; Somerville et al., 2000). Therefore different amounts of 18:3 and 16:3 fatty acids are found within the glycolipids of different plant species. This is used to distinguish between 18:3 plants whose fatty acids with 3 double bonds are almost entirely C18 fatty acids and the 16:3 plants that contain both C16- and C18-fatty acids having 3 double bonds. In chloroplasts of 18:3 plants, enzymic activities catalyzing the conversion of phosphatidate to diacylglycerol and of diacylglycerol to monogalactosyl diacylglycerol (MGD) are significantly less active than in 16:3 chloroplasts. In leaves of 18:3 plants, chloroplasts synthesize stearoyl-ACP2 in the stroma, introduce the first double bond into the saturated hydrocarbon chain, and then hydrolyze the thioester by thioesterases (FIG.1). Released oleate is exported across chloroplast envelopes into membranes of the cell, probably the endoplasmic reticulum, where it is incorporated into PC. PC-linked oleoyl groups are desaturated in these membranes and subsequently move back into the chloroplast. The MGD-linked acyl groups are substrates for the introduction of the third double bond to yield MGD with two linolenoyl residues. This galactolipid is characteristic of 18:3 plants such as Asteraceae and Fabaceae, for example. In photosynthetically active cells of 16:3 plants which are represented, for example, by members of Apiaceae and Brassicaceae, two pathways operate in parallel to provide thylakoids with MGD. In one embodiment, the plant or part thereof such as a vegetative plant part of the invention produces higher levels of non-polar lipids such as TAG, or total fatty acid (TFA) content, preferably both, than a corresponding plant or part thereof such as a vegetative plant part which lacks the genetic modifications or exogenous polynucleotides. In one example, plants of the invention produce seeds, leaves, or have leaf portions of at least 1 cm2in surface area, stems and/or tubers having an increased non-polar lipid content such as TAG or TFA content, preferably both, when compared to corresponding seeds, leaves, leaf portions of at least 1 cm2in surface area, stems or tubers. In another embodiment, the plant or part thereof such as a vegetative plant part, produce TAGs that are enriched for one or more particular fatty acids. A wide spectrum of fatty acids can be incorporated into TAGs, including saturated and unsaturated fatty acids and short-chain and long-chain fatty acids. Some non-limiting examples of fatty acids that can be incorporated into TAGs and which may be increased in level include: capric (10:0), lauric (12:0), myristic (14:0), palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), vaccenic (18:1), linoleic (18:2), eleostearic (18:3), γ-linolenic (18:3), α-linolenic (18:3ω3), stearidonic (18:4ω3), arachidic (20:0), eicosadienoic (20:2), dihomo-γ-linoleic (20:3), eicosatrienoic (20:3), arachidonic (20:4), eicosatetraenoic (20:4), eicosapentaenoic (20:5ω3), behenic (22:0), docosapentaenoic (22:5ω), docosahexaenoic (22:6ω3), lignoceric (24:0), nervonic (24:1), cerotic (26:0), and montanic (28:0) fatty acids. In one embodiment of the present invention, the plant or part thereof is enriched for TAGs comprising oleic acid, and/or is reduced in linolenic acid (ALA), preferably by at least 2% or at least 5% on an absolute basis. Preferably, the plant or part thereof such as a vegetative plant part of the invention is transformed with one or more exogenous polynucleotides such as chimeric DNAs. In the case of multiple chimeric DNAs, these are preferably covalently linked on one DNA molecule such as, for example, a single T-DNA molecule, and preferably integrated at a single locus in the host cell genome, preferably the host nuclear genome. Alternatively, the chimeric DNAs are on two or more DNA molecules which may be unlinked in the host genome, or the DNA molecule(s) is not integrated into the host genome, such as occurs in transient expression experiments. The plant or part thereof such as a vegetative plant part is preferably homozygous for the one DNA molecule inserted into its genome. Transcription Factors Various transcription factors are involved in plant cells in the synthesis of fatty acids and lipids incorporating the fatty acids such as TAG, and therefore can be manipulated for the Push modification. A preferred transcription factor is WRI1. As used herein, the term “Wrinkled 1” or “WRI1” or “WRL1” refers to a transcription factor of the AP2/ERWEBP class which regulates the expression of several enzymes involved in glycolysis and de novo fatty acid biosynthesis. WRI1 has two plant-specific (AP2/EREB) DNA-binding domains. WRI1 in at leastArabidopsisalso regulates the breakdown of sucrose via glycolysis thereby regulating the supply of precursors for fatty acid biosynthesis. In other words, it controls the carbon flow from the photosynthate to storage lipids. wri1 mutants in at leastArabidopsishave a wrinkled seed phenotype, due to a defect in the incorporation of sucrose and glucose into TAGs. Examples of genes which are transcribed by WRI1 include, but are not limited to, one or more, preferably all, of genes encoding pyruvate kinase (At5g52920, At3g22960), pyruvate dehydrogenase (PDH) Elalpha subunit (At1g01090), acetyl-CoA carboxylase (ACCase), BCCP2 subunit (At5g15530), enoyl-ACP reductase (At2g05990; EAR), phosphoglycerate mutase (At1g22170), cytosolic fructokinase, and cytosolic phosphoglycerate mutase, sucrose synthase (SuSy) (see, for example, Liu et al., 2010; Baud et al., 2007; Ruuska et al., 2002). WRI1 contains the conserved domain AP2 (cd00018). AP2 is a DNA-binding domain found in transcription regulators in plants such as APETALA2 and EREBP (ethylene responsive element binding protein). In EREBPs the domain specifically binds to the 1 lbp GCC box of the ethylene response element (ERE), a promotor element essential for ethylene responsiveness. EREBPs and the C-repeat binding factor CBF1, which is involved in stress response, contain a single copy of the AP2 domain. APETALA2-like proteins, which play a role in plant development contain two copies. Other sequence motifs which may be found in WRI1 and its functional homologs include: 1.(SEQ ID NO: 89)R G V T/S R H R W T G R.2.(SEQ ID NO: 90)F/Y E A H L W D K.3.(SEQ ID NO: 91)D L A A L K Y W G.4.(SEQ ID NO: 92)S X G F S/A R G X.5.(SEQ ID NO: 93)H H H/Q N G R/K W E A R I G R/K V.6.(SEQ ID NO: 94)Q E E A A A X Y D. As used herein, the term “Wrinkled 1” or “WRI1” also includes “Wrinkled 1-like” or “WRI1-like” proteins. Examples of WRI1 proteins include Accession Nos: Q6X5Y6, (Arabidopsis thaliana; SEQ ID NO:22), XP_002876251.1 (Arabidopsis lyratasubsp.Lyrata; SEQ ID NO:23), ABD16282.1 (Brassica napus; SEQ ID NO:24), AD016346.1 (Brassica napus; SEQ ID NO:25), XP_003530370.1 (Glycine max; SEQ ID NO:26), AE022131.1 (Jatropha curcas; SEQ ID NO:27), XP_002525305.1 (Ricinus communis; SEQ ID NO:28), XP_002316459.1 (Populus trichocarpa; SEQ ID NO:29), CB129147.3 (Vitis vinifera; SEQ ID NO:30), XP_003578997.1 (Brachypodium distachyon; SEQ ID NO:31), BAJ86627.1 (Hordeum vulgaresubsp.vulgare; SEQ ID NO:32), EAY79792.1 (Oryza sativa; SEQ ID NO:33), XP_002450194.1 (Sorghum bicolor; SEQ ID NO:34), ACG32367.1 (Zea mays; SEQ ID NO:35), XP_003561189.1 (Brachypodium distachyon; SEQ ID NO:36), ABL85061.1 (Brachypodium sylvaticum; SEQ ID NO:37), BAD68417.1 (Oryza sativa; SEQ ID NO:38), XP_002437819.1 (Sorghum bicolor; SEQ ID NO:39), XP_002441444.1 (Sorghum bicolor; SEQ ID NO:40), XP_003530686.1 (Glycine max; SEQ ID NO:41), XP_003553203.1 (Glycine max; SEQ ID NO:42), XP_002315794.1 (Populus trichocarpa; SEQ ID NO:43), XP_002270149.1 (Vitis vinifera; SEQ ID NO:44), XP_003533548.1 (Glycine max; SEQ ID NO:45), XP_003551723.1 (Glycine max; SEQ ID NO:46), XP_003621117.1 (Medicago truncatula; SEQ ID NO:47), XP_002323836.1 (Populus trichocarpa; SEQ ID NO:48), XP_002517474.1 (Ricinus communis; SEQ ID NO:49), CAN79925.1 (Vitis vinifera; SEQ ID NO:50), XP_003572236.1 (Brachypodium distachyon; SEQ ID NO:51), BAD10030.1 (Oryza sativa; SEQ ID NO:52), XP_002444429.1 (Sorghum bicolor; SEQ ID NO:53), NP_001170359.1 (Zea mays; SEQ ID NO:54), XP_002889265.1 (Arabidopsis lyratasubsp.lyrata; SEQ ID NO:55), AAF68121.1 (Arabidopsis thaliana; SEQ ID NO:56), NP_178088.2 (Arabidopsis thaliana; SEQ ID NO:57), XP_002890145.1 (Arabidopsis lyratasubsp.lyrata; SEQ ID NO:58), BAJ33872.1 (Thellungiella halophila; SEQ ID NO:59), NP_563990.1 (Arabidopsis thaliana; SEQ ID NO:60), XP_003530350.1 (Glycine max; SEQ ID NO:61), XP_003578142.1 (Brachypodium distachyon; SEQ ID NO:62), EAZ09147.1 (Oryza sativa; SEQ ID NO:63), XP_002460236.1 (Sorghum bicolor; SEQ ID NO:64), NP_001146338.1 (Zea mays; SEQ ID NO:65), XP_003519167.1 (Glycine max; SEQ ID NO:66), XP_003550676.1 (Glycine max; SEQ ID NO:67), XP_003610261.1 (Medicago truncatula; SEQ ID NO:68), XP_003524030.1 (Glycine max; SEQ ID NO:69), XP_003525949.1 (Glycine max; SEQ ID NO:70), XP_002325111.1 (Populus trichocarpa; SEQ ID NO:71), CB136586.3 (Vitis vinifera; SEQ ID NO:72), XP_002273046.2 (Vitis vinifera; SEQ ID NO:73), XP_002303866.1 (Populus trichocarpa; SEQ ID NO:74), and CB125261.3 (Vitis vinifera; SEQ ID NO:75). Further examples include Sorbi-WRL1 (SEQ ID NO:76), Lupan-WRL1 (SEQ ID NO:77), Ricco-WRL1 (SEQ ID NO:78), andLupin angustifoliusWRI1 (SEQ ID NO:79). A preferred WRI1 is a maize WRI1 or asorghumWRI1. More recently, a subset of WRI1-like transcription factors have been re-classified as WRI2, WRI3 or WRI4 transcription factors, which are characterised by preferential expression in stems and/or roots of plants rather than in developing seeds (To et al., 2012). Despite their re-classification, these are included in the definition of “WRI1” herein. Preferred WRI1-like transcription factors are those which can complement the function of a wri1 mutation in a plant, particularly the function in developing seed of the plant such as in anA. thalianawri1 mutant. The function of a WRI1-like polypeptide can also be assayed in theN. benthamianatransient assays as described herein. The WRI1 transcription factor may be endogenous to the plant or cell, or exogenous to the plant or cell, for example expressed from an exogenous polynucleotide. The WRI1 transcription factor may be a naturally occurring WRI1 polypeptide or a variant thereof, provided it retains transcription factor activity. The level or activity of an endogenous WRI1 polypeptide may also be increased by increased expression of a MED15 polypeptide (Kim et al., 2016), for example polypeptides whose amino acid sequences are provided as SEQ ID NOs:293 or 295, or of a 14-3-3 polypeptide (Ma et al., 2016), for example SEQ ID NOs:297-304 MED15 polypeptide is thought to assist in directing WRI1 to its target promoters and expression of WRI1 expression itself, while 14-3-3 polypeptides are thought to interact with WRI1 polypeptide to increase the WRI1 effect. As used herein, a “LEAFY COTYLEDON” or “LEC” polypeptide means a transcription factor which is a LEC1, LEC1-like, LEC2, ABI3 or FUS3 transcription factor which exhibits broad control on seed maturation and fatty acid synthesis. LEC2, FUS3 and ABI3 are related polypeptides that each contain a B3 DNA-binding domain of 120 amino acids (Yamasaki et al., 2004) that is only found in plant proteins. They can be distinguished by phylogenetic analysis to determine relatedness in amino acid sequence to the members of theA. thalianapolypeptides having the Accession Nos as follows: LEC2, Accession No. AAL12004.1; FUS3 (also known asFUSCA3), Accession No. AAC35247. LEC1 belongs to a different class of polypeptides and is homologous to a HAP3 polypeptide of the CBF binding factor class (Lee et al., 2003). The LEC1, LEC2 and FUS3 genes are required in early embryogenesis to maintain embryonic cell fate and to specify cotyledon identity and in later in initiation and maintenance of embryo maturation (Santos-Mendoza et al., 2008). They also induce expression of genes encoding seed storage proteins by binding to RY motifs present in the promoters, and oleosin genes. They can also be distinguished by their expression patterns in seed development or by their ability to complement the corresponding mutation inA. thaliana. As used herein, the term “Leafy Cotyledon 1” or “LEC1” refers to a NF-YB-type transcription factor which participates in zygotic development and in somatic embryogenesis. The endogenous gene is expressed specifically in seed in both the embryo and endosperm. LEC1 activates the gene encoding WRI1 as well as a large class of fatty acid synthesis genes. Ectopic expression of LEC2 also causes rapid activation of auxin-responsive genes and may cause formation of somatic embryos. Examples of LEC1 polypeptides include proteins fromArabidopsis thaliana(AAC39488, SEQ ID NO:149),Medicago truncatula(AFK49653, SEQ ID NO:154) andBrassica napus(ADF81045, SEQ ID NO:151),A. lyrata(XP_002862657, SEQ ID NO:150),R. communis(XP_002522740, SEQ ID NO:152),G. max(XP_006582823, SEQ ID NO:153),A. hypogaea(ADC33213, SEQ ID NO:156),Z. mays(AAK95562, SEQ ID NO:155). LEC1-like (L1L) is closely related to LEC1 but has a different pattern of gene expression, being expressed earlier during embryogenesis (Kwong et al., 2003). Examples of LEC1-like polypeptides include proteins fromArabidopsis thaliana(AAN15924, SEQ ID NO:157),Brassica napus(AHI94922, SEQ ID NO:158), andPhaseolus coccineusLEC1-like (AAN01148, SEQ ID NO: 159). As used herein, the term “Leafy Cotyledon 2” or “LEC2” refers to a B3 domain transcription factor which participates in zygotic development and in somatic embryogenesis and which activates expression of a gene encoding WRI1. Its ectopic expression facilitates the embryogenesis from vegetative plant tissues (Alemanno et al., 2008). Examples of LEC2 polypeptides include proteins fromArabidopsis thaliana(Accession No. NP_564304.1, SEQ ID NO:142),Medicago truncatula(Accession No. CAA42938.1, SEQ ID NO:143) andBrassica napus(Accession No. AD016343.1, SEQ ID NO:144). In an embodiment, an exogenous polynucleotide of the invention which encodes a LEC2 comprises one or more of the following:i) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOs:142 to 144, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NOs:142 to 144,ii) nucleotides whose sequence is at least 30% identical to i), andiii) a polynucleotide which hybridizes to one or both of i) or ii) under stringent conditions. As used herein, the term “FUS3” refers to a B3 domain transcription factor which participates in zygotic development and in somatic embryogenesis and is detected mainly in the protodermal tissue of the embryo (Gazzarrini et al., 2004). Examples of FUS3 polypeptides include proteins fromArabidopsis thaliana(AAC35247, SEQ ID NO:160),Brassica napus(XP_006293066.1, SEQ ID NO:161) andMedicago truncatula(XP_003624470, SEQ ID NO:162). Over-expression of any of LEC1, L1L, LEC2, FUS3 and ABI3 from an exogenous polynucleotide is preferably controlled by a developmentally regulated promoter such as a senescence specific promoter, an inducible promoter, or a promoter which has been engineered for providing a reduced level of expression relative to a native promoter, particularly in plants other thanArabidopsis thalianaandB. napuscv. Westar, in order to avoid developmental abnormalities in plant development that are commonly associated with over-expression of these transcription factors (Mu et al., 2008). As used herein, the term “BABY BOOM” or “BBM” refers an AP2/ERF transcription factor that induces regeneration under culture conditions that normally do not support regeneration in wild-type plants. Ectopic expression ofBrassica napusBBM (BnBBM) genes inB. napusandArabidopsisinduces spontaneous somatic embryogenesis and organogenesis from seedlings grown on hormone-free basal medium (Boutilier et al., 2002). In tobacco, ectopic BBM expression is sufficient to induce adventitious shoot and root regeneration on basal medium, but exogenous cytokinin is required for somatic embryo (SE) formation (Srinivasan et al., 2007). Examples of BBM polypeptides include proteins fromArabidopsis thaliana(Accession No. NP_197245.2, SEQ ID NO:145), maize (U.S. Pat. No. 7,579,529),Sorghum bicolor(Accession No. XP_002458927) andMedicago truncatula(Accession No. AAW82334.1, SEQ ID NO:146). In an embodiment, an exogenous polynucleotide of the invention which encodes BBM comprises, unless specified otherwise, one or more of the following:i) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as one of SEQ ID NOs:145 or 146, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical to one or both of SEQ ID NOs: 145 or 146,ii) nucleotides whose sequence is at least 30% identical to i), andiii) a polynucleotide which hybridizes to one or both of i) or ii) under stringent conditions. An ABI3 polypeptide (A. thalianaAccession No. NP_189108) is related to the maize VP1 protein, is expressed at low levels in vegetative tissues and affects plastid development. An ABI4 polypeptide (A. thalianaAccession NP_181551) belongs to a family of transcription factors that contain a plant-specific AP2 domain (Finkelstein et al., 1998) and acts downstream of ABI3. ABI5 (A. thalianaAccession No. NP_565840) is a transcription factor of the bZIP family which affects ABA sensitivity and controls the expression of some LEA genes in seeds. It binds to an ABA-responsive element. Each of the following transcription factors was selected on the basis that they functioned in embryogenesis in plants. Accession numbers are provided in Table 26. Homologs of each can be readily identified in many other plant species and tested as described in Example 9. MYB73 is a transcription factor that has been identified in soybean, involved in stress responses. bZIP53 is a transcription factor in the bZIP protein family, identified inArabidopsis. AGL15 (Agamous-like 15) is a MADS box transcription factor which is natively expressed during embryogenesis. AGL15 is also natively expressed in leaf primordia, shoot apical meristems and young floral buds, suggesting that AGL15 may also have a function during post-germinative development. AGL15 has a role in embryogenesis and gibberellic acid catabolism. It targets B3 domain transcription factors that are key regulators of embryogenesis. MYB115 and MYB118 are transcription factors in the MYB family fromArabidopsisinvolved in embryogenesis. TANMEI also known as EMB2757 encodes a WD repeat protein required for embryo development inArabidopsis. WUS, also known as Wuschel, is a homeobox gene that controls the stem cell pool in embryos. It is expressed in the stem cell organizing center of meristems and is required to keep the stem cells in an undifferentiated state. The transcription factor binds to a TAAT element core motif. GFR2a1 and GFR2a2 are transcription factors at least from soybean. Fatty Acyl Acyltransferases As used herein, the term “fatty acyl acyltransferase” refers to a protein which is capable of transferring an acyl group from acyl-CoA, PC or acyl-ACP, preferably acyl-CoA or PC, onto a substrate to form TAG, DAG or MAG. These acyltransferases include DGAT, PDAT, MGAT, GPAT and LPAAT. As used herein, the term “diacylglycerol acyltransferase” (DGAT) refers to a protein which transfers a fatty acyl group from acyl-CoA to a DAG substrate to produce TAG. Thus, the term “diacylglycerol acyltransferase activity” refers to the transfer of an acyl group from acyl-CoA to DAG to produce TAG. A DGAT may also have MGAT function but predominantly functions as a DGAT, i.e., it has greater catalytic activity as a DGAT than as a MGAT when the enzyme activity is expressed in units of nmoles product/min/mg protein (see for example, Yen et al., 2005). The activity of DGAT may be rate-limiting in TAG synthesis in seeds (Ichihara et al., 1988). DGAT uses an acyl-CoA substrate as the acyl donor and transfers it to the sn-3 position of DAG to form TAG. The enzyme functions in its native state in the endoplasmic reticulum (ER) of the cell. There are three known types of DGAT, referred to as DGAT1, DGAT2 and DGAT3, respectively. DGAT1 polypeptides are membrane proteins that typically have 10 transmembrane domains, DGAT2 polypeptides are also membrane proteins but typically have 2 transmembrane domains, whilst DGAT3 polypeptides typically have none and are thought to be soluble in the cytoplasm, not integrated into membranes. Plant DGAT1 polypeptides typically have about 510-550 amino acid residues while DGAT2 polypeptides typically have about 310-330 residues. DGAT1 is the main enzyme responsible for producing TAG from DAG in most developing plant seeds, whereas DGAT2s from plant species such as tung tree (Vernicia fordii) and castor bean (Ricinus communis) that produce high amounts of unusual fatty acids appear to have important roles in the accumulation of the unusual fatty acids in TAG. Over-expression of AtDGAT1 in tobacco leaves resulted in a 6-7 fold increased TAG content (Bouvier-Nave et al., 2000). Examples of DGAT1 polypeptides include DGAT1 proteins fromAspergillus fumigatus(XP_755172.1; SEQ ID NO:80),Arabidopsis thaliana(CAB44774.1; SEQ ID NO:1),Ricinus communis(AAR11479.1; SEQ ID NO:81),Vernicia fordii(ABC94472.1; SEQ ID NO:82),Vernonia galamensis(ABV21945.1 and ABV21946.1; SEQ ID NO:83 and SEQ ID NO:84, respectively),Euonymus alatus(AAV31083.1; SEQ ID NO:85),Nannochloropsis oceanica(Zienkiewicz et al 2017), yeast (Zulu et al 2017),Caenorhabditis elegans(AAF82410.1; SEQ ID NO:86),Rattus norvegicus(NP_445889.1; SEQ ID NO:87),Homo sapiens(NP_036211.2; SEQ ID NO:88), as well as variants and/or mutants thereof. Examples of DGAT2 polypeptides include proteins encoded by DGAT2 genes fromArabidopsis thaliana(NP_566952.1; SEQ ID NO:2),Ricinus communis(AAY16324.1; SEQ ID NO:3),Vernicia fordii(ABC94474.1; SEQ ID NO:4),Mortierella ramanniana(AAK84179.1; SEQ ID NO:5),Homo sapiens(Q96PD7.2; SEQ ID NO:6) (Q58HT5.1; SEQ ID NO:7),Bos taurus(Q70VZ8.1; SEQ ID NO:8),Mus musculus(AAK84175.1; SEQ ID NO:9), as well as variants and/or mutants thereof. DGAT1 and DGAT2 amino acid sequences show little homology. Expression in leaves of an exogenous DGAT2 was twice as effective as a DGAT1 in increasing oil content (TAG). Further,A. thalianaDGAT2 had a greater preference for linoleoyl-CoA and linolenoyl-CoA as acyl donors relative to oleoyl-CoA, compared to DGAT1. This substrate preference can be used to distinguish the two DGAT classes in addition to their amino acid sequences. Examples of DGAT3 polypeptides include proteins encoded by DGAT3 genes from peanut (Arachis hypogaea, Saha, et al., 2006), as well as variants and/or mutants thereof. A DGAT has little or no detectable MGAT activity, for example, less than 300 pmol/min/mg protein, preferably less than 200 pmol/min/mg protein, more preferably less than 100 pmol/min/mg protein. In an embodiment, an exogenous polynucleotide of the invention which encodes a DGAT1 comprises one or more of the following:i) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOs:1 or 80 to 88, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NOs: 1 or 80 to 88,ii) nucleotides whose sequence is at least 30% identical to i), andiii) a polynucleotide which hybridizes to one or both of i) or ii) under stringent conditions. In an embodiment, an exogenous polynucleotide of the invention which encodes a DGAT2 comprises one or more of the following:i) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOs:2 to 9, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NOs: 2 to 9,ii) nucleotides whose sequence is at least 30% identical to i), andiii) a polynucleotide which hybridizes to one or both of i) or ii) under stringent conditions. As used herein, the term “phospholipid:diacylglycerol acyltransferase” (PDAT; EC 2.3.1.158) or its synonym “phospholipid:1,2-diacyl-sn-glycerol O-acyltransferase” means an acyltransferase that transfers an acyl group from a phospholipid, typically from the sn-2 position of PC, to the sn-3 position of DAG to form TAG and lysophosphocholine (LPC). This reaction is different to DGAT and uses phospholipids as the acyl-donors. Increased expression of PDAT such as PDAT1, which may be exogenous or endogenous to the cell or plant of the invention, increases the production of TAG from PC. The enzyme LPCAT can re-acylate the LPC to form more PC, allowing for continued production of DAG by PDAT. There are several forms of PDAT in plant cells including PDAT1, PDAT2 or PDAT3 (Ghosal et al., 2007). Sequences of exemplary PDAT coding regions and polypeptides are provided herein as SEQ ID NOs:258-261 (SorghumandZea maysPDAT1, Accession Nos XM_002462417.1 and NM_001147943), (Dahlqvist et al., 2000; Fan et al., 2013; Fan et al., 2014) although any PDAT encoding gene can be used. Homologs and naturally occurring variants of PDATs from these or other plant, fungal or algal species can readily be identified and used in the present invention. In an embodiment, the homolog or variant is at least 95% identical, preferably at least 99% identical, to the amino acid sequence of the listed SEQ ID NO or Accession No. The PDAT may be exogenous or endogenous to the plant or part thereof. As used herein, the term “monoacylglycerol acyltransferase” or “MGAT” refers to a protein which transfers a fatty acyl group from acyl-CoA to a MAG substrate, for example sn-2 MAG, to produce DAG. Thus, the term “monoacylglycerol acyltransferase activity” at least refers to the transfer of an acyl group from acyl-CoA to MAG to produce DAG. The term “MGAT” as used herein includes enzymes that act on sn-1/3 MAG and/or sn-2 MAG substrates to form sn-1,3 DAG and/or sn-1,2/2,3-DAG, respectively. In a preferred embodiment, the MGAT has a preference for sn-2 MAG substrate relative to sn-1 MAG, or substantially uses only sn-2 MAG as substrate. As used herein, MGAT does not include enzymes which transfer an acyl group preferentially to LysoPA relative to MAG, such enzymes are known as LPAATs. That is, a MGAT preferentially uses non-phosphorylated monoacyl substrates, even though they may also have low catalytic activity on LysoPA. A preferred MGAT does not have detectable activity in acylating LysoPA. A MGAT may also have DGAT function but predominantly functions as a MGAT, i.e., it has greater catalytic activity as a MGAT than as a DGAT when the enzyme activity is expressed in units of nmoles product/min/mg protein (also see Yen et al., 2002). There are three known classes of MGAT, referred to as, MGAT1, MGAT2 and MGAT3, respectively. Examples of MGAT1, MGAT2 and MGAT3 polypeptides are described in WO2013/096993. As used herein, an “MGAT pathway” refers to an anabolic pathway, different to the Kennedy pathway for the formation of TAG, in which DAG is formed by the acylation of either sn-1 MAG or preferably sn-2 MAG, catalysed by MGAT. The DAG may subsequently be used to form TAG or other lipids. WO2012/000026 demonstrated firstly that plant leaf tissue can synthesise MAG from G-3-P such that the MAG is accessible to an exogenous MGAT expressed in the leaf tissue, secondly MGAT from various sources can function in plant tissues, requiring a successful interaction with other plant factors involved in lipid synthesis and thirdly the DAG produced by the exogenous MGAT activity is accessible to a plant DGAT, or an exogenous DGAT, to produce TAG. MGAT and DGAT activity can be assayed by introducing constructs encoding the enzymes (or candidate enzymes) intoSaccharomyces cerevisiaestrain H1246 and demonstrating TAG accumulation. Some of the motifs that have been shown to be important for catalytic activity in some DGAT2s are also conserved in MGAT acyltransferases. Of particular interest is a putative neutral lipid-binding domain with the concensus sequence FLXLXXXN (SEQ ID NO:14) where each X is independently any amino acid other than proline, and N is any nonpolar amino acid, located within the N-terminal transmembrane region followed by a putative glycerol/phospholipid acyltransferase domain. The FLXLXXXN motif (SEQ ID NO:14) is found in the mouse DGAT2 (amino acids 81-88) and MGAT1/2 but not in yeast or plant DGAT2s. It is important for activity of the mouse DGAT2. Other DGAT2 and/or MGAT1/2 sequence motifs include: 1. A highly conserved YFP tripeptide (SEQ ID NO:10) in most DGAT2 polypeptides and also in MGAT1 and MGAT2, for example, present as amino acids 139-141 in mouse DGAT2. Mutating this motif within the yeast DGAT2 with non-conservative substitutions rendered the enzyme non-functional. 2. HPHG tetrapeptide (SEQ ID NO:11), highly conserved in MGATs as well as in DGAT2 sequences from animals and fungi, for example, present as amino acids 161-164 in mouse DGAT2, and important for catalytic activity at least in yeast and mouse DGAT2. Plant DGAT2 acyltransferases have a EPHS (SEQ ID NO:12) conserved sequence instead, so conservative changes to the first and fourth amino acids can be tolerated. 3. A longer conserved motif which is part of the putative glycerol phospholipid domain. An example of this motif is RXGFX(K/R)XAXXXGXXX(L/V)VPXXXFG(E/Q) (SEQ ID NO:13), which is present as amino acids 304-327 in mouse DGAT2. This motif is less conserved in amino acid sequence than the others, as would be expected from its length, but homologs can be recognised by motif searching. The spacing may vary between the more conserved amino acids, i.e., there may be additional X amino acids within the motif, or less X amino acids compared to the sequence above. One important component in glycerolipid synthesis from fatty acids esterified to ACP or CoA is the enzyme sn-glycerol-3-phosphate acyltransferase (GPAT), which is another of the polypeptides involved in the biosynthesis of non-polar lipids. This enzyme is involved in different metabolic pathways and physiological functions. It catalyses the following reaction: G3P+fatty acyl-ACP or -CoA→LPA+free-ACP or -CoA. The GPAT-catalyzed reaction occurs in three distinct plant subcellular compartments: plastid, endoplasmic reticulum (ER) and mitochondria. These reactions are catalyzed by three different types of GPAT enzymes, a soluble form localized in plastidial stroma which uses acyl-ACP as its natural acyl substrate (PGPAT inFIG.1), and two membrane-bound forms localized in the ER and mitochondria which use acyl-CoA and acyl-ACP as natural acyl donors, respectively (Chen et al., 2011). As used herein, the term “glycerol-3-phosphate acyltransferase” (GPAT; EC 2.3.1.15) and its synonym “glycerol-3-phosphate O-acyltransferase” refer to a protein which acylates glycerol-3-phosphate (G-3-P) to form LysoPA and/or MAG, the latter product forming if the GPAT also has phosphatase activity on LysoPA. The acyl group that is transferred is from acyl-CoA if the GPAT is an ER-type GPAT (an “acyl-CoA:sn-glycerol-3-phosphate 1-O-acyltransferase” also referred to as “microsomal GPAT”) or from acyl-ACP if the GPAT is a plastidial-type GPAT (PGPAT). Thus, the term “glycerol-3-phosphate acyltransferase activity” refers to the acylation of G-3-P to form LysoPA and/or MAG. The term “GPAT” encompasses enzymes that acylate G-3-P to form sn-1 LPA and/or sn-2 LPA, preferably sn-2 LPA. Preferably, the GPAT which may be over-expressed in the Pull modification is a membrane bound GPAT that functions in the ER of the cell, more preferably a GPAT9, and the plastidial GPAT that is down-regulated in the Prokaryotic Pathway modification is a soluble GPAT (“plastidial GPAT”). In a preferred embodiment, the GPAT has phosphatase activity. In a most preferred embodiment, the GPAT is a sn-2 GPAT having phosphatase activity which produces sn-2 MAG. As used herein, the term “sn-1 glycerol-3-phosphate acyltransferase” (sn-1 GPAT) refers to a protein which acylates sn-glycerol-3-phosphate (G-3-P) to preferentially form 1-acyl-sn-glycerol-3-phosphate (sn-1 LPA). Thus, the term “sn-1 glycerol-3-phosphate acyltransferase activity” refers to the acylation of sn-glycerol-3-phosphate to form 1-acyl-sn-glycerol-3-phosphate (sn-1 LPA). As used herein, the term “sn-2 glycerol-3-phosphate acyltransferase” (sn-2 GPAT) refers to a protein which acylates sn-glycerol-3-phosphate (G-3-P) to preferentially form 2-acyl-sn-glycerol-3-phosphate (sn-2 LPA). Thus, the term “sn-2 glycerol-3-phosphate acyltransferase activity” refers to the acylation of sn-glycerol-3-phosphate to form 2-acyl-sn-glycerol-3-phosphate (sn-2 LPA). The GPAT family is large and all known members contain two conserved domains, a plsC acyltransferase domain (PF01553; SEQ ID NO:15) and a HAD-like hydrolase (PF12710; SEQ ID NO:16) superfamily domain and variants thereof. In addition to this, at least inArabidopsis thaliana, GPATs in the subclasses GPAT4-GPAT8 all contain a N-terminal region homologous to a phosphoserine phosphatase domain (PF00702; SEQ ID NO:17), and GPATs which produce MAG as a product can be identified by the presence of such a homologous region. Some GPATs expressed endogenously in leaf tissue comprise the conserved amino acid sequence GDLVICPEGTTCREP (SEQ ID NO:18). GPAT4 and GPAT6 both contain conserved residues that are known to be critical to phosphatase activity, specifically conserved amino acids in Motif I (DXDX[T/V][L/V]; SEQ ID NO:19) and Motif III (K-[G/S][D/S]XXX[D/N]; SEQ ID NO:20) located at the N-terminus (Yang et al., 2010). Homologues ofArabidopsisGPAT4 (Accession No. NP_171667.1) and GPAT6 (NP_181346.1) include AAF02784.1 (Arabidopsis thaliana), AAL32544.1 (Arabidopsis thaliana), AAP03413.1 (Oryza sativa), ABK25381.1 (Picea sitchensis), ACN34546.1 (Zea Mays), BAF00762.1 (Arabidopsis thaliana), BAH00933.1 (Oryza sativa), EAY84189.1 (Oryza sativa), EAY98245.1 (Oryza sativa), EAZ21484.1 (Oryza sativa), EEC71826.1 (Oryza sativa), EEC76137.1 (Oryza sativa), EEE59882.1 (Oryza sativa), EFJ08963.1 (Selaginella moellendorffii), EFJ11200.1 (Selaginella moellendorffii), NP_001044839.1 (Oryza sativa), NP_001045668.1 (Oryza sativa), NP_001147442.1 (Zea mays), NP_001149307.1 (Zea mays), NP_001168351.1 (Zea mays), AFH02724.1 (Brassica napus) NP_191950.2 (Arabidopsis thaliana), XP_001765001.1 (Physcomitrella patens), XP_001769671.1 (Physcomitrella patens), (Vitis vinifera), XP_002275348.1 (Vitis vinifera), XP_002276032.1 (Vitis vinifera), XP_002279091.1 (Vitis vinifera), XP_002309124.1 (Populus trichocarpa), XP_002309276.1 (Populus trichocarpa), XP_002322752.1 (Populus trichocarpa), XP_002323563.1 (Populus trichocarpa), XP_002439887.1 (Sorghum bicolor), XP_002458786.1 (Sorghum bicolor), XP_002463916.1 (Sorghum bicolor), XP_002464630.1 (Sorghum bicolor), XP_002511873.1 (Ricinus communis), XP_002517438.1 (Ricinus communis), XP_002520171.1 (Ricinus communis), ACT32032.1 (Vernicia fordii), NP_001051189.1 (Oryza sativa), AFH02725.1 (Brassica napus), XP_002320138.1 (Populus trichocarpa), XP_002451377.1 (Sorghum bicolor), XP_002531350.1 (Ricinus communis), and XP_002889361.1 (Arabidopsis lyrata). The soluble plastidial GPATs (PGPAT, also known as ATS1 inArabidopsis thaliana) have been purified and genes encoding them cloned from several plant species such as pea (Pisum sativum, Accession number: P30706.1), spinach (Spinacia oleracea, Accession number: Q43869.1), squash (Cucurbita moschate, Accession number: P10349.1), cucumber (Cucumis sativus, Accession number: Q39639.1) andArabidopsis thaliana(Accession number: Q43307.2). The soluble plastidial GPAT is the first committed step for what is known as the prokaryotic pathway of glycerolipid synthesis and is operative only in the plastid (FIG.1). The so-called prokaryotic pathway is located exclusively in plant plastids and assembles DAG for the synthesis of galactolipids (MGDG and DGMG) which contain C16:3 fatty acids esterified at the sn-2 position of the glycerol backbone. Conserved motifs and/or residues can be used as a sequence-based diagnostic for the identification of GPAT enzymes. Alternatively, a more stringent function-based assay could be utilised. Such an assay involves, for example, feeding labelled glycerol-3-phosphate to cells or microsomes and quantifying the levels of labelled products by thin-layer chromatography or a similar technique. GPAT activity results in the production of labelled LPA whilst GPAT/phosphatase activity results in the production of labelled MAG. As used herein, the term “lysophosphatidic acid acyltransferase” (LPAAT; EC 2.3.1.51) and its synonyms “1-acyl-glycerol-3-phosphate acyltransferase”, “acyl-CoA: 1-acyl-sn-glycerol-3-phosphate 2-O-acyltransferase” and “1-acylglycerol-3-phosphate O-acyltransferase” refer to a protein which acylates lysophosphatidic acid (LPA) to form phosphatidic acid (PA). The acyl group that is transferred is from acyl-CoA if the LPAAT is an ER-type LPAAT or from acyl-ACP if the LPAAT is a plastidial-type LPAAT (PLPAAT). Thus, the term “lysophosphatidic acid acyltransferase activity” refers to the acylation of LPA to form PA. Oil Body Coating Polypeptides Plant seeds and pollen accumulate TAG in subcellular structures called oil bodies which generally range from 0.5-2.5 μm in diameter. As used herein, “lipid droplets”, also referred to as “oil bodies”, are lipid rich cellular organelles for storage or exchange of neutral lipids including predominantly TAG. Lipid droplets can vary greatly in size from about 20 nm to 100 μm. These organelles have a TAG core surround by a phospholipid monolayer containing several embedded proteins which are involved in lipid metabolism and storage as well as lipid trafficking to other membranes, including oleosins if the oil bodies are from plant seeds or floral tissues (Jolivet et al., 2004). They generally consist of 0.5-3.5% protein while the remainder is the lipid. They are the least dense of the organelles in most cells and can therefore be isolated readily by flotation centrifugation. Oleosins represent the most abundant (at least 80%) of the protein in the membrane of oil bodies from seeds. In an embodiment, the oil body coating polypeptide is non-allergenic, or not known to be allergenic, such as to humans. As used herein, the term “allergenic polypeptide” means a polypeptide which is characterised by the presence of two features: (i) its amino acid sequence comprises a region of at least 80 consecutive amino acids whose sequence is at least 35% identical to a sequence of at least 80 consecutive amino acids of a known allergenic protein, and (ii) its amino acid sequence comprises at least 8 consecutive amino acids which are identical in sequence to a region of at least 8 consecutive amino acids of a known allergenic protein. As used herein, a “non-allergenic polypeptide” is a polypeptide which is not an allergenic polypeptide. Preferred non-allergenic polypeptides are polypeptides which do not have each of features (i) and (ii). For clarity, non-allergenic polypeptides may have feature (i) or (ii) but not both (i) and (ii). A subset of non-allergenic polypeptides have feature (i) but not feature (ii); these are less preferred than polypeptides which have neither feature (i) nor (ii). The features described as (i) and (ii) may be determined by carrying out a search using the AllergenOnline database and search facility, available at www.allergenonline.org. Two searches are carried out using the amino acid sequence of the polypeptide of interest, which is used as a query to search the database of known allergen sequences at AllergenOnline. The first search uses a sliding window of 80 amino acids from the polypeptide of interest (amino acids 1-80, 2-81, 3-82 etc), looking for matches of at least 35% identity by the FASTA program (Pearson and Lipman, 1988). The 35% identity for 80 amino acid segments was proposed in a scientific advisory to regulators for evaluating polypeptides in genetically modified crops, see FAO/WHO 2001 and Codex 2003. The segment matching process evaluating segments of 80 amino acids appears to be quite conservative. That is, when this first search is used on its own in classifying polypeptides as potentially allergenic, a positive match at the 35% identity level may mis-classify polypeptides as potentially allergenic when the extent of identity does not have biological significance for allergenicity. Therefore, the second search for an 8-amino acid match is also carried out, and the polypeptide of interest is classified as a potential allergen on the basis of a positive match in both searches, not just one search. When the AllergenOnline database was searched using query sequences, polypeptides including maize WRI1,ArabidopsisDGAT1, palm DGAT1.1, coconut GPAT9,ArabidopsisFatA2,Arabidopsiscaleosin (At2g33380),NannochloropsisLDSP, pepper fibrillin,RhodococcusTadA, and all caleosins tested showed zero matches in the 80 amino acid sliding window search and are therefore classified as non-allergenic. Other sequences including the vanilla U1 oleosin, Chinese tallow LDAP2,Arabidopsissteroleosin (At5g50600), peanut Oleosin3, sesame oleosinH, avocado oleosin, fig oleosin, cucumber oleosin, flax oleosin, soybean oleosin,Brassicaoleosin and potato oleosin all produced one or more matches in the 80 amino acid sliding window search (i.e. at least 35% identity to a known allergenic protein in a region of at least 80 amino acids) but did not provide any matches in the 8 amino acid search. These were therefore classified as non-allergenic according to the definition above. In contrast, sesame oleosinL was identified as an allergenic polypeptide, providing matches in both searches. As used herein, the term “Oleosin” refers to an amphipathic protein present in the membrane of oil bodies in the storage tissues of seeds (see, for example, Huang, 1996; Tai et al., 2002, Lin et al., 2005; Capuano et al., 2007; Lui et al., 2009; Shimada and Hara-Nishimura, 2010) and artificially produced variants (see for example WO2011/053169 and WO2011/127118). Oleosins are of low Mr(15-26,000), corresponding to about 140-230 amino acid residues, which allows them to become tightly packed on the surface of oil bodies. Within each seed species, there are usually two or more oleosins of different Mr. Each oleosin molecule contains a relatively hydrophilic, variable N-terminal domain (for example, about 48 amino acid residues), a central totally hydrophobic domain (for example, of about 70-80 amino acid residues) which is particularly rich in aliphatic amino acids such as alanine, glycine, leucine, isoleucine and valine, and an amphipathic α-helical domain of about 30-40 amino acid residues at or near the C-terminus. The central hydrophobic domain typically contains a proline knot motif of about 12 residues at its center. Generally, the central stretch of hydrophobic residues is inserted into the lipid core and the amphiphatic N-terminal and/or amphiphatic C-terminal are located at the surface of the oil bodies, with positively charged residues embedded in a phospholipid monolayer and the negatively charged ones exposed to the exterior. As used herein, the term “Oleosin” encompasses polyoleosins which have multiple oleosin polypeptides fused together in a head-to-tail fashion as a single polypeptide (WO2007/045019), for example 2×, 4× or 6× oleosin peptides, and caleosins which bind calcium and which are a minor protein component of the proteins that coat oil bodies in seeds (Froissard et al., 2009), and steroleosins which bind sterols (WO2011/053169). However, generally a large proportion (at least 80%) of the oleosins of oil bodies will not be caleosins and/or steroleosins. The term “oleosin” also encompasses oleosin polypeptides which have been modified artificially, such oleosins which have one or more amino acid residues of the native oleosins artificially replaced with cysteine residues, as described in WO2011/053169. Typically, 4-8 residues are substituted artificially, preferably 6 residues, but as many as between 2 and 14 residues can be substituted. Preferably, both of the amphipathic N-terminal and C-terminal domains comprise cysteine substitutions. The modification increases the cross-linking ability of the oleosins and increases the thermal stability and/or the stability of the proteins against degradation by proteases. A substantial number of oleosin protein sequences, and nucleotide sequences encoding therefor, are known from a large number of different plant species. Examples include, but are not limited to, oleosins from sesame, Arabidposis, canola, corn, rice, peanut, castor, soybean, flax, grape, cabbage, cotton, sunflower,sorghumand barley. Examples of oleosins (with their Accession Nos) includeBrassica napusoleosin (CAA57545.1; SEQ ID NO:95),Brassica napusoleosin S1-1 (ACG69504.1; SEQ ID NO:96),Brassica napusoleosin S2-1 (ACG69503.1; SEQ ID NO:97),Brassica napusoleosin S3-1 (ACG69513.1; SEQ ID NO:98),Brassica napusoleosin S4-1 (ACG69507.1; SEQ ID NO:99),Brassica napusoleosin S5-1 (ACG69511.1; SEQ ID NO:100),Arachis hypogaeaoleosin 1 (AAZ20276.1; SEQ ID NO:101),Arachis hypogaeaoleosin 2 (AAU21500.1; SEQ ID NO:102),Arachis hypogaeaoleosin 3 (AAU21501.1; SEQ ID NO:103),Arachis hypogaeaoleosin 5 (ABC96763.1; SEQ ID NO:104),Ricinus communisoleosin 1 (EEF40948.1; SEQ ID NO:105),Ricinus communisoleosin 2 (EEF51616.1; SEQ ID NO:106),Glycine maxoleosin isoform a (P29530.2; SEQ ID NO:107),Glycine maxoleosin isoform b (P29531.1; SEQ ID NO:108),Linum usitatissimumoleosin low molecular weight isoform (ABB01622.1; SEQ ID NO:109),Linum usitatissimumoleosin high molecular weight isoform (ABB01624.1; SEQ ID NO:110),Helianthus annuusoleosin (CAA44224.1; SEQ ID NO:111),Zea maysoleosin (NP_001105338.1; SEQ ID NO:112),Brassica napussteroleosin (ABM30178.1; SEQ ID NO:113),Brassica napussteroleosin SLO1-1 (ACG69522.1; SEQ ID NO:114),Brassica napussteroleosin SL02-1 (ACG69525.1; SEQ ID NO:115),Sesamum indicumsteroleosin (AAL13315.1; SEQ ID NO:116),Sesame indicumOleosinL (Tai et al., 2002; Accession number AF091840; SEQ ID NO:305),Ficus pumilavar.awkeotsangoleosin L-isoform (Accession No. ABQ57397.1; SEQ ID NO: 306),Cucumis sativusoleosinL (Accession No. XP_004146901.1; SEQ ID NO: 307),Linum usitatissimumoleosinL (Accession No. ABB01618.1; SEQ ID NO: 308),Glycine maxoleosinL (Accession No. XP_003556321.2; SEQ ID NO: 309),Ananas comosusoleosinL (Accession No. OAY72596.1; SEQ ID NO: 310),Setaria italicaoleosinL (Accession No. XP_004956407.1; SEQ ID NO: 311),Fragaria vescasubsp.vescaoleosinL (Accession No. XP_004307777.1; SEQ ID NO: 312),Brassica napusoleosinL (Accession No. CDY03377.1; SEQ ID NO: 313),Solanum lycopersicumoleosinL (Accession No. XP_004240765.1; SEQ ID NO: 314),Sesame indicumOleosinH1 (Tai et al., 2002; Accession number AF302807),Vanilla planifolialeaf OleosinU1 (Huang and Huang, 2016; Accession number SRX648194),Persea americanamesocarp OleosinM lipid droplet associated protein (Huang and Huang, 2016; Accession number SRX627420),Arachis hypogaeaOleosin 3 (Parthibane et al., 2012; Accession number AY722696),A. thalianaCaleosin3 (Shen et al., 2014; Laibach et al., 2015; Accession number AK317039),A. thalianasteroleosin (Accession number AT081653),Zea mayssteroleosin (NP_001152614.1; SEQ ID NO:117),Brassica napuscaleosin CLO-1 (ACG69529.1; SEQ ID NO:118),Brassica napuscaleosin CLO-3 (ACG69527.1; SEQ ID NO:119),Sesamum indicumcaleosin (AAF13743.1; SEQ ID NO:120),Zea mayscaleosin (NP_001151906.1; SEQ ID NO:121),Glycine maxcaleosin (AAB71227). Other lipid encapsulation polypeptides that are functionally equivalent are plastoglobulins and MLDP polypeptides (WO2011/127118). In an embodiment, an exogenous polynucleotide of the invention which encodes an oleosin comprises, unless specified otherwise, one or more of the following:i) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOs:95 to 112 or 305 to 314, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NOs: 95 to 112 or 305 to 314,ii) nucleotides whose sequence is at least 30% identical to i), andiii) a polynucleotide which hybridizes to one or both of i) or ii) under stringent conditions. In an embodiment, an exogenous polynucleotide of the invention which encodes a steroleosin comprises, unless specified otherwise, one or more of the following:i) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOs:113 to 117, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NOs: 113 to 117,ii) nucleotides whose sequence is at least 30% identical to i), andiii) a polynucleotide which hybridizes to one or both of i) or ii) under stringent conditions. In an embodiment, the oleosin is oleosinL or an ortholog thereof. OleosinL lacks the about 18 amino acid H-form insertion towards the C-terminus of other oleosins (see, for example, Tai et al., 2002). Thus, OleosinL's can readily be distinguished from other oleosins based on protein alignment. In an embodiment, an exogenous polynucleotide of the invention which encodes an oleosinL comprises, unless specified otherwise, one or more of the following:i) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOs: 305 to 314, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NOs: 305 to 314,ii) nucleotides whose sequence is at least 30% identical to i), andiii) a polynucleotide which hybridizes to one or both of i) or ii) under stringent conditions. In an alternate embodiment, an exogenous polynucleotide of the invention which encodes an oleosin comprises, unless specified otherwise, one or more of the following:i) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOs: 306 to 314, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NOs: 306 to 314,ii) nucleotides whose sequence is at least 30% identical to i), andiii) a polynucleotide which hybridizes to one or both of i) or ii) under stringent conditions, wherein the oleosin is not allergenic, or not known to be allergenic, such as to humans. As used herein, a “lipid droplet associated protein” or “LDAP” means a polypeptide which is associated with lipid droplets in plants in tissues or organs other than seeds, anthers and pollen, such as fruit tissues including pericarp and mesocarp. LDAPs may be associated with oil bodies in seeds, anthers or pollen as well as in the tissues or organs other than seeds, anthers and pollen. They are distinct from oleosins which are polypeptides associated with the surface of lipid droplets in seed tissues, anthers and pollen. LDAPs as used herein include LDAP polypeptides that are produced naturally in plant tissues as well as amino acid sequence variants that are produced artificially. The function of such variants can be tested as exemplified in Example 11. Horn et al. (2013) identified two LDAP genes which are expressed in avocado pericarp. The encoded avocado LDAP1 and LDAP2 polypeptides were 62% identical in amino acid sequence and had homology to polypeptide encoded byArabidopsisAt3g05500 and a rubber tree SRPP-like protein. Gidda et al. (2013) identified three LDAP genes that were expressed in oil palm (Elaeis guineensis) mesocarp but not in kernels and concluded that LDAP genes were plant specific and conserved amongst all plant species. LDAP polypeptides may contain additional domains (Gidda et al., (2013). Genes encoding LDAPs are generally up-regulated in non-seed tissues with abundant lipid and can be identified thereby, but are thought to be expressed in all non-seed cells that produce oil including for transient storage. Horn et al. (2013) shows a phylogenetic tree of SRPP-like proteins in plants. Exemplary LDAP polypeptides are described in Example 11 and Example 17 herein, such asRhodococcus opacusTadA lipid droplet associated protein (MacEachran et al., 2010; Accession number HM625859),Nannochloropsis oceanicaLSDP oil body protein (Vieler et al., 2012; Accession number JQ268559) andTrichoderma reeseiHFBI hydrophobin (Linder et al., 2005; Accession number Z68124). Homologs of LDAPs in other plant species can be readily identified by those skilled in the art. In an embodiment, an exogenous polynucleotide of the invention which encodes a LDAP comprises, unless specified otherwise, one or more of the following:i) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOs: 228, 230 or 232, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NOs: 228, 230 or 232,ii) nucleotides whose sequence is at least 30% identical to i), andiii) a polynucleotide which hybridizes to one or both of i) or ii) under stringent conditions. As used herein, the term a “polypeptide involved in starch biosynthesis” refers to any polypeptide, the downregulation of which in a plant cell below normal (wild-type) levels results in a reduction in the level of starch synthesis and a decrease in the levels of starch. This reduces the flow of carbon from sugars into starch. An example of such a polypeptide is AGPase. As used herein, the term “ADP-glucose phosphorylase” or “AGPase” refers to an enzyme which regulates starch biosynthesis, catalysing conversion of glucose-1-phosphate and ATP to ADP-glucose which serves as the building block for starch polymers. The active form of the AGPase enzyme consists of 2 large and 2 small subunits. The AGPase enzyme in plants exists primarily as a tetramer which consists of 2 large and 2 small subunits. Although these subunits differ in their catalytic and regulatory roles depending on the species (Kuhn et al., 2009), in plants the small subunit generally displays catalytic activity. The molecular weight of the small subunit is approximately 50-55 kDa. Sequences of exemplary AGPase small subunit polypeptides are provided herein as SEQ ID NOs:254-257 (SorghumandZea mays AGPase, Accession Nos XM_002462095.1 and XM_008666513.1) (Sanjaya et al. 2011, Zale et al. 2016). The molecular weight of the large large subunit is approximately 55-60 kDa. The plant enzyme is strongly activated by 3-phosphoglycerate (PGA), a product of carbon dioxide fixation; in the absence of PGA, the enzyme exhibits only about 3% of its activity. Plant AGPase is also strongly inhibited by inorganic phosphate (Pi). In contrast, bacterial and algal AGPase exist as homotetramers of 50 kDa. The algal enzyme, like its plant counterpart, is activated by PGA and inhibited by Pi, whereas the bacterial enzyme is activated by fructose-1, 6-bisphosphate (FBP) and inhibited by AMP and Pi. TAG Lipases and Beta-Oxidation As used herein, the term “polypeptide involved in the degradation of lipid and/or which reduces lipid content” refers to any polypeptide which catabolises lipid, the downregulation of which in a plant cell below normal (wild-type) levels results an increase in the level of oil, such as fatty acids and/or TAGs, in a cell of a transgenic plant or part thereof such as a vegetative part, tuber, beet or a seed. Examples of such polypeptides include, but are not limited to, lipases, or a lipase such as a CGi58 (Comparative Gene identifier-58-Like) polypeptide, a SUGAR-DEPENDENT1 (SDP1) triacylglycerol lipase (see, for example, Kelly et al., 2011) and a lipase described in WO 2009/027335. As used herein, the term “TAG lipase” (EC.3.1.1.3) refers to a protein which hydrolyzes TAG into one or more fatty acids and any one of DAG, MAG or glycerol. Thus, the term “TAG lipase activity” refers to the hydrolysis of TAG into glycerol and fatty acids. As used herein, the term “CGi58” refers to a soluble acyl-CoA-dependent lysophosphatidic acid acyltransferase encoded by the At4g24160 gene inArabidopsis thalianaand its homologs in other plants and “Ict1p” in yeast and its homologs. The plant gene such as that fromArabidopsisgene locus At4g24160 is expressed as two alternative transcripts: a longer full-length isoform (At4g24160.1) and a smaller isoform (At4g24160.2) missing a portion of the 3′ end (see James et al., 2010; Ghosh et al., 2009; US 201000221400). Both mRNAs code for a protein that is homologous to the human CGI-58 protein and other orthologous members of this a/13 hydrolase family (ABHD). In an embodiment, the CGI58 (At4g24160) protein contains three motifs that are conserved across plant species: a GXSXG lipase motif (SEQ ID NO:127), a HX(4)D acyltransferase motif (SEQ ID NO:128), and VX(3)HGF, a probable lipid binding motif (SEQ ID NO:129). The human CGI-58 protein has lysophosphatidic acid acyltransferase (LPAAT) activity but not lipase activity. In contrast, the plant and yeast proteins possess a canonical lipase sequence motif GXSXG (SEQ ID NO:127), that is absent from vertebrate (humans, mice, and zebrafish) proteins, and have lipase and phospholipase activity (Ghosh et al., 2009). Although the plant and yeast CGI58 proteins appear to possess detectable amounts of TAG lipase and phospholipase A activities in addition to LPAAT activity, the human protein does not. Disruption of the homologous CGI-58 gene inArabidopsis thalianaresults in the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy of isolated lipid droplets from cgi-58 loss-of-function mutants showed they contain triacylglycerols with common leaf-specific fatty acids. Leaves of mature cgi-58 plants exhibit a marked increase in absolute triacylglycerol levels, more than 10-fold higher than in wild-type plants. Lipid levels in the oil-storing seeds of cgi-58 loss-of-function plants were unchanged, and unlike mutations in β-oxidation, the cgi-58 seeds germinated and grew normally, requiring no rescue with sucrose (James et al., 2010). Examples of nucleotides encoding CGi58 polypeptides include those fromArabidopsis thaliana(NM_118548.1 encoding NP_194147.2; SEQ ID NO:130),Brachypodium distachyon(XP_003578450.1; SEQ ID NO:131),Glycine max(XM_003523590.1 encoding XP_003523638.1; SEQ ID NO:132),Zea mays(NM_001155541.1 encoding NP_001149013.1; SEQ ID NO:133),Sorghum bicolor(XM_002460493.1 encoding XP_002460538.1; SEQ ID NO:134),Ricinus communis(XM_002510439.1 encoding XP_002510485.1; SEQ ID NO:135),Medicago truncatula(XM_003603685.1 encoding XP_003603733.1; SEQ ID NO:136), andOryza sativa(encoding EAZ09782.1). In an embodiment, a genetic modification of the invention down-regulates endogenous production of CGi58, wherein CGi58 is encoded by one or more of the following:i) nucleotides comprising a sequence set forth as any one of SEQ ID NOs:130 to 136,ii) nucleotides comprising a sequence which is at least 30% identical to any one or more of SEQ ID NOs:130 to 136, andiii) a polynucleotide which hybridizes to one or both of i) or ii) under stringent conditions. Other lipases which have lipase activity on TAG include SUGAR-DEPENDENT1 triacylglycerol lipase (SDP1, see for example Eastmond et al., 2006; Kelly et al., 2011) and SDP1-like polypeptides found in plant species as well as yeast (TGL4 polypeptide) and animal cells, which are involved in storage TAG breakdown. The SDP1 and SDP1-like polypeptides appear to be responsible for initiating TAG breakdown in seeds following germination (Eastmond et al., 2006). Plants that are mutant in SDP1, in the absence of exogenous WRI1 and DGAT1, exhibit increased levels of PUFA in their TAG. As used herein, “SDP1 polypeptides” include SDP1 polypeptides, SDP1-like polypeptides and their homologs in plant species. SDP1 and SDP1-like polypeptides in plants are 800-910 amino acid residues in length and have a patatin-like acylhydrolase domain that can associate with oil body surfaces and hydrolyse TAG in preference to DAG or MAG. SDP1 is thought to have a preference for hydrolysing the acyl group at the sn-2 position of TAG.Arabidopsiscontains at least three genes encoding SDP1 lipases, namely SDP1 (Accession No. NP_196024, nucleotide sequence SEQ ID NO:163 and homologs in other species), SDP1L (Accession No. NM_202720 and homologs in other species, Kelly et al., 2011) and ATGLL (At1g33270) (Eastmond et al, 2006). Of particular interest for reducing gene activity are SDP1 genes which are expressed in vegetative tissues in plants, such as in leaves, stems and roots. Levels of non-polar lipids in vegetative plant parts can therefore be increased by reducing the activity of SDP1 polypeptides in the plant parts, for example by either mutation of an endogenous gene encoding a SDP1 polypeptide or introduction of an exogenous gene which encodes a silencing RNA molecule which reduces the expression of an endogenous SDP1 gene. Such a reduction is of particular benefit in tuber crops such as sugarbeet and potato, and in “high sucrose” plants such as sweetsorghum, sugarcane and and sugarbeet. Genes encoding SDP1 homologues (including SDP1-like homologues) in a plant species of choice can be identified readily by homology to known SDP1 gene sequences. Known SDP1 nucleotide or amino acid sequences include Accession Nos.: inBrassica napus, GN078290 (SEQ ID NO:164), GN078281, GN078283;Capsella rubella, XP_006287072;Theobroma cacao, XP_007028574.1;Populus trichocarpa, XP_002308909 (SEQ ID NO:166);Prunus persica, XP_007203312;Prunus mume,XP_008240737;Malus domestica, XP_008373034;Ricinus communis, XP_002530081;Medicago truncatula, XP_003591425 (SEQ ID NO:167);Solanum lycopersicum, XP_004249208;Phaseolus vulgaris, XP_007162133;Glycine max, XP_003554141 (SEQ ID NO:168);Solanum tuberosum, XP_006351284;Glycine max, XP_003521151;Cicer arietinum, XP_004493431;Cucumis sativus, XP_004142709;Cucumis melo, XP_008457586; Jatropha curcas, KDP26217;Vitis vinifera, CBI30074;Oryza sativa, JaponicaGroup BAB61223;Oryza sativa, Indica Group EAY75912;Oryza sativa, JaponicaGroup NP_001044325;Sorghum bicolor, XP_002458531 (SEQ ID NO:169);Brachypodium distachyon, XP_003567139 (SEQ ID NO:165);Zea mays, AFW85009;Hordeum vulgare, BAK03290 (SEQ ID NO:172);Aegilops tauschii, EMT32802;Sorghum bicolor, XP_002463665;Zea mays, NP_001168677 (SEQ ID NO:170);Hordeum vulgare, BAK01155;Aegilops tauschii, EMT02623;Triticum urartu, EMS67257;Physcomitrella patens, XP_001758169 (SEQ ID NO:171). Preferred SDP1 sequences for use in genetic constructs for inhibiting expression of the endogenous genes are from cDNAs corresponding to the genes which are expressed most highly in the plant cells, vegetative plant parts or the seeds, whichever is to be modified. Nucleotide sequences which are highly conserved between cDNAs corresponding to all of the SDP1 genes in a plant species are preferred if it is desired to reduce the activity of all members of a gene family in that species. In an embodiment, a genetic modification of the invention down-regulates endogenous production of SDP1, wherein SDP1 is encoded by one or more of the following:i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:163 to 174,ii) nucleotides whose sequence is at least 30% identical to any one or more of the sequences set forth as SEQ ID NOs:163 to 174, andiii) a sequence of nucleotides which hybridizes to one or both of i) or ii) under stringent conditions. As shown in the Examples, reduction of the expression and/or activity of SDP1 TAG lipase in plant leaves greatly increased the TAG content, both in terms of the amount of TAG that accumulated and the earlier timing of accumulation during plant development, in the context of co-expression of the transcription factor WRI1 and a fatty acyl acyltransferase. In particular, the increase was observed in plants prior to flowering, and was up to about 70% on a weight basis (% dry weight) at the onset of senescence. The increase was relative to the TAG levels observed in corresponding plant leaves transformed with exogenous polynucleotides encoding the WRI1 and fatty acyl acyltransferase but lacking the modification that reduced SDP1 expression and/or activity. Reducing the expression of other TAG catabolism genes in plant parts can also increase TAG content, such as the ACX genes encoding acyl-CoA oxidases such as the Acx1 (At4g16760 and homologs in other plant species) or Acx2 (At5g65110 and homologs in other plant species) genes. Another polypeptide involved in lipid catabolism is PXA1 which is a peroxisomal ATP-binding cassette transporter that is requires for fatty acid import for β-oxidation (Zolman et al. 2001). Export of Fatty Acids from Plastids As used herein, the term “polypeptide which increases the export of fatty acids out of plastids of the cell” refers to any polypeptide which aids in fatty acids being transferred from within plastids of plant cells in a plant or part thereof to outside the plastid, which may be any other part of the cell such as for example the endoplasmic reticulum (ER). Examples of such polypeptides include, but are not limited to, a C16 or C18 fatty acid thioesterase such as a FATA polypeptide or a FATB polypeptide, a C8 to C14 fatty acid thioesterase (which is also a FATB polypeptide), a fatty acid transporter such as an ABCA9 polypeptide or a long-chain acyl-CoA synthetase (LACS). As used herein, the term “fatty acid thioesterase” or “FAT” or “acyl-ACP thioesterase” refers to an enzyme which catalyses the hydrolysis of the thioester bond between an acyl moiety and acyl carrier protein (ACP) in acyl-ACP and the release of a free fatty acid. Such enzymes typically function in the plastids of an organism which is synthesizing de novo fatty acids. As used herein, the term “C16 or C18 fatty acid thioesterase” refers to an enzyme which catalyses the hydrolysis of the thioester bond between a C16 and/or C18 acyl moiety and ACP in acyl-ACP and the release of free C16 or C18 fatty acid in the plastid. The free fatty acid is then re-esterified to CoA in the plastid envelope as it is transported out of the plastid. The substrate specificity of the fatty acid thioesterase (FAT) enzyme in the plastid is involved in determining the spectrum of chain length and degree of saturation of the fatty acids exported from the plastid. FAT enzymes can be classified into two classes based on their substrate specificity and nucleotide sequences, FATA and FATB (EC 3.1.2.14) (Jones et al., 1995). FATA polypeptides prefer oleoyl-ACP as substrate, while FATB polypeptides show higher activity towards saturated acyl-ACPs of different chain lengths such as acting on palmitoyl-ACP to produce free palmitic acid. Examples of FATA polypeptides useful for the invention include, but are not limited to, those fromArabidopsis thaliana(NP_189147),Arachis hypogaea(GU324446),Helianthus annuus(AAL79361),Carthamus tinctorius(AAA33020),Morus notabilis(XP_010104178.1),Brassica napus(CDX77369.1),Ricinus communis(XP_002532744.1) andCamelina sativa(AFQ60946.1). Examples of FATB polypeptides useful for the invention include, but are not limited to, those fromZea mays(AIL28766),Brassica napus(ABH11710),Helianthus annuus(AAX19387),Arabidopsis thaliana(AEE28300),Umbellularia californica(AAC49001),Arachis hypogaea(AFR54500),Ricinus communis(EEF47013) andBrachypodium sylvaticum(ABL85052.1). As used herein, the term “fatty acid transporter” relates to a polypeptide present in the plastid membrane which is involved in actively transferring fatty acids from a plastid to outside the plastid. Examples of ABCA9 (ABC transporter A family member 9) polypeptides useful for the invention include, but are not limited to, those fromArabidopsis thaliana(Q9FLT5),Capsella rubella(XP_006279962.1),Arabisalpine (KFK27923.1),Camelina sativa(XP_010457652.1),Brassica napus(CDY23040.1) andBrassica rapa(XP_009136512.1). As used herein, the term “acyl-CoA synthetase” or “ACS” (EC 6.2.1.3) means a polypeptide which is a member of a ligase family that catalyzes the formation of fatty acyl-CoA by a two-step process proceeding through an adenylated intermediate, using a non-esterified fatty acid, CoA and ATP as substrates to produce an acyl-CoA ester, AMP and pyrophosphate as products. As used herein, the term “long-chain acyl-CoA synthetase” (LACS) is an ACS that has activity on at least a C18 free fatty acid substrate although it may have broader activity on any of C14-C20 free fatty acids. The endogenous plastidial LACS enzymes are localised in the outer membrane of the plastid and function with fatty acid thioesterase for the export of fatty acids from the plastid (Schnurr et al., 2002). InArabidopsis, there are at least nine identified LACS genes (Shockey et al., 2002). Preferred LACS polypeptides are of the LACS9 subclass, which inArabidopsisis the major plastidial LACS. Examples of LACS polypeptides useful for the invention include, but are not limited to, those fromArabidopsis thaliana(Q9CAP8),Camelina sativa(XP_010416710.1),Capsella rubella(XP_006301059.1),Brassica napus(CDX79212.1),Brassica rapa(XP_009104618.1),Gossypium raimondii(XP_012450538.1) andVitis Vinifera(XP_002285853.1). Homologs of the above mentioned polypeptides in other species can readily be identified by those skilled in the art. Polypeptides Involved in Diacylglycerol (DAG) Production Levels of non-polar lipids in, for example, vegetative plant parts can also be increased by reducing the activity of polypeptides involved in diacylglycerol (DAG) production in the plastid in the plant parts, for example by either mutation of an endogenous gene encoding such a polypeptide or introduction of an exogenous gene which encodes a silencing RNA molecule which reduces the expression of a target gene involved in diacylglycerol (DAG) production in the plastid. As used herein, the term “polypeptide involved in diacylglycerol (DAG) production in the plastid” refers to any polypeptide in the plastid of plant cells in a plant or part thereof that is directly involved in the synthesis of diacylglycerol. Examples of such polypeptides include, but are not limited to, a plastidial GPAT, a plastidial LPAAT or a plastidial PAP. GPATs are described elsewhere in the present document. Examples of plastidial GPAT polypeptides which can be targeted for down-regulation in the invention include, but are not limited to, those fromArabidopsis thaliana(BAA00575),Capsella rubella(XP_006306544.1),Camelina sativa(010499766.1),Brassica napus(CDY43010.1),Brassica rapa(XP_009145198.1),Helianthus annuus(ADV16382.1) andCitrus unshiu(BAB79529.1). Homologs in other species can readily be identified by those skilled in the art. LPAATs are described elsewhere in the present document. As the skilled person would appreciate, plastidial LPAATs to be targeted for down-regulation for reducing DAG synthesis in the plastid are not endogenous LPAATs which function outside of the plastid such as those in the ER, for example being useful for producing TAG comprising medium chain length fatty acids. Examples of plastidial LPAAT polypeptides which can be targeted for down-regulation in the invention include, but are not limited to, those fromBrassica napus(ABQ42862),Brassica rapa(XP_009137939.1),Arabidopsis thaliana(NP_194787.2),Camelina sativa(XP_010432969.1),Glycine max(XP_006592638.1) andSolanum tuberosum(XP_006343651.1). Homologs in other species of the above mentioned polypeptides can readily be identified by those skilled in the art. As used herein, the term “phosphatidic acid phosphatase” (PAP) (EC 3.1.3.4) refers to a protein which hydrolyses the phosphate group on 3-sn-phosphatidate to produce 1,2-diacyl-sn-glycerol (DAG) and phosphate. Examples of plastidial PAP polypeptides which can be targeted for down-regulation in the invention include, but are not limited to, those fromArabidopsis thaliana(Q6NLA5),Capsella rubella(XP_006288605.1),Camelina sativa(XP_010452170.1),Brassica napus(CDY10405.1),Brassica rapa(XP_009122733.1),Glycine max(XP_003542504.1) andSolanum tuberosum(XP_006361792.1). Homologs in other species of the above mentioned polypeptides can readily be identified by those skilled in the art. Levels of TAG in, for example, vegetative plant parts can also be increased by increasing the activity of polypeptides involved in diacylglycerol (DAG) production in the ER in the plant parts. DAG is also produced in the plants and plant parts of the invention by release of the DAG moiety from PC and can be used for synthesis of TAG. This DAG is termed herein “PC-derived DAG”. The release can occur through one or more of the enzymes phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), the reverse reaction of choline:diacylglycerol cholinephospho-transferase (CPT), or phospholipase C (PLC) or phospholipase D (PLD). These enzymes result in DAG production in the ER rather than the plastid. As used herein, the term “phosphatidylcholine:diacylglycerol cholinephosphotransferase” or “PDCT” (EC 2.7.8.2) means an cholinephosphotransferase that transfers a phosphocholine headgroup from a phospholipid, typically PC, to produce DAG, or the reverse reaction to produce PC from DAG. PDCT can therefore interconvert PC and DAG (Lu et al., 2009; Hu et al., 2012). Thus, the two substrates of the forward reaction are cytidine monophosphate (CMP) and phosphatidylcholine and the two products are CDP-choline and DAG. PDCT belongs to the phosphatidic acid phosphatase-related protein family and typically possesses lipid phosphatase/phosphotransferase (LPT) domains. InArabidopsis thaliana, PDCT is encoded by the ROD1 (At3g15820) and ROD2 (At3g15830) genes (Lu et al., 2009). Homologous genes are readily identified in other plant species (Guan et al., 2015). Sequences of exemplary PDCT coding regions and polypeptides are provided herein as SEQ ID NOs:262-265 (SorghumandZea maysPDCT, Accession Nos XM_002437214 and EU973573.1), although any PDCT encoding gene can be used. Exemplary PDCT polypeptides have the amino acid sequences provided by Accession Nos. NP_566527.1 (Arabidopsis), XP_010487422.1 (Camelina sativa), XP_003531718.1 (Glycine max), XP_013695400.1 (Brassica napus), XP_012073167.1 (Jatropha curcas), XP_002517643.1 (Ricinus communis) XP_013587626.1 (Brassica oleracea), XP_016725741.1 and XP_016725742.1 (Gossypium hirsutum), AQK82308.1, NP_001145186.1 (Zea mays), and XP_021306179.1 (Sorghum bicolor). Homologs and naturally occurring variants from these or other plant, fungal or algal species can readily be identified and used in the present invention. In an embodiment, the homolog or variant is at least 95% identical, preferably at least 99% identical, to the amino acid sequence of the listed Accession No. In an embodiment, the PDCT is other thanA. thalianaPDCT (Lu et al., 2009). Increased expression of PDCT, which may be exogenous or endogenous to the cell or plant of the invention and which is preferably expressed from an exogenous polynucleotide, increases the flow of esterified acyl groups from PC to DAG and thereby increases the TTQ in the total fatty acid content and the level of TAG in vegetative plant parts or cells of the invention. Alternatively, decreasing the level of PDCT activity in the cell or plant by mutation in the gene or by a silencing RNA molecule reduces the production of PC from DAG, the reverse PDCT reaction, and allows for more of the de novo DAG to be used for TAG synthesis. The PDCT enzyme provides for the transfer of 18:1 from DAG into PC for desaturation and also for the reverse transfer of the polyunsaturated fatty acids 18:2 and 18:3 from PC into DAG which can be used in TAG synthesis. Fatty acid labelling experiments indicate that de novo DAG and PC-derived DAG are represented by two separate pools of DAG (Bates 2016), perhaps kept spatially separated in the ER. Some reports suggest that PC-derived DAG might be the predominant form of DAG used in TAG synthesis (Bates and Browse, 2011). As used herein, the term “CDP-choline:diacylglycerol cholinephospho-transferase” or “CPT”, (EC 2.7.8.2) means an enzyme that catalyses the transfer of a choline group from CDP-choline to DAG, forming PC and CMP. This forward reaction results in the net synthesis of PC from DAG. CPT also catalyses the reverse reaction, forming DAG from PC, possibly allowing for the equilibration of DAG and PC levels in the cell.Arabidopsiscontains two genes (AtAAPT1 and AtAAPT2, Accession Nos AAC61768.1 and AAC61769.1) that encode CPT enzymes. Mutations in either gene affect membrane homeostasis and the double mutant is lethal (Liu et al., 2015). Exemplary CPT enzymes have the amino acid sequences provided by the following Accession Nos: XP_010495346.1 and XP_019084815.1 (Camelina sativa); XP_013731103.1 and XP_013720632.1 (Brassica napus); XP_013585950.1 (Brassica oleracea); XP_015572083.1 (Ricinus communis); XP_016679754.1 (Gossypium hirsutum); XP_010687532.1 (Beta vulgaris); XP_012081980.1 (Jatropha curcas); XP_011070871.1 (Sesamum indicum); NP_001151915.1 and XP_008649199.1 (Zea mays); XP_002451408.1 and XP_021305900.1 (Sorghum bicolor). Homologs and naturally occurring variants from these or other plant, fungal or algal species can readily be identified and used in the present invention. In an embodiment, the homolog or variant is at least 95% identical, preferably at least 99% identical, to the amino acid sequence of the listed Accession No. Phospholipases are enzymes that hydrolyze phospholipids into products such as phosphatidic acid (PA), DAG, free fatty acids (FFA) or lysophospholipids (LPL), depending on the class of phospholipase. As used herein, the term “phospholipase C” or “PLC” means an enzyme which catalyses the cleavage of a phospholipid to form DAG and a phosphorylated headgroup, where the cleavage occurs at the ester linkage between the phosphate group and the glycerol backbone of the phospholipid. The phosphorylated headgroup is phosphocholine when the phospholipid is PC. PLCs are distinct from phospholipase D enzymes which cleave the headgroup of phospholipids to form phosphatidic acid (PA) rather than DAG as product, and from phospholipase A1 and phospholipase A2 which produce FFA from the phospholipids. PLCs are membrane-associated enzymes found widely in plants, animals and prokaryoyes. PLCs can be divided into three classes, the phosphatidylinositol-specific phospholipases C (PI-PLC), the phosphatidylcholine-specific phospholipases C (PC-PLC), and the PLC which hydrolyze glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-PLC), according to their substrate specificity range (Pokotylo et al., 2013, Hong et al., 2016). Multidomain animal PI-PLCs are G-protein activated enzymes regulating calcium levels and protein kinase C, and are therefore key components of the regulatory systems of cellular growth and development. The PI-PLCs are less preferred in the present invention. PC-PLCs, in plants also known as non-specific PLCs (NPC) have broader substrate ranges and are typically most active on PC, and are therefore preferred in the present invention. PC-PLCs have been identified in bacteria (Titball 1993), fungi (Morelle et al., 2005) and plants (Hong et al., 2016), the plant ones being more preferred. Six PC-PLC genes have been identified inArabidopsis(Wang, 2001; Nakamura et al., 2005), nine genes in soybean (Huang et al., 2010), five genes in rice (Singh et al., 2013) and multiple copies in diverse plant species. The plant PC-PLCs are classified in several sub-groups, namely the NPC1, NPC2, NPC3-5 and NPC6 sub-groups (Pokotylo et al., 2013) based on homology to theArabidopsisamino acid sequences, also having differing (but overlapping) substrate specificities and tissue distributions. For example,ArabidopsisNPC4 showed activity towards PC and PE, slight activity towards PS, but not PA and PIP2. NPC5 was able to cleave PC and PE, whereas NPC3 demonstrated lysophosphatidic acid (LPA) phosphatase activity resulting in MAG production as well as cleaving PC. NPC4 was expressed in mature leaves, and NPC6 was expressed in most tissues. TheArabidopsisPLCs have between 510 and 540 amino acid residues. In plants, PLCs are involved in lipid remodelling and the plant responses to phosphate deprivation and osmotic, salt and heat stresses, amongst other functions. Examples of PLCs include those identified, with amino acid sequences in the following Accession Nos., fromArabidopsis: NPC1, NP_172203.2; NPC2, NP_180255.1; NPC4, NP_566206.1; NPC5, NP_566207.1; NPC6, NP_190430.2; fromCamelina sativa, NPC1, XP_010457889.1; NPC2, XP_010473071.1; NPC4, XP_010463802.1; NPC5, XP_010485694.1; NPC6, XP_010426358.1; fromBrassica napusNPC1, XP_013687149.1; NPC2, XP_013744020.1; NPC4, XP_013682889.1; NPC6, XP_002511167.1; fromRicinus communisNPC1, XP_002525632.1; NPC4, XP_002524007.1; NPC6, XP_002511167.1; fromGossypium hirsutumNPC1, XP_016715492.1; NPC2, XP_016745351.1; NPC4, XP_016697678.1; NPC6, XP_016734150.1; fromBeta vulgarisNPC1, XP_010685575.1; NPC2, XP_010673757.1; NPC4, XP_010691936.1; fromZea maysNPC1 homolog, NP_001170209.1; fromOryza sativaNPC1, XP_015631592.1; fromGlycine maxNPC1, XP_003551286.1; NPC2, XP_003556783.1; NPC4, XP_003521010.1; NPC6, XP_003523153.1; NPC6, XP_003526950.1; fromJatropha curcasNPC2, XP_012065897.1; NPC4, XP_012070293.1; NPC6, XP_012090681.1; fromSolanum tuberosumNPC2, XP_006362756.1; fromElaeis guineensisNPC2, XP_010938556.1; NPC4, XP_010919939.1; fromBrachypodium distachyonNPC2, XP_003565100.1; fromTrifolium subterraneumNPC4, GAU26202.1; NPC6, GAU26767.1; fromMedicago truncatulaNPC4, XP_013444051.1; and fromHelianthus annuusNPC6, XP_0219841571 Homologs and naturally occurring variants from these or other plant, fungal or algal species can readily be identified and used in the present invention. In an embodiment, the homolog or variant is at least 95% identical, preferably at least 99% identical, to the amino acid sequence of the listed Accession No. As used herein, the term “phospholipase D” or “PLD” means an enzyme which catalyses the cleavage of a the phosphodiesteric linkage of a headgroup of a membrane phospholipid to form phosphatidic acid (PA) and soluble headgroup, the cleavage occurring at the phosphodiester bond distal to the glycerol backbone of the phospholipid. The soluble headgroup product is choline when the phospholipid is PC. The PA is subsequently converted to DAG by the action of PAP. Many PLDs have been identified in plants, animals, fungi and prokaryotes. All of the plant sepcies examined have a PLD family of at least 10 PLD genes (Wang, 2005), for example theArabidopsisPLD family has 12 genes identified. PLDs are classified in subgroups α, β, γ, δ, ε and ζ based on sequence and enzymatic properties (Hong et al, 2016). PLDs have either a C2 domain of approximately 130 amino acids involved in calcium ion and phospholipid binding or a pleckstrin homology (PH) domain and a phox (PX) homology domain. All examined eukaryotic PLDs contain two duplicated catalytic HKD motifs, separated by more than 300 amino acids inArabidopsisPLDs, but which interact with each other to form the active site (Hong et al, 2013). Exemplary PLDs include those fromArabidopsis: Accession Nos. AAL06337.1, CAJ58441.1; fromCamelina sativa, XP_010465702.1, XP_010430169.1; fromGossypium hirsutum, XP_016724300.1; fromRicinus communis, XP_015573380.1; fromSolanum lycopersicum, XP_004229274.1; fromJatropha curcas, XP_012083994.1; fromGlycine max, XP_003534832.1, NP_001275522.1; fromElaeis guineensis, XP_010921600.1; fromBrassica rapa, XP_009146059.1; fromBeta vulgaris, XP_010693582.1 and XP_016713715.1;Medicago truncatulaXP_003591178.2; and fromBrassica napus, XP_013677646.1. Homologs and naturally occurring variants from these or other plant, fungal or algal species can readily be identified and used in the present invention. In an embodiment, the homolog or variant is at least 95% identical, preferably at least 99% identical, to the amino acid sequence of the listed Accession No. Import of Fatty Acids into Plastids Levels of non-polar lipids in vegetative plant parts can also be increased by reducing the activity of TGD polypeptides in the plant parts, for example by either mutation of an endogenous gene encoding a TGD polypeptide or introduction of an exogenous gene which encodes a silencing RNA molecule which reduces the expression of an endogenous TGD gene. As used herein, a “Trigalactosyldiacylglycerol (TGD) polypeptide” is one which is involved in the ER to chloroplast lipid trafficking (Xu et al., 2010; Fan et al., 2015) and involved in forming a protein complex which has permease function for lipids. Four such polypeptides are known to form or be associated with a TGD permease, namely TGD-1 (Accession No. At1g19800 and homologs in other species), TGD-2 (Accession No At2g20320 and homologs in other species), TGD-3 (Accession No. NM-105215 and homologs in other species) and TGD-4 (At3g06960 and homologs in other species) (US 20120237949). TGD5 is also involved in ER to choroplast lipid trafficking, and down-regulation of TGD5 is associated with increased oil production (US2015/337017; Fan et al., 2015). Sequences of exemplary TGD5 polypeptides are provided herein as SEQ ID NOs:250-253 (SorghumandZea maysTGD5, Accession Nos XM_002442154 and EU972796.1). TGD-1, -2 and -3 polypeptides are thought to be components of an ATP-Binding Cassette (ABC) transporter associated with the inner envelope membrane of the chloroplast. TGD-2 and TGD-4 polypeptides bind to phosphatidic acid whereas TGD-3 polypetide functions as an ATPase in the chloroplast stroma. As used herein, an “endogenous TGD gene” is a gene which encodes a TGD polypeptide in a plant. Mutations in TGD-1 gene inA. thalianacaused accumulation of triacylglycerols, oligogalactolipids and phosphatidic acid (PA) (Xu et al., 2005). Mutations in TGD genes or SDP1 genes, or indeed in any desired gene in a plant, can be introduced in a site-specific manner by artificial zinc finger nuclease (ZFN), TAL effector (TALEN) or CRISPR technologies (using a Cas9 type nuclease) as known in the art. Preferred exogenous genes encoding silencing RNAs are those encoding a double-stranded RNA molecule such as a hairpin RNA or an artificial microRNA precursor. Sucrose Metabolism The TAG levels and/or the TTQ of the total fatty content in the cells, plants and plant parts of the invention can also be increased by modifying sucrose metabolism, particularly in the stems of plants such as sugarcane,SorghumandZea mays. In an embodiment, this is achieved by increasing expression of a sucrose metabolism polypeptide such as invertase or sucrose synthase, or of a sucrose transport polypeptide such as SUS 1, SUS4, SUT2, SUT4, or SWEET. The effect of these polypeptides is to increase the supply of sucrose and its monosaccharide components in the cytosol of the cells and/or to decrease the transfer and/or storage of sucrose in the vacuoles of the cells, particularly in stem cells. Sequences of examples of these polypeptides are provided in SEQ ID NOs:274-292. Invertase such as bCIN, INV2 or INV3 acts to convert sucrose into hexoses which can be exported from the vacuoles into the cytoplasm (McKinley et al., 2016). Increased expression of SUS1 or SUS4 breaks down cytosolic sucrose into hexoses for glycolysis and de novo fatty acid synthesis rather than transfer of the sucrose into vacuoles, such as in stem parenchyma cells (McKinley et al., 2016). Increased expression of sugar transport polypeptides such as tonoplast sucrose exporter, for example SUT2 or SUT4, or SWEET polypeptide releases vacuolar sucrose for cytosolic glycolysis and increases de novo fatty acid biosynthesis (Bihmidine et al., 2016; Qazi et al., 2012; Schneider et al., 2012; Hedrich et al., 2015; Klemens et al., 2013). The TAG levels and/or the TTQ of the total fatty content in the cells, plants and plant parts of the invention can also be increased by reducing the level of TST polypeptides such as TST1 or TST2, particularly in the stems of plants such as sugarcane,SorghumandZea mays. TST polypeptide can be decreased by mutation of the endogenous genes encoding the polypeptide, or by introduction of an exogenous polynucleotide that encodes a silencing RNA molecule. Sequences of exemplary TST cDNAs and polypeptides are provided as SEQ ID NOs:266-273. Fatty Acid Modifying Enzymes As used herein, the term “FAD2” refers to a membrane bound delta-12 fatty acid desturase that desaturates oleic acid (C18:1Δ9) to produce linoleic acid (C18:2Δ9,12). As used herein, the term “epoxygenase” or “fatty acid epoxygenase” refers to an enzyme that introduces an epoxy group into a fatty acid resulting in the production of an epoxy fatty acid. In preferred embodiment, the epoxy group is introduced at the 12th carbon on a fatty acid chain, in which case the epoxygenase is a Δ12-epoxygenase, especially of a C16 or C18 fatty acid chain. The epoxygenase may be a Δ9-epoxygenase, a Δ15 epoxygenase, or act at a different position in the acyl chain as known in the art. The epoxygenase may be of the P450 class. Preferred epoxygenases are of the mono-oxygenase class as described in WO98/46762. Numerous epoxygenases or presumed epoxygenases have been cloned and are known in the art. Further examples of expoxygenases include proteins comprising an amino acid sequence provided in SEQ ID NO:21 of WO 2009/129582, polypeptides encoded by genes fromCrepis paleastina(CAA76156, Lee et al., 1998),Stokesia laevis(AAR23815) (monooxygenase type),Euphorbia lagascae(AAL62063) (P450 type), human CYP2J2 (arachidonic acid epoxygenase, U37143); human CYPIA1 (arachidonic acid epoxygenase, K03191), as well as variants and/or mutants thereof. As used herein, the term, “hydroxylase” or “fatty acid hydroxylase” refers to an enzyme that introduces a hydroxyl group into a fatty acid resulting in the production of a hydroxylated fatty acid. In a preferred embodiment, the hydroxyl group is introduced at the 2nd, 12th and/or 17th carbon on a C18 fatty acid chain. Preferably, the hydroxyl group is introduced at the 12thcarbon, in which case the hydroxylase is a Δ12-hydroxylase. In another preferred embodiment, the hydroxyl group is introduced at the 15th carbon on a C16 fatty acid chain. Hydroxylases may also have enzyme activity as a fatty acid desaturase. Examples of genes encoding Δ12-hydroxylases include those fromRicinus communis(AAC9010, van de Loo 1995);Physaria lindheimeri, (ABQ01458, Dauk et al., 2007);Lesquerella fendleri, (AAC32755, Broun et al., 1998);Daucus carota, (AAK30206); fatty acid hydroxylases which hydroxylate the terminus of fatty acids, for example:A. thalianaCYP86A1 (P48422, fatty acid ω-hydroxylase);Vicia sativaCYP94A1 (P98188, fatty acid ω-hydroxylase); mouse CYP2E1 (X62595, lauric acid co-1 hydroxylase); rat CYP4A1 (M57718, fatty acid ω-hydroxylase), as well as as variants and/or mutants thereof. As used herein, the term “conjugase” or “fatty acid conjugase” refers to an enzyme capable of forming a conjugated bond in the acyl chain of a fatty acid. Examples of conjugases include those encoded by genes fromCalendula officinalis(AF343064, Qiu et al., 2001);Vernicia fordii(AAN87574, Dyer et al., 2002); Punica granatum (AY178446, Iwabuchi et al., 2003) andTrichosanthes kirilowii(AY178444, Iwabuchi et al., 2003); as well as as variants and/or mutants thereof. As used herein, the term “acetylenase” or “fatty acid acetylenase” refers to an enzyme that introduces a triple bond into a fatty acid resulting in the production of an acetylenic fatty acid. In a preferred embodiment, the triple bond is introduced at the 2nd, 6th, 12th and/or 17th carbon on a C18 fatty acid chain. Examples acetylenases include those fromHelianthus annuus(AA038032, ABC59684), as well as as variants and/or mutants thereof. Examples of such fatty acid modifying genes include proteins according to the following Accession Numbers which are grouped by putative function, and homologues from other species: Δ12-acetylenases ABC00769, CAA76158, AAO38036, AAO38032; Δ12 conjugases AAG42259, AAG42260, AAN87574; Δ12-desaturases P46313, ABS18716, AAS57577, AAL61825, AAF04093, AAF04094; Δ12 epoxygenases XP_001840127, CAA76156, AAR23815; Δ12-hydroxylases ACF37070, AAC32755, ABQ01458, AAC49010; and Δ12 P450 enzymes such as AF406732. Silencing Suppressors In an embodiment, a transgenic plant or part thereof of the invention may comprise a silencing suppressor. As used herein, a “silencing suppressor” enhances transgene expression in a plant or part thereof of the invention. For example, the presence of the silencing suppressor results in higher levels of a polypeptide(s) produced an exogenous polynucleotide(s) in a plant or part thereof of the invention when compared to a corresponding plant or part thereof lacking the silencing suppressor. In an embodiment, the silencing suppressor preferentially binds a dsRNA molecule which is 21 base pairs in length relative to a dsRNA molecule of a different length. This is a feature of at least the p19 type of silencing suppressor, namely for p19 and its functional orthologs. In another embodiment, the silencing suppressor preferentially binds to a double-stranded RNA molecule which has overhanging 5′ ends relative to a corresponding double-stranded RNA molecule having blunt ends. This is a feature of the V2 type of silencing suppressor, namely for V2 and its functional orthologs. In an embodiment, the dsRNA molecule, or a processed RNA product thereof, comprises at least 19 consecutive nucleotides, preferably whose length is 19-24 nucleotides with 19-24 consecutive basepairs in the case of a double-stranded hairpin RNA molecule or processed RNA product, more preferably consisting of 20, 21, 22, 23 or 24 nucleotides in length, and preferably comprising a methylated nucleotide, which is at least 95% identical to the complement of the region of the target RNA, and wherein the region of the target RNA is i) within a 5′ untranslated region of the target RNA, ii) within a 5′ half of the target RNA, iii) within a protein-encoding open-reading frame of the target RNA, iv) within a 3′ half of the target RNA, or v) within a 3′ untranslated region of the target RNA. Further details regarding silencing suppressors are well known in the art and described in WO 2013/096992 and WO 2013/096993. Polynucleotides The terms “polynucleotide”, and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide of the invention may be of genomic, cDNA, semisynthetic, or synthetic origin, double-stranded or single-stranded and by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), ribozymes, cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, chimeric DNA of any sequence, nucleic acid probes, and primers. For in vitro use, a polynucleotide may comprise modified nucleotides such as by conjugation with a labeling component. As used herein, an “isolated polynucleotide” refers to a polynucleotide which has been separated from the polynucleotide sequences with which it is associated or linked in its native state, or a non-naturally occurring polynucleotide. As used herein, the term “gene” is to be taken in its broadest context and includes the deoxyribonucleotide sequences comprising the transcribed region and, if translated, the protein coding region, of a structural gene and including sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of at least about 2 kb on either end and which are involved in expression of the gene. In this regard, the gene includes control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals, in which case, the gene is referred to as a “chimeric gene”. The sequences which are located 5′ of the protein coding region and which are present on the mRNA are referred to as 5′ non-translated sequences. The sequences which are located 3′ or downstream of the protein coding region and which are present on the mRNA are referred to as 3′ non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region which may be interrupted with non-coding sequences termed “introns”, “intervening regions”, or “intervening sequences.” Introns are segments of a gene which are transcribed into nuclear RNA (nRNA). Introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns are therefore absent in the mRNA transcript. A gene which contains at least one intron may be subject to variable splicing, resulting in alternative mRNAs from a single transcribed gene and therefore polypeptide variants. A gene in its native state, or a chimeric gene may lack introns. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term “gene” includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above. As used herein, “chimeric DNA” refers to any DNA molecule that is not naturally found in nature; also referred to herein as a “DNA construct” or “genetic construct”. Typically, a chimeric DNA comprises regulatory and transcribed or protein coding sequences that are not naturally found together in nature. Accordingly, chimeric DNA may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. The open reading frame may or may not be linked to its natural upstream and downstream regulatory elements. The open reading frame may be incorporated into, for example, the plant genome, in a non-natural location, or in a replicon or vector where it is not naturally found such as a bacterial plasmid or a viral vector. The term “chimeric DNA” is not limited to DNA molecules which are replicable in a host, but includes DNA capable of being ligated into a replicon by, for example, specific adaptor sequences. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. The term includes a gene in a progeny plant or part thereof such as a vegetative plant part which was introducing into the genome of a progenitor cell thereof. Such progeny cells etc may be at least a 3rdor 4thgeneration progeny from the progenitor cell which was the primary transformed cell, or of the progenitor transgenic plant (referred to herein as a TO plant). Progeny may be produced by sexual reproduction or vegetatively such as, for example, from tubers in potatoes or ratoons in sugarcane. The term “genetically modified”, “genetic modification” and variations thereof, is a broader term that includes introducing a gene into a cell by transformation or transduction, mutating a gene in a cell and genetically altering or modulating the regulation of a gene in a cell, or the progeny of any cell modified as described above. A “genomic region” as used herein refers to a position within the genome where a transgene, or group of transgenes (also referred to herein as a cluster), have been inserted into a cell, or predecessor thereof. Such regions only comprise nucleotides that have been incorporated by the intervention of man such as by methods described herein. A “recombinant polynucleotide” of the invention refers to a nucleic acid molecule which has been constructed or modified by artificial recombinant methods. The recombinant polynucleotide may be present in a cell of a plant or part thereof in an altered amount or expressed at an altered rate (e.g., in the case of mRNA) compared to its native state. In one embodiment, the polynucleotide is introduced into a cell that does not naturally comprise the polynucleotide. Typically an exogenous DNA is used as a template for transcription of mRNA which is then translated into a continuous sequence of amino acid residues coding for a polypeptide of the invention within the transformed cell. In another embodiment, the polynucleotide is endogenous to the plant or part thereof and its expression is altered by recombinant means, for example, an exogenous control sequence is introduced upstream of an endogenous gene of interest to enable the transformed plant or part thereof to express the polypeptide encoded by the gene, or a deletion is created in a gene of interest by ZFN, Talen or CRISPR methods. A recombinant polynucleotide of the invention includes polynucleotides which have not been separated from other components of the cell-based or cell-free expression system, in which it is present, and polynucleotides produced in said cell-based or cell-free systems which are subsequently purified away from at least some other components. The polynucleotide can be a contiguous stretch of nucleotides or comprise two or more contiguous stretches of nucleotides from different sources (naturally occurring and/or synthetic) joined to form a single polynucleotide. Typically, such chimeric polynucleotides comprise at least an open reading frame encoding a polypeptide of the invention operably linked to a promoter suitable of driving transcription of the open reading frame in a cell of interest. With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polynucleotide comprises a polynucleotide sequence which is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO. A polynucleotide of, or useful for, the present invention may selectively hybridise, under stringent conditions, to a polynucleotide defined herein. As used herein, stringent conditions are those that: (1) employ during hybridisation a denaturing agent such as formamide, for example, 50% (v/v) formamide with 0.1% (w/v) bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (2) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1% SDS, and/or (3) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C. Polynucleotides of the invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Polynucleotides which have mutations relative to a reference sequence can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis or DNA shuffling on the nucleic acid as described above). Polynucleotides for Reducing Expression of Genes RNA Interference RNA interference (RNAi) is particularly useful for specifically reducing the expression of a gene, which results in reduced production of a particular protein if the gene encodes a protein. Although not wishing to be limited by theory, Waterhouse et al. (1998) have provided a model for the mechanism by which dsRNA (duplex RNA) can be used to reduce protein production. This technology relies on the presence of dsRNA molecules that contain a sequence that is essentially identical to the mRNA of the gene of interest or part thereof. Conveniently, the dsRNA can be produced from a single promoter in a recombinant vector or host cell, where the sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure. The design and production of suitable dsRNA molecules is well within the capacity of a person skilled in the art, particularly considering Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619, WO 99/53050, WO 99/49029, and WO 01/34815. In one example, a DNA is introduced that directs the synthesis of an at least partly double stranded RNA product(s) with homology to the target gene to be inactivated such as, for example, a SDPJ, TGD, plastidial GPAT, plastidial LPAAT, plastidial PAP, AGPase gene. The DNA therefore comprises both sense and antisense sequences that, when transcribed into RNA, can hybridize to form the double stranded RNA region. In one embodiment of the invention, the sense and antisense sequences are separated by a spacer region that comprises an intron which, when transcribed into RNA, is spliced out. This arrangement has been shown to result in a higher efficiency of gene silencing (Smith et al., 2000). The double stranded region may comprise one or two RNA molecules, transcribed from either one DNA region or two. The presence of the double stranded molecule is thought to trigger a response from an endogenous system that destroys both the double stranded RNA and also the homologous RNA transcript from the target gene, efficiently reducing or eliminating the activity of the target gene. The length of the sense and antisense sequences that hybridize should each be at least 19 contiguous nucleotides, preferably at least 50 contiguous nucleotides, more preferably at least 100 or at least 200 contiguous nucleotides. Generally, a sequence of 100-1000 nucleotides corresponding to a region of the target gene mRNA is used. The full-length sequence corresponding to the entire gene transcript may be used. The degree of identity of the sense sequence to the targeted transcript (and therefore also the identity of the antisense sequence to the complement of the target transcript) should be at least 85%, at least 90%, or 95-100%. The RNA molecule may of course comprise unrelated sequences which may function to stabilize the molecule. The RNA molecule may be expressed under the control of a RNA polymerase II or RNA polymerase III promoter. Examples of the latter include tRNA or snRNA promoters. Preferred small interfering RNA (“siRNA”) molecules comprise a nucleotide sequence that is identical to about 19-25 contiguous nucleotides of the target mRNA. Preferably, the siRNA sequence commences with the dinucleotide AA, comprises a GC-content of about 30-70% (preferably, 30-60%, more preferably 40-60% and more preferably about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the organism in which it is to be introduced, for example, as determined by standard BLAST search. microRNA MicroRNAs (abbreviated miRNAs) are generally 19-25 nucleotides (commonly about 20-24 nucleotides in plants) non-coding RNA molecules that are derived from larger precursors that form imperfect stem-loop structures. miRNAs bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing. Artificial miRNAs (amiRNAs) can be designed based on natural miRNAs for reducing the expression of any gene of interest, as well known in the art. In plant cells, miRNA precursor molecules are believed to be largely processed in the nucleus. The pri-miRNA (containing one or more local double-stranded or “hairpin” regions as well as the usual 5′ “cap” and polyadenylated tail of an mRNA) is processed to a shorter miRNA precursor molecule that also includes a stem-loop or fold-back structure and is termed the “pre-miRNA”. In plants, the pre-miRNAs are cleaved by distinct DICER-like (DCL) enzymes, yielding miRNA:miRNA* duplexes. Prior to transport out of the nucleus, these duplexes are methylated. In the cytoplasm, the miRNA strand from the miRNA:miRNA duplex is selectively incorporated into an active RNA-induced silencing complex (RISC) for target recognition. The RISC-complexes contain a particular subset of Argonaute proteins that exert sequence-specific gene repression (see, for example, Millar and Waterhouse, 2005; Pasquinelli et al., 2005; Almeida and Allshire, 2005). Cosuppression Genes can suppress the expression of related endogenous genes and/or transgenes already present in the genome, a phenomenon termed homology-dependent gene silencing. Most of the instances of homologydependent gene silencing fall into two classes—those that function at the level of transcription of the transgene, and those that operate post-transcriptionally. Post-transcriptional homology-dependent gene silencing (i.e., cosuppression) describes the loss of expression of a transgene and related endogenous or viral genes in transgenic plants. Cosuppression often, but not always, occurs when transgene transcripts are abundant, and it is generally thought to be triggered at the level of mRNA processing, localization, and/or degradation. Several models exist to explain how cosuppression works (see in Taylor, 1997). Cosuppression involves introducing an extra copy of a gene or a fragment thereof into a plant in the sense orientation with respect to a promoter for its expression. The size of the sense fragment, its correspondence to target gene regions, and its degree of sequence identity to the target gene can be determined by those skilled in the art. In some instances, the additional copy of the gene sequence interferes with the expression of the target plant gene. Reference is made to WO 97/20936 and EP 0465572 for methods of implementing co-suppression approaches. Antisense Polynucleotides The term “antisense polynucletoide” shall be taken to mean a DNA or RNA molecule that is complementary to at least a portion of a specific mRNA molecule encoding an endogenous polypeptide and capable of interfering with a post-transcriptional event such as mRNA translation. The use of antisense methods is well known in the art (see for example, G. Hartmann and S. Endres, Manual of Antisense Methodology, Kluwer (1999)). The use of antisense techniques in plants has been reviewed by Bourque (1995) and Senior (1998). Bourque (1995) lists a large number of examples of how antisense sequences have been utilized in plant systems as a method of gene inactivation. Bourque also states that attaining 100% inhibition of any enzyme activity may not be necessary as partial inhibition will more than likely result in measurable change in the system. Senior (1998) states that antisense methods are now a very well established technique for manipulating gene expression. In one embodiment, the antisense polynucleotide hybridises under physiological conditions, that is, the antisense polynucleotide (which is fully or partially single stranded) is at least capable of forming a double stranded polynucleotide with mRNA encoding an endogenous polypeptide, for example, a SDP1, TGD, plastidial GPAT, plastidial LPAAT, plastidial PAP or AGPase mRNA under normal conditions in a cell. Antisense molecules may include sequences that correspond to the structural genes or for sequences that effect control over the gene expression or splicing event. For example, the antisense sequence may correspond to the targeted coding region of endogenous gene, or the 5′-untranslated region (UTR) or the 3′-UTR or combination of these. It may be complementary in part to intron sequences, which may be spliced out during or after transcription, preferably only to exon sequences of the target gene. In view of the generally greater divergence of the UTRs, targeting these regions provides greater specificity of gene inhibition. The length of the antisense sequence should be at least 19 contiguous nucleotides, preferably at least 50 nucleotides, and more preferably at least 100, 200, 500 or 1000 nucleotides. The full-length sequence complementary to the entire gene transcript may be used. The length is most preferably 100-2000 nucleotides. The degree of identity of the antisense sequence to the targeted transcript should be at least 90% and more preferably 95-100%. The antisense RNA molecule may of course comprise unrelated sequences which may function to stabilize the molecule. Recombinant Vectors One embodiment of the present invention includes a recombinant vector, which comprises at least one polynucleotide defined herein and is capable of delivering the polynucleotide into a host cell. Recombinant vectors include expression vectors. Recombinant vectors contain heterologous polynucleotide sequences, that is, polynucleotide sequences that are not naturally found adjacent to a polynucleotide defined herein, that preferably, are derived from a different species. The vector can be either RNA or DNA, and typically is a viral vector, derived from a virus, or a plasmid. Plasmid vectors typically include additional nucleic acid sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic cells, e.g., pUC-derived vectors, pGEM-derived vectors or binary vectors containing one or more T-DNA regions. Additional nucleic acid sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert nucleic acid sequences or genes encoded in the nucleic acid construct, and sequences that enhance transformation of prokaryotic and eukaryotic (especially plant) cells. “Operably linked” as used herein, refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory element (promoter) to a transcribed sequence. For example, a promoter is operably linked to a coding sequence of a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate cell. Generally, promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance. When there are multiple promoters present, each promoter may independently be the same or different. Recombinant vectors may also contain one or more signal peptide sequences to enable an expressed polypeptide defined herein to be retained in the endoplasmic reticulum (ER) in the cell, or transfer into a plastid, and/or contain fusion sequences which lead to the expression of nucleic acid molecules as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion or localisation of a polypeptide defined herein. To facilitate identification of transformants, the recombinant vector desirably comprises a selectable or screenable marker gene. By “marker gene” is meant a gene that imparts a distinct phenotype to cells expressing the marker gene and thus, allows such transformed cells to be distinguished from cells that do not have the marker. A selectable marker gene confers a trait for which one can “select” based on resistance to a selective agent (e.g., a herbicide, antibiotic). A screenable marker gene (or reporter gene) confers a trait that one can identify through observation or testing, that is, by “screening” (e.g., β-glucuronidase, luciferase, GFP or other enzyme activity not present in untransformed cells). Exemplary selectable markers for selection of plant transformants include, but are not limited to, a hyg gene which encodes hygromycin B resistance; a neomycin phosphotransferase (nptII) gene conferring resistance to kanamycin, paromomycin; a glutathione-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides as for example, described in EP 256223; a glutamine synthetase gene conferring, upon overexpression, resistance to glutamine synthetase inhibitors such as phosphinothricin as for example, described in WO 87/05327; an acetyltransferase gene fromStreptomyces viridochromogenesconferring resistance to the selective agent phosphinothricin as for example, described in EP 275957; a gene encoding a 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as for example, described by Hinchee et al. (1988); a bar gene conferring resistance against bialaphos as for example, described in WO91/02071; a nitrilase gene such as bxn fromKlebsiella ozaenaewhich confers resistance to bromoxynil (Stalker et al., 1988); a dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate (Thillet et al., 1988); a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea, or other ALS-inhibiting chemicals (EP 154,204); a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan; or a dalapon dehalogenase gene that confers resistance to the herbicide. Preferably, the recombinant vector is stably incorporated into the genome of the cell such as the plant cell. Accordingly, the recombinant vector may comprise appropriate elements which allow the vector to be incorporated into the genome, or into a chromosome of the cell. Expression Vector As used herein, an “expression vector” is a DNA vector that is capable of transforming a host cell and of effecting expression of one or more specified polynucleotides. Expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and that control the expression of polynucleotides of the present invention. In particular, expression vectors of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation such as promoter, enhancer, operator and repressor sequences. The choice of the regulatory sequences used depends on the target organism such as a plant and/or target organ or tissue of interest. Such regulatory sequences may be obtained from any eukaryotic organism such as plants or plant viruses, or may be chemically synthesized. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in for example, Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987, Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989, and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, a transcription termination site, and/or a polyadenylation signal. A number of constitutive promoters that are active in plant cells have been described. Suitable promoters for constitutive expression in plants include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the Figwort mosaic virus (FMV) 35S, the light-inducible promoter from the small subunit (SSU) of the ribulose-1,5-bis-phosphate carboxylase, the rice cytosolic triosephosphate isomerase promoter, the adenine phosphoribosyltransferase promoter ofArabidopsis, the rice actin 1 gene promoter, the mannopine synthase and octopine synthase promoters, the Adh promoter, the sucrose synthase promoter, the R gene complex promoter, and the chlorophyll α/β binding protein gene promoter. These promoters have been used to create DNA vectors that have been expressed in plants, see for example, WO 84/02913. All of these promoters have been used to create various types of plant-expressible recombinant DNA vectors. For the purpose of expression in source tissues of the plant such as the leaf, seed, root or stem, it is preferred that the promoters utilized in the present invention have relatively high expression in these specific tissues. For this purpose, one may choose from a number of promoters for genes with tissue- or cell-specific, or -enhanced expression. Examples of such promoters reported in the literature include, the chloroplast glutamine synthetase GS2 promoter from pea, the chloroplast fructose-1,6-biphosphatase promoter from wheat, the nuclear photosynthetic ST-LS1 promoter from potato, the serine/threonine kinase promoter and the glucoamylase (CHS) promoter fromArabidopsis thaliana. Also reported to be active in photosynthetically active tissues are the ribulose-1,5-bisphosphate carboxylase promoter from eastern larch (Larix laricina), the promoter for the Cab gene, Cab6, from pine, the promoter for the Cab-1 gene from wheat, the promoter for the Cab-1 gene from spinach, the promoter for the Cab 1R gene from rice, the pyruvate, orthophosphate dikinase (PPDK) promoter fromZea mays, the promoter for the tobacco Lhcb1*2 gene, theArabidopsis thalianaSuc2 sucrose-H30symporter promoter, and the promoter for the thylakoid membrane protein genes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS). Other promoters for the chlorophyll α/β-binding proteins may also be utilized in the present invention such as the promoters for LhcB gene and PsbP gene from white mustard (Sinapis alba). A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals, also can be used for expression of RNA-binding protein genes in plant cells, including promoters regulated by (1) heat, (2) light (e.g., pea RbcS-3A promoter, maize RbcS promoter), (3) hormones such as abscisic acid, (4) wounding (e.g., Wunl), or (5) chemicals such as methyl jasmonate, salicylic acid, steroid hormones, alcohol, Safeners (WO 97/06269), or it may also be advantageous to employ (6) organ-specific promoters. As used herein, the term “plant storage organ specific promoter” refers to a promoter that preferentially, when compared to other plant tissues, directs gene transcription in a storage organ of a plant. For the purpose of expression in sink tissues of the plant such as the tuber of the potato plant, the fruit of tomato, or the seed of soybean, canola, cotton,Zea mays, wheat, rice, and barley, it is preferred that the promoters utilized in the present invention have relatively high expression in these specific tissues. The promoter for β-conglycinin or other seed-specific promoters such as the napin, zein, linin and phaseolin promoters, can be used. Root specific promoters may also be used. An example of such a promoter is the promoter for the acid chitinase gene. Expression in root tissue could also be accomplished by utilizing the root specific subdomains of the CaMV 35S promoter that have been identified. In a particularly preferred embodiment, the promoter directs expression in tissues and organs in which lipid biosynthesis takes place. Such promoters may act in seed development at a suitable time for modifying lipid composition in seeds. Preferred promoters for seed-specific expression include: 1) promoters from genes encoding enzymes involved in lipid biosynthesis and accumulation in seeds such as desaturases and elongases, 2) promoters from genes encoding seed storage proteins, and 3) promoters from genes encoding enzymes involved in carbohydrate biosynthesis and accumulation in seeds. Seed specific promoters which are suitable are, the oilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), theVicia fabaUSP promoter (Baumlein et al., 1991), theArabidopsisoleosin promoter (WO 98/45461), thePhaseolus vulgarisphaseolin promoter (U.S. Pat. No. 5,504,200), theBrassicaBce4 promoter (WO 91/13980), or the legumin B4 promoter (Baumlein et al., 1992), and promoters which lead to the seed-specific expression in monocots such as maize, barley, wheat, rye, rice and the like. Notable promoters which are suitable are the barley 1pt2 or 1pt1 gene promoter (WO 95/15389 and WO 95/23230), or the promoters described in WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, thesorghumkasirin gene, the rye secalin gene). Other promoters include those described by Broun et al. (1998), Potenza et al. (2004), US 20070192902 and US 20030159173. In an embodiment, the seed specific promoter is preferentially expressed in defined parts of the seed such as the cotyledon(s) or the endosperm. Examples of cotyledon specific promoters include, but are not limited to, the FP1 promoter (Ellerstrom et al., 1996), the pea legumin promoter (Perrin et al., 2000), and the bean phytohemagglutnin promoter (Perrin et al., 2000). Examples of endosperm specific promoters include, but are not limited to, the maize zein-1 promoter (Chikwamba et al., 2003), the rice glutelin-1 promoter (Yang et al., 2003), the barley D-hordein promoter (Horvath et al., 2000) and wheat HMW glutenin promoters (Alvarez et al., 2000). In a further embodiment, the seed specific promoter is not expressed, or is only expressed at a low level, in the embryo and/or after the seed germinates. In another embodiment, the plant storage organ specific promoter is a fruit specific promoter. Examples include, but are not limited to, the tomato polygalacturonase, E8 and Pds promoters, as well as the apple ACC oxidase promoter (for review, see Potenza et al., 2004). In a preferred embodiment, the promoter preferentially directs expression in the edible parts of the fruit, for example the pith of the fruit, relative to the skin of the fruit or the seeds within the fruit. In an embodiment, the inducible promoter is theAspergillus nidulansalc system. Examples of inducible expression systems which can be used instead of theAspergillus nidulansalc system are described in a review by Padidam (2003) and Corrado and Karali (2009). In another embodiment, the inducible promoter is a safener inducible promoter such as, for example, the maize 1n2-1 or 1n2-2 promoter (Hershey and Stoner, 1991), the safener inducible promoter is the maize GST-27 promoter (Jepson et al., 1994), or the soybean GH2/4 promoter (Ulmasov et al., 1995). In another embodiment, the inducible promoter is a senescence inducible promoter such as, for example, senescence-inducible promoter SAG (senescence associated gene) 12 and SAG 13 fromArabidopsis(Gan, 1995; Gan and Amasino, 1995) and LSC54 fromBrassica napus(Buchanan-Wollaston, 1994). Such promoters show increased expression at about the onset of senescence of plant tissues, in particular the leaves. For expression in vegetative tissue leaf-specific promoters, such as the ribulose biphosphate carboxylase (RBCS) promoters, can be used. For example, the tomato RBCS1, RBCS2 and RBCS3A genes are expressed in leaves and light grown seedlings (Meier et al., 1997). A ribulose bisphosphate carboxylase promoters expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels, described by Matsuoka et al. (1994), can be used. Another leaf-specific promoter is the light harvesting chlorophyll a/b binding protein gene promoter (see, Shiina et al., 1997). TheArabidopsis thalianamyb-related gene promoter (Atmyb5) described by Li et al. (1996), is leaf-specific. The Atmyb5 promoter is expressed in developing leaf trichomes, stipules, and epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. A leaf promoter identified in maize by Busk et al. (1997), can also be used. In some instances, for example when LEC2 or BBM is recombinantly expressed, it may be desirable that the transgene is not expressed at high levels. An example of a promoter which can be used in such circumstances is a truncated napin A promoter which retains the seed-specific expression pattern but with a reduced expression level (Tan et al., 2011). The 5′ non-translated leader sequence can be derived from the promoter selected to express the heterologous gene sequence of the polynucleotide of the present invention, or may be heterologous with respect to the coding region of the enzyme to be produced, and can be specifically modified if desired so as to increase translation of mRNA. For a review of optimizing expression of transgenes, see Koziel et al. (1996). The 5′ non-translated regions can also be obtained from plant viral RNAs (Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus, Alfalfa mosaic virus, among others) from suitable eukaryotic genes, plant genes (wheat and maize chlorophyll a/b binding protein gene leader), or from a synthetic gene sequence. The present invention is not limited to constructs wherein the non-translated region is derived from the 5′ non-translated sequence that accompanies the promoter sequence. The leader sequence could also be derived from an unrelated promoter or coding sequence. Leader sequences useful in context of the present invention comprise the maize Hsp70 leader (U.S. Pat. Nos. 5,362,865 and 5,859,347), and the TMV omega element. The termination of transcription is accomplished by a 3′ non-translated DNA sequence operably linked in the expression vector to the polynucleotide of interest. The 3′ non-translated region of a recombinant DNA molecule contains a polyadenylation signal that functions in plants to cause the addition of adenylate nucleotides to the 3′ end of the RNA. The 3′ non-translated region can be obtained from various genes that are expressed in plant cells. The nopaline synthase 3′ untranslated region, the 3′ untranslated region from pea small subunit Rubisco gene, the 3′ untranslated region from soybean 7S seed storage protein gene are commonly used in this capacity. The 3′ transcribed, non-translated regions containing the polyadenylate signal ofAgrobacteriumtumor-inducing (Ti) plasmid genes are also suitable. Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide by manipulating, for example, the efficiency with which the resultant transcripts are translated by codon optimisation according to the host cell species or the deletion of sequences that destabilize transcripts, and the efficiency of post-translational modifications. Transfer Nucleic Acids Transfer nucleic acids can be used to deliver an exogenous polynucleotide to a cell and comprise one, preferably two, border sequences and one or more polynucleotides of interest. The transfer nucleic acid may or may not encode a selectable marker. Preferably, the transfer nucleic acid forms part of a binary vector in a bacterium, where the binary vector further comprises elements which allow replication of the vector in the bacterium, selection, or maintenance of bacterial cells containing the binary vector. Upon transfer to a eukaryotic cell, the transfer nucleic acid component of the binary vector is capable of integration into the genome of the eukaryotic cell or, for transient expression experiments, merely of expression in the cell. As used herein, the term “extrachromosomal transfer nucleic acid” refers to a nucleic acid molecule that is capable of being transferred from a bacterium such asAgrobacteriumsp., to a plant cell such as a plant leaf cell. An extrachromosomal transfer nucleic acid is a genetic element that is well-known as an element capable of being transferred, with the subsequent integration of a nucleotide sequence contained within its borders into the genome of the recipient cell. In this respect, a transfer nucleic acid is flanked, typically, by two “border” sequences, although in some instances a single border at one end can be used and the second end of the transferred nucleic acid is generated randomly in the transfer process. A polynucleotide of interest is typically positioned between the left border-like sequence and the right border-like sequence of a transfer nucleic acid. The polynucleotide contained within the transfer nucleic acid may be operably linked to a variety of different promoter and terminator regulatory elements that facilitate its expression, that is, transcription and/or translation of the polynucleotide. Transfer DNAs (T-DNAs) fromAgrobacteriumsp. such asAgrobacterium tumefaciensorAgrobacterium rhizogenes, and man made variants/mutants thereof are probably the best characterized examples of transfer nucleic acids. Another example is P-DNA (“plant-DNA”) which comprises T-DNA border-like sequences from plants. As used herein, “T-DNA” refers to a T-DNA of anAgrobacterium tumefaciensTi plasmid or from anAgrobacterium rhizogenesRi plasmid, or variants thereof which function for transfer of DNA into plant cells. The T-DNA may comprise an entire T-DNA including both right and left border sequences, but need only comprise the minimal sequences required in cis for transfer, that is, the right T-DNA border sequence. The T-DNAs of the invention have inserted into them, anywhere between the right and left border sequences (if present), the polynucleotide of interest. The sequences encoding factors required in trans for transfer of the T-DNA into a plant cell such as vir genes, may be inserted into the T-DNA, or may be present on the same replicon as the T-DNA, or preferably are in trans on a compatible replicon in theAgrobacteriumhost. Such “binary vector systems” are well known in the art. As used herein, “P-DNA” refers to a transfer nucleic acid isolated from a plant genome, or man made variants/mutants thereof, and comprises at each end, or at only one end, a T-DNA border-like sequence. As used herein, a “border” sequence of a transfer nucleic acid can be isolated from a selected organism such as a plant or bacterium, or be a man made variant/mutant thereof. The border sequence promotes and facilitates the transfer of the polynucleotide to which it is linked and may facilitate its integration in the recipient cell genome. In an embodiment, a border-sequence is between 10-80 bp in length. Border sequences from T-DNA fromAgrobacteriumsp. are well known in the art and include those described in Lacroix et al. (2008). Whilst traditionally onlyAgrobacteriumsp. have been used to transfer genes to plants cells, there are now a large number of systems which have been identified/developed which act in a similar manner toAgrobacteriumsp. Several non-Agrobacteriumspecies have recently been genetically modified to be competent for gene transfer (Chung et al., 2006; Broothaerts et al., 2005). These includeRhizobiumsp. NGR234,Sinorhizobium melilotiandMezorhizobium loti. As used herein, the terms “transfection”, “transformation” and variations thereof are generally used interchangeably. “Transfected” or “transformed” cells may have been manipulated to introduce the polynucleotide(s) of interest, or may be progeny cells derived therefrom. Plants The invention also provides a plant or part thereof comprising two or more exogenous polynucleotides and/or genetic modifications as described herein. The term “plant” when used as a noun refers to whole plants, whilst the term “part thereof” refers to plant organs (e.g., leaves, stems, roots, flowers, fruit), single cells (e.g., pollen), seed, seed parts such as an embryo, endosperm, scutellum or seed coat, plant tissue such as vascular tissue, plant cells and progeny of the same. As used herein, plant parts comprise plant cells. As used herein, the terms “in a plant” and “in the plant” in the context of a modification to the plant means that the modification has occurred in at least one part of the plant, including where the modification has occurred throughout the plant, and does not exclude where the modification occurs in only one or more but not all parts of the plant. For example, a tissue-specific promoter is said to be expressed “in a plant”, even though it might be expressed only in certain parts of the plant. Analogously, “a transcription factor polypeptide that increases the expression of one or more glycolytic and/or fatty acid biosynthetic genes in the plant” means that the increased expression occurs in at least a part of the plant. As used herein, the term “plant” is used in it broadest sense, including any organism in the Kingdom Plantae. It also includes red and brown algae as well as green algae. It includes, but is not limited to, any species of flowering plant, grass, crop or cereal (e.g., oilseed, maize, soybean), fodder or forage, fruit or vegetable plant, herb plant, woody plant or tree. It is not meant to limit a plant to any particular structure. It also refers to a unicellular plant (e.g., microalga). The term “part thereof” in reference to a plant refers to a plant cell and progeny of same, a plurality of plant cells, a structure that is present at any stage of a plant's development, or a plant tissue. Such structures include, but are not limited to, leaves, stems, flowers, fruits, nuts, roots, seed, seed coat, embryos. The term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in leaves, stems, flowers, fruits, nuts, roots, seed, for example, embryonic tissue, endosperm, dermal tissue (e.g., epidermis, periderm), vascular tissue (e.g., xylem, phloem), or ground tissue (comprising parenchyma, collenchyma, and/or sclerenchyma cells), as well as cells in culture (e.g., single cells, protoplasts, callus, embryos, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture. As used herein, the term “vegetative tissue” or “vegetative plant part” is any plant tissue, organ or part other than organs for sexual reproduction of plants. The organs for sexual reproduction of plants are specifically seed bearing organs, flowers, pollen, fruits and seeds. Vegetative tissues and parts include at least plant leaves, stems (including bolts and tillers but excluding the heads), tubers and roots, but excludes flowers, pollen, seed including the seed coat, embryo and endosperm, fruit including mesocarp tissue, seed-bearing pods and seed-bearing heads. In one embodiment, the vegetative part of the plant is an aerial plant part. In another or further embodiment, the vegetative plant part is a green part such as a leaf or stem. A “transgenic plant” or variations thereof refers to a plant that contains a transgene not found in a wild-type plant of the same species, variety or cultivar. Transgenic plants as defined in the context of the present invention include plants and their progeny which have been genetically modified using recombinant techniques to cause production of at least one polypeptide defined herein in the desired plant or part thereof. Transgenic plant parts has a corresponding meaning. The plant and plant parts of the invention may comprise genetic modifications, for example gene mutations, and be considered as “non-transgenic” provided they lack transgenes. The terms “seed” and “grain” are used interchangeably herein. “Grain” refers to mature grain such as harvested grain or grain which is still on a plant but ready for harvesting, but can also refer to grain after imbibition or germination, according to the context. Mature grain commonly has a moisture content of less than about 18%. In a preferrd embodiment, the moisture content of the grain is at a level which is generally regarded as safe for storage, preferably between 5% and 15%, between 6% and 8%, between 8% and 10%, or between 10% and 15%. “Developing seed” as used herein refers to a seed prior to maturity, typically found in the reproductive structures of the plant after fertilisation or anthesis, but can also refer to such seeds prior to maturity which are isolated from a plant. Mature seed commonly has a moisture content of less than about 12%. As used herein, the term “plant storage organ” refers to a part of a plant specialized to store energy in the form of for example, proteins, carbohydrates, lipid. Examples of plant storage organs are seed, fruit, tuberous roots, and tubers. A preferred plant storage organ of the invention is seed. As used herein, the term “phenotypically normal” refers to a genetically modified plant or part thereof, for example a plant such as a tragsenic plant, or a storage organ such as a seed, tuber or fruit of the invention not having a significantly reduced ability to grow and reproduce when compared to an unmodified plant or part thereof. Preferably, the biomass, growth rate, germination rate, storage organ size, seed size and/or the number of viable seeds produced is not less than 90% of that of a plant lacking said genetic modifications or exogenous polynucleotides when grown under identical conditions. This term does not encompass features of the plant which may be different to the wild-type plant but which do not effect the usefulness of the plant for commercial purposes such as, for example, a ballerina phenotype of seedling leaves. In an embodiment, the genetically modified plant or part thereof which is phenotypically normal comprises a recombinant polynucleotide encoding a silencing suppressor operably linked to a plant storage organ specific promoter and has an ability to grow or reproduce which is essentially the same as a corresponding plant or part thereof not comprising said polynucleotide. Plants go through a series of growing stages from sowing of a seed, germination and emergence of a seedling, through to flowering, seed setting, physiological maturity and ultimately senescence. These stages are well known and readily defined, for example forSorghumplants as follows. Taking the day the seedling first emerges above the soil as day 0, the vegetative stage of growth is defined herein as from 10 days to initiation of flowering at about 60-70 days, and physiogical maturity is reached at about 100 days, depending on the environmental conditions. The vegetative stage includes the boot leaf stage from about 45 days until the first flowering. The boot leaf is the last leaf formed on the plant, from which the panicle (head) emerges. The “boot leaf stage” is defined as from when the boot leaf has fully emerged to initiation of flowering. As used herein, the term “commencement of flowering” or “initiation of flowering” with respect to a plant refers to the time that the first flower on the plant opens, or the time of onset of anthesis. As used herein, the term “seed set” with respect to a seed-bearing plant refers to the time when the first seed of the plant reaches maturity. This is typically observable by the colour of the seed or its moisture content, well known in the art. As used herein, the term “mature” as it relates to a plant leaf means that it has reached full size but has not begun to show signs of ageing or death such as yellowing and/or sensensce. The skilled person can readily determine whether a leaf of a particular plant can be considered as mature. As used herein, the term “senescence” with respect to a whole plant refers to the final stage of plant development which follows the completion of growth, usually after the plant reaches maximum aerial biomass or height. Senescence begins when the plant aerial biomass reaches its maximum and begins to decline in amount and generally ends with death of most of the plant tissues. It is during this stage that the plant mobilises and recycles cellular components from leaves and other parts which accumulated during growth to other parts to complete its reproductive development. Senescence is a complex, regulated process which involves new or increased gene expression of some genes. Often, some plant parts are senescing while other parts of the same plant continue to grow. Therefore, with respect to a plant leaf or other green organ, the term “senescence” as used herein refers to the time when the amount of chlorophyll in the leaf or organ begins to decrease. Senescence is typically associated with dessication of the leaf or organ, mostly in the last stage of senescence. Senescence is usually observable by the change in colour of the leaf from green towards yellow and eventually to brown when fully dessicated. It is believed that cellular senescence underlies plant and organ senescence. Plants provided by or contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. In preferred embodiments, the plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, rice,sorghum, millet, cassava, barley) or legumes such as soybean, beans or peas. The plants may be grown for production of edible roots, tubers, leaves, stems, flowers or fruit. The plants may be vegetable plants whose vegetative parts are used as food. The plants of the invention may be:Acrocomia aculeata(macauba palm),Arabidopsis thaliana, Aracinis hypogaea(peanut),Astrocaryum murumuru(murumuru),Astrocaryum vulgare(tucumã),Attalea geraensis(Indaiá-rateiro),Attalea humilis(American oil palm),Attalea oleifera(andaiá),Attalea phalerata(uricuri),Attalea speciosa(babassu),Avena sativa(oats),Beta vulgaris(sugar beet),Brassicasp. such asBrassica carinata, Brassica juncea, Brassica napobrassica, Brassica napus(canola),Camelina sativa(false flax),Cannabis sativa(hemp),Carthamus tinctorius(safflower),Caryocar brasiliense(pequi),Cocos nucifera(Coconut),Crambe abyssinica(Abyssinian kale),Cucumis melo(melon),Elaeis guineensis(African palm),Glycine max(soybean),Gossypium hirsutum(cotton),Helianthussp. such asHelianthus annuus(sunflower),Hordeum vulgare(barley),Jatropha curcas(physic nut),Joannesia princeps(arara nut-tree),Lemnasp. (duckweed) such asLemna aequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba(swollen duckweed),Lemna japonica, Lemna minor, Lemna minuta, Lemna obscura, Lemna paucicostata, Lemna perpusilla, Lemna tenera, Lemna trisulca, Lemna turionifera, Lemna valdiviana, Lemna yungensis, Licania rigida(oiticica),Linum usitatissimum(flax),Lupinus angustifolius(lupin),Mauritia flexuosa(buriti palm),Maximiliana maripa(inaja palm),Miscanthussp. such asMiscanthusxgiganteusandMiscanthus sinensis, Nicotianasp. (tabacco) such asNicotiana tabacumorNicotiana benthamiana, Oenocarpus bacaba(bacaba-do-azeite),Oenocarpus bataua(patauã),Oenocarpus distichus(bacaba-de-leque),Oryzasp. (rice) such asOryza sativaandOryza glaberrima, Panicum virgatum(switchgrass),Paraqueiba paraensis(mari),Persea amencana(avocado),Pongamia pinnata(Indian beech),Populus trichocarpa, Ricinus communis(castor),Saccharumsp. (sugarcane),Sesamum indicum(sesame),Solanum tuberosum(potato),Sorghumsp. such asSorghum bicolor, Sorghum vulgare, Theobroma grandiforum(cupuassu),Trifoliumsp.,Trithrinax brasiliensis(Brazilian needle palm),Triticumsp. (wheat) such asTriticum aestivum, Zea mays(corn), alfalfa (Medicago sativa), rye (Secale cerale), sweet potato (Lopmoea batatus), cassava (Manihot esculenta), coffee (Cofeaspp.), pineapple (Anana comosus), citris tree (Citrusspp.), cocoa (Theobroma cacao), tea (Camelliasenensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia(Macadamia intergrifolia) and almond (Prunus amygdalus). In an embodiment, the plant is not aNicotianasp. Other preferred plants include C4 grasses such as, in addition to those mentioned above,Andropogon gerardi, Bouteloua curtipendula, B. gracilis, Buchloe dactyloides, Schizachyrium scoparium, Sorghastrum nutans, Sporobolus cryptandrus;C3 grasses such as Elymuscanadensis, the legumesLespedeza capitataandPetalostemum villosum, the forbAster azureus; and woody plants such asQuercus ellipsoidalisandQ. macrocarpa. Other preferred plants include C3 grasses. In a preferred embodiment, the plant is an angiosperm. In an embodiment, the plant is an oilseed plant, preferably an oilseed crop plant. As used herein, an “oilseed plant” is a plant species used for the commercial production of lipid from the seeds of the plant. The oilseed plant may be, for example, oil-seed rape (such as canola), maize, sunflower, safflower, soybean,sorghum, flax (linseed) or sugar beet. Furthermore, the oilseed plant may be other Brassicas, cotton, peanut, poppy, rutabaga, mustard, castor bean, sesame, safflower,Jatropha curcasor nut producing plants. The plant may produce high levels of lipid in its fruit such as olive, oil palm or coconut. Horticultural plants to which the present invention may be applied are lettuce, endive, or vegetable Brassicas including cabbage, broccoli, or cauliflower. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, tomato, or pepper. In a preferred embodiment, the plant is a member of the family Fabaceae (or Leguminosae) such as alfalfa, clover, peas, lucerne, beans, lentils, lupins, mesquite, carob, soybeans, and peanuts, or a member of the family Poaceae such as corn,sorghum, wheat, barley and oats. In a particularly preferred embodiment, the plant is alfalfa, clover, corn orsorghum, each of which are particularly useful for forage or fodder for animals. In a preferred embodiment, the transgenic plant is homozygous for each and every gene that has been introduced (transgene) so that its progeny do not segregate for the desired phenotype. The transgenic plant may also be heterozygous for the introduced transgene(s), preferably uniformly heterozygous for the transgene such as for example, in F1 progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art. Transformation of Plants Transgenic plants can be produced using techniques known in the art, such as those generally described in Slater et al., Plant Biotechnology—The Genetic Manipulation of Plants, Oxford University Press (2003), and Christou and Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004). As used herein, the terms “stably transforming”, “stably transformed” and variations thereof refer to the integration of the polynucleotide into the genome of the cell such that they are transferred to progeny cells during cell division without the need for positively selecting for their presence. Stable transformants, or progeny thereof, can be identified by any means known in the art such as Southern blots on chromosomal DNA, or in situ hybridization of genomic DNA, enablimg their selection. Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because DNA can be introduced into cells in whole plant tissues, plant organs, or explants in tissue culture, for either transient expression, or for stable integration of the DNA in the plant cell genome. For example, floral-dip (in planta) methods may be used. The use ofAgrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. The region of DNA to be transferred is defined by the border sequences, and the intervening DNA (T-DNA) is usually inserted into the plant genome. It is the method of choice because of the facile and defined nature of the gene transfer. Acceleration methods that may be used include for example, microprojectile bombardment and the like. One example of a method for delivering transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method has been reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) that may be coated with nucleic acids and delivered into cells, for example of immature embryos, by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. In another method, plastids can be stably transformed. Methods disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (U.S. Pat. Nos. 5,451,513, 5,545,818, 5,877,402, 5,932,479, and WO 99/05265). Other methods of cell transformation can also be used and include but are not limited to the introduction of DNA into plants by direct DNA transfer into pollen, by direct injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos. The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., In: Methods for Plant Molecular Biology, Academic Press, San Diego, Calif., (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. The development or regeneration of plants containing the foreign, exogenous gene is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polynucleotide is cultivated using methods well known to one skilled in the art. To confirm the presence of the transgenes in transgenic cells and plants, a polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Northern blot hybridisation, Western blot and enzyme assay. Once transgenic plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant parts, may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics. Preferably, the vegetative plant parts are harvested at a time when the yield of non-polar lipids are at their highest. In one embodiment, the vegetative plant parts are harvested about at the time of flowering, or after flowering has initiated. Preferably, the plant parts are harvested at about the time senescence begins, usually indicated by yellowing and drying of leaves. Transgenic plants formed usingAgrobacteriumor other transformation methods typically contain a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene(s). More preferred is a transgenic plant that is homozygous for the added gene(s), that is, a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by self-fertilising a hemizygous transgenic plant, germinating some of the seed produced and analyzing the resulting plants for the gene of interest. It is also to be understood that two different transgenic plants that contain two independently segregating exogenous genes or loci can also be crossed (mated) to produce offspring that contain both sets of genes or loci. Selfing of appropriate F1 progeny can produce plants that are homozygous for both of the exogenous genes or loci. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Similarly, a transgenic plant can be crossed with a second plant comprising a genetic modification such as a mutant gene and progeny containing both of the transgene and the genetic modification identified. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in Fehr, In: Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987). Tilling In one embodiment, TILLING (Targeting Induced Local Lesions IN Genomes) can be used to produce plants in which endogenous genes comprise a mutation, for example genes encoding an SDP1 or TGD polypeptide, TST, a plastidial GPAT, plastidial LPAAT, phosphatidic acid phosphatase (PAP), or a combination of two or more thereof. In a first step, introduced mutations such as novel single base pair changes are induced in a population of plants by treating seeds (or pollen) with a chemical mutagen, and then advancing plants to a generation where mutations will be stably inherited. DNA is extracted, and seeds are stored from all members of the population to create a resource that can be accessed repeatedly over time. For a TILLING assay, heteroduplex methods using specific endonucleases can be used to detect single nucleotide polymorphisms (SNPs). Alternatively, Next Generation Sequencing of DNA from pools of mutagenised plants can be used to identify mutants in the gene of choice. Typically, a mutation frequency of one mutant per 1000 plants in the mutagenised population is achieved. Using this approach, many thousands of plants can be screened to identify any individual with a single base change as well as small insertions or deletions (1-30 bp) in any gene or specific region of the genome. TILLING is further described in Slade and Knauf (2005), and Henikoff et al. (2004). In addition to allowing efficient detection of mutations, high-throughput TILLING technology is ideal for the detection of natural polymorphisms. Therefore, interrogating an unknown homologous DNA by heteroduplexing to a known sequence reveals the number and position of polymorphic sites. Both nucleotide changes and small insertions and deletions are identified, including at least some repeat number polymorphisms. This has been called Ecotilling (Comai et al., 2004). Genome Editing Using Site-Specific Nucleases Genome editing uses engineered nucleases such as RNA guided DNA endonucleases or nucleases composed of sequence specific DNA binding domains fused to a non-specific DNA cleavage module. These engineered nucleases enable efficient and precise genetic modifications by inducing targeted DNA double stranded breaks that stimulate the cell's endogenous cellular DNA repair mechanisms to repair the induced break. Such mechanisms include, for example, error prone non-homologous end joining (NHEJ) and homology directed repair (HDR). In the presence of donor plasmid with extended homology arms, HDR can lead to the introduction of single or multiple transgenes to correct or replace existing genes. In the absence of donor plasmid, NHEJ-mediated repair yields small insertion or deletion mutations of the target that cause gene disruption. Engineered nucleases useful in the methods of the present invention include zinc finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALEN) and CRISPR/Cas9 type nucleases, and related nucleases. Typically nuclease encoded genes are delivered into cells by plasmid DNA, viral vectors or in vitro transcribed mRNA. A zinc finger nuclease (ZFN) comprises a DNA-binding domain and a DNA-cleavage domain, wherein the DNA binding domain is comprised of at least one zinc finger and is operatively linked to a DNA-cleavage domain. The zinc finger DNA-binding domain is at the N-terminus of the protein and the DNA-cleavage domain is located at the C-terminus of said protein. A ZFN must have at least one zinc finger. In a preferred embodiment, a ZFN would have at least three zinc fingers in order to have sufficient specificity to be useful for targeted genetic recombination in a host cell or organism. Typically, a ZFN having more than three zinc fingers would have progressively greater specificity with each additional zinc finger. The zinc finger domain can be derived from any class or type of zinc finger. In a particular embodiment, the zinc finger domain comprises the Cis2His2type of zinc finger that is very generally represented, for example, by the zinc finger transcription factors TFIIIA or Sp1. In a preferred embodiment, the zinc finger domain comprises three Cis2His2type zinc fingers. The DNA recognition and/or the binding specificity of a ZFN can be altered in order to accomplish targeted genetic recombination at any chosen site in cellular DNA. Such modification can be accomplished using known molecular biology and/or chemical synthesis techniques. (see, for example, Bibikova et al., 2002). The ZFN DNA-cleavage domain is derived from a class of non-specific DNA cleavage domains, for example the DNA-cleavage domain of a Type II restriction enzyme such as FokI (Kim et al., 1996). Other useful endonucleases may include, for example, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AhwI. A transcription activator-like (TAL) effector nuclease (TALEN) comprises a TAL effector DNA binding domain and an endonuclease domain. TAL effectors are proteins of plant pathogenic bacteria that are injected by the pathogen into the plant cell, where they travel to the nucleus and function as transcription factors to turn on specific plant genes. The primary amino acid sequence of a TAL effector dictates the nucleotide sequence to which it binds. Thus, target sites can be predicted for TAL effectors, and TAL effectors can be engineered and generated for the purpose of binding to particular nucleotide sequences. Fused to the TAL effector-encoding nucleic acid sequences are sequences encoding a nuclease or a portion of a nuclease, typically a nonspecific cleavage domain from a type II restriction endonuclease such as FokI (Kim et al., 1996). Other useful endonucleases may include, for example, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AhwI. The fact that some endonucleases (e.g., FokI) only function as dimers can be capitalized upon to enhance the target specificity of the TAL effector. For example, in some cases each FokI monomer can be fused to a TAL effector sequence that recognizes a different DNA target sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme. By requiring DNA binding to activate the nuclease, a highly site-specific restriction enzyme can be created. A sequence-specific TALEN can recognize a particular sequence within a preselected target nucleotide sequence present in a cell. Thus, in some embodiments, a target nucleotide sequence can be scanned for nuclease recognition sites, and a particular nuclease can be selected based on the target sequence. In other cases, a TALEN can be engineered to target a particular cellular sequence. Genome Editing Using Programmable RNA Guided DNA Endonucleases Distinct from the site-specific nucleases described above, the clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas system provides an alternative to ZFNs and TALENs for inducing targeted genetic alterations, via RNA-guided DNA cleavage. CRISPR systems rely on CRISPR RNA (crRNA) and transactivating chimeric RNA (tracrRNA) for sequence-specific cleavage of DNA. Three types of CRISPR/Cas systems exist: in type II systems, Cas9 serves as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA target recognition. CRISPR RNA base pairs with tracrRNA to form a two-RNA structure that guides the Cas9 endonuclease to complementary DNA sites for cleavage. The CRISPR system can be portable to plant cells by co-delivery of plasmids expressing the Cas endonuclease and the necessary crRNA components. The Cas endonuclease may be converted into a nickase to provide additional control over the mechanism of DNA repair (Cong et al., 2013). CRISPRs are typically short partially palindromic sequences of 24-40 bp containing inner and terminal inverted repeats of up to 11 bp. Although isolated elements have been detected, they are generally arranged in clusters (up to about 20 or more per genome) of repeated units spaced by unique intervening 20-58 bp sequences. CRISPRs are generally homogenous within a given genome with most of them being identical. However, there are examples of heterogeneity in, for example, the Archaea (Mojica et al., 2000). Feedstuffs The present invention includes compositions which can be used as feedstuffs. For purposes of the present invention, “feedstuffs” include any food or preparation for animal (including human) consumption and which serves to nourish or build up tissues or supply energy, and/or to maintain, restore or support adequate nutritional status or metabolic function. Feedstuffs of the invention include nutritional compositions for babies and/or young children. As used herein, the term “animal” refers to any eukaryotic organism capable of ingesting plant derived material. In an embodiment, the animal is a ruminant animal (cattle, sheep, goats etc). Alternatively, the animal is a non-ruminant animal. In one embodiment, the animal is a mammal. In an embodiment, the animal is a human. In an embodiment, the animal is a livestock animal such, but not limited to, as cattle, goats, sheep, pigs, horses, poultry such as chickens and the like. In an embodiment, the cattle are diary cattle or beef cattle. In another embodiment, the animal is a fish, for instance fish bred using aquaculture including, but not limited to, salmon, trout, carp, bass, bream, turbot, sole, milkfish, grey mullet, grouper, flounder, sea bass, cod, haddock, Japanese flounder, catfish, char, whitefish, sturgeon, tench, roach, pike, pike-perch, yellowtail, tilapia, eel or tropical fish (such as the fresh, brackish, and salt water tropical fish). The animal may be a crustacean such as, but not limited to, krill, clams, shrimp (including prawns), crab, and lobster. Feedstuffs of the invention may comprise for example, a plant or part thereof such as a vegetative plant part of the invention along with a suitable carrier(s). The term “carrier” is used in its broadest sense to encompass any component which may or may not have nutritional value. As the person skilled in the art will appreciate, the carrier must be suitable for use (or used in a sufficiently low concentration) in a feedstuff, such that it does not have deleterious effect on an organism which consumes the feedstuff. Feedstuffs may comprise plant parts which have been harvested and subsequently processed or treated, for example, by chopping, cutting, drying, pressing or pelleting the plant parts, into a form that is suitable for consumption by the animal, or altered by processes such as drying or fermentation to produce hay or silage. The feedstuff of the present invention comprises a lipid and/or protein produced directly or indirectly by use of the methods, plants or parts thereof disclosed herein. The composition may either be in a solid or liquid form. Additionally, the composition may include edible macronutrients, vitamins, and/or minerals in amounts desired for a particular use. The amounts of these ingredients will vary depending on whether the composition is intended for use with normal individuals or for use with individuals having specialized needs such as individuals suffering from metabolic disorders and the like. Examples of suitable carriers with nutritional value include, but are not limited to, macronutrients such as edible fats, carbohydrates and proteins. Examples of such edible fats include, but are not limited to, coconut oil, borage oil, fungal oil, black current oil, soy oil, and mono- and di-glycerides. Examples of such carbohydrates include, but are not limited to, glucose, edible lactose, and hydrolyzed starch. Additionally, examples of proteins which may be utilized in the nutritional composition of the invention include, but are not limited to, soy proteins, electrodialysed whey, electrodialysed skim milk, milk whey, or the hydrolysates of these proteins. With respect to vitamins and minerals, the following may be added to the feedstuff compositions of the present invention, calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, C, and the B complex. Other such vitamins and minerals may also be added. A feedstuff composition of the present invention may also be added to food even when supplementation of the diet is not required. For example, the composition may be added to food of any type, including, but not limited to, margarine, butter, cheeses, milk, yogurt, chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats, fish and beverages. Additionally, material produced in accordance with the present invention may also be used as animal food supplements to alter an animal's tissue or milk fatty acid composition to one more desirable for human or animal consumption, or to reduce methane production in ruminant animals. Furthermore, feedstuffs of the invention can be used in aquaculture to increase the levels of fatty acids and nutrition in fish for human or animal consumption. Preferred feedstuffs of the invention are the plants, seed and other plant parts such as leaves, fruits and stems which may be used directly as food or feed for humans or other animals. For example, animals may graze directly on such plants grown in the field, or be fed more measured amounts in controlled feeding. The invention includes the use of such plants and plant parts as feed for increasing the polyunsaturated fatty acid levels in humans and other animals. For consumption by non-human animals the feedstuff may be in any suitable form for such as, but not limited to, silage, hay or pasture growing in a field. In an embodiment, the feedstuff for non-human consumption is a leguminous plant, or part thereof, which is a member of the family Fabaceae family (or Leguminosae) such as alfalfa, clover, peas, lucerne, beans, lentils, lupins, mesquite, carob, soybeans, and peanuts. In embodiment, the animal is in a feedlot and/or a shed. In an embodiment, the plant or fraction thereof comprises at least about 5%, at least about 10%, at least about 50%, at least about 75%, at least about 90% or all of the feedstuff. Silage As used herein, “silage” is a relatively high-moisture fodder which has been produced and stored in a process called ensilage and which is typically fed to cattle, sheep or other ruminants. During the storage time, carbohydrates, lipids and proteins in the plant material ferment, producing organic acids, or are broken down oxidatively, or both. The plant material upon harvest and the post-fermentation plant materials are both included in silage as the term is used herein. Silage is typically made from grass crops such as maize,sorghum, oats or other cereals, or from mixed pasture grasses and legumes such as alfalfa or clover, using the green, above-ground parts of the plants. Silage is made either by placing cut vegetation (usually the whole above-ground plant biomass which can include reproductive tissues) in a pit or silo or other means for storage, and compressing it down so as to leave as little air as possible with the plant material. Oxygen is excluded to some extent by covering it with a plastic sheet or by wrapping the plant material tightly within plastic film (baling) to reduce air inflow. Silage is made from plant material with a suitable moisture content, generally about 50% to 60% of the fresh weight, depending on the means of storage and the degree of compression used and the amount of water that will be lost in storage, but not exceeding 75%. Forsorghumand corn, harvest begins when the whole-plant moisture is at a suitable level, ideally a few days before it is ripe. For pasture-type crops, the plants are mowed and allowed to wilt for a day or so until the moisture content drops to a suitable level. Ideally the crop is mowed when in full flower and deposited in the pit or silo on the day of its cutting. At harvesting, or after, the plant material is shredded or chopped by the harvester into pieces typically about 1-5 cm long. The plant material may be placed in large heaps on the ground and compressed to reduce the amount of air, then covered with plastic, or into a silo. Alternatively, the plant material may be baled in plastic wrapping to exclude air, which typically requires a lower moisture content of about 30-40%, but still too damp to be stored as dry hay. The cut or chopped, stored plant material undergoes mostly anaerobic fermentation, which starts about 48 hours after the pit or silo is filled. The fermentation process converts sugars and other carbohydrates such as hemicellulose to organic acids, mostly acetic, propionic, lactic and butyric acids. Fermentation starts after the trapped oxygen is consumed and is essentially complete after about two weeks of storage, or may continue for longer periods. When the plant material is closely packed, the supply of oxygen is limited and the fermentation results in the decomposition of the carbohydrates, some lipids and proteins in the material into the organic acids. This product is named sour silage. If, on the other hand, the fodder is more loosely packed, the main reaction is oxidation which proceeds more rapidly and the temperature rises. If the mass is compressed when the temperature is 60-75 C, the reaction ceases and sweet silage results. Fermentation may be aided by inoculation with specific microorganisms such as lactic acid bacteria to speed fermentation or improve the resulting silage, e.g. withLactobacillus plantarum. Bulk silage is commonly fed to dairy cattle, while baled silage tends to be used for beef cattle, sheep and horses. The advantages of silage as animal feed are several. During fermentation, the silage bacteria act on the cellulose and other carbohydrates in the forage to produce the organic fatty acids, thereby lowering the pH. This inhibits competing bacteria that might cause spoilage and the organic acids thereby act as natural preservatives, improve digestibility and palatability. This preservative action is particularly important during winter in temperate regions, when green forage is unavailable. Silage can be produced using techniques known in the art such as those described in CN 101940272 CN 103461658 CN 101946853, CN 101946853, CN 104381743, U.S. Pat. Nos. 3,875,304 and 6,224,916. Pellets for animal feed can be produced using techniques known in the art such as those described in U.S. Pat. Nos. 3,035,920, 3,573,924 and 5,871,802. Plant Biomass An increase in the total lipid content of plant biomass equates to greater energy content, making its use as a feed or forage or in the production of biofuel more economical. The main components of naturally occurring plant biomass are carbohydrates (approximately 75%, dry weight) and lignin (approximately 25%), which can vary with plant type. The carbohydrates are mainly cellulose or hemicellulose fibers, which impart strength to the plant structure, and lignin, which holds the fibers together. Plant biomass typically has a low energy density as a result of both its physical form and moisture content. This also makes it inconvenient and inefficient for storage and transport without some kind of pre-processing. There are a range of processes available to convert it into a more convenient form including: 1) physical pre-processing (for example, grinding) or 2) conversion by thermal (for example, combustion, gasification, pyrolysis) or chemical (for example, anaerobic digestion, fermentation, composting, transesterification) processes. In this way, the biomass is converted into what can be described as a biomass fuel. Combustion Combustion is the process by which flammable materials are allowed to burn in the presence of air or oxygen with the release of heat. The basic process is oxidation. Combustion is the simplest method by which biomass can be used for energy, and has been used to provide heat. This heat can itself be used in a number of ways: 1) space heating, 2) water (or other fluid) heating for central or district heating or process heat, 3) steam raising for electricity generation or motive force. When the flammable fuel material is a form of biomass the oxidation is of predominantly the carbon (C) and hydrogen (H) in the cellulose, hemicellulose, lignin, and other molecules present to form carbon dioxide (CO2) and water (H2O). The plants of the invention provide improved fuel for combustion by virtue of the increased lipid content. Gasification Gasification is a partial oxidation process whereby a carbon source such as plant biomass, is broken down into carbon monoxide (CO) and hydrogen (142), plus carbon dioxide (CO2) and possibly hydrocarbon molecules such as methane (CH4). If the gasification takes place at a relatively low temperature, such as 700° C. to 1000° C., the product gas will have a relatively high level of hydrocarbons compared to high temperature gasification. As a result it may be used directly, to be burned for heat or electricity generation via a steam turbine or, with suitable gas clean up, to run an internal combustion engine for electricity generation. The combustion chamber for a simple boiler may be close coupled with the gasifier, or the producer gas may be cleaned of longer chain hydrocarbons (tars), transported, stored and burned remotely. A gasification system may be closely integrated with a combined cycle gas turbine for electricity generation (IGCC—integrated gasification combined cycle). Higher temperature gasification (1200° C. to 1600° C.) leads to few hydrocarbons in the product gas, and a higher proportion of CO and H2. This is known as synthesis gas (syngas or biosyngas) as it can be used to synthesize longer chain hydrocarbons using techniques such as Fischer-Tropsch (FT) synthesis. If the ratio of H2to CO is correct (2:1) FT synthesis can be used to convert syngas into high quality synthetic diesel biofuel which is compatible with conventional fossil diesel and diesel engines. Pyrolysis As used herein, the term “pyrolysis” means a process that uses slow heating in the absence of oxygen to produce gaseous, oil and char products from biomass. Pyrolysis is a thermal or thermo-chemical conversion of lipid-based, particularly triglyceride-based, materials. The products of pyrolysis include gas, liquid and a sold char, with the proportions of each depending upon the parameters of the process. Lower temperatures (around 400° C.) tend to produce more solid char (slow pyrolysis), whereas somewhat higher temperatures (around 500° C.) produce a much higher proportion of liquid (bio-oil), provided the vapour residence time is kept down to around is or less. Temperatures of about 275° C. to about 375° C. can be used to produce liquid bio-oil having a higher proportion of longer chain hydrocarbons. Pyrolysis involves direct thermal cracking of the lipids or a combination of thermal and catalytic cracking. At temperatures of about 400-500° C., cracking occurs, producing short chain hydrocarbons such as alkanes, alkenes, alkadienes, aromatics, olefins and carboxylic acid, as well as carbon monoxide and carbon dioxide. Four main catalyst types can be used including transition metal catalysts, molecular sieve type catalysts, activated alumina and sodium carbonate (Maher et al., 2007). Examples are given in U.S. Pat. No. 4,102,938. Alumina (Al2O3) activated by acid is an effective catalyst (U.S. Pat. No. 5,233,109). Molecular sieve catalysts are porous, highly crystalline structures that exhibit size selectivity, so that molecules of only certain sizes can pass through. These include zeolite catalysts such as ZSM-5 or HZSM-5 which are crystalline materials comprising A104and SiO4and other silica-alumina catalysts. The activity and selectivity of these catalysts depends on the acidity, pore size and pore shape, and typically operate at 300-500° C. Transition metal catalysts are described for example in U.S. Pat. No. 4,992,605. Sodium carbonate catalyst has been used in the pyrolysis of oils (Dandik and Aksoy, 1998). As used herein, “hydrothermal processing”, “HTP”, also referred to as “thermal depolymerisation” is a form of pyrolysis which reacts the plant-derived matter, specifically the carbon-containing material in the plant-derived matter, with hydrogen to produce a bio-oil product comprised predominantly of paraffinic hydrocarbons along with other gases and solids. A significant advantage of HTP is that the vegetative plant material does not need to be dried before forming the composition for the conversion reaction, although the vegetative plant material can be dried beforehand to aid in transport or storage of the biomass. The biomass can be used directly as harvested from the field. The reactor is any vessel which can withstand the high temperature and pressure used and is resistant to corrosion. The solvent used in the HTP includes water or is entirely water, or may include some hydrocarbon compounds in the form of an oil. Generally, the solvent in HTP lacks added alcohols. The conversion reaction may occur in an oxidative, reductive or inert environment. “Oxidative” as used herein means in the presence of air, “reductive” means in the presence of a reducing agent, typically hydrogen gas or methane, for example 10-15% H2with the remainder of the gas being N2, and “inert” means in the presence of an inert gas such as nitrogen or argon. The conversion reaction is preferably carried out under reductive conditions. The carbon-containing materials that are converted include cellulose, hemi-cellulose, lignin and proteins as well as lipids. The process uses a conversion temperature of between 270° C. and 400° C. and a pressure of between 70 and 350 bar, typically 300° C. to 350° C. and a pressure between 100-170 bar. As a result of the process, organic vapours, pyrolysis gases and charcoal are produced. The organic vapours are condensed to produce the bio-oil. Recovery of the bio-oil may be achieved by cooling the reactor and reducing the pressure to atmospheric pressure, which allows bio-oil (organic) and water phases to develop and the bio-oil to be removed from the reactor. The yield of the recovered bio-oil is calculated as a percentage of the dry weight of the input biomass on a dry weight basis. It is calculated according to the formula: weight of bio-oil×100/dry weight of the vegetative plant parts. The weight of the bio-oil does not include the weight of any water or solids which may be present in a bio-oil mixture, which are readily removed by filtration or other known methods. The bio-oil may then be separated into fractions by fractional distillation, with or without additional refining processes. Typically, the fractions that condense at these temperatures are termed: about 370° C., fuel oil; about 300° C., diesel oil; about 200° C., kerosene; about 150° C., gasoline (petrol). Heavier fractions may be cracked into lighter, more desirable fractions, well known in the art. Diesel fuel typically is comprised of C13-C22 hydrocarbon compounds. Transesterification “Transesterification” as used herein is the conversion of lipids, principally triacylglycerols, into fatty acid methyl esters or ethyl esters by reaction with short chain alcohols such as methanol or ethanol, in the presence of a catalyst such as alkali or acid. Methanol is used more commonly due to low cost and availability, but ethanol, propanol or butanol or mixtures of the alcohols can also be used. The catalysts may be homogeneous catalysts, heterogeneous catalysts or enzymatic catalysts. Homogeneous catalysts include ferric sulphate followed by KOH. Heterogeneous catalysts include CaO, K3PO4, and WO3/ZrO2. Enzymatic catalysts include Novozyme 435 produced fromCandida antarctica. Transesterification can be carried out on extracted oil, or preferably directly in situ in the vegetative plant material. The vegetative plant parts may be dried and milled prior to being used to prepare the composition for the conversion reaction, but does not need to be. The advantage of direct conversion to fatty acid esters, preferably FAME, is that the conversion can use lower temperatures and pressures and still provide good yields of the product, for example, comprising at least 50% FAME by weight. The yield of recovered bio-oil by transesterification is calculated as for the HTP process. Production of Non-Polar Lipids Techniques that are routinely practiced in the art can be used to extract, process, purify and analyze the lipids such as the TAG produced by plants or parts thereof of the instant invention. Such techniques are described and explained throughout the literature in sources such as, Fereidoon Shahidi, Current Protocols in Food Analytical Chemistry, John Wiley & Sons, Inc. (2001) D1.1.1-D1.1.11, and Perez-Vich et al. (1998). Production of Oil from Vegetative Plant Parts or Seed Typically, vegetative plant parts or plant seeds are cooked, pressed, and/or extracted to produce crude vegetative oil or seedoil, which is then degummed, refined, bleached, and deodorized. Generally, techniques for crushing seed are known in the art. For example, oilseeds can be tempered by spraying them with water to raise the moisture content to, for example, 8.5%, and flaked using a smooth roller with a gap setting of 0.23 to 0.27 mm. Depending on the type of seed, water may not be added prior to crushing. Application of heat deactivates enzymes, facilitates further cell rupturing, coalesces the lipid droplets, and agglomerates protein particles, all of which facilitate the extraction process. Vegetative plant parts can be similarly treated, depending on the moisture content. In an embodiment, the majority of the vegetative oil or seedoil is released by passage through a screw press. Cakes (vegetative plant meal, seedmeal) expelled from the screw press may then be solvent extracted for example, with hexane, using a heat traced column, or not be solvent treated, in which case it may be more suitable as animal feed. Alternatively, crude vegetative oil or seedoil produced by the pressing operation can be passed through a settling tank with a slotted wire drainage top to remove the solids that are expressed with the vegetative oil or seedoil during the pressing operation. The clarified vegetative oil or seedoil can be passed through a plate and frame filter to remove any remaining fine solid particles. Once the solvent is stripped from the crude oil, the pressed and extracted portions are combined and subjected to normal lipid processing procedures (i.e., degumming, caustic refining, bleaching, and deodorization). Extraction of the lipid from vegetative plant parts of the invention uses analogous methods to those known in the art for seedoil extraction. One way is physical extraction, which often does not use solvent extraction. Expeller pressed extraction is a common type, as are the screw press and ram press extraction methods. Mechanical extraction is typically less efficient than solvent extraction where an organic solvent (e.g., hexane) is mixed with at least the plant biomass, preferably after the biomass is dried and ground. The solvent dissolves the lipid in the biomass, which solution is then separated from the biomass by mechanical action (e.g., with the pressing processes above). This separation step can also be performed by filtration (e.g., with a filter press or similar device) or centrifugation etc. The organic solvent can then be separated from the non-polar lipid (e.g., by distillation). This second separation step yields non-polar lipid from the plant and can yield a re-usable solvent if one employs conventional vapor recovery. In an embodiment, the oil and/or protein content of the plant part or seed is analysed by near-infrared reflectance spectroscopy as described in Hom et al. (2007) prior to extraction. If the vegetative plant parts are not to be used immediately to extract the lipid it is preferably processed to ensure the lipid content is retained as much as possible (see, for example, Christie, 1993), such as by drying the vegetative plant parts. Degumming Degumming is an early step in the refining of oils and its primary purpose is the removal of most of the phospholipids from the oil, which may be present as approximately 1-2% of the total extracted lipid. Addition of ˜2% of water, typically containing phosphoric acid, at 70-80° C. to the crude oil results in the separation of most of the phospholipids accompanied by trace metals and pigments. The insoluble material that is removed is mainly a mixture of phospholipids and triacylglycerols and is also known as lecithin. Degumming can be performed by addition of concentrated phosphoric acid to the crude oil to convert non-hydratable phosphatides to a hydratable form, and to chelate minor metals that are present. Gum is separated from the oil by centrifugation. The oil can be refined by addition of a sufficient amount of a sodium hydroxide solution to titrate all of the fatty acids and removing the soaps thus formed. Alkali Refining Alkali refining is one of the refining processes for treating crude oil, sometimes also referred to as neutralization. It usually follows degumming and precedes bleaching. Following degumming, the oil can treated by the addition of a sufficient amount of an alkali solution to titrate all of the fatty acids and phosphoric acids, and removing the soaps thus formed. Suitable alkaline materials include sodium hydroxide, potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide, calcium carbonate and ammonium hydroxide. This process is typically carried out at room temperature and removes the free fatty acid fraction. Soap is removed by centrifugation or by extraction into a solvent for the soap, and the neutralised oil is washed with water. If required, any excess alkali in the oil may be neutralized with a suitable acid such as hydrochloric acid or sulphuric acid. Bleaching Bleaching is a refining process in which oils are heated at 90-120° C. for 10-30 minutes in the presence of a bleaching earth (0.2-2.0%) and in the absence of oxygen by operating with nitrogen or steam or in a vacuum. This step in oil processing is designed to remove unwanted pigments (carotenoids, chlorophyll, gossypol etc), and the process also removes oxidation products, trace metals, sulphur compounds and traces of soap. Deodorization Deodorization is a treatment of oils and fats at a high temperature (200-260° C.) and low pressure (0.1-1 mm Hg). This is typically achieved by introducing steam into the oil at a rate of about 0.1 ml/minute/100 ml of oil. Deodorization can be performed by heating the oil to 260° C. under vacuum, and slowly introducing steam into the oil at a rate of about 0.1 ml/minute/100 ml of oil. After about 30 minutes of sparging, the oil is allowed to cool under vacuum. The oil is typically transferred to a glass container and flushed with argon before being stored under refrigeration. If the amount of oil is limited, the oil can be placed under vacuum for example, in a Parr reactor and heated to 260° C. for the same length of time that it would have been deodorized. This treatment improves the colour of the oil and removes a majority of the volatile substances or odorous compounds including any remaining free fatty acids, monoacylglycerols and oxidation products. Winterisation Winterization is a process sometimes used in commercial production of oils for the separation of oils and fats into solid (stearin) and liquid (olein) fractions by crystallization at sub-ambient temperatures. It was applied originally to cottonseed oil to produce a solid-free product. It is typically used to decrease the saturated fatty acid content of oils. Algae Algae can produce 10 to 100 times as much mass as terrestrial plants in a year and can be cultured in open-ponds (such as raceway-type ponds and lakes) or in photobioreactors. The most common oil-producing algae can generally include the diatoms (bacillariophytes), green algae (chlorophytes), blue-green algae (cyanophytes), and golden-brown algae (chrysophytes). In addition a fifth group known as haptophytes may be used. Groups include brown algae and heterokonts. Specific non-limiting examples algae include the Classes: Chlorophyceae, Eustigmatophyceae, Prymnesiophyceae, Bacillariophyceae. Bacillariophytes capable of oil production include the genera Amphipleura, Amphora, Chaetoceros, Cyclotella, Cymbella, Fragilaria, Hantzschia, Navicula,Nitzschia, Phaeodactylum, andThalassiosira. Specific non-limiting examples of chlorophytes capable of oil production include Ankistrodesmus,Botryococcus, Chlorella, Chlorococcum, Dunaliella, Monoraphidium, Oocystis,Scenedesmus, andTetraselmis. In one aspect, the chlorophytes can beChlorellaorDunaliella. Specific non-limiting examples of cyanophytes capable of oil production include Oscillatoria and Synechococcus. A specific example of chrysophytes capable of oil production includes Boekelovia. Specific non-limiting examples of haptophytes include Isochysis and Pleurochysis. Specific algae useful in the present invention include, for example,Chlamydomonassp. such asChlamydomonas reinhardtii, Dunaliellasp. such asDunaliella salina, Dunaliella tertiolecta, D. acidophila, D. Lateralis. D. martima. D. parva, D. polmorpha, D. primolecta, D. pseudosalina, D. quartolecta. D. viridis, Haematococcussp.,Chlorellasp. such asChlorella vulgaris, Chlorella sorokinianaorChlorellaprotothecoides, Thraustochytrium sp., Schizochytrium sp., Volvox sp,Nannochloropsissp.,Botryococcus brauniiwhich can contain over 60 wt % lipid,Phaeodactylum tricornutum, Thalassiosira pseudonana, Isochrysissp.,Pavlovasp., Chlorococcum sp, Elhpsoidion sp.,Neochlorissp.,Scenedesmussp. Algae of the invention can be harvested using microscreens, by centrifugation, by flocculation (using for example, chitosan, alum and ferric chloride) and by froth flotation. Interrupting the carbon dioxide supply can cause algae to flocculate on its own, which is called “autoflocculation”. In froth flotation, the cultivator aerates the water into a froth, and then skims the algae from the top. Ultrasound and other harvesting methods are currently under development. Lipid may be extracted from the algae by mechanical crushing. When algal mass is dried it retains its lipid content, which can then be “pressed” out with an oil press. Osmotic shock may also be used to release cellular components such as lipid from algae, and ultrasonic extraction can accelerate extraction processes. Chemical solvents (for example, hexane, benzene, petroleum ether) are often used in the extraction of lipids from algae. Enzymatic extraction using enzymes to degrade the cell walls may also be used to extract lipids from algae. Supercritical CO2can also be used as a solvent. In this method, CO2is liquefied under pressure and heated to the point that it becomes supercritical (having properties of both a liquid and a gas), allowing it to act as a solvent. Uses of Plant Lipids The lipids produced by the methods described have a variety of uses. In some embodiments, the lipids are used as food oils. In other embodiments, the lipids are refined and used as lubricants or for other industrial uses such as the synthesis of plastics. In some preferred embodiments, the lipids are refined to produce biodiesel. Biodiesel can be made from oils derived from the plants, algae and fungi of the invention. Use of plant triacylglycerols for the production of biofuel is reviewed in Durrett et al. (2008). The resulting fuel is commonly referred to as biodiesel and has a dynamic viscosity range from 1.9 to 6.0 mm2s−1(ASTM D6751). Bioalcohol may produced from the fermentation of sugars or the biomass other than the lipid left over after lipid extraction. General methods for the production of biofuel can be found in, for example, Maher and Bressler (2007), Greenwell et al. (2010), Karmakar et al. (2010), Alonso et al. (2010), Liu et al. (2010a). Gong and Jiang (2011), Endalew et al. (2011) and Semwal et al. (2011). The present invention provides methods for increasing oil content in vegetative tissues. Plants of the present invention have increased energy content of leaves and/or stems such that the whole above-ground plant parts may be harvested and used to produce biofuel. Furthermore, the level of oleic acid is increased significantly while the polyunsaturated fatty acid alpha linolenic acid (ALA) was reduced. The plants, algae and fungi of the present invention thereby reduce the production costs of biofuel. Biodiesel The production of biodiesel, or alkyl esters, is well known. There are three basic routes to ester production from lipids: 1) Base catalysed transesterification of the lipid with alcohol; 2) Direct acid catalysed esterification of the lipid with methanol; and 3) Conversion of the lipid to fatty acids, and then to alkyl esters with acid catalysis. Any method for preparing fatty acid alkyl esters and glyceryl ethers (in which one, two or three of the hydroxy groups on glycerol are etherified) can be used. For example, fatty acids can be prepared, for example, by hydrolyzing or saponifying TAG with acid or base catalysts, respectively, or using an enzyme such as a lipase or an esterase. Fatty acid alkyl esters can be prepared by reacting a fatty acid with an alcohol in the presence of an acid catalyst. Fatty acid alkyl esters can also be prepared by reacting TAG with an alcohol in the presence of an acid or base catalyst. Glycerol ethers can be prepared, for example, by reacting glycerol with an alkyl halide in the presence of base, or with an olefin or alcohol in the presence of an acid catalyst. The alkyl esters can be directly blended with diesel fuel, or washed with water or other aqueous solutions to remove various impurities, including the catalysts, before blending. Aviation Fuel For improved performance of biofuels, thermal and catalytic chemical bond-breaking (cracking) technologies have been developed that enable converting bio-oils into bio-based alternatives to petroleum-derived diesel fuel and other fuels, such as jet fuel. The use of medium chain fatty acid source, such produced by a cell of the invention, a plant or part thereof of the invention, a seed of of the invention, or a transgenic version of any one thereof, precludes the need for high-energy fatty acid chain cracking to achieve the shorter molecules needed for jet fuels and other fuels with low-temperature flow requirements. This method comprises cleaving one or more medium chain fatty acid groups from the glycerides to form glycerol and one or more free fatty acids. In addition, the method comprises separating the one or more medium chain fatty acids from the glycerol, and decarboxylating the one or more medium chain fatty acids to form one or more hydrocarbons for the production of the jet fuel. Compositions The present invention also encompasses compositions, particularly pharmaceutical compositions, comprising one or more plants, plant parts, lipids, proteins, nitrogen containing molecules, or carbon containing molecules, produced using the methods of the invention. A pharmaceutical composition may additionally comprise an active ingredient and a standard, well-known, non-toxic pharmaceutically-acceptable carrier, adjuvant or vehicle such as phosphate-buffered saline, water, ethanol, polyols, vegetable oils, a wetting agent, or an emulsion such as a water/oil emulsion. The composition may be in either a liquid or solid form. For example, the composition may be in the form of a tablet, capsule, ingestible liquid, powder, topical ointment or cream. Proper fluidity can be maintained for example, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. It may also be desirable to include isotonic agents for example, sugars, sodium chloride, and the like. Besides such inert diluents, the composition can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfuming agents. A typical dosage of a particular fatty acid is from 0.1 mg to 20 g, taken from one to five times per day (up to 100 g daily) and is preferably in the range of from about 10 mg to about 1, 2, 5, or 10 g daily (taken in one or multiple doses). As known in the art, a minimum of about 300 mg/day of fatty acid, especially polyunsaturated fatty acid, is desirable. However, it will be appreciated that any amount of fatty acid will be beneficial to the subject. Possible routes of administration of the pharmaceutical compositions of the present invention include for example, enteral and parenteral. For example, a liquid preparation may be administered orally. Additionally, a homogenous mixture can be completely dispersed in water, admixed under sterile conditions with physiologically acceptable diluents, preservatives, buffers or propellants to form a spray or inhalant. The dosage of the composition to be administered to the subject may be determined by one of ordinary skill in the art and depends upon various factors such as weight, age, overall health, past history, immune status, etc., of the subject. Additionally, the compositions of the present invention may be utilized for cosmetic purposes. The compositions may be added to pre-existing cosmetic compositions, such that a mixture is formed, or a fatty acid produced according to the invention may be used as the sole “active” ingredient in a cosmetic composition. Polypeptides The terms “polypeptide” and “protein” are generally used interchangeably herein. A polypeptide or class of polypeptides may be defined by the extent of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by having a greater % identity to one reference amino acid sequence than to another. The % identity of a polypeptide to a reference amino acid sequence is typically determined by GAP analysis (Needleman and Wunsch, 1970; GCG program) with parameters of a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns two sequences over their entire length, and the extent of identity is determined over the full length of the reference sequence. The polypeptide or class of polypeptides may have the same enzymatic activity as, or a different activity than, or lack the activity of, the reference polypeptide. Preferably, the polypeptide has an enzymatic activity of at least 10% of the activity of the reference polypeptide. As used herein a “biologically active fragment” is a portion of a polypeptide of the invention which maintains a defined activity of a full-length reference polypeptide for example, DGAT activity. Biologically active fragments as used herein exclude the full-length polypeptide. Biologically active fragments can be any size portion as long as they maintain the defined activity. Preferably, the biologically active fragment maintains at least 10% of the activity of the full length polypeptide. With regard to a defined polypeptide or enzyme, it will be appreciated that % identity figures higher than those provided herein will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide/enzyme comprises an amino acid sequence which is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO. Amino acid sequence mutants of the polypeptides defined herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein, or by in vitro synthesis of the desired polypeptide. Such mutants include for example, deletions, insertions, or substitutions of residues within the amino acid sequence. A combination of deletions, insertions and substitutions can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics. Mutant (altered) polypeptides can be prepared using any technique known in the art, for example, using directed evolution or rathional design strategies (see below). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they possess transcription factor, fatty acid acyltransferase or OBC activities. In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series for example, by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site. Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues. Substitution mutants have at least one amino acid residue in the polypeptide removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis to inactivate enzymes include sites identified as the active site(s). Other sites of interest are those in which particular residues obtained from various strains or species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of “exemplary substitutions”. TABLE 1Exemplary substitutions.OriginalExemplaryResidueSubstitutionsAla (A)val; leu; ile; glyArg (R)lysAsn (N)gln; hisAsp (D)gluCys (C)serGln (Q)asn; hisGlu (E)aspGly (G)pro, alaHis (H)asn; glnIle (I)leu; val; alaLeu (L)ile; val; met; ala; pheLys (K)argMet (M)leu; phePhe (F)leu; val; alaPro (P)glySer (S)thrThr (T)serTrp (W)tyrTyr (Y)trp; pheVal (V)ile; leu; met; phe, ala In a preferred embodiment a mutant/variant polypeptide has only, or not more than, one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in Table 1. As the skilled person would be aware, such minor changes can reasonably be predicted not to alter the activity of the polypeptide when expressed in a transgenic plant or part thereof. Mutants with desired activity may be engineered using standard procedures in the art such as by performing random mutagenesis, targeted mutagenesis, or saturation mutagenesis on known genes of interest, or by subjecting different genes to DNA shuffling. EXAMPLES Example 1. General Materials and Methods Expression of Genes in Plant Cells in a Transient Expression System Genes were expressed in plant cells using a transient expression system essentially as described by Voinnet et al. (2003) and Wood et al. (2009). Binary vectors containing the coding region to be expressed by a strong constitutive e35S promoter containing a duplicated enhancer region were introduced intoAgrobacterium tumefaciensstrain AGL1. A chimeric binary vector, 35S:p19, for expression of the p19 viral silencing suppressor was separately introduced into AGL1, as described in WO2010/057246. A chimeric binary vector, 35S:V2, for expression of the V2 viral silencing suppressor was separately introduced into AGL1. The recombinant cells were grown to stationary phase at 28° C. in LB broth supplemented with 50 mg/L kanamycin and 50 mg/L rifampicin. The bacteria were then pelleted by centrifugation at 5000 g for 5 min at room temperature before being resuspended to OD600=1.0 in an infiltration buffer containing 10 mM MES pH 5.7, 10 mM MgCl2and 100 uM acetosyringone. The cells were then incubated at 28° C. with shaking for 3 hours after which the OD600 was measured and a volume of each culture, including the viral suppressor construct 35S:p19 or 35S:V2, required to reach a final concentration of OD600=0.125 added to a fresh tube. The final volume was made up with the above buffer. Leaves were then infiltrated with the culture mixture and the plants were typically grown for a further three to five days after infiltration before leaf discs were recovered for either purified cell lysate preparation or total lipid isolation. Transformation ofSorghum bicolorL. Plant Material Sorghumplants of the inbred cultivar TX-430 (Miller, 1984) were grown in a plant growth chamber (Conviron, PGC-20 flex) at 28±1° C. “day” temperature and 20±1° C. “night” temperature, with a 16 hr photoperiod at a light intensity during the “day” of 900-1000 LUX. Panicles were covered with white translucent paper bags before flowering. Immature embryos were harvested from panicles 12-15 days after anthesis. Panicles were washed several times with water and developing seeds that were uniform in size were isolated and surface-sterilized using 20% commercial bleach mixed with 0.1% Tween-20 for 15-20 min. They were then washed with sterile distilled water 3 times each for 20 min, and blotted dry in a laminar flow hood. Immature embryos (IEs) ranging from 1.4 to 2.5 mm in length were aseptically isolated in the laminar flow hood and used as the starting tissue for preparation of green regenerative tissue. Base Cultivation Media Media used for plant transformation were based on MS (Murashige and Skoog, 1962), supplied by PhytoTechnology Laboratories (M519). The pH of the media was adjusted to 5.8 before sterilization at 121° C. for 15 min. Heat sensitive plant growth regulators and other additives such as Geneticin (G418, Sigma) used as a selection agent, were filter sterilized (0.2 μm) and added to the media after sterilization when the media had cooled to about 55° C. The optimized culture medium composition for the different stages of plant transformation from callus induction to plant regeneration from green tissue induced from immature embryos is presented in Table 2. Cultivation Methods and Materials The isolated IEs ranging from 1.4 to 2.5 mm in length were placed onto callus induction media-osmotic medium (CIM-osmotic medium, Table 2) with their scutellum facing upward. The CIM base medium was modified to improve callus quality and induction frequency from immature embryos, as well as callus regeneration media, by including α-Lipoic acid (1 to 5 mg/1), Melatonin (5 to 10 mg/1) and 2-Aminoidan-2-phosphonic acid HCl (1 to 2 mg/1) unless otherwise stated. For the development of green tissue, immature embryos were incubated under fluorescent light of approximately 45-50 μmol s−1m−2(16 h/day) in a tissue culture room at 24±2° C. After three days of culture, the root and shoot poles of the immature embryos were aseptically separated and re-inoculated on to the same CIM and maintained under the same conditions as described above. They were subcultured every two weeks onto the same CIM for 6 weeks and evaluated for callus quality, callus induction efficiency and transformation efficiency. Callus initiated from IEs in the first 3-4 weeks on CIM were mostly embryogenic and slowly differentiated into embryogenic callus with nodular structures which were coloured from pale to darker green. Embryogenic calli with green nodular structures were selected and maintained on the same medium (CIM) by subculturing every 2 weeks for up to 6 months or more, for use as explants for transformation. This type of tissue is termed herein as “differentiating embryogenic callus” tissue or “DEC” tissue, since this tissue forms nodular structures of differentiating cells which maintain embryogenic and organogenic potential, even though the tissues were really a mixture of callus cells, cells forming nodular structures and granular structures, and intermediate cells which the inventors understood were on the developmental pathway somewhere between callus (which is undifferentiated cells) and the nodular structures. Sometimes, the tissues included early stage (globular) somatic embryos. TABLE 2Media used in DEC tissue induction and transformation of sorghumName of themediumCompositionCulture durationCIM-MS medium powder with vitamins, 4.33 g/l; 2,4-D, 13-4 hrs beforeOsmoticmg/l; BAP, 0.5 mg/l; L-proline, 0.7 g/l; L-Lipoicbombardment;Mediumacid, 1 mg/l; peptone, 0.82 g/l; Myo-inositol, 150o/n postmg/l; Copper sulfate, 0.8 mg/l; Manitol, 36.4 g/l;bombardmentSorbitol, 36.4 g/l; Agar, 8.5 g/l, pH 5.8CIM- preMS medium powder with vitamins, 4.33 g/l; 2,4-D, 13-4daysselectionmg/l; BAP, 0.5 mg/l; L-proline, 0.7 g/l; L-Lipoicmediumacid, 1 mg/l; peptone, 0.82 g/l; Myo-inosito, l 150mg/l; Copper sulfate, 0.8 mg/l; Maltose, 30 g/l; L-cysteine, 50 mg/l; Ascorbic acid, 15 mg/l; Agar, 9g/l, pH 5.8CIM-callusMS medium powder with vitamins, 4.33 g/l; 2,4-D, 14weeksinductionmg/l; BAP, 0.5 mg/l; L-proline, 0.7 g/l; L-Lipoicmedium/G25acid, 1 mg/l; peptone, 0.82 g/l; Myo-inositol, 150mg/l; Copper sulfate, 0.8 mg/l; Maltose, 30 g/l;Geneticin, 25 mg/l; Agar, 9 g/l, pH 5.8SIM-shootMS medium powder with vitamins, 4.33 g/l; BAP,2weeksinduction1.0 mg/l; 2,4-D, 0.5 mg/l; L-proline, 0.7 g/l; L-Lipoicmedium/G25acid, 1 mg/l; peptone, 0.82 g/l; Myo-inositol, 150mg/l; Copper sulfate, 0.8 mg/l; Maltose, 30 g/l;Geneticin, 25 mg/l; Agar, 9 g/l, pH 5.8SRM- shootMS medium powder with vitamins, 4.33 g/l; BAP,2weeksregeneration1.0 mg/l; TDZ, 0.5 mg/l; L-proline, 0.7 g/l; L-Lipoicmedium/G25acid, 1 mg/l; peptone, 0.82 g/l; Myo-inositol, 150mg/l; Copper sulfate, 0.8 mg/l; Maltose, 30 g/l;Geneticin, 25 mg/l; Agar, 9 g/l, pH 5.8SOG-shootMS medium powder with vitamins, 2.2 g/l; L-2weeksout growthproline, 0.7 g/l; L-Lipoic acid, 1 mg/l; peptone, 0.82medium/G30g/l; Myo-inositol, 150 mg/l; Copper sulfate, 0.8 mg/l;Sucrose, 15 g/l; Geneticin, 30 mg/l; Agar, 9 g/l, pH5.8RIM-rootMS medium powder with vitamins, 4.33 g/l; L-proline,4weeksinduction0.7 g/l; L-Lipoic acid, 1 mg/l; peptone, 0.82medium/G15g/l; Myo-inositol, 150 mg/l; Copper sulfate, 0.8 mg/l;sucrose, 15 g/l; IAA, 1 mg/l; IBA, 1 mg/l; NAA, 1mg/l; PVP, 2 g/l; Geneticin, 15 mg/l; Agar 9 g/l, pH5.8 Particle-Bombardment of Green Regenerative DEC Tissues Plasmids containing a selectable marker gene encoding the neomycin phosphotransferase II (NptII) providing resistance to the antibiotic Geneticin, under the control of the pUbi promoter and terminated by the nos 3′ region, were made or obtained for experiments to achieve stable transformation or for co-bombardment with other plasmids. Plasmid DNAs were isolated using a Zymopure™ Maxiprep kit (USA) according to the manufacturer's instructions. As a control vector for transformation, a genetic vector was obtained which contained uidA (GUS) and bar genes designed for expression in plant cells. The uidA gene was under the regulatory control of a maize polyubiquitin promoter (pUbi) and anAgrobacterium tumefaciensoctopine synthase polyadenylation/terminator (ocs 3′) sequence. The sequence between the promoter and the protein coding region included the 5′ UTR and first intron of the Ubi gene. The uidA reporter gene also contained, within its protein coding region, an intron from a castor bean catalase gene which prevented translation of functional GUS protein inAgrobacterium, thereby reducing the background GUS gene expression in inoculated plant tissues. Therefore, any GUS expression would be due to expression of the uidA gene in the plant cells. The bar gene was also under the regulatory control of a pUbi promoter and terminated with anAgrobacteriumnopaline synthase 3′ regulatory sequence (nos 3′). The uidA/bar vector was initially used in experiments to detect transient gene expression in thesorghumDEC tissues. Uniform healthy, green regenerative DEC tissues (4-5 mm in size), produced using methods described above and having been cultured for 6 weeks to 6 months from initiation, were used for microprojectile-mediated transformation (bombardment) with the plasmids. Approximately 15 uniform green DEC tissues (each 4-5 mm) were placed at the centre of a petri dish (90 mm diameter) containing CIM-osmotic medium (Table 2) and incubated in the dark for about 4 hrs prior to bombardment. Bombardment was performed with a PDS-1000 He device (Biorad, Hercules, Calif.) as described by Liu et al. (2014). Post bombardment, the tissues were kept on the same osmotic medium overnight and transferred to pre-selection medium the next morning Green DEC tissues bombarded with the genetic vector plasmid having a selectable marker encoding NptII were transferred to CIM-PS medium for 3-4 days before any selection, with addition to the medium of two compounds as antioxidants, L-cysteine (50 mg/1) and ascorbic acid (15 mg/1) (Table 2). Without the addition of these antioxidants in pre-selection medium, many of the bombarded tissues turned brown, some quite dark brown in colour, and many lost any ability to grow further. After 3-4 days on pre-selection medium, some of the bombarded tissues were subjected to GUS staining and viewed under a microscope to count the distinctive blue (GUS positive) spots, to check that genes had been transferred and could be expressed. The inclusion of the two antioxidants in the pre-selection medium improved the efficiency of the transformation as shown by the transient expression of the GUS gene. Selection and Regeneration of Transgenic Plants with Optimised Conditions Following bombardment and 3-4 days culture on pre-selection medium without selective agent (Geneticin), the bombarded tissues had increased in size from 4-5 mm to about 6-7 mm. These tissues were transferred to selective medium CIM/G25 containing 25 mg/l Geneticin (Table 2) and cultured for a further 4 weeks. When possible, the bombarded tissues were split into 2-6 pieces each, increasing the recovery of independent transformants. All of the tissues were cultured on the media as described in Table 2 and maintained in order to regenerate putative transgenic plants. Plants were regenerated efficiently upon growth on these media. Each bombarded tissue and the shoots obtained from it were subcultured and maintained separately for calculation of the transformation efficiency. Positive transformation was confirmed by PCR on plant genomic DNA isolated from shoot samples, showing the presence of the selectable marker gene. The number of transformants was calculated per input DEC tissue. Transformation efficiencies of about 50% were obtained, expressed as independent transformants per input bombarded tissue. Agrobacterium-Mediated Transformation of Green Regenerative DEC Tissues Uniform healthy, green regenerative DEC tissues (4-5 mm in size) produced using methods described in the foregoing examples and which have been cultured for 6 weeks to 6 months from initiation, are used forAgrobacterium-mediated transformation. Genetic vectors having T-DNA regions containing the genes for transformation were designed and made for transformation of green regenerative DEC tissues usingAgrobacterium-mediated transformation. A control binary vector contained uidA (GUS) and bar genes designed for expression in plant cells. The uidA gene was under the regulatory control of a maize polyubiquitin promoter (pUbi) and anAgrobacterium tumefaciensoctopine synthase polyadenylation/terminator (ocs 3′) sequence. The sequence between the promoter and the protein coding region included the 5′ UTR and first intron of the Ubi gene. The uidA reporter gene also contained, within its protein coding region, an intron from a castor bean catalase gene which prevented translation of functional GUS protein inAgrobacterium, thereby reducing the background GUS gene expression in inoculated plant tissues. Therefore, any GUS expression was due to expression of the uidA gene in the plant cells. The bar gene was also under the regulatory control of a pUbi promoter and terminated with anAgrobacteriumnopaline synthase 3′ regulatory sequence (nos 3′). A suitableAgrobacterium tumefaciensstrain was obtained e.g., AGL1 as described in Lazo et al. (1991) and the genetic vector is introduced into theAgrobacterium tumefaciensstrain by heat shock method. Agrobacteriumcultures harboring the genetic construct are grown in suitable medium e.g., LB medium, and under appropriate conditions to produce anAgrobacteriuminoculum, after which time the uniform healthy, green regenerative DEC tissues are infected withAgrobacteriuminoculum. The infected DEC tissues are blotted on sterile filter paper to remove excessAgrobacteriumand transferred to co-cultivation medium, optionally supplemented with antioxidants, and incubated in the dark at approximately 22-24° C. for 2-4 days. Following incubation, the DEC tissues are treated with an appropriate agent to kill theAgrobacterium, washed in sterile water, transferred to an appropriate medium and allowed to grow. After 4-6 weeks, shoots are excised and cultured on shoot elongation medium, after which time putative transgenic shoots are then detected using appropriate assays. Brassica napusTransformation Brassica napusseeds were sterilized using chlorine gas as described by Kereszt et al. (2007) and germinated on tissue culture medium. Cotyledonary petioles with 2-4 mm stalk were isolated as described by Belide et al. (2013) and used as explants.A. tumefaciensAGL1 (Lazo et al., 1991) cultures containing the binary vector were prepared and cotyledonary petioles inoculated with the cultures as described by Belide et al. (2013). Infected cotyledonary petioles were cultured on MS medium supplemented with 1 mg/L TDZ+0.1 mg/L NAA+3 mg/L AgNO3+250 mg/L cefotaxime, 50 mg/L timentin and 25 mg/L kanamycin and cultured for 4 weeks at 24° C. with 16 hr/8 hr light-dark photoperiod with a biweekly subculture on to the same medium. Explants with green callus were transferred to shoot initiation medium (MS+1 mg/L kinetin+3 mg/L AgNO3+250 mg/L cefotaxime+50 mg/L timentin+25 mg/L kanamycin) and cultured for another 2-3 weeks. Small shoots (˜1 cm) were isolated from the resistant callus and transferred to shoot elongation medium (MS medium with 0.1 mg/L gibberelic acid+3 mg/L AgNO3+250 mg/L cefotaxime+25 mg/L kanamycin) and cultured for another two weeks. Healthy shoots with one or two leaves were selected and transferred to rooting media (½ MS with 1 mg/L NAA+20 mg/L ADS+3 mg/L AgNO3+250 mg/L cefotaxime) and cultured for 2-3 weeks. DNA was isolated from small leaves of resistant shoots using the plant DNA isolation kit (Bioline, Alexandria, NSW, Australia) as described by the manufacturer's protocol. The presence of T-DNA sequences was tested by PCR amplification on genomic DNA. Positive, transgenic shoots with roots were transferred to pots containing seedling raising mix and grown in a glasshouse at 24° C. daytime/16° C. night-time (standard conditions). Purified Leaf Lysate—Enzyme Assays Nicotiana benthamianaleaf tissues previously infiltrated as described above were ground in a solution containing 0.1 M potassium phosphate buffer (pH 7.2) and 0.33 M sucrose using a glass homogenizer. Leaf homogenate was centrifuged at 20,000 g for 45 minutes at 4° C. after which each supernatant was collected. Protein content in each supernatant was measured according to Bradford (1976) using a Wallac1420 multi-label counter and a Bio-Rad Protein Assay dye reagent (Bio-Rad Laboratories, Hercules, Calif. USA). Acyltransferase assays used 100 μg protein according to Cao et al. (2007) with some modifications. The reaction medium contained 100 mM Tris-HCl (pH 7.0), 5 mM MgCl2, 1 mg/mL BSA (fatty acid-free), 200 mM sucrose, 40 mM cold oleoyl-CoA, 16.4 μM sn-2 monooleoylglycerol[14C] (55mCi/mmol, American Radiochemicals, Saint Louis, Mo. USA) or 6.0 μM [14C]glycerol-3-phosphate (G-3-P) disodium salt (150 mCi/mmol, American Radiochemicals). The assays were carried out for 7.5, 15, or 30 minutes. Lipid Analysis Analysis of Oil Content in Seeds When seed oil content or total fatty acid composition was to be determined in small seeds such asArabidopsisseeds, fatty acids in the seeds were directly methylated without crushing of seeds. Seeds were dried in a desiccator for 24 hours and approximately 4 mg of seed was transferred to a 2 ml glass vial containing a Teflon-lined screw cap. 0.05 mg triheptadecanoin (TAG with three C17:0 fatty acids) dissolved in 0.1 ml toluene was added to the vial as internal standard. Seed fatty acids were methylated by adding 0.7 ml of 1N methanolic HCl (Supelco) to the vial containing seed material. Crushing of the seeds was not necessary for complete methylation with small seeds such asArabidopsisseeds. The mixture was vortexed briefly and incubated at 80° C. for 2 hours. After cooling the mixtures to room temperature, 0.3 ml of 0.9% NaCl (w/v) and 0.1 ml hexane was added to the vial and mixed well for 10 minutes in a Heidolph Vibramax 110. The FAME were collected into a 0.3 ml glass insert and analysed by GC with a flame ionization detector (FID) as described below. The peak area of individual FAME were first corrected on the basis of the peak area responses of a known amount of the same FAMEs present in a commercial standard GLC-411 (NU-CHEK PREP, INC., USA). GLC-411 contains equal amounts of 31 fatty acids (% by weight), ranging from C8:0 to C22:6. In case of fatty acids which were not present in the standard, the peak area responses of the most similar FAME was taken. For example, the peak area response of FAMEs of 16:1d9 was used for 16:1d7 and the FAME response of C22:6 was used for C22:5. The corrected areas were used to calculate the mass of each FAME in the sample by comparison to the internal standard mass. Oil is stored mainly in the form of TAG and its weight was calculated based on FAME weight. Total moles of glycerol was determined by calculating moles of each FAME and dividing total moles of FAMEs by three. TAG content was calculated as the sum of glycerol and fatty acyl moieties using a relation: % oil by weight=100×((41×total mol FAME/3)+(total g FAME−(15×total mol FAME)))/g seed, where 41 and 15 are molecular weights of glycerol moiety and methyl group, respectively. Analysis of Fatty Acid Content in Larger Seeds To determine fatty acid composition in single seeds that were larger, such as canola andCamelinaseeds, orSorghumor corn seeds, direct methylation of fatty acids in the seed was performed as forArabidopsisseeds except with breaking of the seed coats. This method extracted sufficient oil from the seed to allow fatty acid composition analysis. To determine the fatty acid composition of total extracted lipid from seeds, seeds were crushed and lipids extracted with CHCl3/MeOH. Aliquots of the extracted lipid were methylated and analysed by GC. Pooled seed-total lipid content (seed oil content) of canola was determined by two extractions of lipid using CHCl3/MeOH from a known weight of desiccated seeds after crushing, followed by methylation of aliquots of the lipids together with the 17:0 fatty acids as internal standard. In the case of larger seeds such asCamelina, the lipid from a known amount of seeds was methylated together with known amount of 17:0 fatty acids as for theArabidopsisoil analysis and FAME were analysed by GC. For TAG quantitation, TAG was fractionated from the extracted lipid using TLC and directly methylated in silica using 17:0 TAG as an internal standard. These methods are described more fully as follows. After harvest at plant maturity, seeds were desiccated by storing the seeds for 24 hours at room temperature in a desiccator containing silica gel as desiccant. Moisture content of the seeds was typically 6-8%. Total lipids were extracted from known weights of the desiccated seeds by crushing the seeds using a mixture of chloroform and methanol (2/1 v/v) in an eppendorf tube using a Reicht tissue lyser (22 frequency/seconds for 3 minutes) and a metal ball. One volume of 0.1M KCl was added and the mixture shaken for 10 minutes. The lower non-polar phase was collected after centrifuging the mixture for 5 minutes at 3000 rpm. The remaining upper (aqueous) phase was washed with 2 volumes of chloroform by mixing for 10 minutes. The second non-polar phase was also collected and pooled with the first. The solvent was evaporated from the lipids in the extract under nitrogen flow and the total dried lipid was dissolved in a known volume of chloroform. To measure the amount of lipid in the extracted material, a known amount of 17:0-TAG was added as internal standard and the lipids from the known amount of seeds incubated in 1 N methanolic-HCl (Supelco) for 2 hours at 80° C. FAME thus made were extracted in hexane and analysed by GC. Individual FAME were quantified on the basis of the amount of 17:0 TAG-FAME. Individual FAME weights, after subtraction of weights of the esterified methyl groups from FAME, were converted into moles by dividing by molecular weights of individual FAME. Total moles of all FAME were divided by three to calculate moles of TAG and therefore glycerol. Then, moles of TAG were converted in to weight of TAG. Finally, the percentage oil content on a seed weight basis was calculated using seed weights, assuming that all of the extracted lipid was TAG or equivalent to TAG for the purpose of calculating oil content. This method was based on Li et al. (2006). Seeds other thanCamelinaor canola seeds that are of a similar size can also be analysed by this method. Canola and other seed oil content can be measured by nuclear magnetic resonance techniques (Rossell and Pritchard, 1991) by a pulsed wave NMS 100 Minispec (Bruker Pty Ltd Scientific Instruments, Germany). The NMR method can simultaneously measured moisture content. Seed oil content can also be measured by near infrared reflectance (NIR) spectroscopy such as using a NIRSystems Model 5000 monochromator. Moisture content can also be measured on a sample from a batch of seeds by drying the seeds in the sample for 18 hours at about 100° C., according to Li et al. (2006). Analysis of Lipids from Leaf Lysate Assays Lipids from the lysate assays were extracted using chloroform:methanol:0.1 M KCl (2:1:1) and recovered. The different lipid classes in the samples were separated on Silica gel 60 thin layer chromatography (TLC) plates (MERCK, Dermstadt, Germany) impregnated with 10% boric acid. The solvent system used to fractionate TAG from the lipid extract was chloroform/acetone (90/10 v/v). Individual lipid classes were visualized by exposing the plates to iodine vapour and identified by running parallel authentic standards on the same TLC plate. The plates were exposed to phosphor imaging screens overnight and analysed by a Fujifilm FLA-5000 phosphorimager before liquid scintillation counting for DPM quantification. Total Lipid Isolation and Fractionation of Lipids from Vegetative Tissues Fatty acid composition of total lipid in leaf and other vegetative tissue samples was determined by direct methylation of the fatty acids in freeze-dried samples. For total lipid quantitation, fatty acids in a known weight of freeze-dried samples, with 17:0 FFA, were directly methylated. To determine total TAG levels in leaf samples, TAG was fractionated by TLC from extracted total lipids, and methylated in the presence of 17:0 TAG internal standard, because of the presence of substantial amounts of polar lipids in leaves. This was done as follows. Tissues including leaf samples were freeze-dried, weighed (dry weight) and total lipids extracted as described by Bligh and Dyer (1959) or by using chloroform:methanol:0.1 M KCl (CMK; 2:1:1) as a solvent. Total lipids were extracted fromN. benthamianaleaf samples, after freeze dying, by adding 900 μL of a chloroform/methanol (2/1 v/v) mixture per 1 cm diameter leaf sample. 0.8 DAGE was added per 0.5 mg dry leaf weight as internal standard when TLC-FID analysis was to be performed. Samples were homogenized using an IKA ultra-turrax tissue lyser after which 500 μL 0.1 M KCl was added. Samples were vortexed, centrifuged for 5 min and the lower phase was collected. The remaining upper phase was extracted a second time by adding 600 μL chloroform, vortexing and centrifuging for 5 min. The lower phase was recovered and pooled into the previous collection. Lipids were dried under a nitrogen flow and resuspended in 2 μL chloroform per mg leaf dry weight. Total lipids ofN. tabacumleaves or leaf samples were extracted as above with some modifications. If 4 or 6 leaf discs (each approx 1 cm2surface area) were combined, 1.6 ml of CMK solvent was used, whereas if 3 or less leaf discs were combined, 1.2 ml CMK was used. Freeze dried leaf tissues were homogenized in an eppendorf tube containing a metallic ball using a Reicht tissue lyser (Qiagen) for 3 minutes at 20 frequency/sec. Separation of Neutral Lipids Via TLC and Transmethylation Known volumes of total leaf extracts such as, for example, 30 μL were loaded on a TLC silica gel 60 plate (1×20 cm) (Merck KGaA, Germany). The neutral lipids were fractionated into the different types and separated from polar lipids via TLC in an equilibrated development tank containing a hexane/DEE/acetic acid (70/30/1 v/v/v/) solvent system. The TAG bands were visualised by primuline spraying, marked under UV, scraped from the TLC plate, transferred to 2 mL GC vials and dried with N2. 750 μL of 1N methanolic-HCl (Supelco analytical, USA) was added to each vial together with a known amount of C17:0 TAG as an internal standard, depending on the amount of TAG in each sample. Typically, 30 μg of the internal standard was added for low TAG samples whilst up to 200 μg of internal standard was used in the case of high TAG samples. Lipid samples for fatty acid composition analysis by GC were transmethylated by incubating the mixtures at 80° C. for 2 hours in the presence of the methanolic-HCl. After cooling samples to room temperature, the reaction was stopped by adding 350 μl H2O. Fatty acyl methyl esters (FAME) were extracted from the mixture by adding 350 μl hexane, vortexing and centrifugation at 1700 rpm for 5 min. The upper hexane phase was collected and transferred into GC vials with 300 μl conical inserts. After evaporation, the samples were resuspended in 30 μl hexane. One μ1 was injected into the GC. The amount of individual and total fatty acids (TFA) present in the lipid fractions was quantified by GC by determining the area under each peak and calculated by comparison with the peak area for the known amount of internal standard. TAG content in leaf was calculated as the sum of glycerol and fatty acyl moieties in the TAG fraction using a relation: % TAG by weigh=100×((41×total mol FAME/3)+(total g FAME−(15×total mol FAME)))/g leaf dry weight, where 41 and 15 are molecular weights of glycerol moiety and methyl group, respectively. Capillary Gas-Liquid Chromatography (GC) FAME were analysed by GC using an Agilent Technologies 7890A GC (Palo Alto, Calif., USA) equipped with an SGE BPX70 (70% cyanopropyl polysilphenylene-siloxane) column (30 m×0.25 mm i.d., 0.25 μm film thickness), an FID, a split/splitless injector and an Agilent Technologies 7693 Series auto sampler and injector. Helium was used as the carrier gas. Samples were injected in split mode (50:1 ratio) at an oven temperature of 150° C. After injection, the oven temperature was held at 150° C. for 1 min, then raised to 210° C. at 3° C.min−1and finally to 240° C. at 50° C.min−1. Peaks were quantified with Agilent Technologies ChemStation software (Rev B.04.03 (16), Palo Alto, Calif., USA) based on the response of the known amount of the external standard GLC-411 (Nucheck) and C17:0-Me internal standard. Quantification of TAG Via Iatroscan One μL of lipid extract was loaded on one Chromarod-SII for TLC-FID Iatroscan™ (Mitsubishi Chemical Medience Corporation—Japan). The Chromarod rack was then transferred into an equilibrated developing tank containing 70 mL of a hexane/CHCl3/2-propanol/formic acid (85/10.716/0.567/0.0567 v/v/v/v) solvent system. After 30 min of incubation, the Chromarod rack was dried for 3 min at 100° C. and immediately scanned on an Iatroscan MK-6s TLC-FID analyser (Mitsubishi Chemical Medience Corporation—Japan). Peak areas of DAGE internal standard and TAG were integrated using SIC-48011 integration software (Version:7.0-E SIC System instruments Co., LTD—Japan). TAG quantification was carried out in two steps. First, DAGE was scanned in all samples to correct the extraction yields after which concentrated TAG samples were selected and diluted. Next, TAG was quantified in diluted samples with a second scan according to the external calibration using glyceryl trilinoleate as external standard (Sigma-Aldrich). Quantification of TAG in Leaf Samples by GC The peak area of individual FAME were first corrected on the basis of the peak area responses of known amounts of the same FAMEs present in a commercial standard GLC-411 (NU-CHEK PREP, Inc., USA). The corrected areas were used to calculate the mass of each FAME in the sample by comparison to the internal standard. Since oil is stored primarily in the form of TAG, the amount of oil was calculated based on the amount of FAME in each sample. Total moles of glycerol were determined by calculating the number of moles of FAMEs and dividing total moles of FAMEs by three. The amount of TAG was calculated as the sum of glycerol and fatty acyl moieties using the formula: % oil by weight=100×((41×total mol FAME/3)+(total g FAME−(15×total mol FAME)))/g leaf dry weight, where 41 and 15 were the molecular weights of glycerol moiety and methyl group, respectively. Soluble Protein Extraction and Quantitation Soluble protein was extracted from 10-20 mg ground fresh plant tissue. Briefly, chlorophyll and soluble sugars were extracted at 80° C. in 50-80% (v/v) ethanol in 2.5 mM HEPES buffer at pH 7.5 and the pellet was retained for soluble protein determination. The pellet was washed in distilled water, resuspended in 400 μl 0.1 M NaOH and heated at 95° C. for 30 min. The soluble protein in the supernatant was determined using a Bradford assay (Bradford, 1976). Soluble protein was also extracted from freshly ground tissue in buffer containing 100 mM Tris-HCl pH 8.0 and 10 mM MgCl2. Quantitation of the soluble protein by Bradford assay gave results similar to those obtained using the extraction with NaOH. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Total protein was extracted from frozen, ground leaf tissue by heating the samples in Laemmli buffer (1:3 w/v) at 95° C. for 10 min. Aliquots of the supernatant, normalised to fresh weight (FW), were separated on a 10% acrylamide gel according to Laemmli (1970). Leaf Nitrogen Content Total nitrogen content (% dry weight, DW) of 2-2.2 mg freeze-dried leaf tissue was determined using a Europa 20-20 isotope ratio mass spectrometer with an ANCA preparation system, comprising a combustion and reduction tube operating at 1000° C. and 600° C., respectively. Carbon and Energy Contents Carbon and energy contents were calculcated based on the amount of TAG, starch and total carbohydrates in wildtype and transgenic leaf tissues (% leaf dry weight). Starch levels (% leaf dry weight) were first converted to glucose equivalents by multiplying by a factor of 180/162 to take into account the loss of water due to chain linkages. Soluble sugars were defined as the difference between the total carbohydrate and starch levels. The carbon and energy contents of TAG and soluble sugars were calculated based on the energy density, molecular weight and carbon contents of triolein (35114 kJ/mol; 885.4 g/mol; 57 mol C/mol) and glucose (28.3 kJ/mol; 180 g/mol; 6 mol C/mol), respectively. The carbon and energy contents of the starch-glucose equivalents were calculated as decribed above for the soluble sugar fraction. In summary, the formulas used to obtain carbon content and energy density of the different carbon metabolic pools are as follows: Carbon content of TAG (mmol C/g leaf dry weight)=(% TAG×57 mol C/mol TAG×1000)/(100×885.4 g/mol TAG) Carbon content of soluble sugars (mmol C/g leaf dry weight)=[(% total carbohydrates−(% starch×180/162))×6 mol C/mol glucose*1000]/(100*180 g/mol glucose) Carbon content of starch (mmol C/g leaf dry weight)=[(% starch×180/162))×6 mol C/mol glucose*1000]/(100*180 g/mol glucose) Energy content of TAG (kJ/g leaf dry weight)=(% TAG×39.66 kJ/g TAG)/100 Energy content of soluble sugars (kJ/g leaf dry weight)=[(% total carbohydrates−(% starch×180/162))×15.57 kJ/mol glucose]/100 Energy content of starch (kJ/g leaf dry weight)=[(% starch×180/162))×15.57 kJ/mol glucose]/100. Example 2. Silencing of a TAG Lipase in Plants Accumulating High Levels of TAG in Leaf Tissue The Sugar Dependent 1 (SDP1) TAG lipase has been demonstrated to play a role in TAG turnover in non-seed tissues ofA. thalianaas well as during seed germination (Eastmond et al., 2006; Kelly et al., 2011; Kelly et al., 2013). SDP1 is expressed in developing seed and the SDP1 polypeptide is also present in mature seed in association with oil bodies. Silencing of the gene encoding SDP1 resulted in a small but significant increase in TAG levels inA. thalianaroots and stems (<0.4% on dry weight basis) while an even smaller increase was observed in leaf tissue (Kelly et al., 2013). To determine whether TAG levels could be increased further in leaf and stem tissues relative to co-expression of AtWRI1 and AtDGAT1, an experiment was designed to silence an endogenous SDP1 gene inN. tabacumplants which were homozygous for a T-DNA having genes for transgenic expression of the WRI, DGAT1 and Oleosin polypeptides (Vanhercke et al., 2014). A BLAST search of theN. benthamianatranscriptome (Naim et al., 2012) using the AtSDP1 nucleotide sequence as query identified a transcript (Nbv5tr6385200, SEQ ID NO:173) with homology to theA. thalianaSDP1 gene. A 713 bp region (SEQ ID NO:174) was selected for hairpin mediated gene silencing. A 3.903 kb synthetic fragment was designed, based on the pHELLSGATE12 vector, which comprised, in order, the enTCUP2 constitutive promoter, the 713 bpN. benthamianaSDP1 fragment in sense orientation flanked by attB1 and attB2 sites, a Pdk intron, a cat intron sequence in reverse orientation, a second 713 bpN. benthamianaSDP1 fragment flanked by attB1 and attB2 sites in reverse (antisense) orientation, and the OCS 3′ region terminator/polyadenylation site (FIG.2). The insert was subcloned into pJP3303 using SmaI and KasI restriction sites and the resulting expression vector was designated pOIL051. This chimeric DNA contains a hygromycin resistance selectable marker gene. pOIL051 was used to produce transformedN. tabacumplants byAgrobacterium-mediated transformation. The starting plant cells were from transgenic plants which were homozygous for the T-DNA of pJP3502 (Vanhercke et al., 2014). Transgenic plants containing the T-DNA from pOIL051 were selected by hygromycin resistance and transferred to soil in the glasshouse or in a controlled environment cabinet for continued growth. Leaf samples were harvested from confirmed double-transformants (TO plants) before flowering, at flowering and at seed setting stages of plant development, and the TAG level in each determined. Transgenic plants containing only low levels of leaf TAG, or TAG at the same level as controls, were identified by means of lipid extraction from leaf samples and analysis by spot TLC and discarded. TAG levels in the remaining population of transformants were quantified by GC as described in Example 1. Before flowering, the majority of these plants exhibited greatly increased TAG levels (>5% of leaf dry weight) in their leaf tissue while 4 plants contained TAG levels above 10% (Table 3). The maximum TAG level observed in leaves of these plants, before flowering, was 11.3% in plant 51-13. As a comparison, the transgenic plants of the parentalN. tabacumline expressing AtWRI1, AtDGAT1 and Oleosin displayed TAG levels of about 2% before flowering and about 6% during flowering (Vanhercke et al., 2014). The addition of the SDP1-inhibitory construct to the AtWRI1 plus AtDGAT1 combination was therefore synergistic for increasing the TAG levels in these plants. Surprisingly, the TAG content in leaves harvested from the doubly-transformed plants at flowering stage was greatly increased, observing 30.5% on a dry weight basis (Table 4), representing a 5-fold increase relative to the plants not silenced for SDP1. To the great amazement of the inventors, the TAG level reached an astonishing 70.7% (% of dry weight) in samples of senescing leaves (green and yellow) at the seed setting stage (Table 5). When NMR was used to measure the oil content of entire leaves from the tobacco plants at seed setting stage, the TAG content in some green leaves that had started senescing was about 43% and in some brown, desiccated leaves was 42%. When such leaves were pressed between two brown paper filters, the exuded oil soaked into the paper and made it translucent, whereas control tobacco leaves did not do so, providing a simple screening method for detecting plants having high oil content. Two primary transformants (#61, #69) containing each of the T-DNAs from pJP3502 and pOIL51 and displaying high TAG levels were analyzed by digital PCR (ddPCR) using a hygromycin gene-specific primer pair to determine the number of pOIL51 T-DNA insertions. The plant designated #61 contained one T-DNA insertion from pOIL51, whereas plant #69 contained three T-DNA insertions from pOIL51. Ti progeny plants of both lines were screened again by ddPCR to identify homozygous, heterozygous and null plants. Progeny plants of plant #61 containing no insertions from pOIL51 (nulls; total of 7) or 2 T-DNA insertions (i.e. homozygous for that T-DNA; total of 12) were selected for further analysis. Similarly, progeny plants of line #69 containing zero T-DNA insertions from pOIL51 (nulls; total of 2) or 2 such insertions (total of 15) or 4 or 5 insertions (total of 5) were maintained for further analysis. The selected T1 plants were grown in the glasshouse at the same time and under the same conditions as control plants. Green leaf tissue samples from the T1 plants before flowering were dried and total fatty acid (TFA) and TAG contents determined by GC analysis. TFA contents of the plants containing both T-DNAs ranged from 4.6% to 16.1% on a dry weight basis including TAG levels in the same leaves of 1.2% to 11.8% on a dry weight basis (FIG.3). This was much greater compared to the plants containing only the T-DNA from pJP3502 and growing alongside under the same conditions and analysed at the same stage of growth, again showing the synergism between reducing TAG lipase activity and the WRI1 plus DGAT combination. Plants containing only the pJP3502 T-DNA contained between 4.2% and 6.8% TFA including TAG levels of 1.4% to 4.1% on a dry weight basis (FIG.3). Wild-type plants contained, on average, about 0.8% TFA including less than 0.5% TAG on a dry weight basis. The fatty acid composition in the total fatty acid content and the TAG content of leaves from each of lines #61 and #69 were similar to the composition in leaves containing only the T-DNA from pJP3502 (parent). Compared to the wild-type control leaves, plants containing both of the T-DNAs from pOIL51 and pJP3502 exhibited increased levels of C16:0, C18:1 and C18:2 fatty acids. This significant shift in fatty acid composition came largely at the expense of C18:3 which was reduced from about 50-55% to about 20-30% as a percentage of the total fatty acid content. TABLE 3TAG levels (% leaf dry weight) and TAG fatty acid composition in leaf tissue fromN.tabacumplants (T0 generation) expressing WRI1, DGAT1 and Oleosin transgenes and super-transformedwith a T-DNA encoding an SDP1 hairpin construct (pOIL051), compared to wild-type(untransformed). Leaf samples were harvested during vegetative stage (before flowering). Lipidsamples also contained 0.0-0.2% C16:3, 0.0-0.4% C20:1; 0.0-0.1% C20:2n-6.LineC14:0C16:0C16:1C18:0C18:1C18:1d11C18:2C18:3n3C20:0C22:0C24:0% TAGWT2.520.20.08.65.60.018.944.20.00.00.00.123-310.066.00.00.034.00.00.00.00.00.00.00.023-290.036.11.45.121.00.823.37.12.41.51.12.9570.147.20.35.419.21.90.021.22.11.21.13.423-10.230.81.94.941.21.013.72.41.91.10.74.0580.131.40.23.812.21.633.613.21.71.00.74.0210.131.90.33.910.71.532.315.21.91.10.84.723-300.234.10.74.929.40.917.55.72.91.81.74.9400.134.40.24.314.31.529.711.81.71.00.75.1220.135.80.24.312.81.529.811.71.81.00.75.1150.137.20.13.98.61.729.316.01.50.80.65.1160.135.20.13.913.91.728.513.61.40.70.65.3250.134.40.23.915.41.827.613.21.60.90.75.4650.126.90.23.819.21.535.79.11.70.80.65.5120.231.70.23.615.91.730.512.81.60.90.75.5280.131.40.23.513.51.732.713.71.50.80.65.6260.131.40.23.513.51.732.713.71.50.80.65.8190.130.50.23.714.91.631.713.71.70.90.75.9300.130.40.23.721.32.231.27.41.60.80.75.960.137.50.24.410.51.731.910.61.50.70.66.040.134.20.23.911.91.732.612.51.40.60.56.1420.130.60.24.517.31.832.79.21.70.90.76.3450.131.60.23.918.21.830.410.51.60.80.66.6560.126.80.24.220.01.534.38.71.91.00.86.7430.128.50.23.818.61.634.19.61.70.90.67.1320.128.10.23.416.81.835.510.61.60.80.67.2700.126.30.23.525.51.831.08.91.30.60.57.4690.130.90.24.015.71.731.712.91.50.70.57.4610.131.00.24.016.41.634.19.51.50.70.57.5200.133.30.13.811.71.631.414.81.50.80.67.8530.133.10.13.818.21.929.810.41.30.60.58.4180.129.40.23.718.41.732.810.91.40.60.59.151-10.129.02.03.617.11.633.89.91.40.70.59.2470.130.50.14.220.31.531.98.31.50.70.59.351-600.130.72.63.415.81.931.211.61.30.70.510.2460.124.80.13.628.81.630.37.91.30.60.510.2480.133.10.13.816.51.730.411.41.40.70.510.751-130.125.42.23.323.81.632.78.31.30.60.411.3 TABLE 4TAG levels (% leaf dry weight) and TAG composition in leaf tissue fromN.tabacumplants(T0 generation) expressing WRI1, DGAT1 and Oleosin transgenes and supertransformedwith a T-DNA encoding an SDP1 hairpin construct (pOIL051). Leaf samples wereharvested during flowering.LineC14:0C16:0C16:1C18:0C18:1C18:1d11C18:2C18:3n3C20:0C22:0C24:0% TAGWT0.214.80.68.59.20.320.044.50.60.30.40.3210.125.72.13.721.21.031.011.71.50.80.68.8560.133.21.44.920.71.026.37.42.11.30.99.2650.124.71.53.828.51.029.07.51.70.90.612.0420.134.01.54.416.81.129.47.62.21.41.113.1280.129.52.43.516.41.228.714.61.51.00.613.2300.119.11.93.331.81.030.69.31.30.70.413.6200.122.41.83.727.40.929.010.81.70.90.714.6190.120.91.73.128.41.031.610.01.40.80.515.7120.124.41.63.622.10.935.18.91.40.80.515.8160.121.51.83.434.91.026.27.91.40.70.516.4570.125.01.74.127.71.028.48.41.60.90.617.2260.122.51.63.528.41.131.27.61.71.00.718.0390.130.02.23.722.71.624.311.61.50.90.718.1700.122.12.13.636.31.024.27.21.40.70.518.3450.121.41.83.734.41.027.56.91.40.80.519.1320.123.31.63.224.41.133.69.01.50.90.619.5180.123.42.13.326.40.930.210.31.40.70.520.620Y0.122.31.63.630.30.928.59.11.60.90.620.8430.128.12.03.521.51.229.910.21.50.90.621.240.127.91.93.726.31.226.29.31.50.80.521.810.123.82.03.730.21.128.18.01.40.70.522.3610.124.22.24.032.01.125.27.81.50.80.623.9600.124.42.23.731.01.125.48.61.50.80.625.0460.123.32.03.732.91.024.09.21.60.90.725.760.131.52.63.519.51.625.512.71.30.70.526.3130.121.81.93.635.11.025.18.11.50.80.526.8690.121.81.64.333.40.826.97.61.70.80.526.9530.127.12.13.524.11.229.49.21.40.80.529.2480.129.52.53.921.11.329.09.21.60.80.629.5470.130.92.53.419.41.528.510.61.30.80.530.5 TABLE 5TAG content (% leaf dry weight) and TAG composition in leaf tissue fromN.tabacumplants (T0 generation) expressing WRI1, DGAT1 and Oleosin transgenes andsupertransformed with a T-DNA encoding an SDP1 hairpin construct (pOIL051). Leafsamples were harvested at seed setting stage. Y = yellow leaf, G = green leaf.TAGSampleC14:0C16:0C16:116:3C18:0C18:1C18:1d11C18:2C18:3n3C20:0C20:1d11C22:0C24:0contentwt0.013.00.00.08.47.00.024.746.80.00.00.00.00.3730.026.61.70.08.59.40.027.025.61.10.00.00.00.6180.115.01.80.04.814.40.443.916.31.70.40.70.53.3410.122.31.20.34.424.00.632.810.81.70.30.80.55.2190.114.51.50.33.021.60.744.810.61.40.40.70.47.2200.126.72.40.14.324.91.025.211.31.90.31.00.89.6300.118.61.50.33.524.80.738.98.91.40.30.70.49.9650.122.21.40.33.530.90.729.18.11.70.31.00.611.3420.123.61.50.24.129.00.930.35.92.00.41.30.812.0320.121.31.30.33.321.40.940.77.11.70.31.00.613.7390.125.81.70.33.627.21.227.28.22.00.41.40.914.0450.123.01.50.13.828.00.932.66.31.80.31.00.614.4130.126.92.80.13.732.61.121.67.61.60.30.80.714.6R450.123.41.50.24.127.80.932.26.11.80.31.00.614.6210.123.11.60.23.527.40.831.28.21.80.31.10.715.090.123.21.40.23.523.30.835.68.51.60.30.90.515.4120.124.51.40.23.422.30.836.27.41.70.31.10.715.940.121.91.80.23.622.80.935.99.51.60.30.90.616.1490.123.51.40.24.025.30.834.36.61.80.31.10.716.8260.122.21.30.23.825.40.835.26.52.10.31.30.817.2160.122.21.80.33.429.90.830.18.11.50.30.90.618.210.127.42.70.14.032.01.222.96.31.60.30.80.718.7700.127.12.70.23.732.61.021.57.61.60.30.80.719.060.130.62.60.23.313.01.432.812.91.40.20.90.621.5470.128.02.10.23.618.51.333.29.91.50.20.90.521.6690.125.42.30.14.332.40.923.57.41.80.30.80.622.5530.123.92.10.23.428.21.130.27.61.50.30.90.523.2460.125.92.70.23.732.01.122.88.21.60.30.80.724.0430.123.71.60.23.122.60.937.67.41.40.20.80.524.0480.127.42.20.14.123.01.131.36.91.90.31.00.724.4280.123.01.40.23.324.81.035.67.31.60.30.90.626.61Y0.124.32.50.13.835.71.122.66.71.60.30.70.628.156G0.125.51.80.23.726.70.929.87.31.80.31.10.733.9570.125.11.90.23.220.11.035.210.01.50.30.90.635.456Y0.224.81.40.24.127.20.831.06.02.00.41.20.839.669Y0.124.72.10.24.132.00.824.37.81.90.30.90.746.5R69Y0.124.72.10.24.132.00.824.47.91.90.30.90.746.8610.126.62.70.13.631.61.123.87.41.40.30.70.549.261Y0.125.82.40.13.732.41.124.36.91.50.30.70.558.1600.124.62.40.23.634.11.024.46.41.50.30.70.570.7 The substantial increase in TFA levels including the TAG levels between the plants containing only the pJP3502 T-DNA and plants containing the T-DNAs from both pOIL51 and pJP3502 was maintained throughout plant development. Control plants containing only the T-DNA from pJP3502 contained 7.7% to 17.5% TAG during flowering while TAG levels ranged from 14.1% to 20.7% on a dry weight basis during seed setting. The TAG content in leaves from plants containing both pJP3502 and pOIL51 T-DNAs varied between 6.3% and 23.3% during flowering and 12.6% and 33.6% during seed setting. Similar changes in fatty acid composition of the TAG fraction at both stages were detected as described earlier for the vegetative growth stage. TAG levels were also found to be increased further in other vegetative tissues of the transgenic plants such as roots and stem. Some root tissues of the transgenicN. tabacumplants transformed with the T-DNA of pOIL051 contained 4.4% TAG, and some stem tissues 7.4% TAG, on a dry weight basis (FIG.4). Wild-type plants andN. tabacumcontaining only the T-DNA from pJP3502 exhibited much lower TAG levels in both tissues. The addition of the hairpin SDP1 construct to decrease expression of the endogenous TAG lipase was clearly synergistic with the genes encoding the transcription factor and biosynthesis of TAG (WRI1 and DGAT) for increasing TAG content in the stems and roots. Of note, TAG levels in the roots were lower compared to stem tissue within the same plant while an inverse trend was observed in wild-type plants andN. tabacumcontaining only the T-DNA from pJP3502. The TAG composition of root and stem tissues exhibited similar changes in C18:1 and C18:3 fatty acids as observed previously in transgenic leaf tissue. C18:2 levels in TAG were reduced in transgenic stem tissue while C16 fatty acids were typically reduced in transgenic root tissues when compared to the wild-type control. Therefore, the inventors concluded that addition of an exogenous gene for silencing the endogenous SDP1 gene to the combination of WRI1 and DGAT increased the total fatty acid content, including the TAG content, at all stages of the plant growth, and acted synergistically with WRI1 and DGAT, particularly in the stems and roots. T1 seeds from the transgenic plants were plated on tissue culture media in vitro at room temperature to test the extent and timing of germination. Germination of T1 seed from three independently transformed lines was the same compared to seed from the transgenic plants transformed only with the T-DNA from pJP3502. Furthermore, early seedling vigour appeared to be unaffected. This was surprising given the role of SDP1 in germination inA. thalianaseeds and the observed defects in germination in SDP1 mutants (Eastmond et al., 2006). To overcome any germination defects if such had occurred, a second construct is designed in which the SDP1 inhibitory RNA is expressed from a promoter which is essentially not expressed, or at low levels, in seed, such as for example a promoter from a photosynthetic gene such as SSU. The inventors consider that it is beneficial to reduce the risk of deleterious effects on seed germination or early seedling vigour to avoid a constitutive promoter, or at least to avoid a promoter expressed in seeds, to drive expression of the SDP1 inhibitory RNA. It was noted that the TO plants with the highest TAG levels had been grown under high light conditions in the controlled environment room (500 micro moles light intensity, 16 hr light/26° C.-8 hr dark/18° C. day cycle) and appeared smaller (about 70% in height relative to the plants transformed with the T-DNA from pJP3502) than the wild-type control plants. The inventors concluded that the combination of transgenes and/or genetic modifications for the “push”, “pull”, “protect” and “package” approaches was particularly favourable for achieving high levels of TAG in vegetative plant parts. In this example, WRI1 provided the “push”, DGAT provided the “pull”, silencing of SDP1 provided the “protect” and Oleosin provided the “packaging” of TAG. Example 3. Senescence-Specific Expression of a Transcription Factor Ectopic expression of master regulators of embryo and seed development such as LEC2 have been reported to increase TAG levels in non-seed tissues (Santos-Mendoza et al., 2005; Slocombe et al., 2009; Andrianov et al., 2010). However, constitutive over-expression of LEC2 in plants transformed with a 35S-LEC2 gene resulted in unwanted pleiotropic effects on plant development and morphology including somatic embryogenesis and abnormal leaf structures (Stone et al., 2001; Santos-Mendoza et al., 2005). To test whether limiting LEC2 expression to the leaf senescence stage of plant development, i.e. after plants had fully grown and reached their full biomass, would minimize undesirable phenotypic effects but still increase leaf lipid levels, a chimeric DNA was designed and made for expression of LEC2 under the control of aA. thalianasenescence specific promoter from the SAG12 gene (U37336; Gan and Amasino, 1995). To make the genetic construct, a 3.635 kb synthetic DNA fragment was made comprising, in order, anA. thalianaSAG12 senescence-specific promoter, the LEC2 protein coding sequence and aGlycine maxLectin gene terminator/polyadenylation region. This fragment was inserted between the SacI and NotI restriction sites of pJP3303. This construct was designated pOIL049 and tested in leaves ofN. tabacumplants which were stably transformed with genes encoding WRI1, DGAT1 and Oleosin polypeptides, containing the T-DNA from pJP3502. UsingAgrobacterium-mediated transformation methods, the pOIL049 construct was used to transformN. tabacumplant cells which were homozygous for the T-DNA of pJP3502. Transgenic plants comprising the genes from pOIL049 were selected by hygromycin resistance and were grown to maturity in the glasshouse. Samples are taken from transgenic leaf tissue at different stages of growth including at leaf senescence and contain increased TAG levels compared to theN. tabacumpJP3502 parent line. A total of 149 independent TO plants (i.e. primary transformants) were obtained. Upper green leaves of all plants and the lower brown, fully senesced leaves of selected events were sampled at the seed setting stage of plant development and TAG contents were quantified by TLC-GC. The number of pOIL49 T-DNA insertions in selected plants was determined by ddPCR using a hygromycin gene-specific primer pair. A TAG level of 30.2% on a dry weight basis was observed in green leaf tissue harvested at seed setting stage. TAG levels in brown leaves were lower in most of the plants sampled. However, three plants (#32b, #8b and #23c) displayed greater TAG levels in brown senesced leaf tissue than in the green expanding leaves. These plants contained 1, 2 or 3 T-DNA insertions from pOIL49. T1 progeny of plants #23c and #32b were screened by ddPCR to identify nulls, heterozygous and homozygous plants for the T-DNA from pOIL049. Progeny plants of plant #23c containing zero T-DNA insertions from pOIL049 (nulls; total of 7) or two T-DNA insertions of the T-DNA from pOIL049 (homozygous; total of 4) were selected for further analysis. Similarly, progeny plants of plant #32b containing zero insertions (nulls; total of 6) or two insertions (homozygous; total of 9) were maintained for further analysis. Green leaf tissue was sampled before flowering and TFA and TAG contents were determined by GC. Wild-type plants and plants transformed with the T-DNA from pJP3502 were the same as before (Example 2) and were grown alongside in the same glasshouse. TFA levels in leaves of the transformants containing the T-DNA from pOIL049 ranged from 5.2% to 19.5% on a dry weight basis before flowering (FIG.5). TAG levels in the same tissues ranged from 0.8% to 15.4% on a dry weight basis. This was considerably greater than in plants containing only the T-DNA from pJP3502. TAG levels in plants containing the T-DNAs from pJP3502 and pOIL049 further increased to 38.5% and 34.9% during flowering and seed setting, respectively. When the fatty acid composition of the total fatty acid content was analysed for leaves homozygous for the T-DNA from pOIL049, increased levels of C18:2 and reduced levels of C18:3 were observed (FIG.5) while the percentages of C16:0 and C18:1 remained about the same relative to leaves transformed only with the T-DNA from pJP3502. These data demonstrated that the addition of a second transcription factor gene under the control of a non-constitutive promoter to provide developmentally-regulated expression was able to further increase TAG levels in vegetative tissues of a plant. The data also indicated that the senescence-specific promoter SAG12 had some expression in the green tissue prior to senescence of the leaves. TAG levels were much increased in stem tissue when compared to both wild-typeN. tabacumplants and transgenic plants containing the T-DNA from pJP3502 alone. Some stem tissues of the transgenicN. tabacumplants transformed with the T-DNA from pOIL049 contained 4.9% TAG on a dry weight basis (FIG.6). On the other hand, TAG levels in root tissue exhibited large variation between the three pOIL049 plants with some root tissues containing 3.4% TAG. Of note, TAG levels in roots were lower compared to stem tissue within the same plant while an inverse trend was observed in wild-type plants andN. tabacumcontaining only the T-DNA from pJP3502. The TAG composition of root and stem tissues exhibited similar changes in C18:1 and C18:3 fatty acids as observed previously in transgenic leaf tissue. C18:2 levels in TAG were reduced in transgenic stem tissue while C16 fatty acids were typically reduced in transgenic root tissues when compared to the wild-type control. Corresponding genetic constructs are made encoding other transcription factors under the control of the SAG12 promoter, namely LEC1, LEC1 like, FUS3, ABI3, ABI4 and ABI5 and others (see Example 9). For example, additional constructs were made for the expression of the monocot transcription factorZea maysLEC1 (Shen et al., 2010) orSorghum bicolorLEC1 (Genbank Accession No. XM_002452582.1) under the control of monocot-derived homolog of theA. thalianaSAG12 promoter such as the maize SEE1 promoter (Robson et al., 2004). Further constructs are made for expression of the transcription factors under developmentally controlled promoters, for example which are preferentially expressed at flowering (e.g. day length sensitive promoters), Phytochrome promoters, Chryptochrome promoters, or in plant stems during secondary growth such as a promoter from a CesA gene. These constructs are used to transform plants, and plants which produce at least 8% TAG in vegetative parts are selected. Example 4. Analysis of Transgenic Plants Plant Material and Growth Conditions Plants of three TAG accumulating transgenic lines were grown in growth cabinets or in a glasshouse under controlled conditions:1. Plants over-expressing genes encoding WRI1, DGAT and oleosin (Vanhercke et al, 2014), designated here as HO plants, being plants of the T2 generation which were homozygous for the introduced T-DNA from pJP3052.2. T1 plants transformed with an RNAi construct to silence the SDP1 TAG lipase as well as the T-DNA from pJP3502, encoding the WRI1, DGAT and oleosin polypeptides, from two independent transformed lines. See Example 2. These plants were designated SDP1.3. T1 plants transformed with a genetic construct for over-expressing the transcription factor LEC2 from the SAG12 promoter, as well as the T-DNA from pJP3502 encoding the WRI1, DGAT and oleosin polypeptides. See Example 3. These plants were designated LEC2, and were progeny from a single TO plant. Wild-type plants (WT, of cultivar Wisconsin 38) were used as control plants and grown at the same time and under the same conditions as the transgenic plants. For vegetative samples, WT and HO tobacco plants were grown in PGC20/PGC20FLEX plant growth cabinets (Conviron) at ambient CO2concentrations with 250-450 μmol m−2s−1illumination from fluorescent bulbs. Plants were grown under 12 hr light/25° C.: 12 hr dark/20° C. daily cycles. Plants from which samples were to be harvested at 49 days after sowing (DAS) were grown in 1.25 litre pots in soil with osmocote fertiliser. Plants from which samples were to be harvested at 69 DAS were grown in 4 litre pots in soil and watered every 14 days with aquasol fertiliser. For all assays, samples were taken from four plants of each genotype. For samples to be harvested at seed-setting stage of growth, WT, HO, SDP1 and LEC2 plants were grown in a glasshouse without artificial light (n=3, 3, 8, 6, respectively). For all analyses, leaf discs were harvested from leaves at the end of the growth phase with light, snap frozen and stored at −80° C. until analysis. TAG Levels and Fatty Acid Composition TAG levels were measured in leaves of mature WT, HO, SDP1 and LEC2 plants. The fatty acid composition in TAG of the leaves was also determined. The data are shown in Table 6 for the LEC2 and SDP1 plants. Starch and Sugar Levels Starch and soluble sugar levels were measured in leaf tissue sampled from the wild-type (WT) and transgenic HO, SDP1 and LEC2 plants. In general, an inverse correlation was found between TAG and starch levels in leaf tissue on a dry weight basis in the leaves having both T-DNAs (FIG.7andFIG.8). In contrast, leaf soluble sugars levels were about the same in the transgenic plants as in the wild-type plants, suggesting that there was no significant bottleneck in the conversion from sugars to TAG. An effect of the leaf position in the plants was observed in wild-type plants where starch levels tended to increase from lower leaf to higher leaf position. No such effect was detected in the transgenic plants. Carbon and Energy Contents The amounts of carbon and energy in the TAG, starch and sugar contents in leaves of the HO, SDP1 and LEC2 plants were measured and compared to wild-type plants, on a dry weight basis. The data (Table 7) showed that each of the carbon and energy contents increased in the HO plants and increased even further in the SDP1 and LEC2 plants relative to the WT plants. The increase was seen for the sum of TAG, starch and soluble sugars, as well as for the sum of TAG and starch. It was concluded that the increase in carbon content by increasing the TAG content more than compensated for the reduced starch content. Therefore, the transgenic plants exhibited increased total carbon content and increased total energy content on a dry weight basis. Nitrogen and Soluble Protein Contents Nitrogen and protein contents were measured in leaf samples of the transformed and control plants as described in Example 1, for plants at 69 DAS. The third leaf from the top of each of the WT and HO tobacco plants, which leaves were not yet fully expanded and therefore still growing, had the same nitrogen content at about 3.0% by DW. Older (lower) leaves on each plant were also analysed. In the WT plants, the leaf nitrogen content decreased with leaf age, whereas the nitrogen content was relatively maintained in older HO leaves with less of a decline compared to the WT plants. For example, older leaves such as leaf 11 from the top of the HO plants had more than twice as much nitrogen (2.9%) compared to the corresponding leaves in WT plants (1.3%;FIG.9a). A similar trend was observed for total soluble protein with twice as much soluble protein detected in older HO leaves compared to WT (10.4 and 5.0 μg/mg FW, respectively;FIG.9B). The same trends were observed when soluble protein samples were electrophoresed by SDS-PAGE, after normalising sample loading according to leaf fresh weights. TABLE 6TAG levels (% leaf dry weight) and fatty acid composition in TAG of selectedN.tabacumprimary transformants over-expressing LEC2 (pOIL049) or a silencing construct targeted againstthe gene encoding SDP1 TAG lipase (pOIL051). Both constructs were transformed independentlyinto a previously establishedN.tabacumtransgenic line over-expressing genes encoding WRI1,DGAT1 and OLEOSIN (Vanhercke et al., 2014).%TransgeneLineLeafC16:0C16:1C18:0C18:1Δ9C18:2Δ9,12C18:3Δ9,12,15OtherTAGLEC2#8green28.33.13.219.830.510.25.08.3#8brown31.12.43.716.532.47.76.212.6LEC2#23green26.65.50.15.448.07.37.214.3#23brown26.25.22.53.547.08.17.528.7LEC2#32green28.01.04.319.729.210.96.75.8#32brown22.33.12.419.739.07.26.314.0SDP1#60brown28.03.83.135.919.75.83.626.4SDP1#61brown27.73.73.434.020.76.54.027.9SDP1#69yellow-green28.13.43.629.523.38.13.932.9 TABLE 7Carbon and energy contents of TAG, starch and soluble sugarsin leaf tissues of wild-type and transgenicN.tabacumplants.CarbonEnergy(mmol C/gDW)(kJ/g DW)*SolubleSolubleTAGStarchsugarsTAGStarchsugarswt10.0815.411.330.057.200.62wt60.0812.011.290.055.610.60wt70.0911.101.370.065.180.64HO #210.695.431.246.592.540.58HO #58.6310.781.865.325.040.87HO #79.518.221.475.863.840.69SDP1 #69-119.562.831.6112.051.320.75SDP1 #69-6021.443.112.0313.211.450.95SDP1 #69-9116.780.681.7110.330.320.80LEC2 #32-2114.990.570.889.230.270.41LEC2 #32-2919.190.860.8211.820.400.39*assuming 2803 kJ/mol for glucose and 35114 kJ/mol for triolein (Sanjaya et al., 2011) In both WT and HO plants, leaf protein content increased with plant age (FIG.10, Table 8). In younger plants (49 DAS), soluble protein content was slightly but not significantly higher in older leaves from HO plants compared to WT. By 69 DAS, this difference was significant with an 87% increase in old HO leaves compared to WT (p<0.05, t-test). It was concluded that the leaves of the HO plants had significantly increased nitrogen and protein contents relative to the corresponding leaves in the WT plants. In this context, a “corresponding leaf” meant a leaf of the same age of a plant grown under the same conditions. TABLE 8Leaf soluble protein content in WT and HO tobacco (μg/mgFW). Range includes young, mature and older leavesof younger (49 DAS) and older (69 DAS) plants.WTHOPlant age49 DAS69 DAS49 DAS69 DASRange4.3-9.93.0-15.22.5-11.37.9-19.1 Nitrogen Content in SDP1 and LEC2 Plants The transgenic plants designated LEC2 and SDP1 exhibited increased TAG accumulation compared to the HO plants throughout growth (Examples 2 and 3), increasing with plant age. Leaf samples of the transgenic plants grown in growth cabinets or in the glasshouse were assayed for nitrogen, protein and carbon contents. The LEC2 and SDP1 plants exhibited each of increased leaf carbon content, leaf nitrogen content and soluble protein content relative to the WT plants (Table 9). At the seed setting stage of growth, the LEC2 and SDP1 leaves had between 50% and 100% more nitrogen than WT leaves. The leaf soluble protein content increased between 40% and 87% in LEC2 and SDP1 leaves, respectively, relative to the WT leaves. Leaf carbon content also increased. Despite moderate increases in leaf carbon content (16% to 21%) in LEC2 and SDP1 lines, the greater relative increase in leaf nitrogen content decreased the carbon to nitrogen ratio by up to 40% compared to WT leaves. Total Dietary Fibre (TDF) Analysis of the total dietary fibre of WT, SDP1 and LEC2 in mature leaves obtained at flowering showed that WT leaves had a TDF content of 27%, SDP1 leaves had a TDF content of 15.9% (59% reduction when compared to WT), and LEC2 leaves had a TDF content of 17.9% (34% reduction when compared to WT). TABLE 9TAG content (% dry weight), carbon content, nitrogen content andsoluble protein content of WT, LEC2 and SDP1 tobacco leaves. ForTAG analysis n = 3-8 and for C, N and soluble protein n = 2-5.SolubleTAGNitrogenCarbonproteincontentcontentcontentC:N ratocontentWT0.170.5043.0886:11.47LEC224.570.8552.0851:12.06SDP128.521.0749.9260:12.75 Upregulation of Genes Involved in Photosynthesis The observations described above on the increase in carbon and energy contents in the transgenic plants led the inventors to consider whether the plants might exhibit an increase in photosynthetic capacity, related to the altered carbon allocation between starch and TAG. Therefore, the transcriptome of the HO plants was determined and compared to the transcriptome from WT plants grown under the same conditions. RNA was isolated from plants at the flowering stage, converted to cDNA and the full transcriptomes were determined. When the resultant sequence libraries were compared for the frequency of representation of individual genes, numerous genes involved in photosynthesis were observed to be up-regulated (over-expressed) in the HO plants. Table 10 lists representative genes which were up-regulated. From this, it was concluded that the capacity for photosynthesis was increased in the transgenic plants. Effects of Modifying Photoperiod and Light Intensity The growth conditions were modified compared to those described above, in order to test the effect of increasing or decreasing the photoperiod from the 12 hrs, and of increasing light intensity. In one growth chamber using high light intensity and long photoperiod, the CO2concentration was also increased above the ambient. The following conditions were tested, in each case plants were grown in PGC20/PGC20FLEX plant growth cabinets (Conviron) at 25° C. during the light period, 20° C. during the dark period and leaf samples were harvested at seed-setting stage of growth from leaf Nos. 9, 15 and 20 counting from the bottom of each plant. Leaf 9 was therefore the oldest of the sampled leaves, leaf 15 intermediate, and leaf 20 the youngest leaf sampled. Leafs were assayed for total fatty acid (TFA) content as described in Example 1. TABLE 10Listing of genes related to photosynthesis and whose expression was up-regulated.Unigene1og2FClogCPMLRPValueArabidopsis AnnotationNicotiana Annotationc72304_g1.036.3114.040.000179411PSBP-2 photosystem IIPREDICTED: Oxygen-evolving enhancer protein2_i2subunit P-2 chr2:2-1, chloroplastic (LOC104217148)13118937-13120090c63827_g2.310.9521.084.40E−06NAPREDICTED: PsbQ-like protein 1, chloroplastic1_i2(LOC104213138) variant X1c72304_g1.192.4315.209.66E−05PSBP-1, OEE2, PSII-P,PREDICTED: Oxygen-evolving enhancer protein2_i6OE23 photosystem II2-2, chloroplastic (LOC104220111)subunit P-1 chr1:2047825-2049418c72995_g3.023.42101.497.18E−24NAPREDICTED: Ferredoxin, root R-B1-like1_i1(LOC104250181), transcript variant X2c66865_g1.302.6912.050.000518939NAPREDICTED: Photosystem II repair protein1_i2_1PSB27-H1, chloroplastic (LOC104235950)c64448_g1.068.9113.350.000258132NAPREDICTED: Ferredoxin (LOC104243179),1_i1_1mRNAc65326_g1.178.4520.854.97E−06NAPREDICTED: Oxygen-evolving enhancer protein1_i13-2, (LOC104238927)c68151_g0.788.009.370.002201414PSAF photosystem IPREDICTED: Photosystem I reaction center1_i1subunit F chr1:subunit III, (LOC104229855)11214824-11216037c70874_g0.776.0011.610.000655078PSAF photosystem IPREDICTED: Photosystem I reaction center1_i3subunit F chr1:subunit III, LOC104227234)11214824-11216037c72380_g0.828.8115.309.19E−05ATPC1 ATPase, F1PREDICTED: ATP synthase gamma chain,2_i1complex, gammachloroplastic (LOC104212794)subunit protein chr4:2350498-2352018c84022_g1.256.5419.659.29E−06NAPREDICTED: Plastocyanin A′/A″2_i1(LOC104226609)c80197_g0.666.279.600.00194393PSBO-1, OEE1,PREDICTED: Oxygen-evolving enhancer protein1_i1_1OEE33, OE33,1, chloroplastic (LOC104219516)PSBO1, MSP-1PS II oxygen-evolving complex 1c80359_g0.746.418.910.002843257PSBP-2photosystemPREDICTED: oxygen-evolving enhancer protein2_i3II subunit P-2 chr2:2-2, (LOC104220111), variant X213118937-13120090c84616_g0.958.5313.930.000189474NAPREDICTED: Oxygen-evolving enhancer protein2_i13-2, chloroplastic-like (LOC104238927)c60857_g2.132.7661.065.54E−15NAPREDICTED: Ferredoxin, root R-B2-like1_i2_1(LOC104216941), transcript variant X1c66431_g2.041.1026.672.41E−07NAPREDICTED: Plastocyanin (LOC104222137),3_i1mRNAc70844_g0.728.7710.450.001227902PSBO-1, OEE1,PREDICTED: Oxygen-evolving enhancer protein1_i1OEE33, OE33,1, chloroplastic (LOC104219516), mRNAPSBO1, MSP-1PS II oxygen-evolving complex 1chr5:26568653-26570278c72588_g0.589.388.500.003545489NAPREDICTED: Photosystem I reaction center1_i1_1subunit XI, chloroplastic (LOC104221829)c63567_g0.968.5015.817.01E−05PSAO photosystemPREDICTED: Photosystem I subunit O-like3_i1I subunit O chr1:(LOC104237017), transcript variant X12640813-2641828c79260_g1.766.4268.731.13E−16PSBO-1, OEE1,PREDICTED: Oxygen-evolving enhancer protein1_i1OEE33, OE33,1, chloroplastic-like (LOC104210963), mRNAPSBO1, MSP-1PS II oxygen-evolving complex 1chr5:26568653-26570278c70844_g1.166.1024.557.23E−07PSBO2, PSBO-2,PREDICTED: Oxygen-evolving enhancer protein1_i5OEC33 photosystem1, (LOC104210963)II subunit O-2 chr3:18890876-18892426c72502_g0.864.1912.140.000493881NAPREDICTED: Oxygen-evolving enhancer protein1_i11, (LOC104210963)c79863_g1.262.2313.310.000263854NAPREDICTED: Plastocyanin A′/A″1_i3_1(LOC104226609)c72380_g0.858.5720.426.23E−06ATPC1 ATPase, F1PREDICTED: ATP synthase gamma chain,1_i1_2complex, gammachloroplastic (LOC104212794)subunit protein chr4:2350498-2352018c66717_g0.6110.7610.670.001090419NAPREDICTED: Photosystem II 10 kDa2_i2polypeptide, chloroplastic (LOC104224572)c60043_g3.401.7278.269.04E−19NAPREDICTED: Ferredoxin, root R-B2-like4_i1(LOC104216941), transcript variant X1c64427_g0.709.078.590.003381803NAPREDICTED: Photosystem II reaction center W1_i1_1protein, (LOC104244017)1. Control conditions: 8 plants were grown with 300 μmol m−2s−1illumination from fluorescent bulbs, with a 12-hour photoperiod;2. Increased light intensity: 7 plants were grown with 700 μmol m−2s−1illumination from fluorescent bulbs, with a 12-hour photoperiod;3. Reduced photoperiod: 9 plants were grown with 700 μmol m−2s−1illumination from fluorescent bulbs, with a 8-hour photoperiod;4. Increased light intensity and photoperiod: 10 plants were grown with 700 μmol m−2s−1illumination from fluorescent bulbs, with a 12-hour photoperiod, at 700 ppm CO2concentration. The average data for leaves 9, 15 and 20 of each genotype are plotted inFIG.11. Increased light intensity alone did not significantly affect the TFA levels. Decreasing the photoperiod from 12 hrs to 8 hrs decreased the levels of TFA but to a surprisingly small extent. That is, even reducing the amount of light received each 24 hours by 33% had remarkably small effect. The most dramatic results observed were from the test using an increased photoperiod under increased light intensity and increased CO2concentration. The TFA levels increased dramatically, reaching 50% (w/w dry weight) and above in leaves of the LEC2 plants. Since the TFA assays measured only the fatty acid components of lipids, this meant that the total lipid level was even higher in these leaves. Example 5. Modifying Traits in Vegetative Parts of Monocotyledonous Plants Chimeric DNA constructs were designed to increase oil content in monocotyledonous plants, for example the C4 plantS. bicolor(sorghum), by expressing a combination of genes encoding WRI1,Z. maysLEC1 (Accession number AAK95562; SEQ ID NO:155), DGAT and Oleosin in the transgenic plants. Several pairs of constructs for biolistic co-transformation were designed and produced by restriction enzyme-ligation cloning, as follows. The genetic construct pOIL136 was a binary vector containing three monocot expression cassettes, namely a selectable marker gene encoding phosphinothricin acetyltransferase (PAT) for plant selection, a second cassette for expressing DGAT and a third for expressing Oleosin. pJP136 was first produced by amplifying an actin gene promoter fromOryza sativa(McElroy et al., 1990) and inserting it as a blunt-ClaI fragment into pORE04 (Coutu et al., 2007) to produce pOIL094. pOIL095 was then produced by inserting a version of theSesamum indicumOleosin gene which had been codon optimised for monocot expression into pOIL094 at the KpnI site. pOIL093 was produced by cloning a monocot codon optimised version of theUmbelopsis ramannianaDGAT2a gene (Lardizabal et al., 2008) as a SmaI-KpnI fragment into a vector already containing aZea maysUbiquitin gene promoter. pOIL134 was then produced by cloning the NotI DGAT2a expression cassette from pOIL093 into pOIL095 at the NotI sites. pOIL141 was produced by inserting the selectable marker gene coding for PAT as a BamHI-SacI fragment into a vector containing theZ. maysUbiquitin promoter. Finally, pOIL136 was produced by cloning theZ. maysUbiquitin::PAT expression cassette as a blunt-AscI fragment into the ZraI-AscI of pOIL096. The genetic construct pOIL136 therefore contained the following expression cassettes: promoterO. sativaActin::S. indicumOleosin, promoterZ. maysUbiquitin::U. ramannianaDGAT2a and promoterZ. maysUbiquitin::PAT. A similar vector pOIL197, containing NPTII instead of PAT was constructed by subcloning of theZ. maysUbiquitin::NPTII cassette from pUKN as a HindIII-SmaI fragment into the AscI (blunted) and HindIII sites of pJP3343. The resulting vector, pOIL196, was then digested with HindIII (blunted) and AgeI. The resulting 3358 bp fragment was cloned into the ZraI-AgeI sites of pOIL134, yielding pOIL197. A set of constructs containing genes encoding theZ. maysWRI1 (ZmWRT) or the LEC1 (ZmLEC1) transcription factors under the control of different promoters were designed and produced for biolistic co-transformation in combination with pOIL136 or pOIL197 to test the effect of promoter strength and cell specificity on the function of WRI1 or LEC1, or both if combined, when expressed in vegetative tissues of a C4 plant such assorghum. This separate set of constructs did not contain a selectable marker gene, except for pOIL333 which contained NPTII as selectable marker. The different promoters tested were as follows. TheZ. maysUbiquitin gene promoter (pZmUbi) was a strong constitutive monocot promoter while the enhanced CaMV 35S promoter (e35S) having a duplicated enhancer region was reported to result in lower transgene expression levels (reviewed in Girijashankar and Swathisree, 2009). Whilst theZ. maysphosphoenolpyruvate carboxylase (pZmPEPC) gene promoter was active in leaf mesophyl cells (Matsuoka and Minami, 1989), the site of photosynthesis in C4 plant species, theZ. maysRubisco small subunit (pZmSSU) gene promoter was specific for the bundle sheath cell layer (Nomura et al., 2000; Lebrun et al., 1987), the cells where carbon fixation takes place in C4 plants. The expression of theZ. maysgene encoding the SEE1 cysteine protease (Accession number AJ494982) was identified as similar to that of theA. thalianaSAG12 senescence-specific promoter during plant development. Therefore a 1970 bp promoter from the SEE1 gene (SEQ ID NO:207) was also selected to drive expression of the genes encoding theZ. maysWRI1 and LEC1 transcription factors. Further, the promoter from the gene encoding Aeluropuslittoraliszinc finger protein A1SAP (Ben Saad et al., 2011; Accession number DQ885219; SEQ ID NO:208), the promoter from the gene encoding theSaccharumhybrid DIRIGENT (DIR16) (Damaj et al., 2010; Accession number GU062718; SEQ ID NO:246), the promoter from the gene encoding theSaccharumhybrid 0-Methyl transferase (OMT) (Damaj et al., 2010; Accession number GU062719; SEQ ID NO:247), the A1 promoter allel from the gene encoding theSaccharumhybrid R1MYB1 (Mudge et al., 2009; Accession number JX514703.1; SEQ ID NO:248), the promoter from the gene encoding theSaccharumhybrid Loading Stem Gene 5 (LSG5) (Moyle and Birch, 2013; Accession number JX514698.1; SEQ ID NO:249) and the promoter from the sucrose-responsive ArRolC gene fromA. rhizogenes(Yokoyama et al., 1994; Accession number DQ160187; SEQ ID NO:209) were also selected for expression of ZmWRI1 expression in stem tissue. Therefore, each of these promoters was individually joined upstream of the ZmWRI1 or ZmLEC1 coding regions, as follows. An intermediate vector, pOIL100, was first produced by cloning theZ. maysWRI1 coding sequence and a transcription terminator/polyadenylation region, flanked by AscI-NcoI sites, into the same sites in the binary vector pJP3343. The different versions of the constructs for WRI1 expression were based on this vector and were produced by cloning the various promoters into pOIL100. pOIL101 was produced by cloning a XhoI-SalI fragment containing the e35S promoter with duplicated enhancer region into the XhoI site of pOIL100. pOIL102 was produced by cloning a HindIII-AvrII fragment containing theZ. maysUbiquitin gene promoter into the HindIII-XbaI sites of pOIL100. pOIL103 was produced by cloning a HindIII-NcoI fragment containing aZ. maysPEPC gene promoter into the HindIII-NcoI sites of pOIL100. pOIL104 was produced by cloning a HindIII-AvrII fragment containing aZ. maysSSU gene promoter into the HindIII-AvrII sites of pOIL100. A synthetic fragment containing theZ. maysSEE1 promoter region flanked by HindIII-XhoI unique sites was synthesized. This fragment was cloned upstream of theZ. maysWRI1 protein coding region using the HindIII-XhoI sites in pOIL100. The resulting vector was designated pOIL329. A synthetic fragment containing theA. littoralisA1SAP promoter region flanked by XhoI-XbaI unique sites was synthesized. This fragment was cloned upstream of theZ. maysWRI1 coding region using the XbaI-XhoI sites in pOIL100. The resulting vector was designated pOIL330. A synthetic fragment containing theA. rhizogenesArRolC promoter region flanked by PspOMI-XhoI unique sites was synthesized. This fragment was cloned upstream of theZ. maysWRI1 coding region using the PspOMI-XhoI sites in pOIL100. The resulting vector was designated pOIL335. Finally, a binary vector (pOIL333) containing theZ. maysSEE1::ZmLEC1 expression cassette was obtained in three steps. First, a 35S::GUS expression vector was constructed by amplifying the GUS coding region with flanking primers containing AvrII and KpnI sites. The resulting fragment was subsequently cloned into the SpeI-KpnI sites of pJP3343. The resulting vector was designated pTV111. Next, the 35S promoter region of pTV111 was replaced by theZ. maysSEE1 promoter. To this end, theZ. maysSEE1 sequence was amplified using flanking primers containing HindIII and XhoI unique sites. The resulting fragment was cut with the respective restriction enzymes and subcloned into the SalI-HindIII sites of pTV111. The resulting vector was designated pOIL332. Next the ZmLEC1 coding sequence was amplified using flanking primers containing NotI and EcoRV sites. This resulting fragment was subcloned into the respective sites of pOIL332, yielding pOIL333. A 2673 bp synthetic fragment containing theSaccharumDIR16 promoter region flanked by HindIII-XbaI sites was synthesized. This fragment was cloned upstream of theZ. maysWRI1 protein coding region using the HindIII-XbaI sites in pOIL100. The resulting vector was designated pOIL337. A 2947 bp synthetic fragment containing theSaccharumOMT promoter region flanked by XhoI-XbaI sites was synthesized. This fragment was cloned upstream of theZ. maysWRI1 protein coding region using the XhoI-XbaI sites in pOIL100. The resulting vector was designated pOIL339. A 1181 bp synthetic fragment containing theSaccharumR1MYB1 promoter region flanked by HindIII-XhoI sites was synthesized. This fragment was cloned upstream of theZ. maysWRI1 protein coding region using the HindIII-XhoI sites in pOIL100. The resulting vector was designated pOIL341. A 4482 bp synthetic fragment containing theSaccharumLSG5 promoter region flanked by XbaIII-SmaI sites was synthesized. This fragment was cloned as an XbaIII-SmaI fragment upstream of theZ. maysWRI1 protein coding region using the StuI-NheI sites in pOIL100. The resulting vector was designated pOIL343. Whole plasmid DNA was prepared from pOIL101, pOIL102, pOIL103, pOIL104, pOIL197 and pOIL136 for biolistic transformation. pOIL197 DNA was then mixed with either pOIL101, pOIL102, pOIL103 or pOIL104 and transformed by biolistic-mediated transformation intoS. bicolor(grainsorghumTX430) differentiating embryonic calli (DEC) tissues as described in Example 1. Alternatively, constructs for expression of the same combinations of genes are transformed separately or co-transformed byAgrobacterium-mediated transformation (Gurel et al., 2009; Wu et al., 2014) into DEC tissues. Twenty-five to fifty transgenic plants were regenerated and selected by antibiotic resistance for the pairs of constructs including pOIL197 with each of pOIL102 (pZmUbi::WRI1), pOIL103 (pZmPEPC::WRI1) and pOIL104 (pSSU::WRI1). Transformations were also carried out with pOIL197 alone and with pOIL102 or pOIL103 alone, and for an “empty vector” control. The presence of the desired transgenes in plants that were resistant to the selective agent was demonstrated by PCR. The copy number of each transgene was also determined by digital PCR. Total leaf lipids were quantified in a first subset of transgenicS. bicolorplants prior to their transfer from MS medium to soil. This preliminary screening suggested slightly elevated total lipid levels in leaf tissue of some events at this very early stage. The line with the highest total lipid content, pOIL136 (2), was further analyzed by thin layer chromatography (TLC) to determine the effect of transgene expression on TAG accumulation. Leaf tissue of this particular line was sampled at vegetative stage following transfer to soil in the glasshouse. When compared to the wildtype and empty vector negative controls, pOIL136 (2) exhibited increased TAG levels in leaf tissue after TLC separation and iodine staining. Subsequent quantification revealed 10-fold increased TAG in the transgenic line compared to the negative controls. The TAG profile was dominated by the polyunsaturated fatty acids linoleic and α-linolenic acid. After confirmed transgenic plants were transferred to soil in pots in the glasshouse, whole leaves were sampled from primary transformants at vegetative stage of growth (i.e. prior to the appearance of the boot leaf), at the boot leaf stage (defined as when the boot leaf has fully emerged, the boot leaf is the last leaf formed on the plant and from which the panicle (head) emerges) and at the mature seed-setting stage. Total fatty acid (TFA) and triacylglycerol (TAG) contents (% leaf dry weight) were quantified by TLC-GC as described in Example 1. TFA levels in wildtype and empty vector negative controls decreased during plant development (Table 11) and were in the range 0.7-3.3% (weight/dry weight). The highest TFA levels were detected prior to the appearance of the boot leaf (termed the vegetative stage of growth) and were not higher than 3.3%. TAG levels in the same plants were consistently low in the range 0-0.2% during the entire plant life cycle (Table 11). Both the TFA content and the TAG content had fatty acid compositions of predominantly C16:0, C18:2Δ9,12(LA) and C18:3Δ9,12,15(ALA). In particular, ALA was present at about 50-75% of the TFA content, reflecting the use of this fatty acid in wild-type plastid membranes. ALA also was the main fatty acid in the very small amount of TAG present in the wild-type leaves. Thirty-five confirmed transgenic plants which had been transformed with pOIL197 or pOIL136, each vectors comprising both pZmUbi:DGAT and pZmUbi:Oleosin genes in addition to the selectable marker genes, were analysed at the vegetative, boot leaf and mature seed setting stages. The data are presented in Tables 12-14. Generally, the pOIL197 and pOIL136 primary transformants displayed increased TFA and TAG accumulation compared to the negative control lines, but only to about double for the TFA level compared to the controls. The highest TFA levels were detected at the vegetative stage of growth (Table 12). Similar to the wild-type and negative control lines, TFA levels decreased with progressing plant age (Tables 13 and 14). Maximum TFA levels at vegetative, boot leaf and mature seed setting stages equalled 5%, 4.5% and 2.1%, respectively. The highest TAG levels detected varied between 0.9 and 1.9% depending on the age of the plant at the time of sampling (Table 13), so were increased up to 10-fold relative to the very low levels in the wild-type leaves (Table 11). The TFA composition remained largely unchanged at the different stages and was dominated by ALA. The TAG composition displayed a higher degree of variation between the different transgenic lines. Compared to the fatty acid composition of the TFA content, the levels of stearic acid, oleic acid and LA (18:2Δ9,12) consistently increased in TAG throughout all plant stages investigated. Nine primary transgenic plants made by transformation with pOIL102 (pZmUbi:WRI1) were generated by co-bombardment of pOIL102 and pUKN, containing the NPTII selectable marker gene. Tables 15-17 show the data for TFA and TAG contents and fatty acid compositions were measured at the three growth stages. When compared to the plants transformed with the constructs encoding DGAT2 and Oleosin (pOIL197 or pOIL136), TFA and TAG levels in the pOIL102 transgenic events were generally lower. Indeed, levels of TFA and TAG were similar to the levels in the wild-type and negative control plants. Maximum TFA levels at vegetative, boot leaf and mature seed setting stages were 2.6%, 2.5% and 2.0%, respectively (Tables 15-17). Maximum TAG levels observed were 0.2%, 0.4% and 0.9% at vegetative, boot leaf and mature seed setting stages, respectively. Thirty-six primary transgenic plants made by co-bombardment with both pOIL197 (pZmUbi:DGAT and pZmUbi:Oleosin) and pOIL102 (pZmUbi:WRI1) and confirmed to have integrated both genetic constructs were analysed for TFA and TAG contents and fatty acid composition at the three growth stages. The data are presented in Tables 18-20. Some of the plants exhibited greatly increased TFA and TAG levels compared to the transformations with single pOIL197, pOIL136 or pOIL102 vectors. Maximum TFA levels at vegetative, boot leaf and mature seed setting stages in the pOIL102+pOIL197 population equalled 7.2%, 6.4% and 6.1%, respectively (Tables 18-20). Importantly, the maximum observed TAG levels increased during plant development from 2.7% (vegetative stage) to 3.5% (boot leaf stage) and 4.3% (mature seed setting stage) (Tables 18-20). Compared with the data obtained for the separate transformations with the DGAT and WRI1 transgenes, this exemplified the synergism for co-expressing DGAT and WRI1 transgenes to increase non-polar lipid accumulation in vegetative plant tissues. High levels of TAG and TFA were in most cases associated with a substantial reduction in the C18:3Δ9,12,15content, which was reduced by about 50% in the lines with the highest levels of TAG. Thirty-six primary transformants containing both pOIL197 (pZmUbi:DGAT and pZmUbi:Oleosin) and pOIL103 (pZmPEPC:WRI1) were analysed for TFA and TAG contents and fatty acid composition during the three stages of plant development. The data are presented in Tables 21-23. Some plants with this gene combination exhibited the highest TFA and TAG levels detected in this experimental series. TFA levels were observed at vegetative, boot leaf and mature seed setting stages in the pOIL103+pOIL197 population at 8.3%, 8.3% and 4.5%, respectively (Tables 21-23). TAG levels were observed at vegetative, boot leaf and mature seed setting stages at 2.3%, 6.6% and 3.0%, respectively (Tables 21-23). Of note, the highest TAG (6.6%) and TFA (8.3%) levels amongst all transgenic lines were detected in event TX-03-31 at boot leaf stage. While C18:3Δ9,12,15typically dominated the TFA fraction, TAG compositions in this population displayed a high degree of variability. Of note, some events exhibited increases in levels of palmitic acid (C16:0) and/or linoleic acid (LA, C18:2Δ9,12) at the expense of ALA. Indeed, the ALA level in both TFA and TAG contents was reduced below 40% in some events, less than 30% in selected events. The ALA level in TAG was less than 20% in some selected events. Plants containing the higher levels of TFA and TAG were propagated by separating tillers and transplanting them into soil in new pots. The tillers produced new roots and continued to grow. When leaf samples of the new plants were analysed, TAG levels of 8.3% in a TFA level of 9.3% were observed. Sixteen primary transformants containing both pOIL197 (pZmUbi:DGAT and pZmUbi:Oleosin) and pOIL104 (pSSU:WRI1) were analysed for TFA and TAG contents and fatty acid composition. Leaves of primary transformants containing both pOIL197 and pOIL104 T-DNA regions, sampled at vegetative stage of growth were observed with 4.1% and 5.9% TFA (Table 24). Surprisingly, the highest TFA levels detected in this population were accompanied by a relatively low TAG content. TAG levels in pOIL104+pOIL197 transgenic plants at vegetative and boot leaf stages reached only to 0.6% and 2.8%. Increased TAG levels were typically associated with a reduction in C18:3Δ9,12,15and an increase in both palmitic acid and LA. The TFA and TAG levels in many independent transformed plants are shown schematically inFIG.19. Expression levels of the WRI1 and DGAT1 genes in a number of plants were measured by a RT-PCR method. It was observed that plant TX-03-31 which had a relatively high TTQ had the highest level of expression of DGAT amongst the tested plants. It was concluded that high levels of DGAT expression were beneficial for increasing the TAG level and also the TTQ. Perhaps the most surprising and unexpected conclusion drawn from the large amount of data in this Example was the relatively high level of TFA accompanied by the low levels of TAG, except in a few exceptional plants such as plant TX-03-31 (Table 22). That is, although substantially much increased fatty acid synthesis was occurring, much of the increased fatty acid was not appearing as TAG. This conclusion was completely the opposite of what had been observed with the WRI1+DGAT transgenic plants forNicotianaincluding tobacco. To quantitate this in thesorghumplants, the quotient of the TAG to TFA level was calculated for all of the above mentioned transgenicsorghumpopulations (Tables 11-24). The TAG/TFA Quotient (TTQ) parameter was calculated as the level of TAG (%) divided by the level of TFA (%), each as a percentage of the dry weight of the plant material (leaf in this case). It was observed that for many of thesorghumlines, the TTQ was in the range of 0.01 to 0.6. Addition of one or more further genetic modifications to the plants which provide for a reduction in the level of SDP1, TGD or TST, or an increase in the levels of one or more of PDAT, PDCT or CPT polypeptides increases the TTQ to above 0.6 for a larger proportion of the plant lines. In particular, reduction in TAG lipase in the plants increases the TTQ to up to 0.95. Due to the large difference in absolute TFA and TAG levels in many transgenic lines, the inventors selected two pOIL102+pOIL197 events for quantification of the major neutral and polar lipid classes, to determine the type of lipid in which the high level of fatty acids was present. The types of lipid were separated by TLC and quantitated. At the vegetative stage of growth, TX-02-8 and TX-02-19 contained 4.5% and 7.2% TFA, respectively (Table 18). TAG content was only slightly increased in the TX-02-8 leaves while the levels of phosphatidylcholine (PC, a phospholipid) and the galactolipid MGDG were comparable to the negative controls. TX-02-19 exhibited increased TAG, PC and MGDG levels, indicating an increase in both neutral and polar lipid classes. A more detailed lipid analysis was performed on the TX-03-8 plant (boot leaf stage) and TX-03-28 (vegetative stage) (FIG.13). A wildtype (flowering) and empty vector transformant (vegetative stage) served as controls for comparison. Despite differences in plant age at the time of sampling, leaves of both transgenic plants contained increased levels of TFA and total polar lipids. TX-03-28 contained up to 3.4% TAG at vegetative stage while TAG levels in TX-03-8 were only slightly increased at boot leaf stage. Both transgenic lines exhibited surprisingly large increases in the amounts of the galactolipids MGDG and DGDG. Increases in different polar lipid classes, the phospholipids PC, PG, PE, PA, PS, PI, were less pronounced but still significant (FIG.13B). Further investigation by LC-MS revealed increased levels of C18:0, C18:2Δ9,12and C18:3Δ9,12,15in the free fatty acid fraction of both transgenic lines, suggesting a flux through PC via acyl editing prior to lipolysis. DAG molecular species in transgenic leaf tissues that were increased included 34:2 (likely C16:0/C18:2Δ9,12), 34:3 (likely C16:0/C18:3Δ9,12,15), 36:4 (likely C18:2Δ9,12/C18:2Δ9,12and C18:1Δ9/C18:3Δ9,12,15) and 36:5 (likely C18:2Δ9,12/C18:3Δ9,12,15). The enrichment of poly-unsaturated fatty acids in the DAG fraction matched with the TAG composition and suggested PC-derived DAG as the precursor to TAG synthesis. Similar changes in PC and PE molecular species were observed in both transgenic plants while PI species mainly had C16:0 and C18 fatty acids. PG molecular species were highly enriched in C16:0, reflecting their plastidial synthesis via the prokaryotic pathway. Galactolipids in both transgenic lines were mainly derived from the eukaryotic lipid pathway i.e. enriched in C18 fatty acids. The major MGDG molecular species was 36:6 (likely C18:3Δ9,12,15/C18:3Δ9,12,15), serving as a substrate for DGDG 36:6 synthesis. A second major DGDG species in both transgenic lines, 34:3 (likely C16:0/C18:3Δ9,12,15), was also likely from extra-plastidial origin. TAG molecular species consisting of C16/C16/C18 (48:X), C16/C16/C18 (50:X) and C16/C18/C18 (52:x) were increased in transgenic leaf tissues. Interestingly, 54:8 (likely C18:2Δ9,12/C18:3Δ9,12,15/C18: 3Δ9,12,15) and 54:9 (likely C18:3Δ9,12,15/C18:3Δ9,12,15/C18:3Δ9,12,15) were reduced compared to the negative controls. Taken together, these results suggest increased flux of acyl chains into TAG via PC in the transgenic lines whilst galactolipid biosynthesis mainly occured via the eukaryotic pathway. These data also led the inventors to understand that reduction of TGD activity or increases in PDCT and/or CPT in the plants in addition to the present transgenes would likely enhance the TFA and TAG levels. The chimeric DNA constructs forAgrobacterium-mediated transformation are used to transformZea mays(corn) as described by Gould et al. (1991). Briefly, shoot apex explants are co-cultivated with transgenicAgrobacteriumfor two days before being transferred onto a MS salt media containing kanamycin and carbenicillin. After several rounds of sub-culture, transformed shoots and roots spontaneously form and are transplanted to soil. The constructs are similarly used to transformHordeum vulgare(barley) andAvena sativa(oats) using transformation methods known for these species. Briefly, for barley, theAgrobacteriumcultures are used to transform cells in immature embryos of barley (cv. Golden Promise) according to published methods (Tingay et al., 1997; Bartlett et al., 2008) with some modifications in that embryos between 1.5 and 2.5 mm in length are isolated from immature caryopses and the embryonic axes removed. The resulting explants are co-cultivated for 2-3 days with the transgenicAgrobacteriumand then cultured in the dark for 4-6 weeks on media containing timentin and hygromycin to generate embryogenic callus before being moved to transition media in low light conditions for two weeks. Calli are then transferred to regeneration media to allow for the regeneration of shoots and roots before transfer of the regenerated plantlets to soil. Transformed plants are obtained and grown to maturity in the glasshouse. TABLE 11TFA and TAG levels, fatty acid composition and TTQ in wild-type (WT) andempty vector (EV) negative controls during different stages of plant development.TAGorStageLineTFAC16:0C18:0C18:1C18:2C18:3OtherTFATAGTTQVegWT1TFA9.91.20.78.875.44.01.7VegWT1TAG22.53.63.031.837.41.60.00.027VegWT2TFA12.01.70.78.573.04.22.2VegWT2TAG12.13.22.129.052.31.40.10.028VegWT3TFA15.31.50.710.069.82.72.7VegWT3TAG17.46.52.627.238.08.30.00.000VegWT6TFA12.21.80.57.772.85.13.3VegWT6TAG18.86.83.717.444.78.50.10.017VegEV1TFA13.02.10.99.670.63.82.0VegEV1TAG6.52.81.619.251.418.50.20.090VegEV3TFA12.11.90.99.472.73.02.1VegEV3TAG9.73.82.325.157.61.60.10.056BLEV1TFA17.61.91.514.759.05.41.5BLEV1TAG17.56.53.730.735.65.90.00.031BLWT3TFA14.43.92.411.162.65.61.1BLWT3TAG9.44.84.019.161.21.60.20.153MSSWT3TFA14.23.92.210.263.65.91.2MSSWT3TAG15.312.53.918.243.96.20.10.067MSSEV3TFA16.55.01.612.750.613.60.7MSSEV3TAG13.411.42.619.650.03.00.10.192Veg: Vegetative;BL, Boot leaf stage of growth;MSS, Mature seed setting stage TABLE 12TFA and TAG levels, fatty acid composition and TTQ in sorghum leavestransformed with pOIL197 or pOIL136 (pZmUbi:DGAT; pZmUbi:Oleosin) duringthe vegetative stage of growth. The lines are listed in order of increasing TFA levels.TAGorLineTFAC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-197-18TFA16.33.71.713.359.75.30.7TX-197-18TAG13.95.02.722.253.03.30.10.188TX-197-12TFA15.42.51.613.856.710.01.0TX-197-12TAG12.64.03.528.647.83.40.10.106TX-197-04TFA12.83.71.59.265.47.41.2TX-197-04TAG8.05.03.116.665.32.10.20.169TX-136-03TFA13.92.22.011.765.54.81.2TX-136-03TAG12.13.84.227.850.51.60.10.064TX-197-06TFA13.82.71.710.963.37.51.2TX-197-06TAG9.84.03.522.656.63.40.10.107TX-197-20TFA15.32.61.512.161.47.21.2TX-197-20TAG13.54.23.325.550.33.10.10.085TX-136-24TFA12.22.01.610.969.04.31.5TX-136-24TAG11.73.33.023.355.92.80.40.243TX-197-16TFA14.42.21.713.561.17.21.9TX-197-16TAG14.83.53.225.347.95.30.40.235TX-197-05TFA12.22.31.39.968.26.12.0TX-197-05TAG10.44.32.921.058.72.70.10.070TX-197-17TFA14.02.22.419.555.46.52.1TX-197-17TAG13.73.34.433.840.44.40.60.264TX-197-22TFA11.91.70.98.571.65.42.1TX-197-22TAG11.54.32.423.955.22.80.10.041TX-197-21TFA10.81.60.97.973.35.52.4TX-197-21TAG9.93.82.624.257.02.50.10.045TX-197-10TFA10.51.50.89.372.85.22.7TX-197-10TAG9.02.82.426.655.63.70.20.078TX-197-50TFA12.91.81.010.868.15.32.8TX-197-50TAG14.74.42.523.148.86.60.30.107TX-197-07TFA10.51.40.810.171.85.42.8TX-197-07TAG9.62.92.531.349.64.10.20.067TX-197-48TFA13.21.81.211.467.05.42.8TX-197-48TAG10.13.12.525.553.15.60.30.104TX-197-08TFA11.41.11.412.468.15.62.9TX-197-08TAG15.93.76.145.223.25.80.10.027TX-197-13TFA10.81.60.78.073.55.42.9TX-197-13TAG10.53.62.224.151.28.40.10.037TX-197-15TFA10.51.30.78.973.05.62.9TX-197-15TAG9.62.82.226.955.33.30.20.067TX-136-02TFA12.51.51.314.366.14.32.9TX-136-02TAG14.02.62.727.348.45.00.70.245TX-197-19TFA10.91.40.89.173.04.83.1TX-197-19TAG11.13.02.327.352.63.60.20.063TX-197-40TFA9.91.10.58.277.43.03.1TX-197-40TAG15.46.32.327.146.72.20.00.008TX-197-47TFA11.92.00.77.373.05.23.2TX-197-47TAG10.43.62.419.760.13.80.10.028TX-197-49TFA12.01.72.116.063.15.13.2TX-197-49TAG13.53.86.636.931.97.30.30.085TX-197-28TFA11.11.30.48.075.63.53.2TX-197-28TAG17.54.91.322.347.46.60.10.024TX-197-14TFA9.81.20.810.272.85.23.3TX-197-14TAG9.42.73.539.439.55.50.10.045TX-197-51TFA12.52.01.010.668.35.63.4TX-197-51TAG14.04.52.322.449.87.00.40.122TX-136-01TFA12.51.51.313.369.12.33.4TX-136-01TAG15.03.12.827.844.96.40.80.234TX-197-11TFA10.21.10.911.271.15.53.5TX-197-11TAG12.23.34.643.330.06.60.10.034TX-197-33TFA10.91.40.48.075.73.63.5TX-197-33TAG14.04.71.620.453.06.30.10.025TX-136-25TFA13.12.40.611.567.54.93.8TX-136-25TAG15.84.41.221.149.77.80.80.202TX-197-09TFA10.51.30.79.473.05.13.8TX-197-09TAG11.53.52.430.448.43.90.20.047TX-197-30TFA11.81.70.68.973.04.03.8TX-197-30TAG15.34.11.622.051.35.70.20.051TX-197-23TFA10.51.41.414.167.55.14.3TX-197-23TAG13.13.03.736.338.75.30.80.175TX-197-37TFA10.32.02.418.662.83.95.0TX-197-37TAG12.94.06.238.731.66.71.20.230 TABLE 13TFA and TAG levels, fatty acid composition and TTQ in sorghum leavestransformed with pOIL197 or pOIL136 (pZmUbi:DGAT; pZmUbi:Oleosin) duringthe boot leaf stage of growth. The lines are listed in order of increasing TFA levels.TAGorLineTFAC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-197-14TFA12.75.22.014.457.78.11.2TX-197-14TAG8.87.13.122.754.73.60.30.266TX-197-15TFA14.55.02.314.755.87.71.2TX-197-15TAG12.77.13.221.051.74.30.30.262TX-197-19TFA13.13.22.014.360.96.41.2TX-197-19TAG10.64.33.424.454.03.20.20.203TX-136-03TFA14.11.81.712.665.04.81.2TX-136-03TAG14.54.34.532.942.21.60.10.045TX-197-08TFA14.43.51.314.262.24.41.2TX-197-08TAG13.75.22.722.450.55.50.30.211TX-197-11TFA14.13.82.015.057.08.21.3TX-197-11TAG10.34.83.022.855.93.10.30.267TX-136-24TFA15.52.22.216.958.15.21.3TX-136-24TAG14.73.34.032.442.92.70.20.164TX-136-02TFA12.31.51.414.765.74.41.5TX-136-02TAG13.92.73.028.746.65.10.70.444TX-197-30TFA13.12.31.39.365.18.82.0TX-197-30TAG10.03.02.215.065.34.50.40.223TX-197-46TFA13.22.50.87.971.24.52.0TX-197-46TAG17.318.63.214.742.53.70.10.033TX-197-45TFA13.62.70.66.771.74.52.1TX-197-45TAG22.717.74.412.938.63.60.10.030TX-197-39TFA12.63.61.19.066.27.42.1TX-197-39TAG9.54.01.612.866.75.50.60.291TX-197-22TFA13.62.00.87.371.34.92.1TX-197-22TAG13.83.31.814.264.62.30.10.056TX-197-34TFA12.03.21.29.667.95.92.2TX-197-34TAG9.14.62.318.463.22.30.40.190TX-197-50TFA13.02.51.19.166.87.52.5TX-197-50TAG11.44.62.115.359.86.90.50.183TX-197-43TFA12.42.30.78.071.94.72.5TX-197-43TAG11.04.41.815.762.34.80.20.065TX-197-32TFA12.52.11.19.070.05.32.5TX-197-32TAG12.83.72.116.160.35.00.60.220TX-197-33TFA12.12.70.77.971.05.62.5TX-197-33TAG11.14.81.415.462.44.90.30.130TX-197-41TFA12.81.90.78.172.83.72.6TX-197-41TAG15.15.92.416.753.76.30.20.065TX-197-36TFA12.22.00.87.771.65.62.6TX-197-36TAG11.43.41.613.965.64.10.40.158TX-197-42TFA12.42.10.88.270.36.32.7TX-197-42TAG12.45.42.317.857.15.00.20.060TX-197-51TFA13.62.11.09.966.86.62.7TX-197-51TAG13.14.63.018.853.47.00.50.175TX-197-49TFA15.22.91.09.365.36.32.7TX-197-49TAG17.35.02.016.752.76.30.50.192TX-197-48TFA13.02.31.08.868.56.42.8TX-197-48TAG13.04.72.216.158.06.00.40.144TX-197-38TFA12.22.01.07.772.15.02.9TX-197-38TAG11.23.42.214.963.84.50.50.160TX-197-35TFA12.81.80.98.569.46.62.9TX-197-35TAG12.72.91.714.563.34.90.70.227TX-197-40TFA12.71.90.77.773.93.12.9TX-197-40TAG16.34.73.320.852.42.60.10.031TX-197-47TFA13.92.40.66.972.23.92.9TX-197-47TAG24.619.85.210.734.84.90.00.017TX-136-01TFA11.61.41.314.167.24.33.3TX-136-01TAG14.62.93.029.544.15.90.70.199TX-197-44TFA13.52.11.414.763.15.13.4TX-197-44TAG14.44.33.125.045.08.20.80.245TX-136-25TFA13.62.20.710.867.45.23.4TX-136-25TAG16.64.21.420.151.56.11.00.286TX-197-28TFA11.51.30.47.875.33.63.4TX-197-28TAG17.44.51.619.550.26.90.10.035TX-197-37TFA12.63.46.317.454.16.24.5TX-197-37TAG13.45.010.127.440.23.91.90.426 TABLE 14TFA and TAG levels, fatty acid composition and TTQ in sorghum leaves transformedwith pOIL197 or pOIL136 (pZmUbi:DGAT; pZmUbi:Oleosin) during the matureseed setting stage of growth. The lines are listed in order of increasing TFA levels.TAGorLineTFAC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-197-13TFA15.26.62.612.044.718.81.0TX-197-13TAG10.27.02.720.455.64.10.10.131TX-197-22TFA16.04.32.38.554.614.31.0TX-197-22TAG13.87.63.512.759.72.70.20.153TX-197-19TFA13.65.31.112.456.411.21.1TX-197-19TAG10.88.31.618.655.25.50.20.209TX-197-18TFA14.24.92.611.252.914.11.1TX-197-18TAG10.67.82.918.956.23.50.20.148TX-136-24TFA15.14.61.512.757.68.51.2TX-136-24TAG11.35.22.118.956.56.10.20.191TX-197-15TFA13.26.51.114.457.67.31.3TX-197-15TAG9.28.21.721.154.05.80.30.239TX-197-10TFA12.87.61.615.250.012.81.3TX-197-10TAG8.97.71.922.653.95.00.40.301TX-197-11TFA13.55.81.714.057.18.01.3TX-197-11TAG9.06.72.220.356.94.90.30.242TX-197-33TFA14.84.91.812.654.910.91.3TX-197-33TAG12.36.22.621.351.36.30.50.372TX-197-20TFA15.43.81.19.462.77.61.3TX-197-20TAG21.913.93.917.636.46.30.10.043TX-197-21TFA14.83.61.313.061.06.31.4TX-197-21TAG24.914.94.522.727.35.70.00.026TX-197-09TFA15.65.01.815.153.68.91.5TX-197-09TAG13.66.12.621.351.74.70.40.277TX-197-38TFA13.94.21.412.059.78.71.6TX-197-38TAG12.36.42.721.649.77.40.40.230TX-197-32TFA14.23.61.513.358.58.91.7TX-197-32TAG12.35.22.722.150.57.30.50.279TX-197-17TFA14.43.51.512.457.011.32.0TX-197-17TAG14.04.91.517.052.110.40.70.333TX-197-40TFA13.33.51.28.663.99.52.1TX-197-40TAG13.57.92.216.256.04.20.10.042TX-197-16TFA13.94.71.213.854.112.32.1TX-197-16TAG10.95.91.718.651.811.10.90.444 TABLE 15TFA and TAG levels, fatty acid composition and TTQ in sorghum leavestransformed with pOIL102 (pZmUbi:WRI1) during the vegetative stage of growth.TAGorLineTFAC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-102-1TFA17.32.43.115.655.85.91.2TX-102-1TAG13.52.65.628.743.56.10.20.182TX-102-6TFA12.41.41.19.671.73.82.0TX-102-6TAG21.213.44.627.332.31.30.00.015TX-102-4TFA11.21.00.77.776.43.02.2TX-102-4TAG11.33.32.023.759.60.00.00.019TX-102-8TFA10.21.20.57.277.93.02.3TX-102-8TAG11.63.40.023.261.80.00.00.013TX-102-5TFA11.11.60.98.874.33.32.4TX-102-5TAG17.112.20.027.543.20.00.00.015TX-102-2TFA11.41.51.09.473.53.22.4TX-102-2TAG13.72.93.631.248.60.00.00.018TX-102-3TFA11.81.51.08.873.33.72.6TX-102-3TAG17.13.74.429.944.00.90.00.016TX-102-7TFA12.11.41.09.372.43.82.6TX-102-7TAG20.915.04.826.431.61.30.00.013 TABLE 16TFA and TAG levels, fatty acid composition and TTQ in sorghum leavestransformed with pOIL102 (pZmUbi:WRI1) during the boot leaf stage of growth.TAGorLineTFAC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-102-8TFA16.94.22.312.357.76.50.9TX-102-8TAG14.56.213.525.736.83.40.20.243TX-102-4TFA17.14.22.012.557.56.70.9TX-102-4TAG10.54.43.020.059.62.60.20.182TX-102-1TFA16.64.33.915.450.79.11.1TX-102-1TAG10.74.45.321.954.13.60.30.273TX-102-5TFA16.74.11.711.660.25.81.1TX-102-5TAG11.75.52.821.456.12.50.10.118TX-102-6TFA17.83.815.917.038.86.61.5TX-102-6TAG19.67.029.425.413.94.70.40.267TX-102-2TFA15.01.91.719.156.55.91.7TX-102-2TAG10.61.92.730.251.23.40.40.258TX-102-7TFA15.03.17.013.956.14.92.4TX-102-7TAG16.16.520.528.024.44.50.30.111TX-102-3TFA14.43.59.513.450.98.22.5TX-102-3TAG16.96.723.924.722.55.20.40.150 TABLE 17TFA and TAG levels, fatty acid composition and TTQ in sorghum leaves transformedwith pOIL102 (pZmUbi:WRI1) during the mature seed setting stage of growth.TAGorLineTFAC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-102-5TFA17.05.21.811.253.211.51.0TX-102-5TAG15.77.63.519.849.73.80.10.090TX-102-8TFA17.15.02.612.550.012.81.0TX-102-8TAG18.09.44.621.541.54.90.10.096TX-102-1TFA17.25.22.617.745.511.91.0TX-102-1TAG13.36.84.026.543.85.60.20.203TX-102-9TFA15.95.11.612.953.810.81.1TX-102-9TAG14.07.23.224.148.33.20.10.089TX-102-4TFA17.45.33.112.048.413.71.1TX-102-4TAG15.46.24.122.048.14.20.10.092TX-102-6TFA18.24.76.318.640.911.31.5TX-102-6TAG18.47.614.531.721.06.80.20.147TX-102-2TFA14.46.829.718.818.811.42.0TX-102-2TAG12.39.140.321.87.49.00.90.456 TABLE 18TFA and TAG levels, fatty acid composition and TTQ in sorghum leavestransformed with pOIL102 (pZmUbi:WRI1) and pOIL197 (pZmUbi:DGATand pZmUbi:Oleosin) during the vegetative stage of growth. The lines arelisted in order of increasing TFA levels.TAGorLineTFAC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-02-28TFA12.02.40.69.571.24.42.2TX-02-28TAG11.65.01.416.161.14.80.20.081TX-02-18TFA12.92.30.810.069.64.42.2TX-02-18TAG11.14.91.921.758.22.20.10.059TX-02-37TFA8.71.20.47.079.13.72.3TX-02-37TAG18.36.50.024.045.75.50.00.013TX-02-29TFA12.02.60.57.572.35.12.4TX-02-29TAG10.03.81.314.566.14.30.10.041TX-02-126TFA13.21.50.610.070.54.12.6TX-02-126TAG17.43.31.622.349.85.60.20.085TX-02-23TFA11.02.90.45.973.16.82.6TX-02-23TAG11.13.91.612.966.73.90.10.048TX-02-38TFA19.62.03.120.547.86.92.7TX-02-38TAG28.43.45.831.721.59.32.20.832TX-02-24TFA10.92.50.46.374.55.32.8TX-02-24TAG16.15.22.411.658.36.40.10.033TX-02-25TFA10.92.10.68.972.25.32.9TX-02-25TAG9.54.31.515.761.77.30.30.099TX-02-31TFA9.31.20.68.776.43.73.1TX-02-31TAG24.57.24.533.730.20.00.00.007TX-02-129TFA11.31.40.69.074.03.83.2TX-02-129TAG18.75.02.228.138.77.20.10.026TX-02-34TFA10.11.30.810.273.44.23.3TX-02-34TAG14.03.42.528.246.35.60.30.098TX-02-127TFA11.31.60.46.677.32.73.4TX-02-127TAG14.65.61.916.652.98.50.00.012TX-02-09TFA11.92.10.68.773.53.33.5TX-02-09TAG12.45.01.821.956.03.00.10.024TX-02-131TFA11.01.40.38.175.93.23.5TX-02-131TAG16.94.91.121.348.86.90.10.023TX-02-33TFA8.61.10.58.478.13.43.5TX-02-33TAG19.95.93.028.734.97.50.00.010TX-02-36TFA9.51.30.811.073.54.03.6TX-02-36TAG13.73.82.633.741.74.60.30.071TX-02-35TFA9.21.30.46.877.94.33.6TX-02-35TAG21.67.72.020.539.39.00.00.012TX-02-10TFA12.32.03.420.856.64.74.0TX-02-10TAG18.54.07.838.523.67.51.00.250TX-02-30TFA14.93.81.914.359.06.14.1TX-02-30TAG18.67.64.124.833.711.20.90.223TX-02-12TFA13.71.60.810.169.04.74.5TX-02-12TAG10.54.21.726.255.51.90.10.024TX-02-08TFA16.62.21.911.063.94.54.5TX-02-08TAG22.65.66.424.234.27.00.20.039TX-02-27TFA10.91.20.58.975.82.74.6TX-02-27TAG19.06.02.727.839.25.30.00.011TX-02-13TFA14.61.51.112.965.44.44.6TX-02-13TAG11.95.13.834.039.55.70.30.062TX-02-05TFA14.31.40.912.166.64.75.2TX-02-05TAG10.43.04.042.735.84.10.20.031TX-02-21TFA13.81.00.610.967.95.75.3TX-02-21TAG9.03.21.223.159.34.20.60.121TX-02-07TFA15.61.70.68.668.94.65.5TX-02-07TAG21.86.43.624.634.88.80.10.019TX-02-11TFA21.01.90.68.962.35.25.6TX-02-11TAG28.410.53.822.827.17.40.20.027TX-02-14TFA15.42.41.811.564.64.25.7TX-02-14TAG17.06.06.132.132.66.10.20.029TX-02-16TFA19.81.64.225.843.55.05.7TX-02-16TAG25.72.57.538.818.66.92.70.481TX-02-01TFA13.91.40.610.569.14.65.8TX-02-01TAG9.43.32.429.951.93.10.10.012TX-02-02TFA15.21.80.810.567.34.45.8TX-02-02TAG12.73.73.335.639.15.60.20.036TX-02-06TFA17.71.50.79.466.34.26.1TX-02-06TAG25.63.93.023.935.28.40.20.033TX-02-04TFA12.81.31.011.868.74.56.3TX-02-04TAG17.94.03.732.735.95.80.10.013TX-02-19TFA11.91.81.515.664.54.77.2TX-02-19TAG10.93.95.241.930.67.50.70.097 TABLE 19TFA and TAG levels, fatty acid composition and TTQ in sorghum leavestransformed with pOIL102 (pZmUbi:WRI1) and pOIL197 (pZmUbi:DGAT andpZmUbi:Oleosin) during the boot leaf stage of growth. The lines are listed inorder of increasing TFA levels.TAGorLineTFAC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-02-27TFA17.33.81.410.160.17.21.0TX-02-27TAG11.94.42.119.461.20.80.20.164TX-02-21TFA15.92.32.019.353.37.31.2TX-02-21TAG12.63.72.727.051.03.00.40.318TX-02-01TFA15.24.25.114.753.27.51.3TX-02-01TAG11.75.69.326.142.94.50.30.199TX-02-12TFA15.33.22.013.658.96.91.3TX-02-12TAG13.74.23.625.150.42.90.10.111TX-02-33TFA15.94.31.010.159.79.11.4TX-02-33TAG14.35.42.718.954.74.00.10.107TX-02-13TFA15.45.111.419.439.19.51.4TX-02-13TAG12.96.520.325.228.66.40.50.389TX-02-36TFA16.23.41.812.358.57.81.4TX-02-36TAG15.45.83.321.548.95.10.30.209TX-02-37TFA13.33.51.39.965.36.71.4TX-02-37TAG9.63.63.820.460.62.10.20.137TX-02-18TFA14.63.01.49.865.55.71.4TX-02-18TAG12.55.64.320.654.82.30.10.077TX-02-34TFA16.62.22.217.654.76.71.4TX-02-34TAG14.12.84.130.344.74.10.30.231TX-02-31TFA13.33.11.810.164.77.01.5TX-02-31TAG5.41.83.217.871.10.70.30.171TX-02-29TFA13.23.21.18.268.65.61.6TX-02-29TAG10.54.72.918.162.01.80.10.082TX-02-35TFA17.83.46.514.050.38.01.6TX-02-35TAG18.85.319.128.422.46.10.20.108TX-02-09TFA14.03.30.99.966.06.01.6TX-02-09TAG11.24.71.919.658.73.90.10.036TX-02-24TFA12.93.50.67.967.37.71.8TX-02-24TAG10.73.51.611.869.03.40.10.044TX-02-126TFA13.82.71.19.966.46.01.8TX-02-126TAG12.84.32.117.058.65.20.50.247TX-02-23TFA13.62.70.78.968.35.81.9TX-02-23TAG10.03.32.218.263.92.40.10.047TX-02-07TFA17.52.310.917.544.57.31.9TX-02-07TAG21.03.924.527.415.28.00.40.225TX-02-28TFA12.82.90.57.768.47.82.0TX-02-28TAG13.05.51.211.164.34.80.10.063TX-02-04TFA13.62.91.212.165.34.92.1TX-02-04TAG12.04.42.421.655.93.60.40.206TX-02-25TFA12.22.80.59.468.86.32.5TX-02-25TAG10.34.21.015.462.56.60.40.159TX-02-05TFA13.63.63.214.759.85.12.5TX-02-05TAG12.25.57.026.843.45.10.60.220TX-02-14TFA15.95.730.912.726.08.92.8TX-02-14TAG17.98.542.614.97.88.41.40.514TX-02-131TFA12.61.40.68.373.13.92.9TX-02-131TAG16.03.91.918.053.96.30.20.061TX-02-129TFA12.11.61.010.470.54.32.9TX-02-129TAG12.83.62.522.053.65.50.30.106TX-02-08TFA17.62.65.617.251.25.83.0TX-02-08TAG24.45.915.829.315.88.80.60.183TX-02-02TFA17.93.17.215.549.66.73.1TX-02-02TAG23.76.517.722.819.69.70.60.194TX-02-11TFA25.14.19.016.336.39.13.2TX-02-11TAG33.36.613.920.916.09.31.10.341TX-02-127TFA11.41.60.38.975.42.43.5TX-02-127TAG21.05.81.420.647.43.90.10.016TX-02-30TFA16.43.13.717.153.85.94.0TX-02-30TAG21.35.07.627.130.58.50.90.236TX-02-19TFA13.52.725.422.630.85.04.2TX-02-19TAG14.03.334.327.016.64.82.30.548TX-02-06TFA24.04.814.319.629.77.74.8TX-02-06TAG29.76.919.223.013.47.72.70.555TX-02-10TFA22.03.310.322.733.77.96.3TX-02-10TAG24.84.112.927.022.48.83.50.551TX-02-38TFA24.84.413.924.523.78.76.4TX-02-38TAG21.55.38.625.239.30.02.50.392 TABLE 20TFA and TAG levels, fatty acid composition and TTQ in sorghum leavestransformed with pOIL102 (pZmUbi:WRI1) and pOIL197 (pZmUbi:DGATand pZmUbi:Oleosin) during the mature seed setting stage of growth.TAGorLineTFAC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-02-18TFA15.65.51.113.254.310.30.8TX-02-18TAG14.27.72.522.749.23.70.10.133TX-02-31TFA15.64.41.611.155.911.40.9TX-02-31TAG12.36.33.219.856.22.20.20.163TX-02-37TFA14.84.71.810.357.510.81.0TX-02-37TAG9.65.83.220.658.62.10.10.147TX-02-12TFA16.43.81.713.054.810.21.0TX-02-12TAG15.16.23.321.050.04.40.30.258TX-02-29TFA14.94.71.19.860.09.51.1TX-02-29TAG14.412.72.417.550.62.40.10.125TX-02-01TFA14.94.61.410.559.09.51.3TX-02-01TAG15.86.43.117.354.33.10.10.083TX-02-23TFA14.34.61.48.263.58.01.3TX-02-23TAG9.96.12.613.248.519.80.10.104TX-02-09TFA14.14.50.98.465.07.01.4TX-02-09TAG16.411.72.414.051.63.80.10.052TX-02-24TFA15.14.30.911.659.38.81.5TX-02-24TAG14.57.42.323.848.04.10.10.094TX-02-28TFA14.33.50.78.665.97.01.5TX-02-28TAG16.313.21.912.452.04.20.10.074TX-02-34TFA15.23.81.415.354.210.11.5TX-02-34TAG13.35.82.222.347.09.50.50.347TX-02-27TFA14.82.80.98.967.55.11.5TX-02-27TAG15.911.93.520.246.51.90.10.049TX-02-33TFA14.64.12.413.357.28.41.5TX-02-33TAG12.57.34.121.949.15.20.30.200TX-02-07TFA12.34.20.88.469.94.41.5TX-02-07TAG10.65.52.317.860.82.90.10.042TX-02-05TFA15.46.57.021.739.310.21.6TX-02-05TAG13.18.311.430.128.98.30.60.376TX-02-08TFA18.42.82.514.452.59.51.9TX-02-08TAG25.06.27.623.727.310.20.20.128TX-02-127TFA12.62.80.67.571.94.52.4TX-02-127TAG11.95.02.114.663.42.90.10.026TX-02-38TFA41.914.119.68.71.514.23.0TX-02-38TAG25.39.932.516.12.713.41.10.365TX-02-02TFA16.56.828.215.121.212.23.5TX-02-02TAG16.59.839.116.57.110.91.70.496TX-02-06TFA25.34.812.024.319.314.34.0TX-02-06TAG27.16.214.727.812.411.82.60.658TX-02-30TFA17.04.16.720.243.38.74.3TX-02-30TAG19.65.811.427.426.19.72.20.509TX-02-14TFA13.37.356.16.28.88.36.1TX-02-14TAG13.78.760.16.14.37.14.30.706 TABLE 21TFA and TAG levels, fatty acid composition and TTQ in pOIL103 + pOIL197primary transformants at vegetative setting stage.TFAorLineTAGC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-03-07TFA22.63.31.312.751.88.42.4TX-03-07TAG29.45.63.320.931.39.40.10.056TX-03-02TFA17.72.61.19.164.05.42.4TX-03-02TAG20.25.12.816.447.77.70.20.079TX-03-01TFA16.92.51.28.765.74.92.8TX-03-01TAG18.85.43.317.347.77.50.30.096TX-03-52TFA13.81.40.88.970.64.52.9TX-03-52TAG23.24.32.319.642.87.80.20.082TX-03-47TFA14.11.60.66.573.53.73.0TX-03-47TAG20.63.93.818.048.65.20.10.023TX-03-17TFA15.31.40.57.172.13.63.0TX-03-17TAG29.44.02.016.841.66.30.10.039TX-03-05TFA23.22.00.68.161.24.93.0TX-03-05TAG43.94.41.614.428.96.80.20.053TX-03-53TFA19.61.91.110.961.05.63.1TX-03-53TAG35.33.93.120.530.76.50.30.082TX-03-19TFA20.81.80.69.163.93.93.1TX-03-19TAG39.94.41.817.226.99.80.20.056TX-03-10TFA27.84.31.121.038.07.83.1TX-03-10TAG35.27.31.726.319.89.71.40.442TX-03-48TFA21.42.10.89.062.04.83.2TX-03-48TAG39.14.52.315.631.76.80.20.062TX-03-61TFA16.71.31.817.657.45.33.2TX-03-61TAG19.04.62.933.928.910.70.20.047TX-03-32TFA15.61.50.710.767.34.13.2TX-03-32TAG28.84.03.426.529.28.10.10.032TX-03-40TFA15.01.30.69.169.84.23.3TX-03-40TAG27.63.72.225.232.19.20.20.057TX-03-49TFA17.31.50.58.068.04.83.3TX-03-49TAG35.06.91.918.426.411.30.10.015TX-03-21TFA13.11.30.67.873.73.53.3TX-03-21TAG20.34.13.323.143.06.30.10.029TX-03-62TFA18.01.11.913.659.85.53.3TX-03-62TAG26.24.85.730.224.98.30.20.051TX-03-26TFA14.01.50.57.972.33.83.4TX-03-26TAG22.83.83.222.840.56.90.10.023TX-03-36TFA19.71.60.88.963.75.23.5TX-03-36TAG37.13.92.317.130.59.00.30.075TX-03-50TFA16.71.30.89.366.75.23.5TX-03-50TAG35.93.94.021.925.29.20.10.026TX-03-23TFA19.51.60.36.167.15.43.5TX-03-23TAG39.04.31.213.932.79.00.20.044TX-03-45TFA15.01.60.36.271.95.03.5TX-03-45TAG27.14.70.814.141.711.60.30.087TX-03-34TFA20.61.70.811.060.35.63.5TX-03-34TAG36.13.92.121.627.58.90.20.068TX-03-51TFA12.31.30.79.372.93.63.6TX-03-51TAG23.84.82.626.732.29.90.10.034TX-03-63TFA15.71.31.816.959.74.73.7TX-03-63TAG23.93.82.931.726.611.10.20.049TX-03-41TFA21.01.70.68.063.74.93.7TX-03-41TAG44.73.81.715.227.47.10.20.067TX-03-20TFA10.71.50.79.074.73.33.7TX-03-20TAG14.14.02.324.147.38.20.20.061TX-03-29TFA20.31.90.911.061.24.73.7TX-03-29TAG37.14.43.121.227.56.70.20.054TX-03-25TFA12.11.50.56.575.93.53.8TX-03-25TAG17.67.22.716.648.57.30.10.030TX-03-33TFA24.12.20.913.053.26.63.8TX-03-33TAG40.64.31.720.923.39.10.60.168TX-03-22TFA22.31.71.213.854.56.53.9TX-03-22TAG37.93.32.223.423.59.81.00.245TX-03-46TFA24.41.70.79.957.36.04.0TX-03-46TAG45.23.21.417.624.68.00.60.148TX-03-11TFA25.42.81.020.842.97.24.0TX-03-11TAG33.44.81.528.821.69.91.40.337TX-03-18TFA20.82.70.913.956.25.54.1TX-03-18TAG33.67.12.724.821.510.30.30.078TX-03-57TFA12.91.41.815.863.44.64.2TX-03-57TAG14.52.57.641.524.99.00.50.127TX-03-58TFA13.01.51.815.763.34.84.2TX-03-58TAG16.43.44.935.331.28.80.60.148TX-03-54TFA22.81.91.016.451.26.85.0TX-03-54TAG36.03.51.726.121.611.11.20.245TX-03-28TFA28.32.21.016.844.17.65.4TX-03-28TAG40.93.31.423.422.48.62.30.434TX-03-31TFA22.22.22.025.241.66.85.6TX-03-31TAG30.93.63.034.718.59.32.30.410TX-03-04TFA24.33.40.610.555.45.87.0TX-03-04TAG36.16.52.215.931.38.00.10.016TX-03-08TFA22.61.90.66.863.84.38.3TX-03-08TAG46.44.64.211.126.77.00.10.017 TABLE 22TFA and TAG levels, fatty acid composition and TTQ in pOIL103 + pOIL197primary transformants at boot leaf stage.TFAorLineTAGC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-03-20TFA12.22.61.710.367.55.72.1TX-03-20TAG9.43.63.318.163.02.50.40.217TX-03-54TFA13.63.53.012.161.56.42.1TX-03-54TAG14.16.97.022.543.56.00.40.207TX-03-61TFA23.93.11.719.043.98.32.2TX-03-61TAG31.46.63.428.319.610.80.40.159TX-03-02TFA14.93.02.812.160.66.62.2TX-03-02TAG14.85.55.620.646.76.80.50.222TX-03-53TFA18.53.78.915.443.110.42.3TX-03-53TAG20.16.816.724.523.38.60.60.275TX-03-01TFA13.43.03.012.561.86.42.3TX-03-01TAG13.95.57.523.042.67.40.40.164TX-03-47TFA12.82.11.67.570.75.32.4TX-03-47TAG14.85.15.019.352.13.70.10.050TX-03-07TFA18.42.87.615.647.18.52.5TX-03-07TAG25.86.418.725.515.28.50.30.127TX-03-05TFA21.42.31.49.759.16.12.6TX-03-05TAG36.45.63.917.128.48.60.40.168TX-03-49TFA18.13.78.213.252.04.92.6TX-03-49TAG24.18.218.320.918.89.70.50.212TX-03-34TFA19.02.76.015.450.66.42.6TX-03-34TAG24.810.510.923.920.69.30.80.287TX-03-32TFA18.22.21.612.460.25.42.8TX-03-32TAG20.814.63.221.431.58.50.60.204TX-03-04TFA18.83.15.813.450.38.62.9TX-03-04TAG26.77.514.623.119.09.10.30.118TX-03-23TFA18.91.71.07.963.27.32.9TX-03-23TAG25.04.62.518.139.610.20.20.070TX-03-25TFA14.51.80.46.473.53.43.0TX-03-25TAG20.35.11.012.353.67.70.30.110TX-03-18TFA21.12.91.217.846.310.73.0TX-03-18TAG22.65.94.531.122.613.30.40.143TX-03-50TFA16.52.66.112.953.98.03.0TX-03-50TAG20.219.912.919.620.66.80.70.217TX-03-60TFA20.22.90.814.155.76.23.1TX-03-60TAG30.56.21.621.630.29.90.60.202TX-03-21TFA12.31.70.56.874.44.43.2TX-03-21TAG16.14.71.613.157.07.50.20.067TX-03-40TFA17.11.40.48.068.24.93.2TX-03-40TAG34.54.40.914.539.85.90.40.112TX-03-62TFA25.32.91.714.747.97.63.3TX-03-62TAG40.35.63.522.318.79.50.60.171TX-03-36TFA19.52.02.011.458.36.83.5TX-03-36TAG31.24.04.420.029.411.00.60.160TX-03-63TFA25.43.62.618.242.08.23.5TX-03-63TAG33.16.13.824.921.610.41.40.383TX-03-45TFA16.41.40.58.169.14.53.5TX-03-45TAG30.84.61.416.240.76.30.20.058TX-03-17TFA14.21.80.86.971.25.23.6TX-03-17TAG18.74.52.213.552.88.30.40.120TX-03-57TFA18.73.41.513.855.86.83.6TX-03-57TAG23.46.33.021.036.210.11.20.330TX-03-11TFA29.16.42.122.433.07.13.6TX-03-11TAG30.68.52.827.019.711.41.90.510TX-03-48TFA27.13.73.720.637.27.63.7TX-03-48TAG31.25.05.527.123.08.12.10.569TX-03-29TFA20.12.31.713.455.57.13.7TX-03-29TAG33.05.04.124.326.47.20.40.104TX-03-26TFA15.31.60.45.971.35.53.9TX-03-26TAG25.24.61.713.349.75.50.30.074TX-03-10TFA28.66.82.121.833.07.73.9TX-03-10TAG31.08.52.926.718.612.21.90.491TX-03-58TFA16.32.61.314.560.35.04.1TX-03-58TAG20.45.22.824.339.28.21.10.278TX-03-08TFA19.82.00.76.664.95.94.1TX-03-08TAG34.85.22.714.334.58.50.20.051TX-03-33TFA27.42.41.516.346.06.44.2TX-03-33TAG39.25.42.321.920.810.51.60.386TX-03-22TFA19.82.83.111.853.49.14.2TX-03-22TAG28.45.35.419.438.33.21.20.287TX-03-41TFA18.12.63.111.158.07.14.8TX-03-41TAG27.86.06.819.334.95.30.70.139TX-03-46TFA24.62.00.67.957.47.44.9TX-03-46TAG44.74.21.313.431.45.01.10.220TX-03-28TFA28.52.11.323.433.711.06.2TX-03-28TAG36.02.93.129.618.510.03.70.596TX-03-31TFA33.42.94.328.625.55.58.3TX-03-31TAG38.03.64.930.614.88.16.60.789 TABLE 23TFA and TAG levels, fatty acid composition and TTQ in pOIL103 + pOIL197primary transformants at mature seed setting stage.TFAorLineTAGC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-03-52TFA15.56.74.314.348.710.61.2TX-03-52TAG12.77.98.321.441.18.70.40.315TX-03-51TFA15.66.44.313.652.08.01.5TX-03-51TAG13.78.98.218.641.29.50.40.296TX-03-07TFA20.64.213.118.132.111.91.6TX-03-07TAG25.57.823.724.59.39.10.40.227TX-03-04TFA23.93.74.016.538.813.11.7TX-03-04TAG35.36.19.524.614.79.80.20.110TX-03-54TFA16.65.06.716.145.89.91.7TX-03-54TAG16.67.212.422.734.26.90.40.245TX-03-21TFA14.44.21.010.062.77.71.8TX-03-21TAG12.86.61.916.555.46.70.20.133TX-03-08TFA19.33.67.116.445.38.31.9TX-03-08TAG23.57.316.224.619.68.70.40.213TX-03-02TFA16.44.87.522.039.79.51.9TX-03-02TAG15.16.414.030.325.68.70.60.334TX-03-34TFA24.24.95.218.232.415.22.0TX-03-34TAG27.17.38.626.618.112.30.60.298TX-03-17TFA16.94.22.010.655.211.12.2TX-03-17TAG19.76.73.718.441.410.00.20.107TX-03-26TFA19.33.40.89.957.69.12.2TX-03-26TAG23.96.72.018.239.39.90.30.129TX-03-32TFA23.23.91.715.544.511.22.3TX-03-32TAG29.06.33.824.825.610.60.50.206TX-03-41TFA19.74.96.321.736.510.92.3TX-03-41TAG20.97.611.629.320.310.50.80.331TX-03-49TFA21.15.714.919.327.511.42.3TX-03-49TAG22.68.323.724.712.08.80.90.375TX-03-25TFA17.93.20.68.762.67.12.6TX-03-25TAG21.96.31.514.247.78.30.40.149TX-03-40TFA20.83.40.85.859.79.62.7TX-03-40TAG27.66.30.48.646.310.80.70.238TX-03-36TFA22.84.22.615.745.29.52.9TX-03-36TAG27.17.15.022.925.112.90.80.282TX-03-10TFA28.45.31.721.530.312.73.3TX-03-10TAG32.77.82.325.218.613.31.90.570TX-03-46TFA27.53.71.712.241.413.43.7TX-03-46TAG36.45.11.815.129.112.41.60.420TX-03-48TFA26.75.06.524.724.712.34.5TX-03-48TAG28.66.17.628.217.612.03.00.679 TABLE 24TFA and TAG levels, fatty acid composition and TTQ in pOIL104(pSSU:WRI1) + pOIL197 (pZmUbi:DGAT and pZmUbi:Oleosin) primarytransformants at vegetative setting stage.TFAorLineTFAC16:0C18:0C18:1C18:2C18:3n3OtherTFATAGTTQTX-04-02TFA12.61.71.211.368.44.72.7TX-04-02TAG19.28.23.929.435.14.20.00.008TX-04-25TFA12.41.50.78.172.74.73.1TX-04-25TAG21.911.75.319.838.33.00.10.020TX-04-11TFA13.52.00.57.270.86.03.2TX-04-11TAG17.43.93.313.655.26.50.10.019TX-04-27TFA13.11.71.59.867.86.03.2TX-04-27TAG18.23.62.624.144.66.80.40.134TX-04-24TFA12.91.90.67.672.24.83.3TX-04-24TAG24.211.43.717.340.53.10.10.017TX-04-16TFA13.02.90.88.970.04.53.4TX-04-16TAG22.58.14.922.337.54.60.10.023TX-04-30TFA13.01.61.38.770.25.23.5TX-04-30TAG18.53.82.622.546.95.80.30.072TX-04-10TFA18.92.71.08.360.88.33.5TX-04-10TAG34.05.53.217.730.09.50.10.034TX-04-13TFA13.02.00.76.472.75.13.5TX-04-13TAG16.25.03.614.855.94.50.10.017TX-04-19TFA19.42.20.69.962.75.23.5TX-04-19TAG30.24.33.124.833.24.40.10.025TX-04-06TFA11.61.61.011.269.65.13.6TX-04-06TAG14.14.53.427.940.69.40.10.036TX-04-14TFA12.93.33.38.665.76.13.6TX-04-14TAG20.38.94.021.440.25.30.10.024TX-04-04TFA10.71.80.68.074.34.63.9TX-04-04TAG11.010.13.817.256.31.70.20.044TX-04-15TFA17.42.41.112.260.26.54.0TX-04-15TAG28.55.62.223.331.68.90.60.160TX-04-08TFA17.51.91.915.157.56.14.0TX-04-08TAG28.04.54.829.523.59.70.50.130TX-04-22TFA13.13.51.412.963.95.34.1TX-04-22TAG17.17.84.329.533.38.00.60.150TX-04-09TFA13.72.44.120.453.85.54.1TX-04-09TAG17.45.39.538.120.69.10.60.158 The chimeric DNA constructs forAgrobacterium-mediated transformation are used to transformZea mays(corn) as described by Gould et al. (1991). Briefly, shoot apex explants are co-cultivated with transgenicAgrobacteriumfor two days before being transferred onto a MS salt media containing kanamycin and carbenicillin. After several rounds of sub-culture, transformed shoots and roots spontaneously form and are transplanted to soil. The constructs are similarly used to transformHordeum vulgare(barley) andAvena sativa(oats) using transformation methods known for these species. Briefly, for barley, theAgrobacteriumcultures are used to transform cells in immature embryos of barley (cv. Golden Promise) according to published methods (Tingay et al., 1997; Bartlett et al., 2008) with some modifications in that embryos between 1.5 and 2.5 mm in length are isolated from immature caryopses and the embryonic axes removed. The resulting explants are co-cultivated for 2-3 days with the transgenicAgrobacteriumand then cultured in the dark for 4-6 weeks on media containing timentin and hygromycin to generate embryogenic callus before being moved to transition media in low light conditions for two weeks. Calli are then transferred to regeneration media to allow for the regeneration of shoots and roots before transfer of the regenerated plantlets to soil. Transformed plants are obtained and grown to maturity in the glasshouse. Example 6. Modifying Traits in Dicotyledonous Plants Oil content in the dicotyledonous plant speciesTrifolium repens(clover), a legume commonly used as a pasture species, was increased by expressing the combination of WRI1, DGAT and Oleosin genes in vegetative parts. The construct pJP3502 was used to transformT. repensbyAgrobacterium-mediated transformation (Larkin et al., 1996). Briefly, the genetic construct pJP3502 was introduced intoA. tumefaciensvia a standard electroporation procedure. The binary vector also contained a 35S:NptII selectable marker gene within the T-DNA. The transformedAgrobacteriumcells were grown on solid LB media supplemented with kanamycin (50 mg/L) and rifampicin (25 mg/L) and incubated at 28° C. for two days. A single colony was used to initiate a fresh culture. Following 48 hours vigorous culture, theAgrobacteriumcells was used to treatT. repens(cv. Haifa) cotyledons that had been dissected from imbibed seed as described by Larkin et al. (1996). Following co-cultivation for three days the explants were exposed to 25 mg/L kanamycin to select transformed shoots and then transferred to rooting medium to form roots, before transfer to soil. Six transformed plants containing the T-DNA from pJP3502 were obtained and transferred to soil in the glasshouse. Increased oil content was observed in the non-seed tissue of some of the plants, with one plant showing greater than 4-fold increase in TAG levels in the leaves. Such plants are useful as animal feed, for example by growing the plants in pastures, providing feed with an increased energy content per unit weight (energy density) and resulting in increased growth rates in the animals. The construct pJP3502 is also used to transform other leguminous plants such as alfalfa (Medicago sativa) and barrel medic (Medicago truncatula) by the method of Wright et al. (2006) to obtain transgenic plants which have increased TAG content in vegetative parts. The transgenic plants are useful as pasture species or as hay or silage as a source of feed for animals such as, for example, cattle, sheep and horses, providing an increased energy density in the feed. Example 7. Modification of Plastidial GPAT Expression Over-Expression of Plastidial GPAT in Plant Cells A number of experiments were performed to test the hypothesis that the presence of a highly active 16:3 prokaryotic pathway in a plant (i.e. a so-called 16:3 plant) would provide much lower TAG levels in vegetative tissues upon introduction of the gene combination on pJP3502, relative to 18:3 plants. These experiments are described in the following Examples. Initially, the inventors tested whether the high level TAG accumulation observed in transgenicN. benthamianacould be disrupted by over-expression of a plastidial GPAT, increasing the flux in the prokaryotic pathway. A coding region for expression of theArabidopsis thalianaplastidial GPAT, ATS1 (Nishida et al., 1993), was amplified by RT-PCR fromA. thalianatotal RNA and cloned as an EcoRI-PstI fragment into the binary expression vector pJP3343 under the control of the 35S promoter to produce the constitutive expression vector pOIL098. The effect of over-expressing a plastidial GPAT in a high oil leaf background is determined by infiltration of the chimeric vector pOIL098 into high oil leaf tissue. The high oil leaf tissue is generated either by co-infiltration of WRI1 and DGAT binary expression vectors (Example 1) or by infiltrating pOIL098 into leaves of aNicotianaplant stably transformed with the T-DNA from pJP3502 or another high oil vector. Oil content is expected to be reduced in the infiltrated leaf spots co-expressing the ATS1-encoding gene. This is determined by analysing TFA and TAG as proportions of sample dry mass. This is also determined by observing incorporation of labelled acetate into fatty acids produced by microsomes or leaf lysates made from infiltrated leaf spots. Oil Accumulation in a Plastidial GPAT Mutant ofArabidopsis thaliana The ats1 mutant ofA. thalianahas a disruptive mutation in the gene encoding plastidial GPAT which reduced plastidial GPAT activity to a level of only 3.8% of the wild-type (Kunst et al., 1988). Non-seed TAG accumulation levels, at least in leaves, stems and roots, in both parental and ats1 mutantA. thalianais tested and compared. The T-DNA of the pJP3502 construct for over-expression of the combination of genes encoding WRI1, DGAT and Oleosin is introduced by transformation into plants of both genotypes. The gene combination in the T-DNA of pJP3502 increases fatty acid synthesis in both plant backgrounds. However, the accumulation of TAG in the ats1 mutant is expected to be significantly higher on average than in the transgenic plants derived from the wild-type (parental) genotype due to the reduction in plastidial GPAT activity and therefore the reduced flux of fatty acids into the plastidial prokaryotic pathway. The ratio of the fatty acids C16:3 to C18:3 is significantly reduced in leaves of the ats1 mutant, both transformed and untransformed. Silencing the Gene Encoding Plastidial GPAT in Plant Cells In addition to genetically modifying a plant by introducing a mutation in a gene encoding a plastidial GPAT, the flux of fatty acids through the prokaryotic 16:3 pathway can be reduced and thereby increase oil content in vegetative parts by silencing the plastidial GPAT. This is demonstrated by producing a transgenic cassette having a constitutive or leaf-specific promoter expressing an RNA hairpin corresponding to a region of the gene encoding the plastidial GPAT from the selected species. As an example, an RNAi hairpin expression cassette is produced using the 581 bp SalI-EcoRV fragment of theA. thalianaplastidial GPAT cDNA sequence (NM_179407, SEQ ID NO:177). A region of any gene encoding a plastidial GPAT which has a high degree of sequence identity to the nucleotide sequence of NM_179407 can also be used to construct a gene for expression of a hairpin RNA for silencing an endogenous plastidial GPAT gene. A hpRNAi construct containing a 732 bp fragment (SEQ ID NO:210) of theN. benthamianaplastidial GPAT flanked by SmaI and KasI unique sites was designed for stable transformation intoN. tabacum. The synthesizedN. benthamianaplastidial GPAT fragment was subcloned into the SmaI-KasI sites of pJP3303, resulting in pOIL113. It is expected that reducing plastidial fatty acid retention will result in an increase in TAG accumulation, particularly when combined with a “Push” component such as over-expression of a transcription factor such as WRI1, or by a “Pull” component such as a DGAT or PDAT, and/or reduced SDP1 or TGD activity. Inactivation of the gene encoding a plastidial GPAT or indeed any gene can be achieved using CRISPR/Cas9 methods. For example, inactivation of the gene encodingA. thalianaplastidial GPAT (Accession No. NM_179407) can be carried out by CRISPR/Cas9/sgRNA-mediated gene disruption and subsequent mutagenesis by non homologous end joining (NHEJ) DNA repair. Before targeted DNA cleavage, Cas9 stimulates DNA strand separation and allows a sgRNA to hybridize with a specific 20 nt sequence in the targeted gene. This positions the target DNA into the active site of Cas9 in proper orientation in relation to a PAM (tandem guanosine nucleotides) binding site. This positioning allows separate nuclease domains of Cas9 to independently cleave each strand of the target DNA sequence at a point 3-nt upstream of the PAM site. The double-strand break then undergoes error-prone NHEJ DNA repair during which deletions or insertions of a few nucleotides occur and result in inactivation of the plastidial GPAT gene. SgRNA sequences targeting theA. thalianaGPAT gene are identified and selected through the use of the CRISPRP web tool (Xie et al., 2014). The 20 nt target sequence can be any 20 nt sequence within the target gene, including within non-coding regions of the gene such as a promoter or intron, provided that it is a specific sequence within the genome. The sequence can be inserted into a binary vector containing the CRISPR/Cas9/sgRNA expression cassette and kanamycin plant selectable marker (Jiang et al., 2013) and transformed into the plant cells byAgrobacterium-mediated transformation. Transgenic T1 plants can be screened for mutations in the plastidial GPAT gene by PCR amplification and DNA sequencing. Example 8. Increasing Expression of Thioesterase in Plant Cells De novo fatty acid synthesis takes place in the plastids of eukaryotic cells where the fatty acids are synthesized while bound to acyl carrier protein as acyl-ACP conjugates. Following chain elongation to C16:0 and C18:0 acyl groups and then desaturation to C18:1 while linked to ACP, the fatty acids are cleaved from the ACP by thioesterases and enter the eukaryotic pathway by export from the plastids and transport to the ER where they participate in membrane and storage lipid biogenesis. In chloroplasts, the export process has two steps: firstly, acyl chains are released as free fatty acids by the enzymatic activity of acyl-ACP thioesterases (fatty acyl thioesterase; FAT), secondly by reaction with CoA to form acyl-CoA esters which is catalysed by long chain acyl-CoA synthetases (LACS).A. thalianacontains 3 fatty acyl thioesterases which can be distinguished based on their acyl chain specificity. FATA1 and FATA2 preferentially hydrolyze unsaturated acyl-ACPs while saturated acyl-ACP chains are typically cleaved by FATB. To explore the effect upon total fatty acid content, TAG content, and fatty acid composition of the co-expression of a thioesterase and genes encoding the WRI1 and/or DGAT polypeptides, chimeric genes were made for each of the threeA. thalianathioesterases by insertion of the coding regions into the pJP3343 binary expression vector for transient expression inN. benthamianaleaf cells from the 35S promoter. Protein coding regions for theA. thalianaFATA1 (Accession No. NP_189147.1, SEQ ID NO:193) and FATA2 (Accession No. NP_193041.1, SEQ ID NO:194) thioesterases were amplified from silique cDNA using primers containing EcoRI and PstI sites and subsequently cloned into pJP3343 using the same restriction sites. The resulting expression vectors were designated pOIL079 and pOIL080, respectively. The protein coding region of theA. thalianaFATB gene (Accession No. NP_172327.1, SEQ ID NO:195) was amplified using primers containing NotI and SacI flanking sites and cloned into the corresponding restriction sites of pJP3343, resulting in pOIL081. Constructs pOIL079, pOIL080 and pOIL081 are infiltrated intoN. benthamianaleaf tissue, either individually or in combination with constructs containing the genes for theA. thalianaWRI1 transcription factor (AtWRI1) (pJP3414) and/or DGAT1 acyltransferase (AtDGAT1) (pJP3352). For comparison, chimeric genes encoding theCocos nuciferaFatB1 (CnFATB1) (pJP3630),C. nuciferaFatB2 (CnFATB2) (pJP3629) were introduced intoN. benthamianaleaf tissue in parallel with theArabidopsisthioesterases, to compare the effect of the FatB polypeptides having MCFA specificity to theArabidopsisthioesterases which do not have MCFA specificity. All of the infiltrations included a chimeric gene for expression of the p19 silencing suppressor as described in Example 1. The negative control infiltrated only the p19 T-DNA. A synergistic effect was observed between thioesterase expression and WRI1 and/or DGAT over-expression on TAG levels inN. benthamianaleaves. Expression of the thioesterase genes without the WRI1 or DGAT genes significantly increased TAG levels above the low level in the negative control (p19 alone). For example, expression of the coconut FATB2 thioesterase resulted in an 8.2-fold increase in TAG levels in the leaves compared to the negative control. Co-expression of theA. thalianaWRI1 transcription factor with each of the thioesterases further increased TAG levels compared to the AtWRI1 control. Co-expression of each of the coconut thioesterases CnFATB1 and CnFATB2 with WRI1 resulted in higher TAG levels than each of the threeA. thalianathioesterases with WRI1. Interestingly, the converse was observed when theA. thalianaDGAT1 acyltransferase was co-expressed in combination with a thioesterase and WRI1. This suggested a better match in acyl-chain specificity of theA. thalianathioesterases and theA. thalianaDGAT1 acyltransferase, resulting in a greater flux of acyl-chains from the acyl-ACP into TAG. The non-MCFA thioesterases were also considerably more effective in elevating the percentage of oleic acid in the total fatty acid content in the leaves. Co-expression of the AtWRI1, AtDGAT1 and AtFATA2 resulted in the greatest level of TAG in the leaves, providing a level which was 1.6-fold greater than when AtWRI1 and AtDGAT1 were co-expressed without the thioesterase. These experiments confirmed the synergistic increase in oil synthesis and accumulation when both WRI1 and DGAT were co-expressed as well as showing the further synergistic increase obtained by adding a thioesterase to the combination. Three different binary expression vectors were constructed to test the effect of co-expression of genes encoding WRI1, DGAT1 and FATA on TAG levels and fatty acid composition in stably transformedN. tabacumleaves. The vector pOIL121 contained an SSU::AtWRI1 gene for expression of AtWRI1 from the SSU promoter, a 35S::AtDGAT1 gene for expression of AtDGAT from the 35S promoter, and an enTCUP2::AtFATA2 gene for expression of AtFATA2 from the enTCUP2 promoter which is a constitutive promoter. These genetic constructs were derived from pOIL38 by first digesting the DNA with NotI to remove the gene coding for theS. indicumoleosin. The protein coding region of theA. thalianaFATA2 gene was amplified and flanked with NotI sites using pOIL80 DNA as template. This fragment was then inserted into the NotI site of pOIL38. pOIL121 then served as a parent vector for pOIL122 which contained an additional enTCUP2::SDP1 hairpin RNA cassette for RNAi-mediated silencing of the endogenous SDP1 gene in the transgenic plants. To do this, the entireN. benthamianaSDP1 hairpin cassette was isolated from pOIL51 (Example 2) as an SfoI-SmaI fragment and cloned into the SfoI site of pOIL121, producing pOIL122 (FIG.14). A third vector, pOIL123, containing the SSU::WRI1 and 35S::DGAT1 genes and the enTCUP2::SDP1 hairpin RNA gene was obtained in a similar way by cloning the enTCUP2::SDP1 hairpin RNA cassette as a SfoI-SmaI fragment into the SfoI site of pOIL36. In summary, the vectors contained the gene combinations:pOIL121: SSU::AtWRI1, 35S::AtDGAT1, enTCUP2::AtFATA2.pOIL122: SSU::AtWRI1, 35S::AtDGAT1, enTCUP2::AtFATA2, enTCUP2::SDP1 hairpin.pOIL123: SSU::AtWRI1, 35S::AtDGAT1, enTCUP2::SDP1 hairpin. The three constructs were each used to produce transformedN. tabacumplants (cultivar Wi38) byAgrobacterium-mediated transformation. Co-expression of theA. thalianaFATA2 thioesterase or silencing of the endogenous SDP1 TAG lipase in combination with AtWRI1 and AtDGAT1 expression each resulted in further elevated TAG levels compared to expression of AtWRI1 and AtDGAT1 in the absence of both of the thioesterase gene and the SDP1-silencing gene. The greatest TAG yields were obtained using pOIL122 by the combined action of all four chimeric genes. It is noted thatN. benthamianais an 18:3 plant. The same constructs pOIL079, pOIL080 and pOIL081 are used to transformA. thaliana, a 16:3 plant. The inventors conceived of the model that increasing plastidial fatty acid export such as by increased fatty acyl thioesterase activity reduces acyl-ACP accumulation in the plastids, thereby increasing fatty acid biosynthesis as a result of reduced feedback inhibition on the acetyl-CoA carboxylase (ACCase) (Andre et al., 2012; Moreno-Perez et al., 2012). Thioesterase over-expression increases export of acyl chains from the plastids into the ER, thereby providing an efficient link between so-called ‘Push’ and ‘Pull’ metabolic engineering strategies. Example 9. The Effect of Different Transcription Factor Polypeptides on Plant Traits Previously reported experiments with WRI1 and DGAT (Vanhercke et al., 2013) used a synthetic gene encodingA. thalianaAtWRI1 (Accession No. AAP80382.1) and a synthetic gene encoding AtDGAT1, also fromA. thaliana(Accession No. AAF19262; SEQ ID NO: 1). To compare other WRI polypeptides with AtWRI1 for their ability to combine with DGAT to increase oil content, other WRI coding sequences were identified and used to generate constructs for expression inN. benthamianaleaves. Nucleotide sequences encoding theA. thalianaWRI3 (Accession No. AAM91814.1, SEQ ID NO:196) and WRI4 (Accession No. NP_178088.2, SEQ ID NO:197) transcription factors (To et al., 2012) were synthesized and inserted as EcoRI fragments into pJP3343 under the control of the 35S promoter. The resulting binary expression vectors were designated pOIL027 and pOIL028, respectively. The coding sequence for the oat (Avena sativa) WRI1 (AsWRIL SEQ ID NO:198) was PCR amplified from a vector provided by Prof. Sten Stymne (Swedish University of Agricultural Sciences) using flanking primers containing additional EcoRI sites. The amplified fragment was inserted into pJP3343 resulting in pOIL055. A WRI1 candidate sequence fromS. bicolor(Accession No. XP_002450194.1, SEQ ID NO:199) was identified by a BLASTp search on the NCBI server using theZea maysWRI1 amino acid sequence (Accession No. NP_001137064.1, SEQ ID NO:200) as query. The protein coding region of theS. bicolorWRI1 gene (SbWRI1) was synthesized and inserted as an EcoRI fragment into pJP3343, yielding pOIL056. A gene candidate encoding a WRI1 was identified from the Chinese tallow (Triadica sebifera; TsWRIL SEQ ID NO:201) transcriptome (Uday et al., submitted). The protein coding region was synthesized and inserted as an EcoRI fragment into pJP3343 resulting in pOIL070. The pJP3414 and pJP3352 binary vectors containing the coding sequences for expression of theA. thalianaWRI1 and DGAT1 polypeptides were as described by Vanhercke et al. (2013). Plasmids containing the various WRI coding sequences were introduced intoN. benthamianaleaf tissue for transient expression using a gene encoding the p19 viral suppressor protein in all inoculations as described in Example 1. The genes encoding the WRI polypeptides were either tested alone or in combination with the DGAT1 acyltransferase gene, the latter to provide greater TAG biosynthesis and accumulation. The positive control in this experiment was the combination of the genes encodingA. thalianaWRI1 transcription factor and AtDGAT1. All infiltrations were done in triplicate using three different plants and TAG levels were analyzed as described in Example 1. Expression of most of the individual WRI polypeptides in the absence of exogenously added DGAT1 resulted in increased, yet still low, TAG levels (<0.23% on dry weight basis) in infiltrated leaf spots, compared to the control which had only the p19 construct (FIG.15). The exception was TsWRI1 which, by itself, did not appear to increase TAG levels significantly. In addition, differences in TAG levels produced by expression of the different WRI transcription factors on their own were not great. Both AsWRI1 and SbWRI1 yielded TAG levels similar to AtWRI1 on its own. Analysis of the TAG fatty acid composition revealed only minor changes except for increased C18:1Δ9 levels from expression of AtWRI3 in the infiltrated leaf tissues (Table 25). TABLE 25TAG fatty acid composition inN. benthamianaleaf samples infiltrated with different chimeric genes for expression of WRI(n = 3). All samples were also infiltrated with the P19 construct. The TAG samples also contained 0.1-0.4% C14:0; 0.5-1.2%C16:3 and; 0.1-0.7% C18:1411.InfiltratedgenesC16:0C16:1C18:0C18:1C18:2C18:3n3C20:0C20:1C22:0C24:0Control (P19)33.6 ± 4.70.5 ± 0.48.9 ± 2.24.7 ± 0.616.9 ± 1.032.2 ± 7.81.1 ± 0.20.8 ± 1.50.00.0WRI135.5 ± 3.40.7 ± 0.25.2 ± 0.85.4 ± 1.317.1 ± 1.033.1 ± 2.70.8 ± 0.10.5 ± 0.60.3 ± 0.00.0WRI327.3 ± 1.60.9 ± 0.24.8 ± 0.310.2 ± 1.516.1 ± 1.037.8 ± 1.20.8 ± 0.10.6 ± 0.70.1 ± 0.20.0WRI430.1 ± 0.41.0 ± 0.45.2 ± 0.84.6 ± 0.617.2 ± 0.438.1 ± 1.60.8 ± 0.11.3 ± 1.30.00.0AsWRI35.7 ± 3.01.7 ± 0.45.3 ± 0.76.5 ± 0.315.4 ± 0.431.6 ± 1.60.8 ± 0.10.4 ± 0.70.3 ± 0.10.0SbWRI37.4 ± 0.81.9 ± 0.34.8 ± 0.37.0 ± 1.215.2 ± 0.330.8 ± 0.30.8 ± 0.10.4 ± 0.60.3 ± 0.00.0TsWRI34.5 ± 4.80.09.4 ± 8.25.9 ± 1.716.0 ± 0.729.3 ± 12.40.0n.d.0.00.0Control (P19)31.0 ± 2.10.9 ± 0.18.7 ± 1.38.0 ± 2.324.9 ± 1.522.1 ± 4.72.0 ± 0.10.00.6 ± 0.60.2 ± 0.4WRI1 + DGAT27.7 ± 0.10.3 ± 0.07.0 ± 0.117.2 ± 0.727.9 ± 0.914.7 ± 0.32.4 ± 0.20.3 ± 0.01.1 ± 0.10.8 ± 0.2WRI3 + DGAT30.0 ± 0.80.6 ± 0.15.9 ± 0.413.9 ± 2.921.5 ± 1.121.3 ± 0.82.8 ± 0.10.2 ± 0.01.8 ± 0.11.0 ± 0.2WRI4 + DGAT27.0 ± 0.50.2 ± 0.18.5 ± 0.25.8 ± 0.723.9 ± 0.825.2 ± 1.33.5 ± 0.10.2 ± 0.02.1 ± 0.21.7 ± 0.2AsWRI + DGAT33.8 ± 0.51.1 ± 0.15.5 ± 0.912.2 ± 1.626.0 ± 1.916.3 ± 1.32.2 ± 0.20.2 ± 0.01.2 ± 0.10.8 ± 0.1SbWRI + DGAT34.6 ± 0.51.3 ± 0.15.6 ± 0.413.9 ± 1.623.6 ± 1.315.8 ± 0.62.2 ± 0.10.2 ± 0.01.2 ± 0.10.9 ± 0.1TsWRI + DGAT25.4 ± 0.50.2 ± 0.09.4 ± 0.17.7 ± 1.027.0 ± 1.322.1 ± 2.43.6 ± 0.20.2 ± 0.01.8 ± 0.21.3 ± 0.2 In contrast, differences in TAG yields from expression of the different WRI polypeptides were more pronounced upon co-expression with the AtDGAT1 acyltransferase. This again demonstrated the synergistic effect of WRI1 and DGAT co-expression on TAG biosynthesis in infiltratedN. benthamianaleaf tissue, as reported by Vanhercke et al. (2013). Intermediate TAG levels were observed upon co-expression of DGAT1 with AtWRI3, AtWRI4 and TsWRI1 expressing vectors while levels obtained with the AsWRI1 and AtWRI1 were significantly lower. In a result that could not have been predicted beforehand, the highest TAG yields were obtained with co-expression of DGAT with SbWRI1, even though the assay was done in dicotyledonous cells. TAG fatty acid composition analysis revealed increased levels of C18:1Δ9and decreased levels of C18:3Δ9,12,15(ALA) in the case of SbWRI1, AsWRI1 and the AtWRI1 positive control. Unlike AtWRI1, however, expression of AsWRI1 and SbWRI1 both displayed increased C16:0 levels compared to the p19 negative control. Interestingly, AtWRI3 infiltrated leaf samples exhibited a distinct TAG profile with C18:1Δ9being enriched while C16:0 and ALA were only slightly affected. This experiment showed that theS. bicolorWRI1 transcription factor, SbWRI1, was superior to AtWRI1 when co-expressed with DGAT to increase TAG levels in vegetative plant parts. The inventors also concluded that a transcription factor, for example a WRI1, from a monocotyledonous plant could function well in a dicotyledonous plant cell, indeed might even have superior activity compared to a corresponding transcription factor from a dicotyledonous plant. Likewise, a transcription factor from a dicotyledonous plant could function well in a monocotyledonous plant cell. Use of Other Transcription Factors Genetic constructs were prepared for expression of each of 14 different transcription factors in plant cells to test their ability to function for increasing TAG levels in combination with other genes involved in TAG biosynthesis and accumulation. These transcription factors were candidates as alternatives for WRI1 or for addition to combinations including one or more of WRI1, LEC1 and LEC2 transcription factors for use in plant cells, particularly in vegetative plant parts. Their selection was largely based on their reported involvement in embryogenesis (reviewed in Baud and Lepiniec (2010), and Ikeda et al. (2006)), similar to LEC2. Experiments were therefore carried out to assay their function, using theN. benthamianaexpression system (Example 1), as follows. Nucleotide sequences of the protein coding regions of the following transcription factors were codon optimized for expression inN. benthamianaandN. tabacum, synthesized and subcloned as NotI-SacI fragments into the respective sites of pJP3343:A. thalianaFUS3 (pOIL164) (Luerssen et al., 1998; Accession number AAC35247; SEQ ID NO:160),A. thalianaLEC1L (pOIL165) (Kwong et al. 2003; Accession number AAN15924; SEQ ID NO:157),A. thalianaLEC1 (pOIL166) (Lotan et al., 1998; Accession number AAC39488; SEQ ID NO:149),G. maxMYB73 (pOIL167) (Liu et al., 2014; Accession number ABH02868; SEQ ID NO:212),A. thalianabZIP53 (pOIL168) (Alonso et al., 2009; Accession number AAM14360; SEQ ID NO:213),A. thalianaAGL15 (pOIL169) (Zheng et al., 2009; Accession number NP_196883; SEQ ID NO:214),A. thalianaMYB118 (Accession number AAS58517; pOIL170; SEQ ID NO:215), MYB115 (Wang et al., 2002; Accession number AAS10103; pOIL171; SEQ ID NO:216),A. thalianaTANMEI (pOIL172) (Yamagishi et al., 2005; Accession number BAE44475; SEQ ID NO:217),A. thalianaWUS (pOIL173) (Laux et al., 1996; Accession number NP_565429; SEQ ID NO:218),A. thalianaBBM (pOIL174) (Boutilier et al., 2002; Accession number AAM33893, SEQ ID NO:145),B. napusGFR2a1 (Accession number AFB74090; pOIL177; SEQ ID NO:219) and GFR2a2 (Accession number AFB74089; pOIL178; SEQ ID NO:220) (Liu et al. (2012)). In addition, a codon optimized version of theA. thalianaPHR1 transcription factor involved in adaptation to high light phosphate starvation conditions was similarly subcloned into pJP3343 (pOIL189) (Nilsson et al (2012); Accession number AAN72198; SEQ ID NO:221). These transcription factors are summarised in Table 26. As a screening assay to determine the function of these transcription factors, the genetic constructs and a gene encoding DGAT1 were co-infiltrated intoN. benthamianaleaf cells as described in Example 1, either with or without a gene encoding WRI1. Total lipid content and fatty acid composition of the leaf cells were analysed 5 days post-infiltration. Among the various embryogenic transcription factors tested, only overexpression of FUS3 resulted in significantly increased TAG levels inN. benthamianaleaf tissue when compared to DGAT and DGAT1+WRI1 control infiltrations (Table 27). TABLE 26Additional transcription factors and thegenetic constructs for their expressionTranscriptionLength (aminoAccessionPlasmidfactorSpeciesacid)numberpOIL164FUS3A. thaliana312AAC35247pOIL165LEC1LA. thaliana234AAN15924pOIL166LEC1A. thaliana208AAC39488pOIL167MYB73G. max74ABH02868pOIL168bZIP53A. thaliana146AAM14360pOIL169AGL15A. thaliana268NP_196883pOIL170MYB118A. thaliana437AAS58517pOIL171MYB115A. thaliana359AAS10103pOIL172TANMEIA. thaliana386BAE44475pOIL173WUSA. thaliana292NP_565429pOIL174BBMA. thaliana584AAM33803pOIL177GFR2a1B. napus453AFB74090pOIL178GFR2a2B. napus461AFB74089pOIL189PHR1A. thaliana409AAN72198 TABLE 27TAG level (% leaf dry weight) and fatty acid profile of infiltratedN. benthamianaleaves.C16:0C16:1C18:0C18:1C18:2C18:3TAGP1927.1 ± 1.50.3 ± 0.19.6 ± 1.74.4 ± 1.222.4 ± 4.030.5 ± 0.90.0P19 + DGAT126.3 ± 1.00.1 ± 0.010.7 ± 0.63.7 ± 0.726.1 ± 1.626.4 ± 1.40.2 ± 0.0P19 + DGAT1 + FUS324.1 ± 1.00.1 ± 0.06.3 ± 0.45.2 ± 1.627.9 ± 1.830.0 ± 1.80.6 ± 0.1P19 + DGAT1 + LEC1L26.0 ± 1.40.1 ± 0.010.3 ± 0.83.9 ± 1.026.6 ± 2.126.4 ± 0.70.2 ± 0.0P1930.3 ± 0.70.012.4 ± 0.76.8 ± 0.922.9 ± 0.226.0 ± 0.90.0P19 + DGAT125.8 ± 1.10.010.1 ± 0.44.4 ± 0.926.1 ± 1.326.2 ± 1.40.2 ± 0.0P19 + DGAT1 + WRI122.7 ± 0.90.010.1 ± 0.414.9 ± 0.527.9 ± 1.318.5 ± 0.80.3 ± 0.1P19 + DGAT1 + FUS323.9 ± 0.70.2 ± 0.17.6 ± 0.45.3 ± 0.729.1 ± 0.826.8 ± 0.70.4 ± 0.1P19 + DGAT1 + LEC124.9 ± 0.40.1 ± 0.211.1 ± 0.24.0 ± 0.125.9 ± 0.526.1 ± 0.60.1 ± 0.0P19 + DGAT1 + MYB7325.8 ± 0.30.010.9 ± 0.74.3 ± 1.026.2 ± 0.825.2 ± 1.80.1 ± 0.0P1934.2 ± 4.90.010.6 ± 3.18.3 ± 4.119.5 ± 1.423.2 ± 0.80.1 ± 0.1P19 + DGAT127.7 ± 0.10.3 ± 0.19.9 ± 1.14.2 ± 0.326.4 ± 1.822.5 ± 0.40.2 ± 0.1P19 + DGAT1 + WRI124.8 ± 1.00.2 ± 0.08.8 ± 1.014.7 ± 0.627.6 ± 1.017.2 ± 0.30.4 ± 0.1P19 + DGAT1 + bZIP5329.3 ± 0.80.1 ± 0.28.7 ± 0.42.9 ± 0.322.0 ± 0.525.9 ± 0.50.1 ± 0.1P19 + DGAT1 + AGL1529.2 ± 1.40.2 ± 0.04.9 ± 0.97.0 ± 1.919.8 ± 0.830.0 ± 1.30.3 ± 0.1P19 + DGAT1 + MYB11831.6 ± 1.70.2 ± 0.15.8 ± 1.24.8 ± 0.820.7 ± 0.328.2 ± 1.60.2 ± 0.1P1927.4 ± 1.20.06.9 ± 1.04.8 ± 2.620.0 ± 1.539.0 ± 4.10.1 ± 0.0P19 + DGAT126.0 ± 1.10.08.0 ± 0.64.2 ± 1.622.3 ± 2.433.9 ± 4.30.2 ± 0.0P19 + DGAT1 + WRI123.4 ± 0.80.1 ± 0.18.5 ± 0.617.0 ± 2.423.3 ± 1.823.3 ± 4.30.5 ± 0.1P19 + DGAT1 + MYB11526.3 ± 0.40.1 ± 0.16.6 ± 0.32.8 ± 0.422.5 ± 1.835.7 ± 2.90.2 ± 0.0P19 + DGAT1 + TANMEI25.6 ± 0.90.1 ± 0.28.5 ± 1.22.6 ± 0.521.9 ± 2.035.3 ± 3.80.2 ± 0.0P19 + DGAT1 + WUS24.3 ± 0.90.1 ± 0.15.5 ± 0.61.7 ± 0.216.8 ± 1.647.9 ± 3.30.2 ± 0.0P1930.5 ± 1.30.08.1 ± 0.98.2 ± 6.021.8 ± 1.228.3 ± 7.30.1 ± 0.1P19 + DGAT1 + WRI125.9 ± 1.70.2 ± 0.08.3 ± 0.719.9 ± 2.824.5 ± 1.116.0 ± 0.60.8 ± 0.1P19 + DGAT1 + WRI1 + BBM27.7 ± 0.70.2 ± 0.06.7 ± 0.221.2 ± 0.719.8 ± 0.518.5 ± 0.60.5 ± 0.1P19 + DGAT1 + WRI1 + GFR2a129.2 ± 1.30.4 ± 0.06.1 ± 0.112.9 ± 1.524.3 ± 0.420.9 ± 0.50.4 ± 0.1P19 + DGAT1 + WRI1 + GFR2a229.9 ± 2.40.4 ± 0.15.5 ± 0.613.5 ± 2.723.0 ± 0.521.3 ± 1.20.5 ± 0.1P19 + DGAT1 + WRI1 + PHR126.2 ± 0.30.2 ± 0.04.9 ± 0.07.6 ± 0.219.2 ± 0.336.0 ± 0.70.3 ± 0.0P1932.0 ± 1.91.6 ± 2.711.1 ± 2.75.5 ± 2.223.3 ± 1.125.4 ± 3.30.0P19 + DGAT1 + WRI127.5 ± 1.20.7 ± 0.86.8 ± 0.416.6 ± 2.126.7 ± 0.816.5 ± 0.31.2 ± 0.2P19 + DGAT1 + WRI1 + FUS323.6 ± 1.12.1 ± 3.56.5 ± 0.513.3 ± 0.932.1 ± 2.615.6 ± 1.51.6 ± 0.1 For stable transformation of plants using genes encoding the alternative transcription factors, the following binary constructs are made. The genes for expression of the transcription factors use either the SSU promoter or the SAG12 promoter. Over-expression of embryogenic transcription factors such as LEC1 and LEC2 has been shown to induce a variety of pleotropic effects, undesirable in the present context, including somatic embryogenesis (Feeney et al. (2012); Santos-Mendoza et al. (2005); Stone et al. (2008); Stone et al. (2001); Shen et al. (2010)). To minimize possible negative impact on plant development and biomass yield, tissue or developmental-stage specific promoters are preferred over constitutive promoters to drive the ectopic expression of master regulators of embryogenesis. Example 10. Stem-Specific Expression of a Gene Encoding a Transcription Factor Leaves ofN. tabacumplants expressing transgenes encoding WRI1, DGAT and Oleosin contain about 16% TAG at seed setting stage of development. However, the TAG levels were much lower in stems (1%) and roots (1.4%) of the plants (Vanhercke et al., 2014). The inventors considered whether the lower TAG levels in stems and roots were due to poor promoter activity of the Rubisco SSU promoter used to express the gene encoding WRI1 in the transgenic plants. The DGAT transgene in the T-DNA of pJP3502 was expressed by the CaMV35S promoter which is expressed more strongly in stems and roots and therefore was unlikely to be the limiting factor for TAG accumulation in stems and roots. In an attempt to increase TAG biosynthesis in stem tissue, a construct was designed in which the gene encoding WRI1 was placed under the control of anA. thalianaSDP1 promoter. A 3.156 kb synthetic DNA fragment was synthesized comprising 1.5 kb of theA. thalianaSDP1 promoter (SEQ ID NO: 175) (Kelly et al., 2013), followed by the coding region for theA. thalianaWRI1 polypeptide and theG. maxlectin terminator/polyadenylation region. This fragment was inserted between the SacI and NotI sites of pJP3303. The resulting vector was designated pOIL050, which was then used to transform cells from theN. tabacumplants homozygous for the T-DNA from pJP3502 byAgrobacterium-mediated transformation. Transgenic plants were selected for hygromycin resistance and a total of 86 independent transgenic plants were grown to maturity in the glasshouse. Samples were taken from transgenic leaf and stem tissue at seed setting stage and contain increased TAG levels compared to theN. tabacumparental plants transformed with pJP3502. Example 11. Effect of Oil Body Protein Expression on Plant Traits N. tabacumplants transformed with the T-DNA of pJP3502 and expressing transgenes encodingA. thalianaWRI1, DGAT1 andS. indicumOleosin had increased TAG levels in vegetative tissues. As shown in Example 2 above, when the endogenous gene encoding SDP1 TAG lipase was silenced in those plants, the leaf TAG levels further increased, which indicated to the inventors that substantial TAG turnover was occurring in the plants that retained SDP1 activity. Therefore, the level of expression of the transgenes in the plants was determined. While Northern hybridisation blotting confirmed strong WRI1 and DGAT1 expression and some oleosin mRNA expression, expression analysis by digital PCR and qRT-PCR detected only very low levels of oleosin transcripts. The expression analysis revealed that the gene encoding the Oleosin was poorly expressed compared to the WRI1 and DGAT1 transgenes. From these experiments, the inventors concluded that the oil bodies in the leaf tissue were not completely protected from TAG breakdown because of inadequate production of Oleosin protein when encoded by the T-DNA in pJP3502. To improve stable accumulation of TAG throughout plant development, several pJP3502 modifications were designed in which the Oleosin gene was substituted. These modified constructs were as follows.1. pJP3502 contains a gene (SEQ ID NO:176 provides the sequence of its complement) encoding theS. indicumoleosin which was poorly expressed. That gene has an internal UBQ10 intron which might be reducing the expression level. To test this, a 502 bp synthetic DNA fragment containing theS. indicumoleosin gene and lacking the internal UBQ10 intron was synthesized and inserted into pJP3502 as a NotI fragment, to substitute the oleosin gene containing the intron in pJP3502. The resultant plasmid was designated pOIL040.2. The Rubisco small subunit (SSU) promoter driving expression of the oleosin gene in pJP3502 was replaced by the constitutive enTCUP2 promoter. To this end, a 232 lbp fragment containing the enTCUP2 promoter, Oleosin protein coding region,G. maxlectin terminator/polyadenylation region and the first 643 bp of the downstream SSU promoter driving wri1 expression was synthesized and subcloned into the AscI and SpeI sites of pJP3502 resulting in pOIL038.3. A similar strategy was followed for the expression of an engineered version of theS. indicumoleosin gene containing 6 introduced cysteine residues (o3-3) under the control of the enTCUP2 promoter (Winichayakul et al., 2013). A 2298 bp fragment containing the enTCUP2 promoter, Oleosin o3-3 protein coding region,G. maxlectin terminator/polyadenylation region and the first 643 bp of the downstream SSU promoter driving wri1 expression was synthesized and subcloned into the AscI and SpeI sites of pJP3502 resulting in pOIL037.4. The NotI sites flanking theS. indicumoleosin gene in pJP3502 were used to exchange the protein coding region for one encoding peanut Oleosin3 (Accession No. AAU21501.1) (Parthibane et al., 2012a; Parthibane et al., 2012b). A 528 bp fragment containing the oleosin3 gene, flanked by NotI sites, was synthesized and subcloned into the respective site of pJP3502. The resulting vector was designated pOIL041.5. Similarly, a 1077 bp NotI flanked fragment containing the gene coding for theA. thalianasteroleosin (Arab-1) (Accession No. AAM10215.1) (Jolivet et al., 2014) was synthesized and subcloned into the NotI site of pJP3502, resulting in pOIL043.6. TheNannochloropsisoceanic lipid droplet surface protein (LDSP) (Accession No. AFB75402.1) (Vieler et al., 2012) was synthesized as a 504 bp NotI-flanked fragment and subcloned into the NotI site of pJP3502, yielding pOIL044.7. Finally, theA. thalianacaleosin (CLO3) (Accession No. 022788.1) (Shimada et al., 2014) was synthesized as a 612 bp NotI flanked fragment and subcloned into pJP3502, resulting in pOIL042. Each of these constructs was introduced intoN. benthamianaleaf cells as described in Example 1. Transient expression of both pJP3502 and pOIL040 inN. benthamianaleaf tissue resulted in elevated TAG levels and similar changes in the TAG fatty acid profile but pOIL040 increased the TAG level more (1.3% compared to 0.9%). Each of the constructs pOIL037, pOIL038, pOIL041, pOIL042 and pOIL043 were used to stably transformN. tabacumplants (cultivar W38) byAgrobacterium-mediated methods. Transgenic plants were selected on the basis of kanamycin resistance and are grown to maturity in the glasshouse. Samples are taken from transgenic leaf tissue at different stages during plant development and contain increased TAG levels compared to wild-typeN. tabacumandN. tabacumplants transformed with pJP3502. Cloning and Characterisation of LDAP Polypeptides fromSapium sebiferum Oleosins are not highly expressed in non-seed oil accumulating plant tissues such as the mesocarp of olive, oil palm, and avocado (Murphy, 2012). Instead, lipid droplet associated proteins (LDAP) have been identified in these tissues that may play a similar role to that of oleosin in seed tissues (Horn et al., 2013). The inventors therefore considered it possible that oleosin might not be the optimal packaging protein to protect the accumulated oil from TAG lipase or other cytosolic enzyme activities in vegetative tissues of plants. LDAP polypeptides were therefore identified and evaluated for enhancement of TAG accumulation, as follows. The fruit of Chinese tallow tree,Sapium sebiferum, a member of the family Euphorbiaceae, was of particular interest to the inventors as it contains an oil-rich tissue outside of the seed. A recent study (Divi et al, submitted for publication) indicated that this oleoginous tissue, called a tallow layer, might be derived from the mesocarp of its fruit. Therefore, the inventors queried the transcriptome ofS. sebiferumfor LDAP sequences. A comparative analysis of expressed genes in the fruit coat and seed tissues revealed a group of three previously unidentified LDAP genes which were highly expressed in the tallow layer. Nucleotide sequences encoding the three LDAPs were obtained by RT-PCR using RNAs derived from tallow tissue using three pairs of primers. The primer sequences were based on the DNA sequences flanking the entire coding region of each of the three genes. The primer sequences were: for LDAP1, 5′-TTTTAACGATATCCGCTAAAGG-3′ (SEQ ID NO: 236) and 5′-AATGAATGAACAAGAATTAAGTC-3′ (SEQ ID NO: 237) AT-3′; LDAP2, 5′-CTTTTCTCACACCGTATCTCCG-3′ (SEQ ID NO: 238) and 5′-AGCATGATATA CTTGTCGAGAAAGC-3′ (SEQ ID NO: 239); LDAP3, 5′-GCGACAGTGTAGCGTTTT-3′ (SEQ ID NO: 240) and 5′-ATACATAAAATGAAAACTATTGTGC-3′ (SEQ ID NO: 241). Analysis of theS. sebiferumtranscriptome revealed multiple orthologs for each of the LDAP genes, including eight LDAP1, six LDAP2, and six LDAP3 genes, with less than 10% sequence divergence within each gene family. The putative peptide sequences were aligned and a phylogenetic tree was constructed using Genious software (FIG.16), together with LDAPs homologs from other plant species, including two from avocado (Pam), one from oil palm, one fromPartheniumargentatum (Par), two fromArabidopsis(Ath), five fromTaraxacum brevicorniculatum(Tbr), three from Hevea brasihensis (Hbr), as presented inFIG.16. The phylogenetic tree was revealed that the SsLDAP3 shared greater amino acid sequence identity to the LDAP1 and LDAP2 polypeptides from avocado and the LDAP from oil palm, while the SsLDAP1 and SsLDAP2 polypeptides were more divergent. Genetic Constructs for Over-Expression of LDAP In order to test the function of the LDAPs fromS. sebiferum, expression vectors were made to express each of these polypeptides under the control of the 35S promoter in leaf cells. The full length SsLDAP cDNA sequences were inserted into the pDONR207 destination vector by recombination reactions, replacing the CcdB and Cm(R) regions of the destination vector with the SsLDAP cDNA fragments. Following confirmation by restriction digestion analysis and DNA sequencing, the constructs were introduced intoAgrobacterium tumefaciensstrain AGL1 and used for both transient expression inN. benthamianaleaf cells and stable transformation ofN. tabacum. The expression of each of the three SsLDAP genes under the transcriptional control of the 35S promoter inN. benthamianaleaves in combination with the expression of 35S::AtDGAT1 and 35S::AtWRI1 yielded substantially higher levels of TAG accumulation relative to the cells infiltrated with the 35S::AtDGAT1 and 35S::AtWRI1 genes without the LDAP construct. The TAG level was increased about 2-fold above the TAG level in the control cells. A significant increase in the level of α-linolenic acid (ALA) and a reduced level of saturated fatty acids was observed in the cells receiving the combination of genes, relative to the control cells. Co-Localisation of YFP Fused LDAP Polypeptides with Lipid Droplets in Leaf Cells In order to characterise SsLDAPs in vivo and observe their dynamic behaviour, expression constructs were made for expression of fusion polypeptides consisting of the LDAP polypeptides fused to yellow fluorescent protein (YFP). For each fusion polypeptide, the YFP was fused in-frame to the C-terminus of the SsLDAP. The full open reading frame of each of the three LDAP genes without a stop codon, at its 3′ end, was fused to the YFP sequence and the chimeric genes inserted into pDONR207. Following confirmation of the resultant constructs by restriction digestion and DNA sequencing, the constructs were introduced intoA. tumefaciensstrain AGL1 and used for both transient expression inN. benthamianaleaf cells and stable transformation ofN. tabacum. Three days following infiltration of the leaf cells with the LDAP-YFP constructs, leaf discs from the infiltrated zones were stained with Nile Red, which positively stained lipid droplets, and observed under a confocal microscope to detect both the red stain (lipid droplets) and fluorescence from the YFP polypeptide. Co-localisation of LDAP-YFP with the lipid droplets was observed, indicating that the LDAP associated with the lipid droplets in the leaf cells. Example 12. Silencing of TGD Genes in Plants Li-Beisson et al. (2013) estimated that inArabidopsisleaves (a 16:3 plant), approximately 40% of the fatty acids synthesized in chloroplasts enter the prokaryotic pathway, whereas 60% were exported to enter the eukaryotic pathway. After they were desaturated in the ER, about half of these exported fatty acids are returned to the plastid to support galactolipid synthesis for thylakoid membranes. The transport (import) of the fatty acids as DAG or phospholipids into the plastid involves TGD1, a permease-like protein of the inner chloroplast envelope. TheArabidopsisABC lipid transporter comprising TGD1, 2, and 3 proteins was identified by Benning et al. (2008 and 2009) and more recently by Roston et al. (2012). This protein complex is localized in the inner chloroplast envelope membrane and is proposed to mediate the transfer of phosphatidate across this membrane. TGD2 polypeptide is a phosphatidic-binding protein, and TGD3 an ATPase. A novelArabidopsisprotein, TGD4, was identified by a genetic approach (Xu et al., 2008) and inactivation of the TGD4 gene also blocked lipid transfer from the ER to plastids. Recent biochemical data indicate that TGD4 is phosphatidate binding protein residing in the outer chloroplast envelope membrane (Wang and Benning, 2012). Xu et al. (2005) described leaky tgd1 alleles inA. thalianaresulting in reduced plant growth and high occurrence of embryo abortion. Leaf tissue ofA. thalianatgd1 mutants contained increased TAG levels, likely as cytosol oil droplets. In addition, elevated TAG levels were also found in roots of tgd1 mutants. No difference in seed oil content was detected. Similar TAG accumulation in leaf tissue has been reported forA. thalianatgd2 (Awai et al., 2006), tgd3 (Lu et al., 2007) and tgd4 mutants (Xu et al., 2008). All tgd mutant alleles were either sufficiently leaky or severely impairing in plant development. TGD1 Silencing A silencing construct directed against the TGD1 plastidial importer was generated based on a full length mRNA transcript identified in theN. benthamianatranscriptome. A 685 bp fragment was amplified fromN. benthamianaleaf cDNA while incorporating a PmlI site at the 5′ end. The TGD1 fragment was first cloned into pENTR/D-TOPO (Invitrogen) and subsequently inserted into the pHELLSGATE12 destination vector via LR cloning (Gateway). The resulting expression vector was designated pOIL025 and is transiently expressed inN. benthamianato assess the effect of TGD1 gene silencing on leaf TAG levels. The TGD1 hairpin construct is placed under the control of theA. nigerinducible alcA promotor by subcloning as a PmlI-EcoRV fragment into the NheI (klenow)-SfoI sites of pOIL020 (below). The resulting vector, designated pOIL026, is super-transformed into a homozygousN. tabacumpJP3502 line to further increase leaf oil levels. Further constructs are made for expressing hairpin RNA for reducing expression of the TGD-2, -3, -4 and -5 genes. Transformed plants are produced using these constructs and oil content determined in the transformants. The transformed plants are crossed with the transformants generated with pJP3502 or other combinations of genes as described above. Example 13. Expression of Gene Combinations in Potato Tubers Construction of pJP3506 A genetic construct containing three genes for expression in potato tubers was made and used for potato transformation. This construct was designated as pJP3506 and was based on an existing vector pJP3502 (WO2013/096993) with replacement of promoters to provide for tuber-specific expression. pJP3506 contained (i) an NPTII kanamycin resistance gene driven by 35S promoter with duplicated enhancer region (e35S) as the selectable marker gene and three gene expression cassettes, which were (ii) 35S::AtDGAT1 encoding theArabidopsis thalianaDGAT1, (iii) B33::AtWRI1 encoding theArabidopsis thalianaWRI1, and (iv) B33::sesame oleosin, encoding the oleosin fromSesame indicum. The nucleotide sequences encoding these polypeptides were as in pJP3502. The patatin B33 promoter (B33) was a tuber specific promoter derived fromSolanum tuberosum, which was provided by Dr Alisdair Fernie, Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany. A circular plasmid map of pJP3506 is presented inFIG.17. TheS. tuberosumPatatin B33 promoter sequence used in the pJP3506 construct was a truncated version having 183 nucleotides deleted from the 5′ end and 261 nucleotides deleted from the 3′ end relative to GenBank Accession No. X14483. The nucleotide sequence of the patatin B33 promoter as used in pJP3506 is given as SEQ ID NO: 202. Transformation of Potato Potato seedlings (Solanum tuberosum) of cultivar Atlantic which had been grown asceptically in tissue culture were purchased from Toolangi Elite, Victorian Certified Seed Potato Authority (ViCSPA), Victoria, Australia. Stem internodes were excised into pieces of approximately 1 cm in length under a suspension ofAgrobacterium tumefaciensstrain LBA4404 containing pJP3506. TheAgrobacteriumcells had been grown to an OD of 0.2 and diluted with an equal volume MS medium. ExcessAgrobacteriumsuspension was removed by brief blotting the stem pieces on sterile filter paper, which were then plated onto MS medium and maintained at 24° C. for two days (co-cultivation). The internodes were then transferred onto fresh MS medium supplemented with 200 μg/L NAA, 2 mg/L BAP and 250 mg/L Cefetaxime. Selection of transgenic calli was initiated 10 days later when the internodes were transferred onto fresh MS medium supplemented with 2 mg/L BAP, 5 mg/L GA3, 50 mg/L kanamycin and 250 mg/L Cefetaxime. Shoots regenerated from calli were excised and placed onto plain MS medium for root induction prior to transplanting into a 15 cm diameter pot containing potting mix and grown in the greenhouse until plant maturity including tuber growth. DNA Extraction and Molecular Identification of the Transgenic Plants by PCR Disks of about 1 cm in diameter were obtained from potato leaves from the plants in the greenhouse. These were placed in a deep-well microtiter plate and freeze dried for 48 hr. The freeze dried leaf samples were then ground into powder by adding a steel ball bearing to each well and shaking the plate in a Reicht tissue lyser (Qiagen) at a maximum frequency of 28/sec for 2 min each side of the microtiter plate. 375 μL of extraction buffer containing 0.1 M Tris-HCl pH8.0, 0.05 M EDTA and 1.25% SDS was added to each well containing the powdered leaf tissue. Following 1 hr incubation at 65° C., 187 μL of 6M ammonium acetate was added to each well and the mixtures stored at 4° C. for 30 min prior to centrifugation of the plates for 30 min at 3000 rpm. 340 μL supernatant from each well was transferred into a new deep well microtiter plate containing 220 μL isopropanol and maintained for 5 min at room temperature prior to centrifugation at 3000 rpm for 30 min. The precipitated DNA pellets were washed with 70% ethanol, air dried and resuspended in 225 μL H2O per sample. Two μL from each leaf sample DNA preparation was added to a 20 μL PCR reaction mix using the HotStar PCR system (Qiagen). A pair of oligonucleotide primers based on 5′ and 3′ sequences from theArabidopsis thalianaWRI1 gene, codon-optimized for tobacco, was used in the PCR reactions. Their sequences were: Nt-Wri-P3: 5′-CACTCGTGCTTTCCATCATC-3′ (SEQ ID NO: 203) and Nt-Wri-P1: 5′-GAAGGCTGAGCAACAAGAGG-3′(SEQ ID NO: 204). A pair of oligonucleotide primers based on theArabidopsis thalianaDGAT1 gene, codon-optimized for tobacco, was also used in a separate PCR reaction on each DNA sample. Their sequences were: Nt-DGAT-P2: 5′-GGCGATTTTGGATTCTGC-3′ (SEQ ID NO: 205) and Nt-DGAT-P3: 5′-CCCAACCCTTCCGTATACAT-3′ (SEQ ID NO: 206). Amplification was carried out with an initial cycle at 95° C. for 15 min, followed by 40 cycles of 95° C. for 30 sec, 57° C. for 30 sec and 72° C. for 60 sec. The PCR products were electrophoresed on a 1% agarose gel to detect specific amplification products. Lipid Analysis of Potato Tubers Thin slices of tubers harvested from regenerated potato plants, for confirmed transgenic plants and non-transformed controls, were freeze-dried for 72 hr and analysed for lipid content and composition. Total lipids were extracted from the dried tuber tissues using chloroform:methanol:0.1 M KCl (2:1:1 v/v/v) as follows. The freeze-dried tuber tissues were first homogenized in chloroform:methanol (2:1, v/v) in an eppendorf tube containing a metallic ball using a Reicht tissue lyser (Qiagen) for 3 min at a frequency of 29 per sec. After mixing each homogenate with a Vibramax 10 (Heidolph) at 2,000 rpm for 15 min, 1/3 volume of 0.1 M KCl solution was added to each sample and mixed further. Following centrifugation at 10,000 g for 5 min, the lower phase containing lipids from each sample was collected and evaporated completely using N2flow. Each lipid preparation was dissolved in 3 μL of CHCl3per milligram of tuber dry weight. Aliquots of the lipid preparations were loaded on a thin layer chromatography (TLC) plate (20 cm×20 cm, Silica gel 60, Merck) and developed in hexane:diethyl ether:acetic acid (70:30:1, v/v/v). The TLC plate was sprayed with Primuline and visualized under UV to show lipid spots. TAG and PL were recovered by scraping the silica of the appropriate bands and converted to fatty acid methyl esters (FAME) by incubating the material in 1 N methanolic-HCl (Supelco, Bellefonte, Pa.) at 80° C. for 2 hr together with known amount of Triheptadecanoin (Nu-Chek PREP, Inc. USA) as internal standard for lipid quantification. FAME were analysed by GC-FID (7890A GC, Agilent Technologies, Palo Alto, Calif.) equipped with a 30 m BPX70 column (0.25 mm inner diameter, 0.25 mm film thickness, SGE, Austin, USA) as described previously (Petrie et al., 2012). Peaks were integrated with Agilent Technologies Chem Station software (Rev B.04.03). Among the approximately 100 individual transgenic lines regenerated, analysis of lipids derived from young potato tubers of about 2 cm in diameter revealed increased levels in total lipids, TAG and phospholipids fractions in tubers from many of the transgenic plants, with a range observed between no increase to substantial increases. The first analysis of the potato tuber lipids indicated that a typical wild-type potato tuber at its early stage of development (about 2 cm in diameter) contained about the 0.03% TAG on dry weight basis. The content of total lipids was increased to 0.5-4.7% by weight (dry weight) in tubers of 21 individual transgenic plants, representing 16 independently transformed lines (Table 29). Tubers of line #69 showed the highest TAG accumulation at an average 3.3% on dry weight basis. This was approximately a 100-fold increase relative to the wild-type tubers at the same developmental stage. Tubers of the same transgenic line also accumulated the highest observed levels of phospholipids at 1.0% by weight in the young tubers on a dry weight basis (Table 30). The enhanced lipid accumulation was also accompanied by an altered fatty acid composition in transgenic tubers. The transgenic tubers consistently accumulated higher percentages of saturated and monounsaturated fatty acids (MUFA) and lower level of polyunsaturated fatty acids (PUFA) in both the total fatty acid content and in the TAG fraction of the total fatty acid content (Table 29), particularly a reduced level of 18:3 (ALA) which was reduced from about 17% in the wild-type to less than 10% in the transgenic tubers. The level of oleic acid (18:1) in the total fatty acid content increased from about 1% in the wild-type to more than 5% in many of the lines and more than 15% in some of the tubers. Although palmitic acid levels were increased, the stearic acid (18:0) levels decreased in the best transgenic lines (Tables 28 and 29). TABLE 28Total lipid yield (% weight of potato tuber dry weight) and its fatty acid composition in representative young potatotubers transformed with the T-DNA of pJP3506, prior to flowering of the plants. Tubers of line 65 were equivalentto the wild-type (non-transgenic) tubers.SampleC14:0C16:0C16:1C16:3C18:0C18:1C18:1d11C18:2C18:3C20:0C20:1C22:0C24:0% TFA4-20.216.10.20.05.80.511.755.55.52.10.20.61.51.4190.218.10.20.05.80.412.952.25.92.00.20.71.51.527-20.218.90.30.06.50.55.555.08.02.00.20.82.10.727-40.219.00.30.06.55.40.557.07.91.60.00.51.10.627-50.217.80.60.06.42.20.457.611.71.50.00.41.20.727-60.218.70.40.06.96.30.555.98.21.60.00.40.90.8550.217.80.60.06.47.90.555.78.61.40.00.30.71.0650.219.40.40.05.71.20.553.617.20.90.00.01.00.5690.319.80.10.03.216.50.953.23.71.10.30.40.64.7780.219.50.50.05.34.90.554.711.71.20.00.41.00.9830.216.70.40.06.57.30.556.28.51.70.60.50.91.395-10.321.00.20.13.115.20.852.84.21.10.20.30.73.095-20.421.30.30.14.17.11.056.17.31.20.20.30.72.795-30.421.40.30.04.38.50.954.57.41.30.00.30.71.51000.419.00.50.05.47.60.855.57.31.40.50.50.91.01040.218.00.20.06.10.56.856.17.62.30.10.61.50.91060.219.70.20.14.60.910.754.15.71.70.10.61.31.3 TABLE 29TAG yield (% weight of potato tuber dry weight) and its fatty acid composition in representative young potatotubers, transformed with the T-DNA of pJP3506, prior to flowering of the plants.SampleC14:0C16:0C16:1C16:3C18:0C18:1C18:1d11C18:2C18:3C20:0C20:1C22:0C24:0% TAGWT0.413.40.00.04.65.50.559.915.70.00.00.00.00.034-20.315.40.20.07.00.616.452.53.12.60.20.61.10.5190.216.30.10.07.218.00.550.93.61.90.20.40.60.827-20.019.00.00.011.29.80.052.84.42.80.00.00.00.227-40.417.40.00.010.29.40.055.44.72.60.00.00.00.227-50.017.90.00.012.54.40.054.97.13.20.00.00.00.127-60.017.10.00.09.910.60.055.04.92.50.00.00.00.2550.317.60.50.08.512.50.652.55.21.90.00.00.60.5650.018.10.00.012.00.00.055.614.40.00.00.00.00.0690.320.10.60.03.820.31.049.42.21.30.20.30.53.3780.019.10.00.08.29.40.052.58.42.40.00.00.00.2830.316.40.20.08.711.10.653.45.42.60.00.50.70.595-10.321.70.40.13.618.51.050.12.80.90.20.20.32.295-20.623.40.40.05.110.11.251.95.31.40.00.00.50.995-30.317.20.30.07.70.611.649.78.92.50.00.01.10.11000.018.80.50.08.012.00.854.03.92.00.00.00.00.41040.317.70.00.08.40.611.052.14.73.20.00.71.30.31060.420.10.30.05.415.51.151.83.61.40.00.00.40.7 TABLE 30Phospholipids yield (% weight of potato tuber dry weight) and its fatty acid composition in representative youngpotato tubers, transformed with the T-DNA of pJP3506, prior to flowering.SampleC14:0C16:0C16:1C16:3C18:0C18:1C18:1d11C18:2C18:3C20:0C20:1C22:0C24:0% PL4-20.221.20.20.04.60.43.857.89.30.90.00.01.70.3190.122.70.20.04.45.10.354.98.90.71.00.51.20.427-20.221.00.30.05.22.80.456.99.30.91.30.41.40.427-40.022.90.00.06.02.30.057.28.81.10.00.01.60.327-50.019.60.50.05.01.20.058.712.61.00.00.01.40.427-60.022.90.00.06.32.60.056.39.31.20.00.01.50.3550.121.20.40.05.12.10.057.811.40.70.00.01.00.4650.021.40.40.05.91.10.053.215.71.00.00.01.30.3690.221.50.20.02.33.70.661.97.90.60.00.40.81.0780.022.10.40.04.42.70.455.612.20.80.00.01.30.4830.221.10.30.05.02.90.457.110.70.80.00.41.10.595-10.224.80.50.02.63.50.659.17.60.60.00.00.60.695-20.322.10.00.02.72.10.661.09.60.70.00.00.90.695-30.223.20.50.03.13.60.757.79.30.70.00.00.90.51000.023.30.50.04.63.00.457.29.00.80.00.01.10.41040.021.30.00.04.82.70.058.310.11.00.00.01.70.41060.223.20.20.03.83.00.657.18.60.71.00.41.10.4 The transgenic potato plants were maintained in the glasshouse to allow for continued growth of the tubers. Larger tubers of line #69 contained greater levels of TFA and TAG than the tubers of about 2 cm in diameter. Further increased levels of TFA and TAG are obtained in potato tubers by addition of a chimeric gene that encodes a silencing RNA for down-regulating the expression of the endogenous SDP1 gene, in combination with the WRI1 and DGAT genes. Further Gene Combinations for Transformation of Potato Total RNA from fresh developing potato (Solanum tuberosumL. cv. Atlantic) tubers was extracted by the TRIzol method (Invitrogen). Selected regions of the cDNAs encoding potato AGPase small subunit and SDP1 were obtained through RT-PCR using the following primers: st-AGPs1: 5′-ACAGACATGTCTAGACCCAGATG-3′ (SEQ ID NO: 242), st-AGPa1: 5′-CACTCTCATCCCAAGTGAAGTTGC-3′ (SEQ ID NO: 243); st-SDP1-s1: 5′-CTGAGATGGAAGTGAAGCACAGATG-3′ (SEQ ID NO: 244), and st-SDP1-a1: 5′-CCATTGTTAGTCCTTTCAGTC-3′ (SEQ ID NO: 245). The PCR products were then purified and ligated to pGEMT Easy. Following verification by DNA sequencing, the cloned PCR products were either directly used as the target gene sequence to make a hairpin RNAi construct or fused by overlapping PCR. Three PCR fragments (SDP1, AGPase, SDP+AGP) were subsequently cloned into the pKannibal vector that contained specific restriction sites to clone the desired fragment in sense and antisense orientation. The restriction sites selected were BamHI and HindIII for cloning the fragment in the sense orientation and KpnI and XhoI for inserting the fragment in the antisense orientation. Primers sets used for amplification of the three target gene fragments were altered by addition of restriction sites which direct the fragment into cloning sites of pKannibal. The expression cassettes containing the target DNA fragment between the 35S promoter and OCS terminator in pKannibal were released with NotI and cloned into a binary vector pWBVec2 with hygromycin as the plant selectable marker. Such binary vectors were introduced intoA. tumefaciensAGL1 strain and used for potato transformation as described above. Example 14. Modifying Traits in Monocotyledonous Plants Expression in Endosperm The oil content in the endosperm of the monocotyledonous plant speciesTriticum aestivum(wheat) and therefore in the grain of the plants was increased by expressing a combination of genes encoding WRI1, DGAT and Oleosin in the endosperm during grain development using endosperm-specific promoters. The construct (designated pOIL-Endo2) contained the chimeric genes: (a) the promoter of the Glu1 gene ofBrachypodium distachyon::protein coding region of theZea maysgene encoding the ZmWRI1 polypeptide (SEQ ID NO:35)::terminator/polyadenylation region from theGlycine maxlectin gene, (b) the promoter of the Bx17 glutenin gene ofTriticum aestivum::protein coding region of theA. thalianagene encoding the AtDGAT1 polypeptide (SEQ ID NO:1)::terminator/polyadenylation region from theAgrobacterium tumefaciensNos gene, (c) the promoter of the GluB4 gene ofOryza sativa::protein coding region of theSesame indicumgene encoding the Oleosin polypeptide:terminator/polyadenylation region from theGlycine maxlectin gene and (d) a 35S promoter::hygromycin resistance coding region as a selectable marker gene. The construct was used to transform immature embryos ofT. aestivum(cv. Fielder) byAgrobacterium-mediated transformation. The inoculated immature embryos were exposed to hygromycin to select transformed shoots and then transferred to rooting medium to form roots, before transfer to soil. Thirty transformed plants were obtained which set T1 seed and contained the T-DNA from pOIL-Endo2. Mature seeds were harvested from all 30 plants, and 6 seed of each family cut in half. The halves containing the embryo were stored for later germination; the other half containing mainly endosperm was extracted and tested for oil content. The T-DNA inserted into the wheat genome was still segregating in the T1 seeds from these plants, so the T1 seeds were a mixture of homozygous transformed, heterozygous transformed and nulls for the T-DNA. Increased oil content was observed in the endosperm of some of the grains, with some grains showing greater than a 5-fold increase in TAG levels. The endosperm halves of six wild-type grains (cv. Fielder) had a TAG content of about 0.47% by weight (range 0.37% to 0.60%), compared to a TAG content of 2.5% in some grains. Some families had all six grains with TAG in excess of 1.7%; others were evidently segregating with both wild type and elevated content of TAG. In endosperms with elevated TAG content the fatty acid composition was also altered, showing increases in the percentages of oleic acid and palmitic acid, and a decrease in the percentage of linoleic acid (Table 31). The T1 grain germinated without difficulty at the same rate as the corresponding wild-type grain and plants representing both high oil and low oil individuals from 14 T0 families were grown to maturity. These plants were fully male and female fertile. TABLE 31Fatty acid composition (% of total fatty acids) of TAG content and thetotal TAG content (% oil by weight of half endosperms) in transgenicwheat endospermSampleC14:0C16:0C16:1C16:3C18:0C18:1C18:1d11Control 10.316.90.10.01.615.60.6Control 20.316.00.10.11.615.10.6F5.30.120.10.10.12.623.50.6F16.30.119.10.10.12.824.20.6% oil bySampleC18:2C18:3n3C20:0C20:1C22:0C24:0wt.Control 160.44.00.10.40.00.00.5Control 261.34.30.10.30.00.00.49F5.348.52.40.80.70.30.42.5F16.348.12.90.70.50.30.41.8 220 T2 seed from 22 selected T1 plants were analysed, plus 40 plants from 3 different parental Fielder plants. In most cases ten T2 seed from each T1 plant were tested. Some of the selected T1 plants were nulls with wild type endosperm TAG levels. Some of the results for endosperm half seed analyses are represented inFIG.18. The high endosperm oil T1 plants produced T2 grain many of which had increased endosperm oil, whereas the control Fielder and null segregant T1 plants produced grain with similar levels of endosperm oil (total fatty acid, TFA). The grain is useful for preparing food products for human consumption or as animal feed, providing grain with an increased energy content per unit weight (energy density) and resulting in increased growth rates in the animals such as, for example, poultry, pigs, cattle, sheep and horses. The construct pOIL-Endo2 is also used to transform corn (Zea mays) and rice (Oryza sativa) to obtain transgenic plants which have increased TAG content in endosperm and therefore in grain. Expression in Leaves and Stems A series of binary expression vectors was designed forAgrobacterium-mediated transformation ofsorghum(S. bicolor) and wheat (Triticum aestivum) to increase the oil content in vegetative tissues. The starting vectors for the constructions were pOIL093-095, pOIL134 and pOIL100-104 (see Example 5). Firstly, a DNA fragment encoding theZ. maysWRI1 polypeptide was amplified by PCR using pOIL104 as a template and primers containing KpnI restriction sites. This fragment was subcloned downstream of the constitutiveOryza sativaActin1 promoter of pOIL095, using the KpnI site. The resulting vector was designated pOIL154. The DNA fragment encoding theUmbelopsis ramannianaDGAT2a under the control of theZ. maysubiquitin promoter (pZmUbi) was isolated from pOIL134 as a NotI fragment and inserted into the NotI site of pOIL154, resulting in pOIL155. An expression cassette consisting of the PAT coding region under the control of the pZmUbi promoter and flanked at the 3′ end by theA. tumefaciensNOS terminator/polyadenylation region was constructed by amplifying the PAT coding region using pJP3416 as a template. Primers were designed to incorporate BamHI and SacI restriction sites at the 5′ and 3′ ends, respectively. After BamHI+SacI double digestion, the PAT fragment was cloned into the respective sites of pZLUbi 1 casNK. The resulting intermediate was designated pOIL141. Next, the PAT selectable marker cassette was introduced into the pOIL155 backbone. To this end, pOIL141 was first cut with Nod, blunted with Klenow fragment of DNA polymerase I and subsequently digested with AscI. This 2622 bp fragment was then subcloned into the ZraI-AscI sites of pOIL155, resulting in pOIL156. Finally, the Actin1 promoter driving WRI1 expression in pOIL156 was exchanged for theZ. maysRubisco small subunit promoter (pZmSSU) resulting in pOIL157. This vector was obtained by PCR amplification of theZ. maysSSU promoter using pOIL104 as a template and flanking primers containing AsiSI and PmJI restriction sites. The resulting amplicon was then cut with SpeI+MluI and subcloned into the respective sites of pOIL156. These vectors therefore contained the following expression cassettes:pOIL156: promoterO. sativaActin1::Z. maysWRI1, promoterZ. maysUbiquitin::U. rammanianaDGAT2a and promoterZ. maysUbiquitin::PATpOIL157: promoterZ. maysSSU::Z. maysWRI1, promoterZ. maysUbiquitin::U. rammanianaDGAT2a andZ. maysUbiquitin::PAT. A second series of binary expression vectors containing theZ. maysSEE1 senescence promoter (Robson et al., 2004, see Example 5),Z. maysLEC1 transcription factor (Shen et al., 2010) and aS. bicolorSDP1 hpRNAi fragment were constructed as follows. First, a matrix attachment region (MAR) was introduced into pORE04 by AatII+SnaBI digest of pDCOT and subcloning into the AatII+EcoRV sites of pORE04. The resulting intermediate vector was designated pOIL158. Next, the PAT selectable marker gene under the control of theZ. maysUbiquitin promoter was subcloned into pOIL158. To this end, pOIL141 was first digested with NotI, treated with Klenow fragment of DNA polymerase I and finally digested with AscI. The resulting fragment was inserted into the AscI+ZraI sites of pOIL158, resulting in pOIL159. The original RK2 oriV origin of replication in pOIL159 was exchanged for the RiA4 origin by SwaI+SpeI restriction digestion of pJP3416, followed by subcloning into the SwaI+AvrII sites of pOIL159. The resulting vector was designated pOIL160. A 10.019 kb ‘Monocot senescence part1’ fragment containing the following expression cassettes was synthesized:O. sativaActin1::A. thalianaDGAT1, codon optimized forZ. maysexpression,Z. maysSEE1::Z. maysWRI1,Z. maysSEE1::Z. maysLEC1. This fragment was subcloned as a SpeI-EcoRV fragment into the SpeI-StuI sites of pOIL160, resulting in pOIL161. A second 7.967 kb ‘Monocot senescence part2’ fragment was synthesized and contains the following elements: MAR,Z. maysUbiquitin::hpRNAi fragment targeted againstS. bicolor/T. aestivumSDP1, empty cassette under the control of theO. sativaActin1 promoter. The sequences of twoS. bicolorSDP1 TAG lipases (Accession Nos. XM_002463620; SEQ ID NO:233 and XM_002458486; SEQ ID NO:169) and oneT. aestivumSDP1 sequence (Accession No. AK334547) (SEQ ID NO: 234) were obtained by a BLAST search with theA. thalianaSDP1 sequence (Accession No. NM_120486). A synthetic hairpin construct (SEQ ID NO:235) was designed including four fragments (67 bp, 90 bp, 50 bp, 59 bp) of theS. bicolorXM_002458486 sequence that showed highest degree of identity with theT. aestivumSDP1 sequence. In addition, a 278 bp fragment originating from theS. bicolorXM_002463620 SDP1 lipase was included to increase silencing efficiency against bothS. bicolorSDP1 sequences. The ‘Monocot senescence part2’ fragment is subcloned as a BsiWI-EcoRV fragment into the BsiWI-Fsp1 sites of pOIL161. The resulting vector is designated pOIL162. The genetic constructs pOIL156 pOIL157, pOIL161 and pOIL162 are used to transformS. bicolorandT. aestivumusingAgrobacterium-mediated transformation. Transgenic plants are selected for hygromycin resistance and contain elevated levels of TAG and TFA in vegetative tissues compared to untransformed control plants. Such plants are useful for providing feed for animals as hay or silage, as well as producing grain, or may be used to extract oil. Further genetic constructs are made for expression of combinations of polypeptides in leaves and stems of monocotyledonous plants, including the C4-photosynthesis plantsS. bicolorandZ. mays. Several constructs are made containing genes for expression of WRI1, DGAT and oleosin, with each gene under the control of a constitutive promoter such as a maize Ubiquitin gene promoter or a rice actin gene promoter, and containing an NPTII gene as selectable marker gene. In one particular construct, the WRI1 issorghumWRI1. In another, the oleosin is SiOleosinL (see Example 17). In other particular constructs, the oleosin gene is replaced with a gene encoding either LDAP2 or LDAP3 fromS. sebiferum(Example 11). These constructs are used as the “core constructs” for transformation ofS. bicolorandZ. maysand are deployed on their own or in combination with genetic constructs for expression of a hairpin RNA targeting one or more SDP1 genes insorghumor maize (see above), a construct encoding Lec2 under the control of a SEE1 promoter (senescence specific), or both. Another construct is made comprising three genes, namely for expression of a hairpin RNA targeting the endogenous TGD5 gene to reduce its expression, a FatA fatty acyl thioesterase and a PDAT, which is used to increase the level of TAG and/or the TTQ parameter for plants transformed with this construct. Example 15. Extraction of Oil Extraction of Lipid from Leaves Transgenic tobacco leaves which had been transformed with the T-DNA from pJP3502 were harvested from plants grown in a glasshouse during the summer months. The leaves were dried and then ground to 1-3 mm sized pieces prior to extraction. The ground material was subject to soxhlet (refluxing) extraction over 24 hours with selected solvents, as described below. 5 g of dried tobacco leaf material and 250 ml of solvent was used in each extraction experiment. Hexane Solvent Extraction Hexane is commonly used as a solvent commercially for oil extraction from pressed oil seeds such as canola, extracting neutral (non-polar) lipids, and was therefore tried first. The extracted lipid mass was 1.47 g from 5 g of leaf material, a lipid recovery of 29% by weight. 1H NMR analysis of the hexane extracted lipid in DMSO was preformed. The analysis showed typical signals for long chain triglyceride fatty acids, with no aromatic products being present. The lipid was then subjected to GCMS for identification of major components. Direct GCMS analysis of the hexane extracted lipid proved to be difficult as the boiling point was too high and the material decomposed in the GCMS. In such situations, a common analysis technique is to first make methyl esters of the fatty acids, which was done as follows: 18 mg lipid extract was dissolved in 1 mL toluene, 3 mL of dry 3N methanolic HCL was added and stirred overnight at 60° C. 5 mL of 5% NaCl and 5 mL of hexane were added to the cooled vial and shaken. The organic layer was removed and the extraction was repeated with another 5 mL of hexane. The combined organic fractions were neutralized with 8 mL of 2% KHCO3, separated and dried with Na2SO4. The solvent was evaporated under a stream of N2 and then made up to a concentration of lmg/mL in hexane for GCMS analysis. The main fatty acids present were 16:0 (palmitic, 38.9%) and 18:1 (oleic, 31.3%). FA16:016:118:018:118:220:022:0% wt38.94.66.431.32.51.50.6 Acetone Solvent Extraction Acetone was used as an extraction solvent because its solvent properties should extract almost all lipid from the leaves, i.e. both non-polar and polar lipids. The acetone extracted oil looked similar to the hexane extracted lipid. The extracted lipid mass was 1.59 g from 5 g of tobacco leaf, i.e. 31.8% by weight. 1H NMR analysis of the lipid in DMSO was performed. Signals typical of long chain triglyceride fatty acids were observed, with no signal for aromatic products. Hot Water Solvent Extraction Hot water was attempted as an extraction solvent to see if it was suitable to obtain oil from the tobacco leaves. The water extracted material was gel like in appearance and gelled when cooled. The extracted mass was 1.9 g, or 38% by weight. This material was like a thick gel and was likely to have included polar compounds from the leaves such as sugars and other carbohydrates. The 1H NMR analysis of the material in DMSO was preformed. The analysis showed typical signals for long chain triglyceride fatty acids, with no aromatic products being extracted. The left over solid material was extracted with hexane, yielding 20% of lipid by weight, indicating that the water extraction had not efficiently extracted non-polar lipids. Ethanol Solvent Extraction Ethanol was used as an extraction solvent to see if it was suitable to obtain oil from the tobacco leaves. The ethanol extracted lipid was similar in appearance to both the water- and hexane-extracted lipid, being yellow-red in colour, had a gel-like appearance and gelled when cooled. The extracted lipid mass was 1.88 g from 5 g tobacco, or 37.6% by weight. The ethanol solvent would also have extracted some of the polar compounds in the tobacco leaves. Ether Solvent Extraction Diethyl ether was attempted as an extraction solvent since it was thought that it might extract less impurities than other solvents. The extraction yielded 1.4 g, or 28% by weight. The ether extracted lipid was similar to the hexane extracted material in appearance, was yellowish in colour, and it did appeared a little cleaner than the hexane extract. While the diethyl ether extraction appeared to have given the cleanest oil, the NMR analysis showed a mixture of more organic compounds. Example 16. Feed Rations for Dairy Cows Leaves and stems fromsorghumor corn plants comprising increased TAG and TFA contents are harvested and chopped into pieces 1-2 cm in size. The processed plant parts are ensiled for at least two weeks and then mixed with other components to produce a feedstuff for dairy cows. The feed mixture for dairy cows comprises: 7.5-10 kg ofsorghumor corn silage comprising increase TAG and TFA, 4-5 kg of alfalfa hay, 1 kg brewers grain (about 67% digestible dry matter), 1-2 kg seed meal (canola or soy) or cottonseed, 0.5 kg molasses and mineral supplements such as calcium, phosphorus, magnesium and sulfur. Lipid is optimally present at 5-7% of the total dry matter. Additional amino acids such as lysine and methionine or non-protein nitrogen supplies such as urea may be added, depending on the total protein content. The feedstuff has increased energy density, increased feed value, increased nutritive value and increased digestibility relative to a corresponding feedstuff made with an equivalent amount of wild-typesorghumor corn silage. The increased lipid in the high-oilsorghumor corn silage results in an additional milk production of up to 3 litres per day and an increase of 0.33% in milk fat for each kilogram of lipid eaten. A heifer will eat the equivalent of about 2.3% of her body weight daily and an adult dry cow will eat the equivalent of about 1.5% of her body weight daily. For example, a 300 kg heifer can eat up to 7 kg dry matter and an adult, dry cow weighing 470 kg will eat about the same amount. Lactating cows have greater feed intakes, up to about 4% of body weight per day. Indeed, feed intake on a weight basis tends to increase with feed quality and palatability. Example 17. Expression of Oil Body Proteins in Plant Vegetative Tissues A protein coding region encoding aRhodococcus opacusTadA lipid droplet associated protein (MacEachran et al. 2010; Accession number HM625859), codon optimized for expression in dicotyledonous plants such asNicotiana benthamiana, was synthesised as a NotI-SpeI DNA fragment. The fragment was inserted downstream of the 35S promoter in pJP3343 using the NotI-SpeI sites. The resultant plasmid was designated pOIL380. A protein coding region encoding aSesame indicumOleosinL lipid droplet associated protein (Tai et al. 2002; Accession number AF091840; SEQ ID NO:305) was synthesised as a NotI-SacI DNA fragment and inserted downstream of the 35S promoter in pJP3343 using the same sites. The resultant plasmid was designated pOIL382. A protein coding region encoding aSesame indicumOleosinH1 lipid droplet associated protein (Tai et al., 2002; Accession number AF302807) was synthesised as a NotI-SacI DNA fragment and cloned downstream of the 35S promoter in pJP3343 using the same sites. The resultant plasmid was designated pOIL383. A variant of the protein coding region encodingS. indicumOleosinH1 having three amino acid substitutions to remove ubiquitination sites (K130R, K143R, K145R) (Hsiao and Tzen, 2011) was generated by targeted mutagenesis. The coding region was inserted downstream of the 35S promoter in pJP3343 as a NotI-SacI fragment. The resultant plasmid was designated pOIL384. A protein coding region encoding aVanilla planifolialeaf OleosinU1 lipid droplet associated protein (Huang and Huang, 2016; Accession number SRX648194) was codon optimized for expression inN. benthamiana, synthesised as a SpeI-EcoRI DNA fragment and inserted downstream of the 35S promoter in pJP3343 using the same sites. The resultant plasmid was designated pOIL386. A protein coding region encoding aPersea americanamesocarp OleosinM lipid droplet associated protein (Huang and Huang 2016; Accession number SRX627420) was codon optimized for expression inN. benthamiana, synthesised as a SpeI-EcoRI DNA fragment and inserted downstream of the 35S promoter in pJP3343 using the same restriction sites. The resultant plasmid was designated pOIL387. A protein coding region encoding anArachis hypogaeaOleosin 3 lipid droplet associated protein (Parthibane et al., 2012a; Accession number AY722696) was codon optimized for expression inN. benthamiana, flanked by NotI sites and inserted into the binary expression vector pJP3502. The resulting plasmid, pOIL041, was digested with NotI and the resultant 520 bp DNA fragment was inserted downstream of the 35S promoter of pJP3343. The resultant plasmid was designated pOIL190. Similarly, the protein coding region for theA. thalianaCaleosin3 lipid droplet associated protein (Shen et al., 2014; Laibach et al., 2015; Accession number AK317039) was codon optimized for expression inN. benthamiana, flanked by NotI sites and inserted into pJP3502. The resulting plasmid, pOIL042, was digested with NotI and the resulting 604 bp DNA fragment was inserted downstream of the 35S promoter of pJP3343. The resultant plasmid was designated pOIL191. A protein coding region encoding anA. thalianasteroleosin lipid droplet associated protein (Accession number AT081653) was codon optimized for expression inN. benthamiana, flanked by NotI sites and inserted into pJP3502. The resultant plasmid, pOIL043, was digested with NotI and the resultant 1069 bp DNA fragment was inserted downstream of the 35S promoter of pJP3343. The resultant plasmid was designated pOIL192. A protein coding region encoding aNannochloropsis oceanicaLSDP oil body protein (Vieler et al., 2012; Accession number JQ268559) was codon optimized for expression inN. benthamiana, flanked by NotI sites and inserted into the pJP3502 binary expression vector. The resultant plasmid, pOIL044, was digested with NotI and the 496 bp DNA fragment was inserted downstream of the 35S promoter of pJP3343. The resultant plasmid was designated pOIL193. A protein coding region encoding aTrichoderma reeseiHFBI hydrophobin (Linder et al., 2005; Accession number Z68124) was codon optimized for expression inN. benthamiana, flanked by NotI sites and inserted into pJP3502. The resultant plasmid, pOIL045, was digested with NotI and the 313 bp DNA fragment was inserted downstream of the 35S promoter of pJP3343. The resultant plasmid was designated pOIL194. An ER-targeted variant of theTrichoderma reeseiHFBI hydrophobin was created by amending the KDEL ER retention peptide to the C-terminus (Gutierrew et al., 2013). This variant was codon optimized for expression inN. benthamianaand cloned as a NotI fragment into pJP3502, resulting in pOIL046. Subsequently, pOIL046 was digested with NotI and the 325 bp fragment was inserted into pJP3343. The resulting vector was designated pOIL195. Each of the genetic constructs encoding the lipid droplet associated polypeptides were introduced intoN. benthamianaleaves in combination with genetic constructs encoding WRI1, DGAT1 and p19 as described in Example 1 with some minor modifications.Agrobacterium tumefacienscultures containing the gene coding for the p19 silencing suppressor protein and the chimeric genes of interest were mixed such that the final OD600 of each culture was equal to 0.125 prior to infiltration. Samples being compared were located on the same leaf. After infiltration,N. benthamianaplants were grown for a further five days before leaf discs were harvested, pooled across three leaves from the same plant, freeze-dried, weighed and stored at −80° C. Total lipids were extracted from freeze-dried tissues using chloroform:methanol:0.1 M KCl (2:1:1 v/v/v) and aliquots loaded on a thin layer chromatography (TLC) plate and developed in hexane:diethyl ether:acetic acid (70:30:1, v/v/v). TAG was recovered, converted to FAME in the presence of a known amount of triheptadecanoin (Nu-Chek PREP, Inc. USA) as internal standard for lipid quantitation, and analysed by GC-FID. The assays showed a range of TAG levels compared to the WRI1+DGAT1 control. Some constructs encoding lipid droplet associated polypeptides increased the TAG level relative to the control in some assays whereas others did not. A consistent and statistically significant increase in TAG content was observed when the construct expressing SiOleosinL (pOIL382) was introduced (FIG.20); this construct was superior to all the others tested in these assays. An increase in the levels of C18:2 and C18:1 and a decrease in C16:0 was also observed in the TAG for this construct, relative to the p19+WRI1+DGAT1 control (FIG.20). Microscopic analyses to visualise lipid droplets in the leaf cells expressing SiOleosinL showed a decrease in lipid droplet size and an increase in abundance compared to the control. Further assays were carried out using radiolabelled [14C]-acetate to measure the rate of TAG synthesis for the different gene combinations including each of the lipid droplet associated polypeptides. The [14C]-acetate was infiltrated into the same leaf tissues at 3 days post-infiltration of the genetic constructs i.e. after the genes had been expressed for three days. Three hours later, leaf discs were harvested and total lipids in the tissues were extracted and fractionated by TLC. The amount of radioactivity in different lipid types was quantitated using a Fujifilm FLA-5000 phosphorimager. These assays demonstrated an increase in TAG synthesis rates in the leaves expressing SiOleosinL (pOIL382) as well as an increase in PC and PA synthesis rates over the three hours in leaves expressing SiOleosinL. In contrast, the genetic constructs encoding SiOleosinH, vanilla leaf and avocado mesocarp oleosins did not show a significant effect on TAG synthesis rate or content. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. 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11859194 | DETAILED DESCRIPTION OF THE INVENTION Most life is ultimately sustained by photosynthesis and its rate limiting carbon fixing enzyme, Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme incorporates CO2into plant carbohydrates during photosynthesis. Atmospheric oxygen competes with CO2as a substrate for Rubisco, giving rise to photorespiration and making Rubisco the rate-limiting step in photosynthesis under certain conditions. Although the relatively simple cyanobacterial Rubisco is amenable to in vitro assembly, the hexadecameric higher plant enzyme has been refractory to such manipulation, due to poor understanding of its assembly pathway. In accordance with the present invention, we created transgenic maize lines with enhanced Rubisco abundance. The data presented herein show that overexpression of Rubisco assembly factors and subunits will confer a physiological advantage to maize, specifically under cold stress conditions where Rubisco activity is limiting. Rubisco content has previously been increased 24-30% in transgenic rice, on a leaf area basis, through overexpression of endogenous SS (Suzuki et al. 2007) or a sorghum SS gene (Ishikawa et al. 2011). In both cases Rubisco activity was slightly decreased and the photosynthetic rate was not improved, even when Rubisco kcat was significantly increased. In maize, we previously overexpressed a nucleus-transplanted copy of the LS gene (LSN), and SS, both under control of the ubiquitin promoter (UBI-LS-SS). While both transgenes were expressed at a high level and nucleus-encoded LS was readily incorporated into holoenzyme, no overall change in Rubisco abundance was observed (Wostrikoff et al. 2012). Thus, in C3 rice increasing Rubisco abundance did not confer a photosynthetic advantage, and in C4 maize simply increasing subunit expression did not affect holoenzyme accumulation. From genetic analysis, we now know that apart from LS and SS, BSD2, RAF1, CPS2/Cpn60 and RAF2 are all required for Rubisco assembly and/or stability in maize. Taking advantage of this knowledge we have added ubiquitin promoter-driven expression of RAF1, or both BSD2 and RAF1 to the UBI-LS-SS line, which as shown below increases LS, and presumably holoenzyme abundance. Ubi-RAF2 transgenics can also be generated and can be introgressed into this background for a potential incremental increase above what we have observed. The following definitions are provided to facilitate an understanding of the present invention. As used herein, “genetically altered” means the modified expression of at least two, three or four of RAF1, a BSD2, LS, SS protein or both, or functional mutants thereof resulting from one or more genetic modifications; the modifications including but not limited to: recombinant gene technologies, induced mutations, and breeding stably genetically modified plants to produce progeny and seed comprising the altered gene product. A “modified plant” is a plant that has been treated with an agent that increases expression of endogenous levels of at least SS/RAF1 when compared to wild type untreated plants. Alternatively the plant may be treated with an agent that elevates expression of SS/RAF1 and LS. In yet another embodiment, the treatment results in elevated levels of SS/RAF1/LS and BSD2. A “transgenic plant” refers to a plant whose genome has been altered by the introduction of at least one heterologous nucleic acid molecule. Transgenic plants comprising altered RAF1 protein are provided herein. The term “decreased” is intended to mean that the measurement of a parameter is changed by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more when compared to the measurement of that parameter in a suitable control. The term “increased” is intended to mean that the measurement of a parameter is changed by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more when compared to the measurement of that parameter in a suitable control. The terms “inhibit,” “inhibition,” “inhibiting”, “reduced”, “reduction” and the like as used herein refer to any decrease in the expression or function of a target gene product, including any relative decrement in expression or function up to and including complete abrogation of expression or function of the target gene product. The terms “promote,” “upregulate,” “increase”, and “overexpress”, and the like as used herein refer to any increase in the expression or function of a target gene product, including any relative increase in expression or function of the target gene product. The term “expression” as used herein in the context of a gene product refers to the biosynthesis of that gene product, including the transcription and/or translation of the gene product. Inhibition of expression or function of a target gene product (i.e., a gene product of interest) can be in the context of a comparison between any two plants, for example, expression or function of a target gene product (e.g., protein) in a genetically altered plant versus the expression or function of that target gene product in a corresponding wild-type plant. Expression levels can also be used to refer to increases or decreases in protein levels due to alterations in stability or assembly of such proteins. Inhibition of expression or function of the target gene product can be in the context of a comparison between plant cells, organelles, organs, tissues, or plant parts within the same plant or between plants, and includes comparisons between developmental or temporal stages within the same plant or between plants. Any method or composition that down-regulates expression of a target gene product, either at the level of transcription, translation, or stability or down-regulates functional activity of the target gene product can be used to achieve inhibition of expression or function of the target gene product. The term “inhibitory sequence” encompasses any polynucleotide or polypeptide sequence that is capable of inhibiting the expression of a target gene product, for example, at the level of transcription or translation, or which is capable of inhibiting the function of a target gene product. Exemplary constructs encoding such inhibitory sequences are disclosed herein. When the phrase “capable of inhibiting” is used in the context of a polynucleotide inhibitory sequence, it is intended to mean that the inhibitory sequence itself exerts the inhibitory effect; or, where the inhibitory sequence encodes an inhibitory nucleotide molecule (for example, hairpin RNA, miRNA, or double-stranded RNA polynucleotides), or encodes an inhibitory polypeptide (i.e., a polypeptide that inhibits expression or function of the target gene product), following its transcription (for example, in the case of an inhibitory sequence encoding a hairpin RNA, miRNA, or double-stranded RNA polynucleotide) or its transcription and translation (in the case of an inhibitory sequence encoding an inhibitory polypeptide), the transcribed or translated product, respectively, exerts the inhibitory effect on the target gene product (i.e., inhibits expression or function of the target gene product). Conversely, the terms “increase”, “increased”, and “increasing” in the context of the methods of the present invention refer to any increase in the expression or function of a gene product, including any relative increment in expression or function. In many instances the nucleotide sequences for use in the methods of the present invention, are provided in transcriptional units for transcription in the plant of interest. A transcriptional unit is comprised generally of a promoter and a nucleotide sequence operably linked in the 3′ direction of the promoter, optionally with a terminator. “Operably linked” refers to the functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. The expression cassette will include 5′ and 3′ regulatory sequences operably linked to at least one of the sequences of the invention. Generally, in the context of an over expression cassette, operably linked means that the nucleotide sequences being linked are contiguous and, where necessary to join two or more protein coding regions, contiguous and in the same reading frame. In the case where an expression cassette contains two or more protein coding regions joined in a contiguous manner in the same reading frame, the encoded polypeptide is herein defined as a “heterologous polypeptide” or a “chimeric polypeptide” or a “fusion polypeptide”. The cassette may additionally contain at least one additional coding sequence to be co-transformed into the organism. Alternatively, the additional coding sequence(s) can be provided on multiple expression cassettes. With regard to nucleic acids used in the invention, the term “isolated nucleic acid” is sometimes employed. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5′ and 3′ directions) in the naturally occurring genome of the organism from which it was derived. For example, the “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote. An “isolated nucleic acid molecule” may also comprise a cDNA molecule. An isolated nucleic acid molecule inserted into a vector is also sometimes referred to herein as a recombinant nucleic acid molecule. With respect to RNA molecules, the term “isolated nucleic acid” primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form. By the use of the term “enriched” in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that “enriched” does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased. It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term “purified” in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level, this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process, which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones, yields an approximately 10−6-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. Thus the term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest. The term “probe” as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically. The term “primer” as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3′ terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3′ hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product. Polymerase chain reaction (PCR) has been described in U.S. Pat. Nos. 4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which are incorporated by reference herein. The term “vector” relates to a single or double stranded circular nucleic acid molecule that can be infected, transfected or transformed into cells and replicate independently or within the host cell genome. A circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that are targeted by restriction enzymes are readily available to those skilled in the art, and include any replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element. A nucleic acid molecule of the invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together. Many techniques are available to those skilled in the art to facilitate transformation, transfection, or transduction of the expression construct into a prokaryotic or eukaryotic organism. The terms “transformation”, “transfection”, and “transduction” refer to methods of inserting a nucleic acid and/or expression construct into a cell or host organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest, microinjection, PEG-fusion, and the like. The term “promoter element” describes a nucleotide sequence that is incorporated into a vector that, once inside an appropriate cell, can facilitate transcription factor and/or polymerase binding and subsequent transcription of portions of the vector DNA into mRNA. In one embodiment, the promoter element of the present invention precedes the 5′ end of the recombinant nucleic acid molecule such that the latter is transcribed into mRNA. Host cell machinery then translates mRNA into a polypeptide. Those skilled in the art will recognize that a nucleic acid vector can contain nucleic acid elements other than the promoter element and the RAF1 and or BSD2 coding nucleic acid molecule. These other nucleic acid elements include, but are not limited to, origins of replication, ribosomal binding sites, nucleic acid sequences encoding drug resistance enzymes or amino acid metabolic enzymes, and nucleic acid sequences encoding secretion signals, localization signals, or signals useful for polypeptide purification. A “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, plastid, phage or virus that is capable of replication largely under its own control. A replicon may be either RNA or DNA and may be single or double stranded. An “expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism. As used herein, the terms “reporter,” “reporter system”, “reporter gene,” or “reporter gene product” shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is readily measurable, e.g., by biological assay, immunoassay, radio immunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods. GFP is exemplified herein. The nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, and is operatively linked to the necessary control elements for the expression of the reporter gene product. The required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional-termination signals and the like. The term “selectable marker gene” refers to a gene that when expressed confers a selectable phenotype, such as herbicide tolerance, on a transformed plant cell. The terms “recombinant plant,” or “transgenic plant” refer to plants, which have a new combination of genes or nucleic acid molecules. A new combination of genes or nucleic acid molecules can be introduced into a plant using a wide array of nucleic acid manipulation techniques available to those skilled in the art. The term “isolated protein” or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins, with which it would naturally be associated, so as to exist in “substantially pure” form. “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into, for example, immunogenic preparations or pharmaceutically acceptable preparations. A “specific binding pair” comprises a specific binding member (sbm) and a binding partner (bp), which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules. Examples of specific binding pairs are antigens and antibodies, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples. Further, the term “specific binding pair” is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule. In embodiments in which the specific binding pair comprises nucleic acid sequences, they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long. Preparation of SS, RAF1, LS, and BSD2 Encoding Nucleic Acid Molecules and Creation of Transgenic Plants Containing the Same Nucleic acid molecules of the invention encoding desired polypeptides may be prepared by two general methods: (1) synthesis from appropriate nucleotide triphosphates, or (2) isolation from biological sources. Both methods utilize protocols well known in the art. The availability of nucleotide sequence information, such as the DNA sequences encoding LS, SS, RAF1 and/or BSD2, enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramidite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be used directly or purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Specific probes/primers for identifying such sequences as the LS, SS, RAF1 or BSD2 encoding sequence may be between 15 and 40 nucleotides in length. For probes/primers longer than those described above, the additional contiguous nucleotides are provided the nucleic acid sequences in GenBank for these proteins. Additionally, cDNA or genomic clones having homology with LS, SS, RAF1 and/or BSD2 may be isolated from other species using oligonucleotide probes corresponding to predetermined sequences within the target nucleic acids of the invention. Alternatively, the cDNA may be amplified by reverse transcriptase after making cDNA from the pool. The sequenced maize genome database provides the full length of cDNA and CDS. Such homologous sequences encoding the proteins of intereset may be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations may be performed, according to the method of Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989), using a hybridization solution comprising: 5×SSC, 5×Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42° C. for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2×SSC and 1% SDS; (2) 15 minutes at room temperature in 2×SSC and 0.1% SDS; (3) 30 minutes 1 hour at 37° C. in 1×SSC and 1% SDS; (4) 2 hours at 42-65° C. in 1×SSC and 1% SDS, changing the solution every 30 minutes. One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al., 1989) is as follows: Tm81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63(% formamide)−600/#bp in duplex. As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tmis 57° C. The Tmof a DNA duplex decreases by 11.5° C. with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42° C. The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25° C. below the calculated Tmof the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20° C. below the Tmof the hybrid. In regards to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A high stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. A very high stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes. Also encompassed within the scope of the invention are transgenic plants containing the aforementioned RAF1- and/or BSD2 encoding nucleic acids, or fragments or derivatives thereof. The following example is provided to illustrate certain embodiments of the invention. It is not intended to limit the invention in any way. Example I Transgenic Plant Performance Under Chilling Temperatures Due to the strong limitation Rubisco imposes on CO2assimilation at low temperatures, increasing Rubisco content may be a solution to enhance acclimation to cold in maize, as acclimation involving changes in Rubisco content does occur in some cool tolerant C4 species, such asMiscanthus(Dwyer et al. 2007, Sage and Kubien 2007). To our knowledge, this has never been directly been tested. We show that by overexpression of Rubisco produces high enough concentrations to avoid being limiting at low temperatures. The maize ubiquitin promoter was capable of overexpressing cold tolerance genes in rice under stress conditions (Ito et al. 2006), and should therefore be equally effective in maize grown at 14° C. Additionally, Rubisco activase, Cpn60, LS and SS maintain high average mRNA expression during chilling (Spence 2012). This suggests that decreases in Rubisco content in response to chilling occur post-transcriptionally or post-translationally, i.e. at the translational or protein stability level. Thus in principle, by increasing Rubisco content at least 40% we should be able to compensate for the ˜40% decrease in Rubisco accumulation seen in maize under chilling temperatures. Chloroplast-encoded LS interacts with the chaperonin complex to correctly fold the newly synthesized protein (Native LS). Nucleus-encoded SS is refolded after being translocated from the cytosol via the Tic-Toc complex. The data presented here show that several proteins, RAF2, RAF1 and/or BSD2 are involved in refolding imported SS, and also in forming assembly intermediates that capture folded LS once released from the chaperonin complex. In the absence of RAF2, RAF1 or BSD2, LS is subject to aggregation and proteolysis. Marginal holoenzyme assembly does occur in the absence of RAF2, thus it is possible that its role can be bypassed by RAF1 and BSD2. A schematic drawing of this complex is shown inFIG.1. We employed a transgenic approach to highly express Rubisco subunits and assembly proteins. SeeFIG.2wherein combinations of constructs A+B, C+D, A+C, and A+B+C were created. The results shown inFIGS.3and4demonstrate that Rubisco is assembled as the holoenzyme in bundle sheath cells. Total rubisco migrated at 550 KD, indicating that Rubisco is soluble and assembled into the hexadecameric form. PEPC (present in M cells only) and ME (only present in BS cells) were used to evaluate cell separation purity. From this, LS appears to be accumulating only in BS cells. To see if there was an obvious whole plant phenotype, we germinated UBI-RAF1, UBI-RAF1-SS, UBI-BSD2-LSN-SS-RAF1 and WT plants under 25° C. day/20° C. night conditions under high light (500 μmol photons m-2s-1). At day 18 after sowing we introduced chilling temperatures (14° C. day/12° C. night) for two weeks. 14° C. was used because maize has been shown to lose a significant proportion of its ability to assimilate CO2at and below that temperature (Naidu and Long 2004). We observed that a significant LS increase was found in the UBI-RAF1-SS (C+D) and the UBI-BSD2-LSN-SS-RAF1 (A+B+C) lines (FIG.5).FIG.6shows leaves before and after plants were exposed to 14° C. days/12° C. nights for 0, 1 or 2 weeks. The WT in this experiment was inbred B73, which is slightly less vigorous than the transgenic lines, which were created in the Hi-II hybrid background. Irrespective of size, the WT and UBI-RAF1 became increasingly chlorotic, whereas UBI-RAF1-SS and UBI-BSD2-LSN-SS-RAF1 largely maintained their chlorophyll content. At the end of the experiment (FIG.7), WT and UBI-RAF1 plants appeared both chlorotic and stunted compared to the lines which had been found to harbor increased Rubisco at normal growth temperatures (FIG.5). The comparison between UBI-RAF1 and UBI-RAF1-SS/UBIBSD2-LSN-SS-RAF1 is particularly informative, because all are Hi-II derivatives propagated by selfing or outcrossing to other Hi-II lines, and their statures were similar at the outset of the experiment. LS accumulation was measured in plants pre- and post cold stress, as shown inFIG.8. We found that in the WT control, Rubisco decreased ˜40% at chilling temperatures, in agreement with previous observations (Naidu et al. 2003, Spence 2012, Wang et al. 2008a). On the other hand, the decrease in Rubisco was mitigated in both UBI-RAF1-SS and UBI-BSD2-LSN-SSRAF1 transgenic plants. Quantification of the gels inFIG.8indicates that these lines have comparable levels of Rubisco accumulation in the cold, to what WT plants possess at optimal growth temperatures. Thus, the apparent tolerance to chilling can be correlated with maintenance of sufficient Rubisco content. This correlation of high expression of Rubisco LS protein with maize tolerance to chilling temperatures provides an indication that the transgenic plants described are resistant to other abiotic stresses and provides the means to create such plants. Gas exchange can be used to measures the rate of photosynthetic carbon assimilation. We performed additional studies to ascertain whether maintenance of rubisco content at low temperatures is important for sustaining photosynthetic capacity.FIG.9shows that the transgenic plants of the invention with higher rubisco content show increased CO2assimilation at 14° C.FIG.10shows that excess carbon fixed appears to be incorporated into biomass during chilling stress. The results presented herein show that rubisco content can be increased by overexpression of the subunits and assembly factors and that the overexpressed rubisco was present as holoenzyme in bundle sheath chloroplasts. Increased Rubisco content correlates with much higher photosynthetic rates and increased biomass and leaf area under chilling conditions. Notably, increasing Rubisco appears to lessen the effects of chilling stress in higher plants, and in maize in particular. REFERENCES (1) Long, S. P. (1983)Plant Cell Environ.,6, 345-363.(2) Wang, D., Portis, A. R., Jr., Moose, S. P. and Long, S. P. (2008)Plant Physiol.,148, 557-567.(3) Naidu, S. L. and Long, S. P. (2004)Planta,220, 145-155.(4) Naidu, S. L., Moose, S. P., AK, A. L.-S., Raines, C. A. and Long, S. P. (2003)Plant Physiol.,132, 1688-169(5) Spence, A. K. (2012)Plant Biology. Urbana, Illinois: Univ. Illinois Urbana-Champaign, pp. 146.(6) Suzuki et al. (2007)Plant Cell Physiol.,48:626-637.(7) Whitney et al. (2015)Proc Natl Acad Sci USA.112: 3564-3569. While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope of the present invention, as set forth in the following claims. | 33,820 |
11859195 | DETAILED DESCRIPTION OF THE INVENTION 1. Definitions The following terms are utilized throughout this application:Constitutive Promoter: Promoters referred to herein as “constitutive promoters” actively promote transcription under most, but not necessarily all, environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcript initiation region and the 1′ or 2′ promoter derived from T-DNA ofAgrobacterium tumefaciens, and other transcription initiation regions from various plant genes, such as the maize ubiquitin-1 promoter, known to those of skill.Domain: Domains are fingerprints or signatures that can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can comprise conserved (1) primary sequence, (2) secondary structure, and/or (3) three-dimensional conformation. Generally, each domain has been associated with either a family of proteins or motifs. Typically, these families and/or motifs have been correlated with specific in-vitro and/or in-vivo activities. A domain can be any length, including the entirety of the sequence of a protein. Detailed descriptions of the domains, associated families and motifs, and correlated activities of the polypeptides of the instant invention are described below. Usually, the polypeptides with designated domain(s) can exhibit at least one activity that is exhibited by any polypeptide that comprises the same domain(s).Drought: Plant species vary in their capacity to tolerate drought conditions. For each species, optimal growth can be achieved if a certain level of water is always available. Other factors such as temperature and soil conditions have a significant impact on the availability of water to the plant. “Drought” can be defined as the set of environmental conditions under which a plant will begin to suffer the effects of water deprivation, such as decreased photosynthesis, loss of turgor (wilting) and decreased stomatal conductance. This drought condition results in a significant reduction in yield. Water deprivation may be caused by lack of rainfall or limited irrigation. Alternatively, water deficit may also be caused by high temperatures, low humidity, saline soils, freezing temperatures or water-logged soils that damage roots and limit water uptake to the shoot. Since plant species vary in their capacity to tolerate water deficit, the precise environmental conditions that cause drought stress can not be generalized. However, drought tolerant plants produce higher biomass and yield than plants that are not drought tolerant. Differences in physical appearance, recovery and yield can be quantified and statistically analyzed using well known measurement and analysis methods.Endogenous: The term “endogenous,” within the context of the current invention refers to any polynucleotide, polypeptide or protein sequence which is a natural part of a cell or organisms regenerated from said cell.Exogenous: “Exogenous,” as referred to within, is any polynucleotide, polypeptide or protein sequence, whether chimeric or not, that is initially or subsequently introduced into the genome of an individual host cell or the organism regenerated from said host cell by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and includeAgrobacterium-mediated transformation (of dicots—e.g. Salomon et al.EMBO J.3:141 (1984); Herrera-Estrella et al.EMBO J.2:987 (1983); of monocots, representative papers are those by Escudero et al.,Plant J.10:355 (1996), Ishida et al.,Nature Biotechnology14:745 (1996), May et al.,Bio/Technology13:486 (1995)), biolistic methods (Armaleo et al.,Current Genetics17:97 1990)), electroporation, in planta techniques, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a T0for the primary transgenic plant and T1for the first generation. The term “exogenous” as used herein is also intended to encompass inserting a naturally found element into a non-naturally found location.Flood: Plant species vary in their capacity to tolerate flooding. Some plants, such as rice, are cultivated in water while plants such as corn do not tolerate flooding. “Flood,” as referred to within, is the state of water saturation at which soils become hypoxic or anoxic, thus limiting respiration in the root. Reduced respiration damages roots and can limit the permeability of roots to water, resulting in decreased leaf water potential and wilting. Since plant species vary in their capacity to tolerate flooding, the precise environmental conditions that cause flood stress can not be generalized. However, flood tolerant plants are characterized by their ability to retain their normal appearance or recover quickly from flood. Such flood tolerant plants produce higher biomass and yield than plants that are not flood tolerant. Differences in physical appearance, recovery and yield can be quantified and statistically analyzed using well known measurement and analysis methods.Functionally Comparable Proteins: This phrase describes those proteins that have at least one characteristic in common. Such characteristics include sequence similarity, biochemical activity, transcriptional pattern similarity and phenotypic activity. Typically, the functionally comparable proteins share some sequence similarity or at least one biochemical and within this definition, homologs, orthologs and analogs are considered to be functionally comparable. In addition, functionally comparable proteins generally share at least one biochemical and/or phenotypic activity.Functionally comparable proteins will give rise to the same characteristic to a similar, but not necessarily to the same degree. Typically, comparable proteins give the same characteristics where the quantitative measurement due to one of the comparables is at least 20% of the other; more typically, between 30 to 40%; even more typically, between 50-60%; even more typically, 70 to 80%; even more typically between 90 to 100%.Heterologous sequences: “Heterologous sequences” are those that are not operatively linked or are not contiguous to each other in nature. For example, a promoter from corn is considered heterologous to anArabidopsiscoding region sequence. Also, a promoter from a gene encoding a growth factor from corn is considered heterologous to a sequence encoding the corn receptor for the growth factor. Regulatory element sequences, such as UTRs or 3′ end termination sequences that do not originate in nature from the same gene as the coding sequence originates from, are considered heterologous to said coding sequence. Elements operatively linked in nature and contiguous to each other are not heterologous to each other. On the other hand, these same elements remain operatively linked but become heterologous if other filler sequence is placed between them. Thus, the promoter and coding sequences of a corn gene expressing an amino acid transporter are not heterologous to each other, but the promoter and coding sequence of a corn gene operatively linked in a novel manner are heterologous.High Temperature: Plant species vary in their capacity to tolerate high temperatures. Very few plant species can survive temperatures higher than 45° C. The effects of high temperatures on plants, however, can begin at lower temperatures depending on the species and other environmental conditions such as humidity and soil moisture. “High temperature” can be defined as the temperature at which a given plant species will be adversely affected as evidenced by symptoms such as decreased photosynthesis. Since plant species vary in their capacity to tolerate higyh temperature, the precise environmental conditions that cause high temperature stress can not be generalized. However, high temperature tolerant plants are characterized by their ability to retain their normal appearance or recover quickly from high temperature conditions. Such high temperature tolerant plants produce higher biomass and yield than plants that are not high temperature tolerant. Differences in physical appearance, recovery and yield can be quantified and statistically analyzed using well know measurement and analysis methods.Inducible Promoter: An “inducible promoter” in the context of the current invention refers to a promoter which is regulated under certain conditions, such as light, chemical concentration, protein concentration, conditions in an organism, cell, or organelle, etc. A typical example of an inducible promoter, which can be utilized with the polynucleotides of the present invention, is PARSK1, the promoter from theArabidopsisgene encoding a serine-threonine kinase enzyme, and which promoter is induced by dehydration, abscissic acid and sodium chloride (Wang and Goodman,Plant J.8:37 (1995)). Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light.Low Temperature: Plant species vary in their capacity to tolerate low temperatures. Chilling-sensitive plant species, including may agronomically important species, can be injured by cold, above-freezing temperatures. At temperatures below the freezing-point of water most plant species will be damaged. Thus, “low temperature” can be defined as the temperature at which a given plant species will be adversely affected as evidenced by symptoms such as decreased photosynthesis and membrane damage (measured by electrolyte leakage). Since plant species vary in their capacity to tolerate low temperature, the precise environmental conditions that cause low temperature stress can not be generalized. However, low temperature tolerant plants are characterized by their ability to retain their normal appearance or recover quickly from low temperature conditions. Such low temperature tolerant plants produce higher biomass and yield than plants that are not low temperature tolerant. Differences in physical appearance, recovery and yield can be quantified and statistically analyzed using well known measurement and analysis methods.Plant seeds vary considerably in their ability to germinate under low temperature conditions. Seeds of most plant species will not germinate at temperatures less than 10° C. Once seeds have imbibed water they become very susceptible to disease, water and chemical damage. Seeds that are tolerant to low temperature stress during germination can survive for relatively long periods under which the temperature is too low to germinate. Since plant species vary in their capacity to tolerate low temperature during germination, the precise environmental conditions that cause low temperature stress during germination can not be generalized. However, plants that tolerate low temperature during germination are characterized by their ability to remain viable or recover quickly from low temperature conditions. Such low temperature tolerant plants produce, germinate, become established, grow more quickly and ultimately produce more biomass and yield than plants that are not low temperature tolerant. Differences in germination rate, appearance, recovery and yield can be quantified and statistically analyzed using well known measurement and analysis methods.Master pool: The “master pools” discussed in these experiments are a pool of seeds from five different transgenic plants transformed with the same exogenous gene.Misexpression: The term “misexpression” refers to an increase or a decrease in the transcription of a coding region into a complementary RNA sequence as compared to the wild-type. This term also encompasses expression of a gene or coding region for a different time period as compared to the wild-type and/or from a non-natural location within the plant genome.Percentage of sequence identity: “Percentage of sequence identity,” as used herein, is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the polynucleotide or amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and WatermanAdd. APL. Math.2:482 (1981), by the homology alignment algorithm of Needleman and WunschJ. Mol. Bio.48:443 (1970), by the search for similarity method of Pearson and LipmanProc. Natl. Acad Sci.(USA) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLASTi PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. The term “substantial sequence identity” between polynucleotide or polypeptide sequences refers to polynucleotide or polypeptide comprising a sequence that has at least 80% sequence identity, preferably at least 85%, more preferably at least 90% and most preferably at least 95%, even more preferably, at least 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using the programs.Query nucleic acid and amino acid sequences were searched against subject nucleic acid or amino acid sequences residing in public or proprietary databases. Such searches were done using the Washington University Basic Local Alignment Search Tool Version 1.83 (WU-Blast2) program. The WU-Blast2 program is available on the internet from Washington University. A WU-Blast2 service forArabidopsiscan also be found on the internet. Typically the following parameters of WU-Blast2 were used: Filter options were set to “default,” Output format was set to “gapped alignments,” the Comparison Matrix was set to “BLOSUM62,” Cutoff Score (S value) was set to “default,” the Expect (E threshold) was set to “default,” the Number of best alignments to show was set to “100,” and the “Sort output” option was set to sort the output by “pvalue.”Plant Promoter: A “plant promoter” is a promoter capable of initiating transcription in plant cells and can drive or facilitate transcription of a nucleotide sequence or fragment thereof of the instant invention. Such promoters need not be of plant origin. For example, promoters derived from plant viruses, such as the CaMV35S promoter or fromAgrobacterium tumefacienssuch as the T-DNA promoters, can be plant promoters. A typical example of a plant promoter of plant origin is the maize ubiquitin-1 (ubi-1) promoter known to those of skill.Specific Promoter: In the context of the current invention, “specific promoters” refers to promoters that have a high preference for being active in a specific tissue or cell and/or at a specific time during development of an organism. By “high preference” is meant at least 3-fold, preferably 5-fold, more preferably at least 10-fold still more preferably at least 20-fold, 50-fold or 100-fold increase in transcription in the desired tissue over the transcription in any other tissue. Typical examples of temporal and/or tissue specific promoters of plant origin that can be used with the polynucleotides of the present invention, are: SH-EP fromVigna mungoand EP-Cl fromPhaseolus vulgaris(Yamauchi et al. (1996)Plant Mol Biol.30(2):321-9.); RCc2 and RCc3, promoters that direct root-specific gene transcription in rice (Xu et al.,Plant Mol. Biol.27:237 (1995) and TobRB27, a root-specific promoter from tobacco (Yamamoto et al. (1991)Plant Cell3:371).Stringency: “Stringency” as used herein is a function of probe length, probe composition (G+C content), and salt concentration, organic solvent concentration, and temperature of hybridization or wash conditions. Stringency is typically compared by the parameter Tm, which is the temperature at which 50% of the complementary molecules in the hybridization are hybridized, in terms of a temperature differential from Tm. High stringency conditions are those providing a condition of Tm−5° C. to Tm−10° C. Medium or moderate stringency conditions are those providing Tm−20° C. to Tm−29° C. Low stringency conditions are those providing a condition of Tm−40° C. to Tm−48° C. The relationship of hybridization conditions to Tm(in ° C.) is expressed in the mathematical equation Tm=81.5−16.6(log10[Na+])+0.41(% G+C)−(600/N) (1) where N is the length of the probe. This equation works well for probes 14 to 70 nucleotides in length that are identical to the target sequence. The equation below for Tmof DNA-DNA hybrids is useful for probes in the range of 50 to greater than 500 nucleotides, and for conditions that include an organic solvent (formamide). Tm=81.5+16.6 log {[Na+]/(1+0.7[Na+])}+0.41(% G+C)−500/L 0.63(% formamide) (2) where L is the length of the probe in the hybrid. (P. Tijessen, “Hybridization with Nucleic Acid Probes” in Laboratory Techniques in Biochemistry and Molecular Biology, P. C. vand der Vliet, ed., c. 1993 by Elsevier, Amsterdam.) The Tmof equation (2) is affected by the nature of the hybrid; for DNA-RNA hybrids Tmis 10-15° C. higher than calculated, for RNA-RNA hybrids Tmis 20-25° C. higher. Because the Tmdecreases about 1° C. for each 1% decrease in homology when a long probe is used (Bonner et al.,J. Mol. Biol.81:123 (1973)), stringency conditions can be adjusted to favor detection of identical genes or related family members. Equation (2) is derived assuming equilibrium and therefore, hybridizations according to the present invention are most preferably performed under conditions of probe excess and for sufficient time to achieve equilibrium. The time required to reach equilibrium can be shortened by inclusion of a hybridization accelerator such as dextran sulfate or another high volume polymer in the hybridization buffer. Stringency can be controlled during the hybridization reaction or after hybridization has occurred by altering the salt and temperature conditions of the wash solutions used. The formulas shown above are equally valid when used to compute the stringency of a wash solution. Preferred wash solution stringencies lie within the ranges stated above; high stringency is 5-8° C. below Tm. medium or moderate stringency is 26-29° C. below Tmand low stringency is 45-48° C. below Tm.Superpool: As used in the context of the current invention, a “superpool” refers to a mixture of seed from 100 different “master pools”. Thus, the superpool contains an equal amount of seed from 500 different events, but only represents 100 transgenic plants with a distinct exogenous nucleotide sequence transformed into them, because the master pools are of 5 different events with the same exogenous nucleotide sequence transformed into them.T0: As used in the current application, the term “T0” refers to the whole plant, explant or callus tissue inoculated with the transformation medium.T1: As used in the current application, the term T1refers to either the progeny of the T0plant, in the case of whole-plant transformation, or the regenerated seedling in the case of explant or callous tissue transformation.T2: As used in the current application, the term T2refers to the progeny of the T1plant. T2progeny are the result of self-fertilization or cross pollination of a T1plant.T3: As used in the current application, the term T3refers to second generation progeny of the plant that is the direct result of a transformation experiment. T3progeny are the result of self-fertilization or cross pollination of a T2plant. 2. Important Characteristics of the Polynucleotides and Polypeptides of the Invention The polynucleotides and polypeptides of the present invention are of interest because when they are misexpressed (i.e. when expressed at a non-natural location or in an increased or decreased amount) they produce plants with modified water use efficiency. “Water use efficiency” is a term that includes various responses to environmental conditions that affect the amount of water available to the plant. For example, under high heat conditions water is rapidly evaporated from both the soil and from the plant itself, resulting in a decrease of available water for maintaining or initiating physiological processes. Likewise, water availability is limited during cold or drought conditions or when there is low water content in the soil. Interestingly, flood conditions also affect the amount of water available to the plant because it damages the roots and thus limits the plant's ability to transport water to the shoot. As used herein, modulating water use efficiency is intended to encompass all of these situations as well as other environmental situations that affect the plant's ability to use and/or maintain water effectively (e.g. osmotic stress, salinity, etc.). The polynucleotides and polypeptides of the invention, as discussed below and as evidenced by the results of various experiments, are useful for modulating water use efficiency. These traits can be used to exploit or maximize plant products for agricultural, ornamental or forestry purposes in different environment conditions of water supply. Modulating the expression of the nucleotides and polypeptides of the present invention leads to transgenic plants that will require less water and result in better yield in high heat and/or drought conditions, or that have increased tolerance levels for an excess of water and result in better yield in wet conditions. Both categories of transgenic plants lead to reduced costs for the farmer and better yield in their respective environmental conditions. 3. The Polynucleotides and Polypeptides of the Invention The polynucleotides of the invention, and the proteins expressed thereby, are set forth in the Sequence Listing. Some of these sequences are functionally comparable proteins. Functionally comparable proteins are those proteins that have at least one characteristic in common. Such characteristics can include sequence similarity, biochemical activity and phenotypic activity. Typically, the functionally comparable proteins share some sequence similarity and generally share at least one biochemical and/or phenotypic activity. For example, biochemical functionally comparable proteins are proteins that act on the same reactant to give the same product. Another class of functionally comparable proteins is phenotypic functionally comparable proteins. The members of this class regulate the same physical characteristic, such as increased drought tolerance. Proteins can be considered phenotypic functionally comparable proteins even if the proteins give rise to the same physical characteristic, but to a different degree. The polypeptides of the invention also include those comprising the consensus sequences described in Tables 1-4, 2-8, 3-9, 5-8, 6-8, 7-6, 8-7, 10-6 and 11-6. A consensus sequence defines the important conserved amino acids and/or domains within a polypeptide. Thus, all those sequences that conform to the consensus sequence are suitable for the same purpose. Polypeptides comprised of a sequence within and defined by one of the consensus sequences can be utilized for the purposes of the invention namely to make transgenic plants with improved water use efficiency, including improved tolerance to heat or high or low water conditions. 4. Use of the Polynucleotides and Polypeptides to Make Transgenic Plants To use the sequences of the present invention or a combination of them or parts and/or mutants and/or fusions and/or variants of them, recombinant DNA constructs are prepared which comprise the polynucleotide sequences of the invention inserted into a vector, and which are suitable for transformation of plant cells. The construct can be made using standard recombinant DNA techniques (Sambrook et al. 1989) and can be introduced to the species of interest byAgrobacterium-mediated transformation or by other means of transformation as referenced below. The vector backbone can be any of those typical in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs and PACs and vectors of the sort described by(a) BAC: Shizuya et al., Proc. Natl. Acad. Sci. USA 89: 8794-8797 (1992); Hamilton et al., Proc. Natl. Acad. Sci. USA 93: 9975-9979 (1996);(b) YAC: Burke et al., Science 236:806-812 (1987);(c) PAC: Sternberg N. et al., Proc Natl Acad Sci USA. Jan; 87(1):103-7 (1990);(d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al., Nucl Acids Res 23: 4850-4856 (1995);(e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et al., J. Mol Biol 170: 827-842 (1983); or Insertion vector, e.g., Huynh et al., In: Glover NM (ed) DNA Cloning: A practical Approach, Vol. 1 Oxford: IRL Press (1985); T-DNA gene fusion vectors: Walden et al., Mol Cell Biol 1: 175-194 (1990); and(g) Plasmid vectors: Sambrook et al., infra. Typically, the construct comprises a vector containing a sequence of the present invention with any desired transcriptional and/or translational regulatory sequences, such as promoters, UTRs, and 3′ end termination sequences. Vectors can also include origins of replication, scaffold attachment regions (SARs), markers, homologous sequences, introns, etc. The vector may also comprise a marker gene that confers a selectable phenotype on plant cells. The marker typically encodes biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, bleomycin, hygromycin, or herbicide resistance, such as resistance to glyphosate, chlorosulfuron or phosphinotricin. A plant promoter is used that directs transcription of the gene in all tissues of a regenerated plant and may be a constitutive promoter, such as p326 or CaMV35S. Alternatively, the plant promoter directs transcription of a sequence of the invention in a specific tissue manner (tissue-specific promoter) or is otherwise under more precise environmental control (inducible promoter). Various plant promoters, including constitutive, tissue-specific and inducible, are known to those skilled in the art and can be utilized in the present invention. Typically, preferred promoters to use in the present invention are those that are induced by heat or low water conditions Such as the RD29a promoter (Kasuga et al.,Plant Cell Physiol.45:346 (2004) and Yamaguchi-Shinozaki and Shinozaki,Mol Gen Genet.236: 331 (1993)) or other DRE-containing (dehydration-responsive elements) promoters (Liu et al, Cell 10: 1391 (1998)). Another preferred embodiment of the present invention is the use of root specific promoters such as those present in the AtXTH17, AtXTH18, AtXTH19 and AtXTH20 genes ofArabidopsis(Vissenberg et al. (2005)Plant Cell Physiol46:192) or guard cell specific promoters such as TGG1 or KST1 (Husebye et al. (2002)Plant Physiol128:1180; Plesch et al. (2001)Plant J28:455). Alternatively, misexpression can be accomplished using a two component system, whereby the first component comprises a transgenic plant comprising a transcriptional activator operatively linked to a promoter and the second component comprises a transgenic plant comprising a sequence of the invention operatively linked to the target binding sequence/region of the transcriptional activator. The two transgenic plants are crossed and the sequence of the invention is expressed in their progeny. In another alternative, the misexpression can be accomplished by transforming the sequences of the two component system into one transgenic plant line. Any promoter that functions in plants can be used in the first component, such as those discussed above. Suitable transcriptional activator polypeptides include, but are not limited to, those encoding HAP1 and GAL4. The binding sequence recognized and targeted by the selected transcriptional activator protein (e.g. a UAS element) is used in the second component. Transformation Nucleotide sequences of the invention are introduced into the genome or the cell of the appropriate host plant by a variety of techniques. These techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g. Weising et al.,Ann. Rev. Genet.22:421 (1988); and Christou, Euphytica, v. 85, n.1-3:13-27, (1995). Processes for the transformation and regeneration of monocotyledonous and dicotyledonous plants are known to the person skilled in the art. For the introduction of DNA into a plant host cell a variety of techniques is available. These techniques include transformation of plant cells by injection (e.g. Newell, 2000), microinjection (e.g. Griesbach (1987)Plant Sci.50 69-77), electroporation of DNA (e.g. Fromm et al. (1985)Proc. Natl Acad. Sci. USA82:5824 and Wan and Lemaux, Plant Physiol. 104 (1994), 37-48), PEG (e.g. Paszkowski et al. (1984)EMBO J.3:2717), use of biolistics (e.g. Klein et al. (1987)Nature327:773), fusion of cells or protoplasts (Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A. Pühler, P. Stadler, eds., Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge), via T-DNA usingAgrobacterium tumefaciens(e.g. Fraley et al. (Crit. Rev. Plant. Sci. 4, 1-46 and Fromm et al., Biotechnology 8 (1990), 833-844) orAgrobacterium rhizogenes(e.g. Cho et al. (2000)Planta210:195-204) or other bacterial hosts (e.g. Brootghaerts et al. (2005) Nature 433:629-633), as well as further possibilities. In addition, a number of non-stable transformation methods well known to those skilled in the art may be desirable for the present invention. Such methods include, but are not limited to, transient expression (e.g. Lincoln et al. (1998)Plant Mol. Biol. Rep.16:1-4) and viral transfection (e.g. Lacomme et al. (2001) In “Genetically Engineered Viruses” (C. J. A. Ring and E. D. Blair, Eds). Pp. 59-99, BIOS Scientific Publishers, Ltd. Oxford, UK). Seeds are obtained from the transformed plants and used for testing stability and inheritance. Generally, two or more generations are cultivated to ensure that the phenotypic feature is stably maintained and transmitted. One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. The nucleic acids of the invention can be used to confer the trait of increased tolerance to heat and/or low water conditions, without reduction in fertility, on essentially any plant. The nucleotide sequences according to the invention encode appropriate proteins from any organism, in particular from plants, fungi, bacteria or animals. The process according to the invention can be applied to any plant, preferably higher plants, pertaining to the classes of Angiospermae and Gymnospermae. Plants of the subclasses of the Dicotylodenae and the Monocotyledonae are particularly suitable. Dicotyledonous plants belong to the orders of the Magniolales, Illiciales, Laurales, Piperales Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Comales, Proteales, Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales. Monocotyledonous plants belong to the orders of the Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales, LiWales, and Orchidales. Plants belonging to the class of the Gymnospermae are Pinales, Ginkgoales, Cycadales and Gnetales. The method of the invention is preferably used with plants that are interesting for agriculture, horticulture, biomass for bioconversion and/or forestry. Examples are tobacco, oilseed rape, sugar beet, potato, tomato, cucumber, pepper, bean, pea, citrus fruit, apple, pear, berries, plum, melon, eggplant, cotton, soybean, sunflower, rose, poinsettia,petunia, guayule, cabbage, spinach, alfalfa, artichoke, corn, wheat, rye, barley, grasses such as switch grass or turf grass, millet, hemp, banana, poplar,eucalyptustrees, conifers. Homologs Encompassed by the Invention Sequences of the invention include proteins comprising at least about a contiguous 10 amino acid region preferably comprising at least about a contiguous 20 amino acid region, even more preferably comprising at least about a contiguous 25, 50, 75 or 100 amino acid region of a protein of the present invention. In another preferred embodiment, the proteins of the present invention include between about 10 and about 25 contiguous amino acid region, more preferably between about 20 and about 50 contiguous amino acid region, and even more preferably between about 40 and about 80 contiguous amino acid region. Due to the degeneracy of the genetic code, different nucleotide codons may be used to code for a particular amino acid. A host cell often displays a preferred pattern of codon usage. Nucleic acid sequences are preferably constructed to utilize the codon usage pattern of the particular host cell. This generally enhances the expression of the nucleic acid sequence in a transformed host cell. Any of the above described nucleic acid and amino acid sequences may be modified to reflect the preferred codon usage of a host cell or organism in which they are contained. Modification of a nucleic acid sequence for optimal codon usage in plants is described in U.S. Pat. No. 5,689,052. Additional variations in the nucleic acid sequences may encode proteins having equivalent or superior characteristics when compared to the proteins from which they are engineered. It is understood that certain amino acids may be substituted for other amino acids in a protein or peptide structure (and the nucleic acid sequence that codes for it) without appreciable change or loss of its biological utility or activity. The amino acid changes may be achieved by changing the codons of the nucleic acid sequence. It is well known in the art that one or more amino acids in a native sequence can be substituted with other amino acid(s), the charge and polarity of which are similar to that of the native amino acid, i.e., a conservative amino acid substitution, resulting in a silent change. Conservative substitutes for an amino acid within the native polypeptide sequence can be selected from other members of the class to which the amino acid belongs (see below). Amino acids can be divided into the following four groups: (1) acidic (negatively charged) amino acids, such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids, such as arginine, histidine, and lysine; (3) neutral polar amino acids, such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. In a further aspect of the present invention, nucleic acid molecules of the present invention can comprise sequences that differ from those encoding a protein or fragment thereof selected from the group consisting of the sequences present in the Sequence Listing due to the fact that the different nucleic acid sequence encodes a protein having one or more conservative amino acid changes. In another aspect, biologically functional equivalents of the proteins or fragments thereof of the present invention can have about 10 or fewer conservative amino acid changes, more preferably about 7 or fewer conservative amino acid changes, and most preferably about 5 or fewer conservative amino acid changes. In a preferred embodiment, the protein has between about 5 and about 500 conservative changes, more preferably between about 10 and about 300 conservative changes, even more preferably between about 25 and about 150 conservative changes, and most preferably between about 5 and about 25 conservative changes or between 1 and about 5 conservative changes. 5. Experiments Confirming the Usefulness of the Polynucleotides and Polypeptides of the Invention 5.1 Procedures The nucleotide sequences of the invention were identified by use of a variety of screens for modified water conditions, including heat and/or low water conditions. These screens are recognized by those skilled in the art to be predictive of nucleotide sequences that provide plants with improved water use efficiency including improved tolerance to heat and/or low water conditions because they emulate the different environmental conditions that can result from increased heat and/or low water conditions. These screens generally fall into two categories (1) soil screens and (2) in vitro screens. Soil screens have the advantage of assaying the response of the entire plant to particular conditions, such as drought or high heat. On the other hand, in vitro screens have the advantage of relying on defined media and so allow more defined manipulation of growth conditions. “Surrogate” in vitro screens use particular chemicals to alter the water available to the plant by manipulating the concentrations and/or components of the growth media. For example, the ability of the plant to maintain the water concentration within its cells, which can occur during times of low water in the soil, can be tested by growing plants on high sucrose media. Such a screen thus allows one to separate the effects of water loss from roots from, for example, the water loss from leaves during high heat conditions. Each of the screens used is described in more detail below. In general, the screens used to identify the polynucleotides and polypeptides of the invention were conducted using superpools ofArabidopsisT2transformed plants. The T1plants were transformed with a Ti plasmid containing a particular SEQ ID NO in the sense orientation relative to a constitutive promoter and harboring the plant-selectable marker gene phosphinothricin acetyltransferase (PAT), which confers herbicide resistance to transformed plants. For surrogate screens, seed from multiple superpools (1,200 T2seeds from each superpool) were tested. T3seed were collected from the resistant plants and retested on all other surrogate screens. The results of the screens conducted for each SEQ ID NO can be found in the Examples below. 5.1.1. Mannitol Screens for mannitol resistant seedlings are surrogate screens for drought (see Quesada et al., Genetic analysis of salt-tolerant mutants inArabidopsis thaliana. Genetics. 2000 154:421-36). Seeds are sterilized in 30% household bleach for 5 minutes and then washed with double distilled deionized water three times. Sterilized seed is stored in the dark at 4° C. for a minimum of 3 days before use. Manitol media is prepared by mixing 375 ml sterile 1 mM mannitol with 375 ml sterile 1×MS. Approximately 1200 seeds are evenly spaced per PEG plate before incubating at 22° C. for 14 days. Putative mannitol-resistant seedlings are transferred to MS with 3% sucrose for recovery. Approximately one week later, resistant seedlings are transferred to soil and sprayed with Finale. Finale resistant plants are genotyped as described below. DNA is isolated from each plant and used in PCR reactions using the following cycling conditions 95° C. for 30 sec, five cycles of 51° C. for 30 sec, 72° C. for 1.15 min, 95° C. for 30 sec, 25 cycles of 48° C. for 30 sec, 72° C. for 1.15 min, 72° C. for 7 min and 4° C. hold. Aliquots of the reaction product are analyzed on a 1.2% agarose gel stained with ethidium bromide. T3Seed from those plants containing the expected PCR product are collected and retested on 375 mM mannitol media. 5.1.2. Polyethylene Glycol (PEG) Screens for PEG resistant seedlings are surrogate screens for drought (see van der Weele et al., Growth ofArabidopsis thalianaseedlings under water deficit studied by control of water potential in nutrient-agar media.J Exp Bot.2000 51(350):1555-62). Seeds are sterilized in 30% household bleach for 5 minutes and then washed with double distilled deionized water three times. Sterilized seed is stored in the dark at 4° C. for a minimum of 3 days before use. 18% PEG media is prepared by mixing 360 ml of hot sterile 50% PEG with 400 ml of hot sterile 0.5×MS media. Approximately 1200 seeds are evenly spaced per PEG plate before incubating at 22° C. for 14 days. Putative PEG-resistant seedlings are transferred to MS with 0.01% Finale. One week later, resistant seedlings are transferred to soil. Three days later the seedlings are genotyped as described below. DNA is isolated from each plant and used in PCR reactions using the following cycling conditions 95° C. for 30 sec, five cycles of 51° C. for 30 sec, 72° C. for 1.15 min, 95° C. for 30 sec, 25 cycles of 48° C. for 30 sec, 72° C. for 1.15 min, 72° C. for 7 min and 4° C. hold. Aliquots of the reaction product are analyzed on a 1.2% agarose gel stained with ethidium bromide. T3Seed from those plants containing the expected PCR product are collected and retested using 20% PEG media. 5.1.3 Soil Drought Soil drought screens identify plants with enhanced tolerance to drought and enhanced recovery after drought. Seeds are planted in holed flats containing Zonolite vermiculite that are placed in no-holed flats. Flats are watered with 3 L of Hoagland's solution and covered with a plastic dome before being placed at 4° C. After 4 days, the flats are moved to the Greenhouse (22° C.) and grown 2 weeks, with 1.5 L Hoagland's solution being added every 4 days, or when top of vermiculite is dry. The final application of Hoagland's solution is 4 days prior to the end of the 2 weeks. After 2 weeks, 1 L Hoagland's solution is added to the no-holed flat. After 10 days plants are wilted but still green. Green, turgid plants are transplanted to 4″ square pots containing 60% sunshine mix #5 and 40% thermo-o-rock vermiculite, with Osmocote (1 tbsp/8 L) and Marathon (1 tbsp/8 L). The soil is moistened and the pots sub-irrigated with water. They are grown under a plastic dome for one day, then the plastic dome is removed for the remaining growth period. To assess plants for enhanced recovery after drought the green wilted plants remaining in the flats are sub-irrigated with 2.5 L Hoagland's solution and cover with cleared with a plastic dome. The following day, the dome is removed and green survivors transplanted to 4″ square pots containing 60% sunshine mix #5, 40% Therm-o-rock vermiculite, Osmocote (1 tbsp/8 L) and marathon (1 tbsp/8 L). The soil is moistened and the pots sub-irrigated with water. They are grown under a plastic dome for one day, then the plastic dome is removed for the remaining growth period. DNA from a leaf from each plant is transferred to FTA paper via pressure and an aliquot of the DNA containing paper used in PCR reactions using the following cycling conditions 94° C. for 10 min, five cycles of 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 3 min, five cycles of 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 3 min, 30 cycles of 94° C. for 30 sec, 53° C. for 30 sec, 72° C. for 3 min, 72° C. for 7 min and 4° C. hold. Aliquots of the reaction product are analyzed on a 1% agarose gel stained with ethidium bromide. T3Seed from those plants containing the expected PCR product are collected and retested using 50 seeds from each line. 5.1.4. Heat High heat screens identify plants with enhanced tolerance to heat and enhanced recovery after heat. Seeds are sterilized in 30% household bleach for 5 minutes and then washed with double distilled deionized water three times. Sterilized seed is stored in the dark at 4° C. for a minimum of 3 days before use. MS media, pH 5.7 is prepared. Approximately 12 seeds are evenly spaced per MS plate before incubating in the vertical position at 22° C. for 14 days. Under these conditions, the plates are exposed to 12,030 LUX from above and 3,190 LUX from the bottom. On day 15 the plates are transferred to a 22° C. oven, which increased temperature in 5° C. increments to 45° C. The duration of treatment at 45° C. was based on complete and homogenous wilting of 100 wild-type seedlings (˜10 plates). After exposure to 45° C., seedlings were placed in the horizontal position at 23° C. for recovery, where they remained for 4-11 days. Heat recovery was assessed based on vigor and greenness and continued growth after treatment. Leaves of control and non-resistant plants become wilted and yellowed after only 2 days, completely bleaching after an additional 4 days. All wild type (WS) and non-heat resistant plants die by day 6. DNA is isolated from each plant and used in PCR reactions using the following cycling conditions 95° C. for 30 sec, five cycles of 51° C. for 30 sec, 72° C. for 1.15 min, 95° C. for 30 sec, 25 cycles of 48° C. for 30 sec, 72° C. for 1.15 min, 72° C. for 7 min and 4° C. hold. Aliquots of the reaction product are analyzed on a 1.2% agarose gel stained with ethidium bromide. T3Seed from those plants containing the expected PCR product are collected and retested. To differentiate between natural acquired thermo tolerance of recovered T3events, seeds were sterilized and stratified in parallel to wildtype seed that was never heat treated and wild-type controls previously heat treated with the T2events. 15 day old seedlings were heat shocked in the dark at 45° C. for 5 hours, as described above. Duration of treatment at 45° C. was based on complete and homogenous wilting of pre-heat treated wild-type seed and un-pretreated wild-type controls. After exposure to seedlings were returned to the permissive temperature of 23° C. for recovery where they remained for another 7 days. Thermo tolerance was assessed based on prolonged greenness and continued growth after treatment. 5.1.5. Heat (Soil) Seeds are sown in pots containing soil of the following composition: 60% autoclaved Sunshine Mix #5, 40% vermiculite with 2.5 Tbsp Osmocote and 2.5 Tbsp 1% granular Marathon per 25 L of soil. After sowing, pots are covered with plastic propagation domes and seed is placed at 4° C. in the dark for at least 3 days. Pots are then returned to the greenhouse (long day light conditions of 16 hours), covered with 55% shade cloth and provided a normal watering regime. After 7 days, seedlings were transferred to a 36° C. growth chamber under continuous light and allowed to grow until harvest. Plants were watered minimally so as to allow for some drying of the top soil similar to that in heat-induced drought conditions in the field. Plants are sprayed with a mixture of 3 ml Finale in 48 oz of water. Spraying is repeated every 3-4 days until only transformants remain. The remaining transformants were weeded to a maximum of 5 evenly spaced transformants per pot. T3 seed was recovered and tested for thermotolerance and recovery as described above. 5.1.6. High Sucrose Screens for germination and growth on limited nutrients and 9% sucrose are surrogate screens for the altered carbon/nitrogen balance frequently associated with drought (see Laby et al., TheArabidopsissugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response.Plant Journal23: 587-596). Seeds are sterilized in 30% household bleach for 5 minutes and then washed with double distilled deionized water three times. Sterilized seed is stored in the dark at 4° C. for a minimum of 3 days before use. MS media containing 9% sucrose is prepared. Approximately 1200 seeds are evenly spaced per MS-sucrose plate before incubating at 22° C. for 9 days. Putative sucrose-resistant green seedlings are transferred to MS plates. After one week of recovery, resistant the seedlings are genotyped as described below. DNA from a leaf from each plant is transferred to FTA paper via pressure and an aliquot of the DNA containing paper used in PCR reactions using the following cycling conditions 94° C. for 10 min, five cycles of 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 3 min, five cycles of 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 3 min, 30 cycles of 94° C. for 30 sec, 53° C. for 30 sec, 72° C. for 3 min, 72° C. for 7 min and 4° C. hold. Aliquots of the reaction product are analyzed on a 1% agarose gel stained with ethidium bromide. T3Seed from those plants containing the expected PCR product are collected and retested using 9% sucrose MS media. 5.1.7. ABA Screens for ABA resistant seedlings are surrogate screens for drought. Seeds are sterilized in 30% household bleach for 5 minutes and then washed with double distilled deionized water three times. Sterilized seed is stored in the dark at 4° C. for a minimum of 3 days before use. MS media containing 1.5 μM ABA is prepared. Approximately 1200 seeds are evenly spaced per PEG plate before incubating at 22° C. for 14 days. Putative ABA-resistant seedlings are transferred to MS with 0.01% Finale. One week later, resistant seedlings are transferred to soil. Three days later the seedlings are genotyped as described below. DNA is isolated from each plant and used in PCR reactions using the following cycling conditions 95° C. for 30 sec, five cycles of 51° C. for 30 sec, 72° C. for 1.15 min, 95° C. for 30 sec, 25 cycles of 48° C. for 30 sec, 72° C. for 1.15 min, 72° C. for 7 min and 4° C. hold. Aliquots of the reaction product are analyzed on a 1.2% agarose gel stained with ethidium bromide. T3Seed from those plants containing the expected PCR product are collected and retested using 1.5 μM ABA MS media. 5.1.8. Procedure for Identifying Functional Homologs and Consensus Sequences The isolated sequence of the invention was compared to the sequences present in the various gene banks. Pairwise comparisons were conducted and those sequences having the highest percent identity to the query sequence identified as functional homologs. A multi-pairwise alignment was generated using the amino acid query sequence and the amino acid sequence of the functional homologs. This allowed identification of the conserved regions or domains of the polypeptide. Using the conserved regions as a guide, a consensus sequence was generated. This consensus sequence indicates the critical amino acid residues and those can be either substituted and/or deleted without impacting the biological function of the protein. 5.2 Results The results of the above experiments are set forth below wherein each individual example relates to all of the experimental results for a particular polynucleotide/polypeptide of the invention. Example 1—Ceres cDNA 12331850 Clone 11830, Ceres cDNA 12331850, encodes a full-length glycosyl hydrolase family 17 protein, which has similarity to elicitor inducible chitinase Nt-SubE76 GI:11071974 from (Nicotiana tabacum(C-terminal homology only) and β-1,3-glucanase. Ectopic expression of Ceres cDNA 12331850 under the control of the CaMV35S or 32449 promoter induces the following phenotypes:Germination on high concentrations of polyethylene glycol (PEG), mannitol, and abscissic acid (ABA).Continued growth on high concentration of PEG, mannitol, and ABA. Generation and Phenotypic Evaluation of T1Lines Containing 35S::cDNA 12331850. Wild-typeArabidopsisWassilewskija (WS) plants were transformed with a Ti plasmid containing cDNA 12331850 in the sense orientation relative to the CaMV35S constitutive promoter. The Tiplasmid vector used for this construct, CRS338, contains PAT and confers herbicide resistance to transformed plants. Ten independently transformed events were selected and evaluated for their qualitative phenotype in the T1generation. No positive or negative phenotypes were observed in the T1plants. Screens of Superpools on High PEG, Mannitol, and ABA as Surrogate Screens for Drought Tolerance. Seeds from 13 superpools (1,200 T2seeds from each superpool) from the CaMV35S or 32449 over-expression lines were tested on 3 drought surrogate screens (high concentrations of PEG, mannitol, and ABA) as described above. T3seeds were collected from the resistant plants and analyzed for resistance on all three surrogate drought screens. Once cDNA 12331850 was identified in resistant plants from each of the three surrogate drought screens, the five individual T2events containing this cDNA (ME01297) were screened on high PEG, mannitol, and ABA to identify events with the resistance phenotype. Superpools (SP) are referred to as SP1, SP2 and so on. The letter following the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA) and the number following the letter refers to a number assigned to each plant obtained from that screen on that superpool. For example, SP1-M18 is the 18thplant isolated from a mannitol screen of Superpool 1. Results: Qualitative Analysis of 13 Superpools on PEG, Mannitol, and ABA Resistant candidates were selected based on increased size compared to the largest wild-type control. All three screens resulted in a decrease in germination for both wildtype and superpools as compared to seeds on control media. Wild-type seeds that germinated on any of the screens were small and never developed any first leaves even after 40 days. To ensure that even slightly tolerant individuals were not omitted, seedlings that showed any growth and greening whatsoever were recovered and transferred to soil for assessment in the T3generation. All recovered candidates showed signs of vigorous re-growth on soil, although the development was slightly delayed as compared to unstressed plants, presumably because of the transient exposure to stress. The plants transferred to soil were sprayed with BastaRto eliminate any false-positives, or any lines where the BastaRmarker was suppressed. All of the BastaR-resistant candidates flowered and set seed. Resistant seedlings were recovered from Superpools 1, 7 and 11 on all screens (Table 1-1). TABLE 1-1Number of BastaRseedlings identified on several screens.SuperpoolPromoter18% PEG375 mM Mannitol1.5 uM ABASP135S101311SP235S065SF335S0210SP435S040SP535S005SP635S004SP735S148SP835S041SP9324490012SP10324490014SP11324491531SP1232449306SP1332449021 Qualitative and Quantitative Analysis of 5 Independent Events Representing 35S::cDNA 12331850 on PEG, Mannitol, and ABA Screens. Seedlings that survived transfer to soil and which were BastaR-resistant were subjected to PCR and sequencing. At least one resistant plant in each of the three screens contained 35S::cDNA 12331850 (ME01297) making this a good candidate for further testing. T2seeds from the 5 independent transformants that contain this clone and that were used in the pooling process were tested for BastaRresistance and for stress tolerance in the 3 surrogate drought screens. To identify two independent events of 35S::cDNA 12331850 showing PEG, mannitol and ABA resistance, 36 seedlings from each of five events, ME01297-01, 02, 03, 04, and 05 were screened as previously described. Simultaneously, BastaRsegregation was assessed to identify events containing a single insert segregating in a 3:1 (R:S) ratio as calculated by a chi-square test (Table 1-2). All of the events segregated for a single functional insert. TABLE 1-2Basta segregation for ME01297 individual eventsEventResistantSensitiveTotalProbability of Chi-test*ME01297-012214360.05429ME01297-022610360.70031ME01297-032511360.44142ME01297-042214360.05429ME01297-052412360.24821*Chi-test to determine whether actual ratio of resistant to sensitive differs from the expected 3:1 ratio. Events ME01297-02 and 03 were chosen as the two events because they had the strongest and most consistent resistance to PEG, mannitol and ABA. Resistance was observed for ME01297-01, 04, and 05 although not in expected ratios in all three screens (data not shown). The controls were sown the same day and on the same plate as the individual events. The transgenic control in each of these plates is a segregant, from this ME line or another ME line being tested, that failed to show resistance to the particular stress. The PEG, mannitol and ABA (Table 1-3) segregation ratios observed for ME01297-02 and 03 are consistent with the presence of a single insert, similar to what we observed for Basta resistance (Table 1-1). On 18% PEG, the resistant seedlings from these two events show some root growth and they are green with new leaves emerging. The mannitol resistant seedlings also showed more root and shoot growth than the sensitive seedlings. The ABA resistant seedlings showed a slight increase in growth. The phenotype of the resistant seedlings is unique to each of the screens. TABLE 1-3Segregation of Resistance to PEG, mannitol and ABA inME01297-02 and ME01297-03 Progeny.PEGmannitolABAProba-Prob-Proba-bilityabilitybilityofofofRSChi-testRSChi-testRSChi-testME01297-49230.17447250.05750220.27602ME01297-58140.27651210.41456160.58603Expected541854185418(3:1segregation) Qualitative and Quantitative Analysis of Progeny of T2Plants Isolated on High Concentrations of PEG, Mannitol, and ABA Screens. Progeny from T2plants that were recovered from the three screens and which contained cDNA 12331850 (SP1-A1, SP1-P1, and SP1-M18) were analyzed and also found to be resistant to PEG, mannitol and ABA indicating that resistance is transmitted to the next generation. Taken together, 1) the isolation of resistant seedlings containing cDNA 12331850 from all three screens, 2) the inheritance of this resistance in a subsequent generation, and 3) the fact that the progeny from two or more events from the original transformation also segregated for resistance to the stresses, provide strong evidence that cDNA 12331850 when over-expressed can provide tolerance to osmotic stress. This gene is annotated as a glycosyl hydrolase family 17, which has similarity to elicitor inducible chitinase Nt-SubE76 GI:11071974 fromNicotiana tabacum(C-terminal homology only). TIGR also notes that the protein contains similarity to beta-1,3-glucanase. FIG. 1 provides the results of the consensus sequence (SEQ ID NOs: 112-120) analysis based on Ceres cDNA 12331850. Example 2—Ceres cDNA 12334963 Clone 35743, Ceres cDNA 12334963, encodes a full-length putative hypothetical protein. Ectopic expression of Ceres cDNA 12334963 under the control of the 35S promoter induces the following phenotypes:Germination on high concentrations of polyethylene glycol (PEG), mannitol, and abscissic acid (ABA).Continued growth on high PEG, mannitol, and ABA. Generation and Phenotypic Evaluation of T1Lines Containing 35S::cDNA 12334963. Wild-typeArabidopsisWassilewskija (WS) plants were transformed with a Ti plasmid containing cDNA 12334963 in the sense orientation relative to the CaMV35S constitutive promoter. The Tiplasmid vector used for this construct, CRS338, contains PAT and confers herbicide resistance to transformed plants. Ten independently transformed events were selected and evaluated for their qualitative phenotype in the T1generation. No positive or negative phenotypes were observed in the T1plants. Screens of Superpools on High PEG, Mannitol, and ABA as Surrogate Screens for Drought Tolerance. Seeds from 13 superpools (1,200 T2seeds from each superpool) from the CaMV35S or 32449 over-expression lines were tested on 3 drought surrogate screens (high concentrations of PEG, mannitol and ABA) as described above. T3seeds were collected from the resistant plants and analyzed for resistance on all three surrogate drought screens. Once cDNA 12334963 was identified in resistant plants from each of the three surrogate drought screens, the five individual T2events containing this cDNA (ME01467) were screened on high PEG, mannitol and ABA to identify events with the resistance phenotype. Superpools (SP) are referred to as SP1, SP2 and so on. The letter following the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA) and the number following the letter refers to a number assigned to each plant obtained from that screen on that superpool. For example, SP1-M18 is the 18thplant isolated from a mannitol screen of Superpool 1. Results: Qualitative Analysis of 13 Superpools on PEG, Mannitol, and ABA. Resistant candidates were selected based on increased size when compared to the largest wild-type control seedlings. All three screens resulted in a decrease in germination for both wildtype and superpools compared to seeds on control media. Wild-type seeds that germinated on any of the screens were small and never developed any first leaves even after 40 days. To ensure that even slightly tolerant individuals were not omitted, seedlings that showed any growth and greening whatsoever were recovered and transferred to soil for assessment in the T3generation. All recovered candidates showed signs of vigorous re-growth on soil, although the development was slightly delayed as compared to unstressed plants, presumably because of the transient exposure to stress. The plants transferred to soil were sprayed with BastaRto eliminate any false-positives, or any lines where the BastaRmarker was suppressed. All of the BastaR-resistant candidates flowered and set seed. Resistant seedlings were recovered from Superpools 1, 7 and 11 on all screens (Table 2-1). TABLE 2-1Number of stress-tolerant and BastaRseedlings identified ondraught surrogate screens.SuperpoolPromoter18% PEG375 mM Mannitol1.5 uM ABASP135S101311SP235S065SP335S0210SP435S040SP535S005SP635S004SP735S148SP835S041SP9324490012SP10324490014SP113244915313SP1232449306SP1332449021 Qualitative and Quantitative Analysis of 5 Independent Events Representing 35S::cDNA 12334963 on PEG, Mannitol, and ABA Seedlings that survived transfer to soil and which were BastaR-resistant were subjected to PCR and sequencing. At least one resistant plant in each of the three osmotic screens contained 35S::cDNA 12334963 (ME01467) making this a good candidate for further testing. T2seeds from the 5 independent transformants that contain this clone and that were used in the pooling process were tested for BastaRresistance and for stress tolerance in the 3 surrogate drought screens. To identify two independent events of 35S::cDNA 12334963 showing PEG, mannitol and ABA resistance, 36 seedlings from each of five events, ME01467-01, 02, 03, 04, and 05 were screened as previously described. Simultaneously, Basta segregation was assessed to identify events containing a single insert segregating in a 3:1 (R:S) ratio as calculated by a Chi-square test (Table 2-2). All of the lines segregated for a single functional insert. TABLE 2-2BastaRsegregation for ME01467 individual eventsEventResistantSensitiveTotalProbability of Chi-test*ME01467-01288360.70031ME01467-02297360.44142ME01467-032412360.24821ME01467-04288360.70031ME01467-05279361*Chi-test to determine whether actual ratio of resistant to sensitive differs from the expected 3:1 ratio. Events ME01467-03 and 05 were chosen as the two events because had the strongest and most consistent resistance to PEG, mannitol and ABA. Resistance was observed for ME01467-01, 02, and 04, although not in expected ratios in all three screens (data not shown). The controls were sown the same day and in the same plate as the individual events. The transgenic control in each of these plates is a segregant, from this ME line or another ME line being tested, that failed to show resistance to the particular osmotic stress. The PEG (Tables 2-3 and 2-4), mannitol (Tables 2-5 and 2-6) and ABA (Tables 2-7 and 2-8) segregation ratios observed for ME01467-03 and 05 are consistent with the presence of a single insert as demonstrated by Chi-Square. This result is similar to the observation for BastaRresistance (Table 2-2). On 18% PEG, the resistant seedlings from these two events showed some root growth but they were also green with emergence of new leaves at day 14. The mannitol-resistant seedlings showed more root and shoot growth than the PEG resistant seedlings. The resistant seedlings on ABA show the least amount of growth, and very little root growth relative to the mannitol and PEG screens. The phenotype TABLE 2-3Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01467-03 containing 35S::cDNA 12334963 on PEG.EventObservedExpectedχ2Probability or Chi-TestPEG Resistant242401.0PEG Sensitive88032320 of the resistant seedlings is unique on each of the screens. TABLE 2-4Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01467-05 containing 35S::cDNA 12334963 on PEG.EventObservedExpectedχ2Probability of Chi-TestPEG Resistant26270.0370.7PEG Sensitive1090.11136360.148 TABLE 2-5Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01467-03 containing 35S::cDNA 12334963 on mannitol.EventObservedExpectedχ2Probability of Chi-TestMannitol Resistant29270.1480.4Mannitol Sensitive790.44436360.592 TABLE 2-6Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01467-05 containing 35S::cDNA 12334963 on mannitol.EventObservedExpectedχ2Probability of Chi-TestMannitol Resistant182730.0005Mannitol Sensitive1899363612 TABLE 2-7Chi-square analysis assuming a 3:1 (R:S) ratio for progenyof ME01467-03 containing 35S::cDNA 12334963 on ABA.ProbabilityEventObservedExpectedχ2of Chi-TestABA Resistant2526.250.05950.626ABA Sensitive108.750.17935350.239 TABLE 2-8Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01467-05 containing 35S::cDNA 12334963 on ABA.EventObservedExpectedχ2Probability of Chi-TestABA Resistant22270.9260.054ABA Sensitive1492.7836363.706 Qualitative and Quantitative Analysis of Progeny of T2Plants Isolated on PEG, Mannitol and ABA Progeny of the T2plants that were recovered from the three screens and which contained cDNA 12334963 (SP1-A12, SP1-P9, and SP1-M4) were analyzed and found to be resistant to PEG, mannitol and ABA, indicating that resistance is transmitted to the next generation. Taken together, 1) the isolation of resistant seedlings containing cDNA 12334963 from all three surrogate screens for drought, 2) the inheritance of this resistance in the next generation, and 3) the fact that the progeny from two or more events from the original transformation also segregated for resistance to these osmotic stresses, provides strong evidence that cDNA 12334963, when over-expressed, can provide tolerance to drought, freezing and other osmotic stresses. FIG. 2 provides the results of the consensus sequence (SEQ ID NOs: 121-129) analysis based on Ceres cDNA 12334963. Example 3—Ceres cDNA 12333678 Clone 26006, Ceres cDNA 12333678, encodes a full-length glycosyl hydrolase. Ectopic expression of Ceres cDNA 12333678 under the control of the CaMV35S promoter induces the following phenotypes:Germination on high concentrations of polyethylene glycol (PEG), mannitol and abscissic acid (ABA).Continued growth on high PEG, mannitol and ABA. Generation and Phenotypic Evaluation of T1Lines Containing 35S::cDNA 12333678. Wild-typeArabidopsisWassilewskija (WS) plants were transformed with a Ti plasmid containing cDNA 12333678 in the sense orientation relative to the CaMV35S constitutive promoter. The Tiplasmid vector used for this construct, CRS338, contains PAT and confers herbicide resistance to transformed plants. Ten independently transformed events were selected and evaluated for their qualitative phenotype in the T1generation. No positive or negative phenotypes were observed in the T1plants. Screens of Superpools on High PEG, Mannitol and ABA as Surrogate Screens for Drought Tolerance. Seeds from 13 superpools (1,200 T2seeds from each superpool) from the CaMV35S or 32449 over-expression lines were tested on 3 drought surrogate screens (high concentrations of PEG, mannitol and ABA) as described above. T3seeds were collected from the resistant plants and analyzed for resistance on all three surrogate drought screens. Once cDNA 12333678 was identified in resistant plants from each of the three surrogate drought screens, the five individual T2events containing this cDNA (ME01334) were screened on high PEG, mannitol and ABA to identify events with the resistance phenotype. Superpools (SP) are referred to as SP1, SP2 and so on. The letter following the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA) and the number following the letter refers to a number assigned to each plant obtained from that screen on that superpool. For example, SP1-M18 is the 18thplant isolated from a mannitol screen of Superpool 1. Results: Qualitative Analysis of 13 Superpools on PEG, Mannitol and ABA. Resistant candidates were selected based on increased size when compared to the largest wild-type control seedlings. All three screens resulted in a decrease in germination for both wildtype and superpools as compared to seeds on control media. Wild-type seeds that germinated on any of the screens were small and never developed any first leaves even after 40 days. To ensure that even slightly tolerant individuals were not omitted, seedlings that showed any growth and greening whatsoever were recovered and transferred to soil for assessment in the T3generation. All recovered candidates showed signs of vigorous re-growth on soil, although the development was slightly delayed compared to unstressed plants, presumably because of the transient exposure to stress. The plants transferred to soil were sprayed with B BastaRto eliminate any false-positives, or any lines where the BastaRmarker was suppressed. All of the BastaR—resistant candidates flowered and set seed. Resistant seedlings were recovered from Superpools 1, 7 and 11 on all screens (Table 3-1). TABLE 3-1Number of stress-tolerant and BastaRseedlings identified ondrought surrogate screens.SuperpoolPromoter18% PEG375 mM Mannitol1.5 uM ABASP135S101311SP235S065SP335S0210SP435S040SP535S005SP635S004SP735S148SP835S041SP9324490012SP10324490014SP113244915313SP1232449306SP1332449021 Qualitative and Quantitative Analysis of 5 Independent Events Representing 35S::cDNA 12333678 on PEG, Mannitol and ABA Seedlings that survived transfer to soil and which were BastaR—resistant were subjected to PCR and sequencing. At least one resistant plant in each of the three osmotic screens contained 35S::cDNA 12333678 (ME01334). T2seeds from the 5 independent transformants that contain this clone and that were used in the pooling process were tested for BastaRresistance and for stress tolerance in the 3 surrogate drought screens. To identify two independent events of 35S::cDNA 12333678 showing PEG, mannitol, and ABA resistance, 36 seedlings from each of four events, ME01334-01, 02, 03, and 04 were screened as previously described. Simultaneously, BastaRsegregation was assessed to identify events containing a single insert segregating in a 3:1 (R:S) ratio as calculated by a Chi-square test (Table 3-2). All of the events tested segregated for a single functional insert. TABLE 3-2BastaRsegregation for ME01334 individual eventsProbabilityEventResistantSensitiveTotalof Chi-test*ME01334-01288360.70031ME01334-022214360.05429ME01334-03315360.12366ME01334-042412360.24821ME01334-5Insufficient seedsto test*Chi-test to determine whether actual ratio of resistant to sensitive differs from the expected 3:1 ratio. Events ME01334-01 and 04 were chosen as the two events because they had the strongest and most consistent resistance to PEG, mannitol and ABA. Resistance was observed for ME01334-02 and 03 although not in expected ratios in all three screens (data not shown). The controls were sown the same day and in the same plate as the individual events. The transgenic control in each of these plates is a segregant, from this ME line or another ME line being tested, that failed to show resistance to the particular stress. The PEG (Tables 3-3 and 3-4), mannitol (Tables 3-5 and 3-6) and ABA (Tables 3-7 and 3-8) segregation ratios observed for ME01334-01 and 01 are consistent with the presence of a single insert as demonstrated by Chi-Square. This is similar to that observed for BastaRresistance (Table 3-2). On 18% PEG, the resistant seedlings from these two events show some root growth but they are also green with new leaves emerging at day 14. The mannitol-resistant seedlings showed more root and shoot growth than the PEG resistant seedlings. The resistant seedlings on ABA show the least amount of growth, and very little root growth relative to the mannitol and PEG screens. The phenotype of the resistant seedlings is unique on each of the screens. TABLE 3-3Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME013341-01 containing 35S::cDNA 12333678 on PEG.EventObservedExpectedχ2Probability of Chi-TestPEG Resistant2626.250.0020.922PEG Sensitive98.750.00735350.009 TABLE 34Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01334-04 containing 35S::cDNA 12333678 on PEG.EventObservedExpectedχ2Probability of Chi-TestPEG Resistant272701.0PEG Sensitive99036360 TABLE 3-5Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01334-01 containing 35S::cDNA 12333678 on mannitol.ProbabilityEventObservedExpectedχ2of Chi-TestMannitol Resistant1423.253.680.0001Mannitol Sensitive177.7511.04313114.72 TABLE 3-6Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01334-04 containing 35S::cDNA 12333678 on mannitol.EventObservedExpectedχ2Probability of Chi-TestMannitol Resistant1622.51.880.006Mannitol Sensitive47.55.6330307.51 TABLE 3-7Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01334-01 containing 35S::cDNA 12333678 on ABA.EventObservedExpectedχ2Probablifty of Chi-TestABA Resistant242401.0ABA Sensitive88032320 TABLE 3-8Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01334-04 containing 35S::cDNA 12333678 on ABA.EventObservedExpectedχ2Probability of Chi-TestABA Resistant1925.51.6570.01ABA Sensitive158.54.9734346.627 Qualitative and Quantitative Analysis of Progeny of T2Plants Isolated on PEG, Mannitol and ABA Progeny from T2plants that were recovered from the three screens and contained cDNA 12333678 (SP1-A2, SP1-P3, and SP1-M19) were analyzed and also found to be resistant to PEG, mannitol and ABA indicating that resistance is transmitted to the next generation. Taken together, 1) the isolation of resistant seedlings containing cDNA 12333678 from all three surrogate screens for drought, 2) the inheritance of this resistance in the next generation, and 3) the fact that the progeny from two or more events from the original transformation also segregated for resistance to these osmotic stresses, provides strong evidence that cDNA 12333678 when over-expressed provides tolerance to osmotic stress. This gene is annotated as an alpha/beta hydrolase, a probable acetone-cyanohydrin lyase. Acetone-cyanohydrin lyase is involved in the catabolism of cyanogenic glycosides. FIG. 3 provides the results of the consensus sequence (SEQ ID NOs: 130-146) analysis based on Ceres cDNA 12333678. Example 4—Ceres cDNA 12384873 Clone 34419, Ceres cDNA 12384873, encodes a full-length strictosidine synthase. Ectopic expression of Ceres cDNA 12384873 under the control of the CaMV35S promoter induces the following phenotypes:Germination on high concentrations of polyethylene glycol (PEG), mannitol, and abscissic acid (ABA).Continued growth on high PEG, mannitol, and ABA. Generation and Phenotypic Evaluation of T1Lines Containing 35S::cDNA 12384873 Wild-typeArabidopsisWassilewskija (WS) plants were transformed with a Tiplasmid containing cDNA 12384873 in the sense orientation relative to the CaMV35S constitutive promoter. The Tiplasmid vector used for this construct, CRS338, contains PAT and confers herbicide resistance to transformed plants. Ten independently transformed events were selected and evaluated for their qualitative phenotype in the T1generation. No positive or negative phenotypes were observed in the T1plants. Screens of Superpools on High PEG, Mannitol, and ABA as Surrogate Screens for Drought Tolerance. Seeds from 13 superpools (1,200 T2seeds from each superpool) from the CaMV35S or 32449 over-expression lines were tested on 3 drought surrogate screens (high concentrations of PEG, mannitol, and ABA) as described above. T3seeds were collected from the resistant plants and analyzed for resistance on all three surrogate drought screens. Once cDNA 12384873 was identified in resistant plants from each of the three surrogate drought screens, the five individual T2events containing this cDNA (ME01490) were screened on high PEG, mannitol and ABA to identify events with the resistance phenotype. Superpools (SP) are referred to as SP1, SP2 and so on. The letter following the hyphen refers to the screen (P=PEG, M=mannitol and A=ABA) and the number following the letter refers to a number assigned to each plant obtained from that screen on that superpool. For example, SP1-M18 is the 18thplant isolated from a mannitol screen of Superpool 1. Qualitative Analysis of 13 Superpools on PEG, Mannitol, and ABA. Resistant candidates were selected based on increased size when compared to the largest wild-type control seedlings. All three screens resulted in a decrease in germination for both wildtype and superpools as compared to seeds on control media. Wild-type seeds that germinated on any of the screens were small and never developed any first leaves even after 40 days. To ensure that even slightly tolerant individuals were not omitted, seedlings that showed any growth and greening whatsoever were recovered and transferred to soil for assessment in the T3generation. All recovered candidates showed signs of vigorous re-growth on soil, although the development was slightly delayed as compared to unstressed plants, presumably because of the transient exposure to stress. The plants transferred to soil were sprayed with BastaRto eliminate any false-positives, or any lines where the BastaRmarker was suppressed. All of the BastaR—resistant candidates flowered and set seed. Resistant seedlings were recovered from Superpools 1, 7 and 11 on all screens (Table 4-1). TABLE 4-1Number of stress-tolerant and BastaRseedlings identified ondrought surrogate screens.SuperpoolPromoter18% PEG375 mM Mannito1.5 uM ABASP135S101311SP235S065SP335S0210SP435S040SP535S005SP635S004SP735S148SP835S041SP9324490012SP10324490014SP11324491531SP1232449306SP1332449021 Qualitative and Quantitative Analysis of 5 Independent Events Representing 35S::cDNA 12384873 on PEG, Mannitol and ABA Seedlings that survived transfer to soil and which were BastaR-resistant were subjected to PCR and sequencing. At least one resistant plant in each of the three osmotic screens contained 35S::cDNA 12384873 (ME01490 g. T2seeds from the 5 independent transformants that contain this clone and that were used in the pooling process were tested for BastaRresistance and for stress tolerance in the 3 surrogate drought screens. To identify two independent events of 35S::cDNA 12384873 showing PEG, mannitol and ABA resistance, 36 seedlings from each of five events, ME01490-01, 02, 03, 04, and 05 were screened as previously described. Simultaneously, BastaRsegregation was assessed to identify events containing a single insert segregating in a 3:1 (R:S) ratio as calculated by a Chi-square test (Table 4-2). Three of the events segregated for a single functional insert (-01,-02 and -04). For the other two events one segregated for two independent inserts (-03) and one segregated for a deficiency of BastaRseedlings (-05). TABLE 4-2BastaRsegregation for ME01490 individual eventsEventResistantSensitiveTotalProbability of Chi-test*ME01490-01*2412360.24821ME01490-02*2610360.70031ME01490-03351360.00208**ME01490-04288360.70031ME01490-052115360.02092***Chi-test to determine whether actual ratio of resistant to sensitive differs from the expected 3:1 ratio.**Significantly different than a 3:1 (R:S) ratio Events ME01490-01 and -02 were chosen as the two events because they had the strongest and most consistent resistance to PEG, mannitol and ABA. Resistance was observed for ME01490-03, -04, and -05 although not in expected ratios in all three screens (data not shown). The controls were sown the same day and in the same plate as the individual lines. The transgenic control in each of these plates is a segregant, from this ME line or another ME line being tested, that failed to show resistance to the particular stress. The PEG (Tables 4-3 and 4-4), mannitol (Tables 4-5 and 4-6) and ABA (Tables 4-7 and 4-8) segregation ratios observed for ME01490-01 and -02 are consistent with the presence of single insert, as demonstrated by Chi-square. This result is similar to that observed for BastaRresistance (Table 4-2). TABLE 4-3Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01490-01 containing 35S::cDNA 12384873 on PEG.EventObservedExpectedχ2Probability of Chi-TestPEG Resistant29270.1480.441PEG Sensitive790.44436360.592 TABLE 4-4Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01490-02 containing 35S::cDNA 12384873 on PEG.EventObservedExpectedχ2Probability of Chi-TestPEG2424.750.02270.763ResistantPEG98.250.068Sensitive33330.0907 TABLE 4-5Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01490-01 containing 35S::cDNA 12384873 on mannitol.EventObservedExpectedχ2Probability of Chi-TestMannitol2226.250.6880.097ResistantMannitol138.752.06Sensitive35352.748 TABLE 4-6Chi-square analysis assuming a 3:1 (R:S) ratio for progenyof ME01490-02 containing 35S::cDNA 12384873 on mannitol.ProbabilityEventObservedExpectedχ2of Chi-TestMannitol Resistant2023.250.4540.178Mannitol Sensitive117.751.36331311.817 TABLE 4-7Chi-square analysis assuming a 3:1 (R:S) ratio for progenyof ME01490-01 containing 35S::cDNA 12384873 on ABA.ProbabilityEventObservedExpectedχ2of Chi-TestABA Resistant28270.0370.7ABA Sensitive890.14836361.85 TABLE 4-8Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01490-02 containing 35S::cDNA 12384873 on ABA.ProbabilityEventObservedExpectedχ2of Chi-TestABA Resistant23270.5930.124ABA Sensitive1391.7836362.373 Qualitative and Quantitative Analysis of Progeny of T2Plants Isolated on PEG, Mannitol and ABA Progeny of the T2plants that were recovered from the three screens and contained cDNA 12384873 (SP1-A18, SP1-P14, SP1-M5, SP1-M6 and SP1-M7) were analyzed and also found to be resistant to PEG, mannitol and ABA indicating that resistance is transmitted to the next generation. Taken together, 1) the isolation of resistant seedlings containing cDNA 12384873 from all three surrogate screens for drought, 2) the inheritance of this resistance in the next generation, and 3) the fact that the progeny from two or more events from the original transformation also segregated for resistance to these osmotic stresses, provides strong evidence that cDNA 12384873 when over-expressed can provide tolerance to osmotic stress. Clone 34419 encodes the first 29 amino acids of a strictosidine synthase protein, and then a frame shift results in a novel stretch of 63 amino acids on the 3′ end of the protein. Example 5—Ceres cDNA 12659859 Ceres cDNA 12659859 encodes a FAD-linked oxidoreductase family, a probable berberine bridge enzyme fromArabidopsis thaliana. Ectopic expression of Ceres cDNA 12659859 under the control of the CaMV35S promoter induces the following phenotypes:Germination on high concentrations of polyethylene glycol (PEG), mannitol and abscissic acid (ABA).Continued growth on high concentration of PEG, mannitol and ABA. Generation and Phenotypic Evaluation of T1Lines Containing 35S::cDNA 12659859. Wild-typeArabidopsisWassilewskija (WS) plants were transformed with a Ti plasmid containing cDNA 12659859 in the sense orientation relative to the CaMV35S constitutive promoter. The Tiplasmid vector used for this construct, CRS338, contains PAT and confers herbicide resistance to transformed plants. Ten independently transformed events were selected and evaluated for their qualitative phenotype in the T1generation. No positive or negative phenotypes were observed in the T1plants. Screens of Superpools on High PEG, Mannitol, and ABA as Surrogate Screens for Drought Tolerance. Seeds from 13 superpools (1,200 T2seeds from each superpool) from the CaMV35S or 32449 over-expression lines were tested on 3 drought surrogate screens (high concentrations of PEG, mannitol, and ABA) as described above. T3seeds were collected from the resistant plants and analyzed for resistance on all three surrogate drought screens. Once cDNA 12659859 was identified in resistant plants from each of the three surrogate drought screens, the five individual T2events containing this cDNA (SR01010) were screened on high PEG, mannitol, and ABA to identify events with the resistance phenotype. Superpools (SP) are referred to as SP1, SP2 and so on. The letter following the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA) and the number following the letter refers to a number assigned to each plant obtained from that screen on that superpool. For example, SP1-M18 is the 18thplant isolated from a mannitol screen of Superpool 1. Qualitative and Quantitative Analysis of 2 Independent Events Representing 12659859 (SR01010) on PEG, Mannitol and ABA To identify two independent events of 35S::cDNA 12659859 showing PEG, mannitol, and ABA resistance, 36 seedlings from each of three events, SR01010-01, 02, and 03 were screened as previously described. BastaRsegregation was assessed to identify lines containing a single insert segregating in a 3:1 (R:S) ratio as calculated by a Chi-square test (Table 5-1). Two of three T2generation events (01 and 03) segregated for a single insert although the segregation ratio for -01 is also not different than a 15:1 (R:S) ratio. TABLE 5-1BastaRsegregation for SR01010 individual eventsProbabilityEventResistantSensitiveTotalof Chi-test*SR01010-01324360.05429SR01010-02323350.0248**SR01010-032412360.24821SR01010-01-1279361SR01010-03-12013330.05619*Chi-test to determine whether actual ratio of resistant to sensitive differs from the expected 3:1 ratio.**Significantly different than a 3:1 (R:S) ratio Lines SR0101-01 and -03 were chosen as the two events because they had a strong and consistent resistance to PEG, mannitol and ABA. Resistance was observed for SR01010-02 although not in expected ratios in all three screens (data not shown). The controls were sown the same day and in the same plate as the individual lines. The PEG (Tables 5-2 and 5-3), mannitol (Tables 5-4 and 5-5) and ABA (Tables 5-6 and 5-7) segregation ratios observed for SR01010-01 and -03 are consistent with the presence of single insert as demonstrated by chi-square, similar to what we observed for BastaRresistance (Table 5-1). The progeny from one resistant T2plant from each of these two events was tested in the T3generation in the same manner. Resistance to PEG, mannitol and ABA was also observed in the T3generation. Taken together, the segregation of resistant seedlings containing cDNA 12659859 from two events on all three drought surrogate screens and the inheritance of this resistance in a subsequent generation, provide strong evidence that cDNA 12659859 when over-expressed can provide tolerance to drought. TABLE 5-2Chi-square analysis assuming a 3:1 (R:S) ratio for progenyof SR01010-01T2containing 35S::cDNA 12659859 on PEG.EventObservedExpectedχ2ProbabilityPEG Resistant3126.25.860.064PEG Sensitive48.752.57935353.438 TABLE 5-3Chi-square analysis assuming a 3:1 (R:S) ratio for progenyof SR01010-03 T2containing 35S::cDNA 12659859 on PEG.EventObservedExpectedχ2ProbabilityPEG Resistant2827.037.700PEG Sensitive89.1113636.148 TABLE 5-4Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01010-01T2containing 35S::cDNA 12659859 on mannitol.EventObservedExpectedχ2ProbabilityMannitol Resistant2527.148.441Mannitol Sensitive119.4443636.593 TABLE 5-5Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01010-3 T2containing 35S::cDNA 12659859 on mannitol.EventObservedExpectedχ2ProbabilityMannitol Resistant2120.25.028.739Mannitol Sensitive66.75.0832727.111 TABLE 5-6Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01010-01T2containing 35S::cDNA 12659859 on ABA.EventObservedExpectedχ2ProbabilityABA Resistant3027.333.248ABA Sensitive69136361.333 TABLE 5-7Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01010-03 T2containing 35S::cDNA 12659859 on ABA.EventObservedExpectedχ2ProbabilityABA Resistant272701.0ABA Sensitive99036360 FIG. 4 provides the results of the consensus sequence (SEQ ID NOs: 147-177) analysis based on Ceres cDNA 12659859. Example 6—Ceres cDNA 12723147 Ceres cDNA 12723147 encodes anArabidopsisputative aldo/keto reductase. Ectopic expression of Ceres cDNA 12723147 under the control of the CaMV35S promoter induces the following phenotypes:Germination on high concentrations of polyethylene glycol (PEG), mannitol and abscissic acid (ABA).Continued growth on high concentration of PEG, mannitol and ABA. Generation and Phenotypic Evaluation of T1Lines Containing 35S::cDNA 12723147. Wild-typeArabidopsisWassilewskija (WS) plants were transformed with a Ti plasmid containing cDNA 12723147 in the sense orientation relative to the CaMV35S constitutive promoter. The Tiplasmid vector used for this construct, CRS338, contains PAT and confers herbicide resistance to transformed plants. Ten independently transformed events were selected and evaluated for their qualitative phenotype in the T1generation. No positive or negative phenotypes were observed in the T1plants. Screens of Superpools on High PEG, Mannitol, and ABA as Surrogate Screens for Drought Tolerance. Seeds from 13 superpools (1,200 T2seeds from each superpool) from the CaMV35S or 32449 over-expression lines were tested on 3 drought surrogate screens (high concentrations of PEG, mannitol, and ABA) as described above. T3seeds were collected from the resistant plants and analyzed for resistance on all three surrogate drought screens. Once cDNA 12723147 was identified in resistant plants from each of the three surrogate drought screens, the five individual T2events containing this cDNA (SR01013) were screened on high PEG, mannitol, and ABA to identify events with the resistance phenotype. Superpools (SP) are referred to as SP1, SP2 and so on. The letter following the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA) and the number following the letter refers to a number assigned to each plant obtained from that screen on that superpool. For example, SP1-M18 is the 18thplant isolated from a mannitol screen of Superpool 1. Qualitative and Quantitative Analysis of 2 Independent Events Representing 35S::cDNA 12659859 (SR01010) on PEG, Mannitol and ABA To identify two independent events of 35S::cDNA 12659859 showing PEG, mannitol, and ABA resistance, 36 seedlings from each of two events, SR01013-01 and -02 were screened as previously described. BastaRsegregation was assessed to verify that the lines contained a single insert segregating in a 3:1 (R:S) ratio as calculated by a chi-square test (Table 6-1). Both lines (01 and 02) segregated for a single insert in the T2generation (Table 1) TABLE 6-1BastaRsegregation for SR01013 individual eventsProbabilityEventResistantSensitiveTotalof Chi-test*SR01013-01305350.14323SR01013-02306360.24821SR01013-01-3341360.00248**SR01013-02-2320320.00109***Chi-test to determine whether actual ratio of resistant to sensitive differs form the expected 3:1 ratio.**Significantly different than a 3:1 (R:S) ratio Lines SR01013-01 and -02 were chosen as the two events because they had a strong and consistent resistance to PEG, mannitol and ABA. The controls were sown the same day and in the same plate as the individual lines. The PEG (Tables 6-2 and 6-3), mannitol (Tables 6-4 and 6-5) and ABA (Tables 6-6 and 6-7) segregation ratios observed for SR01013-01 and -02 are consistent with the presence of single insert as demonstrated by chi-square, similar to what we observed for BastaRresistance (Table 6-1). The progeny from one resistant T2plant from each of these two events were tested in the same manner as the T2. Resistance to PEG, mannitol and ABA was also observed in the T3generation. Taken together, the segregation of resistant seedlings containing cDNA 12723147 from two events on all three drought surrogate screens and the inheritance of this resistance in a subsequent generation, provide strong evidence that cDNA 12723147 when over-expressed can provide tolerance to drought. TABLE 6-2Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-01T2containing 35S::cDNA 12723147 on PEG.ProbabilityEventObservedExpectedχ2of Chi-TestPEG Resistant22270.9260.054PEG Sensitive1492.77836363.704 TABLE 6-3Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-02 T2containing 35S::cDNA 12723147 on PEG.ProbabilityEventObservedExpectedχ2of Chi-TestPEG Resistant26270.037.700PEG Sensitive109.1113636.148 TABLE 6-4Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-01 T2containing 35S::cDNA 12723147 on mannitol.ProbabilityEventObservedExpectedχ2of Chi-TestMannitol Resistant2827.037.700Mannitol Sensitive89.1113636.148 TABLE 6-5Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-02 T2containing 35S::cDNA 12723147 on mannitol.ProbabilityEventObservedExpectedχ2of Chi-TestMannitol Resistant18273.0005Mannitol Sensitive1899363612 TABLE 6-6Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-02 T2containing 35S::cDNA 12723147 on ABA.EventObservedExpectedχ2ProbabilityABA Resistant13245.0427.098ABA Sensitive19815.125323220.167 TABLE 6-7Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-02 T2containing 35S::cDNA 12723147 on ABA.EventObservedExpectedχ2ProbabilityABA Resistant13245.0427.098ABA Sensitive19815.125323220.167 FIG. 5 provides the results of the consensus sequence (SEQ ID NOs: 178-200) analysis based on Ceres cDNA 12723147. Example 7—Ceres cDNA 13488750 Clone 125039, Ceres cDNA 13488750, encodes a full-length putative adenylylsulfate (APS) kinase fromArabidopsis thaliana. Ectopic expression of Ceres cDNA 13488750 under the control of the CaMV35S promoter induces the following phenotypes:Continued growth under high heat conditions. Generation and Phenotypic Evaluation of T1Lines Containing 35S::cDNA 13488750. Wild-typeArabidopsisWassilewskija (WS) plants were transformed with a Ti plasmid containing cDNA 13488750 in the sense orientation relative to the CaMV35S constitutive promoter. The Tiplasmid vector used for this construct, CRS338, contains PAT and confers herbicide resistance to transformed plants. Ten independently transformed events were selected and evaluated for their qualitative phenotype in the T1generation. No negative phenotypes were observed in the T1plants although three of the T1 lines produced a small rosette (ME02526-01, 02 and 05). T2and T3lines of events ME02526-01 and ME02526-05 did not show the small rosette phenotype. Screens of Masterpools for Heat Tolerance Via Heat Shock In Vitro. Seeds from 100 masterpools from the CaMV35S or 32449 over-expression lines were tested for heat tolerance in vitro as described above. Once cDNA 13488750 was identified in tolerant plants from the screen, five individual T2events containing this cDNA (ME02526) were screened on soil as described above to identify events with the resistance phenotype. Qualitative Analysis of the T2Masterpool of cDNA 13488750 Plants Heat Shocked on Plates Visual phenotyping of the masterpool containing cDNA 13488750 (ME02526) on agar or soil showed no visible alterations in phenotype (data not shown). After heat-shock at 15 days of age, the ME02526 masterpool showed greater heat recovery as compared to the wild-type control and other transgenic masterpools. Assessment was a measure of “greenness” as well as continued growth at 23° C. after heat shock at 45° C. for between 5 and 8 hours. Immediately after the heat shock stress, the extent of stress-induced damage in the control and ME02526 masterpool appeared comparable. The leaves and cotyledons were wilted and droopy although still green. However, after 4 days of recovery, 2 of 10 plants in the ME02526 masterpool had completely recovered and were growing again. Other seedlings showed some recovery as measured by greenness compared to the wild-type control. Qualitative and Quantitative Analysis of Individual T2Events of cDNA 13488750 Under Continual Heat Treatment on Soil Five independent events of ME02526 were tested on soil as described above. Two of the events (ME02526-04 and -05) showed heat resistance after continual growth at 36° C. Heat resistance was noted as decreased chlorosis compared to wild-type. Segregation frequencies of the transgene under test suggest that these two events contain a single insert, as calculated by a chi-square test (Tables 7-1, 7-2 and 7-3). Ten and 11 plants from events 04 and 05, respectively, showed continued vigor and decreased chlorosis after continuous heat treatment compared to wild-type controls. TABLE 74Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME02526-04 and -05 containing 35S::cDNA 13488750 on Finale.Ob-ProbabilityEventservedExpectedχ2of Chi-TestME02526-04 Finale Resistant29270.5930.44ME02526-04 Finale Sensitive793636ME02526-05 Finale Resistant272701ME02526-05 Finale Sensitive903636 TABLE 7-2Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME02526-04 containing 35S::cDNA 13488750 for thermo tolerance(continual growth at 36° C. on soil).EventObservedExpectedχ2Probability of Chi-TestHeat Tolerant1011.250.5560.456Heat Sensitive53.751515 TABLE 7-3Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME02526-05 containing 35S::cDNA 13488750 for thermo tolerance(continual growth at 36° C. on soil).EventObservedExpectedχ2Probability of Chi-TestHeat Tolerant1111.250.0220.881Heat Sensitive43.751515 The plants that survive the heat treatment show premature bolting and reduced fecundity but much less so than the control plants. Control and ME02526 plants bolt after only 4 days of exposure to 36° C. (11-day old plants), but were more vigorous and less chlorotic than the controls and the height and branch number was comparable to those of wild-type (data not shown). Heat-tolerant lines all showed seed abortion and reduced fecundity (data not shown). Seed abortion and reduced silique size was also prevalent in all wild-type controls (data not shown). Events 04 and 05, which had a thermo tolerant phenotype in the T2generation, were evaluated in greater detail in the T3generation for heat resistance and fecundity after prolonged heat stress on MS plates. Qualitative and Quantitative Analysis of Individual T3Events Under 36° C. Heat Treatment on Plates Seeds from five individuals of the T3generation for ME02526-04 and -05 lines and controls were sterilized, stratified and germinated for 7 days at 23° C. prior to exposure to 36° C. heat stress. The events were evaluated for heat resistance to prolonged heat stress. The thermo tolerant phenotype became apparent after 15 days of 36° C. treatment. T3progeny from ME02526-04 (Table 7-4) and ME02526-05 (Table 7-5) were found to segregate in the expected 3:1 ratio for the thermo tolerant phenotype. TABLE 7-4Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME02526-04 containing 35S::cDNA 13488750 for thermotolerance (continual growth at 36° C.EventObservedExpectedχ2Probability of Chi-TestHeat Tolerant1414.250.0170.89Heat Sensitive54.751919 TABLE 7-5Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME02526-05 containing 35S::cDNA 13488750 for thermotolerance (continual growth at 36° C.EventObservedExpectedχ2Probability of Chi-TestHeat Tolerant1514.250.1570.69Heat Sensitive44.751919 Qualitative Analysis of Individual T3Events Heat Shocked on Plates for Differentiation of Natural Acquired Thermotolerance Plants acquire thermotolerance to lethal high temperatures such as 45° C. if previously exposed to moderately high temperature or if the temperature is raised gradually to an otherwise lethal temperature (Vierling, 1991). To ascertain whether the thermotolerance observed in the T3generation is due to some naturally acquired thermotolerance imparted by heat exposure of the T2parent plant (ME2526-04 and thermotolerance was assessed by comparing pre-heat treated wild-type and transgenic controls and unheated wild-type controls. T3events of ME02526-04 and 05 were heat shocked for 4 hours as described above. ME02526-04 and -05 were able to stay greener longer than both pre-heat treated controls and un-heat treated controls. However, both ME02526 lines and all controls (wild-type and transgenic) failed to elongate and thrive after heat treatment at 45° C. and eventually all died. Un-heat treated wild-type control became chlorotic faster (1-day after treatment) than pre-heat treated wildtype control and pre-heat treated transgenic control suggesting that there is some natural acquired thermotolerance that occurs that is not correlated with the over-expression of 35S::cDNA 13488750. Even with exposure to this lethal temperature, ME02526 was greener after 7 days than controls, indicating that ME02526 has a thermotolerance phenotype that is unrelated to the natural mechanisms of acquired thermotolerance. FIG. 6 provides the results of the consensus sequence (SEQ ID NOs: 201-214) analysis based on Ceres cDNA 13488750. Example 8—Ceres cDNA 13489782 Clone 10044, Ceres cDNA 13489782, encodes a full-length putative 114-amino acid hypothetical protein fromArabidopsis thaliana. Ectopic expression of Ceres cDNA 13489782 under the control of the 32449 promoter induces the following phenotypes:Germination on high concentrations of polyethylene glycol (PEG) and abscissic acid (ABA) andContinued growth on high concentrations of PEG and ABA. Generation and Phenotypic Evaluation of T1Lines Containing 32449::cDNA 13489782 Wild-typeArabidopsisWassilewskija (WS) plants were transformed with a Ti plasmid containing cDNA 13489782 in the sense orientation relative to the CaMV35S constitutive promoter. The Tiplasmid vector used for this construct, CRS338, contains PAT and confers herbicide resistance to transformed plants. Ten independently transformed events were selected and evaluated for their qualitative phenotype in the T1generation. No positive or negative phenotypes were observed in the T1plants. Screens of Superpools on High PEG, Mannitol, and ABA as Surrogate Screens for Drought Tolerance Seeds from 13 superpools (1,200 T2seeds from each superpool) from the CaMV35S or 32449 over-expression lines were tested on 3 drought surrogate screens (high concentrations of PEG, mannitol, and ABA) as described above. T3seeds were collected from the resistant plants and analyzed for resistance on all three surrogate drought screens. Once cDNA 13489782 was identified in resistant plants from each of the three surrogate drought screens, the five individual T2events containing this cDNA (ME00446) were screened on high PEG, mannitol, and ABA to identify events with the resistance phenotype. Superpools (SP) are referred to as SP1, SP2 and so on. The letter following the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA) and the number following the letter refers to a number assigned to each plant obtained from that screen on that superpool. For example, SP1-M18 is the 18thplant isolated from a mannitol screen of Superpool 1. Qualitative Analysis of Superpools on PEG, Mannitol, and ABA. Resistant candidates were selected based on increased size when compared to the largest wild-type control seedlings. All three screens resulted in a decrease in germination for both wildtype and superpools as compared to seeds on control media. Wild-type seeds that germinated on any of the screens were small and never developed any first leaves even after 40 days. To ensure that even slightly tolerant individuals were not omitted, seedlings that showed any growth and greening whatsoever were recovered and transferred to soil for assessment in the T3generation. All recovered candidates showed signs of vigorous re-growth on soil, although the development was slightly delayed as compared to unstressed plants, presumably because of the transient exposure to stress. The plants transferred to soil were sprayed with Basta to eliminate any false-positives, or any lines where the BastaRmarker was suppressed. All of the Basta-resistant candidates flowered and set seed. Resistant seedlings were recovered from Superpools 1, 7 and 11 on all screens (Table 8-1). TABLE 8-1Number of stress-tolerant and BasteRseedlings identified ondrought surrogate screens.SuperpoolPromoter18% PEG375 mM Mannitol1.5 uM ABASP135S101311SP235S065SP335S0210SP435S040SP535S005SP635S004SP735S148SP835S041SP9324490012SP10324490014SP113244915313SP1232449306SP1332449021 We obtained sequence from 3, 3, and 13 plants that were both BastaRand resistant to PEG, mannitol or ABA, respectively. For each of the three surrogate drought screens, one or more plants contained the 32449::clone 10044 (ME00446). The probability of finding a plant containing this cDNA at random in all three screens is 0.03×0.03×0.03. Qualitative and Quantitative Analysis of 6 Independent Events Representing 32449::cDNA 13489782 on PEG, Mannitol and ABA To identify independent events of 32449::cDNA 13489782 showing PEG, mannitol and ABA resistance, 36 seedlings from each of six events, ME00446-01, 02, 03, 04, 05 and 06 were screened as previously described. Simultaneously, BastaRsegregation was assessed to identify lines containing a single insert segregating in a 3:1 (R:S) ratio as calculated by a Chi-square test (Table 8-2). All of the lines segregated for a single functional insert. TABLE 8-2BastaRsegregation for 6 events of ME00446DidProbabilityME LineAssessmentNot GerminateResistantSensitiveTotalof Chi-test00446-01Oct. 30, 20031287350.4945200446-02Oct. 30, 20030288360.7003100446-03Oct. 30, 20031287350.4945200446-04Oct. 30, 20031278350.769700446-05Oct. 30, 200322410340.5524500446-06Oct. 30, 200302511360.44142*Chi-test to determine whether actual ratio of resistant to sensitive differs from the expected 3:1 ratio. Events ME00446-02 and 04 were chosen as the two events for further analysis because they had the strongest and most consistent resistance to PEG and ABA. None of the lines showed mannitol resistance at 375 mM concentration. The controls were sown the same day and in the same plate as the individual lines. The PEG (Tables 8-3 and 8-4) and ABA (Tables 8-5 and 8-6) segregation ratios observed for ME00446-02 and -04 are consistent with the presence of single insert as demonstrated by the Chi-Square test. This is similar to that observed for Basta resistance (Table 8-2). Despite the fact that this line was isolated from all three screens, it was subsequently concluded that it could not be considered mannitol resistant. This is likely due to an overly stringent mannitol concentration. In a superpool screen setting, the seedlings are more densely grown than in an individual line setting. This means that in a superpool screen, there is a lower effective concentration of mannitol. When putative tolerant plant from a superpool is tested as an individual line, the effective concentration it is grown on is actually higher. In the case of ME00446, this difference was enough to invalidate it as mannitol tolerant. In fact, a resistant plant to mannitol was isolated from superpool 11 that corresponds to clone 10044. TABLE 8-3Chi-square analysis assuming a 3:1(R:S) ratio for progenyof ME00446-02 containing 32449::clone 10044 on PEG.Probability ofEventObservedExpectedχ2χ2PEG Resistant3026.950.34520.2557PEG Sensitive57.70.9468Total35351.292 TABLE 8-4Chi-square analysis assuming a 3:1(R:S) ratio for progeny ofME00446-04 containing 32449::clone 10044 on PEG.Probability ofEventObservedExpectedχ2χ2PEG Resistant2627.770.1130.482PEG Sensitive108.230.3813Total36360.4943 TABLE 8-5Chi-square analysis assuming a 3:1(R:S) ratio for progeny ofME00446-02 containing 32449::clone 10044 on ABA.Probability ofEventObservedExpectedχ2χ2ABA Resistant31270.59260.1237ABA Sensitive591.778Total36362.370 TABLE 8-6Chi-square analysis assuming a 3:1(R:S) ratio for progeny ofME00446-04 containing 32449::clone 10044 on ABA.Probability ofEventObservedExpectedχ2χ2ABA Resistant31270.59260.1237ABA Sensitive591.778Total36362.370 Qualitative and Quantitative Analysis of Progeny of T2Plants Isolated on PEG, Mannitol and ABA Progeny from T2plants that were recovered from the three screens and containing clone 10044 (SP11-M13 and SP11-P5) were found to be resistant to PEG and ABA. Taken together, 1) the isolation of resistant seedlings containing clone 10044 from all three surrogate screens for drought, 2) the inheritance of this resistance in the next generation, and 3) the fact that the progeny from two or more events from the original transformation also segregated for resistance to these osmotic stresses, provide strong evidence that clone 10044 when over-expressed provides resistance to osmotic and dehydration stress. FIG. 7 provides the results of the consensus sequence (SEQ ID NOs: 215-222) analysis based on Ceres cDNA 13489782. Example 9—Ceres cDNA 13486759 Clone 10987, corresponding Ceres cDNA 13486759, encodes anArabidopsis251-amino acid expressed protein. Ectopic expression of clone 10987 under the control of the CaMV35S promoter induces the following phenotypes:Germination on high concentrations of polyethylene glycol (PEG), mannitol, and abscissic acid (ABA), andContinued growth on high concentrations of PEG, mannitol, and ABA. Generation and Phenotypic Evaluation of T1Lines Containing 35S::cDNA 13486759 Wild-typeArabidopsisWassilewskija (WS) plants were transformed with a Tiplasmid containing cDNA 13486759 in the sense orientation relative to the CaMV35S constitutive promoter. The Tiplasmid vector used for this construct, CRS338, contains PAT and confers herbicide resistance to transformed plants. Ten independently transformed events were selected and evaluated for their qualitative phenotype in the T1generation. No positive or negative phenotypes were observed in the T1plants. Screens of Superpools on High PEG, Mannitol and ABA as Surrogate Screens for Drought Tolerance Seeds from 13 superpools (1,200 T2seeds from each superpool) from the CaMV35S or 32449 over-expression lines were tested on 3 drought surrogate screens (high concentrations of PEG, mannitol, and ABA) as described above. T3seeds were collected from the resistant plants and analyzed for resistance on all three surrogate drought screens. Once cDNA 13486759 was identified in resistant plants from each of the three surrogate drought screens, the five individual T2events containing this cDNA (ME03316) were screened on high PEG, mannitol, and ABA to identify events with the resistance phenotype. Superpools (SP) are referred to as SP1, SP2 and so on. The letter following the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA) and the number following the letter refers to a number assigned to each plant obtained from that screen on that superpool. For example, SP1-M18 is the 18thplant isolated from a mannitol screen of Superpool 1. Qualitative Analysis of 13 Superpools on PEG, Mannitol and ABA Resistant candidates were selected based on increased size when compared to the largest wild-type control seedlings. All three screens resulted in a decrease in germination for both wildtype and superpools as compared to seeds on control media. Wild-type seeds that germinated on any of the screens were small and never developed any first leaves even after 40 days. To ensure that even slightly tolerant individuals were not omitted, seedlings that showed any growth and greening whatsoever were recovered and transferred to soil for assessment in the T3generation. All recovered candidates showed signs of vigorous re-growth on soil, although the development was slightly delayed as compared to unstressed plants, presumably because of the transient exposure to stress. The plants transferred to soil were sprayed with Basta to eliminate any false-positives, or any lines where the BastaRmarker was suppressed. All of the Basta-resistant candidates flowered and set seed. Resistant seedlings were recovered from Superpools 1, 7 and 11 on all screens (Table 9-1). TABLE 9-1Number of stress-tolerant and BastaRseedlings identified ondrought surrogate screens.SuperpoolPromoter18% PEG375 mM Mannitol1.5 uM ABASP135S101311SP235S065SP335S0210SP435S040SP535S005SP635S004SP735S148SP835S041SP9324490012SP10324490014SP113244915313SP1232449306SP1332449021 We obtained sequence from 3, 3, and 13 plants from Superpool 11 that were both BastaR-resistant and resistant to PEG, mannitol or ABA, respectively. For each of the three osmotic screens, one or more plants contained the 35S::clone 10987 (ME03316), which made it a good candidate for further testing. The probability of finding a plant containing this clone 10987 at random in all three screens is 0.03×0.03. Qualitative and Quantitative Analysis of 5 Independent Events Representing 35S::cDNA 13486759 on PEG, Mannitol and ABA To identify independent events of 35S::cDNA 13486759 showing PEG, mannitol and ABA resistance, 36 seedlings from each of five events, ME03316-01,-02,-03,-04, and -05 were screened as previously described. Simultaneously, BastaRsegregation was assessed to identify events containing a single insert segregating in a 3:1 (R:S) ratio as calculated by a chi-square test (Table 9-2). All of the events segregated for a single functional insert. ME03316-02 could be segregating for two linked or unlinked inserts but the ratios on the surrogate drought screens indicate it is likely to be a single insert. TABLE 9-2BastaRsegregation for 5 individual eventsEventResistantSensitiveTotalProbability of Chi-testME03316-012313360.12366ME03316-02324360.05429ME03316-03306360.24821ME03316-042511360.44142ME03316-05297360.44142*Chi-test to determine whether actual ratio of resistant to sensitive differs from the expected 3:1 ratio. Lines ME03316-01 and 02 were chosen as the two events for further analysis because they had the strongest and most consistent resistance to PEG, mannitol and ABA. Resistance was observed for ME03316-03, 04, and 05 although not in expected ratios in all three screens (data not shown). The controls were sown the same day and on the same plate as the individual lines. The PEG (Tables 9-3 and 9-4), mannitol (Tables 9-5 and 9-6) and ABA (Table 9-7) segregation ratios observed are consistent with the presence of a single insert as demonstrated by chi-square. ME03316-02 seedlings on ABA (Table 9-8) appear to be segregating for two inserts which is still consistent with the ratio observed on BastaR. Both events segregate for a deficiency of resistant TABLE 9-3Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME03316-01 containing 35S::clone 10987 on PEG.EventObservedExpectedχ2Probability of Chi-TestPEG Resistant22270.930.0543PEG Sensitive1492.78Total36363.7 seedlings on mannitol. TABLE 9-3Chi-square analysis assuming a 3:1 (R:S) ratio for progenyof ME03316-02 containing 35S::clone 10987 on PEG.ProbabilityEventObservedExpectedχ2of Chi-TestPEG Resistant2326.250.40240.2046PEG Sensitive128.751.2071Total35351.610 TABLE 9-5Chi-square analysis assuming a 3:1 (R:S) ratio for progenyof ME03316-01 containing 35S::clone 10987 on Mannitol.ProbabilityEventObservedExpectedχ2of Chi-TestMannitol1925.51.6570.01ResistantMannitol158.54.971SensitiveTotal34346.63 TABLE 9-6Chi-square analysis assuming a 3:1 (R:S) ratio for progenyof ME03316-02 containing 35S::clone 10987 on Mannitol.ProbabilityEventObservedExpectedχ2of Chi-TestMannitol1826.252.5930.0013ResistantMannitol178.757.779SensitiveTotal353510.371 TABLE 9-7Chi-square analysis assuming a 3:1 (R:S) ratio for progenyof ME03316-01 containing 35S::clone 10987 on ABA.ProbabilityEventObservedExpectedχ2of Chi-TestABA2725.50.08820.5525Resistant78.50.2647ABASensitiveTotal34340.3529 TABLE 9-8Chi-square analysis assuming a 3:1 (R:S) ratio for progenyof ME03316-02 containing 35S::clone 10987 on ABA.ProbabilityEventObservedExpectedχ2of Chi-TestABA3224.752.1240.0036ResistantABA18.256.371SensitiveTotal33338.495 Qualitative and Quantitative Analysis of Progeny of T2Plants Isolated on PEG, Mannitol and ABA Progeny from T2plants that were recovered from the three screens and containing clone 10987 (SP11-A15, SP11-A16, SP11-P2, and SP11-M10) were found to be resistant to PEG, mannitol and ABA. On PEG, the progeny of SP11-M10 segregated for a deficiency of resistant seedlings similar to the deficiency that noted for the T2seedlings in Tables 9-5 and 9-6. A deficiency of resistant seedlings is also noted for the progeny of SP11P2 on PEG. Taken together, 1) the isolation of resistant seedlings containing clone 10987 from all three surrogate screens for drought, 2) the inheritance of this resistance in the next generation, and 3) the fact that the progeny from two or more events from the original transformation also segregated for resistance to these osmotic stresses, these findings provide strong evidence that clone 10987 when over-expressed can provide tolerance to osmotic stresses. Example 10—Ceres cDNA 13500101 Clone 17206, corresponding to Ceres cDNA 13500101, encodes a putative strictosidine synthase. Ectopic expression of cDNA 13500101 under the control of the CaMV35S promoter induces the following phenotypes:Germination on high concentrations of polyethylene glycol (PEG) and mannitol.Continued growth on high concentrations of PEG and mannitol. Generation and Phenotypic Evaluation of T1Lines Containing 35S::cDNA 13500101. Wild-typeArabidopsisWassilewskija (WS) plants were transformed with a Ti plasmid containing cDNA 13500101 in the sense orientation relative to the CaMV35S constitutive promoter. The Tiplasmid vector used for this construct, CRS338, contains PAT and confers herbicide resistance to transformed plants. Ten independently transformed events were selected and evaluated for their qualitative phenotype in the T1generation. No positive or negative phenotypes were observed in the T1plants. Screens of Superpools on High PEG, Mannitol, and ABA as Surrogate Screens for Drought Tolerance. Seeds from 13 superpools (1,200 T2seeds from each superpool) from the CaMV35S or 32449 over-expression lines were tested on 3 drought surrogate screens (high concentrations of PEG, mannitol, and ABA) as described above. T3seeds were collected from the resistant plants and analyzed for resistance on all three surrogate drought screens. Once cDNA 13500101 was identified in resistant plants from each of the three surrogate drought screens, the five individual T2events containing this cDNA (SR01000) were screened on high PEG, mannitol, and ABA to identify events with the resistance phenotype. Superpools (SP) are referred to as SP1, SP2 and so on. The letter following the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA) and the number following the letter refers to a number assigned to each plant obtained from that screen on that superpool. For example, SP1-M18 is the 18thplant isolated from a mannitol screen of Superpool 1. Qualitative and Quantitative Analysis of 2 Independent Events Representing 35S::cDNA 13500101 (SR01000) on PEG, Mannitol and ABA To identify two independent events of 35S::clone 17206 showing PEG, mannitol, and ABA resistance, 36 seedlings from each of three events, SR01000-01, 02 and 03 were screened as previously described. Basta segregation was assessed to verify that the lines contained a single insert segregating in a 3:1 (R:S) ratio as calculated by a chi-square test (Table 1). Two lines (-01 and -02) segregated for a single insert (Table 1). TABLE 10-1BastaRsegregation for SR01000 individual eventsProbabilityEventResistantSensitiveTotalof Chi-testSR01000-01297360.44142SR01000-022313360.12366SR01000-03350350.00064SR01000-01-01350350.00064SR01000-02-01279361SR01000-03-01360360.00053*Chi-test to determine whether actual ratio of resistant to sensitive differs from the expected 3:1 ratio. Testing of the progeny from the T2resistant plants on the 3 surrogate drought screens showed that lines SR01000-01 and 02 had a strong and consistent resistance to PEG, mannitol, but not to ABA. These were chosen as the two events for further analysis. The controls were sown the same day and in the same plate as the individual lines. The PEG (Tables 10-2 and 10-3), and mannitol (Tables 10-4 and 10-5) segregation ratios observed for SR01000-01 and 02 are consistent with the presence a of single insert as demonstrated by chi-square (Table 10-1). TABLE 10-2Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR91000-01 T2containing 35S::cDNA 13500101 on PEG.ProbabilityEventObservedExpectedχ2of Chi-TestPEG Resistant2626.250.0020.922PEG Sensitive98.750.00735350.009 TABLE 10-3Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01000-02 T2containing 35S::cDNA 13500101 on PEG.ProbabilityEventObservedExpectedχ2of Chi-TestPEG Resistant2724.750.2050.366PEG Sensitive68.250.61433330.818 TABLE 10-4Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01000-01 T2containing 35S::cDNA 1.3500101 on mannitol.ProbabilityEventObservedExpectedχ2of Chi-TestMannitol Resistant28270.0370.700Mannitol Sensitive890.11136360.148 TABLE 10-5Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01000-02 T2containing 35S::cDNA 13500101 on mannitol.ProbabilityEventObservedExpectedχ2of Chi-TestMannitol Resistant30270.3330.248Mannitol Sensitive691.00036361.333 Qualitative and Quantitative Analysis of Progeny of T2Plants Isolated on PEG, Mannitol and ABA The progeny from one resistant T2plant from each of these two events were tested in the same manner as the T2. Resistance to PEG and mannitol persisted in the second generation. Taken together, 1) the isolation of resistant seedlings containing clone 17026 from two of the surrogate screens for drought (PEG and mannitol), 2) the inheritance of this resistance in the next generation, and 3) the fact that the progeny from two or more events from the original transformation also segregated for resistance to these osmotic stresses, these findings provide strong evidence that clone 17026 when over-expressed can provide tolerance to osmotic stresses. FIG. 8 provides the results of the consensus sequence (SEQ ID NOs: 223-240) analysis based on Ceres cDNA 13500101. Example 11—Ceres cDNA 13509011 (12357529) Clone 104691, corresponding to Ceres cDNA 13509011 (12357529), encodes a probable strictosidine synthase enzyme. Ectopic expression of cDNA 13509011 under the control of the CaMV35S promoter induces the following phenotypes:Germination on high concentrations of polyethylene glycol (PEG) and mannitol.Continued growth on high concentration of PEG and mannitol. Generation and Phenotypic Evaluation of T1Lines Containing 35S::cDNA 13509011. Wild-typeArabidopsisWassilewskija (WS) plants were transformed with a Ti plasmid containing cDNA 12334963 in the sense orientation relative to the CaMV35S constitutive promoter. The Tiplasmid vector used for this construct, CRS338, contains PAT and confers herbicide resistance to transformed plants. Ten independently transformed events were selected and evaluated for their qualitative phenotype in the T1generation. No positive or negative phenotypes were observed in the T1plants. Screens of Superpools on High PEG, Mannitol, and ABA as Surrogate Screens for Drought Tolerance. Seeds from 13 superpools (1,200 T2seeds from each superpool) from the CaMV35S or 32449 over-expression lines were tested on 3 drought surrogate screens (high concentrations of PEG, mannitol, and ABA) as described above. T3seeds were collected from the resistant plants and analyzed for resistance on all three surrogate drought screens. Once cDNA 13509011 was identified in resistant plants from each of the three surrogate drought screens, the five individual T2events containing this cDNA (SR01002) were screened on high PEG, mannitol, and ABA to identify events with the resistance phenotype. Superpools (SP) are referred to as SP1, SP2 and so on. The letter following the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA) and the number following the letter refers to a number assigned to each plant obtained from that screen on that superpool. For example, SP1-M18 is the 18thplant isolated from a mannitol screen of Superpool 1. Qualitative and Quantitative Analysis of 2 Independent Events Representing 35S::Clone 104691 (SR01002) on PEG, Mannitol and ABA To identify two independent events of 35S::clone 104691 showing PEG, mannitol and ABA resistance, 36 seedlings from each of three events, SR01002-01, 02, and 03 were screened as previously described. Simultaneously, Basta segregation was assessed to identify lines containing a single insert segregating in a 3:1 (R:S) ratio as calculated by a chi-square test (Table 1). Two lines (01 and 03) segregated for a single insert. TABLE 11-1Basta segregation for SR01002 individual eventsProbabilityEventResistantSensitiveTotalof Chi-testSR01002-012610360.70031SR01002-02238310.91741SR01002-03288360.70031SR01002-01-1360360.00053SR01002-02-1288360.70031SR01002-03-12511360.44142*Chi-test to determine whether actual ratio of resistant to sensitive differs from the expected 3:1 ratio. Testing of the progeny from the resistant T2plants on the 3 surrogate drought screens showed that lines SR01002-01 and 03 had a strong and consistent resistance to PEG, mannitol, but not to ABA. These were chosen as the two events for further analysis. The controls were sown the same day and in the same plate as the individual lines. The PEG (Tables 11-2 and 11-3), and mannitol (Tables 11-4 and 11-5) segregation ratios observed for SR01002-01 and 03 are consistent with the presence of a single insert as demonstrated by chi-square. This is similar to that observed for Basta resistance (Table 11-1). TABLE 11-2Chi-square analysis assuming a 3:1 (R:S) ratio for progenyof SR01092-01 T2containing 35S::clone 104691 on PEG.EventObservedExpectedχ2ProbabilityPEG Resistant2224.750.3060.269PEG Sensitive118.250.91733331.222 TABLE 11-3Chi-square assuming analysis a 3:1 (R:S) ratio for progenyof SR01002-03 T2containing 35S::clone 104691 on PEG.EventObservedExpectedχ2ProbabilityPEG Resistant2425.50.0880.552PEG Sensitive108.50.26534340.353 TABLE 11-4Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01002-01 T2containing 35S::clone 104691 on mannitol.EventObservedExpectedχ2ProbabilityMannitol Resistant30270.3330.248Mannitol Sensitive691.00036361.333 TABLE 11-5Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01002-03 T2containing 35S::clone 104691 on mannitol.EventObservedExpectedχ2ProbabilityMannitol Resistant32270.9260.054Mannitol Sensitive492.7836363.70 Qualitative and Quantitative Analysis of Progeny of T2Plants Isolated on PEG, Mannitol, and ABA Screens. The progeny from one resistant T2plant from each of these two events was tested in the T3 generation in the same manner. Resistance to PEG and mannitol persisted into the next generation. Taken together, 1) the isolation of resistant seedlings containing clone 104691 from two of the surrogate screens for drought (PEG and mannitol), 2) the inheritance of this resistance in the next generation, and 3) the fact that the progeny from two or more events from the original transformation also segregated for resistance to these surrogate drought conditions, these findings provide strong evidence that clone 104691 when over-expressed can provide tolerance to osmotic stresses. FIG. 9 provides the results of the consensus sequence (SEQ ID NOs: 241-262) analysis based on Ceres cDNA 13509011 (12357529). The invention being thus described, it will be apparent to one of ordinary skill in the art that various modifications of the materials and methods for practicing the invention can be made. Such modifications are to be considered within the scope of the invention as defined by the following claims. Each of the references from the patent and periodical literature cited herein is hereby expressly incorporated in its entirety by such citation. | 129,419 |
11859196 | The following is a list of sequences appearing in this document: SEQ ID NO: 1 is a CDS of the At4g29760 gene fromArabidopsis;ATGGCTGAGCGATTATTACAATCTATGTCAAGGGTGGCTGGCCGATGTCATCCAGATTGCGTAAAAGCAAGTGATGAGCAAGAAGATTACCATGCATCTCAAAATGCAGCTTTGGTAGCTCTCAATCTGATTAGCTCTGCAACGTTAATACTGAAACTCCACGCTGAGTTTACTGAGTACTCAGCTCAGTTTTTGATGGACAATGCTGGAAAGGAAGACGACCCGGGAGAAGTGGATCAACAACGCAATCAGGTCACGACCGAAAACTGCCTTCGCTACTTGGCCGAAAACGTTTGGACCAAGAAGGAAAATGGGCAGGGAGGAATGGATCAACAACGCCCTGTGCTCACTGTCAAAGACTGCTTGGAACTTGCTTTTAAAAAAGGGCTGCCGAGAAGAGAACACTGGGCACATTTGGGATGTACCTTCAAGGCTCCCCCATTTGCTTGTCAGATACCTCGCGTTCCTGTGAAAGGAGAAGTGGTTGAGGTTAAGACTTTTGATGAAGCATTCAAGCTGTTGGTGCATCAACCCATTGGAGCAAAACTGCATTTGTTCAGTCCGCAGATTGATAATGTTGGAGAGGGAGTTTACAAAGGCCTCACGACAGGTAATGAAACACACTATGTTGGACTTAGAGATGTGCTAATAGCTTCAGTGGAGGAGTTCGAGGGAGATTCTGTTGCTATTGTGAAGATCTGCTACAAGAAGAAGCTTTCATTTATCAAAGTGTCTTTGAGCGTTAGGTTTCTCTCAGTAGCACATGATGGTGATAAGTCTAAGTTCATAGCGCCAACAGGTCTGCTTGTTGACTTCTGTGTCCCGCGCTTATCTATCAACTAASEQ ID NO: 2 is a CDS of the At4g29770 gene fromArabidopsis;ATGATGGCAATCTCAGAAAAAGGAGTCATGGCAATCTCAGAAAAAGGAGTCATGGCAACGAAAATTGACAAAAACGGCGTCCTTCGAGAGTTAAGGCGACATTTCACTGAGTTTTCTCTACGCGACGTAGATCTGTGTCTCCGGAGTTCATCGCAGATGGAGTCATTGTTAGAATGTTTTGCAATCACGGATGGCAAATGTCATCCCGATTGCTTAAAAGCAAACAATGAGCAAGAAGATTACGATGCATGTCAATCTGCAGCTTTGGTAGCTGTGAGTTTGATTAGCTCTGCACGTGTTATCTTCAAGATCGACTCTAAGTATACTGAGTACTCACCTCAGTATTTGGTGGATAACGTTGGGAAGGAAGAAGTTGAGGGAGAAATGGATCAACCAAGCTGTCAGTACACTGTCGGAAACCTCCTTAGTTACTTGGTGGAAAACGTTTGGACCAAGAAGGAAGTTAGGCAGAGAGAAATGGATCAACAACGCCGTGAGTTCACTGTCAAAGACTGCTTTGAATTTGCTTTTAAAAAAGGGCTTCCAAGAAATGGACATTGGGCGCATGTGGGATGTATATTCCCGGTTCCTCCATTTGCTTGTCAAATACCTCGCGTTCCCATGAAAGGAGAAGTGATTGAGGCTGCAAATGTGAGTGAAGCGTTGAAGCTGGGTATGCAACAACCAGCGGCAGCAAGGCTGCATTTGTTCAGTCCAGAGTTTGATCTTGTTGGAGAGGGTATTTACGATGGCCCGTCAGGTAATGAAACACGATATGTTGGACTTAGAGATGTGCTCATGGTTGAGGCGGAGAAGATCAAGGGAGAAACTGTTTTTACTGTGCAGATATGCTACAAGAAGAAGACTTCATTTGTCAAAGTGTCTACGAGAAGTATGATTCTCCCGCTTAATGGTGACGACGAGTCTCAGGTCACAGAGCCAGCATGTCTACTTGTTGACTTCTGTATCCCACGTTTTTCTATCAACTAASEQ ID NO: 3 is a CDS of the At5g18065 gene fromArabidopsis;ATGGATATGAATCAGCTATTCATGCAATCTATTGCAAACAGTCGTGGACTCTGTCATCCAGATTGCGAAAAAGCAAATAATGAGCGTGAAGATTATGATGCGTCTCAACATGCCGCTATGGTAGCGGTGAATCTGATTAGCTCTGCACGGGTTATCCTCAAGCTTGATGCTGTGTATACTGAGTACTCAGCTCAGTATTTGGTGGATAATGCTGGGAAGGAAGACAACCAGGGAGAAATGGATCAACAAAGCTCTCAGCTCACTCTCCAAAACTTGCTTCAGTATATGGATGAAAATGTCTGGAATAAGAAGGAAGATGTGCAGGGAGAAAGGGAGCAACCACTCACTGTCAAAGACTGCCTTGAATGTGCTTTCAAGTAASEQ ID NO: 4 is a CDS of the At5g18040 gene fromArabidopsis;ATGAATATGATTCAGCGATTCATGCAATCTATGGCAAAGACGCGTGGCCTCTGTCATCCAGATTGCGTAAAAGCAAGTAGTGAGCAAGAAGATTACGATGCGTCTCAGCTCAGTATTTGGTGGATAATGCTGGGAAGGAAGACGACCAGGGAGAAATGGATGAACCAAGCTCTCAGTTCACTATCGAAAACTTGCATCAGTATATGGTGGAAAATGTCTGGAATAAGAGGTAAGATGTGCAGGGAGAGGGAGCAACCACTCACTGTCAAAGACTGCCTTGAATGTGCTTTCAAGAAAGGGCTACCGAGAAGAGAACATTGGGCACATGTGGGATGTACATTCAAGGCTCCCCCATTTGCTTGTCACATACCCCGCGTGCCCATGAAAGGAGAAGTGATTGAGACTAAGAGTTTGGATGAAGCGTTTAAGCTGTTGATTAAACAACCGGTGGGTGCAAGACTCCATGTGTTCAGTCCAGACCTTGATAATOTTGGAGAGGGAGTTTACGAGGGCCTGTCTAGCCTGTCTCGTAAGGAATCACGCTATGTTGGACTTAGGGATGTCATCATAGTTGCAGTGAATAAGTCCGAGGGAAAAACTGTTGCTACTGTGAAGATATGTTACAAGAAGAAGACTTCATTTGTCAAAGTGTGTTTGAGCCGTATGTTTGTCCAGCTTGGTGGTGGCGAGGAGTCTCAGGTGAAAGAGCCAACAGGTCTGCTTGTTGACTTCTGTATCCCACGCTTATCTATCAACTAASEQ ID NO: 5 is a CDS of the Atl g51670 gene fromArabidopsis;ATGGCACTCCCTCCCTATGATCCGAATTTCACATTGGCTTTTTCATACGGTAGACGCGATAATGTCTTTGAGAATGACCCAGAGCACGATGAATCTGCTTCTGCTGCTATCGTAGCGGTTGAGCTGATAAGCTCTGCACGGCTTGCACTTAAGCTGGATAGTGTCCGCACTGAGTACTCAGCTCAGTATTTGGTGGACAAAGCTGGCTCACGCAACCTCAGGCGCAGGCGCAAGCTCACTGTCAAGGACTGCCTTAACTTTGCGTTAAAGAAAGGCGGCATACCGAGAGCAGAAGATTGGCCACCTTTGGGATCTGAGTCAAAGACCCCATCATCGTACGAACCTGCTCTCGTTTCCATGAAAGGAGAAGTGATTGAGCCTAAGGATATGGACGAAGTACCTGAGTTOTTGGTGCATCAATCAGCCGTGGGAGCAAAACTGCATGTGTTCACTCCACACATTGAACTTCAACAAGACGCAATTTACTTGCCTCGTCAGGTGAGTATGCGCGCTACGTTGGACTTAGAGATGGGATAGSEQ ID NO: 6 is a consensus sequence of Kanghan conserved domain B (100% consensus)hTVKDChphAhpSEQ ID NO: 7 is a consensus sequence of Kanghan conserved domain B (80% consensus)LTVKDCLEhAhKXG (where X is Lys or absent)SEQ ID NO: 8 is a consensus sequence of Kanghan conserved domain B (70% consensus)LTVKDCLEhAFKKGSEQ ID NO: 9 is a consensus sequence of Kanghan conserved domain C (80% consensus)VshKGpVlEstshpEsXchhhpQs-huA + LHlFpPph (where X is any amino acid)SEQ ID NO: 10 is a consensus sequence of Kanghan conserved domain C (70% consensus)VsMKGEVIEspsh_EAhcLllcQPlGA + LHlFoPclSEQ ID NO: 11 is a consensus sequence of Kanghan conserved domain A (80% consensus)cppDYDtStpAAhVAlpLISSARlhLKlDuhhTEYSsQaLhDpsutppSEQ ID NO: 12 is a consensus sequence of Kanghan conserved domain A (70% consensus)spphhpShupscChCHPDCXKAssEpEDYDASQpAAhVAVsLISSARlhLKLDusaTEYSAQYLVDNAGpccs (where X is any amino acid or absent)SEQ ID NO: 13 is a CDS of the At1g48180 gene fromArabidopsis:ATGGCACTCCCACCCTATGATCCCAATTTCAAATTTGCATTCTCTCTTGGCACGATTGCGAAACACCAAGATTACGATGAATCTGCTTCTGCTGCTGTTGTAGCGCTTGATCTGATAAGCTCTGCACGGTTTGCACTTAAGCTGGATAGTGTCTATACTGAGTACTCTGCTAAGTATGTGGTGGACAATGCTGCTGGCTCACACAGTGGGCGCAAGCTCACTGTCAAAGACTGTCTTGAGTTTGCCTTAAACAAAGGCGGCATACCGAAAGCAGAAGATTGGCCACGCTTGGGATCTGTGATAACGCCCCCATCATCGTATAAACCTGATCTCGTTTCGATGAAAGGACAAGTGATTGAGCCTCAGACTATTGAGGAAGCATGTGACATGGTGGTGGATCAACCAGTAGGAGCAAAATTGCATGTGTTCAAGCCACACATTGAACTTCAACAAGACGCAAGTGCTATAACTGGCATTTACTCTGCCACCTCAGGTGAGCCACCCACCTATCTCCGACTTACAGATCCCATCATCGTTGGAGTCGAGAAGATCCAAGGGAAGTCTATTGGAACTGTGAAGGTATGGTACAAGAAGTTCATATTTCTGAAAGTGGCTATGAGCAGGTGGTTTCAGTTATACTCTCCGGATGGCACACACACGGGCATAAAGCGAACAGATTACCTTGTTGATTTTTGTGTCCCACGCCTATCCATCGATTAASEQ ID NO: 14 is the polypeptide encoded by SEQ ID NO: 1MAERLLQSMSRVAGRCHPDCVKASDEQEDYHASQNAALVAVNLISSARLILKLDAEFTEYSAQFLMDNAGKEDDPGEVDQQRNQVTTENCLRYLAENVWTKKENGQGGMDQQRPVLTVKDCLELAFKKGLPRREHWAHLGCTFKAPPFACQIPRVPVKGEVVEVKTFDEAFKLLVHQP1GAKLHLFSPQIDNVGEGVYKGLTTGNETHYVGLRDVLIASVEEFEGDSVAIVKICYKKKLSFIKVSLSVRFLSVAHDGDKSKFIAPTGLLVDFCVPRLSINSEQ ID NO: 15 is the polypeptide encoded by SEQ ID NO: 2MMAISEKGVMAISEKGVMATKIDKNGVLRELRRHETEFSLRDVDLCLRSSSQMESLLECFATTDGKCHPDCLKANNEQEDYDACQSAALVAVSLISSARVIFKIDSKYTEYSPQYLVDNVGKEEVEGEMDQPSCQYTVGNLLSYLVENVWTKKEVRQREMDQQRREFTVKDCFEFAFKKGLPRNGHWAHVGCIFPVPPFACQIPRVPMKGEVIEAANVSEALKLGMQQPAAARLHLFSPEFDLVGEGIYDGPSGNETRYVGLRDVLMVEAEKIKGETVETVQICYKKKTSFVKVSTRSMILPLNGDDESQVTEPACLLVDFCIPRESINSEQ ID NO: 16 is the polypeptide encoded by SEQ ID NO: 3MDMNQLFMQSIANSRGLCHPDCEKANNEREDYDASQHAAMVAVNLISSARVILKLDAVYTEYSAQYLVDNAGKEDNQGEMDQQSSQLTLQNLLQYMDENVWNKKEDVQGEREQPLTVKDCLECAFKSEQ ID NO: 17 is the polypeptide encoded by SEQ ID NO: 4MNMIQRFMQSMAKTRGLCHPDCVKASSEQEDYDASQLSIWWIMLGRKTTREKWMNQALSSLSKTCISIWWKMSGIRGKMCREREQPLTVKDCLECAFKKGLPRREHWAHVGCTFKAPPEACHIPRVPMKGEVIETKSLDEAFKLLIKQPVGARLHVFSPDLDNVGEGVYEGLSSLSRKESRYVGLRDVIIVAVNKSEGKTVATVKICYKKKTSFVKVCLSRMFVQLGGGEESQVKEPTGLLVDFCIPRLSINSEQ ID NO: 18 is the polypeptide encoded by SEQ ID NO: 5MALPPYDPNFTLAFSYGRRDNVFENDPEHDESASAAIVAVELISSARLALKLDSVRTEYSAQYLVDKAGSRNLRRRRKLTVKDCLNFALKKGGIPRAEDWPPLGSESKTPSSYEPALVSMKGEVIEPKDMDEVPELLVHQSAVGAKLHVFTPHIELQQDAIYLPRQVSMRATLDLEMGSEQ ID NO: 19 is the polypeptide encoded by SEQ ID NO: 13MALPPYDPNFKFAFSLGTIAKHQDYDESASAAVVALDLISSARFALKLDSVYTEYSAKYVVDNAAGSHSGRKLTVKDCLEFALNKGGIPKAEDWPRLGSVITPPSSYKPDLVSMKGQVIEPQTIEEACDMVVDQPVGAKLHVFKPHIELQQDASAITGIYCGTSGEPASYVGLRDAIIVGVEKIQGKSIGTVKVWYKKFIFLKVAMSRWFQLYSPDGTHTGIKRTDYLVDFCVPRLSMDSEQ ID NOs: 20 and 21 are a primer pair designed to target BnaCO3g77540D(LOC106364365)TAGATTCTGCTGAGAGAGCCGCTAC (SEQ ID NO: 20)GGATCCGTCGACGCACCTATGGGTCCATGCTTTAAC (SEQ ID NO: 21)SEQ ID NOs: 22 and 23 are a primer pair designed to target BnaA08g12920D(LOC106424160)TCATCCAGATTGCCAACGAG (SEQ ID NO: 22)GGATCCGTCGACACGCATCCTCCAGTGTCTTAG (SEQ ID NO: 23)SEQ ID NOs: 24 and 25 are a primer pair designed to target hygromycinTACACAGCCATCGGTCCAGA (SEQ ID NO: 24)GTAGGAGGGCGTGGATATGTC (SEQ ID NO: 25)SEQ ID NOs: 26 and 27 are a primer pair designed to target BnaA07g02270DCGCTACGAGGCACGTACTCAAT (SEQ ID NO: 26)CTCGGTCTTCCCCGGTTTC (SEQ ID NO: 27)SEQ ID NOs: 28 and 29 are a primer pair designed to target BnaA08g12920DGCTTAGAGACGTGATCCTGGTAGC (SEQ ID NO: 28)CCAGTGTGGTGAACATACGGC (SEQ ID NO: 29)SEQ ID NOs: 30 and 31 are a primer pair designed to target BnaA01g07670DGTTTTGTTGGTCTCTTCTCTTTGC (SEQ ID NO: 30)TTCTTAAGAGGCGTTTCAGATGG (SEQ ID NO: 31)SEQ ID NOs: 32 and 33 are a primer pair designed to target BnaC03g77540DTGATTTGGGTTTTGCCTGATAC (SEQ ID NO: 32)GAAACAAACCATAAATGAGTTGCC (SEQ ID NO: 33)SEQ ID NOs: 34 and 35 are a primer pair designed to target BnaC03g77550DCATTTGGGATGTGTCGATTGAG (SEQ ID NO: 34)CCCACGTAGCTTGTTCCGTT (SEQ ID NO: 35)SEQ ID NOs: 36 and 37 are a primer pair designed to target BnaA01g06470DAACACTGTCACGCAGATTGCC (SEQ ID NO: 36)CTGTCCAGGTTAGCTACCATACGA (SEQ ID NO: 37)SEQ ID NOs: 38 and 39 are a primer pair designed to target BnaC01g08490DCGGTATCCAACTCATTCGAAGG (SEQ ID NO: 38)TCAAGTATATACTGGGTTGGCTGC (SEQ ID NO: 39)SEQ ID NOs: 40 to 171 are detailed in the sequence listing. DETAILED DESCRIPTION In the following detailed description, various non-limiting examples are set out of particular embodiments, together with experimental procedures that may be used to implement a wide variety of modifications and variations in the practice of the present invention. For clarity, a variety of technical terms are used herein in accordance with what is understood to be the commonly understood meaning, as reflected in definitions set out below. The term “line” refers to a group of plants that displays very little overall variation among individuals sharing that designation. A “line” generally refers to a group of plants that display little or no genetic variation between individuals for at least one trait. Plants within a group of plants that display little or no genetic variation between individuals may also be referred to as having the same genetic background. A “variety” or “cultivar” includes a line that is used for commercial production. In some aspects,Brassicavarieties may for example be derived from “doubled haploid” (DH) lines, which refers to a line created by the process of microspore embryogenesis, in which a plant is created from an individual microspore. By this process, lines are created that are homogeneous, i.e. all plants within the line have the same genetic makeup. The original DH plant is referred to as DH1, while subsequent generations are referred to as DH2, DH3 etc. Doubled haploid procedures are well known and have been established for several crops. A procedure forB. junceahas been described by Thiagrarajah and Stringham (1993). New lines, varieties or plants may be produced by introducing a heritable change in a parent plant. In this context, a “heritable change” is any molecular alteration, typically a genetic change, that is capable of being passed from one generation of plant to the next. This term is intended to include molecular alterations such as, but not limited to, insertions, deletions, point mutations, frame-shift mutations, inversions, rearrangements, and the introduction of transgenes. There is a wide variety of techniques available for introducing heritable changes to plants and plant cells. Plant “mutagenesis” in the present context is a process in which an agent known to cause alterations in genetic material is applied to plant material, for example the mutagenic agent ethyl methylsulfonate (EMS). A range of molecular techniques such as recombination with foreign or heterologous nucleic acid fragments or gene editing may also be used for mutagenesis. All such methods of introducing nucleic acid sequence changes are included within the term “mutagenesis” as used herein. Plant “regeneration” involves the selection of cells capable of regeneration (e.g. seeds, microspores, ovules, pollen, vegetative parts) from a selected plant or variety. These cells may optionally be subjected to mutagenesis, following which a plant is developed from the cells using regeneration, fertilization, and/or growing techniques based on the types of cells mutagenized. Applicable regeneration techniques are known to those skilled in the art; see, for example, Armstrong et al. (1985); and Close et al. (1987). “Improved characteristics” of a plant means that the characteristics in question are altered in a way that is desirable or beneficial or both in comparison with a reference value or attribute, which in the absence of an express comparator relates to the equivalent characteristic of a wild type strain. Plant “progeny” means the direct and indirect descendants, offspring and derivatives of a plant or plants and includes the first, second, third and subsequent generations and may be produced by self-crossing, crossing with plants with the same or different genotypes, and may be modified by range of suitable genetic engineering techniques. Plant “breeding” includes all methods of developing or propagating plants and includes both intra and inter species and intra and inter line crosses as well as all suitable artificial breeding techniques. Desired traits may be transferred to other lines through conventional breeding methods and can also be transferred to other species through inter-specific crossing. Both conventional breeding methods and inter-specific crossing methods as well as all other methods of transferring genetic material between plants are included within the concept of “breeding”. “Molecular biological techniques” means all forms of anthropomorphic manipulation of a biological molecules, such as nucleic acid sequences, for example to alter the sequence and expression thereof and includes the insertion, deletion, modification or editing of sequences or sequence fragments and the direct or indirect introduction of new sequences into the genome of an organism, for example by directed or random recombination using suitable vectors and/or techniques. “Marker-assisted selection” (MAS) refers to the use of molecular markers to assist in phenotypic selection in the context of plant breeding. A wide variety of molecular markers, such as single nucleotide polymorphisms (SNPs), may for example be used in MAS plant breeding, including the application of next-generation sequencing (NGS) technologies. The term “genetically derived” as used for example in the phrase “an improved characteristic genetically derived from the parent plant or cell” means that the characteristic in question is dictated wholly or in part by an aspect of the genetic makeup of the parent plant or cell, applying for example to progeny of the parent plant or cell that retain the improved characteristic of the parent plant or cell. Various genes and nucleic acid sequences of the invention may be recombinant sequences. The term “recombinant” means that something has been recombined, so that when made in reference to a nucleic acid construct the term refers to a molecule that is comprised of nucleic acid sequences that are joined together or produced by means of molecular biological techniques. Nucleic acid “constructs” are accordingly recombinant nucleic acids, which have been generally been made by aggregating interoperable component sequencers. The term “recombinant” when made in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques. The term “recombinant” when made in reference to the genetic composition or an organism or cell refers to a gamete or progeny with new combinations of alleles that did not occur in the parental genomes. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Referring to a nucleic acid construct as ‘recombinant’ therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and/or repair events. Recombinant constructs of the invention may include a variety of functional molecular or genomic components, as required for example to mediate gene expression or suppression in a transformed plant. In this context, “DNA regulatory sequences,” “control elements,” and “regulatory elements,” refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, and protein degradation signals that regulate gene expression. In the context of the present disclosure, “promoter” means a sequence sufficient to direct transcription of a gene when the promoter is operably linked to the gene. The promoter is accordingly the portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not universally, located in the 5′ non-coding regions of a gene. A promoter and a gene are “operably linked” when such sequences are functionally connected so as to permit gene expression mediated by the promoter. The term “operably linked” accordingly indicates that DNA segments are arranged so that they function in concert for their intended purposes, such as initiating transcription in the promoter to proceed through the coding segment of a gene to a terminator portion of the gene. Gene expression may occur in some instances when appropriate molecules (such as transcriptional activator proteins) are bound to the promoter. Expression is the process of conversion of the information of a coding sequence of a gene into mRNA by transcription and subsequently into polypeptide (protein) by translation, as a result of which the protein is said to be expressed. As the term is used herein, a gene or nucleic acid is “expressible” if it is capable of expression under appropriate conditions in a particular host cell. Promoters may for example be used that provide for preferential gene expression within a specific organ or tissue, or during a specific period of development. For example, promoters may be used that are specific for leaf (Dunsmuir et al., 1983), root tips (Pokalsky et al., 1989), fruit (Peat et al., 1989; U.S. Pat. No. 4,943,674 issued 24 Jul. 1990; International Patent Publication WO-A 8 809 334; U.S. Pat. No. 5,175,095 issued 29 Dec. 1992; European Patent Application EP-A 0 409 629; and European Patent Application EP-A 0 409 625) embryogenesis (U.S. Pat. No. 5,723,765 issued 3 Mar. 1998 to Oliver et al.), or young flowers (Nilsson et al. 1998). Promoters demonstrating preferential transcriptional activity in plant tissues are, for example, described in European Patent Application EP-A 0 255 378 and International Patent Publication WO-A 9 113 980. Promoters may be identified from genes which have a differential pattern of expression in a specific tissue by screening a tissue of interest, for example, using methods described in U.S. Pat. No. 4,943,674 and European Patent Application EP-A 0255378. The disclosure herein includes examples of this embodiment, showing that plant tissues and organs can be modified by transgenic expression of a Kanghan gene. An “isolated” nucleic acid or polynucleotide as used herein refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring). An isolated nucleic acid or polypeptide may contain less than about 50%, less than about 75%, less than about 90%, less than about 99.9% or less than any integer value between 50 and 99.9% of the cellular or biological components with which it was originally associated. A polynucleotide amplified using PCR so that it is sufficiently distinguishable (on a gel for example) from the rest of the cellular components is, for example, thereby “isolated”. The polynucleotides of the invention may be “substantially pure,” i.e., having the high degree of isolation as achieved using a purification technique. In the context of biological molecules “endogenous” refers to a molecule such as a nucleic acid that is naturally found in and/or produced by a given organism or cell. An “endogenous” molecule may also be referred to as a “native” molecule. Conversely, in the context of biological molecules “exogenous” refers to a molecule, such as a nucleic acid, that is not normally or naturally found in and/or produced by a given organism or cell in nature. As used herein to describe nucleic acid or amino acid sequences the term “heterologous” refers to molecules or portions of molecules, such as DNA sequences, that are artificially introduced into a particular host cell, for example by transformation. Heterologous DNA sequences may for example be introduced into a host cell by transformation. Such heterologous molecules may include sequences derived from the host cell. Heterologous DNA sequences may become integrated into the host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination events. Transformation techniques that may be employed include plant cell membrane disruption by electroporation, microinjection and polyethylene glycol based transformation (such as are disclosed in Paszkowski et al. (1984); Fromm et al. (1985); Rogers et al. (1986); and in U.S. Pat. Nos. 4,684,611; 4,801,540; 4,743,548 and 5,231,019), biolistic transformation such as DNA particle bombardment (for example as disclosed in Klein et al. (1987); Gordon-Kamm, et al. (1990); and in U.S. Pat. Nos. 4,945,050; 5,015,580; 5,149,655 and 5,466,587);Agrobacterium-mediated transformation methods (such as those disclosed in Horsch et al. (1984); Fraley et al. (1983); and U.S. Pat. Nos. 4,940,838 and 5,464,763). Transformation systems adapted for use inCamelina sativaare for example described in US Patent Publication 20140223607. Varieties ofCamelina sativaare for example described in US Patent Publication 20120124693, and the subject of seed samples deposited under ATCC Accession No. PTA-11480. Aspects of the present invention involve altering known plant varieties, such asCamelina sativa, to alter endogenous Kanghan genes. Transformed plant cells may be cultured to regenerate whole plants having the transformed genotype and displaying a desired phenotype, as for example modified by the expression of a heterologous Kanghan gene during growth or development. A variety of plant culture techniques may be used to regenerate whole plants, such as are described in Gamborg et al. (1995); Evans et al. (1983); Binding (1985); Klee et al. (1987). Various aspects of the present disclosure encompass nucleic acid or amino acid sequences that are homologous to other sequences. As the term is used herein, an amino acid or nucleic acid sequence is “homologous” to another sequence if the two sequences are substantially identical, as defined herein, and the functional activity of the sequences is conserved (as used herein, sequence conservation or identity does not infer evolutionary relatedness). Nucleic acid sequences may also be homologous if they encode substantially identical amino acid sequences, even if the nucleic acid sequences are not themselves substantially identical, for example as a result of the degeneracy of the genetic code. With reference to biological sequences “substantial homology” or “substantial identity” is meant, in the alternative, a sequence identity of greater than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% up to 100% sequence identity. Homology may refer to nucleic acid or amino acid sequences as the context dictates. In alternative embodiments, sequence identity may for example be at least 75%, at least 90% or at least 95%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the search for similarity method of Pearson and Lipman (1988), and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al. (1990) (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (NCBI) at their Internet site. The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff et al., 1992) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, less than about 0.1, less than about 0.01, or less than about 0.001. An alternative indication that two amino acid sequences are substantially identical is that one peptide is specifically immunologically reactive with antibodies that are also specifically immunoreactive against the other peptide. Antibodies are specifically immunoreactive to a peptide if the antibodies bind preferentially to the peptide and do not bind in a significant amount to other proteins present in the sample, so that the preferential binding of the antibody to the peptide is detectable in an immunoassay and distinguishable from non-specific binding to other peptides. Specific immunoreactivity of antibodies to peptides may be assessed using a variety of immunoassay formats, such as solid-phase ELISA immunoassays for selecting monoclonal antibodies specifically immunoreactive with a protein (see Harlow et al., 1988). An alternative indication that two nucleic acid sequences are substantially identical is that the two sequences hybridize to each other under moderately stringent, or stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel, et al. (eds), 1989). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds), 1989). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. The term “a polynucleotide that hybridizes under stringent (low, intermediate) conditions” is intended to encompass both single and double-stranded polynucleotides although only one strand will hybridize to the complementary strand of another polynucleotide. Washing in the specified solutions may be conducted for a range of times from several minutes to several days and those skilled in the art will readily select appropriate wash times to discriminate between different levels of homology in bound sequences. In alternative embodiments, the invention provides nucleic acids, such as isolated or recombinant nucleic acid molecules, comprising the sequence of a Kanghan allele of the invention. Isolated nucleic acids of the invention may include coding sequences of the invention recombined with other sequences, such as cloning vector sequences. Homology to sequences of the invention may be detectable by hybridization with appropriate nucleic acid probes, by PCR techniques with suitable primers or by other techniques. In particular embodiments there are provided nucleic acid probes which may comprise sequences homologous to portions of the alleles of the invention. Further embodiments may involve the use of suitable primer pairs to amplify or detect the presence of a sequence of the invention, for example a sequence that is associated with an abiotic stress response, such as drought or heat resistance. In alternative embodiments, the invention provides methods for identifying plants, such asCamelina, BrassicaorTriticumplants, with a desirable abiotic stress response, such as drought tolerance and/or heat resistance, or a desired genomic characteristic. Methods of the invention may for example involve determining the presence in a genome of particular Kanghan alleles. In particular embodiments the methods may comprise identifying the presence of: a nucleic acid polymorphism associated with one of the identified alleles; or an antigenic determinant associated with one of the alleles. Such a determination may for example be achieved with a range of techniques, such as PCR amplification of the relevant DNA fragment, DNA fingerprinting, RNA fingerprinting, gel blotting and RFLP analysis, nuclease protection assays, sequencing of the relevant nucleic acid fragment, the generation of antibodies (monoclonal or polyclonal), or alternative methods adapted to distinguish the protein produced by the relevant alleles from other variants or wild type forms of that protein. In selected embodiments, a specific base pair change in a Kanghan allele may for example be used to design protocols for MAS, such as the use of allele-specific probes, markers or PCR primers. For an exemplary summary of allele-specific PCR protocols, see Myakishev et al. (2001) or Tanhuanpaa et al. (1999). In alternative embodiments, for example, various methods for detecting single nucleotide polymorphisms (SNPs) may be used for identifying Kanghan alleles of the invention. Such methods may for example include TaqMan assays or Molecular Beacon assays (Tapp et al., 2000), Invader Assays (Mein et al., 2000) or assays based on single strand conformational polymorphisms (SSCP) (Orita et al., 1989). In alternative embodiments, the invention provides progeny of parent plant lines having altered endogenous or heterologous Kanghan genes, for example progeny ofCamelina sativaparent line which is the subject of ATCC Accession number PTA-11480. Such progeny may for example be selected to have a desired alteration in an abiotic stress response compared to the parent strain, such as improved drought resistance or heat tolerance. In alternative embodiments, a plant seed is provided, such as anArabidopsis, Camelina, TriticumorBrassicaseed. In alternative embodiments, genetically stable plants are provided, such as plants of the genusArabidopsis, Camelina, TriticumorBrassica. In further alternative embodiments the invention provides processes of producing genetically stable plants, such asArabidopsis, Camelina, TriticumorBrassicaplants, for example plants having a desired alteration in an abiotic stress response compared to a reference strain that does not have a particular alteration in a Kanghan gene, such as improved drought resistance or heat tolerance. In various aspects, the invention involves the modulation of the number of copies of an expressible Kanghan coding sequence in a plant genome. By “expressible” it is meant that the primary structure, i.e. sequence, of the coding sequence indicates that the sequence encodes an active protein. Expressible coding sequences may nevertheless not be expressed as an active protein in a particular cell, for example due to gene silencing. This ‘gene silencing’ may for example take place by various mechanisms of homologous transgene inactivation or epigenetic silencing in vivo. Homologous transgene inactivation and epigenetic silencing in transgenic plants has been described in plants where a transgene has been inserted in the sense orientation, with the result that both the gene and the transgene are down-regulated (Napoli et al., 1990; Rajeevkum et al., 2015). In the present invention, the expressible coding sequences in a genome may accordingly not all be expressed in a particular cell, and may in some embodiments result in suppression of Kanghan gene expression. In other aspects, reduction of Kanghan gene expression may include the reduction, including the suppression or elimination (aka knockout), of expression of a nucleic acid sequence that encodes a Kanghan protein, such as a nucleic acid sequence of the invention. By elimination of expression, it is meant herein that a functional amino acid sequence encoded by the nucleic acid sequence is not produced at a detectable level. By suppression of expression, it is meant herein that a functional polypeptide encoded by the nucleic acid sequence is produced at a reduced level relative to the wild type level of expression of the polypeptide. Reduction of Kanghan expression may include the elimination of transcription of a nucleic acid sequence that encodes a Kanghan protein, such as a sequence of the invention encoding a Kanghan protein. By elimination of transcription it is meant herein that the mRNA sequence encoded by the nucleic acid sequence is not transcribed at detectable levels. Reduction of Kanghan activity may also include the production of a truncated amino acid sequence from a nucleic acid sequence that encodes a Kanghan protein, meaning that the amino acid sequence encoded by the nucleic acid sequence is missing one or more amino acids of the functional amino acid sequence encoded by a wild type nucleic acid sequence. In addition, reduction of Kanghan activity may include the production of a variant Kanghan amino acid sequence, meaning that the amino acid sequence has one or more amino acids that are different from the amino acid sequence encoded by a wild type nucleic acid sequence. A variety of mutations may be introduced into a nucleic acid sequence for the purpose of reducing Kanghan activity, such as frame-shift mutations, introduction of premature stop codon(s), substitutions and deletions. For example, mutations in coding sequences may be made so as to introduce substitutions within functional motifs or conserved domains in a Kanghan protein, such as conserved Kanghan protein domains A, B or C. In an alternative aspect, the down-regulation of Kanghan genes may be used to alter a plant response to abiotic stress, for example to enhance drought tolerance. Such down-regulation may be tissue-specific. For example, anti-sense oligonucleotides may be expressed to down-regulate expression of Kanghan genes. The expression of such anti-sense constructs may be made to be tissue-specific by operably linking anti-sense encoding sequences to tissue-specific promoters. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, act to block the translation of mRNA by binding to targeted mRNA and inhibiting protein translation from the bound mRNA. For example, anti-sense oligonucleotides complementary to regions of a DNA sequence encoding a Kanghan protein may be expressed in transformed plant cells during development to down-regulate the expression of the Kanghan gene. Alternative methods of down-regulating Kanghan gene expression may include the use of ribozymes or other enzymatic RNA molecules (such as hammerhead RNA structures) that are capable of catalyzing the cleavage of RNA (as disclosed in U.S. Pat. Nos. 4,987,071 and 5,591,610). Aspects of the invention involve the use of gene editing to alter Kanghan gene sequences. For example, CRISPR-Cas system(s) (e.g., single or multiplexed) can be used to perform plant gene or genome interrogation or editing or manipulation. Kanghan genes may for example be edited for functional investigation and/or selection and/or interrogation and/or comparison and/or manipulation and/or transformation of plant Kanghan genes. This editing may be carried out so as to create, identify, develop, optimize, or confer trait(s) or characteristic(s) to plant(s) or to transform a plant genome, for example to alter an abiotic stress response in a plant, such as a drought or heat tolerance. Gene editing can in this way be used to provide improved production of plants, new plants with new combinations of traits or characteristics or new plants with enhanced traits. Such CRISPR-Cas system(s) can for example be used in Site-Directed Integration (SDI) or Gene Editing (GE) or any Near Reverse Breeding (NRB) or Reverse Breeding (RB) techniques (see the University of Arizona website “CRISPR-PLANT” http://www.genome.arizona.edu/crispr/). Embodiments of the invention can be used in genome editing in plants alone or in combination with other molecular biological techniques, such as RNAi or similar genome editing techniques (see, e.g., Nekrasov, 2013; Brooks, 2014; Shan, 2013; Feng, 2013; Xie, 2013; Xu, 2014; Caliando et al, 2015; U.S. Pat. Nos. 6,603,061; 7,868,149; US 2009/0100536; Morrell et al., 2011). Protocols for targeted plant genome editing via CRISPR/Cas9 are also available in Li et al, 2015. In some embodiments, the invention provides new Kanghan polypeptide sequences, which may be produced from wild type Kanghan proteins by a variety of molecular biological techniques. It is well known in the art that some modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide, to obtain a biologically equivalent polypeptide. As used herein, the term “conserved amino acid substitutions” refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without any appreciable loss or gain of function, to obtain a biologically equivalent polypeptide. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing. Conversely, as used herein, the term “non-conserved amino acid substitutions” refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution causes an appreciable loss or gain of function of the peptide, to obtain a polypeptide that is not biologically equivalent. In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following hydrophilicity values are assigned to amino acid residues (as detailed in U.S. Pat. No. 4,554,101): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Pro (−0.5); Thr (−0.4); Ala (−0.5); His (−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); Ile (−1.8); Tyr (−2.3); Phe (−2.5); and Trp (−3.4). Non-conserved amino acid substitutions may be made were the hydrophilicity value of the residues is significantly different, e.g. differing by more than 2.0. In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser (−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glu (−3.5); Gln (−3.5); Asp (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5). Non-conserved amino acid substitutions may be made were the hydropathic index of the residues is significantly different, e.g. differing by more than 2.0. In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr. Non-conserved amino acid substitutions may be made were the residues do not fall into the same class, for example substitution of a basic amino acid for a neutral or non-polar amino acid. Example 1:ArabidopsisKanghan Genes This Example illustrates that drought tolerance inArabidopsisis conferred by novel QTLs located on three different chromosomes. These genes were identified in an extremely drought tolerantArabidopsisecotype, designated herein as #95. The #95 ecotype was isolated during a series of drought treatment experiments, and assessed as follows. In one assay, 36 plants of ecotype Col and 36 plants of ecotype #95 were used for drought sensitivity testing. At the outset, soil for each pot was dried and weighed to ensure that each pot had the same amount of soil, after which water was added to maintain moisture. Seeds from Col and #95 were first germinated, then sown one seedling per pot separately. The plants were grown in a controlled environment under long-day conditions (16-h-light/8-h-dark cycle) at 23° C., light intensity of 50 gmol m−2s−1and 70% relative humidity (rH). Watering was stopped for both Col and #95 plants three weeks after germination, and all pots were then weighed again, and additional water was supplied to keep every pot at the same weight. Thereafter, drought treatment was initiated and survival days were recorded for both ecotypes. After a period of 15 days without watering, all 36 plants of ecotype Col had died. In contrast, the plants of ecotype #95 retained considerable vigor, and fully recovered to maturity when water supply was resumed. The extreme drought toleranceArabidopsisecotype #95 was particularly evident after withdrawing water for 38 days. Plants of the ecotype Col were all severely wilted due to drought. Ecotype #95, in contrast, still exhibited clear vigor. The F1 progeny between Col and #95 were also sensitive to drought, indicating the recessive nature of the #95 drought resistant trait. In one assay, 27 days after water was withdrawn, the plants were segregated into two groups, those that had died, and those that maintained vigor and were recoverable to full maturity when watering was resumed. In alternative drought tolerance tests of F2 progeny derived from a cross between Col and #95, segregation of F2 population plants after drought treatment (50 days after water withdrawal) was much lower than 3:1. This segregation is consistent with the involvement of major QTL in controlling the drought tolerance trait.FIG.1is graph illustrating the drought tolerance diversity of the F2 generation of theseArabidopsisplants (col×#95). Segregation of the drought tolerance trait from 500 F2 individual lines was calculated by the survival days after drought treatment (cessation of watering). InFIG.1, the survival in days of Col and #95 plants are marked by arrows with legends. The normal distribution for the phenotype of F2 drought tolerance indicates that several QTLs govern the drought tolerance trait. Map based cloning through crossing with ecotype Col, revealed that the drought-related trait was governed by three major QTLs distributed on three different chromosomes. To delineate the underlying genetic components, an F1 generation was developed from the seeds of a cross between Col and #95. The F1 seeds were then used to develop a large F2 population of 5000 lines. The F2 populations showed significant segregation of the drought tolerance trait, with some plants showing significant drought tolerance, and others showing no drought tolerance, which indicated that the drought tolerance trait of #95 was controlled by several QTLs. A fine mapping of the genes was further pursued using 500 lines of this population from which 20 extremely drought tolerant individuals and 20 extremely drought sensitive individuals were selected to conduct a Bulk Segregate Analysis (BSA) with 106 molecular markers which cover all 5 chromosomes ofArabidopsis. Based on this analysis, three major QTLs distributed on three different chromosomes were identified. Specifically, QTL's were identified on chromosomes 1, 4 and 5 of theArabidopsisgenome. The contribution rates of these 3 loci to the observed drought tolerance trait were 13.8%, 29.3%, 37.7%, respectively, explaining in the aggregate more than 80% of the drought tolerance variation between ecotype #95 and Col. Fine mapping was first focused on loci on Chr.4 and Chr.5, which was carried out using 700 extremely drought tolerant individuals from a total of 5000 F2 plants. The candidate genes were narrowed down to two regions of 540 kb on Chr.4 and 189 kb on Chr.5. Single nucleotide polymorphism (SNP) and insertion/deletion (In/del) analysis, as well as expression level analysis based on the TAIR database, was carried out for all of the genes identified in these two regions on Chr.4 and 5. The full genome sequence of ecotype #95 was compared with the full genome sequence ofArabidopsisecotype Columbia (ecotype Col. The three major QTL's associated with drought tolerance on Chr. 1, Chr. 4 and Chr. 5 of ecotype #95 were revealed to harbor members of a protein coding gene family: At1g51670 (also referred to as Kanghan3 or KH3), At4g29760 (also referred to as Kanghan4 or KH4), At4g29770 (also referred to as Kanghan2 or KH2), At5g18065 (also referred to as Kanghan5 or KH5) and At5g18040 (also referred to as Kanghan1 or KH1). An additional member of the gene family was recognized by sequence similarity: At1g48180 (also referred to as Kanghan6 or KH6). This gene family is designated herein as the Kanghan gene family, the first 5 of which have very strong roles in drought tolerance (a GenBank database accession number for a protein encoded by each of the nativeArabidopsisgenes is given after the gene name in brackets): Kanghan1 (At5g18040; NP_197305.1), Kanghan2 (At4g29770; NP 001154277.1), Kanghan3 (At1g51670; NP_175578.2), Kanghan4 (At4g29760; NP_194705.1), Kanghan5 (At5g18065; NP_680172.2), Kanghan6 (At1g48180; NP_175252.1). Analysis of the genomic sequence of Ecotype #95 reveals that mutations within Kanghan family genes are associated with drought tolerance. Specifically, in ecotype #95, all 5 members of the Kanghan family strongly associated with drought tolerance have dramatic mutations. Specifically, four members of the Kanghan gene family (At4g29770, At5g18065, At5g18040 and At1g51670) contain a premature stop codon (seeFIG.2), which is indicative of loss-of-function mutations (null) in ecotype #95 compared to the Col variety. A fifth member of the Kanghan gene family, At4g29760, does not contain a premature stop codon, but 5 amino acid substitutions occur in the coding region of this gene. Among the five Kanghan genes strongly associated with drought tolerance, At5g18040, At4g29770 and At1g51670 are much more highly expressed (over 10 times) in both Col and #95 compared to At5g18065 and At4g29760, suggesting that At5g18040, At4g29770 and At1g51670 may in some circumstances contribute more than the other two genes to drought tolerance trait. Example 2: Reversing Drought Resistance To further illustrate the role of the Kanghan genes in drought tolerance, two full length Kanghan genes (AT5g18040 and At4g29770) fromArabidopsisecotype Col were used to transformArabidopsisecotype #95, including at least 2 kb 5′UTR, 1 kb 3′UTR and CDS. The transformants lost their drought resistance, confirming that the modulation of Kanghan gene expression plays a dramatic role in drought resistance. A further illustration of the dramatic effect of Kanghan genes on drought tolerance was provided by introducing five Kanghan gene alleles from ecotype #95 into ecotype Columbia (Col) by crossing and molecular marker based selection, generation by generation. The 7thgeneration of backcrossed lines was used for self-crossing to provide homozygous plants which contained the five Kanghan gene alleles from #95 strongly associated with drought tolerance. These homozygous plants were subjected to drought treatment. The result was that introduction of the #95 Kanghan gene alleles rendered ecotype Columbia drastically enhanced in its drought tolerance traits. A further illustration of the effect of Kanghan genes on abiotic stress response was provided by measuring the canopy temperatures of Col, #95 and the backcrossed lines bearing the Kanghan alleles. Increased canopy temperature was clearly evident in #95 plants and backcrossed lines, when compared with Col ecotype plants. Further, subjecting seedlings of #95 and Col to heat treatment at 45° C. confirmed heat sensitivity in ecotype #95. As this Example illustrates, functional expression of Kanghan gene family proteins plays a positive role in heat tolerance, and a negative role in drought tolerance. The invention accordingly provides a variety of avenues for modulating abiotic stress response in plants. In some embodiments, this involves balancing Kanghan gene expression to achieve a desired phenotype of abiotic stress response, for example balancing drought and heat tolerance. The negative role of the Kanghan family of genes in drought tolerance serves as a basis for improving plant drought tolerance by down-regulating or silencing members of the Kanghan gene family. This may for example be achieved through a wide variety of techniques, including mutagenesis (TILLing) or targeted gene editing, as discussed above. Example 3: Kanghan Sequence Similarity and Protein Domains TABLE 1BLAST alignments of Kanghan proteins, with AT4G29770 as reference sequence.SequencePercentPercentLength ofMis-SEQ IDAccessionGeneIdentitiesPositivesAlignmentmatchesGapsNO:NP_001154277.1AT4G297701001003290015NP_194705.1AT4G2976060.43273.38278108214NP_197305.1AT5G1804048.22760.99282112417NP_680172.2AT5G1806563.41573.9812342116NP_175252.1AT1G4818035.90750.19259120519NP_175578.2AT1G5167036.62849.4217270418 TABLE 2Continuation of BLAST alignments of Kanghan proteins,with AT4G29770 as reference sequence.SequenceQueryQuerySubjectSubjectMaxSEQ IDAccessionStartEndStartEndE ValueScoreNO:NP_001154277.113291329068515NP_194705.15432932802.53E−11534714NP_197305.15132922521.06E−8025817NP_680172.25617871261.16E−4215516NP_175252.177329212391.45E−3915019NP_175578.280249281628.24E−2095.118 TABLE 3BLAST alignments of Kanghan proteins, with AT1G51670 as reference sequence.PercentPercentLength ofMis-SEQ IDIdentitiesPositivesAlignmentmatchesGapsNO:NP_175578.2AT1G516701001001780018NP_175252.1AT1G4818065.62576.2516048319NP_194705.1AT4G2976043.40753.8518262714NP_001154277.1AT4G2977036.62849.4217270415NP_197305.1AT5G1804045.83360.429647417NP_680172.2AT5G1806543.7551.049621216 TABLE 4Continuation of BLAST alignments of Kanghan proteins,with AT1G51670 as reference sequence.QueryQuerySubjectSubjectMaxSEQ IDStartEndStartEndE ValueScoreNO:NP_175578.2117811786.95E−12736618NP_175252.1116011535.32E−6120119NP_194705.120162191986.40E−2510814NP_001154277.128162802494.46E−2095.115NP_197305.169162771699.09E−1373.617NP_680172.22890311261.36E−0963.216 As set out in the tables above, which alternatively set out BLAST alignments with reference sequences that are the most divergent of the Kanghan genes (AT4G29770 and AT1G51670) the Kanghan gene family may be defined as including genes that encode proteins, that when optimally aligned, have at least 35% identity and/or at least 49% positive alignments, over a length of at least 90 amino acids, with BLOSUM or PAM substitution matrix, with gaps permitted. This Example further illustrates the existence of conserved protein domains encoded by Kanghan family genes, as depicted inFIGS.3through7. Conserved domain A is close to the amino end of the proteins, and as shown inFIGS.4and5, comprises a region that may be defined as having a reasonably high degree of consensus (80%) to the following sequence: cppDYDtStpAAhVAlpLISSARlhLKlDuhhTEYSsQaLhDpsutpp. Alternatively, at a slightly reduced level of consensus, conserved domain A may be defined as comprising a region that is defined as having a reasonably high degree of consensus (70%) to the following sequence: spphhpShupscGhCHPDC-KAssEpEDYDASQpAAhVAVsLISSARlhLKLDusaTEYSAQYLVDNAGpccs. Conserved domain B, as shown inFIGS.4and6, comprises a region that may be defined as having a high degree of consensus (100%) to the following sequence: hTVKDChphAhp. Alternatively, at a reduced level of consensus, conserved domain B may be defined as comprising a region that is defined as having at least 80% identity to the following sequence: LTVKDCLEhAhK-G. Alternatively, at a further reduced level of consensus, conserved domain B may be defined as comprising a region that is defined as having at least 70% identity to the following sequence: LTVKDCLEhAFKKG. Conserved domain C, as shown inFIGS.4and7, comprises a region that may be defined as having at least 80% identity to the following sequence: VshKGpVlEstshpEs.chhhpQs-huA+LHlFpPph. Alternatively, at a reduced level of consensus, conserved domain C may be defined as comprising a region that is defined as having at least 70% identity to the following sequence: VsMKGEVIEspsh-EAhcLllcQP-lGA+LHlFoPcl.FIGS.5,6and7illustrate consensus sequences using a sequence logo, which is a graphical representation of an amino acid or nucleic acid multiple sequence alignment (CLUSTL W). Each logo consists of stacks of symbols, one stack for each position in the sequence. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino or nucleic acid at that position. The width of the stack is proportional to the fraction of valid symbols in that position—positions with many gaps have thin stacks (Crooks et al., 2004; Schneider et al., 1990). Shading of the weblogo images reflects amino acid chemistry (AA). Conserved domain A is absent in Kanghan1 (At5g18040) in Columbia (Col) due to an 82 bp deletion compared to the orthologous gene in other species ofArabidopsis. As shown inFIG.2, the At5g18040 gene in Ecotype #95 contains conserved domain A, before the premature stop codon, so that the existence of this domain on its own does not appear to confer drought tolerance. Conserved domain B is relatively highly conserved in all members of Kanghan gene family in Columbia. In contrast, the premature stop codons of Kanghan1, Kanghan2, Kanghan3, 5 and Kanghan5 cause the loss of conserved domain B in #95. Accordingly, this absence of this domain is closely associated with the drought tolerance trait. Conserved domain C and the tasi-RNA target site are not present in Kanghan5 (At5g18065) in both Columbia and #95. BLAST searching reveals that Kanghan family genes are widely distributed in Brassicaceae, in addition to the six Kanghan genes inArabidopsis thaliana, there are also 5 members inArabidopsis lyrata,6 members inCaspsella rubella,5 members inBrassica rapa,11 members inBrassica napus,3 members inEutrema salsugineum,1 member inThellugiella parvula, and at least 24 members inCamelina sativa. Most of these Kanghan genes include all three conserved domains, and all of them contain conserved domain B. TABLE 5BLASTP search results identifying plant Kanghan proteins basedon sequence similarity to the protein encoded by AT4G29770.Length ofSEQ IDSeqGeneIdentitiesPositivesAlignmentNO:NP_001154277.1AT4G2977010010032915CAB43652.1hypothetical protein10010028245[Arabidopsis thaliana]NP_567833.1target of trans acting-10010027746siR480/255 [Arabidopsisthaliana]XP_002869410.1hypothetical protein85.5689.8927747ARALYDRAFT_491783[Arabidopsis lyratasubsp.lyrata]XP_006293511.1hypothetical protein73.18883.3327648CARUB_v10023817mg[Capsella rubella]XP_010447809.1PREDICTED:71.32682.827949uncharacterized proteinLOC104730345[Camelina sativa]XP_010438266.1PREDICTED:70.50482.3727850uncharacterized proteinLOC104721886[Camelina sativa]XP_010433066.1PREDICTED:70.92281.5628251uncharacterized proteinLOC104717221[Camelina sativa]XP_010447810.1PREDICTED:68.179.5727952uncharacterized proteinLOC104730347[Camelina sativa]XP_010436343.1PREDICTED:74.19482.6624853uncharacterized proteinLOC104720070[Camelina sativa]XP_002869411.1predicted protein65.23376.3427954[Arabidopsis lyratasubsp.lyrata]XP_010441644.1PREDICTED:66.79178.3626855uncharacterized proteinLOC104724792[Camelina sativa]XP_010494756.1PREDICTED:66.04577.9926856uncharacterized proteinLOC104771851[Camelina sativa]XP_010451117.1PREDICTED:65.10876.2627857uncharacterized proteinLOC104733215[Camelina sativa]XP_010441643.1PREDICTED:62.81676.927758uncharacterized proteinLOC104724791[Camelina sativa]XP_002871796.1predicted protein64.23474.0927459[Arabidopsis lyratasubsp.lyrata]XP_006280936.1hypothetical protein66.41577.3626560CARUB_v10026934mg[Capsella rubella]XP_010438262.1PREDICTED:65.55674.8127061uncharacterized proteinLOC104721884[Camelina sativa]XP_002871797.1predicted protein63.29672.2826762[Arabidopsis lyratasubsp.lyrata]NP_194705.1AT4G2976060.43273.3827814XP_006413298.1hypothetical protein54.37370.7226364EUTSA_v10026005mg[Eutrema salsugineum]XP_013601305.1PREDICTED:53.40970.4526465uncharacterized proteinLOC106308720[Brassica oleraceavar.oleracea]XP_013720359.1PREDICTED:54.44467.7827066uncharacterized protein(protein)LOC106424160170[Brassica napus](cDNA)XP_006412791.1hypothetical protein54.41268.7527267EUTSA_v10027444mg[Eutrema salsugineum]XP_013628081.1PREDICTED:54.85168.2826868uncharacterized proteinLOC106334325[Brassica oleraceavar.oleracea]XP_010436344.1PREDICTED:62.67370.0521769uncharacterized proteinLOC104720071[Camelina sativa]XP_009108974.1PREDICTED:53.33366.6727070uncharacterized proteinLOC103834660 isoformX2 [Brassica rapa]XP_010438269.1PREDICTED:50.15857.4131771uncharacterized proteinLOC104721889[Camelina sativa]CDY23253.1BnaA08g12930D52.23968.2826872[Brassica napus](protein)171(cDNA)XP_006294905.1hypothetical protein49.81163.7726573CARUB_v10023956mg[Capsella rubella]XP_009108973.1PREDICTED:51.49367.1626874uncharacterized proteinLOC103834660 isoformX1 [Brassica rapa]XP_009127652.1PREDICTED:51.51568.5626475uncharacterized proteinLOC103852500[Brassica rapa]XP_013659423.1PREDICTED:52.45365.2826576uncharacterized proteinLOC106364376[Brassica napus]NP_197305.1AT5G18904048.22760.9928217CDX68686.1BnaA01g07670D53.81570.2824978[Brassica napus](protein)165(cDNA)XP_013668007.1PREDICTED:5067.4226479uncharacterized proteinLOC106372351[Brassica napus]XP_013720313.1PREDICTED:50.56266.2926780uncharacterized proteinLOC106424116 isoformX2 [Brassica napus]AAM64385.1unknown [Arabidopsis49.45160.8127381thaliana]XP_013720312.1PREDICTED:50.56266.2926782uncharacterized proteinLOC106424116 isoformX1 [Brassica napus]XP_013659411.1PREDICTED:5269.625083uncharacterized proteinLOC106364365[Brassica napus]CDY23252.1BnaA08g12940D49.06465.1726784[Brassica napus]CDY55618.1BnaC03g77520D47.9465.1726785[Brassica napus](protein)166(cDNA)XP_009102300.1PREDICTED:46.79263.7726586uncharacterized protein(protein)LOC103828450167[Brassica rapa](cDNA)XP_009108975.1PREDICTED:47.9462.5526787uncharacterized proteinLOC103834660 isoformX3 [Brassica rapa]CDY55620.1BnaC03g77540D48.38763.7124888[Brassica napus]XP_010495074.1PREDICTED:49.43458.1126589uncharacterized proteinLOC104772124[Camelina sativa]XP_013674022.1PREDICTED:47.05965.1622190uncharacterized proteinLOC106378439[Brassica napus]XP_010433021.1PREDICTED:40.89255.7626991uncharacterized proteinLOC104717183[Camelina sativa]XP_010438210.1PREDICTED:42.80454.9827192uncharacterized proteinLOC104721842[Camelina sativa]XP_010447759.1PREDICTED:42.85755.6426693uncharacterized proteinLOC104730304[Camelina sativa]XP_006393225.1hypothetical protein42.91252.8726194EUTSA_v10011766mg[Eutrema salsugineum]CDY55622.1BnaC03g77550D43.93956.8226495[Brassica napus](protein)169(cDNA)KFK22930.1hypothetical protein42.33955.2424896AALP_AAs51418U000100[Arabis alpina]XP_010447760.1PREDICTED:42.57855.0825697uncharacterized proteinLOC104730305[Camelina sativa]XP_002894098.1F21D18.8 [Arabidopsis39.14752.3325898lyratasubsp.lyrata]XP_009108976.1PREDICTED:47.54164.4818399uncharacterized proteinLOC103834661[Brassica rapa]XP_010479661.1PREDICTED:39.68353.97252100uncharacterized proteinLOC104758482[Camelina sativa]XP_010462001.1PREDICTED:39.04453.39251101uncharacterized proteinLOC104742681[Camelina sativa]KFK30349.1hypothetical protein42.38754.73243102AALP_AA7G249900[Arabis alpina]XP_006304151.1hypothetical protein39.76853.28259103CARUB_v10010162mg[Capsella rubella]XP_010482049.1PREDICTED:38.67251.95256104uncharacterized proteinLOC104760782[Camelina sativa]NP_680172.2AT5G1806563.41573.9812316XP_010479658.1PREDICTED:36.82252.33258106uncharacterized proteinLOC104758479[Camelina sativa]XP_002891651.1predicted protein38.49251.98252107[Arabidopsis lyratasubsp.lyrata]NP_175252.1AT1G4818035.90750.1925919XP_006304149.1hypothetical protein36.86351.37255109CARUB_v10010150mg[Capsella rubella]XP_002891717.1hypothetical protein37.54951.78253110ARALYDRAFT_892299[Arabidopsis lyratasubsp.lyrata]XP_010471249.1PREDICTED:36.95750.87230111uncharacterized proteinLOC104751067[Camelina sativa]AAF79518.1F21D18.8 [Arabidopsis35.12548.39279112thaliana]XP_006303339.1hypothetical protein36.851.6250113CARUB_v10010206mg[Capsella rubella]XP_010501962.1PREDICTED:34.34849.57230114uncharacterized proteinLOC104779303[Camelina sativa]AAG50884.1unknown protein36.11149.6252115[Arabidopsis thaliana]XP_010442215.1PREDICTED:38.09550.6168116uncharacterized proteinLOC104725285[Camelina sativa]XP_010500744.1PREDICTED:30.03844.49263117uncharacterized proteinLOC104778076[Camelina sativa]KFK24575.1hypothetical protein47.58161.29124118AALP_AAs45078U000200[Arabis alpina]NP_175578.2AT1G5167036.62849.4217218XP_013684707.1PREDICTED:31.81850176120uncharacterized proteinLOC106389038 isoformX1 [Brassica napus]CDY43538.1BnaA01g07060D31.81850176121[Brassica napus]XP_013684772.1PREDICTED:31.81850176122uncharacterized proteinLOC106389038 isoformX2 [Brassica napus]XP_013596364.1PREDICTED:35.53753.72121123uncharacterized proteinLOC106304487 isoformX3 [Brassica oleraceavar.oleracea]XP_013750812.1PREDICTED:33.87150124124uncharacterized proteinLOC106453111 isoformX3 [Brassica napus]XP_013750806.1PREDICTED:33.87150124125uncharacterized proteinLOC106453111 isoformX1 [Brassica napus] Example 4: Modulating Abiotic Stress Response in Wheat with Kanghan Genes This example illustrates a genetic modification of a wild-type wheat by gene gun mediated transformation using a Kanghan gene construct, to modulate an abiotic stress response, in this case conferring heat tolerance. Transgenic constructs for overexpression ofArabidopsisKanghan family genes in wheat were produced using monocot special overexpression vector PANIC5E. This vector was designed for stable transformation and overexpression of heterologous Kanghan genes in wheat. Over expression ofArabidopsisKanghan1 (At5g18040) in one wheat wild type (Fielder) was achieved in this way by gene gun mediated transformation. The construct used to perform this transformation is shown inFIG.8. To illustrate the heat tolerance of the wheat transgenic lines, three-week seedlings of both wild types and T1 transgenic lines were heat treated at 42/38° C. (day/night). After two weeks of heat treatment, recovery at normal growth temperature was performed, and phenotypes observed. Heat tolerance was clearly observed in T1 transformants compared to non-transgenic plants under heat treatment. Non-transgenic plants displayed wilt symptoms or died. The transformants, on the other hand, recovered after transferring to normal growth temperature conditions, and were able to grow normally and transit to reproductive growth. To further illustrate the heat tolerance of the wheat transgenic lines, three week old seedlings of both wild-type and T1 transgenic lines were subjected to 40/38° C. (day/night) for three weeks, followed by a three week recovery period at 25° C. After this recovery period, the transgenic plants fully recovered whereas the control plants failed to recover (FIGS.9A and9B). After a further seven weeks at 25° C., the transgenic plants reached maturity and produced seeds (FIG.9C). Under standard growth conditions of 23° C. day/18° C. night, 16 h photoperiod (16 h light/8 h dark), and 200 μmol m−2 s−1 light intensity wild-type and transgenic plants are visually indistinguishable, however as determined by infrared thermal imaging using FLIR T640 Infrared Camera, the canopy temperature of T1 transgenic wheat plants is significantly lower (FIG.10). These studies illustrate the utility of the Kanghan genes in modulating abiotic stress response in crop species such as wheat, in this case to improve heat tolerance. Example 5: Identifying Kanghan Homologs inBrassica napus A BLAST sequence search was carried out on available genome and transcript data fromBrassica napusto identify potential homologues of at4g29770 (SEQ ID NOs: 2 and 15), at4g29760 (SEQ ID NOs: 1 and 14), at5g18040 (SEQ ID NOs: 4 and 17), at5g18065 (SEQ ID NOs: 3 and 16), at1g51670 (SEQ ID NOs: 5 and 18), and at1g48180 (SEQ ID NOs: 13 and 19). The potential candidates identified are provided in Table 6. TABLE 6Homologs ofArabidopsis thalianaKanghan family genes inBrassica napus.Homologs ofat4g29770, at4g29760, at5g18040, andHomologs of at1g51670 andat5g18065at1g48180BnaA01g06470D (SEQ ID NO: 79)BnaC01g08520D (SEQ ID NO: 63)BnaA07g02270D (SEQ ID NO: 86)BnaC01g08490D (SEQ ID NO: 77)BnaA08g12920D (SEQ ID NO: 66)BnaA01g07060D (SEQ ID NO: 121)BnaA08g12930D (SEQ ID NO: 72)BnaA08g12940D (SEQ ID NO: 84)BnaA01g07670D (SEQ ID NO: 78)BnaC03g77520D (SEQ ID NO: 85)BnaC03g77540D (SEQ ID NO: 88)BnaC03g77550D (SEQ ID NO: 95) A DNA neighbor phylogenetic tree of theBrassica napusKanghan gene candidates and theirArabidopsis thalianacounterparts is provided inFIG.11and a protein neighbor phylogenetic tree is provided inFIG.12. A DNA neighbor phylogenetic tree of theBrassica napusKanghan gene candidates is shown inFIG.13. The candidates indicated by arrows were selected for targeting by RNAi. A sequence alignment of theBrassica napushomologues of at4g29770, at4g29760, at5g18040, and at5g18065 is provided inFIG.20. These homologues may be characterized by their consensus sequences: a first 100% consensus sequence DsucpAshlAssLISstRhhhpLDp.hTpYSsQaLVDNAh . . . p (SEQ ID NO: 126) and a second 100% consensus sequence ptsplhl+tsLthAhKcGlP+ . . . WsHlGsl . . . Ps.h.h.shV.hKGphhEsKp.-tA.cLhppt.luAKLhVFsPph-h . . . tha.G.uG . . . topYVGLRDshlsu.tphps.shhpVplhYKKp.thhpVuhs.hh . . . ppus. pppltP.hLLVDFhlPph.h (SEQ ID NO: 127). A sequence alignment of theBrassica napushomologues of at1g51670 and at1g48180 is provided inFIG.21. These homologues may be characterized by their consensus sequences: a first 100% consensus sequence MAD.HLhPtLTRHRHTVPsISDDFYNYMKLIpKT-PEIMSKLLPILRTIPDSGIQLlp (SEQ ID NO: 128) and a second 100% consensus sequence R-chpL-cQYAVLQYD-HEhVWAVIAAp.l.h (SEQ ID NO: 129). Example 6: TargetingBrassica napusKanghan Genes by RNAi Primer Design Two conserved fragments from 12 putativeBrassica napusKanghan genes, identified based on ClustalW multiple alignment, were used to design two pairs of RNAi primers. The reverse primers were designed to include a BamH1 restriction site and a Sal1 restriction site to facilitate cloning. The first primer pair was designed to target BnaC03g77540D (LOC106364365): RNAiF1 GP438:(SEQ ID NO: 20)TAGATTCTGCTGAGAGAGCCGCTACRNAiR1 GP439:(SEQ ID NO: 21)GGATCCGTCGACGCACCTATGGGTCCATGCTTTAAC The second primer pair was designed to target BnaA08g12920D (LOC106424160): RNAiF2 GP440:(SEQ ID NO: 22)TCATCCAGATTGCCAACGAGRNAiR2 GP441:(SEQ ID NO: 23)GGATCCGTCGACACGCATCCTCCAGTGTCTTAG Production of BnKanghan RNAi Construct and Establishment ofBrassica NapusRNAi Lines To generate a cDNA library ofBrassica napus, total RNA was isolated from 3-week-old leaves of canola wild type ‘Hero’ using the Plant RNeasy Mini Kit (Qiagen). Then, RNA samples were used for library construction using the QuantiTect Reverse Transcription Kit (Qiagen). The primer pairs RNAiF1 GP438 (SEQ ID NO: 20)+RNAiR1 GP439 (SEQ ID NO: 21) and RNAiF2 (SEQ ID NO: 22)+RNAiR2 GP441 (SEQ ID NO: 23) were used separately to amplify fragments from two target BnKanghan genes from the obtained cDNA library. Each of the resulting PCR products was isolated and cloned into the pGEM®-T vector (Promega, USA). A map of the pGEM-T vector is provided inFIG.14. Then, two copies of the Kanghan gene fragments were subcloned into the pCAMBIA 1301-35S-Int-T7 vector in opposite orientations using a Pst1, Sal1 digest and a BamH1, Sac1 digest to generate two RNAi constructs, one for each gene fragment. A map of the pCAMBIA 1301-35S-Int-T7 vector is provided inFIG.15and a partial map of the resulting RNAi constructs is provided inFIG.16. Next, a genetic modification of canola wild type ‘Hero’ was conducted using both of these completed RNAi constructs throughagrobacterium-mediated transformation aimed to obtain increased drought tolerance. Positive transformants were confirmed using a pair of hygromycin specific primers (HptF TACACAGCCATCGGTCCAGA (SEQ ID NO: 24) and HptR GTAGGAGGGCGTGGATATGTC (SEQ ID NO: 25)). A cross was carried out between T1 positive transformants from the two different constructs. In the C2 generation, lines harboring both constructs together were selected for further evaluation of silencing of BnKanghan family genes and drought tolerance traits. To assess the expression level of BnKanghan family genes in transgenic and crossing lines, a number of primer pairs were designed for qRT-PCR assays to assess the expression levels of seven candidate Kanghan genes fromBrassica napus. The targets of these primer pairs are identified in Table 7. In total, 12 lines harboring both RNAi constructs from the C2 generation were selected to detect expression level changes of BnKanghan family genes. Each line tested showed decreases in expression of at least three BnKanghan genes. The most commonly suppressed genes were: BnaA07g02270D, BnaA08g12920D, and BnaC03g77550D, followed by BnaC03g77540D and BnaC01g08490D. TABLE 7qRT-PCR primers targeting BnKanghan family genes.SEQprimerIDproductno.NO:primer sequencelengthKanghan geneGP63526CGCTACGAGGCACGTACTCAAT103BnaA07g02270DGP63627CTCGGTCTTCCCCGCTTTCGP63728GCTTAGAGACGTGATCCTGGTAGC128BnaA08g12920DGP63829CCAGTGTGGTGAACATACGGCGP63930GTTTTGTTGGTCTCTTCTCTTTGC71BnaC01g07670DGP64031TTCTTAAGAGGCGTTTCAGATGGGP64132TGATTTGGGTTTTGCCTGATAC69BnaC03g77540DGP64233GAAACAAACCATAAATGAGTTGCCGP64534CATTTGGGATGTGTCGATTGAG165BnaC03g77550DGP64635CCCACGTAGCTTGTTCCGTTGP64936AACACTGTCACGCAGATTGCC124BnaA01g06470DGP65037CTGTCCAGGTTAGCTACCATACGAGP65538CGGTATCCAACTCATTCGAAGG121BnaC01g08490DGP65639TCAAGTATATACTGGGTTGGCTGC Testing Canopy Temperature and Drought Tolerance ofBrassica napusRNAi Lines Individual lines C2-83-20 and C2-83-10 each showed decreased expression of six BnKanghan genes. These two lines were selected for further drought tolerance measurements. To predict the potential drought tolerance of the C2-83-20 and C2-83-10 lines, the canopy temperatures were measured using an infrared camera. In comparison to wild type plants, higher canopy temperatures were observed for the transgenic plants (FIG.17) indicating a lower leaf water potential. These RNAi phenotypes are similar to loss-of-function alleles of At Kanghan genes inArabidopsis, which suggests a similar role for the BnKanghan genes in canola. To assess the drought tolerance of the transgenic plants, four weeks-old plants of both wild type and these two transgenic lines were subjected to drought treatment. The same amount of soil and water were applied to each individual plant before treatment, and then the water supply was stopped. After two weeks of drought treatment, recovery by re-watering of the plants was performed. The resulting phenotypes are shown inFIGS.18and19. Increased drought tolerance was clearly observed in transgenic lines compared to wild type plants under drought conditions that lead to wilt symptoms or death of the wild type plants. The transformants, on the other hand, recovered after being transferred to normal watering conditions and were able to grow up normally and transit to reproductive growth. This demonstrates that silencing of BnKanghan family genes in crop species, such as canola, can improve drought tolerance. Example 7: TargetingBrassica napusKanghan Genes by CRISPR The pan-genome architecture ofBrassica napuswas recently released (Song et al., 2020), providing a possibility to identify all the members of Kanghan gene family inB. napus. Furthermore, through CRISPR/Cas genome editing technologies, the knock-out of designated member(s) of Kanghan gene family can used to generate non-GMOB. napuslines with high abiotic stress resistance traits. Identifying Kanghan Homologs inBrassica napusand Other Brassicaceae Species Genome-wide identification of Kanghan gene family numbers was performed in multiple Brassicaceae species, in which the whole genome sequence information has been released. Kanghan homologs inA. thaliana, A. lyrata, A. helleri, B. napus, B. oleraceaandB. rapawere identified and a phylogenetic tree was built based on their protein sequence (FIG.22). The genes included in the phylogenetic tree are identified in Table 8. Pairwise analysis between each member will be conducted to check the closest homologs for each member in different species. After confirmation of their phylogenic relationship, CRISPR/Cas knock-out will be designed to target different combinations of homologs inB. napus. TABLE 8Sequences used to produce the phylogenetictree provided in FIG. 22Gene nameSpeciesSEQ ID NO:BnaA08g12920DBrassica napus66Bra010276Brassica rapa144B03g175440Brassica oleracea68BnaC03g77550DBrassica napus95BnaA08g12930DBrassica napus72Bra039897Brassica rapa86BnaA07g02270DBrassica napus86BnaC03g77520DBrassica napus85Bra010278Brassica rapa147B01g011940Brassica oleracea65BnaA01g07670DBrassica napus78Bra011210Brassica rapa75g04250Arabidopsis halleri150At4g29760 (KH4)Arabidopsis thaliana14g04249Arabidopsis halleri151AI scaffold 0007 1135Arabidopsis lyrata54fgenesh2 kg.7 1216Arabidopsis lyrata47AT4G29770.1g04248Arabidopsis halleri154At4g29770 (KH2)Arabidopsis thaliana15At5g18040 col (KH1)Arabidopsis thaliana17At5g18040 95 (KH1)Arabidopsis thaliana155AI scaffold 0006 1720Arabidopsis lyrata59gl3293Arabidopsis halleri157At5g18065 (KH3)Arabidopsis thaliana16AI scaffold 0006 1721Arabidopsis lyrata62g13290Arabidopsis halleri159fgenesh1 pm.C scaffoldArabidopsis lyrata981003033g17457Arabidopsis halleri161At1g48180 (KH6)Arabidopsis thaliana19scaffold 105093.1Arabidopsis lyrata110At1g51670 (KH5)Arabidopsis thaliana18AI scaffold 0001 4560Arabidopsis lyrata107g21951Arabidopsis halleri164 Multiplexed Gene Editing Through an Optimized CRISPR/Cas9 Toolkit A multiplexed toolkit (Cermak et al., 2017) has been selected and optimized for application inB. napus. This toolkit could carry up to 12 guide RNAs (gRNAs) to realize the knock-out of multiple target genes through one construct. Reducing the number of constructs will ideally reduce the cost of plant transformations and downstream molecular confirmation for gene editing. Targeted gRNA design will be performed through multiple bioinformatic tools to avoid potential off-targets and cover as many as Kanghan homologs as possible. gRNAs targeting conserved regions and specific regions of Kanghan family genes will be confirmed after a genome-wide SNP/indels screening for duplicates and homologs in different subgenomes (AA and CC). The final selected 6 gRNAs will be tandem connected with Csy-type ribonuclease 4 (Csy4) for simultaneous expression through Pol II promoter (FIG.23) using Gibson assembly (Gibson et al., 2009) (FIG.24) through a specific designed primer list (Table 9). The final plasmid for plant transformation will be constructed following Golden Gate® protocol to link Cas9, gRNA cassette and selection markers together into pTRANS_220d backbone (FIG.25), a binary vector for T-DNA insertion with neomycin phosphotransferase II (npt II) selection (FIG.26). TABLE 9Primers for gRNA productionPrimerNameSequenceSEQ ID NO:DG564TGCTCTTCGCGCTGGCAGACATACTGTCCCAC130DG565TCGTCTCCAGCGCACTCGAGCTGCCTATACGGCAGTGAAC131DG566TCGTCTCACGCTTTCAAGGAGTTTTAGAGCTAGAAATAGC132DG567TCGTCTCCCTTTGAAAGAAGCTGCCTATACGGCAGTGAAC133DG568TCGTCTCAAAAGCGTACTCGGTTTTAGAGCTAGAAATAGC134DG569TCGTCTCCCTCTCAGCAGAACTGCCTATACGGCAGTGAAC135DG570TCGTCTCAAGAGAGCTGCTAGTTTTAGAGCTAGAAATAGC136DG571TCGTCTCCGCCGAGTACTCGCTGCCTATACGGCAGTGAAC137DG572TCGTCTCACGGCTCAGTTCCGTTTTAGAGCTAGAAATAGC138DG573TCGTCTCCGCATTGGGCACACTGCCTATACGGCAGTGAAC139DG574TCGTCTCAATGCTCTCTCCTGTTTTAGAGCTAGAAATAGC140DG575TCGTCTCCACCATACGAGCACTGCCTATACGGCAGTGAAC141DG576TCGTCTCATGGTAGCTAACCGTTTTAGAGCTAGAAATAGC142DG577TGCTCTTCTGACCTGCCTATACGGCAGTGAAC143 Generating Transgenic Plants ThroughAgrobacterium-Mediated Transformation Transformation will be conducted in canola cultivar DH12075 using the generated CRISPR/Cas9 construct throughagrobacterium-mediated transformation. Positive transformants will be confirmed using a pair of npt II specific primers in T0 generation transgenic lines. High Throughput Validation for the Gene Editing Mutations in targeted genes from T0 generation will be identified. To detect the editing on all ten KH homologs inB. napusfor hundreds of T0 and T1 generation positive lines, a cost-efficient high throughput detection method is desired. A workflow using droplet digital PCR (ddPCR) assay will be established to achieve a high throughput validation. Fluorescent probes targeting gDNA-associated regions will be designed, and the corresponding primer will be selected based on SNP/Indels information obtained above. Thus, gene editing information of every Kanghan homolog in each transgenic line will be identified. The combination of different mutations in different homologs will provide transgenic materials to investigate knock-out lines of At5g18040 (KH1) homologs, At4g29770 (KH2) homologs, At5g18065 (KH3) homologs, Atg29760 (KH4) homologs, At1g51670 (KH5) homologs and knock-out lines for all Kanghan gene family members in canola, respectively. Non-GMO lines with successful gene-editing in Kanghan gene(s) but without the transformed plasmid will be identified in T1 and T2 generations. While the present application has been described with reference to specific examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the present application is intended to cover various modifications and equivalent arrangements encompassed by the scope of the appended claims. 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